Tree of Life

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The Cindarian Tree of Life Project

Cnidaria ATol

Cnidaria AToL (CnidTol) is a five-year, collaborative project funded by the National Science

Foundation under the "Assembling the Tree of Life" program. The overall goal of CnidToL is to significantly enhance our understanding of evolution in the Phylum Cnidaria. The CnidToL project will use an integrative, multi-level approach to investigating cnidarian evolution. The CnidToL team is comprised of PIs, co-PIs and contractors from eleven laboratories at nine institutions and also includes multiple international and U.S. collaborators within the cnidarian scientific community. Through a collaborative effort, we will generate extensive molecular and morphological datasets that will be used to reconstruct phylogenetic hypothesis of cnidarian relationships. Molecular data will include mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA plus nuclear 28S ribosomal DNA; we anticipate a total of 10+ molecular markers. One aspect of morphology that will be examined in detail is the diversity of nematocysts (stinging cells); in turn the utility of these anatomical data for phylogenetic analyses will be evaluated. In addition, we will develop laboratory culture conditions for several different cnidarian species representing a broad phylogenetic sampling in an effort to develop new model organisms amenable for future in-depth developmental, life-history, and morphological studies. (See the CnidToL homepage)

Training and outreach are important components of the CnidToL project. An online database will include a catalogue of species, bibliography of literature in which they were described, inventory of type specimens, distribution maps, and images (see cnidarian.info). In addition, all of the molecular and morphological datasets generated from this project will be included in the CnidToL database. Undergraduate, graduate, post-doctoral training and outreach to K-12 educators will also be a prominent component to this project.

Overview

Over the next 5 years, we aim to gather large amounts of DNA sequence data accompanied by voucher specimens to assemble as robust a phylogeny of the scyphomedusae (coronates, rhizostomes, semaeostomes) as possible, including understanding the evolution of scyphozoans relative to cubomedusae, hydromedusae, and stauromedusae. Important goals of the project are to establish collaborations that will (1) support new international projects, (2) enable the diverse taxonomic sampling essential to success of the project, and (3) develop the robust global community essential for continuing rapid progress during and after the project.

The following text provides information on the logistics of collaborating on, or contributing to, the project. It is intended as a general guide that should be sufficiently flexible to fit a variety of circumstances. If you have questions or suggestions specific to your circumstances, please write to Mike Dawson mdawson@ucmerced.edu. Our goal is to foster long-term, fruitful, mutually beneficial research partnerships.

Collecting

If medusae are large, a small subsample of tissue (one or a few pieces, total ~10-50 cubic mm volume) can be biopsied from the specimen, then placed in a vial with excess (1 ml) 95% ethanol. The remainder of the specimen should then be placed in excess 4% formalin in ambient salinity water (i.e. seawater if it is a marine species, estuary water if it is an estuarine species).

If medusae are small, samples for DNA and morphological analyses can be taken from different individuals if you can be absolutely sure that the different individuals are the same species. Again, samples for DNA analyses should be placed in a vial with excess (1 ml) 95% ethanol; samples for morphological analysis should be placed in excess 4% formalin in ambient salinity water.

Ideally, several medusae of a range of sizes should be sampled for DNA and morphological analyses. If only one large specimen is found, it can be treated as noted above. If only one small specimen is found, it would be best if roughly one quarter of the animal (not including the mouth) is cut away and placed in 95% ethanol, with the rest placed in 4% formalin.

Please individually label each specimen with an unique identifier and include information on collection locality, GPS coordinates if available, date of collection, your name.

A small sample kit with all materials necessary for DNA preservation can be sent to you free of charge.

If possible, please take photographs of the medusae in the water and/or in tanks; photograph the whole medusae, and when possible also the fine structure of the bell, tentacles, and oral arms.

Additional instructions for sampling and photographing medusae.

Sending Samples

Keep the samples for at least a few weeks after collection to ensure they are well preserved. Please send ethanol-preserved samples for DNA analysis separate from formalin-preserved samples for morphological analysis.

The small vials of 95% ethanol with tissue for DNA analysis can be wrapped in a couple of plastic bags, sealed, put in a padded envelope or box, and mailed safely.

The specimens in 4% formalin should be placed in small, non-breakable (e.g. plastic bag, or plastic bottle), containers with sufficient space to fit the animal and just enough liquid to stop it getting damaged in transit. Remove all air from the container to minimize damage in transit. Make sure the container is well-sealed, then seal it another 3 times in additional bags. Pack the sample with plenty of cushioning inside a box.

Include a note in the box stating the point of origin and destination of the package, and a statement that it is a sample for scientific purposes. This will help it pass through customs easily if the package is checked by a customs officer.

All samples should be mailed to: Dr. Paulyn Cartwright, Department of Ecology and Evolutionary Biology, Haworth Hall, RM 7016, 1200 Sunnyside Ave., University of Kansas, Lawrence, KS 66045, phone: 785/864-4432, fax: 785/864-5860

Taxonomic Scope

All species of scyphomedusae (coronates, rhizostomes, semaeostomes) plus a broad sampling of cubomedusae, hydromedusae, and stauromedusae.

Expenses

CnidToL will cover moderate costs associated with sampling and shipping specimens. This can include (1) a small pre-payment of up to a few hundred US$$ if funds are needed before sampling and shipping, or (2) reimbursement of costs after sampling and shipping. Payment of costs by CnidToL will require providing a budget (for pre-payments), receipts (for reimbursements), institutional information for transfer of funds, and signing a contract with Kansas University [a formality to ensure everybody's obligations to the project are clear]. For information on pre-payments and reimbursements and the contract, please contact the CnidToL project leader Dr. Paulyn Cartwright pcart@ku.edu

Publications

The arrangement proposed is that each colleague and the relevant medusozoan specialist (e.g. Allen Collins, Mike Dawson, Antonio Marques) will write a manuscript describing the medusae that you contribute to the project. This most likely would take the form of a regional summary of collections or a taxonomic paper on which you would be the lead author and to which we could contribute taxonomic expertise, DNA analyses, some writing and/or figure preparation, editing, etc., as appropriate on a paper-by-paper basis. CnidToL would then also use the sequences and vouchers in analyses of higher-level taxa. Thus, if you make many collections in particular taxonomic groups, we can discuss additional co-authorships on systematic/phylogenetic papers to which those samples contribute significantly. Please feel welcome to discuss this or other appropriate arrangements with each CnidToL specialist with whom you collaborate.

Contact

If you are interested in more information or want to discuss potential collaborations and/or contributions to the project, please contact the most relevant point-person listed below. A general description of the framework for collaborations on medusozoans follows the list of cnidarian personnel.

Actiniarians and nematocysts: Meg Daly, daly.66@osu.edu

Culturing new model organisms: Neil Blackstone, neilb@niu.edu

Data analyses: Dan Janies, janies-1@medctr.osu.edu

Database development: Daphne Fautin, fautin@ku.edu

Hydra: Daniel Mart’nez, dmartinez@pomona.edu

Hydrozoans: Paulyn Cartwright, pcart@ku.edu

Hexacorals: Sandra Romano, sromano@uvi.edu

Medusozoans: Allen Collins, CollinsA@si.edu

Molecular marker development: Cliff Cunningham, cliff@duke.edu; Bernie Ball, bernie.ball@duke.edu

Octocorals: Cathy McFadden, mcfadden@hmc.edu

Scyphozoans: Mike Dawson, mdawson@ucmerced.edu


Please ensure that you have all necessary permits for collecting and shipping samples. Please abide by postal regulations (in most cases, mailing very small quantities of ethanol and 4% formalin is OK when packed appropriately). We encourage you to also deposit specimens in 4% formalin with your regional museum. If your collaboration with CnidToL can be used to leverage additional support from your home institution or national funding agencies that will contribute to your own work and to the CnidToL project please contact us for letters of support.

If you have any questions regarding collaboration on the project or about scyphozoan jellyfishes, please do not hesitate to contact Mike Dawson mdawson@ucmerced.edu.

Prepared by P. Cartwright and M. N Dawson

Griffith University Workshop

Griffith Univeristy Workshop

The Cnidarian Tree of Life (CnidToL) Workshop on Scyphozoa
Griffith University, Gold Coast, Australia
28th June, 2007

Program

9:30 a.m. - Welcome

9:40 a.m. - Introduction to the CnidToL project

9:50 a.m. - Aquaria, medusae, and polyps (C. Widmer)

10:30 a.m. - Analyses of polyps (G. Jarms)

11:00 a.m. - Morning tea

11:30 a.m. - Morphometrics on medusae (M.N Dawson)

12:00 p.m. - Lunch

1:00 p.m. - Wet lab: Morphometrics on medusae (M.N Dawson)

2:30 p.m. - Molecular phylogenetics (M.N Dawson)

2:50 p.m. - Afternoon tea

3:15 p.m. - Mitochondrial genomics (J.-S. Lee)

4:00 p.m. - Discussion & FAQ

4:30 p.m. - Close


Global Phylogeography

Phylogeny and Biogeography of Scyphozoa and Discomedusae; Michael N Dawson, et al.

Outline

Until early in this century, Aurelia aurita was considered to be an ecological generalist distributed circumglobally between about 70° N and 50° S. Molecular analyses demonstrated that this was not the case; there are at least 14 species of Aurelia found in different regions of the world, each presumably adapted to local conditions. A similar story can be told about Cyanea capillata, and probably many other widely distributed taxa. The probable preponderance of cryptic species is a problem because we know little about a species unless we know it's boundaries; we cannot know where it's distribution ends, how short or far it disperses, its upper or lower temperature tolerances, how much it eats, what it can eat, it's metabolic rate, grow rate, fecundity, it's ecological impact, whether it blooms, or is invasice, etc. This project - at the nexus of CnidToL and REVSYS - aims to increase our knowledge of species boundaries by sampling many scyphozoan taxa from many locations around the world. The preliminary map of species distributions that results will provide a template for reinterpreting the ecological data that has been collected over many decades on many species in its appropriate taxonomic context. Thus, each person has the potential to know the species they are working with, its true characteristics, and how it differs from other closely (or distantly) related species ... the essence of any comparative biology or study of biodiversity.

Methods

Target species are listed in the Systematics pages of The Scyphozoan (see especially Coronatae, Rhizostomeae, Semaeostomeae), as are the samples collected to date and their locations. If you can contribute any additional specimens, please contact Mike Dawson (mdawson AT ucmerced DOT edu). The protocol for preservation of samples is a slight modification of the standard protocol for CnidToL, as follows.

Collecting up to 10 specimens using your standard procedure, rinse the bell margin or oral arms that you will sample with clean water. Biopsy a piece of tissue rich in dermis that is about half the size of your small finger-nail. Preserve the tissue in excess 95% ethanol (i.e. about 10 times as much ethanol as tissue) in a screw-cap, o-ring, 2 ml vial. Preserve the remainder of the medusa in 4-7% formalin-in-seawater such that the final concentration of formalin is 2-4%. Store the ethanol+tissue in a freezer until you are ready to send it to CnidToL; allow the specimen in formalin to 'fix' for at least 3-4 weeks before sending to CnidToL. When ready, pack the samples robustly (shipping information and details) and send them to one of the two following address (Contact Mike to ask which is most appropriate for the specimens in hand).


Dr. Paulyn Cartwright,
Department of Ecology and Evolutionary Biology,
Haworth Hall, RM 7016, 1200 Sunnyside Ave.,
University of Kansas, Lawrence,
KS 66045, USA

or

Dr. Michael N Dawson
School of Natural Sciences
University of California, Merced
P.O. Box 2039
Merced, CA 95344
USA

Why get involved?

In addition to being able to identify which species you are working with, this project offers multiple opportunities for collaboration and co-authorship. A single species might be collected from multiple locations for a regional phylogeographic study (samples from different environments, or on different sides of a supposed biogeographic boundary are particularly interesting), or multiple species might be collected from a single region (for a biodiversity survey), or multiple species might be collected from multiple regions for a biogeographic study. All of these studies might additionally lead to descriptions of new species or other taxonomic revisions.


Cnidarian Mitogenomics

Emphases on Scyphozoa and Discomedusae; Jae-Seong Lee, Michael N Dawson, et al.

Outline

Comparison of mitochondrial genomes offers one perspective (the whole mitochondrion is typically considered a single genetic 'locus') on the phylogeny of organisms. That perspective is seen with two eyes, one sees the pattern of genome structure, the other the pattern of sequence differences. Our mutual interests in these two aspects led to the initiation of this collaborative project. The goal is to document the changes in genome structure with evolution of Medusozoa and to reconstruct a robust mitochondrial-based hypothesis of phylogeny in Cnidaria, particularly Scyphozoa, with emphasis on Discomedusae (= Semaeostomeae + Rhizostomeae).

Methods

At this stage, we are gathering tissue samples for sequencing. Target species are listed in the table below. Those without brackets have been collected (and sequenced if bold), those in brackets are required (in some cases other taxa will be used instead), and "??" indicates that any species from that family is acceptable and is still needed. If you can contribute any of these specimens, please contact Mike Dawson (mdawson AT ucmerced DOT edu). The protocol for preservation of samples is a slight modification of the standard protocol for CnidToL, as follows.

Target Species for Tisse Sampling
CLASS
ORDER
FAMILY
Source of Genus Species
ANTHOZOA


> 20 in GenBank
STAUROZOA

Eleutherocarpidae
?? (Halyclistus)


Cleistocarpidae
 ?? (Craterolophus)
CUBOZOA

Carybdeidae
 ??


Chirodropidae
 ??
HYDROZOA
LEPTOTHECATA
Aequoreidae
 ?? (Aequorea)

TRACHYMEDUSAE
Rhopalonematidae
 ?? (Aglantha)
SCYPHOZOA
CORONATAE
Periphyllidae
 ?? (Periphylla periphylla)


Linuchidae
 ?? (Linuche)

SEMAEOSTOMEAE
Cyaneidae
 ??


Pelagiidae
Chrysaora (Dactylometra) quinquecirrha**


Ulmaridae
Aurelia aurita*, Aurelia sp.1**

RHIZOSTOMEAE
Cassiopeidae
 ??(Cassiopea)


Catostylidae
 ??


Cepheidae
 ?? (Cotylorhiza)


Lychnorhizidae
 ??


Lobonematidae
 ??


Mastigiidae
 ?? (Mastigias)


Rhizostomatidae
 ??


Stomolophidae
 ??


  • Shao Z., S. Graf, O.Y. Chaga, & D.V. Lavrov (2006) Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): a linear DNA molecule encoding a putative DNA-dependent DNA polymerase. Gene 381: 92-101. [GenBank NC_008446]
    ** Jae-Seong Lee & colleagues (unpublished data)

After collecting the specimen(s) using your standard procedure, place it (each) in a large bucket of seawater filtered through 20-80 um plankton mesh. Keep the medusa there for 24-36 hours so that it clears its gut of prey, thus removing that potential source of contamination. Then, dissect out tissues rich in dermis (ideally gonad, or gastric tissue, or oral arms if they are not greatly thickened by mesoglea) until you have a quantity about half the size of your small finger. Preserve the tissue in excess 95% ethanol (i.e. about 10 times as much ethanol as tissue). Preserve the remainder of the medusa in 4-7% formalin-in-seawater such that the final concentration of formalin is 2-4%. Store the ethanol+tissue for 3-to-4 weeks in a refrigerator; invert the tube a few times to mix it every other day during the first week, then once or twice per week for the remaining few weeks. Finally, remove most of the liquid, pack the samples robustly (see shipping) and send the samples to the following address.


Dr. Michael N Dawson
School of Natural Sciences
University of California, Merced
P.O. Box 2039
Merced, CA 95344
USA


We will sequence whole mtDNA and, separately from several specimens, COI and 16S using existing universal primers which we will compare with a broader sampling of taxa (see Global Phylogeography above) as an error-checking mechanism to make sure there is no contamination of samples.

Why get involved?

Due to the highly technical nature of this project, the small number of samples needed (a few individuals of each of the taxa listed in the table ), and the lack of requirement for additional field data, it is unlikely that people who are not involved in the laboratory or phylogenetic analyses can justifiably be authors on publications arising from this project. (But if you have many months to spare to learn sequencing and phylogenetic analyses, it could be a different story.) So, why get involved? The major reason is that sequencing and comparison of this diversity of mitochondrial genomes will make available an off-the-shelf 'toolkit' of molecular markers for answering questions ranging from those about the highest (class) to the finest (population) levels of biological organization for any and all medusozoa. This will be a dramatic advance, allowing convincing statements about very recent events affecting population connectivity, invasive species, genetic diversity of populations, etc., to be made for the first time. Such knowledge is essential to making strong arguments about many other facets of ecology, such as adaption and selection. The easiest way to apply the new markers to the species in which you're interested is to have it's whole mitochondrion sequenced, because then you know exactly which tools to pull out of the toolbox. So, the question, really, is why not get involved?