Gretchen Lambert 

12001 11th Ave. NW, Seattle, WA 98177

206-365-3734 or

home page:


Number 68                                                                                        November 2011


Thanks so much for the many emails and letters of condolence I received after Charles’s death June 1; they have been a great source of comfort to me and to our daughters, to hear from so many friends and colleagues. Please see below the link to a short biography. Thanks also to all of you who sent the many contributions for this issue and also for your words of encouragement for continuing AN; I am happy to know that AN is still an important resource. There are 162 new publications listed at the end of this newsletter, many abstracts from recent meetings, announcements of upcoming meetings in 2012, and much more! This is the largest issue ever.


*Ascidian News is not part of the scientific literature and should not be cited as such.




1. Charley Lambert’s last publication, a remarkable summing up of his past 20+ years of research, can  be found in the special issue of Molecular Reproduction and Development, Vol. 78, issues 10-11 (Oct. – Nov.), which contains the proceedings of the symposium Mechanisms of egg maturation and fertilization: From sea to land, held at the Friday Harbor Laboratories Sept. 2010. You can download the article from . Signaling pathways in ascidian oocyte maturation: The roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD (pages 726–733).

   I was invited to write a short biography of Charley for this issue; you can find it at and download it if you wish. It includes 2 of my favorite photos I took of him, as well as just a few of the many memorable tributes I received from former students, colleagues and friends.

   The issue also contains several other ascidian papers (see list of Recent Publications at the end of this newsletter).  


2. Two scholarship funds have been set up in Charley’s name:

a) The Charles Lambert Memorial Endowment. Provides assistance to graduate students for research and/or coursework at the Univ. of Washington Friday Harbor Labs that includes cell or developmental biology of marine invertebrates, or any aspect of ascidian biology. Charley and I met at the Labs in 1964, when we went there to take summer classes. Without scholarship help we could not have attended; the rest is history! So we have been very dedicated to helping others attend this fine institution.

b) The Charles Lambert Memorial Scholarship fund. Charley taught at Calif. State Univ. Fullerton Biol. Dept. for 28 years, was the campus Outstanding Professor for 1986 and earned other awards as well. The scholarship provides an annual graduate student award for research in Invertebrate Cell and Developmental Biology, or any aspect of ascidian biology.

   We also set up an online photo album with many photos old and new. It’s also a site where you can add a tribute if you wish. . You can click on an album and then on Slideshow for larger images with labels. We hope that some of you will be inspired to add comments/photos. 


3. From Kevin Heasman, Cawthron Institute, Nelson, New Zealand: the next International Invasive Sea Squirt Conference will be held in Nelson April 11-14, 2012.  

   Invasive ascidians are impacting ecosystems, creating a nuisance for the aquaculture industry, and are a major component of fouling communities. The aim of the fourth conference is to further our knowledge, address the problems associated with invasive species of tunicates and investigate avenues of exclusion and mitigation.

   Details can be found at  Abstract due date is 1 February 2012. The Conference will start on April the 11th with a Taxonomy workshop held by Gretchen Lambert. The following two days (12th and 13th April) will each start with a plenary speaker with the rest of each day focused on presentations and evening poster sessions. On Saturday 14th April a field trip to the Marlborough Sounds to look at fouling, aquaculture and enjoy a day in the spectacular Marlborough Sounds.


4. From Ken Hastings, Montreal Neurological Institute, McGill University, Montreal, Quebec,


    The Sixth International Tunicate Meeting was held in Montréal, Québec, Canada, at McGill University, July 3-7, 2011. 

   Abstract details are at  A number of the abstracts are included below in this newsletter. Click on Program Book from the left-hand menu of the website for the complete list of talks and posters with abstracts. This pdf is downloadable.

   Lifetime Achievement Awards were given to Nori Satoh and Christian Sardet at the final banquet. Both were given personalized copies of Berrill's "The Origin of Vertebrates" in honor of John Berrill who taught at McGill Univ. for many years and published many classic papers on ascidian biology, especially embryology.

   The next ITM meeting is tentatively scheduled for sometime in 2013.


5. The 7th Intl. Conference on Marine Bioinvasions was held 23-25 August in Barcelona, Spain. The meeting included a number of talks on ascidians; the program can be viewed at Two of the abstracts are included in this newsletter.


6. From Mary Carman, Biology Dept., Woods Hole Oceanographic Institution, Woods Hole, MA.

   The papers from the International Invasive Sea Squirt Conference III (held at Woods Hole MA April 2010) have now been published in Aquatic Invasions and can be downloaded from the Open Access website.  Many thanks to the guest editors Andrea Locke and Mark Hanson.


7. From Christina Simkanin and Greg Ruiz: The National Exotic Marine and Estuarine Species Information System (NEMESIS) Exotic Tunicate Database.

   NEMESIS was developed by the Marine Invasions Lab at the Smithsonian Environmental Research Center.  The database includes detailed information on approximately 500 different non-native species that have invaded marine and estuarine environments in the continental U.S., Alaska and Hawaii.  It provides information on the invasion history and current distribution of each species, and summarizes key information on the ecology and known impacts of each invader.  In December 2011, the Marine Invasions Lab will be launching the Tunicate section of NEMESIS, making information on 28 exotic tunicate species accessible through the NEMESIS website (  The Tunicate database contains geographic and ecological information for each of the 28 species, many of which have been introduced to multiple locations throughout the world.


8. The third edition of the Ascidian Workshop in Bocas del Toro Smithsonian Tropical Research Institute (STRI), Panama was held last June 9-30. The workshop was called Advanced Tunicate Biology: Integrating Modern and Traditional Techniques for the Study of Ascidians. Eighteen advanced undergrad, and graduate students, pos-docs, and researchers attended the workshop, funded by the NSF Pan American Advanced Studies Institute (PASI). Because of the nature of the grant, half of the participants were from the U.S., and the rest from other American countries (Canada, Mexico, Brazil, Colombia, Chile, Argentina), The workshop also included one student from Singapore and another from South Africa who obtained other funding.

   Activities included field collecting, taxonomy labs, lectures on recent advances in many aspects of ascidian biology (symbiosis, chemical ecology, evo-devo, population genetics, regeneration, and invasion biology), and plenty of hands-on lab time. Field collecting was carried out by snorkeling almost every other day at various sites including coral reefs, mangroves, seagrass beds and harbor docks. One optional scuba dive trip was also included. In the laboratory, each participant was provided with a stereo dissecting microscope, and a compound microscope was shared by every two participants. This allowed us to study ascidian structures and internal morphological characters, particularly important for taxonomic keys. The last days were reserved for research projects on many interesting subjects, such as larval choice mediated by environmental cues, larval pigment development, the role of artificial substrates in species introduction, climate change effects on introduced species, the use of the ascidian tunic by bivalves, bacteria associated with the tunic, morphological comparison of distant populations of Phallusia nigra (Dr. Hirose brought some samples from Okinawa e.g.), antifeedant properties of ascidian extracts, and variation of egg size in Ascidia curvata and Ascidia sydneiensis from different populations. 

   The workshop was very successful in promoting future research collaborations among attendants and instructors of the course, which was one of its main goals. We are thankful to the staff at Bocas Research Station and its director Rachel Collin for such a warm welcome and being such good hosts! Last but not least, we´d like to mention that the meals at Bocas Station were always good and provided us with the energy to withstand the long schedules of the course.  We had an enjoyable time and great logistics for field and lab activities.  We hope to see some of you in the next edition already planned for 2014!

Rosana Rocha (, Xavier Turon (, Euichi Hirose (, Federico Brown (, Billie Swalla (

  More information on the STRI lab and this and other courses can be found at




1. Billie Swalla’s Lab at the University of Washington is working on a comprehensive phylogeny of the molgulid ascidians. If you have a molgulid species that you would like identified and/or sequenced, please contact me, and send a voucher specimen fixed in 4% formaldehyde (10% seawater formalin) and one sent in 95% ethanol to Professor Billie J. Swalla, Dept. of Biology, 238 Kincaid Hall, Univ. of Washington, Seattle, WA 98195.


2. Ecuadorean Ascidian Studies. Gabriela Agurto1, Stefania Gutierrez2, & Federico Brown1,2.  11Centro Nacional de Acuicultura e Investigaciones Marinas (CENAIM), San Pedro, Ecuador; 2Universidad de los Andes, Bogotá, Colombia.

   Taking advantage of biological diversity in Ecuador, and funded by the Ecuadorean Council for Science and Technology (SENESCYT), we are currently collecting and describing species in continental and insular Ecuadorean coastlines. Ascidian studies in Ecuador are scarce or absent due mainly to a lack of local taxonomic experts. Therefore we plan to begin a survey and broad sampling of ascidian species in El Oro, Guayas, Santa Elena, Manabí, Esmeraldas, and Galápagos Provinces. We are focusing on morphological descriptions of budding in collected colonial ascidians, and studying regeneration abilities of colonial ascidians in culture. The objectives of this project are: (1) collecting and describing ascidian diversity in Ecuador, both morphologically and molecularly; (2) morphological descriptions of budding and regeneration in selected colonial ascidians; (3) establishing a colonial ascidian developmental model system for stem cell and regeneration studies.


3. From Carmen Primo, National Centre for Marine Conservation and Resource Sustainability, Australian Maritime College, Univ. of Tasmania, Launceston, Tasmania, Australia.

   Elsa Vazquez and I are preparing the Ascidiacea section for the "CALM/SCAR-MarBIN Biogeographic Atlas of the Southern Ocean". This will take the form of a collection of maps and synthetic texts presenting the key biogeographic patterns, and their causal processess, of the main benthic and pelagic/nektonic taxa in the Southern Ocean south of the Subtropical Convergence. There will be a hard copy in large format, as well as a dynamic online version on SCAR-MarBIN. Illustrative maps will be the key elements of the AAtlas, which is not intended to be a Treatise of the Southern Ocean Biogeography.


4. From Tom Ermak, Sea Peach BioScience, 65 Hagen Road, Newton Centre, MA

   I started two ascidian websites this year. The first one is my consulting/microscopy website, Sea Peach Bioscience (, and the other is an image library called Tunicarium ( containing a collection of my histology and EM micrographs.  My autoradiography/histology slide set from the 1970s is still in excellent condition and I have started to digitally re-photograph many of the images from the slides. In late spring, I set up a photo-microscopy facility in my home office with bright field and stereo-zoom photomicroscopes. During the summer, I participated in the Massachusetts marine invasive species monitoring program ( surveying invasive invertebrates (including 6 ascidian species) and algae at marinas in Salem Sound and Cape Cod Bay, MA.  Some of the stereo-zoom microscopic images of live colonial ascidians have been added to Tunicarium.


5. From Edwin L. Cooper and David Yao, Lab. of Comp. Neuroimmunology, Dept. of Neurobiology, David Geffen School of Medicine, UCLA, Los Angeles, CA
Diving for drugs: marine tunicate anticancer compounds
The marine biosphere boasts tremendous biodiversity replete with structurally-unique, active and selective secondary metabolites. Bioprospecting for anti-tumor compounds has been rewarding, and tunicates have been especially successful in yielding prospective cancer therapies. These compounds are now subjected to clinical trials in Europe and the US. With the ongoing search for potent and specific cancer drugs, this review redirects attention to this promising – yet still novel and unexplored – source of potential pharmaceuticals. We discuss marine-derived anti-tumor drugs, their structures, and their various types and levels of anti-tumor activities. [submitted to
Drug Discovery Today]


5. From Christina Simkanin, Dept. of Biology, Univ. of Victoria, British Columbia, Canada

   Biotic resistance to the infiltration of natural habitats by an invasive ascidian: examining the role of predation in the distribution of Botrylloides violaceus.

   Anthropogenic marine habitats, such as marinas and breakwaters, are frequently colonized by non-indigenous species (NIS). Comparative studies show that few sessile NIS spread to nearby natural benthos, but little is known about the processes affecting infiltration of these habitats. In British Columbia, the invasive colonial ascidian Botrylloides violaceus is widespread, but it is largely restricted to man-made structures at marinas and aquaculture facilities.  To determine whether predation by native species, an element of biotic resistance, is influencing the abundance and distribution of B. violaceus in natural benthic habitats, we conducted a series of predator exclusion experiments.  Established adult colonies and newly settled juvenile B. violaceus were transplanted onto replicate PVC panels and deployed at marina pilings and adjacent natural rocky reefs at two locations. Panels were assigned to three treatments at each location: full cage (1 cm or 0.15 cm mesh), partial cage, and no cage.  Survival was significantly lower in uncaged treatments for both juvenile and adult ascidians, suggesting predation can limit the abundance and distribution of this species. This trend did not vary between piling and rocky reef habitats however, and predation rates were similar for both life stages.  Our results suggest that adult and juvenile B. violaceus are vulnerable to predation in both habitats studied and floating habitats, such as marina docks, may provide a refuge from predation for this species.  If larvae of B. violaceus can disperse from dock floats into adjacent natural rock, biotic resistance, through predation by native species, may mediate the likelihood of successful infiltration.  


6. From V.K. Meenakshi: D.Shanmugapriya, Assistant Prof. of  Chemistry, is doing her Ph.D, entitled “Studies on some ascidians of Tuticorin coast” under the guidance of  Dr. Dr.S.Gopalakrishnan, Dept. of Pharmaceutical Chemistry, Manonmanium Sundaranar University, Tirunelveli and coguidance of Dr. V.K. Meenakshi, Dept. of Zoology, A.P.C.Mahalaxmi College for Women, Tuticorin, Tamilnadu, India.

   GC-MS analysis, HPTLC technique and pharmacological studies are in progress in the simple ascidian Phallusia nigra.  GC-MS analysis of the methanolic extract of the animal revealed the presence of eleven components. Methyl 3-bromo-1-adamantaneacetate (24.65%), n-Hexadecanoic acid (24.45%), 11-Hexadecen-1-ol,(Z)-(17.64%)  and 2,6-Dimethyl-6-trifluoroacetoxyoctane (9.82%) are some of the components found to have antimicrobial, antifungal, antioxidant, hypocholesterolemic and hemolytic properties. These chemical components have not been reported elsewhere from ascidians especially Phallusia nigra and hence can be considered as a first report. HPTLC analysis of the flavonoids  present in the simple  ascidian Phallusia nigra showed the presence of five flavonoids such as Gallic acid, Ferulic acid, Caffeic acid, Isoquercitrin and quercetin.  Pharmacological properties like anti pyretic and analgesic activity of the methanolic extract of Phallusia nigra indicates the presence of  biomedical components.   


7. From Gretchen Lambert: In July I participated in a Navy 5 day biological survey of San Diego Bay with several other taxonomists; we sampled a number of marina floating docks and examined many settlement panels both wood and plastic that had been in place for a year and were thus heavily fouled. The emphasis was on documentation of invasive species; with Charley’s and my long familiarity with the ascidians of southern California we were looking forward to resurveying many of our sites after an absence of 11 years or more, and therefore I wanted to honor this commitment. I invited a colleague and 2 graduate students working on southern Calif. ascidians to participate in the survey, and at its completion the 4 of us surveyed a number of marinas in nearby Mission Bay.

    Of the 17 non-native ascidian species we originally documented, all but one are still present and thriving. Very surprisingly, we did not find any new ascidian introductions.

[Lambert, C. C. and Lambert, G. 1998. Non-indigenous ascidians in southern California harbors and marinas. Mar. Biol. 130: 675-688.

Lambert, C. C. and Lambert, G. 2003. Persistence and differential distribution of nonindigenous ascidians in harbors of the Southern California Bight. Mar. Ecol. Prog. Ser. 259: 145-161.

Cohen, A. N., Harris, L. H., Bingham, B. L., Carlton, J. T., Chapman, J. W., Lambert, C. C., Lambert, G., Ljubenkov, J. C., Murray, S. N., Rao, L. C., Reardon, K. and Schwindt, E. 2005. Rapid Assessment Survey for exotic organisms in southern California bays and harbors, and abundance in port and non-port areas. Biol. Invasions 7: 995-1002.

Lambert, G. 2007. The nonindigenous ascidian Molgula ficus in California. Cah. Biol. Mar. 48: 95-102.]




1.Society for Integrative & Comparative Biology (SICB) Annual Meeting, January 3-7, 2012, Charleston, SC.

Experimental whole body regeneration among botryllid ascidian species in San Francisco Bay. Chow, B., Wray, M., Villines, B., Pinnick, G., Sheets, E., Spaulding, J., Cohen, C. S. San Francisco State Univ.

    Regeneration processes show a broad phyletic distribution and dramatic variation in potential across diverse organisms. Colonial ascidians in the family Botryllidae are the only chordates known to be capable of whole body regeneration (WBR). WBR may occur following the artificial removal of zooids and buds, leaving behind ampullar fragments that may ultimately lead to production of new functional zooids. Prior work on botryllid WBR has relied on laboratory assays, primed by application of retinoic acid, and aimed at elucidating intrinsic differences in mechanism through morphological observations. We compared the regenerative abilities of three botryllid species (Botryllus schlosseri, Botrylloides violaceus, and Botrylloides sp.) at three separate locations in San Francisco Bay. All three species were successful in regenerating complete zooids from ampullar fragments in the field without exogenous application of retinoic acid; however, success rate varied among sites and species. B. violaceus had significantly lower success overall with complete regeneration occurring at only one site. Conversely, B. schlosseri and Botrylloides sp. showed success at both early and later stages at all three sites. Mean time to regeneration for successful individuals was significantly longer between B. violaceus and the other two species and field regeneration times were longer than published laboratory studies with RA. This study shows that WBR occurs in the field and varies among species and populations, thus potentially affecting population viability following disruptive processes such as predation, senescence, or intentional human removal.


2. 35th Scientific Meeting of the Assoc. of Marine Laboratories of the Caribbean, May 23-27, 2011, Univ. of Costa Rica, San Jose, Costa Rica.

Diversity and Distribution of Tunicata (Urochordata) of Tobago. Linda Cole, Smithsonian Institution National Museum of Natural History, Dept. of Invertebrate Zoology, Wash., D.C.

   The beautiful island of Tobago is the southernmost Caribbean island. The sister island of Trinidad, it belongs to the Republic of Trinidad and Tobago. Thirty two species of tunicates were collected from Tobago from depths of 40 meters or less and they are listed. Tunicates listed in this work are from collections made in 1956, 1991, 1993, 2002 and 2006 and although specimens were collected from the Atlantic Ocean side of the island as well as the Caribbean Sea side, all species turned out to be typical Caribbean species. [poster]


3. ColEvol (Colombian Evolutionary Biology) Meeting, 28- 29 July 2011, Medellín, Colombia.

Identificación y caracterización de la gemación en ascidias de Colombia y Panamá.

Pilar Andrea Endara  Pinillos, Federico D. Brown. Laboratorio de Biología del Desarrollo Evolutiva (EvoDevo), Depto. de Ciencias Biológicas, Univ. de los Andes, Bogotá D.C., Colombia.

   El presente estudio hace un registro de especies de la clase Ascidiacea por primera vez en Colombia. El muestreo, se realizó en sitios de manglares a no más de dos metros de profundidad, en tres diferentes localidades de la costa Caribe colombiana: la isla Barú en el Archipiélago Corales del Rosario, la isla Titipán que hace parte del Archipiélago de San Bernardo e Inca Inca en las cercanías de Santa Marta. Se identificó mediante claves taxonómicas y se encontraron siete especies ya registradas en Venezuela y Panamá y una posible especie nueva. Las especies compartidas en los tres países de referencia en el Caribe fueron Rhopalaea abdominalis, Botrylloides nigrum, Pyura vittata y Symplegma brakenhielmi.  Esta última, al parecer es ubicua, pues se encontró en las tres localidades colombianas  y también está  en Bocas del Toro, Panamá y La Restinga, Venezuela. Adicionalmente se hicieron observaciones de la gemación de las ascidias se mapeó este carácter en la filogenia de estos organismos para analizar la relación con la evolución de su colonialidad. Se colectaron ascidias con gemación por estrobilación y paleal, pero no se colectó ninguna ascidia con gemación estolonial. La estrobilación abdominal es un rasgo característico de la familia Didemnidae, y la gemación paleal ocurre en las Botryllidae. La colonialidad está relacionada a cambios en rasgos del desarrollo y reproductivos, como la viviparidad, la miniaturización del cuerpo, diferencia en la diferenciación de los órganos adultos en las larvas de ascidias solitarias y coloniales y mayor potencial de regeneración. Estos cambios modulares en el desarrollo, podrían generar también diferencias en los mecanismos de gemación en cada grupo de ascidias coloniales. De este modo, se relacionó la agregación de los individuos de una colonia con el desarrollo los diferentes tipos de gemación. Se encontró que el índice de agregación, i.e. la relación entre el área que ocupan los zooides adultos en la colonia y la distancia entre estos, es mayor en aquellas especies en que se ha registrado dos tipos de gemación, paleal y vascular, es decir, en miembros de la familia Botryllidae. 


4. 6th International Tunicate Meeting, Montreal (Canada), July 3-7, 2011.


a) Determining the native region of the putatively invasive ascidian Didemnum vexillum. Lauren Stefaniak, Huan Zhang, Adriaan Gittenberger, Kirsty Smith, Kent Holsinger, Senjie Lin, Robert Whitlatch.

   Over the past thirty years, many new populations of a colonial ascidian, recently identified as Didemnum vexillum, have been recorded in most temperate coastal regions of the world. These newly established populations affect aquaculture operations and infest both natural rocky habitats and cobble/gravel substrates. The earliest sample thought to be D. vexillum was collected in Mutsu Bay, Japan in 1926, although it was not described as a species at the time. Because D. vexillum was not described until 2002, populations discovered before that time were misidentified, often as species native to the region of discovery. The combination of incomplete historical records and misidentification means that the native range of D. vexillum is not conclusively known. To determine which portion of the current known range of D. vexillum is within its native region, we sequenced two genes, co1 (mitochondrial) and tho2 (nuclear), from 353 samples of D. vexillum from around the world. Both population genetics (co1 only) and phylogenetics (co1 and tho2) were used to identify a potential native region based on comparisons of the amount and type of variation present in each region where D. vexillum is currently found (Eastern North America, Japan, New Zealand, Western Europe, and Western North America).


b) Mitogenomics reveals a remarkably high intra-specie substitution rate in the ascidian Botryllus schlosseri. Francesca Griggio (1), Ayelet Voskoboynik (2), Fabio Iannelli (1), Dmitry Pushkarev (2), Carmela Gissi (1)* (1) Dip. Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy (2) Institute of Stem Cell Biology and Regenerative Medicine, Stanford Univ. School of Medicine, Stanford, USA.
   The leitmotiv of mitochondrial genome (mtDNA) evolution in ascidians is the hypervariability of many genomic features (such as gene order, nucleotide substitution rate and tRNA gene content) even at short phylogenetic distances. As consequence, the ascidian mtDNA has been proved to unambiguously discriminate between two cryptic species of Ciona intestinalis. Here, we describe the mtDNA of a model species, the colonial ascidian Botryllus schlosseri. Two specimens, one from California and one from Italy, have been sequenced. We found that the mtDNA of B. schlosseri has a novel gene order, completely different from other ascidians, and encodes for peculiar tRNA-like structures. Surprisingly, the sequence divergence between the two specimens is up to one order of magnitude higher (depending on the functional mt region) than the one measured in other ascidian intra-species comparisons. However, this value is lower than the one measured within genera, and between the two cryptic C. intestinalis species. Thus, based on mtDNA, the Californian and Italian B. schlosseri specimens appear to belong to a single species that is characterized by a remarkably high nucleotide substitution rate.


c) Comparative analysis of neural related microRNA during the development of Amphioxus and ascidians. Roberta Pennati (1)*, Roberta De Santis (1), Mario Pestarino (2),  Carmela Gissi (3), David S. Horner (3), Fiorenza De Bernardi (1), Simona Candiani (2).

(1) Dept. of Biology, Univ. of Milano; (2) Dept of Biology, Univ. of Genova; (3) Dept of Biotechnology, Univ. of Milano, Italy.

   MicroRNAs (miRNAs) are small non-coding RNAs that act as posttranscriptional and translational regulators of gene expression. Recent studies suggested that increased morphological complexity in metazoans is correlated with the number and function of miRNAs. We analyzed and compared the spatio-temporal expression of miRNA in the amphioxus Branchiostoma floridae and in two ascidian species, Phallusia mammillata and Ciona intestinalis. We focused our attention on 6 conserved miRNA families known to be expressed exclusively or preferentially in the nervous system of invertebrates and vertebrates. Interestingly, some of the analyzed miRNA showed a conserved pattern of expression among the analyzed species, while others differ regarding the spatial (CNS vs. PNS) or the temporal (early vs. late) expression pattern. These data can help to elucidate the evolution of nervous system among chordates.


d) A collaborative study of non-indigenous ascidians on the south coast of Newfoundland. Don Deibel (, J. Ben Lowen, Kevin C. K. Ma, Cynthia H. McKenzie, Matthew L. Rise, Gavin Applin, Jennifer Hall and Ray Thompson.  Ocean Sciences Centre, Mem. Univ. of Newfoundland, St. John's, Canada.

   In 2007, a harbour monitoring program was established linking researchers from Memorial University, the federal Department of Fisheries and Oceans (DFO) and the provincial Department of Fisheries and Aquaculture. Shortly thereafter, two non-indigenous ascidian species, Botryllus schlosseri and Botrylloides violaceus, were discovered in several harbours on the south coast of insular Newfoundland. Although B. schlosseri had been known in Newfoundland since 1975, this was the first report of B. violaceus. Since both of these species are well known pests of aquaculture operations elsewhere, we designed an interdisciplinary research program focused on factors predicting invasion fitness and future spread in the cold coastal waters of Newfoundland. Our results are intended to inform the early warning, zonal closure, and mitigation decisions of government. The seasonal life cycle of B. schlosseri is being determined in Arnold's Cove, including somatic growth and the rates and timing of asexual and sexual reproduction (poster of Lowen et al.) and larval recruitment (poster of Ma et al.). Diver surveys are being conducted in Belleoram harbour to assess the effectiveness of trial efforts by DFO to remove B. violaceus. Since mitigation efforts are more effective if applied at an early stage of invasion, an early warning tool kit is being developed consisting of TaqMan molecular gene probes sufficiently sensitive to detect a single egg or larva (poster by Applin et al.). B. schlosseri has been found in several harbours, with high colour morphotype and genetic diversity, while B. violaceus has been found only in a single harbour, with much less colour and genetic diversity.


e) Seasonal variability in larval recruitment of the non-indigenous ascidian Botryllus  schlosseri in Arnold’s Cove, Newfoundland. Kevin C. K. Ma (, J. Ben Lowen, Don Deibel, and Cynthia H. McKenzie.   Ocean Sciences Centre, Mem. Univ. of  Newfoundland, St. John's, Canada.

   Viable populations of Botryllus schlosseri, a potentially invasive species that has been disrupting shellfish aquaculture in Atlantic Canada, are fouling wharf structures and boat hulls in the subarctic waters of insular Newfoundland. At monthly intervals from March to December, settlement plates (aluminum, PVC, and wood) were moored at 3 subtidal depths (1, 2.5, and 4 m from the surface) at 3 floating docks in Arnold’s Cove. 12 megapixel images of settlement plates were analysed for the presence of larvae and juvenile colonies using “ImageJ.” Environmental data (in situ fluorescence, salinity, and temperature) were also collected 4 times per day using a moored YSI sonde. Larval settlement occurred from late August to October, coincident with maximum seasonal temperatures. Larval recruitment rates were highest at 1 m depth and at the most sheltered mooring site (from 11-31 larvae m-2d‑1). Unexpectedly, larvae readily settled on PVC at higher rates than on aluminum and wood substrates. Thus, the continued use of PVC plates to track recruitment of invasive ascidians within the Atlantic Zone Monitoring Program is recommended. Future management of B. schlosseri can target mitigation efforts before peak recruitment in September. Also, risk of regional spread can be minimised by identifying sheltered harbours and boat hulls that are susceptible to settlement and fouling.


f) Life-history constraints affecting invasion success in the ascidian Botryllus schlosseri. J. Ben Lowen ( ), Don Deibel, Kevin Ma, Cynthia H. McKenzie, Ray Thompson. Ocean Sci. Centre, Mem. Univ. of Newfoundland, St. John’s,                                                                                                  


   Temperature limits for somatic growth and sexual reproduction may strongly influence the invasive ability of Botryllus schlosseri. However, somatic growth and sexual reproduction rates of B. schlosseri have yet to be determined in a sub-arctic environment with an extended 'over wintering' period. To address this lack of information, we analyzed the life-history strategies of B. schlosseri in Arnold's Cove, NL. We specifically focused on somatic growth, sexual reproduction, and survival in 12 cohorts of marked colonies (n=472) observed bi-weekly during 2010. Somatic growth commenced in June, but at slower rates during June- July than has been documented in more southern latitudes. Somatic growth rates were maximal from August to mid-September when temperatures were >17oC. The window for sexual reproduction in Arnold’s Cove (i.e., late July to September) is also much shorter than in more southerly latitudes. These data reflect time constraints imposed by the short “growing season” in Newfoundland in comparison to the more southerly range of B. schlosseri. The life-time reproductive output of colonies recruiting at different times of the year was in turn affected by these time constraints, with a dominant colony morphotype (by frequency and size) also evident.


g) Ascidian photosymbionts: Do we know who’s hiding in the tunic? López-Legentil S, Turon X, Song B.

   Symbiotic interactions between ascidians and microbes are only beginning to be explored. We reviewed the existing literature on ascidian-cyanobacteria symbiosis and added our own research to the field by characterizing the cyanobacterial populations of some didemnid species from the Bahamas. So far, 91 species have been shown to contain photosymbionts, mostly from the genera Prochloron (Prochlorales) and Synechocystis (Chroococcales). To identify the photosymbionts present in Trididemnum solidum, T. cyanophorum, Lissoclinum fragile, and L. aff. fragile, we sequenced a fragment of the cyanobacterial 16S rRNA gene and the entire 16S-23S rRNA internal transcribed spacer region (ITS), and observed symbiont morphology by transmission electron (TEM) and confocal microscopy (CM). Similar to previous results for other ascidians, Trididemnum spp. mostly contained symbionts from the Prochloron-Synechocystis group. However, sequence analysis of the symbionts in Lissoclinum revealed two major clades. The first clade could not be associated with any known cyanobacterium (<93% max. identity), while the second clade matched closest to Acaryochloris marina (95% max. identity). Further observations using CM revealed the presence of both chl d and phycobiliproteins within these symbiont cells, which are characteristic of Acaryochloris species. Moreover, these symbionts were also observed by TEM in both the adult and larvae of L. fragile, indicating vertical transmission to the progeny. ITS gene sequences displayed much higher variability than 16S rRNA sequences, allowing a precise determination of the genetic diversity and host specificity of symbiont populations among ascidian species. Our results demonstrate that there is still much to learn on ascidian symbiosis, and that further work is necessary to assess the diversity, dynamics and significance of the microbial symbionts hiding in ascidian tunics.


h) Protochordate VCBPs are involved in the host-microbial dialogue of the gut.

Larry J. Dishaw, Stefano Giacomellid, Daniela Melillod, Ivana Zucchetti, Robert N. Haire, Lenina Natale, Nicola A. Russo, Rosaria De Santis, Gary W. Litman, Maria Rosaria Pinto.

Statione Zoologica Anton Dohrn, Naples, Italy.

   In all metazoans, despite an enormous variation in developmental morphology, an endodermally derived gut plays a central role not only in host metabolism but in the molecular dialogue between host and microbes. The typical gut consists of an outer epidermal case and an inner epithelial layer separated by a thin tissue space, which in many cases is populated by immunocytes. Innate immunity provides immediate and, in many animals, the exclusive form of recognition and defense within the gut. Throughout phylogeny, a variety of protein structures have been recruited by innate immunity to facilitate host discrimination of dietary antigens, and transient or symbiotic microbes. Variable region-containing chitin-binding proteins (VCBPs) of protochordates have been described in amphioxus and proposed to function in gut immunity. Here we describe VCBPs in Ciona intestinalis, a urochodate model of development and immunity. In Ciona, VCBPs are expressed exclusively by distinct cells of the gut epithelium and both expressed and recognized by specific amoebocytes. Using native and recombinant forms of VCBPs, as well as immunogold histochemistry, we show that secretory VCBPs bind bacteria in the gut lumen and enhance phagocytosis in amoebocytes opposite the epithelium. This is the first demonstation of an immunoglobulin-type molecule functioning in the molecular dialogue between host and gut microflora in non-vertebrate species.


i) Crossing the thin line between introduced and invasive species: factors shaping the distribution and invasive potential of the solitary ascidian Styela plicata. M.C. Pineda*1, S. Lopez-Legentil1, M. Rius2, C. McQuaid3, X. Turon4.  1Animal Biology Dept., Univ. of Barcelona, Spain; 2Dept. of Evolution & Ecol., Univ. of CA, Davis, CA;  3Dept. of Zool. & Entomology, Rhodes Univ., Grahamstown, S. Africa; 4Centre for Advanced Studies of Blanes (CEAB, CSIC), Blanes, Spain.

   There is a thin line between introduced and invasive species. However, since their invasive potential is uncertain, introduced species commonly receive less attention. The aim of this study was to use an integrative approach to assess the invasive potential of the solitary ascidian Styela plicata. Demographic and biological parameters were studied using four different approaches: 1) A global phylogeography using two genetic markers; 2) An assessment of the S. plicata reproductive cycle in two Mediterranean populations over a 2-year period; 3) An assessment of the susceptibility of S. plicata to changes in salinity, pollutant concentration, and temperature; and 4) An assessment of the stress response of S. plicata to natural environmental fluctuations over time, through quantification of hsp70 gene expression. Taken together, our results showed that S. plicata has been present in all studied oceans for a long time, and that the present distribution of this species appears to be regulated by high tolerance to pollution, and high sensitivity to low salinity and high temperature events. Proliferation and survival of S. plicata over time is ensured by a protracted reproductive period and rapid growth. This study highlights the importance of multidisciplinary approaches for understanding the interaction among the many factors shaping the invasive potential of introduced species.


j) Wild and cultured edible tunicates: a review. Mary R. Carman1 and Gretchen Lambert2.

1Biology Dept., Woods Hole Oceanog. Instit., Woods Hole, MA

2Univ. of Washington Friday Harbor Labs, Friday Harbor, WA 98250

   Most tunicate species are not edible but some solitary stolidobranchs in the Styelidae and

Pyuridae families are harvested wild or cultured. The main species are Halocynthia aurantium, H. roretzi, Microcosmus hartmeyeri, M. sabatieri, M. sulcatus, M. vulgaris, Polycarpa pomaria, Pyura chilensis, Styela clava, and S. plicata, and they may be eaten raw, cooked, dried or pickled. Historically the Maoris ate Pyura pachydermatina in New Zealand and aboriginal people ate P. praeputialis in Australia, though it is now only used for fishing bait. There is a large market for cultured tunicates, especially among Asian populations. S. clava and S. plicata have become extremely abundant in many countries as non-native introductions; they could easily be harvested and sold as seafood, as could common species that have not previously been consumed such as Herdmania pallida. Disease and overexploitation can reduce cultured product and wild populations. Recently, the disease ‘soft tunic syndrome’ caused up to a 70% loss of H. roretzi crop in Korea, while harvesting wild P. chilensis reduced their richness three fold in some parts of Chile. Most aquaculture operations are located in bays with urban runoff where pollutants including heavy metals and toxic substances could accumulate in tunicates. Natural disasters like tsunamis will also negatively impact aquaculture. Nevertheless, with proper controls and monitoring, certain edible tunicate species that are currently an underutilized food in many parts of the world could be easily cultivated or the huge numbers of invaders could be harvested and marketed.


k) Overwintering of the tunicate Didemnum vexillum in coastal New England. Page C. Valentine1, Mary R. Carman2, and Dann S. Blackwood1. 1US Geological Survey, Woods Hole, MA 02543; 2Woods Hole Oceanog. Instit., Woods Hole, MA 02543.

   The invasive colonial tunicate Didemnum vexillum flourishes in the coastal waters of New England. Colonies regress in winter when water temperatures can reach 0 oC and lower. To document this process, in May 2008 we set out 6 vertically-oriented, dark gray PVC settlement plates (12 x 12 cm) at 3 m water depth in Woods Hole, MA as substrates for D. vexillum. The plates were naturally covered with D. vexillum by the start of observations on November 3, 2008 (11.7 °C), by which time cloacal canals and apertures were clogged with fecal pellets indicating that feeding by the colonies had ceased. Plates were inspected at 8-9 day intervals until April 28, 2009. By December 9 (5.5 °C), zooid thoraces were tightly contracted. By December 29 (4.9 °C), the outer surfaces of cloacal canals had fallen off, exposing pockets of loose fecal pellets. From January 19 (0.4 °C) to April 8 (5.9 °C), colony tissues were either patches of white, spicule-laden, intact surface tissue or patches of disintegrating tissue, where the orange internal abdominal organs of the zooids were exposed. By April 14 (6.6 °C), the white tissue began to enlarge and oral siphons with tentacles were visible. Tissue regeneration signaled the end of the overwintering stage. By then, 91 to 100 % of the original colonies had disintegrated and fallen off the plates. By April 28 (9.8 °C), oral siphons were open, cloacal apertures had formed and were expelling fecal material, and the zooids were feeding. The response of D. vexillum to falling temperatures can result in a significant loss in areal coverage, but the effect is temporary, as we observed the colonies grow and spread as waters warmed.


l) Didemnum vexillum in eelgrass habitat. MR Carman1; DW Grunden2; P Colarusso3; MM Chintala4; DS Blackwood5; BH Becker6; K Smith7.  1Biol. Dept., Woods Hole Oceanog. Inst., Woods Hole, MA; 2Oak Bluffs Shellfish Dept., Oak Bluffs, MA; 3US Envl. Protection Agency, Boston, MA; 4US Envl. Protection Agency, Atlantic Ecology Division, Narragansett, RI; 5US Geol. Survey, Woods Hole, MA; 6Point Reyes Natl. Seashore, Point Reyes Station, CA; 7Cawthron Institute, Nelson, New Zealand.

   Seagrasses may be facilitating the spread of invasive tunicates by providing substrate and a dispersal mechanism (rafting). During the past 3 years, we observed D. vexillum in the

northwest Atlantic (Massachusetts) and eastern Pacific (California) utilizing eelgrass

(Zostera marina) as substrate for the first time ever. In some cases D. vexillum encapsulated plants to such an extent that they could no longer naturally defoliate or release seed, and upright shoots collapsed from the weight of the tunicates. Genetic analysis of D. vexillum specimens indicated that there is no genetic difference between colonies on eelgrass versus other substrate. Eelgrass that has been colonized by tunicates had fewer leaves, grew at a slower rate and had higher concentrations of sugar in its leaves. We speculate that the plants are reacting to loss of light by producing less new tissue and stockpiling the carbon it does produce. Tunicate growth affects the ability of eelgrass to function as a complex habitat. The presence of tunicates on eelgrass inhibits its use by bay scallops (Argopecten irradians) and herbivorous grazers such as snails.


m) Evidence for positive selection on an allorecognition locus in Botryllus schlosseri. Nydam, M.K. (, Taylor, A.A., and De Tomaso, A.W.


5. 82th Annual Meeting of the Zoological Society of Japan, Asahikawa, Japan, 21-23 September 2011.

Organization of the neural complex of Symplegma viride. Hiromichi Koyama1 and Shigeki Fujiwara2, 1College of Nursing, School of Medicine, Yokohama City Univ.; 2Dept. of Applied Science, Kochi Univ.

   We examined the ultrastructure of the cerebral ganglion of Symplegma viride with a transmission electron microscope. The cerebral ganglion is enveloped by a fibrous sheath about 3 microns thick. This sheath covers the nerve fiber bundles emanating from the cerebral ganglion. Each nerve fiber is also covered by thin fibrous mesh. The nucleus of the large somata contains chromatin clusters, some of which demarcate the internal nuclear membrane. There are mitochondria, Golgi complex, centriole, and many vesicles in the cytoplasm. The rough ER in large neuronal somata is less extensive than that of Polyandrocarpa misakiensis. The vesicles with electron-dense content vary in size, 100 - 300 nm in diameter. There is a hithero undescribed organelle in ascidian neurons, and we named it multigranular body. The multigranular body is irregular in shape and occupied by electron-transparent matrix, which contains several electron dense granules.


6. Western Soc. of Naturalists' 2011 Annual Meeting, Vancouver, WA, Nov. 10-13, 2011.

a) Contrasting effects of flow on adult and juvenile ascidian life history stages, including the global invasive Didemnum vexillum. Tren Kauzer1,2, Joseph D. Spaulding2, C. Sarah Cohen2. Calif. State Univ., San Luis Obispo1; Biology Dept and Romberg Tiburon Center for Environmental Studies, San Francisco State Univ.2

   The colonial ascidian Didemnum vexillum has shown an incredible propensity to invade marine ecosystems and poses a threat to both native species and the aquaculture industry. For sessile filter feeders, flow is a critical element influencing survival and growth, determining success across life history stages.  However, few studies have quantified the relationship between flow and the recruitment, growth and survival of colonial filter feeders. In this study we evaluated the growth of D. vexillum and larval recruitment under different local flow conditions in Half Moon Bay, California, USA. D. vexillum colonies were subjected to four different manipulated flow conditions over the course of three weeks, and growth and recruitment were measured.  A direct relationship was seen between inferred increases in flow and adult growth, and an inverse relationship was seen between inferred increases in flow and recruitment.  These trends suggest that optimal flow conditions differ for juveniles and adults.  Significant differences in growth were seen between colonies subjected to almost no flow and the other three conditions.  The adults' ability to grow over a range of flow conditions adds to its danger as a biological invader.  Understanding how adult and juvenile ascidians behave under different flow conditions could be used to determine which marine environments are most vulnerable to invasion and could help those who wish to eradicate or control its spread. [poster presentation]


b) The importance of early life-history stages in ecological succession. Rius M, Potter E, Aguirre JD, Stachowicz JJ.  Dept. of Evolution and Ecology, Univ. of Calif., Davis CA

   Ecological succession is a crucial process for understanding the spatial distribution of species and the maintenance of species diversity. In marine systems, succession has long been studied in fouling communities, but the focus has been on understanding interactions between adults or effects of adults on new recruits. Despite considerable advances, it is still not clear, for example, how competitively inferior species persist in places with low disturbance intensity and frequency. Here, we studied ecological interactions during early life-history stages to assess their influence on successional dynamics of the fouling community in Spud Point (Bodega Bay, California), a sheltered marina where population and reproductive phenologies have been studied over 10 years. During the period when community development and breeding thrived, we conducted additive and replacement design experiments in the laboratory covering interactions from gamete release to post-metamorphic stages. We also placed new metamorphs in the field in various combinations to examine effects of early life-history neighbors on longer-term success. The solitary ascidian Ascidia ceratodes is the clear competitive dominant in this system, yet it does not monopolize the space and coexists with other competitively inferior species. Similarly, the non-indigenous Ciona intestinalis has been slow to invade the system and has not established monocultures as it has elsewhere, despite the fact that it is likely to be competitively dominant. This study seeks to answer whether solitary ascidians are not more dominant because their early life-history stages are susceptible to predation and competition, even though as adults they are generally resistant to both.


7. 7th Intl. Conference on Marine Bioinvasions, Barcelona, Spain, 23-25 August 2011.


a) Coming and going: Temporal genetic variability of the introduced sea squirt Styela plicata. Valero, C., Pérez-Portela, R., López-Legentil, S.

  Introduced species may have an important impact on the natural ecosystem, disrupting ecological processes such as succession and species composition. Styela plicata is a solitary ascidian commonly found in harbors and marinas from tropical and temperate waters. This species is believed to have spread worldwide travelling on ship hulls. In this study, we determined the temporal genetic structure between and within cohorts of S. plicata. Samples were collected from the decks of the Center for Marine Science (UNC Wilmington, USA) between February 2007 and July 2009. We used the 454 Genome Sequencer GS-FLX next-generation sequencing platform to obtain 159,832 genomic reads averaging 278 bp. These sequences were then analyzed for repeat motives using Phobos v 3.3.12. We obtained over 100 potential microsatellite regions and from these, we isolated 8 polymorphic microsatellites. Our results showed that there were genetic differences among cohorts during the 2.5 years of study. In addition, there was a clear shift in the genetic structure of the population between May and July. This shift may be due to the high mortality event recorded every June, followed by the arrival of new recruits in July. Our results suggest that the dispersion and re-colonization of Styela plicata in coastal waters of North Carolina is an ongoing process. The most parsimonious explanation is an arrival of new recruits carried by the many ships that cross the Intracostal Waterway, which runs parallel to the coast from New Jersey to Texas.


b) A threat around the corner? Assessing the invasive potential and biogeographic boundaries of an introduced ascidian. M.C. Pineda*, Animal Biology Dept., Univ. of Barcelona, Spain; S. Lopez-Legentil, Animal Biol. Dept., Univ. of Barcelona, Spain; M Rius, Dept. of Evolution & Ecol., Univ. of Calif., Davis, CA; C.D. McQuaid, Dept. of Zool. & Entomology, Rhodes Univ., Grahamstown, S. Africa; X. Turon, Centre for Advanced Studies of Blanes (CEAB, CSIC), Spain. 

   There is a thin line between introduced and invasive species. However, since their invasive potential is uncertain, introduced species commonly receive less attention. The aim of this study was to use an integrative approach to assess the invasive potential and biogeography of marine introduced species. For this, we used as a model organism the solitary ascidian Styela plicata (Lesueur, 1823), a species that is present in harbours and on submerged structures around the world. Nevertheless, this species is not considered invasive, as there are few demonstrations of introduced populations outcompeting native species. Demographic and biological parameters were studied using four different approaches: 1) A global phylogeography using two genetic markers; 2) An assessment of the S. plicata reproductive cycle in two Mediterranean populations over a 2-year period; 3) An assessment of the susceptibility of S. plicata to changes in salinity, pollutant concentration, and temperature; and 4) An assessment of the stress response of S. plicata to natural environmental fluctuations over time, through quantification of hsp70 gene expression. The results of the phylogeographic study showed that S. plicata has been present in all the studied oceans for a long time, and that its populations have been shaped by recurrent and ongoing colonization events through ship traffic. The study assessing the reproductive cycle of this species showed that S. plicata has a protracted reproductive period and rapid growth. Experiments using embryos and larvae showed that early life-history stages of this species were highly tolerant to a common pollutant (copper), while low salinities (as found in estuarine conditions) and high temperatures seriously impaired larval development. Finally, low salinities and high temperatures in the field significantly increased hsp70 gene expression in this species. Taken together, our results indicate that the present distribution of this species appears to be regulated by low salinity and high temperature events in estuaries, while it shows high tolerance of pollution in harbours and marinas. Considering that many potentially suitable coastal areas remain to be colonized, our results suggest that S. plicata has the potential to proliferate and extend beyond its current boundaries. This study highlights the importance of multidisciplinary approaches for understanding the interaction among the many factors shaping the invasive potential of introduced species.


8. International Human Microbiome Congress, Vancouver, March 2011

A protochordate model of gut microbial-immune dynamics. Larry J. Dishaw, M. Gail Mueller, Jaime Flores-Torres, Daniela Melillo, Ivana Zucchetti, Rosaria De Santis, Maria Rosaria Pinto, and Gary W. Litman. Statione Zoologica Anton Dohrn, Naples, Italy.

   Most encounters between host and microbe involve complex symbiotic relationships, of which many are governed at the surface of gut epithelium. This single cell layer of gut epithelium is phylogenetically ancient and is thought to possess fully developed immunological capabilities. Ciona intestinalis, which is a descendant of the last common ancestor of all vertebrates and lacks adaptive immunity, is a potentially valuable new model for studying gut microbial-immune dynamics (GMID) and barrier defenses. A variety of immunological phenomena are well characterized in Ciona, including the expression of relevant innate immune molecules such as toll-like receptors, complement C3, and variable domain-containing chitin- binding proteins. We find that the Ciona gut is populated with diverse microbial communities and is predominated by gamma-proteobacteria. As with vertebrate models, the Ciona diet plays a significant role in microbial composition; presumably relevant dysbiosis is induced via starvation. The Ciona gut epithelium is responsive to microbial associated molecular patterns (MAMPs), and a distinct, hemocyte- rich, gut-associated tissue actively engages the microbiata. GMID can be investigated in Ciona against the background of a competent innate immune system and in the absence of central components of vertebrate adaptive immunity.


9. 9th Congress of the Southern African Society for Systematic Biology, Grahamstown, South Africa, 19-21 January 2011.

Detecting taxonomic boundaries of a species complex: the case of a widespread marine invertebrate. Rius M, Teske PR.

   Coastal environments are among the most heavily invaded ecosystems in the world. A large proportion of the coastal invertebrate fauna has poorly resolved taxonomic status and unknown historical distributions, which makes it difficult to distinguish between invasive species and long-established, but previously overlooked, cryptic species. We address this dilemma by studying the widespread marine ecosystem engineer Pyura stolonifera, a large solitary ascidian found in Africa, Australasia and South America. The taxonomic status of different populations of this species is disputed, especially since there is evidence for several distinct morphological and genetic units that point towards the existence of multiple cryptic species. While some researchers still recognize P. stolonifera as a single species, others treat the different populations as distinct species. Here, we present a revision of the P. stolonifera species complex based on a morphological examination and a phylogenetic study of samples from all regions where there are reliable reports of this taxon. We recognize four species that are morphologically distinct, one of which is new to science. In addition, at least five cryptic species can be further subdivided into regional genetic lineages. Strong evidence for both cryptic native diversity and the existence of invasive populations allows us to considerably refine our view of its native versus introduced status within the different coastal communities it dominates. Cryptic diversity in widespread species, and the taxonomic confusion arising from it, represents a major challenge for managing biodiversity.




1. Examination of secondary metabolites and inorganic acids as chemical defenses against predation and fouling in Antarctic and sub-tropical ascidians. Gil Koplovitz, Dept. of Biology, Univ. of Alabama at Birmingham. Ph.D. dissertation; advisor Dr. J.B. McClintock.

   Palatability of fresh outer tissues of 12 species of ascidians from the Western Antarctic Peninsula was evaluated using the sympatric, omnivorous fish Notothenia coriiceps and sea star Odontaster validus as model predators.  All ascidians were unpalatable to fish, while 58% were unpalatable to sea stars.  Lipophilic and hydrophilic extracts of 11 ascidian species were incorporated into food pellets and tested in fish and sea star bioassays.  Only the lipophilic extract from Distaplia colligans caused feeding deterrence in either predator.  Organic extracts were also examined in food pellets using the sympatric, omnivorous amphipod Gondogeneia antarctica.  Only the lipophilic extract of Distaplia cylindrica was deterrent.  Five species of ascidians had acidic tunics.  Acidified food pellets were deterrent against sea stars but not fish. 

   The secondary metabolites from a similar suite of Antarctic ascidian species were tested against sympatric marine bacteria and diatoms from the Western Antarctic Peninsula. All ascidians had lipophilic and hydrophilic extracts assayed against twenty bacterial strains and against a sympatric diatom (Syndroposis sp.). Only the lipophilic extract of D. colligans showed broad-spectrum antimicrobial activity. At least one extract from all but one ascidian taxa caused significant diatom mortality.

   The palatability of five species of ascidians commonly found in sub-tropical seagrass habitats were evaluated using the sympatric, omnivorous pinfish Lagodon rhomboides as a model predator. Fresh outer tissues of three of the ascidian species were unpalatable to fish. Food pellets containing organic extracts of these species did not deter feeding by the fish.  The toughness of the tunic of all five ascidian species was evaluated using a penetrometer.  Tunic toughness is likely to explain the lack of palatability of two of the three species.  Acidity is unlikely to explain deterrence in the third species as fish consumed acidified food pellets. 

   Despite their lack of palatability to sea stars and fish, organic chemical defenses against predation are uncommon in both Antarctic and Sub-tropical ascidians.  Toughness and inorganic chemicals appear more important.  Despite the lack of antibacterial defenses in Antarctic ascidians, secondary metabolites appear to play a potential role in preventing fouling by diatoms.  


2. Chemical screening and pharmacological evaluation of ascidians. S. Gomathy, Dept. of Zoology, A.P.C.Mahalaxmi College for Women, Tuticorin, Tamilnadu, India.  Ph.D. dissertation; advisor Dr.V.K. Meenakshi.

   The simple ascidian Microcosmus exasperatus was subjected to qualitative chemical screening, HPTLC and GC - MS analysis. This screening revealed the presence of various chemical constituents like alkaloids, terpenoids, steroids, tannins, saponins, flavonoids, quinones, anthraquinones, proteins, carbohydrates and lipids.  HPTLC profile for flavonoids showed the presence of gallic acid, ferulic acid, caffeic acid, isoquercitrin and quercetin which are being reported for the first time from the whole body extract of  Microcosmus exasperatus. Twenty biologically active constituents were identified in the ethanolic extract by GC-MS analysis. Of these 20 constituents, the following seven constituents: n-hexadecanoic acid, tetradecanoic acid, trichloroacetic acid, hexadecyl ester, 26-Nor-5-cholesten-3á-ol-25-one, 6,9,12-Octadecatrienoic acid, phenylmethyl ester, (Z,Z,Z)- ), Cholestan-3-ol  and 2-piperidinone, showed biological activities such as antioxidant, antimicrobial, anti-nflammatory, hepatoprotective, hypoglycemic, antiasthmatic, anticancer, antipyretic and antiandrogenic.


3. Identificación taxonómica y caracterización de la gemación en ascidias de Colombia y Panamá.  Pilar Andrea Endara  Pinillos. Laboratorio de Biología del Desarrollo Evolutiva (EvoDevo), Depto. de Ciencias Biológicas, Univ. de los Andes, Bogotá D.C., Colombia. Undergraduate thesis; advisor Dr. Federico Brown.

   Este estudio presenta el primer registro para Colombia de las especies de tunicados de la clase Ascidiacea. El muestreo se realizó en tres diferentes ubicaciones o estaciones de la costa Caribe colombiana: la playa de Cholón en la isla de Barú, la isla Titipán que hace parte del Archipiélago de San Bernardo e Inca Inca en la bahía de Gaira en Santa Marta. De las siete especies encontradas, seis ya han sido registradas en Venezuela o Panamá y un organismo colectado en Colombia no ha sido descrito una posible nueva especie. Observaciones detalladas de la gemación y la agregación de las colonias de ascidias fueron registradas fotográficamente. Mediante el cálculo de un índice de agregación simple (IA) se discute su relación con la gemación y la evolución de los estilos de vida solitario y colonial en el grupo. 


4. Determination of genetic differences between cohorts of the introduced ascidian Styela plicata. Claudio Valero, Master in Biodiversity, University of Barcelona. Advisers: Rocío Pérez-Portela and Susanna López-Legentil

   Introduced species may have an important impact on the natural ecosystem, disrupting ecological processes such as succession and species composition. Styela plicata is a solitary ascidian commonly found in harbors and marinas from tropical and temperate waters. This species is believed to have spread worldwide travelling on ship hulls. In this study, we isolated microsatellite loci from shotgun DNA genome pyrosequencing and performed a preliminary study of the temporal genetic structure between and within cohorts of S. plicata. Samples were collected from the Center for Marine Science docks (UNC Wilmington, USA) between February 2007 and July 2009. We used the 454 Genome Sequencer GS-FLX platform (Roche) to obtain 159,832 genomic reads averaging 278 bp. The resulting sequences were analyzed for repeat motives using Phobos v 3.3.12. Over a hundred potential microsatellite regions were detected, and from these, we selected and tested 18 primer pairs. Eight polymorphic microsatellites were finally isolated and optimized for further analyses. Preliminary analyses of S. plicata’s cohort structure showed that there were few genetic differences during the 2.5 years of study. However, a clear shift in the genetic structure of the population was detected between May 07 and July 07, which could be attributed a mass mortality event recorded in June. New recruits appeared to re-colonize the area in July, carried by the many ships that cross the Intracostal Waterway, where the Center for Marine Science docks are located. Our results indicated that the genetic structure of the analyzed population was quite constant over time, with occasional re-colonization events in summer.


5. Diversity, invasibility, and resource use in marine fouling communities of San Francisco Bay. Safra Altman, Univ. of Georgia Odum Sch. of Ecology & UGA Marine Institute. Ph.D. dissertation, Univ. of Maryland.

   Invasive species threaten the biodiversity of estuaries worldwide. To examine the relationships between biodiversity, invasibility, and invasion success, I conducted field surveys and experiments in San Francisco Bay marine fouling communities, including 1) surveys to estimate alpha, gamma, and beta diversity of native, non-native and cryptogenic components of the community; 2) experiments to assess the influence of diversity and resource availability on short-term recruitment of novel non-indigenous species (NIS) into test communities and subsequent community development over time; and 3) an experiment to explore the role of facilitative interactions of NIS in the diversity-invasibility relationship. Surveys (10-24 sites) showed that non-native alpha diversity was significantly greater than native or cryptogenic alpha diversity, beta diversity was significantly greater for native and cryptogenic species than for NIS, and gamma diversity was similar for NIS and native species. These results indicate that native species had high turn over from site to site while NIS were spread throughout the Bay. Experiments showed that on short time scales (2-4 weeks), the effect of initial diversity on the density of recruitment of NIS was significant and negative, with no effect of resource level (increased open space). Changes in community composition over time (2-24 weeks) also indicated significant inverse relationships between percent cover of NIS and diversity of the initial community with no evidence of a resource effect. Abundant NIS occupied less space in communities with higher initial diversity. However, the same NIS occupied (i.e., had invaded) all experimental communities regardless of starting diversity. Additional experiments revealed that recruitment to secondary substrates did not vary significantly with invasive species diversity or resource availability. When total recruitment to primary and secondary substrates were combined, there was no longer a significant relationship between diversity and recruitment. Analysis of secondary settlement patterns revealed that some NIS, such as Bugula neritina, were facilitating recruitment and settlement of additional NIS. In contrast, other species, such as Clathria prolifera and Botryllus schlosseri, inhibited secondary settlement of NIS. The influence of diversity and primary resource availability on secondary settlement did not appear to affect settlement on facilitative species, but reduced settlement on inhibitive species.




Abbott, C. L., Ebert, D., Tabata, A. and Therriault, T. W. 2011. Twelve microsatellite markers in the invasive tunicate, Didemnum vexillum, isolated from low genome coverage 454 pyrosequencing reads. Conservation Genetic Resources 3: 79-81.

Aiello, A., Fattorusso, E., Imperatore, C., Irace, C., Luciano, P., Menna, M., Santamaria, R. and Vitalone, R. 2011. Zorrimidazolone, a bioactive alkaloid from the non-indigenous Mediterranean stolidobranch Polyandrocarpa zorritensis. Marine Drugs 9: 1157-1165.

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