Into The Deep: A Phylogenetic Approach To The Bivalve Subclass Protobranchia

Molecular Phylogenetics and Evolution 69 (2013) 188204
Contents lists available at SciVerse ScienceDirect
Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev
Into the deep: A phylogenetic approach to the bivalve subclass

Protobranchia
Prashant P. Sharma a,, John D. Zardus b, Elizabeth E. Boyle c, Vanessa L. Gonzlez d, Robert M. Jennings c,
Erin McIntyre d, Ward C. Wheeler a, Ron J. Etter c, Gonzalo Giribet d
a
Division of Invertebrate Zoology, American Museum of Natural History, 200 Central Park West, New York, NY 10024, USA
Department of Biology, The Citadel, 171 Moultrie Street, Charleston, SC 29409, USA
Biology Department, University of Massachusetts, Boston, MA 02125, USA
d
Museum of Comparative Zoology & Department of Organismic and Evolutionary Biology, Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA
b
c
a r t i c l e
i n f o
Article history:
Received 18 February 2013
Revised 14 May 2013
Accepted 21 May 2013
Available online 4 June 2013
Keywords:
Mollusca
Bivalvia
Molecular phylogeny
Protobranch gill
Tree alignment problem
End-Permian
Extinction
a b s t r a c t
A molecular phylogeny of Protobranchia, the subclass of bivalve mollusks sister to the remaining Bivalvia,
has long proven elusive, because many constituent lineages are deep-sea endemics, which creates methodological challenges for collecting and preserving genetic material. We obtained 74 representatives of
all 12 extant protobranch families and investigated the internal phylogeny of this group using sequence
data from ve molecular loci (16S rRNA, 18S rRNA, 28S rRNA, cytochrome c oxidase subunit I, and histone
H3). Model-based and dynamic homology parsimony approaches to phylogenetic reconstruction unanimously supported four major clades of Protobranchia, irrespective of treatment of hypervariable regions
in the nuclear ribosomal genes 18S rRNA and 28S rRNA. These four clades correspond to the superfamilies
Nuculoidea (excluding Sareptidae), Nuculanoidea (including Sareptidae), Solemyoidea, and Manzanelloidea. Salient aspects of the phylogeny include (1) support for the placement of the family Sareptidae with
Nuculanoidea; (2) the non-monophyly of the order Solemyida (Solemyidae + Nucinellidae); (3) and the
non-monophyly of most nuculoid and nuculanoid genera and families. In light of this rst family-level
phylogeny of Protobranchia, we present a revised classication of the group. Estimation of divergence
times in concert with analyses of diversication rates demonstrate the signature of the end-Permian
mass extinction in the phylogeny of extant protobranchs.
2013 Elsevier Inc. All rights reserved.
1. Introduction
Among the poorest known molluscan groups is the subclass
Protobranchia, a bivalve lineage that has diversied and colonized
the deepest oceans, with numerous cosmopolitan species at abyssal depths (Allen and Sanders, 1996; Etter et al., 2011; Zardus et al.,
2006). Of the ca. 750 protobranch species (Table 1; Zardus, 2002),
most are deposit feeders in soft sediments, but two lineages host
chemoautotrophic, sulde-oxidizing bacteria, with concomitant
reductions of the hosts alimentary system (Cavanaugh, 1983;
Gustafson and Reid, 1988; Yamanaka et al., 2008; Oliver et al.,
2011; Oliver and Taylor, 2012). The incidence of doubly uniparental inheritance (i.e., mitochondrial heteroplasmy), once thought to
occur only in Autobranchia, has been discovered very recently in a
protobranch species, suggesting an earlier origin of this
exceptional mode of mitochondrial transmission in bivalves
(Doucet-Beaupr et al., 2010; Boyle and Etter, 2013). Protobranchs
have a probable Cambrian origin (Cope, 1996, 1997; but see Carter
Corresponding author.
E-mail address: psharma@amnh.org (P.P. Sharma).
1055-7903/$ - see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ympev.2013.05.018
et al., 2000), with several lineages radiating thereafter in the deep

sea, where they constitute the dominant group of bivalves (Allen,
1978, 1979).
Early studies on bivalve phylogenetics based on nucleotide sequence data frequently recovered non-monophyly of Protobranchia and suggested an early split into Opponobranchia (the clade
Nuculida + Solemyida) and Foliobranchia (Nuculanida + Autobranchia) (e.g., Giribet and Wheeler, 2002; Giribet and Distel, 2003;
Giribet, 2008; Wilson et al., 2010). More recently, the monophyly
of Protobranchia has become well established on the basis of larger
molecular analyses (Kocot et al., 2011; Smith et al., 2011; Sharma
et al., 2012), consistent with a compelling number of morphological characters that have traditionally united the protobranch bivalves. These characters include the eponymous protobranch gill,
which resembles the putatively plesiomorphic gill of patellogastropods; the palp proboscides (absent in the solemyoids, likely a consequence of obligate chemosymbiosis, as with reductions of the
alimentary system); and characteristic taxodont dentition, consisting of a series of identical or very similar vertical teeth (Coan et al.,
2000). Additionally, protobranchs are distinguished from other
Bivalvia in having a pericalymma larva (e.g., Drew, 1899; Gustafson
P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Table 1
Diversity and sampling of extant protobranch families.
Family
Described species
Sampled species
Solemyidae
Nucinellidae
Nuculidae
Sareptidae
Bathyspinulidae
Malletiidae
Neilonellidae
Nuculanidae
Phaseolidae
Siliculidae
Tindariidae
Yoldiidae
29
20
167
7
19
60
39
214
3
6
30
158
6
2
10
3
5
3
3
17
1
3
2
9
and Reid, 1986; Zardus and Morse, 1998). By contrast, the autobranch bivalves bear a typical veliger larva, comparable to gastropod counterparts (Jablonski and Lutz, 1983).
Several classications of Protobranchia have been proposed, but
most agree on division into three orders, Nuculida Dall, 1889, Solemyida Dall, 1889 (divided into Solemyoidea Gray, 1840 and Manzanelloidea Chronic, 1952), and Nuculanida Carter, Campbell and
Campbell, 2000 (Bieler et al., 2010). However, the monophyly of
Manzanelloidea has been questioned (Oliver and Taylor, 2012)
and the number of families and their constituent genera remains
in ux (Table 1). Protobranch phylogenetic study is still in its infancy, as little morphological and molecular work has focused on
this basal clade of bivalves. Due to the increasing predominance of
protobranchs with depth, this group of bivalves has gured prominently in studies on speciation in the deep sea (Allen, 1971; Etter
et al., 2005), with recent efforts highlighting discovery of species
from extreme environments (e.g., Oliver et al., 2011; Oliver and Taylor, 2012) or the nature of endosymbiosis with sulde-oxidizing
bacteria (e.g., Taylor and Glover, 2010; Oliver and Taylor, 2012).
The presence of chemosymbiosis in Nucinellidae has been inferred
(Reid, 1990, 1998; Taylor and Glover, 2010), and corroborated by
both anatomical and molecular data (Oliver and Taylor, 2012).
Resolution within Protobranchia has been analysis-dependent,
but previous studies have supported the sister relationship of Solemyidae to the clade (Nuculida + Nuculanida), albeit without sampling Manzanelloidea (Smith et al., 2011; Sharma et al., 2012). The
relationships of Nucinellidae and Solemyidae were reviewed by
Oliver and Taylor (2012; see also Pojeta, 1988), and a small analysis
of Solemyidae was recently published (Taylor et al., 2008).
Although with limited taxon sampling, Taylor et al.s (2008) study
addresses the taxonomy of Solemyidae and considerably advances
our knowledge of these bivalves, supporting the reciprocal monophyly of Acharax and Solemya, and the monophyly of the subgenus
Solemyarina. Analysis of an 18S rRNA dataset of the solemyid genus
Acharax has similarly revealed aspects of diversication among
Indo-Pacic species (Neulinger et al., 2006). Barring these few advances, protobranch internal phylogeny remains largely unknown,
because few families have been included in previous sampling efforts. For example, Manzanelloidea has heretofore not been represented in a molecular phylogenetic analysis.
The state of protobranchiate phylogenetics is in marked contrast
to that of major groups within Autobranchia, many of which have
been investigated using molecular data and have demonstrably stable phylogenies (e.g., pterioids: Tmkin, 2010; palaeoheterodonts:
Graf and Cummings, 2006; anomalodesmatans: Harper et al.,
2006; veneroids: Mikkelsen et al., 2006; heterodonts: Taylor et al.,
2007). In part, the recalcitrance to include Protobranchia in molecular phylogenetic datasets is attributable to operational challenges
stemming from their habitat; protobranch tissues suitable for
molecular techniques are notoriously difcult to obtain for some
groups because of the great depths that these bivalves inhabit.
189
Inherent to the task is the difculty of identifying living (or recently

expired) and minute (often less than 3 mm) specimens that require
several hours to raise from the deep sea via dredging (Boyle et al.,
2004). Moreover, the solubility of calcium carbonate at great depths
is such that for some specimens, only the periostracum remains by
the time the specimen is recovered. The mainly deep-sea solemyid
genus Acharax (see Yamanaka et al., 2008 for a shallow example) is
particularly susceptible to this phenomenon, hence is rarely obtained alive (Coan et al., 2000; Neulinger et al., 2006). Many Acharax
also burrow deeply and are capable of swimming when disturbed,
hampering collecting efforts.
To redress this long-standing lacuna in bivalve phylogeny, we
assembled a multilocus dataset to infer a protobranch phylogeny,
which required multiple collecting campaigns extending over a
decade. Our taxon sampling encompasses for the rst time all extant families of Protobranchia described heretofore, including the
enigmatic Sareptidae. On the basis of this phylogeny, we present
an updated classication of the protobranchs.
2. Materials and methods
2.1. Species sampling
Specimens of Protobranchia were collected by the authors and
multiple other individuals over several collecting campaigns. Rare
species were largely obtained by deep-sea dredging. Data collected
in previous studies (e.g., Giribet and Wheeler, 2002; Giribet and
Distel, 2003; Passamaneck et al., 2004) were additionally accessed
from GenBank. Collected specimens were stored in 96% EtOH.
Sequenced specimens consisted of seven Solemyidae, two Nucinellidae, 48 Nuculanoidea, and 17 Nuculoidea (including Sareptidae). These spanned all 12 recognized families of extant
Protobranchia sensu Bieler et al. (2010). Outgroup taxa for the
study consisted of three Gastropoda, three Pteriomorphia, and nine
Heterodonta. However, we have previously observed that ribosomal-dominated datasets consistently result in non-monophyly
of Protobranchia (Giribet and Wheeler, 2002; Giribet and Distel,
2003; Wilson et al., 2010; reviewed in Sharma et al., 2012) (Supplementary Fig. 1). Given that the monophyly of protobranch bivalves and the sister relationship of Solemyidae to the remaining
Protobranchia have been demonstrated recently using nuclear
genes and phylogenomic approaches (Smith et al., 2011; Sharma
et al., 2012), and that this study is concerned only with internal
relationships, we limited outgroup sampling to the subset of gastropods for principal analyses. The full list of specimens included
in our study is found in Table 2; collecting data are provided in
Supplementary Table 1.
2.2. Molecular methods
Total DNA was extracted from dissected tissues or whole animals using Qiagens DNeasy tissue kit (Valencia, CA, USA). Formalin-xed tissues were extracted following the protocol of Boyle
et al. (2004). Puried genomic DNA was used as a template for
PCR amplication. Molecular markers consisted of two mitochondrial genes (16S rRNA and cytochrome c oxidase subunit I), two nuclear ribosomal genes (18S rRNA and 28S rRNA), and one nuclear
protein-encoding gene (histone H3). Primer sequences and obtained fragment lengths are indicated in Supplementary Table 2.
Polymerase chain reactions (PCR), visualization by agarose gel
electrophoresis, and direct sequencing were conducted for most
specimens as described by Sharma and Giribet (2009). For rare
specimens, PCR was conducted using illustra Ready-To-Go
PCR Beads (GE Healthcare, Little Chalfont, UK). Chromatograms obtained from the automatic sequencer were read and sequences
MANZANELLOIDEA
Huxley munita (Dall, 1898)
Nucinella sp.
Family
Source
18S rRNA
28S rRNA
COI
Manzanellidae
Manzanellidae
BivAToL-137
MNHN; MCZ MAL-379095/DNA101571
KC429323
KC429324
KC429412-13
KC429414
KC429089
Solemyidae
Solemyidae
Solemyidae
Solemyidae
Solemyidae
Solemyidae
Solemyidae
MCZ DNA106839 / CASIZ 188907

MCZDNA106719
MCZ MAL-379147/DNA106718
GenBank
BivAToL-73
MCZ MAL-379150
BivAToL-17
KC984714
KC984715
KC984719
AF117737
KC984717
KC984718
AF120524
KC984828
KC984793
KC984795
KC984794
KC984796
KC429415
Bathyspinulidae
Bathyspinulidae
Bathyspinulidae
Bathyspinulidae
Bathyspinulidae
Maletiidae
Maletiidae
Maletiidae
Maletiidae
Neilonellidae
Neilonellidae
Neilonellidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Nuculanidae
Phaseolidae
Siliculidae
Siliculidae
Siliculidae
Tindariidae
Tindariidae
Yoldiidae
Yoldiidae
Yoldiidae
Yoldiidae
AMNH PRTB001
AMNH PRTB002
UMASS 3D534.2
AMNH PRTB003
AMNH PRTB004
BivAToL-217
UMASS Ice142.5
UMASS 3D534.6
AMNH PRTB005
AMNH PRTB006
GenBank
BivAToL-218
MCZ DNA100657
GenBank
MNHN; MCZ DNA105568
MNHN; MCZ DNA105566
MNHN; MCZ DNA105567
AMNH PRTB007
UMASS 3D534.3
UMASS Ice142.3
MCZ DNA105564
DIVA2; MCZ DNA104865
AMNH PRTB008
GenBank
Protostome AToL
MCZ MAL-379011/DNA100065
MCZ MAL-379111/DNA100121
GenBank
BivAToL-134.1a
UMASS 3D534.2
AMNH PRTB009
BivAToL-100
UMASS EN_10UC1
AMNH PRTB010
MNHN; MCZ DNA105569
UMASS 3D561.13
AMNH PRTB011
MNHN;MCZ DNA105565
MCZ MAL-378912/DNA104864
MCZ MAL-379181/DNA101624
MCZ MAL-379182/DNA100119/BivAToL-19
MCZ MAL-379185/DNA100120
KC993875
KC993876
KC984712
KC993877
KC993878
KC429320
KC984697
KC984698
KC993879
KC993881
AF207645
KC984695
KC984691
KC984701
KC984700
KC984710
KC984711
KC984685
KC984688
DQ279938
AF120529
AY070111
AF207644
AY145385
KC984693
KC984687
KC984692
KC429321
KC984705
KC984686
KC984703
KC984694
KC984702
KC993882
KC984699
KC984696
KC429322
AF207643
KC984841
KC984806
KC429409
KC984809
KC984810
KC984837
KC984838
AF207652
KC984822
KC984819
AB103131
KC984825
KC984824
KC984821
KC984843
KC984839
KC984804
KC984805
KC984820
KC984800
DQ279961
AF120586
AY070124
AF207651
AY145419
KC984801
KC984799
KC984802
KC429410
KC984798
KC984836
KC984840
KC984803
KC984812
KC984823
KC984811
KC984808
KC429411
AF207650
16S rRNA
histone H3
KC429157
KC429158
KC984781
KC984743
KC984671
KC984672
KC984673
KC984744
KC984745
U56852
KC984674
KC984675
JQ728447
KC984780
KC984778
AY070146
KC984733
KC993870
KC993871
KC993874
KC993869
KC993868
KC984669
KC993872
AF207656
KC984732
KC984659
KC984666
KC984739
KC993873
KC984738
KC984740
DQ280018
AF120643
AY070138
KC984737
KC984735
KC984736
KC984667
DQ280030
KC984664
KC984665
KC984661
KC984663
KC984734
KC984731
KC984730
KC429088
AF207655
KC984779
KC993889
KC984773
KC429154
KC984759
KC984758
KC993888
KC993887
KC984756
KC984753
KC993885
KC993884
KC993886
KC984792
KC984770
KC984771
KC984772
KC984769
KC984763
DQ280002
KC984765
AY070148
KC984764
KC984766
KC984761
KC984785
KC429155
KC984783
KC984767
KC984762
KC984760
KC984755
KC984757
KC984754
KC429156
SOLEMYOIDEA
Acharax bartschii (Dall, 1908)
Acharax gadirae Oliver, Rodrigues & Cunha, 2011
Solemya elarraichensis Oliver, Rodrigues & Cunha, 2011
Solemya pervernicosa Kuroda, 1948
Solemya velesiana Iredale, 1931
Solemya velum Say, 1822
Solemya velum Say, 1822
NUCULANOIDEA
Bathyspinula calcar (Dall, 1908)
Bathyspinula latovae (Knudsen, 1967)
Bathyspinula hilleri (Allen & Sanders, 1982)
Tindariopsis agatheda (Dall, 1890)
Tindariopsis sulcata (Gould, 1852)
Clencharia abyssorum (Verrill & Bush, 1898)
Malletia cuneata (A) Jeffreys, 1876
Malletia cuneata (B) Jeffreys, 1876
Malletia johnsoni Clarke, 1961
Neilonella salicensis (Seguenza, 1877)
Neilonella subovata (Verrill & Bush, 1897)
Neilonella whoii Allen & Sanders, 1996
Adrana scaphoides Rehder, 1939
Jupiteria sematensis (Suzuki & Ishizuka, 1943)
Jupiteria sp.
Jupiteria sp.
Jupiteria sp.
Ledella ecaudata (Pelseneer, 1903)
Ledella jamesi Allan & Hannah, 1989
Ledella pustulosa (Jeffreys, 1876)
Ledella sp.
Ledella ultima (Smith, 1885)
Nuculana conceptionis (Dall, 1896)
Nuculana minuta (Mller, 1776)
Nuculana minuta (Mller, 1776)
Nuculana pella (Linnaeus, 1767)
Nuculana pernula (Mller, 1779)
Propeleda cf. carpenteri
Propeleda cf. longicaudata
Scaeoleda caloundra (Iredale, 1929)
Lametila abyssorum Allen & Sanders, 1973
Silicula rouchi Lamy, 1911
Silicula sp.
Silicula sp.
Tindaria kennerlyi (Dall, 1897)
Tindaria sp.
Megayoldia sp.
Yoldia eightsi (Jay, 1839)
Yoldia limatula (Say, 1831)
Yoldia myalis (Couthouy, 1838)
190
Table 2
List of species and gene fragments included in phylogenetic analyses.
Yoldia scissurata Dall, 1897

Yoldiella americana Allen, Sanders & Hannah, 1995
Yoldiella inconspicua inconspicua Verrill & Bush, 1898
Yoldiella orcia (Dall, 1916)
Yoldiella cf. valleri
Yoldiidae
Yoldiidae
Yoldiidae
Yoldiidae
Yoldiidae
AMNH PRTB012
UMASS 3D8369.2
UMASS Icel42.7.1
AMNH PRTB013
AMNH PRTB014
KC984706
KC984707
KC984689
KC984690
KC993880
KC984797
KC984842
KC984807
KC984832
KC984831
KC984729
KC984726
KC984727
KC984728
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
Nuculidae
GenBank
BivAToL-205
UMASSEN 14CC1
AMNHPRTB015
UMASS EN_3aAC8
MCZ MAL-379107/DNA105848
MCZ MAL-379010/DNA103781
BivAToL-215/MCZ DNA101159
AMNH PRTB016
GenBank
MCZ MAL-379108/DNA100067
MCZ MAL-379109/DNA100104
MCZ MAL-379098/DNA100117
MCZ MAL-379099/DNA100118
BivAToL-189/Protostome AToL T68
AF120527
KC429319
KC984722
KC984716
KC984721
KC984684
KC984724
KC984723
KC984720
AF120526
KC984713
AF120525
KC984725
AF207642
AF120584
KC429408
KC984814
KC984829
KC984817
KC984826
KC984813
KC984818
KC984830
AF120583
KC984816
KC984827
AF120582
KC984815
DQ279960
Sareptidae
Sareptidae
Sareptidae
UMASS EN_18aLC1
UMASS EN_10RC1
UMASS EN_18aXC1
KC984704
KC984708
KC984709
KC984834
KC984833
KC984835
OUTGROUPS
AUTOBRANCHIA
Arcopsis adamsi (Dall, 1886)
Lima lima (Linnaeus, 1758)
Pinna carnea Gmelin, 1791
Eucrassatella cumingii (Adams, 1854)
Neotrigonia lamarckii (Gray, 1838)
Unio pictorum (Linnaeus, 1758)
Cardiomya sp.
Lyonsia oridana Conrad, 1849
Thracia sp.
Dreissena polymorpha (Pallas, 1771)
Solen vaginoides Lamarck, 1818
Thyasira exuosa (Montagu, 1803)
Noetiidae
Limidae
Pinnidae
Crassatellidae
Trigoniidae
Unionidae
Cuspidariidae
Lyonsiidae
Thraciidae
Dreissenidae
Solenidae
Thyasiridae
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
GenBank
KC429327
KC429339
KC429337
KC429350
KC429345
KC429349
KC429362
KC429353
KC429356
AF120552
KC429399
KC429367
KC429419-20
KC429434
KC429431-32
KC429448
KC429443
KC429447
KC429463-64
KC429451
KC429454-56
KC429513-14
KC429507
KC429469
KC429092
KC429101
KC429099
KC429110
KC429105
KC429109
KC429118
AF120654
KC429115
KC429149
GASTROPODA
Crepidula fornicata (Linnaeus, 1758)
Haliotis tuberculata Linnaeus, 1758
Siphonaria pectinata (Linnaeus, 1758)
Calyptraeidae
Haliotidae
Siphonariidae
GenBank
GenBank
GenBank
AY377660
AY145418
X91973
AY145406
AY145418
DQ256744
AF353154
AY377729
AF120638
KC429087
KC984748
KC984749
KC984747
KC984750
KC984742
KC984741
AF120641
KC984746
DQ280017
KC429241
KC984680
KC984681
KC984678
KC984682
KC984683
KC984676
KC984677
AY377617
KC984679
DQ280029
KC984670
KC984790
KC984787
KC984788
KC984789
KC993883
KC984782
KC984751
KC984774
KC984775
KC984752
KC984768
KC984776
AY070147
KC984777
DQ280001
KC984784
KC984786
KC984791
KC429245
KC429257
KC429255
KC429267
KC429262
KC429266
KC429276
KC429268
KC429271
DQ280038
KC429308
KC429162
KC429174
KC429172
KC429187
KC429182
KC429186
KC429198
KC429191
KC429194
KC429234
KC429230
KC429200
AY377625
AY377622
AY377627
AY377778
AY377775
AY377780
KC429122
NUCULOIDEA
Acila castrensis (Hinds, 1843)
Acila castrensis (Hinds, 1843)
Brevinucula verrilli (Dall, 1886)
Ennucula cf. cardara
Ennucula granulosa (Verrill, 1884)
Ennucula tenuis expansa (Montagu, 1808)
Leionucula cf. cumingi
Nucula atacellana Schenck, 1939
Nucula profundorum Smith, 1885
Nucula proxima Say, 1822
Nucula sulcata Bronn, 1831
SAREPTOIDEA
Pristigloma cf. alba
Pristigloma cf. nitens
Pristigloma sp.
KC984662
KC984668
191
192
assembled using the sequence editing software Sequencher

(Gene Codes Corporation, Ann Arbor, MI, USA). Sequence data were
edited in Se-Al v. 2.0a11 (Rambaut, 1996).
resulting sub-optimal likelihood trees to the unconstrained ML

topology obtained using the same dataset. To generate the null distribution, 500 resampling replicates were conducted.
2.3. Phylogenetic Analyses
2.5. Estimation of divergence times
Maximum likelihood (ML) and Bayesian inference (BI) analyses

were conducted on static alignments, which were inferred as follows. Sequences of ribosomal genes were aligned using MUSCLE
v. 3.6 (Edgar, 2004) with default parameters, and subsequently
treated with GBlocks v. 0.91b (Castresana, 2000) to cull positions
of ambiguous homology. Sequences of protein encoding genes
were aligned using MUSCLE v. 3.6 with default parameters as well,
but alignments were additionally conrmed using protein sequence translations prior to treatment with GBlocks v.0.91b. The
size of data matrices for each gene prior and subsequent to treatment with GBlocks v. 0.91b is provided in Supplementary Table 3.
ML analyses were conducted using RAxML ver. 7.2.7 (Stamatakis, 2006). For the maximum likelihood searches, a unique General
Time Reversible (GTR) model of sequence evolution with corrections for a discrete gamma distribution (GTR + C) was specied
for each data partition, and 100 independent searches were conducted. Nodal support was estimated via the rapid bootstrap algorithm (250 replicates) using the GTR-CAT model (Stamatakis et al.,
2008). Bootstrap resampling frequencies were thereafter mapped
onto the optimal tree from the independent searches.
BI analysis was performed using MrBayes v. 3.1.2 (Huelsenbeck
and Ronquist, 2005) with a unique GTR model of sequence evolution, corrections for a discrete gamma distribution and a proportion of invariant sites (GTR + C + I) specied for each partition, as
selected in jModeltest v. 0.1.1 (Guindon and Gascuel, 2003; Posada,
2008) under the Akaike information criterion (AIC) (Posada and
Buckley, 2004) (Supplementary Table 3). Default priors were used
starting with random trees. Two runs, each with three hot and one
cold Markov chains, were executed until the average deviation of
split frequencies reached <0.01 (107 generations). Convergence
diagnostics were assessed using Tracer ver. 1.5 (Rambaut and
Drummond, 2009).
Parsimony analyses were based on a direct optimization (DO)
approach (Wheeler, 1996) using POY v. 4.1.2 (Varn et al., 2010).
Tree searches were performed using the timed search function in
POY, i.e., multiple cycles of (a) building Wagner trees, (b) subtree
pruning and regrafting (SPR) and (c) tree bisection and reconnection (TBR), (d) ratcheting (Nixon, 1999), and (e) tree-fusing (Goloboff, 1999, 2002). Timed searches of 24 h were run on 24 processors
under a mixed parameter set, such that ribosomal genes were
weighted using the parameter set 3221 (indel opening cost = 3;
transversions = transitions = 2; indel extension cost = 1) and protein-encoding genes were weighted using the parameter set 121
(indel cost = 2; transversion cost = 2; transition cost = 1). The design of this parameter set follows previous exploration of invertebrate datasets (Sharma et al., 2011). Nodal support for the optimal
parameter set was estimated via jackkning (250 replicates) with a
probability of deletion of e1 (Farris et al., 1996).
Ages of clades were inferred using BEAST v. 1.7.4 (Drummond

et al., 2006; Drummond and Rambaut, 2007). We specied a unique GTR model of sequence evolution with corrections for a discrete gamma distribution and a proportion of invariant sites
(GTR + C + I) for each partition (as with BI analysis). An uncorrelated lognormal clock model was inferred for each partition, and
a Yule speciation process was assumed for the tree prior. We selected the uncorrelated lognormal model because its accuracy is
comparable to an uncorrelated exponential model, but it has narrower 95% highest posterior density intervals. Additionally, the
variance of the uncorrelated lognormal model can better accommodate data that are already clock-like (Drummond et al., 2006).
Priors were sequentially optimized in a series of iterative test runs;
the command les are available upon request from the authors.
Two Markov chains were run for 5 107 generations, sampling
every 5000 generations. Convergence diagnostics were assessed
using Tracer ver. 1.5 (Rambaut and Drummond, 2009).
Fossil taxa were used for divergence time calibration, as follows: We constrained the diversication of Nucinellidae using
the early Jurassic (Hettangian stage, 201.6197 Ma) fossil Nucinella
liasina (Bistram, 1903), which is inferred to be the earliest crown
nucinellid (Conti, 1954). To account for uncertainty in estimation
of the fossil age we applied a normal distribution prior to this node
with a mean of 197 Ma and standard deviation of 5 Myr. While the
Permian species Manzanella cryptodontaChronic, 1952 is the type of
the superfamily, our examination of the holotype suggests that this
specimen may not be a protobranch and thus was not used in the
estimation of divergence times. The systematics of Manzanella
have been addressed elsewhere (Oliver and Taylor, 2012). Diversication of Solemyoidea was constrained using the Ordovician fossil
Ovatoconcha (Cope, 1999); a normal distribution prior with a mean
of 475 Ma and standard deviation of 10 Myr was applied to this
node. Nuculida was constrained using the same prior distribution
as for Solemyoidea, on the basis of several Arenig fossils (Cope,
2004). Finally, the age of the ingroup was constrained using a uniform prior bounding the maximum age of Protobranchia at
545 Ma, based on the age of the earliest crown-group bivalve,
Fordilla troyensis (Pojeta et al., 1973; Pojeta and Runnegar, 1974;
Pojeta, 2000; Parkhaev, 2008).
2.4. Likelihood-based tests of topology

To assess the strength of phylogenetic evidence for the placement of key taxa, ShimodairaHasegawa (SH) tests were conducted using RAxML v. 7.2.7 (Shimodaira and Hasegawa, 1999).
We enforced topological constraints consistent with alternative
hypotheses of taxon placement for three scenarios: (1) the monophyly of Solemyida (=Solemyidae + Nucinellidae), (2) the monophyly of Opponobranchia (=Solemyida + Nuculida), and (3) the
monophyly of Nuculida, including Sareptidae (i.e., the traditional
denition of this order). For each evaluation, we compared the
2.6. Diversication through time

Likelihood analyses of speciation and extinction were conducted using Laser v. 2.3 (Rabosky, 2006) on the dated tree topology. To overcome the effect of incomplete sampling of extant
lineages and the associated rate slowdown artifact (Cusimano
and Renner, 2010), branching times younger than 65 Myr were
culled. Six models were tted to the truncated protobranch chronogram: a pure-birth (Yule) model, a birth-death model, a logistic
density dependence model (DDL), an exponential density dependence model (DDX), a two-rate Yule model, and a three-rate Yule
model. The DAICrc was calculated as the difference in AIC scores
of the best-t rate-constant and best-t rate-variable model.
To compare the empirical net diversication curve to a scenario
corresponding to the end-Permian mass extinction, we simulated
500 phylogenies with 700 extant taxa using TreeSim v. 1.6 (Stadler,
2012), under a constant-rate birth-death model with parameters
equal to the initial model inferred by LASER v. 2.3 at the base of
the protobranch radiation. Seven hundred taxa reect an approximation of the number of valid extant protobranch species
described, which we treated as a proxy for total extant diversity. To

simulate the effect of mass extinction, we induced a single nonselective cull of 99% of lineages at time t = 250, with no subsequent
modication of evolutionary rates. To simulate the effect of sampling error, we randomly sampled subtrees of 60 taxa from the
700-taxon trees. Sixty taxa were subsampled to enable comparison
to the empirical chronogram.
3. Results
3.1. Maximum likelihood
ML analysis of the ve-gene, 77-ingroup taxon dataset using
RAxML v. 7.2.7 resulted in a tree topology with ln L = 39026.88
(Supplementary Fig. 2). However, 12 of the ingroup taxa were
demonstrably unstable in their phylogenetic placement due to
large amounts of missing data. We therefore constructed a smaller
and denser dataset excluding these 12 terminals, using the same
methods for inferring tree topology. Analysis of the 65-taxon dataset resulted in topologies with ln L = 38412.02 (Fig. 2).
Major aspects of the ML topology include the mutual monophyly of the solemyoid genera Solemya and Acharax; the nonmonophyly of Solemyida (=Solemyidae + Nucinellidae) (BS = 88);
and the monophyly of the sister clades Nuculida (excluding Sareptidae) and Nuculanida (including Sareptidae). These relationships
193
received signicant nodal support (bootstrap resampling frequency [BS] > 70), irrespective of inclusion of taxa with missing
data. The ML topology also weakly supported the monophyly of
Nuculida + Nuculanida (BS = 75).
All exemplars of Sareptidae (traditionally placed in Nuculida)
were recovered as nested within Nuculanida, and closely related
to Yoldia eightsi (BS = 71). Henceforth we refer to Nuculida sensu
stricto as the clade that does not include Sareptidae, i.e., the traditionally dened superfamily Nuculoidea.
Except Solemyidae and Sareptidae, few of the families or genera
represented by multiple specimens were recovered as monophyletic. Among nuculids, both Nucula and Ennucula were polyphyletic. Among nuculanids, Nuculana was rendered paraphyletic
due to the inclusion of a Jupiteria (BS = 99); Propeleda and Silicula
formed a grade (without signicant support) sister to Nuculana + Jupiteria; and Yoldiidae was recovered as a polyphyletic
assemblage of at least three lineages. The inclusion of taxa that
had signicant amounts of missing data mostly resulted in the
recovery of generic non-monophyly (e.g., Jupiteria, Tindaria, Bathyspinula) and/or signicantly depressed nodal support, with the
exception of the genus Neilonella (BS = 95) (Supplementary Fig. 2).
3.2. Bayesian inference
Runs of MrBayes v.3.1.2 reached stationarity in <106 generations; 2.5 106 generations (25%) runs were discarded as burnin.
Fig. 1. Exemplars of Protobranchia. (A) Solemya velesiana (Solemyidae); (B) Huxleyia munita (Nucinellidae); (C) same as (B), detail of hinge dentition; (D) Yoldiella orcia
(Yoldiidae); (E) Pristigloma cf. nitens (Sareptidae); (F) Scaeoleda caloundra (Nuculanidae); (G) Leionucula cumingi (Nuculidae); (H) Nucula tenuis expansa (Nuculidae); (I) Ledella
ultima (Nuculanidae).
194
Fig. 2. Phylogenetic relationships of Protobranchia based on maximum likelihood analysis of ve genes (ln L = 38412.02). Numbers on nodes indicate bootstrap resampling
frequencies. Colors in tree topology correspond to major lineages (red: Solemyidae; orange: Nucinellidae; green: Nuculoidea sensu stricto; blue: Nuculanoidea + Sareptoidea).
(For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
BI analysis recovered a topology highly congruent with respect to

the monophyly of Solemyoidea (posterior probability [PP] = 0.98),
Nucinellidae (PP = 1.00), Nuculida sensu stricto (PP = 1.00) and
Nuculanida (PP = 1.00) (Fig. 3). Both the non-monophyly of Solemyida and the monophyly of Nuculida + Nuculanida were weakly
supported (PP = 0.94 and PP = 0.91, respectively). Minor topological differences exist between the ML and BI topologies (e.g., the
placement of Scaeoleda caloundra and Nuculana pella), but these
differences were unsupported in both topologies.
3.3. Direct optimization

Iterative rounds of tree-fusing and driven searches using direct
optimization in POY v. 4.1.2 resulted in a single most parsimonious
tree with a length of 18,827 weighted steps. In general, the
parsimony DO tree was very similar to the topologies based on
static alignments, particularly with respect to derived relationships
(Fig. 4). Two major topological differences are (1) the placement of
Nucinellidae, which was recovered as sister to Sareptidae + Nuculanida
195
Fig. 3. Phylogenetic relationships of Protobranchia based on Bayesian inference analysis of ve genes. Numbers on nodes indicate posterior probabilities. Colors in tree
correspond to major lineages (as in Fig. 2).
(jackknife resampling frequency [JF] = 67), and (2) the placement

of Sareptidae, which was recovered sister to the remaining Nuculanida (JF < 50). Nodal support values for other relationships (e.g.,
monophyly of the four major clades; mutual monophyly of Solemya
and Acharax) were generally comparable to those based on modelbased approaches.
3.4. ShimodairaHasegawa tests
The unconstrained ML topology of 65-ingroup taxa (ln
L = 38412.02) was not signicantly better than a topology consistent with the Solemyida hypothesis (ln L = 38421.95 for best suboptimal tree) at a = 0.05 (Fig. 5). However, the unconstrained ML
topology was found to be signicantly better than topologies consistent with either Opponobranchia (ln L = 38430.90 for best suboptimal tree) or Nuculida sensu lato., i.e., including Sareptidae (ln
L = 38631.41 for best suboptimal tree), at a = 0.05 and a = 0.01,

respectively (Fig. 5).
3.5. Estimation of divergence times
Diversication of major lineages using BEAST for the 147-taxon
dataset is estimated as follows: Protobranchia, 522.7 Ma (95% highest posterior density interval [HPD] 507.1539.1 Ma); Solemyoidea,
465.8 Ma (95% HPD 448.1482.9 Ma); Nucinellidae, 196.7 Ma (95%
HPD 187.1206.6 Ma); Nuculida sensu stricto, 259.4 Ma (95% HPD
181.3332.0 Ma); Sareptidae + Nuculanida, 456 Ma (95% HPD
438.6473.2 Ma); Nuculanida, 282.3 Ma (95% HPD 201.9
368.8 Ma) (Fig. 6). Most aspects of the dated topology are comparable to ML and BI results, particularly the placement of Nucinellidae.
However, as in the DO topology, Sareptidae is recovered sister to the
remaining Nuculanida with high support (PPBEAST = 1.00).
196
Fig. 4. Phylogenetic relationships of Protobranchia based on parsimony under direct optimization of ve genes. Numbers on nodes indicate jackknife resampling frequencies.
Colors in tree correspond to major lineages (as in Fig. 2). (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this
article.)
3.6. Analyses of diversication rates

The log-lineage through time plot obtained from the chronogram corresponded to an anti-sigmoidal curve (Fig. 8A), consistent with either an upturn in diversication after a period of
low cladogenetic potential, or a cryptic mass extinction (Crisp
and Cook, 2009). The timing of the upturn in diversication corresponded to ca. 250 Ma, the timing of the end-Permian
extinction. Of the six competing diversication models, the optimal was a Yule-three-rate model (lnL = 102.95; AIC = 215.89),
incorporating a diversication rate slowdown at 456 Ma and

rate acceleration at 260 Ma (Table 3). With respect to the
Yule-three-rate model, all suboptimal models differed by
DAIC > 5.
Comparison of the empirical log-lineage through time plot of
Protobranchia to trees simulated under (1) constant net diversication with 700 extant species, (2) a single cull of 99% at time
t = 250 Ma, and (3) taxon sampling of 60 extant species indicated
that the empirical chronogram is not signicantly different from
the simulated null distribution (p > 0.05) (Fig. 8B).
197
Fig. 5. Comparisons of tree topologies using Shimodaira-Hasegawa tests. Colors in tree correspond to major lineages (as in Fig. 2). Open circles indicate constrained nodes.
Fig. 6. Evolutionary timetree of Protobranchia inferred from BEAST analysis of all molecular data. Colored bars indicate 95% highest posterior density intervals for nodes of
interest. Black text adjacent to selected nodes indicates median ages; red text indicates posterior probabilities (for selected nodes). Asterisks indicate posterior probability of
1.00. Open circles indicate calibrated nodes. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
4. Discussion
The use of various algorithmic approaches for investigating protobranch phylogeny was prompted by the challenging nature of
this multilocus dataset. For example, the genus Acharax is known
to have hypervariable regions within the small ribosomal subunit
(18S rRNA; Neulinger et al., 2006). In the present study, we observed that both sampled species of Acharax bear highly variable
regions in the large ribosomal subunit (28S rRNA) as well, adding
hundreds of nucleotide characters to static alignments. Similarly,
many species were distinguished in available sequence data only
within length variable regions of ribosomal genes (e.g., the three
198
Table 3
Fit of models to the protobranch log-lineage through time curve, truncated at 65 Ma.
Boldface text indicates optimal model; parameters of Yule-3-rate model indicate
speciation rates (k) and shift points in time.
Model
Parameters
lnL
AIC
Pure birth
Birthdeath
DDL
DDX
Yule-2-rate
Yule-3-rate
1
2
2
2
3
5
109.66
109.14
109.66
109.53
107.82
102.95
221.31
222.28
223.31
223.06
221.63
215.89
Yule-3-rate model parameters:

k1 0:01734; k2 0:00083; k3 0:00799.
shift1 = 455.66 Ma; shift2 = 259.60 Ma.
Pristigloma, discussed below). Indel characters are inherently informative for analyses under direct optimization, inasmuch as POY
v.4.1.2 can incorporate both indel opening and extension parameters. Both RAxML v.7.2.7 and MrBayes v.3.1.2 incorporate rapid
heuristic algorithms and sophisticated modeling of substitution
events, although neither can distinguish indels from missing data
(but see Simmons and Ochoterrena, 2000 for a common workaround). Consequently, model-based approaches may constitute
an unsatisfactory compensation for the loss of information that
transpires when phylogenetic signal resides exclusively in
length-variable regions (e.g., Lindgren and Daly, 2007). The protobranch dataset we generated thus presented an opportune case
where both static and dynamic homology approaches could elucidate different aspects of protobranch phylogeny.
analyses of molecular data, and (3) the apomorphic hinge structure

of nucinellids, we reafrm the validity of Nucinellidae as a fourth
and distinct clade of protobranchs.
All model-based inferences of tree topology recover a clade
comprised of Nuculida and Nuculanida (the traditional Palaeotaxodonta sensu Newell, 1969; Nuculoida sensu Sanders and Allen, 1973; Beesley et al., 1998), which bear prominent hinge plates
and a characteristic taxodont dentition (Figs. 2, 3 and 6). This clade
is additionally supported by the presence of small gills that are situated posteriorly, and large labial palps and palp proboscides. Previous efforts toward higher-level bivalve phylogeny supported a
clade of Solemyida + Nuculida (Waller, 1998; Carter et al., 2000;
Giribet and Wheeler, 2002; Giribet and Distel, 2003), a hypothesis
formalized by the name Opponobranchia (Giribet, 2008). Comparative assessment of the strength of the Opponobranchia hypothesis
indicates that the Palaeotaxodonta topology is signicantly better
than a topology consistent with Opponobranchia, albeit only at
a = 0.05 (Fig. 5). The sister group relationship of Solemyoidea to
the remaining protobranchs is also supported by recent phylogenetic and phylogenomic datasets (Kocot et al., 2011; Smith et al.,
2011; Sharma et al., 2012), though Nucinellidae was not sampled
in those studies. As the present study constitutes the most comprehensive sampling of protobranch bivalves for phylogenetic analysis, we tentatively favor the palaeotaxodont hypothesis, but
advocate reassessment of this topology by sampling of nucinellids
in future phylogenomic analysis and/or re-evaluation using a suite
of non-overlapping molecular markers (e.g., Sharma et al., 2012).
4.2. Sareptidae is a lineage of Nuculanida
4.1. Higher-level relationships of Protobranchia

All phylogenetic analyses based on molecular sequence data
unambiguously recover with signicant support the division of Protobranchia into four clades, corresponding to Solemyidae, Nucinellidae, Nuculida, and Nuculanida (Figs. 24 and 6, Supplementary
Fig. 2). Barring the placement of Sareptidae within or sister to
Nuculanida, and the non-monophyly of Solemyida (=Solemyidae + Nucinellidae)both results insensitive to algorithmic treatmentthe constituent families and genera of these four clades are
consistent with the traditional classication of the protobranchs.
Part of the discordant phylogenetic signal for a monophyletic
Solemyida appears to stem from the nuclear ribosomal genes; analyzed on their own for the present species sampled, these will recover Solemyida, albeit without signicant nodal support
(Supplementary Fig. 3). Although model-based algorithmic approaches to the entire molecular dataset support the paraphyly
of Solemyida (Figs. 2 and 3), the resulting topology is not signicantly better than a suboptimal Solemyida topology (Fig. 5). Morphologically, a sister relationship of Solemyidae and Nucinellidae
is consistent with the large, bipinnate protobranch gill and the reduced (or absent) palp proboscides in these two superfamilies.
However, these characters may be attributable to habitat and/or
the incidence of chemosymbiosis in both lineages, and thus constitute either convergence or protobranch symplesiomorphies (Oliver
and Taylor, 2012). Moreover, the ultrastructure of the nucinellid
shell suggests afnity to Nuculanida (Coan et al., 2000), an observation consistent with the direct optimization topology (Fig. 4).
Nucinellidae therefore constitutes a curious lineage with
ambiguous afnities to other protobranchs. The uniqueness of this
clade, whose fossil record extends to the Permian (Chronic, 1952),
is evident in its hinge structure, which is strong, short, and consists
of one to two prominent lateral teeth and several cardinal taxodont
teeth (Fig. 1C). By contrast, the hinge plate of true Solemyoidea is
weak and edentate. Given (1) the lack of unambiguous morphological synapomorphies uniting solemyids and nucinellids, (2) the
absence of signicant nodal support uniting these families in
One of the smaller and more curious families of protobranchs is

Sareptidae Stoliczka, 1870, one of the two families of Nuculoidea,
with ca. 10 known species (Huber, 2010). As currently understood
by Bieler et al. (2010), Sareptidae includes the nominal genus Sarepta Adams, 1860 and the genera Pristigloma Dall, 1900 and Setigloma Schileyko, 1983, and therefore the new Sareptidae concept
includes Pristiglomidae and Setiglomidae as junior synonyms.
However, this taxonomic assignment is not without controversy
and is not based on any recent phylogenetic analysis. For example,
Coan et al. (2000) used the superfamily Pristiglomoidea Sanders
and Allen, 1973 to include the single family Pristiglomidae Sanders
and Allen, 1973, with the genera Pristigloma, Setigloma, and Pseudoglomus Dall, 1898, the latter now in Malletiidae, although it probably does not belong there. They treated Pristiglomoidea as a rank
comparable to Nuculoidea and Nuculanoidea (in their Nuculoidea).
Sanders and Allen (1973), when they proposed Pristiglomidae, also
included the genus Microgloma Sanders and Allen, 1973 in this
family, and removed Pristigloma from Nuculanacea to be placed
in Nuculacea, formalizing the transfer of the new family to the
current Nuculida. Allen and Hannah (1986) also included Pseudoglomus in Pristiglomidae, but treated Sarepta as a member of Yoldiidae Allen and Hannah, 1986, in their Nuculanacea. Ockelmann
and Warn (1998) transferred Microgloma to Nuculanidae and discuss the possible synonymy of Pristiglomidae with Sareptidae, as
well as cast doubts on the position of Pristiglomidae. The most
recent bivalve compendium uses Sareptoidea as a superfamily,
comparable to Nuculoidea, Solemyoidea, Manzanelloidea and
Nuculanoidea, without arranging them (Huber, 2010).
Sareptidae are distinguished from both Nuculoidea and Nuculanoidea in having few hinge teeth, often of chevron shape, and being
greatly miniaturized. The miniaturization of sareptids appears to
be achieved by smaller cell size and lowered reproductive output
(Sanders and Allen, 1973). A relationship between part of Sareptidae (Pristigloma) and Nuculoidea has thus often been suggested,
largely on the basis of the shell shape (a rounded posterior end,
resulting in antero-posterior [AP] symmetry), disposition of major
organs, and the absence of a pallial sinus. It is therefore surprising

that we obtain all sareptid species (genus Pristigloma) as nested
within or sister to Nuculanoidea across all topologies (Figs. 24
and 6), and with signicant support in SH tests (Fig. 5). Moreover,
Sareptidae is recovered sister to Yoldia eightsi, a genus of Yoldiidae
that includes species with rounded posterior ends and chevron
teeth, in the probabilistic analyses (BS = 71; PP = 0.99), as suggested by the placement of Sarepta by Allen and Hannah (1986),
but sister group to Nuculanida in the direct optimization and
BEAST analyses (JF < 50; PPBEAST = 1.00). The three Pristigloma species are sufciently closely related that their sequences are rendered identical upon removal of length-variable regions.
However, analysis of divergence time suggests that this lineage,
if indeed sister to true Nuculanida, is ancient, with an origin dating
to the Ordovician and/or Early Silurian (Fig. 6).
We therefore re-examined the morphological characters supporting the placement of Sareptidae within Nuculida and found
these unconvincing. Sanders and Allen (1973) contend that Pristigloma shared many characters with true Nuculida, such as the anterior (rather than posterior) inhalant current, the lack of mantle
fusion, anterior mucus glands of the mantle, absence of siphons,
transversely oriented ctenidia, and the large, broad palp. However,
many of these features may result from the dramatic miniaturization of sareptids and the atypical AP symmetry of the shell in this
cryptic family. Reduction of hinge tooth number and the pallial sinus may also represent further effects of miniaturization in Sareptidae. Many species of Nuculanoidea (e.g., Yoldiella capsa, Yoldiella
subcircularis, Neilonella mexicana) also do not have the marked AP
asymmetry characteristic of most nuculanids. The absence of the
pallial sinus, mantle fusion, and siphons in Pristigloma and true
nuculoids is also unsatisfactory, insofar as absence of a character
may not constitute a sound basis for diagnosis given prevalence
of homoplasy. Moreover, the placement of Pristigloma in all of
our topologies is consistent with their lack of nacreous shell layers,
which occur only in modern Nuculida (Carter, 1990). Given the
number of morphological and molecular sequence characters
shared by Sareptidae and various clades of Nuculanida, we consider Sareptidae to constitute a lineage of Nuculanida (Table 4).
As a conservative measure, we maintain this lineage in the superfamily Sareptoidea for the present, due to the basal (i.e., nonnested) placement of Sareptidae in the direct optimization and dated tree topologies (Fig. 4), but recognize that additional molecular
data should be gathered, specically from the rare genus Sarepta.
4.3. Systematic validity of protobranch families and genera
Under either approach to alignment, few of the genera and families within the four major clades of protobranchs were recovered
as monophyletic. A notable exception is the monophyly of Solemyidae and the mutual monophyly of the genera Solemya and
Acharax, which were invariably supported in all phylogenetic analyses, including under parametric treatment of hypervariable regions in POY v.4.1.2 (Figs. 24). The deep-sea genus Acharax is
known to form at least two clusters of species, as inferred from
18S rRNA sequences (Neulinger et al., 2006). Both species of Acharax sampled here correspond to the JAC clade dened by Neulinger
et al. (2006), as inferred from multiple sequence alignments (data
not shown). The 28S rRNA sequences of Acharax bartschii and A.
gadirae bear numerous hypervariable regions as well. The signicance of the elongated insertions in the ribosomal array of Acharax
is not known, but obtaining more 28S rRNA sequences from other
species of this genus may prove useful for corroborating the clusters delimited by Neulinger et al. (2006).
Within the palaeotaxodont genera, only Neilonella was monophyletic (Acila and Malletia were represented by multiple conspecics, and thus cannot test generic monophyly) (Figs. 24).
199
Table 4
Proposed classication of Protobranchia.
Subclass Protobranchia Pelseneer, 1889
Order Solemyida Dall, 1889
Superfamily Solemyoidea Gray, 1840
Family Solemyidae Gray, 1840
Superfamily Manzanelloidea Chronic, 1952
Family Manzanellidae Chronic, 1952
Family Nucinellidae Vokes, 1956
Order Nuculida Dall, 1889
Superfamily Nuculoidea Gray, 1824
Family Nuculidae Gray, 1824
Order Nuculanida Carter, Campbell & Campbell, 2000
Superfamily Nuculanoidea H. Adams & A. Adams, 1858
Family Bathyspinulidae Coan and Scott, 1997
Family Malletiidae H. Adams & A. Adams, 1858
Family Neilonellidae Schileyko, 1989
Family Nuculanidae H. Adams & A. Adams, 1858
Family Phaseolidae Scarlato & Starobogatov, 1971
Family Siliculidae Allen & Sanders, 1973
Family Tindariidae Verrill & Bush, 1897
Family Yoldiidae Dall, 1908
Superfamily Sareptoidea Stocliczka, 1870
Family Sareptidae Stocliczka, 1870
Among nuculoids, Nucula is a triphyletic assemblage, owing to

the placement of Acila, Ennucula, and Leionucula (Figs. 24). The systematic validity of Ennucula has been in question for some time, as
this genus is distinguished from Nucula only by the absence of crenulations on the ventral interior surface of the shell (Maxwell, 1988;
Kilburn, 1999). The arrangement of clades in the nuculoid phylogeny suggests that absence of ventral crenulations is a symplesiomorphy within this subfamily (these do not occur in Ennucula or
Brevinucula) and/or has been lost repeatedly in unrelated lineages.
These results herald future revision of nuculoid genera.
Among nuculanoids (Fig. 7), the genus Nuculana is represented
by three species and is a somewhat coherent entity, save for the
inclusion of Jupiteria, an erstwhile subgenus of Nuculana (Allen
and Hannah, 1986) (BS = 99; PP = 1.00; JF = 69; Figs. 24). Our results therefore suggest that Jupiteria should once again be synonymized with Nuculana. Similarly, the genus Ledella (Nuculanidae;
Ledellinae in Allen and Hannah, 1986) is largely coherent in ML
and BI topologies, but for the inclusion of Bathyspinula hilleri
(Bathyspinulidae)heretofore a subfamily of Nuculanidae (Coan
and Scott, 1997; Coan et al., 2000; Spinulinae in Allen and Hannah,
1986) (Figs. 2 and 3). The placement of Bathyspinula within Ledella
is supported by multiple nuclear gene tree topologies (Boyle, 2011).
Marked morphological similarities between Bathyspinula and many
Ledella that also bear an attenuate rostrum (e.g., Ledella robusta, Ledella ultima) is dissuasive of a distinction between the two families,
much less the genera Ledella and Bathyspinula (Boyle, 2011).
Another pair of genera with greatly asymmetrical shells and/or
recurved rostra are Propeleda (Nuculanidae) and Silicula (Siliculidae), which form a grade sister to the Nuculana + Jupiteria clade
(Figs. 24). As nodal support for the non-monophyly of these genera is not signicant, we cannot dismiss the possibility that they
are systematically valid. However, support for the inclusion of Silicula with a clade of Propeleda + Nuculana + Jupiteria is strong
(BS = 98; PP = 1.00; JF = 93), and this clade appears nested within
other nuculanids, disputing the validity of the family Siliculidae
as an entity separate from Nuculanidae.
Of all the nuculanoid families, Yoldiidae (represented here by
the genera Yoldia, Yoldiella, and Megayoldia) appears to be in direst
need of dissolution, having been recovered as a polyphyletic
assemblage across all topologies (Figs. 24). At least two Yoldiella
are supported as members of a clade with Malletia and Megayoldia.
In model-based analyses, Yoldia eightsi is sister to Pristigloma
(BS = 71; PP = 0.99), whereas another three species of Yoldia form
200
Fig. 7. Interfamilial relationships within Nuculanoidea based on ML analysis.
a clade with the malletiid Clencharia abyssorum and the nuculanids

(BS = 88; PP = 1.00; Figs. 2 and 3). Clades of Yoldiidae are somewhat more coherent in the direct optimization topology, but are
still recovered as triphyletic (Fig. 4). The type genus Yoldia is not
monophyletic in any of the analyses (Figs. 24). The great variation
in yoldiid shell morphology accords with the incoherent position of
yoldiids among malletiids and nuculanids across the phylogeny.
Yoldiidae may have a rounded or truncated posterior shell margin,
and the resilifer may be large or small (Coan et al., 2000; Huber,
2010). Consistent with the placement of Malletia as derived Yoldiidae, both yoldiids and malletiids have large labial palps with narrow palp proboscides. Only the absence of the resilifer in
Malletiidae distinguishes this lineage from nuculanids and yoldiids. But as with the absence of AP asymmetry in Sareptidae or
the lack of ventral crenulations in Ennucula (discussed above),
the absence of a character is a demonstrably poor justication
for dening derived clades in Protobranchia. The only character
system that reasonably distinguishes Nuculanidae, Malletiidae
and Yoldiidae is the alimentary system (e.g., Sanders and Allen,
1985; Allen, 1992; Allen et al., 1995), but this is also in need of
re-examination at the family and genus-level.
The placement of Neilonellidae, Phaseolidae and Tindariidae as
derived lineages within the Nuculanidae-complex (nested among
Nuculanidae, Malletiidae and Yoldiidae) further highlights inconsistencies in the present classication of Protobranchia. The addition of
terminals with signicant missing data does lend support to some
groups (e.g., Neilonella is still recovered as monophyletic; Fig. 7),
but mostly casts additional doubt upon the validity of several genera
(e.g., Tindaria, Jupiteria, Malletia). Meanwhile, many protobranch
genera remain to be sampled for testing familial and generic relationships. Forthcoming efforts are therefore anticipated to redene
and reestablish a classication of Nuculida and Nuculanida.
4.4. Protobranch phylogeny retains the signature of the end-Permian

mass extinction
One of the ideas that has dominated paleontological and evolutionary thinking for several decades is the Sepkoski Curve, the outcome of detailed tabulation of fossil lineages through the
stratigraphic record (Sepkoski, 1981; Sepkoski and Bambach,
1981). Observing the phenomenon of early bursts in radiation, tandem plateaus of stability, and abrupt declines, Sepkoski quantitatively described three evolutionary faunasthe Cambrian, the
Paleozoic, and the Moderncomprising distinct assemblages of
taxa associated with particular geological periods (Sepkoski,
1978, 1979, 1981). An important component of transitions from
one fauna to the next are mass extinctions, many of which both dene certain geological periods and precede rapid diversication of
the ensuing faunal assemblage (Raup and Sepkoski, 1982, 1984).
The single greatest episode of these is the end-Permian mass
extinction ca. 254 Ma. Estimated to have extinguished 9599% of
marine species and approximately 75% of families of terrestrial
vertebrates, the end-Permian event radically altered the composition of Earths biota. The marine realm, theretofore dominated by
such Palaeozoic lineages as crinoids, bryozoans, brachiopods,
belemnites, ammonites, and trilobites, subsequently bore spectacular radiations of bivalves, gastropods, and echinoidsconstituents of the Modern fauna.
Mass extinctions are measured by the persistence and decline
of fossil lineages, but their effects on the phylogenies of extant taxa
are largely inferred through theory and simulations (Rabosky and
Lovette, 2008; Crisp and Cook, 2009). One of the characteristic features of a simulated mass extinction on evolutionary history is to
engender long branches in a tree topology. This is observed as
a log-lineage through time (LTT) plota visualization of net
diversication rate through timeof anti-sigmoidal shape (Crisp

and Cook, 2009). Generally, the greater the extinction event, the
greater will be the curvature of the LTT plot (Crisp and Cook,
2009). The intermittent plateau of net diversication rate is a consequence of extinction, and subsequent upturn in diversication
rate corresponds to recovery from the extinction event. The ampli-
201
tude of lineage loss during the end-Permian extinction is therefore

expected to give rise to a characteristic net diversication rate
curve for lineages that originated prior to, and survived, this event.
However, an anti-sigmoidal curve is infrequently observed in
empirical studies, principally because a large number of extant lineages that diversied before the extinction event is necessary to
Fig. 8. (A) Log-lineage through time (LTT) plot inferred from molecular dating of Protobranchia. Shading and rates indicate parameters of optimal model; note truncation of
post-Cretaceous branching times. Inset: Schematic of Yule-three-rate model tted to data by Laser v. 2.3. (B) Simulated LTT plots (in gray) corresponding to a constant
speciation process interrupted by a 99% cull at time t = 250 (as shown in inset schematic). Apparent downturn in net diversication rate as time t ? 0 is caused by simulation
of sampling limitations. The observed LTT plot of Protobranchia is shown in red.
202
observe such a curve. Many taxa with ancient origins have too few
extant species to infer diversication through time (e.g., nautiloids,
Lindgren et al., 2004; horseshoe crabs, Obst et al., 2012), too few
fossils to calibrate dated phylogenies reliably (e.g., most soft-bodied invertebrates; Kawauchi et al., 2012), or are survived by a single lineage that diversies in the wake of the mass extinction
(revenant clades, sensu Sharma and Wheeler, 2013). As an example,
all crown-group Crinoidea are inferred to have diversied immediately after the end-Permian, engendering a characteristic long
branch subtending a clade of ca. 620 extant species (Rouse et al.,
2013). Additionally, simulation studies have demonstrated that
prolonged extinction events can cause shifts in root ages, causing
diversications of extant taxa to appear younger, even with complete taxon sampling; this effect is especially pronounced for small
clades (Yedid et al., 2012; Sharma and Wheeler, 2013).
In the case of protobranch bivalves, early diversication gave
rise to all extant superfamilial lineages prior to the Silurian, but
diversication of most of their constituent clades occurred in the
Mesozoic (Fig. 6). The long branches subtending crown-group
superfamilies engender an LTT plot with a characteristic anti-sigmoidal curve, with upturn in diversication toward the end of
the Permian (260 Ma; Table 3; Fig. 8A). The diversication rate
estimated for the middle portion (the saddle of the anti-sigmoidal curve) of protobranch evolutionary history is remarkably low
under the optimal model (0.0008 lin/Myr; Fig. 8A). However,
although recovered as optimal, the Yule-three-rate model is only
designed to infer three phases of pure speciation, with no mechanism for parameterizing either intrinsic extinction (l) or extrinsic
diversity culls, such as mass extinction events. To test whether
protobranch evolutionary history is discernible from a constant
diversication process experiencing mass extinction, we employed
simulations of an end-Permian event-like process to generate a
null distribution for comparison. The empirical protobranch LTT
is indistinguishable from such a null distribution (Fig. 8B). Some
deviation from the simulated evolutionary histories occurs in the
Recent, likely stemming from assumptions made for the purpose
of generating a tractable null distribution (e.g., actual clade diversity approximately equal to described number of extant species;
equal pre- and post-extinction diversication rates).
Taken together with the fossil record of the group, these analyses indicate that the phylogeny of extant Protobranchia retains the
signature of the end-Permian mass extinction, consistent with predictions from theory and simulations (Crisp and Cook, 2009). Protobranchia provide a compelling contrast in this regard to such
groups as crinoids, which similarly arose in the Cambrian and have
a comparable number of extant species, yet were survived by a single lineage through the end-Permian (Rouse et al., 2013). In concert
with denser sampling of the protobranch tree of life, future investigation of this extinction signature should incorporate direct measurements of speciation and extinction rates from the protobranch
fossil record, particularly for gauging post-extinction recovery in
the Recent and improving inference of evolutionary history
through modelling approaches.
5. Conclusion
We comprehensively sampled the families of Protobranchia,
and generated a molecular phylogeny of this bivalve subclass
based on a multilocus dataset that is largely insensitive to algorithmic approaches. All tree topologies obtained distinguish Nucinellidae from Solemyidae with support and indicated that Sareptidae is
more closely allied to Nuculanida than to Nuculida, either as a derived, miniaturized family (probabilistic approaches) or as a basal
lineage (direct optimization and BEAST analyses). Forthcoming
systematic revisions of Nuculida and Nuculanida are imperative
for establishing a new classication of these orders based on natural, monophyletic groups. Estimation of divergence times and analysis of diversication rates reveal characteristic hallmarks of mass
extinction in the evolutionary history of protobranchs.
Acknowledgments
Sampling and sequencing were facilitated by two NSF-funded
AToL Grants to G.G. (NSF #DEB-0732903: Collaborative Research:
AToL: Phylogeny on the half-shellAssembling the Bivalve Tree
of Life and NSF #EF-0531757: AToL: Collaborative Proposal:
Assembling the Protostome Tree of Life). G.G. acknowledges Victoriano Urgorri for his invitation to the 2009 DIVA-Artabria cruise to
the Banco de Galicia on board of the Spanish Governmenr R/V Sarmiento de Gamboa, and Nerida Wilson and Greg Rouse for their
invitation to a SCRIPPS sampling in the Cortes Bank in 2007 onboard of the UNOLS vessel R/V Robert Gordon Sproul. Marina da
Cunha generously provided samples from the mud volcanoes of
the Gulf of Cdiz. Nstor Ardila (Universidad de los Andes), Cruz
Palacn (Universitat de Barcelona), Jess Troncoso (Universidade
de Vigo), Maria Israelson (Sweden), and Elizabeth Kools and Terry
Gosliner (California Academy of Sciences) helped in securing samples for this study. Sampling and sequencing conducted by the Etter lab was supported by NSF Grants OCE0726382 and
OCE1130541. We thank Lisa Levin and Christina Frieder for their
invitation to collect samples on the SD-SeaFEx cruise (MV1209
supported by UC Ship Funds), Saskia Brix for the invitation to collect samples on the IceAGE cruise, Katrin Linse for bivalves from
the ANDEEP cruises, and Pedro Martinez for bivalves from the
DIVA3 cruise. We also thank the crews of the R/V Endeavor (cruise
ENN447), FS Meteor and R/V Melville for their advice and help
while sampling deep-water habitats. Christina Frieder generously
provided a Propeleda from her Chile margin samples. Editor Suzanne Williams and two reviewers provided comments that helped
rene this article. P.P.S. was supported by the National Science
Foundation Postdoctoral Research Fellowship in Biology under
Grant No. DBI-1202751.
Appendix A. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ympev.
2013.05.018.
References
Allen, J.A., 1971. Evolution and functional morphology of the deep water
protobranch bivalves of the Atlantic. In: Uda, M. (Ed.), Proceedings of the
Joint Oceanographic Assembly. Japan Society for the Promotion of Science,
Tokyo, pp. 251253.
Allen, J.A., 1978. Evolution of the deep sea protobranch bivalves. Phil. Trans. Roy.
Soc. Lond. B 284, 387401.
Allen, J.A., 1979. The adaptations and radiation of deep-sea bivalves. Sarsia 64, 19
27.
Allen, J.A., 1992. The evolution of the hindgut of deep-sea protobranch bivalves. Am.
Malacol. Bull. 9, 187191.
Allen, J.A., Hannah, F.J., 1986. A reclassication of the recent genera of the subclass
Protobranchia (Mollusca: Bivalvia). J. Conchol. 32, 225249.
Allen, J.A., Sanders, H.L., 1996. The zoogeography, diversity and origin of the deepsea protobranch bivalves of the Atlantic: the epilogue. Prog. Oceanogr. 38, 95
153.
Allen, J.A., Sanders, H.L., Hannah, F.J., 1995. Studies on the Deep-Sea Protobranchia
(Bivalvia); the Subfamily Yoldiellinae. Bull. Nat. Hist. Mus. 61, 1190.
Beesley, P.L., Ross, G.J.B., Wells, A. (Eds.), 1998. Mollusca: The Southern Synthesis,
Fauna of Australia, vol. 5. CSIRO Publishing, Melbourne.
Bieler, R., Carter, J.G., Coan, E.V., 2010. Classication of bivalve families. Malacologia
52, 113133.
Boyle, E.E., 2011. Evolutionary Patterns in Deep-Sea Mollusks. Doctoral dissertation,
University of Massachusetts Boston.
Boyle, E.E., Etter, R.J., 2013. Heteroplasmy in a deep-sea protobranch bivalve
suggests an ancient origin of doubly uniparental inheritance of mitochondria in
Bivalvia. Mar. Biol. 160, 413422.

Boyle, E.E., Zardus, J.D., Chase, M.R., Etter, R.J., Rex, M.A., 2004. Strategies for
molecular genetic studies of preserved deep-sea macrofauna. Deep-Sea Res. I
51, 13191336.
Carter, J.G., 1990. Evolutionary signicance of shell microstructure in the
Palaeotaxodonta, Pteriomorphia and Isolibranchia. In: Carter, J.G. (Ed.),
Skeletal Biomineralization: Patterns Processes and Evolutionary Trends, vol. 1.
Van Nostrand Reinhold, New York, pp. 135301.
Carter, J.G., Campbell, D.C., Campbell, M.R., 2000. Cladistic perspectives on early
bivalve evolution. In: Harper, E.M., Taylor, J.D., Crame, J.A. (Eds.), The
Evolutionary Biology of the Bivalvia. The Geological Society of London,
London, pp. 4779.
Castresana, J., 2000. Selection of conserved blocks from multiple alignments for
their use in phylogenetic analysis. Mol. Biol. Evol. 17, 540552.
Cavanaugh, C.M., 1983. Symbiotic chemoautotrophic bacteria in marine
invertebrates from sulphide-rich habitats. Nature 302, 5861.
Chronic, H., 1952. Molluscan fauna form the Permian Kaibab formation, Walnut
Canyon, Arizona. Bull. Geol. Soc. Amer. 63, 95166.
Coan, E.V., Scott, P.H., 1997. Checklist of the marine bivalves of the northeastern
Pacic Ocean. Santa Barbara Mus. Nat. History, Contrib. Sci. 1, 28.
Coan, E.V., Valentich Scott, P., Bernard, F.R., 2000. Bivalve seashells of western North
America. Marine bivalve mollusks from Arctic Alaska to Baja California. Santa
Barbara Museum of Natural History Monographs 2. Santa Barbara Museum of
Natural History, Santa Barbara.
Conti, S., 1954. Stratigra e paleontologia della Val Solda (Lago di Lugano). Memorie
Descrittive della Carta Geologica dItalia 30, 1248.
Cope, J.C.W., 1996. The early evolution of the Bivalvia. In: Taylor, J.D. (Ed.), Origin
and Evolutionary Radiation of the Mollusca. Oxford University Press, Oxford, pp.
361370.
Cope, J.C.W., 1997. The early phylogeny of the class Bivalvia. Palaeontology 40, 713
746.
Cope, J.C.W., 1999. Middle Ordovician bivalves from Mid-Wales and the Welsh
borderland. Palaeontology 42, 467499.
Cope, J.C.W., 2004. Bivalve and rostroconch mollusks. In: Webby, B.D., Droser, M.L.
(Eds.), The Great Ordovician Biodiversication Event. Columbia University
Press, New York, pp. 196208.
Crisp, M.D., Cook, L.G., 2009. Explosive radiation or cryptic mass
extinction? Interpreting signatures in molecular phylogenies. Evolution 63,
22572265.
Cusimano, N., Renner, S.S., 2010. Slowdowns in diversication rates from real
phylogenies may not be real. Syst. Biol. 59, 458464.
Doucet-Beaupr, H., Breton, S., Chapman, E.G., Blier, P.U., Bogan, A.E., Stewart, D.T.,
Hoeh, W.R., 2010. Mitochondrial phylogenomics of the Bivalvia (Mollusca):
searching for the origin and mitogenomic correlates of doubly uniparental
inheritance of mtDNA. BMC Evol. Biol. 10 (1), 50.
Drew, G.A., 1899. Some observations on the habits, anatomy and embryology of
members of the Protobranchia. Anat. Anz. 15, 493519.
Drummond, A.J., Ho, S.Y.W., Phillips, M.J., Rambaut, A., 2006. Relaxed phylogenetics
and dating with condence. PLoS Biol. 4, e88.
Drummond, A.J., Rambaut, A., 2007. BEAST: Bayesian evolutionary analysis by
sampling trees. BMC Evol. Biol. 7, 214.
Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and
high throughput. Nucl. Acids Res. 32, 17921797.
Etter, R.J., Boyle, E.E., Glazier, A., Jennings, R.M., Dutra, E., Chase, M.R., 2011.
Phylogeography of a pan-Atlantic abyssal protobranch bivalve: implications for
evolution in the Deep Atlantic. Mol. Ecol. 20, 829843.
Etter, R.J., Rex, M.A., Chase, M.R., Quattro, J.M., 2005. Population
differentiation decreases with depth in deep-sea bivalves. Evolution 59,
14791491.
Farris, J.S., Albert, V.A., Kllersj, M., Lipscomb, D., Kluge, A.G., 1996. Parsimony
jackkning outperforms neighbor-joining. Cladistics 12, 99124.
Giribet, G., 2008. Bivalvia. In: Ponder, W.F., Lindberg, D.R. (Eds.), Phylogeny
and Evolution of the Mollusca. University of California Press, Berkeley, pp.
105142.
Giribet, G., Distel, D.L., 2003. Bivalve phylogeny and molecular data. In: Lydeard, C.,
Lindberg, D.R. (Eds.), Molecular Systematics and Phylogeography of Mollusks.
Smithsonian Books, Washington, pp. 4590.
Giribet, G., Wheeler, W., 2002. On bivalve phylogeny: a high-level analysis of the
Bivalvia (Mollusca) based on combined morphology and DNA sequence data.
Invertebr. Biol. 121, 271324.
Goloboff, P.A., 1999. Analyzing large data sets in reasonable times: solutions for
composite optima. Cladistics 15, 415428.
Goloboff, P.A., 2002. Techniques for analyzing large data sets. In: Desalle, R., Giribet,
G., Wheeler, W.C. (Eds.), Techniques in Molecular Systematics and Evolution.
Brikhuser Verlag, Basel, pp. 7079.
Graf, D.L., Cummings, K.S., 2006. Palaeoheterodont diversity (Mollusca: Trigonioida:
Unionoida): what we know and what we wish we knew about freshwater
mussel evolution. Zool. J. Linn. Soc. 148, 343394.
Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate
large phylogenies by maximum likelihood. Syst. Biol. 52, 696704.
Gustafson, R.G., Reid, R.G.B., 1986. Development of the pericalymma larva of
Solemya reidi (Bivalvia: Cryptodonta: Solemyidae) as revealed by light and
electron microscopy. Mar. Biol. 93, 411427.
Gustafson, R.G., Reid, R.G.B., 1988. Association of bacteria with larvae of the gutless
protobranch bivalve Solemya reidi (Cryptodonta: Solemyidae). Mar. Biol. 97,
389401.
203
Harper, E., Dreyer, H., Steiner, G., 2006. Reconstructing the Anomalodesmata
(Mollusca: Bivalvia): morphology and molecules. Zool. J. Linn. Soc. 148, 395
420.
Huber, M., 2010. Protobranchia. In: Compendium of Bivalves. ConchBooks,
Hackenheim, Germany, pp. 80105.
Huelsenbeck, J.P., Ronquist, F., 2005. Bayesian analyses of molecular evolution using
MrBayes. In: Nielsen, R. (Ed.), Statistical Methods in Molecular Evolution.
Springer, New York.
Jablonski, D., Lutz, R.A., 1983. Larval ecology of marine benthic invertebrates:
paleobiological implications. Biol. Rev. 58, 2189.
Kawauchi, G.Y., Sharma, P.P., Giribet, G., 2012. Sipunculan phylogeny based on six
genes, with a new classication and the descriptions of two new families. Zool.
Scr. 41, 186210.
Kilburn, R.N., 1999. The family Nuculidae (Bivalvia: Protobranchia) in South Africa
and Mozambique. Ann. Natal Mus. 40, 245268.
Kocot, K.M., Cannon, J.T., Todt, C., Citarella, M.R., Kohn, A.B., Meyer, A., Santos, S.R.,
Schander, C., Moroz, L.L., Lieb, B., Halanych, K.M., 2011. Phylogenomics reveals
deep molluscan relationships. Nature 477, 452456.
Lindgren, A.R., Daly, M., 2007. The impact of length-variable data and alignment
criterion on the phylogeny of Decapodiformes (Mollusca: Cephalopoda).
Cladistics 23, 464476.
Lindgren, A.R., Giribet, G., Nishiguchi, M.K., 2004. A combined approach to the
phylogeny of Cephalopoda (Mollusca). Cladistics 20, 454486.
Maxwell, P.A., 1988. Comments on A reclassication of the Recent genera of the
subclass Protobranchia (Mollusca: Bivalvia) by J. A. Allen and F. 1. Hannah
(1986). J. Conchol. 33, 8596.
Mikkelsen, P.M., Bieler, R., Kappner, I., Rawlings, T.A., 2006. Phylogeny of
Veneroidea (Mollusca: Bivalvia) based on morphology and molecules. Zool. J.
Lin. Soc. 148, 439521.
Neulinger, S.C., Sahling, H., Shling, J., Imhoff, J.F., 2006. Presence of two
phylogenetically distinct groups in the deep-sea mussel Acharax (Mollusca:
Bivalvia: Solemyidae). Mar. Ecol. Prog. Ser. 312, 161168.
Newell, N.D., 1969. Classication of Bivalvia. In: Moore, R. (Ed.), Treatise on
Invertebrate Paleontology, Part N, Mollusca 6, vol. 1. Boulder-Lawrence:
Geological Society of America & University of Kansas, Bivalvia.
Nixon, K.C., 1999. The Parsimony Ratchet, a new method for rapid parsimony
analysis. Cladistics 15, 407414.
Obst, M., Faurby, S., Bussarawit, S., Funch, P., 2012. Molecular phylogeny of extant
horseshoe crabs (Xiphosura, Limulidae) indicates Paleogene diversication of
Asian species. Mol. Phylogenet. Evol. 62, 2126.
Ockelmann, K., Warn, A., 1998. Taxonomy of and biological notes on the bivalve
genus Microgloma, with comments on protobranch nomenclature. Ophelia 48,
124.
Oliver, G., Rodrigues, C.F., Cunha, M.R., 2011. Chemosymbiotic bivalves from the
mud volcanoes of the Gulf of Cadiz, NE Atlantic, with descriptions of new
species of Solemyidae, Lucinidae and Vesicomyidae. ZooKeys 113, 138.
Oliver, P.G., Taylor, J.D., 2012. Bacterial symbiosis in the Nucinellidae (Bivalvia:
Solemyida) with descriptions of two new species. J. Moll. Stud. 78, 8191.
Parkhaev, P.Y., 2008. The Early Cambrian radiation of Mollusca. In: Ponder, W.F.,
Lindberg, D.R. (Eds.), Phylogeny and Evolution of the Mollusca. University of
California Press, Berkeley, pp. 3369.
Passamaneck, Y.J., Schander, C., Halanych, K.M., 2004. Investigation of molluscan
phylogeny using large-subunit and small-subunit nuclear rRNA sequences. Mol.
Phylogenet. Evol. 32, 2538.
Pojeta Jr., J., 1988. The origin and Paleozoic diversication of solemyid pelecypods.
New Mex. Bureau Mines Min. Res. Mem. 44, 201222.
Pojeta Jr., J., 2000. Cambrian Pelecypoda (Mollusca). Am. Malacol. Bull. 15, 157166.
Pojeta Jr., J., Runnegar, B., 1974. Fordilla troyensis and the early history of pelecypod
mollusks. Am. Sci. 62, 706711.
Pojeta Jr., J., Runnegar, B., Kriz, J., 1973. Fordilla troyensis Barrande: the oldest known
pelecypod. Science 180, 866868.
Posada, D., 2008. JModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25,
12531256.
Posada, D., Buckley, T., 2004. Model selection and model averaging in
phylogenetics: advantages of Akaike information criterion and Bayesian
approaches over likelihood ratio tests. Syst. Biol. 53, 793808.
Rabosky, D.L., 2006. LASER: a maximum likelihood toolkit for detecting temporal
shifts in diversication rates from molecular phylogenies. Evol. Bioinform. 2,
247250.
Rabosky, D.L., Lovette, I.J., 2008. Explosive evolutionary radiations: decreasing
speciation or increasing extinction through time? Evolution 62, 18661875.
Rambaut, A.E., 1996. Se-Al Sequence Alignment Editor. University of Oxford, UK
Program. <http://evolve.zoo.ox.ac.uk/software.
Rambaut, A., Drummond, A.J., 2009. Tracer v. 1.5. Program. <http://
beast.bio.ed.ac.uk/Tracer/>.
Raup, D.M., Sepkoski Jr., J.J., 1982. Mass extinctions in the marine fossil record.
Science 215, 15011503.
Raup, D.M., Sepkoski Jr., J.J., 1984. Periodicity of extinctions in the geologic past.
Proc Natl. Acad. Sci. USA 81, 801805.
Reid, R.G.B., 1990. Evolutionary implications of sulphide oxidizing symbioses in
bivalves. In: Morton, B. (Ed.), The Bivalvia. Hong Kong University Press, Hong
Kong, pp. 127140.
Reid, R.G.B., 1998. Subclass Protobranchia. In: Beesley, P.L., Ross, G.J.B., Wells, A.
(Eds.), Mollusca: The Southern Synthesis, Fauna of Australia, vol. 5. CSIRO
Publishing, Melbourne, pp. 235247.
204
Rouse, G.W., Jermiin, L.S., Wilson, N.G., Eeckhaut, I., Lanterbecq, D., Oji, T., Young,
C.M., Browning, T., Cisternas, P., Helgen, L.E., Stuckey, M., Messing, C.G., 2013.
Fixed, free, and xed: The ckle phylogeny of extant Crinoidea (Echinodermata)
and their Permian-Triassic origin. Mol. Phylogenet. Evol. 66, 161181.
Sanders, H.L., Allen, J.A., 1973. Studies on deep-sea Protobranchia (Bivalvia);
Prologue and the Pristiglomidae. Bull. Mus. Comp. Zool. 145, 237262.
Sanders, H.L., Allen, J.A., 1985. Studies on the deep-sea Protobranchia (Bivalvia); the
family Malletiidae. Bull. Brit. Mus. 49, 195238.
Sepkoski Jr., J.J., 1978. A kinetic model of Phanerozoic taxonomic diversity. I.
Analysis of marine orders. Paleobiology 4, 223251.
Sepkoski Jr., J.J., 1979. A kinetic model of Phanerozoic taxonomic diversity. II. Early
Phanerozoic families and multiple equilibria. Paleobiology 5, 222251.
Sepkoski Jr., J.J., 1981. A factor analytic description of the Phanerozoic marine fossil
record. Paleobiology 7, 3653.
Sepkoski Jr., J.J., Bambach, R.K., 1981. Phanerozoic marine diversity and the fossil
record. Nature 293, 435437.
Sharma, P., Giribet, G., 2009. Sandokanid phylogeny based on eight molecular
markersthe evolution of a southeast Asian endemic family of Laniatores
(Arachnida, Opiliones). Mol. Phylogenet. Evol. 52, 432447.
Sharma, P.P., Wheeler, W.C., 2013. Revenant clades in historical biogeography: the
geology of New Zealand predisposes endemic clades to root age shifts. J.
Biogeogr. http://dx.doi.org/10.1111/jbi.12112.
Sharma, P.P., Gonzlez, V.L., Kawauchi, G.Y., Andrade, S.C., Guzmn, A., Collins, T.M.,
Glover, E.A., Harper, E.M., Healy, J.M., Mikkelsen, P.M., Taylor, J.D., Bieler, R.,
Giribet, G., 2012. Phylogenetic analysis of four nuclear protein-encoding genes
largely corroborates the traditional classication of Bivalvia (Mollusca). Mol.
Sharma, P.P., Vahtera, V., Kawauchi, G.Y., Giribet, G., 2011. Running WILD: the case
for exploring mixed parameter sets in sensitivity analysis. Cladistics 27, 538
549.
Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of log-likelihoods with
applications to phylogenetic inference. Mol. Biol. Evol. 16, 11141116.
Simmons, M.P., Ochoterrena, H., 2000. Gaps as characters in sequence-based
phylogenetic analyses. Syst. Biol. 49, 369381.
Smith, S.A., Wilson, N.G., Goetz, F.E., Feehery, C., Andrade, S.C.S., Rouse, G.W.,
Giribet, G., Dunn, C.W., 2011. Resolving the evolutionary relationships of
molluscs with phylogenomic tools. Nature 480, 364367, See also: Smith, S. A.,
Wilson, N. G., Goetz, F. E., Feehery, C., Andrade, S. C. S., Rouse, G. W., Giribet, G. &
Dunn, C. W. (2013). Corrigendum: Resolving the evolutionary relationships of
molluscs with phylogenomic tools. Nature 493, 708.
Stadler, T., 2012. Package TreeSim ver. 1.6. Simulating Trees Under the Birth-death
Model.
Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic

analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688
2690.
Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the
RAxML Web servers. Syst. Biol. 57, 758771.
Taylor, J.D., Glover, E.A., 2010. Chemosymbiotic bivalves. In: Kiel, S. (Ed.), The Vent
and Seep Biota, Topics in Geobiology, vol. 33. Springer Press, pp. 107135.
Taylor, J.D., Glover, E.A., Williams, S.T., 2008. Ancient shallow water
chemosymbiotic bivalves: systematics of Solemyidae of eastern and southern
Australia. Mem. Queensl. Mus. 54, 75104.
Taylor, J.D., Williams, S.T., Glover, E.A., Dyal, P., 2007. A molecular phylogeny of
heterodont bivalves (Mollusca: Bivalvia: Heterodonta): new analyses of 18S and
28S rRNA genes. Zool. Scr. 36, 587606.
Tmkin, I., 2010. Molecular phylogeny of pearl oysters their relatives (Mollusca,
Bivalvia, Pterioidea). BMC Evol. Biol. 10, 342.
Varn, A., Vinh, L.S., Wheeler, W.C., 2010. POY version 4: phylogenetic analysis
using dynamic homologies. Cladistics 26, 7285.
Waller, T.R., 1998. Origin of the molluscan class Bivalvia and a phylogeny of major
groups. In: Johnston, P.A., Haggart, J.W. (Eds.), Bivalves: An Eon of Evolution
Palaeobiological Studies Honoring Norman D. University of Calgary Press,
Calgary, Newell, pp. 145.
Wheeler, W.C., 1996. Optimization alignment: the end of multiple sequence
alignment in phylogenetics? Cladistics 12, 19.
Wilson, N.G., Rouse, G.W., Giribet, G., 2010. Assessing the molluscan hypothesis
Serialia (Monoplacophora + Polyplacophora) using novel molecular data. Mol.
Yamanaka, T., Mizota, C., Matsuyama-Serisawa, K., Kakegawa, T., Miyazaki, J.-I.,
Mampuku, M., Tsutsumi, H., Fujiwara, Y., 2008. Stable isotopic characterization
of carbon, nitrogen and sulfur uptake of Acharax japonica from central Japan.
Plankton Benthos Res. 3, 3641.
Yedid, G., Stredwick, J., Ofria, C.A., Agapow, P.-M., 2012. A comparison of the effects
of random and selective mass extinctions on erosion of evolutionary history in
communities of digital organisms. PLoS ONE 7, e37233.
Zardus, J.D., 2002. Protobranch bivalves. Adv. Mar. Biol. 42, 165.
Zardus, J.D., Etter, R.J., Chase, M.R., Rex, M.A., Boyle, E.E., 2006. Bathymetric and
geographic population structure in the pan-Atlantic deep-sea bivalve
Deminucula atacellana (Schenck, 1939). Mol. Ecol. 15, 639651.
Zardus, J.D., Morse, M.P., 1998. Embryogenesis, morphology and ultrastructure of
the pericalymma larva of Acila castrensis(Bivalvia: Protobranchia: Nuculoida).
Invertebr. Biol. 117, 221244.

Into The Deep: A Phylogenetic Approach To The Bivalve Subclass Protobranchia

Uploaded by

Copyright:

Available Formats

Into The Deep: A Phylogenetic Approach To The Bivalve Subclass Protobranchia

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Into The Deep: A Phylogenetic Approach To The Bivalve Subclass Protobranchia

Uploaded by

Copyright:

Available Formats

Molecular Phylogenetics and Evolution 69 (2013) 188204

Contents lists available at SciVerse ScienceDirect

Molecular Phylogenetics and Evolution

Into the deep: A phylogenetic approach to the bivalve subclass

et al., 2000), with several lineages radiating thereafter in the deep

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Inherent to the task is the difculty of identifying living (or recently

MCZ DNA106839 / CASIZ 188907

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Yoldia scissurata Dall, 1897

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

assembled using the sequence editing software Sequencher

resulting sub-optimal likelihood trees to the unconstrained ML

2.3. Phylogenetic Analyses

2.5. Estimation of divergence times

Maximum likelihood (ML) and Bayesian inference (BI) analyses

Ages of clades were inferred using BEAST v. 1.7.4 (Drummond

2.4. Likelihood-based tests of topology

2.6. Diversication through time

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

described, which we treated as a proxy for total extant diversity. To

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

BI analysis recovered a topology highly congruent with respect to

3.3. Direct optimization

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

(jackknife resampling frequency [JF] = 67), and (2) the placement

L = 38631.41 for best suboptimal tree), at a = 0.05 and a = 0.01,

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

3.6. Analyses of diversication rates

incorporating a diversication rate slowdown at 456 Ma and

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Yule-3-rate model parameters:

analyses of molecular data, and (3) the apomorphic hinge structure

4.1. Higher-level relationships of Protobranchia

One of the smaller and more curious families of protobranchs is

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

organs, and the absence of a pallial sinus. It is therefore surprising

Among nuculoids, Nucula is a triphyletic assemblage, owing to

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Fig. 7. Interfamilial relationships within Nuculanoidea based on ML analysis.

a clade with the malletiid Clencharia abyssorum and the nuculanids

4.4. Protobranch phylogeny retains the signature of the end-Permian

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

diversication rate through timeof anti-sigmoidal shape (Crisp

tude of lineage loss during the end-Permian extinction is therefore

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

P.P. Sharma et al. / Molecular Phylogenetics and Evolution 69 (2013) 188204

Stamatakis, A., 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic

You might also like