Received: 5 November 2019 
|  Revised: 6 February 2020 
DOI: 10.1111/zsc.12419
ORIGINAL ARTICLE
Molecular phylogeny of protobranch bivalves and systematic
implications of their shell microstructure
Kei Sato1,2  | Yasunori Kano3
Takenori Sasaki2
1
Waseda University, Tokyo, Japan
2
The University Museum, The University of
Tokyo, Tokyo, Japan
3
Atmosphere and Ocean Research Institute,
The University of Tokyo, Chiba, Japan
4
National Institute of Technology,
Wakayama College, Gobo, Japan
5
Japan Agency for Marine-Earth Science
and Technology, Yokosuka, Japan
Correspondence
Yasunori Kano, Atmosphere and Ocean
Research Institute, The University of
Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba
277-8564, Japan.
Email: kano@aori.u-tokyo.ac.jp
Funding information
Japan Society for the Promotion of
Science, Grant/Award Number: 18H02494,
24654167 and 26291077
1  |   IN T RO D U C T ION
The Protobranchia represent an intriguing group of Bivalvia
in terms of their early evolution, unique anatomy, lar-
val development and ecological diversification in the deep
Zoologica Scripta. 2020;00:1–15. wileyonlinelibrary.com/journal/zsc © 2020 Royal Swedish Academy of Sciences     1
|  Accepted: 1 March 2020
  | Davin H. E. Setiamarga2,4  | Hiromi K. Watanabe5  |
Abstract
Higher systematics and evolutionary history of Protobranchia, a subclass of Bivalvia,
have long been controversial due to paucity of prominent shell characters and diffi-
culties in collecting live material for diverse taxa. Here, we evaluate the reliability of
shell microstructure for protobranch higher systematics by reconstructing a molecu-
lar phylogeny of the subclass. Relationships were assessed using the nuclear (18S
rRNA, 28S rRNA and histone H3) and mitochondrial (16S rRNA and cytochrome c
oxidase subunit 1) gene sequences from 89 in-group species. Maximum likelihood
reconstruction with the nuclear markers recognized five superfamilies (Nuculoidea,
Solemyoidea, Manzanelloidea, Nuculanoidea and Sareptoidea) as the in-group
clades of the monophyletic Protobranchia. Sareptoidea is herein redefined to com-
prise Sarepta and Setigloma in the sole family Sareptidae, whereas Pristigloma and
its monotypic Pristiglomidae are transferred from this superfamily to Nuculanoidea,
both in the order Nuculanida. Mapping of shell microstructure characters on the tree
confirmed their conservativeness at superfamily level when only living species were
taken into account. The Nuculoidea have shells with the outer prismatic and mid-
dle/inner nacreous structures; Solemyoidea are characterized by either the radially
elongate simple prismatic structure or the reticulate structure in the outer shell layer;
Manzanelloidea, Nuculanoidea and Sareptoidea have shells of homogeneous, fibrous
prismatic and/or fine complex crossed lamellar structures, all of which lack large
structural units. Our Bayesian time calibration, on the contrary, suggested frequent
loss of nacre in the Paleozoic and Mesozoic history of Protobranchia, at least once
each in Nuculoidea, Manzanelloidea, Solemyoidea and Sareptoidea in the Paleozoic,
and perhaps multiple times in Nuculanoidea by the Mesozoic.
KEYWORDS
Bivalvia, fossil, nacre, Protobranchia, Sareptoidea, time calibration
sea (Zardus, 2002). They first appeared at the latest in the
Early Ordovician (Cope, 2004) and were considered a prob-
able stem group of Bivalvia (Cox et al., 1969; Zong-Jie &
Sánchez, 2012). The early ontogeny of protobranchs is com-
monly characterized by a lecithotrophic pericalymma larva,
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2       SATO et al.
in contrast to a veliger larva found in many other groups of
bivalves (Zardus & Martel, 2002). The traditional classifica-
tion of the extant Protobranchia recognizes five superfamilies
in three orders: Nuculanoidea and Sareptoidea in the order
Nuculida, Solemyoidea and Manzanelloidea in Solemyida,
and Nuculanoidea in Nuculanida (Bouchet, Rocroi, Bieler,
Carter, & Coan, 2010; Coan & Valentich-Scott, 2012; Huber,
2010). The Nuculida and Nuculanida are generally deposit
feeders (Stanley, 1970; Zardus, 2002). The Solemyida host
chemoautotrophic bacteria within their gills (Distel, 1998;
Stewart & Cavanaugh, 2006); the origin of their chemosym-
biosis-based lifestyle is estimated to date back to the early
Ordovician (Oliver & Taylor, 2012). Protobranchs have
changed little in their morphologies and habitats (Allen, 1983)
and remained important components of marine soft-sediment
assemblages over time (Rhoads & Young, 1970).
The higher systematics of Protobranchia has been chang-
ing and it is yet to be established. As expressed by Allen
(1985), the gross shell morphology of protobranchs is “never
extravagant” with few characters suitable for the recognition
of natural groups. Moreover, the primary association of pro-
tobranchs with the deep sea (Allen, 1978) and infaunal hab-
itat (Stanley, 1970) hinders the collection of fresh material
for soft-part observations and molecular analyses. Such dif-
ficulties have made the in-group relationships and even the
monophyly of this subclass controversial (e.g., Combosch
et al., 2017; Giribet, 2008), whereas recent molecular studies
finally provided support for a monophyletic Protobranchia
(Bieler et al., 2014; González et al., 2015; Lemer, Bieler,
& Giribet, 2019; Sharma et al., 2012; Smith et al., 2011).
Sharma et al. (2013) conducted the most extensive molec-
ular phylogenetic analyses to date for protobranch bivalves.
They pointed out several major issues in the previous higher
systematics of the group and formally reassigned the fam-
ily Sareptidae and its own superfamily Sareptoidea from the
order Nuculida to Nuculanida. The monophyly of Solemyida
(Manzanelloidea and Solemyoidea) was also questioned, but
this and other traditional taxa were provisionally accepted in
their proposed classification of Protobranchia (Sharma et al.,
2013:199).
In addition to these issues on the higher classification,
recent population genetic studies have indicated signifi-
cant genetic divergence within the putative morphologi-
cal species of Protobranchia, hence posing questions on
species-level taxonomy. For example, Neulinger, Sahling,
Süling, and Imhoff (2006) identified two distinct genetic
lineages among the geographic populations of a single mor-
phospecies “Acharax cf. johnsoni.” Genetic differentiation
has also been detected among populations of protobranchs
along depth gradients. Zardus, Etter, Chase, Rex, and Boyle
(2006) showed that genetic divergence among the pop-
ulations of Nucula atacellana is much greater at different
depths within the same basins (the North American, West
European and Argentine basins) than populations at similar
depths and thousands of kilometers apart. These studies sug-
gest that protobranch bivalves include many cryptic species,
which possibly negate the accurate assessment of concho-
logical characters for higher classification if different spec-
imens are used in phenotypic and molecular phylogenetic
analyses for a nominal species.
Molluscan shell microstructure is composed of structural
units of calcium carbonate and minor organic contents. The
components of the shell microstructure are generally con-
servative in evolution and used to diagnose the superfam-
ilies of Protobranchia (Bieler et al., 2014; Zardus, 2002).
Sato and Sasaki (2015) have produced updated descriptions
for the extant Nuculida and Nuculanida and corroborated
this general conservativeness of shell composition. For ex-
ample, the shells of Nuculoidea and Nuculanoidea can be
differentiated by the presence or absence of nacre or mother
of pearl (see also Coan, Valentich-Scott, & Bernard, 2000).
More subtle microstructural differences of shell layers can
be useful in discriminating families, genera and even spe-
cies. For example, solemyid genera and subgenera are diag-
nosed by unique types of structures in the outer layer (Sato,
Nakashima, Majima, Watanabe, & Sasaki, 2013). Nuculids
often show differences among species in the shape and ori-
entation of outer layer prisms (Carter, 1990; Sato & Sasaki,
2015).
However, this generalization based solely on the observa-
tion of extant taxa faces a considerable number of counter-
examples in the fossil record. Many non-nacreous nuculoids
as well as nacre-bearing nuculanoids and solemyids are
known among extinct taxa, which closely match contem-
porary counterparts in shell shapes (Carter, 1990, 2001).
The presence or absence of nacre is not always a clear-cut
distinction even among extant taxa. Although most living
nuculoids have the middle and inner shell layers of entirely
nacreous structure, small patches of homogeneous or fine
complex crossed lamellar structure have been found in the
inner layer of some specimens of Nucula proxima (Carter,
1990). Even more intriguing is Condylonucula maya, where
the middle layer ontogenetically changes its composition
from nacreous to homogeneous (Carter, 2001). On the con-
trary, two extant species of Nuculanoidea were reported
to have nacre (Silicula sp. and Phaseolus sp.; Nevesskaya,
Scarlato, Starobogatov, Eberzin, 1971), while this finding
requires confirmation. These exceptions and intermediate
conditions have prompted debates about the validity of shell
microstructure in the higher systematics of Protobranchia
(e.g., Carter, 1990; Sato & Sasaki, 2015; Taylor, Kennedy,
& Hall, 1969).
In this study, we conducted a phylogenetic reevaluation
of protobranch shell microstructure by taking the follow-
ing three steps: (a) DNA sequencing from the same set of
individuals used for previous microstructural observations
SATO et al.
(Sato & Sasaki, 2015), (b) maximum likelihood and
Bayesian estimation of phylogenetic relationships and di-
vergence times based on the molecular data and fossil re-
cord, and finally (c) the assessment of usefulness of the
shell microstructure by mapping character states on the
molecular phylogeny.
2  |  M ATE R IA L S A N D ME T HODS
2.1  |  Taxon sampling
Thirty-two species of protobranchs were collected from
Japanese and adjacent waters. These included 5 solemy-
oids, 1 manzanelloid, 15 nuculanoideas, 9 nuculoids and
2 sareptoids (sensu Sharma et al., 2013). Most specimens
were boiled at 80–99°C for <1  min to deactivate DNases
(Ueshima, 2002) before fixation in 99% of ethanol. The same
specimens have been used for the observation of shell micro-
structure (see Sato, Nakashima, et al., 2013; Sato & Sasaki,
2015 for details). Voucher information and photographs of
shells are shown as Table S1 and Figure S1.
2.2  |  DNA extraction,
amplification and sequencing
DNA was extracted from the mantle or the whole animal
using the DNeasy Blood and Tissue Kit (Qiagen) and pu-
rified by GeneReleaser (BioVentures, Inc.) following the
manufacturers' protocols. A total of five gene fragments
were amplified, including two nuclear ribosomal genes
(18S rRNA and 28S rRNA), one nuclear protein-coding
gene (histone H3), and two mitochondrial genes (16S rRNA
and cytochrome c oxidase subunit 1: COI). PCR profile
for the mitochondrial genes was as follows: initial dena-
turation for 150  s at 94°C, followed by 30–35 cycles of
denaturation for 120 s at 94°C, annealing for 30 s at 50°C
and elongation for 60 s at 72°C. The final elongation step
was conducted for 180 s at 72°C. PCR conditions for the
nuclear genes were the same as above except that the an-
nealing step was performed in a touch down manner (Don,
Cox, Wainwright, Baker, & Mattick, 1991), with a starting
annealing temperature of 70°C and decreasing it by 1°C in
each subsequent cycle until reaching 65°C, which was fol-
lowed by 25–30 cycles at the final annealing temperature.
The primer sequences are listed in Table S2.
After purification by Illustra ExoProStar (GE
Healthcare UK Limited), sequencing was performed using
GenomeLab DTCS Quick Start Kit (Beckman Coulter Inc.)
and sequencing primers shown in Table S2. Capillary elec-
trophoresis was conducted with a CEQ 2000 sequencer
(Beckman Coulter).
  
|
   3
2.3  |  Additional DNA sequences
Published sequences of the five loci were also obtained for
protobranch and out-group taxa (e.g., Giribet et al., 2006;
Giribet & Wheeler, 2002; Neulinger et al., 2006; Sharma
et al., 2013; Taylor, Glover, & Williams, 2008). The accu-
racy of each sequence fragment was checked by a BLAST
search and by comparison with homologous sequences
from related taxa; species with dubious data were ex-
cluded from subsequent analyses. The sequences were then
double-checked by reconstructing independent gene trees
using the maximum likelihood (ML) method (Figure S2).
The combined new and published sequence data cover a
total of 115 in-group and seven out-group operational taxo-
nomic units (OTUs). The in-group OTUs cover 28 proto-
branch genera and all of five superfamilies and 13 families
in the current classification scheme (Bouchet et al., 2010;
Sharma et al., 2013). A full list of species and specimens
included in this study is shown as Table S3.
2.4  |  Phylogenetic analyses
Sequences of the five genes were aligned individually using
the online version of MAFFT 7 (Katoh & Standley, 2013)
with the alignment strategy set to Q-INS-i for the ribosomal
genes (18S, 28S and 16S) and G-INS-i for the protein-coding
genes (H3 and COI, aligned as amino acids). The resulting
alignments were visually confirmed using MEGA 6 (Tamura,
Stecher, Peterson, Filipski, & Kumar, 2013). Each alignment
was masked to remove ambiguous sites using Gblocks ver-
sion 0.91b (Castresana, 2000) by setting parameters to allow
smaller final blocks, gap positions within final blocks and
less strict flanking positions. The final lengths of the align-
ments after the masking are as follows: 1,658 characters in
the 18S rRNA (88% of 1,882 characters in the initial align-
ment), 1,279 in 28S (33% of 3,931), 324 in H3 (99% of 327),
459 in 16S (70% of 653) and 639 in COI sequences (96% of
663).
Three datasets were generated based on different com-
binations of genes. The first and second datasets comprise
the three nuclear genes (118 OTUs and 3,261 characters)
and the two mitochondrial genes (99 OTUs and 1,098
characters), respectively. The third dataset, thereafter re-
ferred to as the combined dataset, consists of all five genes
that were concatenated to create a matrix of 121 OTUs
and 4,361 characters. Mesquite version 3.61 (Maddison
& Maddison, 2019) was used to concatenate the multiple
gene sequences.
Maximum likelihood analyses of the aligned datasets were
conducted using the GTR + Γ + I substitution model (as sug-
gested by MEGA 6) in RAxML GUI version 1.3 (Silvestro
& Michalak, 2012). Each gene was allowed to have different
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4       SATO et al.
parameters, resulting in a total of five partitions. The robust-
ness of topologies was evaluated by generating 1,000 boot-
strap replicates of the alignment sites.
2.5  |  Divergence time estimation
Divergence dates between protobranch clades were calcu-
lated using the three-gene nuclear dataset (based on the re-
sults of preceding ML analyses; see below) and a relaxed
molecular clock model in BEAST 2.4.5 (Bouckaert et al.,
2014). The GTR + Γ + I model was applied and parameters
were unlinked across three partitions (18S, 28S and H3);
branch lengths and dates were estimated with an uncorrelated
lognormal relaxed-clock model and a Yule prior on the tree.
A single run consisted of 200,000,000 generations (with a
sample frequency of 10,000) produced 20,000 estimates of
divergence dates. The convergence and mixing of the chains
were assessed in Tracer 1.5.0 and first 10,000 estimates were
discarded as burn-ins.
The time calibration was made by setting the crown ages
of Bivalvia, Protobranchia and Solemyida. (a) The Lower
Cambrian (Tommotian and Atdabanian) occurrences of ear-
liest bivalve genera (Pojeta, 2000; Parkhaev, 2008) were used
to roughly represent the first split of the extant lineages of
Bivalvia. This split was constrained to be at least 510 mil-
lion years ago (Mya) with a 95% interval of 511–541 Mya
(lognormal distribution, mean in real space: 10, log SD: 1,
offset: 510). (b) The first split within the extant lineages of
Protobranchia was set at a minimum of 482 Mya with a 95%
upper limit of 513 Mya (lognormal, mean in real space: 10,
log SD: 1, offset: 482), based on the Tremadocian occurrence
of the earliest protobranch family Praenuculidae (Cope,
2004:198, figure 20.2). Lastly, (3) divergence between the
superfamilies Solemyoidea and Manzanelloidea (collectively
the order Solemyida) was set to have a minimum bound of
462 Mya with a 95% upper limit of 493 Mya (lognormal,
mean in real space: 10, log SD: 1, offset: 462). This minimum
bound corresponds the upper boundary of the Whiterockian,
a Middle Ordovician stage with the earliest solemyoid fos-
sil Psiloconcha senecta (see Pojeta, 1988: figure 3). The
three reference nodes for time calibration and the out-group
(Gastropoda) were constrained as monophyletic based on the
results of preceding ML analyses.
2.6  |  Ancestral state reconstruction
Estimation of shell microstructural conditions for ancestral
nodes was carried out using ML method in Mesquite 3.61
and the topology of the time-calibrated BEAST tree. Six or
seven character states were recognized for each of the outer,
middle and inner shell layers of the extant Protobranchia
(see Sato & Sasaki, 2015). The Markov k-state 1 (Mk1) pa-
rameter model was used for reconstruction at each ancestral
node with equal probability for any particular character state
change.
3  |  RESULTS
Three ML trees were reconstructed based on the nuclear
(18S  +  28S  +  H3), mitochondrial (16S  +  COI) and com-
bined (18S  +  28S  +  H3  +  16S  +  COI) datasets (Figure  1
and Figures  S3 and S4). The monophyly of the subclass
Protobranchia was recovered in two out of the three trees, al-
beit with low support values: bootstrap support (BS) of 65%
and 47% by the nuclear and combined gene datasets, respec-
tively. A separate ML analysis based on the same nuclear
genes with two additional taxa from the bivalve subclass
Pteriormorphia supported the monophyly of Protobranchia
with a higher BS value (81%) but resulted in lower support
for many of in-group nodes (Figure S5).
The monophyly of each Nuculoidea, Solemyoidea
and Manzanelloidea was strongly supported in all trees
(BS  ≥  94%). By contrast, the conventional Nuculanoidea
and Sareptoidea (sensu Bouchet et al., 2010; Sharma et al.,
2013) were supported solely by the mitochondrial dataset
(≥97%). The nuclear and combined trees instead recognized
two phylogenetically distant clades in the conventional
Sareptoidea: three species of Pristigloma were nested within
the Nuculanoidea (100%/75% by nuclear/combined data-
sets), whereas Sarepta speciosa and Setigloma japonica were
recovered as the sister clade to this Nuculanoidea including
Pristigloma (98%/100%). The latter two datasets recov-
ered the order Solemyida (Solemyoidea + Manzanelloidea)
as monophyletic and the Nuculoidea as the sister group to
all other Protobranchia, both with a higher support value
in the nuclear tree (82%/71%) than in the combined tree
(50%/63%).
At the generic level, Acila, Brevinucula and Huxleyia were
each monophyletic (100%) while Nucula and Ennucula were
non-monophyletic in all trees. Solemya was either mono-
phyletic (95%/100% in the nuclear/combined trees) or para-
phyletic with respect to Acharax (60% in the mitochondrial
tree). Many families and genera as currently circumscribed
in Nuculanoidea were non-monophyletic, whereas support
values were generally low for early divergences within the
superfamily.
The independent genes showed significant incongru-
ence in terms of the position of Pristigloma. A single 16S
sequence of P. nitens (KC984670; Sharma et al., 2013) rep-
resented the only mitochondrial data available for the genus.
This 16S sequence suggested a very close affinity between
Pristigloma and Setigloma (100%; Figure S2d), whereas each
of the nuclear-gene trees (18S, 28S and H3) recovered all
SATO et al.   
   5
|

F I G U R E 1   Phylogenetic relationships of Protobranchia based on the maximum likelihood analysis of nuclear-gene sequences
(18S + 28S + H3). Numbers on nodes indicate bootstrap values in percent. Five superfamilies as redefined in the present study are marked with
different colors (blue, Nuculoidea; purple, Manzanelloidea; red, Solemyoidea; green, Sareptoidea; orange, Nuculanoidea)

study species of the former genus (including P. nitens) within
the Nuculanoidea, far distant from the latter genus (Figures
S2a–c, S6). The independent COI-gene tree did not contain
any Pristigloma (Figure S2e).
The three-gene nuclear dataset was utilized to explore the
divergence dates of major protobranch clades by setting three
fossil-based priors in BEAST (Figure 2). The overall topol-
ogy from this BEAST analysis was nearly identical to that of
the ML reconstruction and most basal nodes were recovered
with maximum support (Bayesian posterior probability of 1).
The posterior mean age and 95% highest posterior density
(HPD) intervals for the first splits within superfamilies were
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6       SATO et al.

F I G U R E 2   Time-calibrated BEAST phylogeny of protobranch bivalves inferred from nuclear-gene sequences (18S + 28S + H3) and
calibration priors on the ages of three basal nodes (shown as dark blue error bars). Four branches with dots were constrained to be monophyletic.
Numerals on branches denote Bayesian posterior probabilities (BPP); asterisks denote maximum value (1.00). Light blue bars show estimated nodal
ages (in million years ago, Mya) and 95% credibility intervals (HPD)

as follows: Nuculoidea (mean: 259.2 Mya, 95% HPD interval:
177.0–354.9), Manzanelloidea (233.9 Mya, 119.2–350.3),
Solemyoidea (248.3 Mya, 164.0–337.1), Sareptoidea exclud-
ing Pristigloma (96.6 Mya, 33.4–176.0) and Nuculanoidea
including Pristigloma (279.6 Mya, 201.7–352.8).
Figure 3 and Figure S8 show character-state distributions
and reconstructed ancestral states for protobranch shell mi-
crostructure. Reconstructed states with significant proba-
bilities (p  >  .95) are summarized for nodes at the order or
superfamily level in Table S4.
SATO et al.
4  |   D IS C U SSION
4.1  |  Phylogenetic position and generic
composition of Sareptoidea—utility of
mitochondrial data questioned?
The most significant finding of the present molecular analy-
sis concerns the phylogenetic position and generic composi-
tion of Sareptoidea. We conclude that the family Sareptidae
(including the genera Sarepta and Setigloma) and its own
superfamily Sareptoidea belong to the order Nuculanida,
whereas another putative sareptid genus Pristigloma to be the
independent Pristiglomidae in Nuculanoidea, another super-
family in Nuculanida (Table 1).
In their molecular analysis for protobranch bivalves,
Sharma et al. (2013) included three species of the genus
Pristigloma to represent this superfamily. The position of
the genus differed fundamentally in their trees inferred
with different methods of analysis: nested within or sister
to the Nuculanoidea (in RAxML and MrBayes or POY and
BEAST reconstructions, respectively; Sharma et al., 2013:
figures 2–4, 6). As a conservative measure, they maintained
Sareptoidea as an independent superfamily by assuming that
the latter, basal placement of Pristigloma more plausibly re-
flected the evolutionary history of the Protobranchia (their
Table S4).
The present reconstruction of single-gene trees revealed
that the above inconsistency was most probably attributable
to a single 16S sequence from P. nitens, which represented
the only mitochondrial data available for Sareptoidea in
Sharma et al. (2013). This sequence pointed toward the
exclusion of Pristigloma from Nuculanoidea (Figure S2d),
while the opposite was suggested in all nuclear-gene trees
with this and other congeneric species (Figures S2a–c, S5,
S6). The nuclear markers used were not only 18S and 28S
in the same rRNA gene cluster, but also the entirely in-
dependent histone H3 gene. It seems thus likely that the
alleged 16S sequence of P. nitens does not provide suitable
information for the phylogenetic affinity of this species or
the genus Pristigloma.
Bivalve mitochondrial genomes are known to exhibit a
number of uncommon features that may prevent compar-
ison of homologous sequences from different lineages or
individuals. The most unique aspect is the doubly unipa-
rental inheritance (DUI) in many bivalve groups (Doucet-
Beaupré et al., 2010; Plazzi, Puccio, & Passamonti, 2016),
including the Protobranchia (Gusman, Lecomte, Stewart,
Passamonti, & Breton, 2016). Male bivalves with DUI are
heteroplasmic and contain both female- and male-transmit-
ted mitochondrial genomes that might have diverged from
each other in a very early ancestor (Doucet-Beaupré et al.,
2010). Another potential obstacle in homologous sequence
comparison is gene duplication within a single genome. For
  
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   7
example, female-transmitted mitochondrial genomes of some
bivalves have two copies of COII or atp8 gene (Breton et al.,
2014; Stöger & Schrödl, 2013). A more general caution for
reconstructing mitochondrial phylogenies is the possible
presence of nuclear pseudogenes of mitochondrial origin
(NUMT; Richly & Leister, 2004; see also Lee, Hong, Kim,
& O'Foighil, 2007).
We, therefore, dismiss the mitochondrial markers as po-
tentially disturbing signals in the present phylogenetic re-
construction. In the following lines, the evolutionary trends
of the Protobranchia are discussed based on the nucle-
ar-gene tree (Figure 1). Exclusion of the mitochondrial data
significantly increased resolution and nodal support values
for the entire subclass (see Figure S4 and trees in Sharma
et al., 2013). Disturbing signals in the mitochondrial data-
set might also explain the polyphyletic Protobranchia in
the latest five-gene phylogeny of the Bivalvia (Combosch
et al., 2017).
Five genera, that is, Sarepta, Pristigloma, Setigloma,
Microgloma and Pseudoglomus, have been relevant to the
discussion on sareptoid systematics (Table  1). Ockelmann
and Warén (1998) documented similarities in hinge teeth and
ligament morphology between the type species of Sarepta
and Setigloma (Sarepta speciosa and Setigloma japonica).
For Setigloma, Schileiko (1983) instead assumed its kin-
ship to Pristigloma, also based on the hinge teeth and liga-
ment; this contradicted a previous classification by Sanders
and Allen (1973), in which Pristiglomidae was proposed as
a new family to accommodate Pristigloma and Microgloma
near the Nuculidae. The latter two genera share the pres-
ence of an anterior rather than a posterior inhalant current
and an anterior position of mucus glands of the mantle and
the absence of siphons, a large palp and an internal liga-
ment (Sanders & Allen, 1973). More recently, Huber (2010)
considered Sarepta, Setigloma and Pristigloma to be the
members of Sareptidae by combining the ideas of Schileiko
(1983) and Ockelmann and Warén (1998). This classification
scheme was followed by Sharma et al. (2013), who treated
Pristigloma as the Sareptidae without having molecular data
for the type genus Sarepta.
Based on our phylogenetic reconstruction using sequences
from the type species of Sarepta, we conclude that the fam-
ily Sareptidae and its own superfamily Sareptoidea belong to
the order Nuculanida as proposed by Sharma et al. (2013),
whereas Pristigloma to be the independent Pristiglomidae in
Nuculanoidea, another superfamily in Nuculanida. The other
possible sareptids (Microgloma and Pseudoglomus) have
never been subjected to molecular-based phylogeny and we,
therefore, refrain from drawing any conclusions. The iden-
tification of additional anatomical characteristics is also re-
quired to better understand relationships within Sareptidae,
together with the reconfirmation of mitochondrial sequences
of Pristigloma.
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8       SATO et al.

F I G U R E 3   Shell microstructural compositions mapped on the nuclear-gene tree of Protobranchia. Filled circles indicate nodes with
meaningful bootstrap support (≥80%). Photographs of shells are shown for species with stars; microstructural compositions for these species were
characterized in Sato, Nakashima, et al. (2013) and Sato and Sasaki (2015). Microstructural data for six species without a star were retrieved from
earlier studies and reviewed in Sato, Nakashima, et al. (2013). Asterisk denotes Manzanelloidea. The condition of the myostracum (M.) is classified
into clearly present (black square) or absent (open) or indistinct (gray). CCL, complex crossed lamellar structure; CP, composite prismatic; FP,
fibrous prismatic; Hom., homogeneous; IFP, irregular fibrous prismatic; INDCP, irregular non-denticular composite prismatic; N, nacreous; NDCP,
non-denticular composite prismatic; RESP, radially elongate simple prismatic; SP, simple prismatic

4.2  |  Higher level relationships within monophyletic group based on morphological charac-
Protobranchia ters (e.g., Cope, 1996; Morton, 1996), but this had not
been supported by molecular data for a long time (e.g.,
Protobranchia has traditionally been treated as a Combosch et al., 2017; Giribet & Distel, 2003; Wilson,
 SATO et al.

T A B L E 1   Previous and present taxonomic treatments of five protobranch genera with potential affinities to Sareptidae

Cox et al. Sanders & Allen & Hannah Ockelmann & Coan et al. Sharma et
(1969) Allen (1973) Schileiko (1983) (1986) Warén (1998) (2000) Huber (2010) al. (2013) This study
Sarepta Nuculanida Nuculanida
Adams, 1860 Nuculanoidea – – Nuculanoidea Nuculoidea? – Sareptoidea Sareptoidea Sareptoidea
Nuculanidae Yoldiidae Sareptidae Sareptidae Sareptidae Sareptidae
Setigloma Nuculida Nuculanida Nuculanida
Schileiko, 1983 – – Nuculanoidea – Nuculoidea? Pristiglomoidea Sareptoidea Sareptoidea Sareptoidea
Pristiglomidae Sareptidae*1 Pristiglomidae Sareptidae Sareptidae Sareptidae
Pristigloma Nuculida Nuculanida Nuculanida
Dall, 1900 Nuculanoidea Nuculoidea Nuculanoidea Nuculoidea Nuculoidea? Pristiglomoidea Sareptoidea Sareptoidea Nuculanoidea
Nuculanidae Pristiglomidae Pristiglomidae Pristiglomidae Pristiglomidae?*2 Pristiglomidae Sareptidae Sareptidae Pristiglomidae
Microgloma
Allen & Sanders, – Nuculoidea – Nuculoidea Nuculanoidea – Nuculanoidea – –
1973 Pristiglomidae Pristiglomidae Nuculanidae Yoldiidae
Pseudoglomus Nuculida
Dall, 1898 Nuculanoidea – – Nuculoidea Nuculanoidea Pristiglomoidea Nuculanoidea – –
Malletiidae Pristiglomidae Fam. undet.*3 Pristiglomidae Malletiidae
*1 Setigloma considered to be a probable synonym of Sarepta. *2 Pristiglomidae considered to be a potential synonym of Sareptidae. *3 Possible position in Malletiidae, Neilonellidae or Tindariidae.
  
|   9
 |
10       SATO et al.
Rouse, & Giribet, 2010). Recent molecular studies have
finally supported their monophyly, based on phylogenetic
(Bieler et al., 2014; Smith et al., 2011) and phylogenomic
data (González et al., 2015; Lemer et al., 2019). Our com-
bined nuclear-gene analyses similarly recovered the mono-
phyletic Protobranchia with bootstrap probabilities of
65%–81% (Figure 1 and Figure S5).
The higher systematics of Protobranchia has been in-
consistent from study to study (Sharma et al., 2012: figure
1). Two major hypotheses for order to superfamily-level
relationships are the Cryptodonta–Paleotaxodonta (Cope,
1996; Newell, 1965) and Opponobranchia–Nuculanida
(Giribet, 2008) concepts, with the main difference lying in
the position of Nuculoidea. The former divides Protobranchia
into Cryptodonta (Solemyoidea and Manzanelloidea;
= Solemyida) and Paleotaxodonta (Nuculoidea and
Nuculanoidea), while the latter concept recognizes
Opponobranchia [Nuculida (Nuculoidea)  +  Solemy(o)ida
(Solemyoidea and Manzanelloidea)] and Nuculan(o)ida
(Nuculanoidea). The tree topologies in Sharma et al. (2013)
were closer to the Cryptodonta–Paleotaxodonta concept but
without a monophyletic Solemyida (see their figure 5). The
latest phylogenomic studies also favor this concept, albeit
with conflicts between the analyses of different data matrices
(González et al., 2015; Lemer et al., 2019).
The phylogenetic reconstructions in Bieler et al. (2014)
and the present study do not support either of the two hy-
potheses, with the Nuculoidea being sister to the rest of
protobranchs (Figure  1). The fossil record agrees with this
topology; the oldest representatives of protobranch bivalves
were in the shell form of the Nuculoidea (e.g., Allen, 1985;
Cope, 1996, 2004; Waller, 1998; Zardus, 2002; Zong-Jie &
Sánchez, 2012). Certain nuculoids retain the most ances-
tral condition among extant protobranchs also in the mode
of feeding; the settled juvenile of Nucula collects food with
the foot (Mortimer, 1962) as suggested in the hypothetical
earliest bivalve (Morton, 1996). Moreover, a recent study
on the sperm ultrastructure of the Protobranchia shows
the presence of radiating plates in the acrosomal vesicle
of Solemyda and Nuculanida, but not in the Nuculida, po-
tentially suggesting their relationships (Healy, Mikkelsen,
Giribet, & Bieler, 2017:24). The present phylogeny is also
concordant with the fossil record in recovering the clade
Solemyoidea  +  Manzanelloidea as the order Solemyida
(Cope, 2000; Pojeta, 1988). A number of shared anatomical
traits (Allen & Sanders, 1969) as well as chemosymbiotic
lifestyles (Oliver & Taylor, 2012) further support the close
relationship of the two superfamilies.
Regardless, the higher systematics of Protobranchia re-
mains contentious. The need of a deeper phylogenomic focus
on this group has been stressed (Lemer et al., 2019), especially
including members of the so-far unsampled Manzanelloidea
and Sareptoidea.
4.3  |  Families and genera
Superfamilies in Protobranchia have been relatively well es-
tablished by recent studies with the exception of Sareptoidea
(see above). Families and genera, however, require further
taxonomic revisions, as outlined below.
1. Superfamily Nuculoidea. The species of the only ex-
tant family Nuculidae have been classified into about
a dozen genera based on the size, outline and sculp-
ture of the shell (e.g., Coan & Valentich-Scott, 2012).
Four genera (sensu Huber, 2010) are included in the
present phylogeny; Acila and Brevinucula each formed
a strongly supported clade (BS  =  100%), while Nucula
and Ennucula were polyphyletic (Figure  1).
2. Superfamily Solemyoidea. The sole family Solemyidae
consists of two reciprocally monophyletic genera
(Figure  1), which are distinguished by the position
of the ligament: internal in Solemya while external in
Acharax (e.g., Bailey, 2011; Cox et al., 1969; Pojeta,
1988; Sato, Watanabe, & Sasaki, 2013). Huber (2010)
had classified several species of Acharax (herein repre-
sented by A. japonica) as the members of his subgenus
Pseudacharax in Solemya, by attaching importance to
their ovate shells with a marginal fringe as in Solemya
s.s. Taylor et al. (2008) proposed different diagnoses for
the subgeneric classification of Solemya, which would
place S. pusilla and S. flava in Solemya s.s., S. velesiana
in Solemyarina and S.  elarraichensis, S.  pervernicosa
and S. velum in Petrasma (see Figure 1). Neither of the
subgeneric classifications was supported in the present
phylogeneric reconstruction.
3. Superfamily Manzanelloidea. The only extant fam-
ily Nucinellidae includes two genera, Nucinella and
Huxleyia (Oliver & Taylor, 2012), which collectively
formed a clade sister to the Solemyoidea in our phy-
logeny (Figure  1). The fossil record suggests that the
Manzanelloidea have diverged from the Solemyoidea
by the Permian period, when the extinct Manzanella ap-
peared as a possible ancestor of living manzanelloids
(Cope, 2000; Pojeta, 1988).
4. Superfamily Nuculanoidea. None of the widely rec-
ognized families (i.e., Nuculanidae, Bathyspinulidae,
Malletiidae, Neilonellidae, Phaseolidae, Siliculidae,
Tindariidae and Yoldiidae) formed a monophyletic
group in the present reconstruction (Figure S7). Of
these, Nuculanidae, Malletiidae, Siliculidae, Tindariidae
and Yoldiidae were each rendered as non-monophyletic
with a meaningful (≥80%) bootstrap value. Although
there still exists considerable phylogenetic uncertainty,
morphological diagnoses and included taxa need to be
revised for nuculanoid families and genera (see also
Sharma et al., 2013).
SATO et al.
4.4  |  Character mapping of shell
microstructure and their systematic
implications
The primary objective of this study is to test the useful-
ness of shell microstructural characters for the system-
atic classification of living and fossil protobranchs. We
mapped character-state distributions on our nuclear-gene
tree by using the same specimens or specimen lots for
both phylogeny and shell microstructure (30 species; see
Sato, Nakashima, et al., 2013; Sato & Sasaki, 2015), sup-
plemented by previous descriptions of microstructure for
six sequenced species (Carter, 1990; Kobayashi, 1971;
Schmidt, 1922; Taylor et al., 1969). This helps postulate
putative synapomorphies for clades and evaluate diagnos-
tic character states for superfamilies, families and genera
(Figure 3 and Figure S8).
1. Superfamily Nuculoidea. Recent species are characterized
by having a prismatic outer layer and nacreous middle
and inner layers. The presence of nacre is a unique di-
agnostic feature for nuculoids in Protobranchia, whereas
exceptions exist among Paleozoic and Mesozoic fossils
(see below). More subtle microstructural differences in
the outer prismatic layer seem to at least partly reflect
phylogenetic relationships among species (Figure 3), and
thus, would be useful as diagnostic characters for nu-
culoid genera and subgenera. The middle layer also
exhibits two types of microstructure: columnar and sheet
nacreous. However, the sheet nacre is patchily distributed
among nuculoids and its systematic value (and polarity
between the two types) is uncertain until more species
are incorporated into a rigorous phylogenetic reconstruc-
tion. The presence or absence of the myostracum also
shows a patchy distribution in the present phylogenetic
tree.
2. Superfamily Solemyoidea. The crown members of this
clade are diagnosed by the presence of the radially elon-
gate simple prismatic (RESP) or reticulate structure, both
of which are unknown in any other molluscan groups
(Sato, Nakashima, et al., 2013). The present phylogenetic
reconstruction suggests that the RESP structure represents
the plesiomorphic condition for the extant Solemyoidea.
The only study taxon with the reticulate structure (Acharax
johnsoni) is nested among other solemyoids (Figure  3).
More specifically, A.  johnsoni is closest to A.  japonica
with the RESP type-C structure, which rather resembles
the reticulate structure in having complex, branching
prisms (Sato, Nakashima, et al., 2013). The lineage lead-
ing to A.  johnsoni might have acquired the organic-rich
reticulate structure as an adaptation to their deep-sea che-
mosynthetic habitats (see Cai et al., 2006; Sasaki, Okutani,
& Fujikura, 2005).
  
|
   11
3. Superfamily Manzanelloidea. The shell microstructures
of the two superfamilies of Solemyida differ signifi-
cantly from each other. The homogeneous structure of
Manzanelloidea, composed of indistinct granular crystals,
is too simple to postulate its homology or homoplasy with
that of Nuculanida (Figure 3). Nonetheless, the known ex-
istence of nacre in some Paleozoic solemyoids suggests
neither RESP nor homogeneous was the condition in the
common ancestor of Solemyida (see below).
4. Superfamilies Nuculanoidea and Sareptoidea. The highly
supported clade Nuculanida is characterized by shell
microstructures composed of granular to fine acicular
crystals. Such microstructures can be categorized into ho-
mogeneous, fibrous prismatic (FP) or fine complex crossed
lamellar (CCL) structure, although there is no clear-cut
distinction between the three (Sato & Sasaki, 2015). The
patchy distribution of species with the FP or the fine CCL
structure in the reconstructed phylogeny may, therefore,
reflect the evolutionary plasticity of crystal shapes. The
overall inconclusiveness of the tree topology (Figure S7)
does not allow us to rigorously discuss the homology and
polarity of these conditions.
4.5  |  Extinct taxa and repeated loss of nacre
As discussed above, the living members of Nuculida
(= Nuculoidea), Solemyoidea and Nuculanida
(Sareptoidea + Nuculanoidea) are recovered as robust clades
in the present molecular phylogeny with each clade sharing
diagnostic microstructural conditions. The Nuculida are most
readily differentiated from other extant protobranchs in hav-
ing nacre or mother-of-pearl structure (Figure 3). However,
nacre-bearing species existed among the Carboniferous
Solemyoidea and the Ordovician to Triassic Nuculanida
(Carter, 1990, 2001; Carter et al., 2015; Vendrasco, Checa,
Heimbrock, & Baumann, 2013) (previous records of nacre
in the Cretaceous Nuculanida seem to be erroneous; K. Sato,
unpublished data). We consider that many of these fossils
belong to the stem groups of the crown Solemyoidea and
Nuculanida. Several studies have shown that the nacre-
ous structure is a costly material and has gradually lost its
“market share” in the course of molluscan evolution (e.g.,
Cartwright & Checa, 2007; Frýda, Klicnarová, Frýdová, &
Mergl, 2010; Vendrasco et al., 2013).
Our phylogeny-based time calibration suggests that
the most recent common ancestor (MRCA) of the extant
Solemyoidea lived in the Permian or Triassic period (mean:
248 Mya; Figure  2). This postdates the Carboniferous oc-
currence of the nacre-bearing “Acharax” trapezoids (Carter,
1990) and implies the lack of the nacreous structure al-
ready in the MRCA. The conventional classification of cer-
tain fossils into Acharax seems to reflect the plesiomorphic
 |
12       SATO et al.
shell shape and ornamentation of the genus among the
Solemyoidea, rather than their positions in the crown group
(see Pojeta, 1988). The situation is less conclusive in the case
of Nuculanida. The MRCA of the extant Nuculanida was cal-
culated to have occurred sometime between the Silurian and
Carboniferous (mean: 372 Mya, Devonian), when nacreous
and non-nacreous Nuculanida co-existed (Carter, 1990, 2001;
Carter et al., 2015; Vendrasco et al., 2013). The presence or
total absence of nacre among the crown Nuculanida, there-
fore, cannot be deduced from the present time calibration.
A nacre-bearing MRCA, however, would be plausible, given
that the nacreous shells of the Triassic Dacryomya, Phestia
and Ryderia closely resemble the non-nacreous shells of cer-
tain living nuculanoids in general appearance and sculpture
(see Cox et al., 1969).
Conversely, a non-nacreous lineage seems to have arisen
from the otherwise nacre-bearing Nuculida. Six species of
Palaeonucula from the Upper Carboniferous to Middle
Jurassic had non-nacreous shells, which, however, look
very similar to the nacre-bearing shells of extant nuculoids
(Carter, 1990). The MRCA of Nuculida or Nuculoidea
was calculated to have occurred sometime between the
Carboniferous and Early Jurassic (mean: 259 Mya, Permian;
Figure 2). We, therefore, cannot infer whether the non-nacre-
ous Palaeonucula originated from the stem or crown group
nuculoids.
Summing up, the mapping of shell-microstructure char-
acters on the present molecular phylogeny confirmed their
conservativeness at superfamily level, but this was only when
living species were taken into account. The Bayesian time
calibration suggested frequent loss of nacre in the Paleozoic
and Mesozoic evolution of Protobranchia, at least once each
in the Paleozoic Nuculoidea, Manzanelloidea, Solemyoidea
and Sareptoidea and probably multiple times in the stem and
crown linages of Nuculanoidea by the Mesozoic. Further ob-
servations of Paleozoic and Mesozoic fossils in combination
with more rigorous phylogenetic reconstruction would en-
able to better trace the evolutionary history of protobranch
shell microstructure.
ACKNOWLEDGMENTS
We are very grateful to Prof. Kazuyoshi Endo, Prof. Toshihiro
Kogure (University of Tokyo, UT), Dr. Takanobu Tsuihiji
(National Museum of Nature and Science, Tokyo, NSMT)
and two anonymous reviewers for their valuable comments
and suggestions on earlier drafts of this manuscript. We also
thank Dr. Koji Seike (National Institute of Advanced Industrial
Science and Technology, Japan), Mr. Hisanori Kohtsuka
(UT), Dr. Takuma Haga (NSMT), Dr. Robert G. Jenkins
(Kanazawa University) who kindly donated protobranch
specimens. The first and second authors (K. S. and Y. K.) par-
ticipated in research cruises through the courtesy of Prof. Koji
Inoue, Prof. Kazuhiro Kogure, Dr. Toshiro Saruwatari (UT)
and Prof. Jun Hashimoto (Nagasaki University). This research
was supported by Grants-in-Aid for Scientific Research (Nos.
24654167, 26291077 and 18H02494) from the Japan Society
for the Promotion of Science.
ORCID
Yasunori Kano  https://orcid.org/0000-0002-9527-5116
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How to cite this article: Sato K, Kano Y, Setiamarga
DHE, Watanabe HK, Sasaki T. Molecular phylogeny
of protobranch bivalves and systematic implications
of their shell microstructure. Zool Scr. 2020;00:1–15.
https://doi.org/10.1111/zsc.12419

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