Barcoding of Marine Nematodes

1
Barcoding of Marine Nematodes
Birgit H M MELDAL1, Robin M FLOYD2, Brian J
Elce1, Mark L BLAXTER3, Craig R Smith4, Melanie C
AUSTEN5, Alex D ROGERS2, P John D LAMBSHEAD1 1
Nematode Research Group, Department of Zoology,
The Natural History Museum, Cromwell Road,
London, SW7 5BD, UK. bire_at_nhm.ac.uk 2 Natural
Environment Research Council, British Antarctic
Survey, High Cross, Madingley Road, Cambridge,
CB3 0ET, UK. rmfl_at_bas.ac.uk 3 Institute of
Evolutionary Biology, School of Biological
Sciences, University of Edinburgh, EH9 3JT, UK 4
Department of Oceanography, University of Hawaii,
Honolulu, Hawaii 96822, U.S.A. 5 Plymouth Marine
Laboratory, Prospect Place, The Hoe, Plymouth,
PL1 3DH, UK.
Abstract Marine Nematodes occur in virtually
every marine benthic habitat and they are
therefore a useful tool for biodiversity
estimates and biomonitoring. However, species
have only been described from a few global
regions, mainly from shallow sublittoral and
intertidal habitats of northern Europe and
analyses using alpha taxonomy are time consuming
and labour intensive. Barcoding of nematodes
promises to be a tool for quick and easy
assessments of biodiversity of any marine
habitat. However, in order to place sequences
from unidentified marine nematodes into the
phylogenetic framework of the Nematoda a database
for sequences from known, identified nematodes is
required. A working database has been initiated
for adding unidentified nematode sequences from
environmental samples but data is still lacking
from a number of major groups of marine
nematodes. Here, we demonstrate the use of a
skeleton of known sequences for placing
unidentified nematodes into a phylogenetic
framework.

• Methods
• Sequencing of marine nematodes from environmental
  samples without identification, e.g. deep-sea
  mining sites (KAPLAN project, Floyd,
  unpublished).
• Alignment of unidentified sequences to framework
  of sequences from identified marine nematodes,
  e.g. from British coastal samples (Meldal, 2004).
• Neighbourhood-joining analysis to establish
  unique clades (MOTUs) (see Box 1).

MOTU0018
MOTU0029
MOTU0007

• Results so far
• Sequences from identified marine nematodes are
  only available from British coastal habitats
  (Meldal, 2004 Cook et al, in press Bhadury et
  al., 2005).
• 64 MOTUs of 84 specimens from the deep Pacific,
  but no alpha taxonomy due to sampling process
  (KAPLAN, Floyd, this study).
• Pacific MOTUs fit into current framework but no
  exact matches to identified species could be
  found (Figure 1).
• K-dominance curve (Figure 2, Box 2) shows that
  nematode communities from morphological and
  molecular sampling efforts show strong
  similarities but the MOTU method picks up more
  singletons as depicted by a stronger curve
  between 40 60 dominance).

MOTU0003

• Why Barcode Marine Nematodes?
• Free-living marine nematodes are a target
  organism for environmental and biodiversity
  monitoring as they are cosmopolitan (found in all
  sedimentary habitats), abundant (commonly 105 to
  106 specimens m-2 from the deep-sea to shallow
  water) and have high species richness (20-80
  species m-2). In the deep-sea, nematodes
  represent more than half of the metazoa so are
  the key taxon for conservation monitoring
  (Lambshead, 2004).
• A major obstacle is the immature state of marine
  nematode taxonomy. Only 4,000 species have been
  identified out of an estimated 100,000 to 1
  million species (Lambshead, 2004). Few marine
  nematode taxonomists are available to make
  morphological descriptions so molecular barcoding
  is an effective alternative.
• Where taxonomy is underdeveloped, molecular
  barcoding represents an objective alternative to
  subjective taxonomic opinion, which is helpful
  for legal purposes. MOTU analysis (see Box 1)
  allows estimations of diversity and abundance
  using a standard methodology, without specialist
  training in the taxonomy of the organismal group
  in question.

MOTU0014
MOTU0008

• Future Research
• The primary task is to calibrate molecular
  barcoding against current morphology-based
  taxonomic and environmental data, which mainly
  comes from North-west Europe. This involves
  testing MOTUs against the morphological species
  concept currently employed both in individual
  specimens and environmental samples.
• The molecular database must be populated by
  barcoding a wide variety of identified marine
  taxa globally from a number of diverse habitats.
• Fauna from key areas of immediate importance such
  as the Central Equatorial Pacific mining zone
  must be comprehensively barcoded.
• Finally, a practical monitoring system, probably
  using phylochips, needs to be developed.

Figure 1 Neighbourhood-joining tree of sequences
from identified and unidentified (KAPLAN)
nematodes including number of substitutions for
internal branches and MOTUs of gt1 individuals.
References Bhadury, P., Austen, M. C., Smerdon,
G. R., Bilton, D. T., Lambshead, P. J. D.,
Rogers, A. D. (2005). Identification of marine
nematodes- a molecular approach. This
Conference. Cook, A. A., Bhadury, P., Debenham,
N. J., Meldal, B. H. M.m Blaxter, M. L., Smerdon,
G., Austen, M. C., Lambshead, P. J. D., Rogers,
A. D. (in press). Denaturing gradient gel
electrophoresis (DGGE) as a tool for the
identification of marine nematodes. Marine
Ecology Progress Series Floyd, R., Abebe, E.,
Papert, A., Blaxter, M. (2002). Molecular
barcodes for soil nematode identification.
Molecular Ecology 11, 839-850. Lambshead, P. J.
D. (2004). Marine nematode biodiversity. In Z.
X. Chen, W. Y. Chen, S. Y. Chen D. W. Dickson
(Eds). Nematology Advances and Perspectives Vol
1 Nematode Morphology, Physiology and Ecology.
CABI Publishing, pp. 436-467. Lambshead, P. J.
D., Platt, H. M., Shaw, K. M. (1983). Detection
of differences among assemblages of marine
benthic species based on an assessment of
dominance and diversity. Journal of Natural
History 17859-874 Meldal, B. H. M. (2004).
Phylogenetic Systematics of the Phylum Nematoda
Evidence from Molecules and Morphology. Ph. D.
Thesis, University of Southampton, UK, 326 pp.
Box 1 Molecular Operational Taxonomic Units
(MOTUs) An automated pairwise clustering
algorithm based on nucleotide identity of
individual specimens. Unique MOTUs are separated
by gt2bp variations to allow for sequencing
errors.
Figure 2 K dominance curve for 21
morphological cores (yellow) and 1 molecular core
(blue) from the central, Eastern Pacific deep-sea.
Box 2 K Dominance A graphical representation
of diversity used where samples are of a similar
size. Lower curves, extending further to the
right higher diversity (Lambshead et al., 1983)
Acknowledgements This work was supported by the
J. M. Kaplan Fund, the Natural Environment
Research Council of the UK (Grant
NER/A/S/2000/01331) and a Research Development
Grant from BHP Billiton. The work is also a
contribution to the Marine Genomics Network of
Excellence, funded by the EU Framework 6. Thanks
go to Paul de Ley for providing some unpublished
sequences.