Management plan
The new nematode AToL project will build directly on the experience and worldwide collaborative links established by the preceding one (see Results from Prior Support) by (1) continuing to expand and upgrade the shared Nematol website as a primary tool for data archiving and exchange, (2) utilizing the priority taxon lists, morphological matrices, lists of immediate collaborators and lists of nematode providers as starting points for obtaining the species prioritized for multigene analysis as well as for building comprehensive morphological character matrices to resolve the outstanding polytomies; (3) integrating the expertise of new CoPIs Abebe, Subbotin and Hyman with targeted focus on other important data sources including mitochondrial and developmental character suites. As before, we will engage the wider nematological community by scheduling Tree of Life sessions during the annual or biannual meetings of professional nematological societies, and we will provide timely progress reviews and opportunities for brainstorming and discussion during annual project-specific workshops.
Eyualem Abebe (EA), Co-PI, Elizabeth City State University (ECSU). EA will be responsible for providing specimens for the Solexa sequencing work to be done at UNH of target taxa from the orders Monhysterida (relevant to resolution of Clades III, IV and V), Chromadorida and Desmodorida (both relevant to resolution of basal topology of the phylum-wide tree)., within the class Chromadorea. From the scientific side, EA will be engaged in generating data on early developmental processes on selected representative taxa specifically on mechanisms of axis specification and the correlated segregation of determinant components such as P-granules. Also, EA will select, train, guide and mentor two undergraduate students at ECSU.
James Baldwin (JB), Co-PI, University of California, Riverside. JB (with support from a postdoc to be hired, a graduate student and participants Burr, Giblin-Davis Poiras, and Yushin) will have primary responsibility for developing morphological data matrices focused on resolution of features shown in the previous ATOL to be particularly informative across major branches. Beyond conventional light microscopy these will include, as needed, additional tools of SEM, TEM and confocal microscopy of selected representatives. JB will direct particular attention to morphology in resolving Secernentea sensu lato and its radiations, including Cephalobina and Tylenchina as well as selected species from other clades provided by other PIs. JB will also co-ordinate supply and identification for PIs addressing other aspects of the project of representative taxa of Clade IV (excluding animal parasites to be coordinated by SN) and oversee curation and deposition of voucher specimens from workshops; this will include VCE of selected type and other specimens of special relevance to the project from the UCR nematode collections.
Paul De Ley (PDL), Co-PI, University of California, Riverside. PDL with ½ time support from assistant specialist Dr. Irma Tandingan De Ley and 1/5 time support from postdoc Dr. Oleksandr Holovachov will have primary responsibility for carrying out Solexa sequencing at UCR of 40 reference species from all clades coordinated by the three UCR CoPIs (Plectida, most Clade IV animal parasites excepted and all Clade I representatives), as well as co-ordinating supply and identification for PIs addressing other aspects of the project of representative taxa of Plectida (relevant to resolution of Clades III, IV and V), Dorylaimida and Mononchida (members of Clade I and relevant to resolution of basal topology of the phylum-wide tree). For these species PDL will furthermore assemble a morphological character matrix (in coordination with R. Peña Santiago and V. Peneva). Where necessary (in case of absence in Solexa contigs) PDL will specifically target for PCR amplification and traditional sequencing selected orthologous gene sets needed for completing the multigene sequence matrix. PDL will also supervise graduate student Melissa Yoder in creating and testing image browsing tools for the Nematol database as part of her dissertation research on new approaches to image- and video-based identification of nematodes.
David Fitch (DF), Co-PI, New York University, sequencing facility director, Director of NYU Rhabditid Collection. DF will oversee phylogenetic analysis of Clade V. This will involve identifying representative taxa for multilocus sequencing and for supply to PIs addressing other aspects of the project. For these species DF will furthermore perform the following tasks: sequence cDNAs of orthologous gene sets, assemble a morphological character matrix (in coordination with W. Sudhaus and A. von Lieven), and study male tail development using MH27 antibody staining. Using material provided by other PIs, MH27 antibody techniques will also be extended to analyse male tail development in selected species from other clades. The DF lab will prepare and send material to UNH for Solexa sequencing of 20 Clade V species. If necessary (in case of absence in Solexa contigs) DF will specifically target for PCR amplification and traditional sequencing those orthologs needed for completing the multigene sequence matrix, and especially the par group of genes, lin-44/Wnt, RNAP2, tpi-1, lin-41 and tlp-1, as preliminary data suggest these will help resolution within Clade V at least. DF will also perform in-depth, within-clade phylogenetic analyses for Clade V using a cluster-computing facility available at NYU.
Bradley C. Hyman (BCH), Co-P.I., UCR. BCH will oversee the mitochondrial genome component of the project, including coordinating the mitochondrial pan-ortholog sequence acquisition at both UNH and UCR with whole mtDNA sequencing conducted at UCR. He will work with SAN and WKT to conduct gene-by-gene mtDNA phylogenies and with PD to assess syntenic relations of mitochondrial genes at key nodes, e.g. the relationship of outgroup orders to the Chromadorea. BCH will also co-ordinate supply and identification for PIs addressing other aspects of the project of representative taxa of Mermithida (inside Clade I). BCH and PDL will furthermore act as co-major professors for Samantha Lewis' Ph.D. dissertation research on mitochondrial genetics and phylogenetics of nematodes.
Steve Nadler (SN), Co-PI, University of California, Davis. SN will organize workshops (at
Bodega Bay Marine Lab) in years 2 and 4. In year 2 (with working group coordinator J. Lambshead), the
workshop will focus on stem lineages of the Chromadoria (Fig. 1); in year 4 (with working group
coordinator M. Blaxter), the workshop will focus on Spirurina (Fig. 1, mainly vertebrate parasites). SN
(and graduate student to be recruited) will be responsible for 33% of the 18S rRNA sequencing, with
sequencing reactions to be performed in the UCD automated sequencing facility. SN will facilitate
phylogenetic analyses of molecular datasets representing clades consisting of fewer taxa, using computer
resources at UCD, including the parallel-computing cluster available in M. Sanderson’s lab.
Steve Nadler (SN), Co-PI, University of California, Davis, and Curator of the UC Davis Nematode Collection. SN will oversee phylogenetic analysis of Clade III and co-coordinate with Sergei Subbotin (SS), Co-PI, University of California, Davis, to oversee phylogenetic analysis of Clade IV. This will involve identifying representative taxa for multilocus sequencing, including Solexa sequencing of 40 taxa at UCD. For these species SN will also: undertake amplification and traditional sequencing for those orthologs needed for completing the multigene sequence matrix (any not captured by Solexa) and along with SS complete the LSU rRNA sequencing of species as required for Clades III and IV. SN and SS will also perform in-depth, within-clade phylogenetic analyses for Clade IV, and SN will undertake similar analyses for Clade III. SN and SS will participate (along with other Co-PIs) in the phylum-wide analyses of Nematoda, helping to coordinate and complete analyses of molecular datasets.
W. Kelley Thomas (WKT), P.I., Director of the Hubbard Center for Genome Studies, UNH ( WKT will provide oversight for the project, including organization/coordination of the collaborating institutions and annual reporting of progress. Research activities of WKT will focus on the modifications and expansions of the Nematol database ( and maintenance and development of bioinformatic pipelines for the analysis of the EST sequence data and discovery of orthologous gene sets for mutligene phylogenetic analysis. As part of this initiative, UNH will host two workshops (years 1 and 3).
Oversight of Nematol Database:Nematol has been a focal point for the previous NematolNematode AToL grant. The database structure has allowed for the linking of diverse morphological information, DNA sequences, and other associated data. The initial phase of this Nematol project has continued to grow and evolve in response to the needs of the PIs and the broader nematological community. The database has become a resource for other projects (Byron USDA #2005-35302-16089, UCR NSF DEB0315829, Lambshead and Creer (NERC program UK), RGD, , We will look these up…NSF DEB0315829). We have recently become a repository for one of the larger, international nematode biodiversity study groups (Hans Helder, Wageningen). The ongoing design and development of the database in response to users. Ongoing efforts most recently include our addition of the Nemalog resource, a relational database for the discovery orthologous genes. We have maintained this database for over 5 years and have recently and significantly upgraded the server and storage capacity. The primary database manager (Fangning Liu) was the principle designer of Nematol and has worked in this capacity for over 5 years.
Bioinformatics: Development of specific bioinformatic pipelines for the efficient use of massively parallel sequencing data for the discovery of pan orthologs from large numbers of nematode species. WKT and the Hubbard Center for Genome Studies actively train computer science students, several of which have participated and will continue to participate in the Nematode Tree of Life Project.
Data availability:The nematode ToL database will be maintained with an open access policy. Users will be able to access completed taxa in the database at several points. Sequence data will be accessible by BLAST. The database will also be searchable by taxonomic queries and through links embedded in phylogenetic tree output files. Consequently, all sequences from completed taxa and all associated alignments can be accessed, downloaded, and utilized by the scientific community. The database is specifically designed to allow users to follow the data from chromatograms and finished sequences through alignments and phylogenetic analysis. The data can be downloaded at any point in the process. In addition, feedback by users will be sought through the database access point. The open format and feedback by users is a critical mechanism for refining the user-interface of the database. All finished sequence data will be made searchable immediately upon generation.
Monitoring and directing progress through annual workshops:
The PIs and closely involved international collaborators will hold annual meetings each year specifically for strategic planning, tactical updates and evaluation of progress. During the 3 successive years of work, each workshop will have a specific emphasis on different components of the multiple methods we will employ.
Workshop 1 will be organized to coordinate project startup and to introduce all participants to the principles and protocols of our novel techniques for producing multigene data. Special attention will be given to data formats and standardization, new and improved functions in the Nematol database, and first results obtained with targeted PCR amplifications of selected pan-orthologous genes. It will be held at UNH and attended by all CoPIs and selected members of their respective research teams.
Workshop 2 will focus on the results obtained to date for mitochondrial gene sequences, mtDNA gene order, as well as ultrastructural and confocal analyses of selected taxa. It will also address results obtained to date in the clades coordinated by the CoPIs at UCR and UCDavis: Clades I, III, IVa as well as the order Plectida. Additionally it will review progress and address troubleshooting of the Gene Analyzer results obtained to date at UCR and UCDavis (primarily with taxa from the aforementioned clades). This workshop will be held at UCR, and it will include not only the CoPIs and their directly involved team members but also the international collaborators with immediately relevant taxonomic and morphological expertise.
Workshop 3 will focus on the results of developmental studies conducted at NYU and ECSU, as well as the clades coordinated by the PI at UNH and CoPIs at both of the other two east coast campuses mentioned: Clades II, Monhysterida and Clade V. This workshop will also coordinate our comprehensive analysis and data integration across the project and among all participating teams, so as to provide the basis for collective publication of the the conclusive and collaborative summary of all preceding work. This will be organized at UNH and attended by all CoPIs and most relevant team members, as well as by international collaborators with directly relevant expertise.
Timeline:
There are 7 major research efforts to be undertaken as proposed.
- There will be three workshops. The first workshop will focus on the generation and analysis of Solexa based EST data. This workshop will be held at UNH in the third quarter of 2009. This workshop is intended to bring together expertise in Solexa based sequencing and the use of informatics tools developed at UNH. The second workshop will be held at UC Riverside and focus on phylogenetic analysis with the multigene datasets and a special emphasis on mtDNA gene order. The final workshop will be held at UNH and focus on the integration of major branching orders and the developmental studies conducted at NYU and ECSU.
- Bioinformatic analysis: Two major informatics pipelines are proposed. The first involves further modifications to our Nemalogs pipeline to best accommodate the Solexa-based unigenes. This will be complete in the first quarter. The major informatics efforts in the first year will be ongoing optimization of the pipeline for Solexa assembly and clustering analysis. These are dynamic processes that will change as read-lengths increase and taxon representation increases.
- Solexa Sequencing: The Solexa Sequencing will be conducted in the first two years of the proposal. Sequencing will be conducted Clade by Clade to maximize the opportunities for analysis.
- Mitochondrial gene assembly and gene order analysis: As mitochondrial sequencing and gene order will use the products of Solexa sequencing these will follow with the same species.
- Phylogenetic inference:
- Developmental Evolutionary analysis:
- Morphological analysis:
There will be a figure in this section for the timeline which will be generated from the excel sheet sent around earlier…
with the figure added we will have 5 pages so it may not be necessary to revive the list of international collaborators (red section below)
Milestones:
By Workshop 1: First 20 taxa sequenced and orthologs generated
By Workshop 2:100 taxa sequenced, major
By Workshop 3:160 taxa sequenced
We’ve arranged our milestones to coincide with our three workshops as our best mechanism for being able to evaluate as a group where we are in respect to these milestones.
We need feed back here about where the PIs see their projects/milestones fitting in to this. We will make this into more of a figure/diagram when we have this feedback.
Collaborators: a worldwide consortium to support nematode systematics.
To develop the systematic resources for this proposal, a letter of inquiry was sent to virtually all
scientists with expertise in nematode systematics. This letter outlined the goals of this proposal and
invited scientists to participate in this worldwide effort to develop a robust phylogeny for the phylum. The
response was overwhelmingly positive. More than 35 scientists with expertise on nematodes committed to
participating in the project (Table 3). Members of this consortium will participate in the research by
supplying and identifying optimal taxa to represent the phylum, and by participating in workshops and
working groups to develop the specimen database, gather morphological information, prepare vouchers,
and obtain specimens for molecular analysis. These collaborators will build a network and infrastructure
that will contribute to the development of nematode phylogenetics long after the granting period.
Possibly move this to the Nematol website and refer to it…
Table. 3 Worldwide consortium committed to supporting this nematode ToL project.
Investigator Institution Taxon and expertise
Adams, Byron U. Florida, Gainesville Tylenchida, Dorylaimida, species delimitation
Adamson, Martin U. of British Columbia Oxyurids, vertebrate parasites
Aleshin, Vladimir and
V.V. Malakhov
Moscow State University Adenophorea, marine nematodes
Blaxter, Mark University of Edinburgh All nematodes, outgroups / sequence
Borgonie, Gaetan Ghent University, Belgium All nematodes / cell lineage
Bostrom, Sven and
Bjorn Sohlenius
Swedish Museum of Natural
History
Cephalobids and panagrolaimids
Carta, Lynn USDA, Beltsville Tylenchida, Rhabditina
Coomans, August Ghent University, Belgium Dorylaimida, Mononchida
Decraemer, Wilfrida Royal Belgian Institute for
Natural Sciences
Triplonchida, marine nematodes
Fagerholm, Hans-Peter Abo Akademi, Finland Ascaridoids, Spirurids, fish nematodes
Ferris, Virginia Purdue U. Tylenchida, some Dorylaimida
Gardner, Scott U. Nebraska Various vertebrate parasites
Gasser, Robin U. Melbourne, Australia “Strongylida”, various vertebrate parasites;
Giblin-Davis, Robin U. Florida Diplogasterida, Insect associates
Hoberg, Eric USDA Beltsville Vertebrate parasites; host-parasite biogeography
Hope, Duane Smithsonian Marine nematodes
Hugot, Jean-Pierre Museum National d’Histoire
Naturelle, Paris
Oxyurids, host-parasite co-speciation, supertrees
Hunt, David CABI Bioscience, UK Rhigonematida
Lambshead, John London M. N. H. Marine nematodes
Moens, Maurice Ghent University/ Belgium Sequence
Olafsson, Emil Stockholm University Ecology of marine nematodes
Overstreet, Robin GCRL, Mississippi Parasites of marine fishes
Paggi, Lia U. Rome, La Sapienza Marine mammal parasites