SUPPLEMENTARY INFORMATION
Tetraodon naming conventions, natural habitat and phylogeny
Tetraodon nigroviridis (Marion de Procé, 1822) is a pufferfish that lives in the rivers,
estuaries and mangroves of Indonesia, Indochina, Malaysia and the Philippines. It is also
occasionally caught in full sea water, and has thus a diverse habitat. Adults measure between
3 cm and 10 cm and possess two fused upper and lower teeth in a beak like shape (Tetraodon
means “four teeth” in latin). It is often confused with Tetraodon fluviatilis (Hamilton, 1822)
in the aquariophilist market. However T. fluviatilis is larger, has a markedly different pattern
of black spots on its back (larger and more symmetrical) and lives in Sri Lanka and in India.
T. nigroviridis is often caught from the wild although it is also sold to the aquariophilist
market by fishermen who breed them in artificial ponds by the rivers.
Since T. nigroviridis and T. fluviatilis are morphologically similar and since their
habitat may overlap, it is not excluded that hybrid individuals also exist on the aquariophilist
market which is the most convenient source of Tetraodon for research purposes. For this
reason, we conducted an extensive study of molecular markers that could unambiguously
differentiate T. nigroviridis and T. fluviatilis. We amplified and sequenced the mitochondrial
cytochrome b gene region (306 bp1) and the complete hypervariable control region (D-loop;
808-815 bp among the taxa studied). The sequence data was obtained for 26 fish (4 T.
fluviatilis, 17 T. nigroviridis, 5 T. biocellatus) and multiple alignments were constructed using
the ED program from the MUST package2 and PILEUP of the GCG package (Wisconsin
Package Version 9.1, Genetics Computer Group (GCG), Madison, Wisc) (Fig. S1A and S1B).
They provided 107 variable positions for the cytochrome b dataset (out of 306 positions)
among which 70 are informative for parsimony analysis. For the D-loop dataset, a total of 428
variable positions including 40 insertions/deletions were observed out of 863 positions, and
390 of these variations are parsimony-informative. Reconstruction of phylogenetic trees (Fig.
S2) by the method of Maximum Parsimony was done by the PAUP program 3 (version 4.0)
included in the GCG package.
Results confirm the monophyletic clustering of the Tetraodon species. Groupings of
the sequences are in agreement with specimen morphology, except for specimens quoted as T.
sp. Specimens “a” (T. Crnogorac-Jurcevic, I. C. R. F., London, personal communication) and
“m” presented the T. nigroviridis morphotype whereas specimen “g” and “h” presented the T.
fluviatilis morphotype. Therefore, it appears that discrimination of T. fluviatilis/T. nigroviridis
on the basis of simple morphological characters such as body colour and pattern alone is not
sufficient. However, molecular markers such as the cytochrome b and control region
sequences help in grouping the specimens into a defined species group. Diagnostic characters
for each species with reference to the T. nigroviridis haplotype are provided in the alignments
(Fig. S1A and S1B). Further methodological details and discussions on Tetraodon
phylogenies are available on the Tetraodon web site: http://www.genoscope.org/tetraodon