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J. Moll. Stud. (2004) 70: 187–202 © The Malacological Society of London 2004
INTRODUCTION
Out of the five bivalve families known to possess endosymbioses
with sulphide-oxidizing chemoautotrophic bacteria (Fisher,
1990; Reid, 1990; Distel, 1998), the Lucinidae are by far the most
diverse, live in the greatest variety of marine habitats and are
geographically the most widespread. The biology of Lucinidae
has attracted considerable attention for the probable obligate
association with sulphide-oxidizing chemosymbiotic bacteria
housed within the ctenidia and upon which the bivalves are
nutritionally highly dependent (Distel & Felbek, 1987; Reid,
1990; Frenkiel & Mouëza, 1995; Frenkiel, Gros & Mouëza, 1996;
Distel, 1998; Gros, Frenkiel & Mouëza, 1998; Taylor & Glover,
2000). Despite this interest in their biology, systematic studies
have been neglected and details of relationships, both within
the family and to other bivalves, are confused.
In order to establish a phylogenetic framework for the
Lucinidae that is independent of shell characters, we have
undertaken a molecular analysis using 18S rRNA and 28S rRNA
genes of species both within the family and their putative rela-
tives. This is based upon sequences from 31 species in 19 genera
from around the world, including representatives from most of
the major groups recognized by Chavan (1969) and Bretsky
(1976). Our primary aim was to establish the monophyly of
Lucinidae and determine the relationships of its component
genera with a particular focus on the position of Anodontia. At
the same time we wanted to test the hypothesis that the families
currently classified within the Lucinoidea form a monophyletic
group and, for this purpose, we included species of Ungulinidae,
Thyasiridae and Fimbriidae. An objective here was to test the
ideas of Reid & Brand (1986) and Hickman (1994) in which the
presence of bacterial chemosymbiosis was considered pleisio-
morphic for the Lucinoidea, but subsequently lost in the
Ungulinidae and partially lost in the Thyasiridae.
The Lucinidae are currently classified within the superfamily
Lucinoidea (Chavan, 1969; Amler, 1999) along with six other
families, Fimbriidae, Thyasiridae, Ungulinidae, Mactromyidae,
Cyrenoididae and Paracyclidae. Of these, only the Lucinidae,
Fimbriidae, Thyasiridae and Ungulinidae have received much
attention. Some classifications (Skelton & Benton, 1993)
include the Ordovician fossil Babinka (Babinkidae) within the
Lucinoidea but others place it within a separate superfamily
Babinkoidea (Chavan, 1969), later classified by Amler (1999) in
the order Actinodontoida.
The position of Lucinoidea in relation to other major groups
of bivalves is also poorly understood. On morphological
grounds the superfamily has been regarded as a basal hetero-
dont clade (Salvini-Plawen & Steiner, 1996), with Morton
(1996) regarding it as a sister taxon to the Crassatelloidea and
Galeommatoidea with this combined clade as a sister group to
all other heterodont bivalves. Similarly, from fossil evidence
Bretsky (1976) and Johnston (1993) considered the Lucinoidea
related to the Crassatelloidea. The few molecular results for
Lucinidae published to date suggest a position within the
MOLECULAR PHYLOGENY OF THE LUCINOIDEA (BIVALVIA):
NON-MONOPHYLY AND SEPARATE ACQUISITION OF
BACTERIAL CHEMOSYMBIOSIS
SUZANNE T.WILLIAMS, JOHN D. TAYLOR AND EMILY A. GLOVER
Department of Zoology, Natural History Museum, London SW7 5BD, UK
(Received 13 June 2003; accepted 1 October)
ABSTRACT
The bivalve superfamily Lucinoidea is usually considered to comprise six separate families: Lucinidae,
Thyasiridae, Ungulinidae, Fimbriidae, Mactromyidae and Cyrenoididae. Chemoautotrophic chemo-
symbiosis with sulphide-oxidizing bacteria is present in all studied species of Lucinidae, Fimbriidae and
many, but not all, Thyasiridae. However, it is absent from Ungulinidae. The Mactromyidae are likely
to be an entirely fossil group with doubtful affinities with Lucinoidea. The Cyrenoididae are poorly
investigated, but anatomical features suggest they are unrelated to lucinoids. To investigate phylo-
genetic relationships within the Lucinoidea and test hypotheses concerning the evolution of the
chemosymbiosis, a molecular study was made using sequences of 18S and 28S rRNA genes. The study
incorporated species of Ungulinidae (two genera, two species), Thyasiridae (three species), Fimbriidae
(one species) and many Lucinidae (31 species, 19 genera) as well as a range of outgroups representing
major groups of heterodont and palaeoheterodont bivalves. The results demonstrate that the mono-
phyly of the Lucinoidea is not supported. The Ungulinidae and Thyasiridae are unrelated to the
Lucinidae. Ungulina and Diplodonta of the Ungulinidae group with a clade comprising Veneroidea,
Arcticoidea and Mactroidea. The three Thyasira species analysed form a monophyletic branch in a
basal position among the heterodont bivalves. The only member of the Fimbriidae examined, Fimbria
fimbriata, groups within the Lucinidae and separation as a family is not supported. The Lucinidae form
a monophyletic group within which several distinct and well-supported clades and lineages are recog-
nized: the Myrtea clade, the ‘Anodontia’ clade, Fimbria lineage, Phacoides pectinatus lineage, and two
clades comprising all other lucinids. The implication of non-monophyly of the superfamily Lucinoidea
is that Thyasiridae represent an independent acquisition of bacterial chemosymbiosis and this is
reflected in major morphological differences from the Lucinidae.
Correspondence: J. D. Taylor; e-mail: jdt@nhm.ac.uk
heterodont bivalves, excluding the Carditoidea and Crassatel-
loidea (Steiner & Hammer, 2000; Giribet & Wheeler, 2002;
Giribet & Distel, 2004; Dreyer, Steiner & Harper, 2004).
The families of Lucinoidea
The Lucinidae are the most diverse, morphologically varied and
well studied of the lucinoidean families. Typically they have
single thickened demibranchs and vestigial labial palps with all
species studied to date possessing chemosymbiotic bacteria in
the ctenidia (Reid, 1990; Taylor & Glover, 2000). There are
many shallow water species, but some such as Lucinoma are
frequent to depths of 1700 m and more (Salas & Woodside,
2002) and diverse lucinid faunas from around 1000 m are
known (R. von Cosel & P. Bouchet, personal communication).
The Lucinidae are geologically the oldest of the lucinoidean
families; the earliest fossil with convincing lucinid features is
Iliona prisca (Hisinger, 1837) from the Silurian (Liljedahl, 1991)
but pre-Mesozoic records are infrequent.
Although at the present day there are just two living species of
Fimbria, species assigned to the family Fimbriidae were much
more diverse and abundant during the Mesozoic (Monari,
2003). Aspects of the biology and anatomy of Fimbria fimbriata
were described by Allen & Turner (1970) and Morton (1979)
and they both concluded that Fimbria shared many morpho-
logical features with Lucinidae. Both of these studies predated
the discovery of chemautotrophic symbioses in bivalves, but the
presence of bacteria in Fimbria ctenidia was subsequently report-
ed by Janssen (1992). Fimbria are today associated with tropical,
shallow-water, carbonate sands and this was also the usual
habitat of the Mesozoic taxa (e.g. Dubar, 1948; Monari, 2003).
Thyasiridae comprises 11 genera of small, largely offshore and
deeper water bivalves (Payne & Allen, 1991; Oliver & Killeen,
2002), some living as deep as 7326 m (Fujikura et al., 1999).
Chemautotrophic symbiosis has been demonstrated in some
species but is lacking in others (Dando & Southward, 1986;
Southward, 1986; Herry & Le Pennec, 1987; Dando & Spiro,
1993). Some thyasirids possess ctenidia with two demibranchs,
the outer being smaller, while others have only single, inner
demibranchs. Although Chavan (1969) cites the geological
range as extending from the Trias onwards, unambiguously
identifiable thyasirids date only from the Cretaceous (Albian)
(Kauffman, 1967; Skelton & Benton, 1993).
The family Ungulinidae comprises around 12 living genera,
some with general similarity of shell features to Lucinidae.
Although they occur in shallow water, remarkably little is known
of their biology. They possess ctenidia with unthickened, paired
demibranchs (Allen, 1958) and no chemosymbiotic bacteria
have been reported (Southward, 1986; J. D. Taylor & E. A.
Glover, unpublished observations). Ungulinidae have been
recognized from the Upper Cretaceous onwards (Chavan
1969). Doubt concerning the position of Ungulindae within the
Lucinoidea was suggested by 18S rDNA sequence data for a
single species Diplodonta subrotundata (Steiner & Hammer,
2000).
Another family, the Mactromyidae, comprising a disparate
group of fossil genera from the Palaeozoic and Mesozoic, has
also been included within the Lucinoidea (Chavan 1959, 1969;
Amler, 1999) but the reasons for this placement are unclear.
Two Recent species from Australia, Bathycorbis despecta (Hedley,
1904) and B. percostata (Hedley, 1911), both from deep water,
have been included in the Mactromyidae on the basis of sup-
posed similarities of hinge teeth (Chavan, 1959). Alternatively,
Cotton (1961) included Bathycorbis within the Lucinidae. Little
is known of these small (c. 5 mm in length) bivalves and their
affinities are uncertain but we see no reason to include them
within the Mactromyidae. Johnston (1993) erected a new
family, Paracyclidae, for the Palaeozoic genus Paracyclas that was
formerly included within the Mactromyidae. Finally, Skelton &
Benton (1993) excluded all other Palaeozoic taxa from the
family.
The brackish and freshwater family Cyrenoididae, com-
prising the single genus Cyrenoida, is also frequently included
within the Lucinoidea (Dall, 1901; Chavan, 1969; Boss, 1982).
Alternatively, it was placed near the Corbiculidae by Thiele
(1943) and elevated to separate superfamily status (Cyrenoid-
oidea) by Olsson (1961). The shells resemble those of some
Ungulinidae, and Boss (1982) considered them closely related,
but little is known of their biology or anatomy. We have exam-
ined specimens of Cyrenoida rosea (d’Ailly, 1896) from Nigeria
that have ctenidia with both inner and outer demibranchs
present, large, paired labial palps and fused, medium-long
posterior siphons. Unfortunately, no material was available for
molecular analysis, but none of the morphological characters
suggest a relationship to Lucinoidea.
Relationships within the Lucinidae
Currently, the Lucinidae includes around 50 living genera, but
it is clear from work over the last few years that lucinids are con-
siderably more diverse than previously recognized, especially
from tropical areas (Oliver, 1986; Glover & Taylor, 1997, 2001;
Taylor & Glover, 1997a, b, 2002). The two most recent general
taxonomic revisions, the generic treatment of Chavan (1969)
and the study by Bretsky (1976), largely of North American taxa,
are rather incongruent with each other. Furthermore, ideas of
relationships between the genera are confused and no rigorous
phylogenetic analysis has been attempted. The only phylogenet-
ic trees for the Lucinidae presented in an explicit form are those
of Chavan (1938: 242) and Bretsky (1976); the latter was based
on a phenetic analysis of shell characters (Bretsky, 1970). In
both of these phylogenies, details of branching patterns are
often confusing or conflicting. Finally, Chavan (1969), in the
most widely used classification, grouped all fossil and Recent
genera of Lucinidae into four subfamilies, Lucininae,
Myrteinae, Milthinae and Divaricellinae.
Previous hypotheses of relationships within the Lucinidae
were based entirely on shell characters such as sculpture, hinge
teeth, ligament structure and position, and size and shape of the
anterior adductor muscle scar. However, the homologies and
polarities of the various characters, such as sculptural features
and ligament structure, are poorly resolved. For example,
Chavan (1969) grouped all genera possessing divaricate shell
sculpture into the subfamily Divaricellinae, but Bretsky (1976)
and Dekker & Goud (1994) considered that divaricate sculpture
probably developed independently in several lineages. Many
lucinids have few obvious shell characters, lacking sculpture and
hinge teeth, and classification is difficult. For instance, the
morphological simplicity of the shell in the Anodontia group is
probably responsible for the differing opinions on its systematic
position (Chavan, 1969; Bretsky, 1976).
Anatomical characters, such as gill structure, degree of
mantle fusion and form of posterior apertures, type of mantle
gills, shape of the foot and sperm morphology have consider-
able potential in phylogenetic analyses, but as yet few species
have been studied in any detail (Allen, 1958, Narchi & Farani
Assis, 1980; Reid & Brand, 1986; Distel & Felbeck, 1987; Mouëza
& Frenkiel, 1995; Frenkiel et al., 1996; Taylor & Glover, 1997a,
2000).
MATERIAL AND METHODS
A total of 68 taxa were included in the 18S rRNA phylogeny and
46 in the combined 28S rRNA and 18S rRNA phylogeny
(Table 1). In total, 40 sequences corresponding to taxa thought
to belong to the superfamily Lucinoidea were included in the
S. T. WILLIAMS ET AL.
188
analyses (three Thyasiridae, two Ungulinidae, one Fimbriidae
and 34 Lucinidae).
DNA extraction, amplification and sequencing
DNA was extracted from mantle tissue, using the method of
Winnepenninckx, Backeljau & De Wachter (1993) with minor
modifications. Tissue was taken from ethanol, blotted dry and
soaked for up to 2 h in 0.01 TE buffer to remove ethanol. The
tissue was then placed in a microfuge tube containing 300 l of
CTAB extraction buffer pre-heated to 60°C (100 mM Tris–HCl
pH 8.0, 1.4 mM NaCl, 20 mM EDTA, 2% w/v CTAB, 2% w/v PVP
40,000 MW, and 0.2% -mercaptoethanol added just before
use). The tissue was ground using a plastic pestle and incubated
overnight at 60°C with 20 l of proteinase K (20 mg/ml). The
tissue extract was then extracted at least twice with chloroform
and precipitated with two volumes of ethanol in the presence of
sodium acetate and finally re-suspended in 0.01 TE buffer.
Diluted total gDNA (15–80 ng) was used in individual poly-
merase chain reactions (PCR) of 50 l to amplify c. 1,000 bp of
18S rRNA and 700 bp of 28S rRNA. Reactions contained 0.1 M
of a forward and reverse PCR primer (listed in Table 2), 200 M
of each dNTP, 2.5 mM of magnesium chloride for 28S rRNA
amplifications and 3.0 mM for 18S rRNA amplifications,
2.5 units of Qiagen™ Taq DNA polymerase (50 l reaction),
one-fifth volume of ‘Q solution’ and one-tenth volume of
Qiagen™ buffer (10). Thermal cycling was performed with an
initial denaturation for 3 min at 95°C, followed by 40 cycles of
45 s at 94°C, 45 s at a gene-specific annealing temperature, 2 min
at 72°C, with a final extension of 10 min at 72°C. All temperature
changes were ramped 1°C/s. Annealing temperatures were
52°C for 28S rRNA and 54°C for 18S rRNA. Automated sequenc-
ing was performed directly on purified PCR products using
a BigDye Terminator v.3.1 cycle sequencing kit (Applied
Biosystems; www.appliedbiosystems.com). Protocols for cycle
sequencing followed manufacturer’s instructions except that
sequence reaction volumes were reduced to 10 l. Thermal
cycling was performed with an initial denaturation for 5 min at
96°C, followed 25 cycles of 15 s at 96°C, 10 s at 50°C and 4 min at
60°C. Sequence reactions were run on a 377 automated
sequencer (Applied Biosystems).
Sequences were verified by forward and reverse comparisons
where possible. However both 18S rRNA and 28S rRNA
sequences showed intra-individual variation (heterozygous
bases, recognizable by ‘double’ peaks on both forward and
reverse chromatograms) and some regions were difficult to
sequence through, probably because of intra-individual varia-
tion in length (1–2 bp). In this case, good sequence could be
obtained until a certain base was reached, and from then on
multiple sequences were evident in both forward and reverse
chromatograms. Similar patterns of intra-individual variation at
rRNA genes have also been recognized in gastropods (Stothard,
Brémond, Andriamaro et al., 2000, Williams, Reid & Littlewood,
2003) and cephalopods (Bonnaud, Saihi & Boucher-Rodoni,
2002). These regions were checked by multiple overlapping
sequences in a single direction only. Some of this sequence was
not used in analyses, as it was too variable to align unambiguously.
All sequences have been deposited in GenBank (Table 1).
Sequence analysis and phylogenetic reconstruction
Sequences were assembled and edited using Sequencher (v 4.0;
Gene Codes Corporation, Ann Arbor, MI, USA). Sequences
of ribosomal genes were aligned using Clustal X (Thompson,
Higgins & Gibson, 1994; Thompson et al., 1997), with ‘delay
divergent sequence’ set at 95–98%, gap opening penalty at
15–20 and gap extension penalty at 5. Further minor adjust-
ments to improve alignments were made by eye. Regions where
the alignment was ambiguous were excluded from the analyses.
Sites at which an insertion affected only a single individual were
also excluded. Alignments have been deposited with EBI/
EMBL, and are available by anonymous FTP from ftp://ftp.
ebi.ac.uk/pub/databases/embl/align/ or via SRS at http://srs.
ebi.ac.uk, under accession numbers ALIGN_000615, and
ALIGN_000616.
A phylogeny was constructed from both 18S rRNA and 28S
rRNA sequence data sets and from a combined data set using
Bayesian methods (MrBayes v3.0b4; Huelsenbeck & Ronquist,
2001). In the Bayesian analysis, base frequencies were estim-
ated, four chains were used (default temperature) and the
starting tree was random. The substitution model used corres-
ponded to the general time-reversible model, with gamma-
distributed rate variation among sites approximated with four
categories (shape estimated) and allowing for invariant sites
(GTR I). In each case, this was the best model found using
AIC criteria (MrModelTest v 1.1b, J. Nylander, www.ebc.uu.se/
systzoo/staff/nylander.html). The analysis for each data set was
run for 1,500,000 generations, with a sample frequency of 100.
The first 5,000 trees were discarded, so that the final consensus
was based on 10,000 trees. The analysis was repeated two more
times with each data set to confirm that independent analyses
were converging on the same tree and the final tree was comput-
ed from the combined accepted trees from each analysis (a total
of 30,000 trees). Support for nodes was expressed as posterior
probabilities (calculated by MrBayes) and also as bootstrap
support [neighbour-joining (NJ) algorithm with distances
estimated using maximum likelihood (ML) with the GTR I
model, parameters estimated from the MrBayes tree, 10,000
replicates].
Trees resulting from analyses of both the combined data set
and the 18S rRNA are reported, as 18S rRNA sequence were
available on GenBank for many additional outgroup taxa.
Congruence between individual gene trees was assessed by look-
ing for conflicting branches with greater than 95% support
(posterior probability; PP). For comparison, analyses on com-
bined data sets were also performed using parsimony (heuristic
search, simple addition of taxa, TBR algorithm, sites unweight-
ed), and NJ (using ML distances) algorithms (implemented in
PAUP*).
RESULTS
Sequence comparisons
Three data sets were analysed. One combined data set (18S
28S) with 46 taxa (33 new sequences plus 13 GenBank
sequences), and two independent gene sequence data sets: 18S
rRNA gene sequence data set with 68 taxa (35 new sequences
plus 33 GenBank and unpublished sequences provided by D.
Distel) and 28S rRNA gene sequence from 33 taxa (all new
sequences). Although c. 700 bp of 28S rRNA was amplified in
this study, only 425 aligned sites that correspond to the D3
region have been used in combined data set (18S 28S) analy-
ses as this permitted the inclusion of equal length 28S rRNA
sequences from outgroup taxa (predominantly from Giribet
& Wheeler, 2002). Although both 28S and 18S rRNA gene
sequences were available for Thyasira sarsi, only the 18S rRNA
sequence is used in phylogenetic analysis. The 28S rRNA
sequence reported by Giribet and Distel (2004) for this species
did not cluster with the two other Thyasira sequences (as in 18S),
but instead clustered within the lucinids in a preliminary phylo-
genetic analysis of 28S rRNA sequences, suggesting possible
confusion between samples.
Out of a total of 1729 bp of aligned nucleotide sequences used
in the combined data set (425 bp 28S rRNA and 1304 bp 18S
MOLECULAR PHYLOGENY OF LUCINOIDEA
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2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
20
1
2
3
4
5
6
7
8
9
30
1
2
3
4
5
6
7
8
9
40
1
2
3
4
5
6
7
8
9
50
1
2
3
4
5
6
7
8
9
60
1
2
3
4
5
6
7
8
S. T. WILLIAMS ET AL.
190
Table 1. Species sampled, collection locality and date and GenBank accession numbers for the genes sequenced for each taxon.
Species Locality and date of collection 18S rRNA 28S rRNA
Lucinidae
Anodontia alba Link, 1804 1: Lower Matecumbe Key, Florida, USA (2002) AJ581840 AJ581875
2: Guadeloupe, French West Indies, Caribbean From D. Distel
Anodontia bullula (Reeve, 1850) Moreton Bay, Queensland, Australia (1999) AJ581841 AJ581876
Anodontia fragilis (Philippi, 1836) Embiez Island, France (2002) AJ581842 AJ581877
Anodontia omissa (Iredale, 1930) Gunnamatta Bay, New South Wales, Australia (2001) AJ581843 AJ581878
Anodontia ovum (Reeve, 1850) 1: Lizard Island, Great Barrier Reef, Australia (2000) AJ581844 AJ581879
2: Rodrigues, Indian Ocean (2001) AJ581845
Anodontia philippiana (Reeve, 1850) King Bay, Dampier, Western Australia (2002) AJ581846 AJ581880
Anodontia schrammi (Crosse, 1876) Bermuda From D. Distel
Anodontia sp. Moreton Bay, Queensland, Australia (1999) AJ581847 AJ581881
Austriella corrugata (Deshayes, 1843) King Bay, Dampier, Western Australia (2002) AJ581848 AJ581882
Cardiolucina pisiformis (Thiele, 1930) Dampier, Western Australia (2001) AJ581849 AJ581883
Cardiolucina semperiana (Issel, 1869) Safaga, Egypt AJ389655
Chavania striata (Tokunaga, 1906) Lizard Island, Great Barrier Reef, Australia (2000) AJ581850 AJ581884
Codakia rugifera (Reeve, 1850) Ulladulla, New South Wales, Australia (1997) AJ581851 AJ581885
Codakia tigerina (Linnaeus, 1758) Lizard Island, Great Barrier Reef, Australia (2000) AJ581852 AJ581886
Ctena divergens (Reeve, 1850) Safaga, Egypt AJ389656
Ctena orbiculata (Montagu, 1808) Long Key, Florida, USA (2002) AJ581853 AJ581887
Divalinga quadrisulcata (Orbigny, 1842) Guadeloupe, French West Indies, Caribbean (2001) AJ581854 AJ581888
Divaricella irpex (Smith, 1885) Lizard Island, Great Barrier Reef, Australia (2000) AJ581855 AJ581889
Lucina pensylvanica (Linnaeus, 1758) Guadeloupe, French West Indies, Caribbean (2001) AJ581856 AJ581890
Loripes lucinalis (Lamarck, 1818) Weymouth, UK (2000) AJ581857 AJ581891
‘Lucina’ (new genus and species) Kermadec Ridge, New Zealand (2002) AJ581858 AJ581892
‘Lucina’ dalli (Lynge, 1909) Hong Kong (2001) AJ581859 AJ581893
Lucinoma borealis (Linnaeus, 1767) Salcombe, UK (2001) AJ581860 AJ581894
Myrtea spinifera (Montagu, 1803) Banyuls, France (2000) AJ581861 AJ581895
Notomyrtea botanica (Hedley, 1918) Victor Harbour, South Australia (2003) AJ581862 AJ581896
Phacoides pectinatus (Gmelin, 1792) 1: Brazil (2001) AJ581863 AJ581897
2: Guadeloupe, French West Indies, Caribbean (2001) AJ581863
Pillucina pisidium (Dunker, 1860) Gunnamatta Bay, New South Wales Australia (2001) AJ581865 AJ581898
Pillucina vietnamica (Zorina, 1978) Port Douglas, Queensland, Australia (1997) AJ581866 AJ581899
Pseudolucinisca lacteola (Tate, 1897) Esperance, Western Australia (2003) AJ581867 AJ581900
Rasta lamyi (Abrard, 1942) Aqaba, Jordan (2002) AJ581868 AJ581901
Wallucina assimilis (Angas, 1867) Jervis Bay, New South Wales, Australia (1997) AJ581869 AJ581902
Fimbriidae
Fimbria fimbriata (Linnaeus, 1758) AY070116 AY070128
Thyasiridae
Thyasira flexuosa (Montagu, 1803) Plymouth, UK (2002) AJ581870 AJ581903
Thyasira gouldi (Philippi, 1845) Firth of Forth, UK (2001) AJ581871 AJ581904
Thyasira sarsi (Philippi, 1845) AY070117 AY070129
(not used)
Ungulinidae
Ungulina cuneata (Spengler, 1798) Calahonda, Spain (1998) AJ581872 AJ581905
Diplodonta subrotundata Issel, 1869 Safaga, Egypt AJ389654
OUTGROUPS
Unionidae
Elliptio complanata (Lightfoot, 1786) AF117738
Lampsilis cardium Rafinesque, 1820 AF120537
Trigoniidae
Neotrigonia bednalli (Verco, 1907) AF120538
Crassatellidae
Eucrassatella donacina (Lamarck, 1818) Esperance, Western Australia (2003) AJ581873 AJ581906
Astartidae
Astarte castanea (Say, 1822) AF120551 AF120612
rRNA), 583 bp were excluded from the analyses. Most of the
excluded sites were long insertions in the anomalodesmatids or
Anodontia species, which were not found in other taxa. Of the
1146 bp of aligned sequence used to determine phylogenetic
relationships among 46 taxa in the combined data set analyses,
437 sites were variable, of which 289 were phylogenetically
informative (74 phylogenetically informative sites out of 269 bp
of included 28S rRNA sequence, 215/877 bp of 18S rRNA). The
same region of 18S rRNA gene sequence was used in the individ-
ual data set, but because it included more taxa (68 instead of
46 in the combined data set) there were 295 phylogenetically
informative sites out of 877 bp of included sequence. The data
set for 28S rRNA gene alone for the 33 new sequences included
MOLECULAR PHYLOGENY OF LUCINOIDEA
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2
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9
20
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2
3
4
5
6
7
8
9
30
1
2
3
4
5
6
7
8
9
40
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2
3
4
5
6
7
8
9
50
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4
5
6
7
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9
60
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5
6
7
8
Table 1. (continued).
Species Locality and date of collection 18S rRNA 28S rRNA
Carditidae
Carditamera floridana Conrad, 1838 AF229617
Arcticidae
Arctica islandica (Linnaeus, 1767) U93555
Vesicomyidae
Calyptogena magnifica Boss & Turner, 1980 AF120556 AF120617
Veneridae
Mercenaria mercenaria (Linnaeus, 1758) AF106073 AF131019
Venus verrucosa Linnaeus, 1758 AJ007614
Mactridae
Spisula solida (Linnaeus, 1758) L11266
Spisula subtruncata (da Costa, 1778) L11271 AF120615
Cardiidae
Fragum unedo (Linnaeus, 1758) D84664
Tridacnidae
Tridacna gigas (Linnaeus, 1758) D84189
Tridacna maxima (Röding, 1798) Great Barrier Reef, Australia (1996) AJ581874 AJ581907
Semelidae
Abra prismatica (Montagu, 1808) AF120554
Cultellidae
Ensis ensis (Linnaeus, 1758) AF120555 AF120616
Ensiculus cultellus (Linnaeus, 1758) AF229614
Galeommatidae
Galeomma takii Kuroda, 1945 X91969
Galeomma turtoni (Sowerby, 1825) AF120547 AF120608
Myidae
Mya arenaria Linnaeus, 1758 AF120560 AF120621
Gastrochaenidae
Gastrochaena dubia (Pennant, 1777) AF120562 AF120623
Gastrochaena stimpsoni Tryon, 1861 AF229615
Teredinidae
Bankia carinata (Gray, 1827) AF120564 AF120625
Cuspidariidae
Tropidomya abbreviata (Forbes, 1843) AJ389657
Pandoridae
Pandora arenosa Conrad, 1834 AF120539 AF120601
Lyonsidae
Lyonsia floridana Conrad, 1849 AF120540 AF120602
Hiatellidae
Hiatella arctica (Linnaeus, 1767) AF120563 AF120624
Voucher material held in the Natural History Museum, London. GenBank numbers in bold have been sequenced at Natural History Museum, London.
Table 2. Forward (F) and reverse (R) PCR prmers used to amplify portions
of 18S and 28S rRNA genes.
Name Sequence 5–3Source
28S
LSU900 (F) CCG TCT TGA AAC ACG GAC CAA G Olsen et al., 2003
LSU1600 (R) AGC GCC ATC CAT TTT CAG G Williams et al., 2003
18S
18S-5CTG GTT GAT YCT GCC AGT Winnepenninckx et al.,
1998
18S1100 (R) CTT CGA ACC TCT GAC TTT CG Williams et al., 2003
a total of 771 bp of aligned sequence, of which 133 bp were
excluded from analyses. Of the remaining 638 bp of included
sequence, 204 bp were variable, of which 142 were phylo-
genetically informative.
Phylogenetic analyses
All three independent Bayesian runs converged on exactly the
same tree for the 18S rRNA data set and for the combined data
set, and on almost identical trees for the 28S rRNA data set.
The Lucinidae form a well-supported clade in both individual
and combined analyses (Figs 1–3, 5). Likewise the Thyasira
sequences cluster together as do the two ungulinid sequences.
Contrary to taxonomic expectations, the Lucinoidea do not
form a monophyletic clade. Trees with a monophyletic
Lucinoidea or a Lucinidae/Thyasiridae clade or Lucinidae/
Ungulinidae clade did not occur at all in 30,000 trees retained
from an unconstrained Bayesian analysis of either the 18S rRNA
data set or the combined data set. Such a low probability
suggests that, while the exact sister taxa of the Lucinidae may
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Figure 1. Molecular phylogeny of the superfamily Lucinoidea produced by Bayesian analysis (implemented in MrBayes) of partial sequence data from 18S
rRNA gene. Nodal support is indicated in cladogram in Figure 2.
not be well resolved, the superfamily Lucinoidea, as previously
defined, is not monophyletic.
As we were very conservative in our decision about which sites
to include in the analysis, we repeated the Bayesian analysis for
the combined data set including all 583 previously omitted sites.
The tree topology was almost identical with that in Figure 4,
except that Fimbria was sister to the Myrtea clade (with low
support, PP 54%) and this clade was sister to the Anodontia
clade (PP 95%). Where the topology was the same as that in
Figure 4, support for various nodes was increased: the clade with
all the lucinids Ensis Galeomma/Tridacna/Mya/Bankia/
Calyptogena/Mercenaria/Spisula/Ungulina, PP 92%; lucinid
clade B Phacoides, PP 96%; lucinid clade B alone, PP 92%;
Divalinga Austriella, PP 99%; Chavania Wallucina
Loripes Pillucina pisidium, PP 100%; Chavania Wallucina,
PP 100%. The topology of the outgroups was unchanged
except that Hiatella was not sister to the Gastrochaena clade, but
sister to the large clade mentioned above (lucinids Ensis
Galeomma clade), although with only poor support (PP 49%)
and Spisula and Ungulina are sisters (PP 72%).
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Figure 2. Molecular phylogeny of the superfamily Lucinoidea produced by Bayesian analysis (implemented in MrBayes) of partial sequence data from 18S
rRNA gene. The tree topology is that of Figure 1, with collapse of branches with posterior probability <90%. Nodal support is given above branches (posterior
probability/bootstrap using ML distance).
Similar trees to those presented were obtained using altern-
ative tree-building methods (unweighted parsimony and NJ
using ML distances), although both NJ and MP showed some
evidence of long-branch attraction. A strict consensus of 358
trees obtained by parsimony analysis of 18S rRNA gene data set
was completely consistent with the tree in Figure 1, except that it
placed the Phacoides clade as sister to the main Anodontia clade
(ovum/schrammi/philippiana/bullula/omissa) (but with < 50%
bootstrap support). The topology of a NJ tree based on 18S
rRNA sequence data differed slightly from that in Figure 1. The
Anodontia clades are not sister to each other, the smaller clade
falling out in a clade with Fimbria and lucinid clades A and B
(bootstrap 61%). Phacoides pectinatus is sister to lucinid clade
B (bootstrap 55%), as in the combined data tree (Figs 4, 5).
The topology of the strict consensus of 88 trees obtained by
parsimony analysis of the 28S rRNA gene data set differs slightly
from that in Figure 3, but the only incongruence between
branches that have >50% bootstrap support and the tree in
Figure 3 is that Phacoides pectinatus is sister to Tridacna maxima
(bootstrap 52%). As both of these species have long branches,
this is probably an effect of long-branch attraction. The NJ
similarly had Phacoides pectinatus sister to Tridacna maxima
Ungulina cuneata (although with <50% bootstrap support). The
topology of the strict consensus of 24 trees obtained by parsimo-
ny analysis for the combined data set differs slightly from that in
Figure 4, but there are no incongruences between branches that
have >50% bootstrap support and the tree in Figure 5. Similarly
the NJ tree for the combined data set differs slightly from the
tree in Figure 4, but all branches with >50% bootstrap support
are identical to those in those in Figure 5.
DISCUSSION
Monophyly of the Lucinoidea?
The monophyly of the Ungulinidae, Thyasiridae, Fimbriidae
and Lucinidae within the superfamily Lucinoidea has been
tacitly assumed by previous authors but difficulty has been
experienced in incorporating the families into a lucinoid
evolutionary scenario (Fig. 6). From his study of comparative
anatomy within the Lucinoidea, Allen (1958) considered the
ungulinids to be the least derived group followed by the
Thyasiridae and then Lucinidae as the most advanced (Fig. 6A).
McAlester (1966) pointed out that this arrangement was in
apparent contradiction of the fossil record, Lucinidae first
appearing long before the other two families and he proposed a
reversed sequence of the families (Fig. 6B). Later, Boss (1970)
agreed with McAlester and also considered the Lucinidae and
Fimbriidae to be more closely related to each other than to the
Thyasiridae and Ungulinidae (Fig. 6C), suggesting that outer
gill demibranchs had been re-acquired in the latter two families.
Following the discovery of chemosymbiosis within the Lucinidae
and Thyasiridae, Reid & Brand (1986; Reid, 1998) proposed
a scenario, modified by Hickman (1994) (Fig. 6D) in which
chemosymbiosis was plesiomorphic for the Lucinoidea, but
subsequently lost in the Ungulinidae and partially lost in the
Thyasiridae.
The position of the Ungulinidae within the Lucinoidea was
questioned by Steiner & Hammer (2000) on the basis of an18S
rRNA sequence for Diplodonta subrotundata. Our sequences for
Ungulina cuneata confirm this result and the analyses place the
two ungulinid species within a clade containing Arcticoidea,
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Figure 3. A. Molecular phylogeny of the superfamily Lucinoidea produced by Bayesian analysis (implemented in MrBayes) of partial sequence data from
28S rRNA gene. Taxa occur in the same order as in the cladogram. B. Cladogram with the same topology as in A, with collapse of branches with posterior prob-
ability <90%. Nodal support is given above branches (posterior probability/bootstrap using ML distance). Bootstrap support for lucinid clade B (not shown
because PP < 90%) is 56%.
Veneroidea and Mactroidea. Some Ungulinidae have shells
superficially resembling some Lucinidae and the foot is simi-
larly elongate with a differentiated tip. Their anatomy, however,
differs in important features (Duvernoy, 1842; Mittre, 1850;
Allen, 1958). They possess ctenidia with both inner and outer
demibranchs present, the outer about half the size of the inner.
The gill filaments are unthickened and no bacteriocytes or
chemosymbiotic bacteria have been reported. They also possess
large, triangular, labial palps and lack a posterior exhalant tube.
Little is known of the biology of Ungulinidae, some live in
similar reducing habitats to Lucinidae such as seagrass beds but,
others such as Diplodonta orbella (Gould, 1851), from the East
Pacific, nestle in agglutinated ‘nests’ amongst rubble or kelp
holdfasts (Haas, 1943; Coan, Scott & Bernard, 2000). Ungulina
cuneata may be a rock borer (Duvernoy, 1842).
More surprising is the well-supported finding that the three
Thyasira species analysed do not form a monophyletic group
with the Lucinidae. This result is at variance, however, with
the molecular analysis of Giribet & Distel (2004) where
Thyasiridae were identified as a monophyletic group with
Lucinidae and Fimbriidae. In a combined 18S rRNA and 28S
rRNA tree they placed Thyasira sarsi in a monophyletic clade
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8Figure 4. Molecular phylogeny of the superfamily Lucinoidea produced by Bayesian analysis (implemented in MrBayes) of concatenated gene sequence data
from 28S rRNA and 18S rRNA genes. Nodal support is indicated in cladogram in Figure 5.
with Lucinidae and Fimbria, although this position was not
supported in the strict consensus tree. The apparent genetic
similarity between Thyasira and Lucinidae may, however, be
artefactual, because the T. sarsi 28S rRNA sequence used in
their phylogeny is possibly a lucinid sequence.
A number of morphological similarities between Thyasiridae
and Lucinidae have led to their being classified together,
including: general shell shape; the lengthened anterior adductor
muscle slightly detached from the pallial line; the long, exten-
sible, vermiform foot and thickened ctenidia (Allen, 1958;
Bernard, 1972; Scott, 1986; Oliver & Killeen, 2002). Nonethe-
less, there are other major anatomical differences that support
their separation. In gill morphology, some thyasirids (Thyasira,
Parathyasira,Conchocele,Maorithyasira,Axinopsida) have both
inner and outer gill demibranchs present but others (Mendicula,
Axinulus, Adontorhina,Leptaxinus) have inner demibranchs
alone. Many thyasirids possess elaborate, arborescent visceral
pouches occupied by digestive gland and tubules (Bernard,
1972; Payne & Allen, 1991; Oliver & Killeen, 2002). Comparable
structures are absent from Lucinidae but a few species have
simple, dome-like pouches (Taylor & Glover, 1997b). Within
the posterior apertures of thyasirids, mantle fusion forms the
exhalant aperture, which lacks an extensible tube. The inhalant
aperture, if present, is formed only by epithelial adhesion
(Bernard, 1972; Payne & Allen, 1991) rather than the extensive
tissue fusion of lucinids (Allen, 1958: fig. 9; Glover & Taylor,
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Figure 5. Molecular phylogeny of the superfamily Lucinoidea produced by Bayesian analysis (implemented in MrBayes) of concatenated gene sequence data
from 28S rRNA and 18S rRNA genes. The tree topology is that of Figure 4, with collapse of branches with posterior probability <90%. Nodal support is given
above branches (posterior probability/bootstrap using ML distance). *Bootstrap support is 96% for lucinid clade B, excluding Phacoides pectinatus.
2001: fig. 15a). In thyasirids, the labial palps are small, tri-
angular structures with a long proximal oral groove, whereas in
the lucinids the palps are reduced to small swellings at the edge
of the lips with only a short oral groove (Allen, 1958).
All Lucinidae examined to date possess chemosymbiotic
bacteria in the ctenidia, as do some, but not all Thyasiridae.
However, there are major differences in the way in which the
bacteria are housed. In Thyasiridae the bacteria are packed
together within single, large ‘vesicles’ located in the apical part
of the bacteriocytes (Fig. 7A, B) (Southward, 1986; Reid &
Brand, 1986; Herry & LePennec, 1987; Fisher, 1990). There is
some dispute whether the ‘vesicles’ are intra- or extracellular
(see Fisher, 1990). By comparison, in Lucinidae and Fimbria
(Distel & Felbeck, 1987; Janssen, 1992; Gros, Frenkiel & Mouëza,
1996) the individual bacteria are usually housed in separate
vesicles within the bacteriocytes (Fig. 7C, D).
Our molecular data suggest that Thyasira may be more basal
members of the heterodont bivalves than lucinids. Although
the thyasirids cluster between Hiatella and a Eucrassatella/
Carditamera/Astarte clade, further work is needed to determine
their sister group, as this was not the intended focus of this study
and not all appropriate outgroups have been included. Deter-
mination of a sister group to the thyasirids may be further com-
plicated by the fact that the Thyasiridae themselves may not be
monophyletic. At present, extant Thyasiridae are currently
classified into 11 genera (Chavan, 1969), divided by some
authors (Bernard, 1983; Coan et al., 2000) into two subfamilies
Thyasirinae and Axinopsidinae. There seems to be some
anatomical support for this division but, as yet, too few species
have been investigated. The species known to house endosym-
biotic bacteria belong to the genera Thyasira, Conchocele, Para-
thyasira and Maorithyasira within the Thyasirinae (Southward,
1986; Dando & Spiro, 1993; Fujikura et al., 1999; Imhoff et al.,
2003). In the Axinopsidinae, some species of Axinopsida and
Mendicula lack bacteria (Southward, 1986; Reid & Brand, 1986).
However, for many taxa there is no available information.
The monophyly of Fimbria and Lucinidae is supported by our
results. In both the 18S rRNA and combined trees (Figs 1, 2, 4,
5), Fimbria fimbriata falls within a clade with other Lucinidae. Its
exact relationship is only poorly resolved, although the 18S and
combined data trees suggest that it is probably basal to the two
main lucinid clades. Fimbria fimbriata is one of only two living
species of Fimbria (Nicol, 1950), but bivalves resembling Recent
Fimbria have a long fossil history extending to the lower Jurassic
(Dubar 1948; Chavan, 1969; Monari, 2003); the relationships of
these taxa to Recent forms are uncertain. Fimbriidae have been
separated as a distinct family on account of the convex, thick
shells, the large cardinal teeth and the short anterior adductor
muscle only slightly detached from the pallial line. The anatomy
of Fimbria fimbriata has many features in common with species of
Lucinidae, including ctenidial structure, morphology of labial
palps, posterior exhalant tube, mantle gills (Boss, 1970; Allen &
Turner, 1970; Morton 1979) and shell microstructure (Taylor,
Kennedy & Hall, 1973). Symbiotic bacteria contained within
bacteriocytes of the thickened demibranchs (Fig. 7C) are
housed in a similar manner to those of Lucinidae (Janssen,
1992). Molecular evidence places Fimbria firmly within the
monophyletic group Lucinidae and there is no present justifica-
tion for separate familial status.
Finally, we are unable to comment on the status of Mactro-
myidae within the Lucinoidea. The only living taxon attributed
to the family is Bathycorbis but this is known only from dead
shells. However, the assignment of Bathycorbis, to the Mactro-
myidae, and indeed of this whole family of fossil bivalves to the
Lucinoidea, is highly questionable.
Relationships within the Lucinidae
The two most recent systematic treatments of Lucinidae by
Chavan (1969) and Bretsky (1976) form the basis of the discus-
sion below. Chavan’s (1937–8) earlier review of the Lucinidae
has also been taken into account, although some of his ideas
had clearly changed by 1969. Recent and fossil lucinid genera
were divided by Chavan (1969) into four subfamilies, Lucininae,
Myrteinae, Milthinae and Divaricellinae. Later, aided by a
phenetic analysis of shell characters, Bretsky (1970, 1976)
divided the North American lucinids into seven groups or
lineages and presented phylogenetic trees incorporating fossil
taxa for some of these. There are major differences between
these two classifications concerning the assignment of genera to
either the subfamilies or lineages (see Bretsky, 1976: table 2).
For example, Lucinoma was included in the Myrteinae by
Chavan but placed in the Miltha lineage by Bretsky; Anodontia
was included as part of the Milthinae by Chavan, but considered
a separate lineage related to Myrtea by Bretsky.
The results of our molecular analysis based on a subset of 31
taxa from 19 living genera show that the Lucinidae are mono-
phyletic but within the group several well-supported clades can
be recognized, a Myrtea clade, two Anodontia clades and two
lucinid clades (Figs 1–5). The positions of these clades within
the tree and of Fimbria and Phacoides are not well resolved and
require further work using additional genes.
Myrtea clade
In this analysis the two species sequenced (Myrtea spinifera and
Notomyrtea botanica) are monophyletic and distinct from the rest
of Lucinidae. The topology of the 18S rRNA tree suggests that
the Myrtea group may be the sister group to all the remaining
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Figure 6. Schematic diagrams of proposals concerning relationships within
Lucinoidea, modified from Hickman (1994: figs 4–6). A. After Allen (1958:
480), B. From McAlester (1966: fig. 2), C. From Boss (1970: fig. 6). D. From
Reid & Brand (1986: fig. 10; and Hickman (1994: figs 4–6).
lucinids, the topology of the 28S rRNA tree places the Myrtea
group between the two Anodontia clades and the combined data
tree suggests that the Myrtea group plus the Anodontia clades are
sister to all other lucinids. None of these positions is well sup-
ported.
Introducing the new subfamily Myrteinae, Chavan (1969)
listed 11 living and fossil genera, including Myrtea and Goni-
myrtea but also Lucinoma. However, Lucinoma is unrelated,
belonging instead to our lucinid clade A (Figs 1–5). Bretsky
(1976) also recognized a ‘Myrtea lineage’ of five living genera
that included Myrtea, Notomyrtea and Epilucina. We have been
unable to obtain living Epilucina, although on shell characters
alone a close relationship to Myrtea seems unlikely. Myrtea,
Notomyrtea and Gonimyrtea have laterally compressed shells
with commarginal lamellae, often with prominent dorsal
spines, elongate lunules, and hinges with small cardinal and
extended lateral teeth. They are more abundant in offshore,
shelf habitats than in shallow water and we are aware of many
undescribed taxa, especially from tropical locations. Bivalves
with Myrtea-like characters, e.g. Mesolinga and Discomiltha, have
a long fossil history into the lower Jurassic (Dubar, 1948;
Chavan, 1952).
‘Anodontia’ group
Our molecular analysis strongly supports the separation of
Anodontia from all other Lucinidae (Figs 1–5), with the possible
exception of Myrtea (Fig. 3). A single Anodontia clade containing
all the ‘Anodontia’ species as well as Pseudolucinisca lacteola from
southern Australia, is well supported by high posterior prob-
abilities in the tree based on the combined data set and in the
28S rRNA tree (but also including the Myrtea clade), but only
poorly in the tree based on 18S rRNA data only. This clade
divides into two distinct sub-clades, firstly that of Anodontia alba
plus P. lacteola, and secondly that of the other Indo-Pacific Ano-
dontia species, A. schrammi (type species of genus Pegophysema)
from the West Atlantic and A. fragilis (type species of Loripinus)
from the Mediterranean. This second group is well supported
with both posterior probabilities and moderate to high boot-
strap support in all three trees. These species are distinguished
from all other lucinids by the shared presence of unique base
changes and short insertions, particularly the lineage with
A. omissa (type species of Cavatidens) and A. bullula. This molec-
ular separation of the Anodontia group is further corroborated
by a number of distinctive anatomical features. These include
the presence of a mantle septum, digitiform mantle gills, a
S. T. WILLIAMS ET AL.
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Figure 7. Endosymbiotic bacteria in bacteriocytes of Thyasiridae, Fimbria and Lucinidae. Scanning electron microscope images of critical point dried portions
of ctenidia. A, B. Thyasira sp. (West Pacific vent species) with mass of bacteria held in single ‘vesicle’ (B). Scale bars: A5m; B2m. C. Fimbria fimbriata
Lizard Island, Queensland. Scale bar 5m. D.Austriella corrugata Dampier, Western Australia. Scale bar 5 m.
thickened outer mantle fold and a highly fused mantle ventral
to the inhalant aperture, involving both the inner and most of
the middle folds (J. D. Taylor & E. A. Glover, 2000; unpublished
observations).
The smooth, rather featureless shells of the Anodontia group
(including the genera Loripinus,Pegophysema and Cavatidens)
have long caused taxonomic confusion and there have been
differing ideas concerning their systematic position and rela-
tionships. Chavan (1938: 120, 242) thought that the Anodontia
group (Pegophysema and Anodontia) shared a common origin
with Cavilucina and was derived from Monitilora. However, he
later (Chavan, 1969) placed Anodontia and Pegophysema in the
subfamily Milthinae and Monitilora in the Myrteinae. Two differ-
ent views were expressed by Bretsky (1976: figs 7, 8): on p. 244 ‘I
consider it most probable that Loripes is related to the Anodontia
lineage, with Loripes s.s., Anodontia, and Loripinus having a
common ancestry . . .’, but on p. 306 ‘The similarity of sculpture
and outline . . . may indicate a close relationship between the
Myrtea and Anodontia lineages’. Bretsky’s (1976) idea of a
relationship between Loripes and Anodontia is not supported by
our molecular data, however, because of lack of material we
were unable to test the ideas of a relationship between Anodontia
and Miltha or Monitilora.
In proposing the new subfamily Milthinae, Chavan (1969)
included the Recent genera Miltha,Anodontia,Gibbolucina,
Eomiltha,Austriella (as Eamesiella). Some parts of this subfamily
have a long fossil record, for Miltha-like lucinids are present into
the Jurassic (Chavan 1969; Bretsky, 1976) and Eomiltha to the
lower Cenozoic (Bretsky, 1976). However, most of these fossils
are in serious need of reassessment. Although we were unable to
obtain specimens of the rare Miltha,Gibbolucina or Eomiltha for
molecular analysis, our results show that Anodontia and Austriella
are distantly related. Thus, Chavan’s (1969) concept of the sub-
family Milthinae cannot be supported. Bretsky (1976) believed
that Lucinoma was closely related to Miltha, indeed including it
as a subgenus of the latter. Again, in the absence of Miltha from
our analysis we could not test this hypothesis.
The molecular analysis has highlighted a taxonomic problem
concerning the concept of the genus Anodontia and its type
species, A. alba Link, from the western Atlantic. From the
evidence given here the species is apparently genetically distant
from the rest of the ‘Anodontia’ group. This separation is
supported by some morphological features, such as the shape of
the anterior adductor muscle, ligament structure, form of the
mantle gills and septum. Anodontia edentuloides (Verrill, 1870)
from the eastern Pacific and the fossil A. sphericula (Dall &
Ochsner, 1928) are similar in shell morphology to A. alba, and
together they probably form a distinct group. However, the other
western Atlantic species, A. schrammi (sometimes erroneously
referred to as A. philippiana), groups with the Indo-Pacific
species despite often being considered similar to A. alba.
The position of Pseudolunisca lacteola as a sister taxon to
Anodontia alba is surprising on the evidence of shell morph-
ology. It has commarginal lamellae, a large asymmetric lunule
and a prominent cardinal tooth in the right valve, although
lateral teeth are absent. Anatomical information is lacking, but
our observation of a single living specimen show that, unlike
Anodontia alba, there is only a short length of mantle fusion
ventral to the inhalant aperture.
Phacoides pectinatus
In the phylogenetic analysis the position of Phacoides pectinatus is
unclear but it seems likely to be a sister to the lucinid clade B. It
may be the only living species of the genus, living deeply bur-
rowed in sulphide-rich muds often associated with mangroves in
the western Atlantic (Frenkiel et al., 1996). The general anatomy
was described by Narchi and Farani Assis (1980), who illustrated
a line of discrete mantle gills around the anterior ventral mantle
that are unlike those of any other lucinid. Detailed studies of the
ultrastructure of the bacteriocytes (Frenkiel et al., 1996; Liberge,
Gros & Frenkiel, 2001) have also identified unusual features not
observed in other lucinids, such as high haemoglobin concen-
tration, sulphide-oxidizing bodies and large lysosomes. These
morphological peculiarities are reflected in the molecular data.
Phacoides pectinatus has many small indels (1–2 bp) and base
changes that are not shared by other lucinids. These add to the
branch length and do not assist in resolving its relationship with
other lucinids. Bretsky (1976) included Phacoides as a subgenus
of Lucina within her Lucina lineage. [The name Phacoides is now
used in a more restricted sense than in the past following ICZN
(1977) Opinion 1095 which designated Lucina pensylvanica as
the type species of Lucina and Venus jamaicensis Spengler,
1784 Phacoides pectinatus (Gmelin, 1791) as the type species of
Phacoides.]
Lucinidae clades A and B
The remaining lucinids (except Fimbria) in our analysis fall into
two further well-supported groups. The first group (clade A)
comprises Ctena spp., Codakia spp., Lucinoma borealis and
Pillucina vietnamica; the second group (clade B) includes
Cardiolucina spp.,Lucina pensylvanica,Rasta lamyi,Divalinga
quadrisulcata,Chavania striata,Wallucina assimilis,Loripes luci-
nalis,Pillucina pisidium, undescribed new genus and species
from Kermadec Ridge, Divaricella irpex,Austriella corrugata and
‘Lucina’ dalli. Although the two lucinid clades are well resolved,
there is little support for the internal relationships. Additional
sequence data from mitochondrial genes as well as the inclusion
of more taxa in the analysis are needed for improved resolution
of these relationships.
The present molecular data have provided some interesting
new evidence that supports or questions existing ideas con-
cerning relationships within clades A and B and has important
implications for future reclassification of the Lucinidae. For
example, on shell characters, Codakia and Ctena have long been
associated together (Chavan, 1937–8) and our analysis supports
this idea. Further, in a recent systematic revision, Glover &
Taylor (2001) grouped together lucinids of the genera Pillucina,
Wallucina and Chavania based on the presence, amongst other
characters, of a short, deeply inset internal ligament similar to
that of Loripes lucinalis. In the molecular analysis, Loripes,
Wallucina, Chavania and Pillucina pisidium group together but
Pillucina vietnamica clusters with Lucinoma in the other lucinid
clade. This suggests that this form of internal ligament is homo-
plastic. With hindsight, we also observe that characters of the
posterior apertures (papillate inhalant aperture; extent of
mantle fusion) are similar in Lucinoma borealis and Pillucina
vietnamica (Allen, 1958; Glover & Taylor, 2001). Sequences
from Rasta lamyi, a species with unusual shell form and long
periostracal pipes (Glover & Taylor 1997; Taylor & Glover,
1997a), reveal that it nests in lucinid clade B.
Fossil record
Although Lucinidae seemingly have a long fossil record into the
lower Palaeozoic, the pre-Mesozoic record is very patchy and the
fossils mostly poorly preserved. Futhermore, most discussions
of fossil Lucinoidea have assumed the superfamily to be mono-
phyletic and have considered Ungulinidae and Lucinidae
together (e.g. Johnston, 1993). The first possible lucinid is Iliona
prisca from the upper Silurian, possessing an elongate and
detached anterior adductor muscle scar and similarity in shape
to the Recent Eomiltha verhooevei (Deshayes, 1857). Claims of the
Ordovician Babinka as a lucinid ancestor are unconvincing
(McAlester, 1966; Cope, 1997; Taylor & Glover, 2000). Many
MOLECULAR PHYLOGENY OF LUCINOIDEA
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Palaeozoic records of Lucinidae are uncertain, but some fossils
are distinctly lucinid in form. For instance, Paracyclas proavia
(Goldfuss, 1840) from the Devonian of Australia (Johnston,
1993: fig. 82) has a shell morphology strikingly similar to the
Recent Anodontia alba, but differs in the position of the anterior
adductor muscle scar. Nonetheless, other Anodontia-like fossils
do not occur until the Eocene. It is not until the Mesozoic and
Cenozoic that Lucinidae become regularly encountered mem-
bers of benthic assemblages (Chavan, 1952). Notable amongst
these is the largest lucinid, ‘Pseudomiltha’ megameris (Dall, 1901),
from the Eocene of Jamaica that reaches a shell height of 32 cm
(BMNH Pal. Dept. L. 74779). Although fossil lucinids have
been incorporated into evolutionary lineages (Chavan 1937–8;
Bretsky, 1976) all these bivalves need re-evaluation using more
rigorous methods of character analysis.
Concluding remarks
1. Our results conclusively demonstrate that the Thyasiridae,
long regarded as forming a monophyletic clade with the
Lucinidae, are in fact phylogenetically distant and should be
excluded from the Lucinoidea. Thyasiridae thus represent
yet another evolutionary pathway to chemosymbiosis along
with other bivalve groups including Solemyidae, Bathymo-
diolidinae, Vesicomyidae and Lucinidae (Fisher, 1990; Reid,
1990; Distel, 1998). Additionally, Teredinidae and Xylo-
phaginae also have ctenidial endosymbionts that may relate
to their wood-based nutrition (Distel & Roberts, 1997). We
conclude that the behaviour and morphology of thyasirids
do not belong to an evolutionary progression or regression
from the Lucinidae as implied by previous authors (Allen,
1958; Reid & Brand, 1986). Not all thyasirid species possess
chemosymbiotic bacteria and the relationships of the groups
with and without the symbiosis need investigation.
2. Our molecular evidence confirms that the Ungulinidae are
unrelated to the Lucinidae (Steiner & Hammer, 2000) and
should also be removed from the Lucinoidea.
3. Although not sequenced the Cyrenoididae are morpho-
logically distinct and appear unconnected to the lucinoids.
The status of the fossil family Mactromyidae is unresolved.
4. Our results confirm those of Giribet & Distel (2004), show-
ing that Fimbriidae nest within the Lucinidae and we suggest
that their separate familial status is not warranted.
5. Within the Lucinidae, a high phylogenetic diversity has been
revealed with several divergent groups. Notably, the
‘Anodontia’ and Myrtea clades are well separated from other
lucinids. Most other species fall into two large, distinct
clades. Improved resolution within these clades may be
achieved with additional taxa and sequences from other
genes. The currently used classifications of Lucinidae
(Chavan, 1969; Bretsky, 1976) are not supported by our
results.
ACKNOWLEDGEMENTS
We are indebted to the following friends and colleagues for
generously providing tissue samples used in this study: Georgina
Budd and Steve Hawkins (MBA, Plymouth), Stephanie Clark
(Australian Museum), Gonzalo Giribet (Harvard University),
Serge Gofas (University of Malaga), Olivier Gros (Université des
Antilles et de la Guyane), Paul Kingston (Heriot-Watt Uni-
versity), Bruce Marshall (Museum of New Zealand), Graham
Oliver (National Museum of Wales), Phil Rainbow (Natural
History Museum, London), Fred Wells (Western Australian
Museum), Beth Ballment and Lesa Peplow (Australian Institute
of Marine Science). Dan Distel and Gonzalo Giribet generously
provided sequences that were unpublished when this manuscript
was being prepared. Many people provided extensive logistic
support during field collecting including Rudiger Bieler, Paula
Mikkelsen, Gary Kendrick, Brian Morton, Fred Wells and the
Lizard Island Laboratory. We thank Julia Llewellyn-Hughes and
Claire Griffin for expert technical assistance in operating the
automated sequencers. We are grateful to David Reid for sup-
port, encouragement and regular discussion and to Noel Morris
for helpful discussions concerning fossil lucinoideans.
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