Table Of ContentRelationship of some coleoid cephalopods established by 3' end
of the 16S rDNA and cytochrome oxidase III gene sequence comparison
Laure Bonnaud1 Renata Boucher-Rodoni1 and Monique Monnerot2
, ,
1 Biologie des Invertebres marins et Malacologie, URA 699 and GDR 1005 CNRS, Museum National d'Histoire Naturelle, 55 rue Buffon,
75005 Paris, France
2CentredeGenetique moleculaire,Centre Nationalde laRecherche Scientifique,91198 Gif-sur-Yvette, France
Abstract: Phylogenetic relationships forextant cephalopods have been based, so far, mainly on morphology and paleontology. Nucleotide sequence
dataarestillrare.Sequenceanalyses fromthe 3' endofthe 16SrDNAgeneofcephalopodshaveshownthatthisportionofgenecanprovide valuable infor-
mationontaxonomicrelationshipsattheinfrafamiliallevel.Anothermitochondrialgene,cytochromeoxidase III, isinvestigatedtoanalyzehigher(;'. e. ordi-
nal) taxonomic levels. The results obtained by the two gene portions are compared, but the low numberofspecies does not allow adefinitive answeron
interfamilial relationships. The low divergence between nucleotide sequences oftwo populations of Loligo vulgaris Lamarck, 1798, and ofL. reynaudii
Orbigny, 1845,suggeststhatthelatterisnotaclearlydistinctspecies.ThegroupingofthethreefamiliesofSepioidea(Sepiidae, Spirulidae,andSepiolidae)
isnotsupported. IdiosepiidaegroupswiththeoegopsidsquidEnoploteuthisirregardlessoftheanalysis(parsimonyordistance).
The Decapoda are composed of two orders, extracted from frozen or alcohol-preserved tissues accord-
Teuthoidea and Sepioidea. Relationships within these ing to the protocol described in Bonnaud et al. (1994). A
orders are not stabilized basedon morphological characters. portion of 16S and a portion ofCOIII were amplified with
Itis now possible with molecularcharacters to obtain a new universal primers: 984 and 986 for 16S and COIIIa and
type of information which can help to resolve some phylo- COIIIb according to the classification of Simon et al.
genetic relationships. The 3' end of the mitochondrial- l-r- (1991). These portions were cloned in pBS+ (Stratagene)
RNA (16S) was already investigated but with this portion and sequenced (ca. 500 pb each) with the dideoxy chain
ofthe molecule, extensive nucleotide variability leads to an termination (Sanger etai, 1977). The alignments were per-
unresolved phylogeny between the orders (Bonnaud etai, formed by eye, with the aid of secondary structure for 16S
1994). Another mitochondrial gene, coding for cytochrome and of the reading coding frame for COIII. Phylogenetic
oxidase III (COIII), was chosen here to analyze the rela- trees were calculated by distance (Neighbor-Joining)
tionships between some species ofdecapods. The impact of method using the MUST package (Philippe, 1992) and par-
mutations on protein function is very important, and simony method using PAUP 3.1 (Swofford, 1990). These
accordingly, the structure ofsome genes coding forproteins two methods gave similar results and only the trees
should be less variable at the nucleotide level: this is the obtained with the distance method are described here. The
case for the genes coding for cytochrome oxidase subunits, robustness of internal branching was tested by bootstrap-
COI, COII, and COIII. COIII gene analyses were thus ping.
thought to be suitable for solving phylogenetic relation- Transversions (changes of pyrimidine to purine or
ships at hierarchical taxonomic levels higher than those vice versa) are known to be less abundant than transitions
resolved by the 16S gene. For this preliminary study, one (changes of purine to purine or pyrimidine to pyrimidine)
species in each family or suborder was analyzed and the in some vertebrate taxa. When sequences are highly vari-
results obtained with the two gene portions compared. able, transitions can introduce noise in the analyses. As a
consequence, the use oftransversions should lowerthe inci-
dence of homoplasy between distant taxa. Analyses were
MATERIAL AND METHODS
performed using both all the substitutions and only the
transversions. Attributing weight to the transversions
Details on the taxonomic position and origin ofthe instead of removing the transitions did not further change
eight species studied are presented in Table 1. DNA was the results obtained.
AmericanMalacological Bulletin,Vol. 12(1/2)(1996):87-90
87
88 AMER. MALAC. BULL. 12(1/2) (1996)
Table1.Geographicaloriginandsystematicpositionofspeciesstudied.
SPECIES(ORIGIN) SYSTEMATICPOSITION
SepiaofficinalisLinne, 1758(Banyuls) Sepiidae
Sepiettasp.(Banyuls) Sepiolidae SEPIOIDEA
Spirulaspirula(Linne, 1758)(NewCaledonia) Spirulidae
IdiosepiuspygmaeusSteenstrup, 1881 (Australia) Idiosepiidae
EnoploteuthisreticulataRancurel, 1970(Hawaii) Enoploteuthidae(Oegopsidsquid)
LoligovulgarisLamarck, 1798(Roscoff)
L. vulgaris(Banyuls) Loliginidae(Myopsidsquids) TEUTHOIDEA
L. reynaudiiOrbigny, 1845(SouthAfrica)
OctopuscyaneaGray, 1849(NewCaledonia) Octopodidae
RESULTS III gene. The trees obtained with nucleotide sequence
analysis (Figs. 2-3) likewise show a solid grouping of the
Analysisofpartial 16S gene three loliginids. The sequences of the two populations of
Analyses were performed with 131 informative sites Loligo vulgaris differ significantly with this gene (4.9% of
out of 231 variable sites. In agreement with the results nucleotide divergence), and the divergence between L. vul-
obtained previously (Bonnaud et ai, 1994) the tree issued garis "Roscoff and L. reynaudii was 6.3%. Theirgrouping
from the analysis of /-r-RNA gene is unresolved (Fig. 1). was always supported by a very high bootstrap value (100).
No strong relationship can be established among taxa, Another well-supported group is composed of the
except for the Loliginidae. This is correlated with the oegopsid squid Enoploteuthis reticulata and of Idiosepius
sequence identities: the sequences of the two Loligo vul- pygmaeus, the representative of Idiosepiidae, one of the
sepioid families. This same grouping of Idiosepius and
garis populations are identical and that ofL. reynaudii dif-
fers by only 1.1%, a percentage close to the error percent- Enoploteuthis was obtained when taking all the substitu-
age generally accepted after amplification, cloning, or tions into account or only the transversions. Grouping of
sequencing. The general topology ofthe tree appears coher- the three other families of Sepioidea (Sepiidae, Spirulidae,
ent with the classification issued from morphological data and Sepiolidae) is not supported. This was confirmed by
(/'. e. Idiosepius, Sepia, and Sepietta grouped together) but the separation of these three species in the PAUP analyses
none ofthe groupings is strongly supported by a high boot- including all the substitutions or with weighted transver-
strap value. Idiosepius is not more closely related to the sions (data not shown). When taking into account only the
Sepioidea than to the Teuthoidea. It must be stressed that a transversions, Spirula became linked with Loligo with a
complementary analysis with all available species did not bootstrap value of 57.9 with Neighbor-Joining as well as
provide a clearer answer, the substitutions between families with PAUP analyses, and the group excluding Sepietta and
being saturated (Bonnaud etal., 1994): Idiosepius' position Sepia was well supported.
cannot bedetermined.
DISCUSSION AND CONCLUSION
Analysis ofpartial COIII gene
Analyses wereperformed with 166 informative sites It is clear that the two Loligo species are difficult
out of258 variable sites ofa portion ofcytochrome oxidase to distinguish in terms of nucleotide variability with the
Octopuscyanea
—
—Sepiaofficinalis
Idiosepiuspygmaeus
Sepiettasp.
Enoploteuthisreticulata
Spirulaspirula
52.7 —
100 Loligo vulgaris
Loligoreynaudii
Fig. 1.Phylogenetictreefromtheanalysisofthe3' endof/-r-RNAusingallsubstitutions(Neighbor-Joiningmethod).
BONNAUDETAL.: GENE SEQUENCE ANALYSES OF COLEOIDS 89
35.9 Sepiaofficinalis
Spirulaspirula
—Sepiettasp.
77.6 Enoploteuthisreticulata
Idiosepiuspygmaeus
Loligoreynaudii
100
98.3 Loligovulgaris"Baoyuls"
. Loligovulgaris"Roscoff"
Octopuscyanea
48.61 Sepiaofficinalis
Sepiettasp.
Enoploteuthisreticulata
77.7
Idiosepiuspygmaeus
76.5
Spirulaspirula
57.9 ~l7oojr—L"olLLio*ogloigroeyvnualugadriiis"Banyuls"
65.8T_ Loligovulgaris"Roscoff"
Octopuscyanea
Figs.2-3.PhylogenetictreesfromanalysesofpartialCOIII geneusing(2)all thesubstitutions,(3)onlythetransversions(Neighbor-Joiningmethod).
portions of the two mitochondrial genes. The observed of the idiosepiids has rarely been changed: it was always
divergence suggests differences due to geographical separa- considered as a family of the order Sepioidea except by
tion rather than a well-established speciation event. A study Guerra (1992) who raised Idiosepiidae to ordinal rank. The
involving numerous specimens from along the French and idiosepiids are isolated by characters like a dorsal adhesive
African coasts would certainly provide an answer to this organ in adults, the retardation of the tentacle development
hypothesisofclinal variation. in juveniles, and the statocyst structure. They were often
The analysis oftransversions only must be viewed described without shell or gladius. The presence of a glad-
with precaution, especially when the group excluding ius was certainly difficult to detect because of the very
Sepietta and Sepia is considered. It is clear that the number small size of the members of this genus (20 mm maximum
of species was limited. In PAUP analyses of the COIII por- mantle length). Hylleberg and Nateewathana (1991a, b)
tion, Sepietta was linked with Idiosepius and Enoploteuthis found a thin gladius in the specimens examined and sug-
whatever substitutions were considered. The opposing gested that /. pygmaeus might be more closely related to
results from the two methods for the grouping of Sepietta Teuthoidea than to Sepioidea. Steenstrup (1881) created the
reveal that this relation is uncertain and needs confirmation. genus Idiosepius; in his original description he mentioned
The same is true forSpirula grouped with Loligo. The boot- that some specimens of small squids were described by
strap values of 52.7 (with 16S) and 57.9 (with COIII) are early authors (e. g. Lamarck, Orbigny, Ferussac, Blainville,
low. If these values are really significant, they could be Peron, Lesueur) under various names: Cranchia minima
modified by increasing the species sampling, when possi- Ferussac, 1835, Loligo minima Orbigny, 1848, Loligopsis
ble. peronii Lamarck, 1822, Loligo parvula Peron in Blainville,
On the contrary, Idiosepius pygmaeus is always 1823, Sepiola minima Lesueur, 1821. In the opinion of
grouped with the oegopsid irregardless ofthe analysis (par- Steenstrup, these decapods might be idiosepiids. The rea-
simony or distance). This was unexpected because sons which lead these early authors to attribute squid char-
Idiosepiidae was placed by Naef (1916) with the Sepiidae acteristics to Idiosepius could be an indication of the pecu-
and Sepiolidae as members ofthe order Sepioidea. Formost liar position ofthis genus within Decapoda. It is difficult to
authors, Idiosepius is more closely related to the Sepiolidae find morphological criteria which justify linking Idiosepius
and Sepiadariidae than to the Sepiidae or Spirulidae, and its with sepiolids or sepiids and the few existing morphological
phylogenetic position has been questioned so far only with studies, like those of Hylleberg and Nateewathana (1991a,
regard to the first two families (Fig. 4). The taxonomic rank b), do not analyze the taxonomic status of Idiosepius.
,
90 AMER. MALAC. BULL. 12(1/2) (1996)
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ACKNOWLEDGMENTS
familie.DanskeVidenskabeligSelskapSkrifter(6)1:212-242.
We wish to thank Drs. M. Roeleveld and M. Dunning for their Dateofmanuscriptacceptance: 8February 1996
kind help in providing some ofthe samples, and to Dr. P. Rodhouse for
reading the manuscript. The technical helpofMs. A. Saihi and M. Martin
wasgreatlyappreciated.
