John M. Healy

Investigating the Bivalve Tree of Life – an exemplar-based approach combining molecular and novel morphological characters

Abstract. To re-evaluate the relationships of the major bivalve lineages, we amassed detailed morpho-anatomical, ultrastructural and molecular sequence data for a targeted selection of exemplar bivalves spanning the phylogenetic diversity of the class. We included molecular data for 103 bivalve species (up to five markers) and also analysed a subset of taxa with four additional nuclear protein-encoding genes. Novel as well as historically employed morphological characters were explored, and we systematically disassembled widely used descriptors such as gill and stomach ‘types’. Phylogenetic analyses, conducted using parsimony direct optimisation and probabilistic methods on static alignments (maximum likelihood and Bayesian inference) of the molecular data, both alone and in combination with morphological characters, offer a robust test of bivalve relationships. A calibrated phylogeny also provided insights into the tempo of bivalve evolution. Finally, an analysis of the informativeness of morphological characters showed that sperm ultrastructure characters are among the best morphological features to diagnose bivalve clades, followed by characters of the shell, including its microstructure. Our study found support for monophyly of most broadly recognised higher bivalve taxa, although support was not uniform for Protobranchia. However, monophyly of the bivalves with protobranchiate gills was the best-supported hypothesis with incremental morphological and/or molecular sequence data. Autobranchia, Pteriomorphia, Heteroconchia, Palaeoheterodonta, Archiheterodonta, Euheterodonta, Anomalodesmata and Imparidentia new clade ( = Euheterodonta excluding Anomalodesmata) were recovered across analyses, irrespective of data treatment or analytical framework. Another clade supported by our analyses but not formally recognised in the literature includes Palaeoheterodonta and Archiheterodonta, which emerged under multiple analytical conditions. The origin and diversification of each of these major clades is Cambrian or Ordovician, except for Archiheterodonta, which diverged from Palaeoheterodonta during the Cambrian, but diversified during the Mesozoic. Although the radiation of some lineages was shifted towards the Palaeozoic (Pteriomorphia, Anomalodesmata), or presented a gap between origin and diversification (Archiheterodonta, Unionida), Imparidentia showed steady diversification through the Palaeozoic and Mesozoic. Finally, a classification system with six major monophyletic lineages is proposed to comprise modern Bivalvia: Protobranchia, Pteriomorphia, Palaeoheterodonta, Archiheterodonta, Anomalodesmata and Imparidentia.

Phylogenetic analysis of four nuclear protein-encoding genes largely corroborates the traditional classification of Bivalvia (Mollusca)

Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase β, elongation factor-1α, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida+Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta+Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta+Archiheterodonta+Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.

A family-level Tree of Life for bivalves based on a Sanger-sequencing approach

The systematics of the molluscan class Bivalvia are explored using a 5-gene Sanger-based approach including the largest taxon sampling to date, encompassing 219 ingroup species spanning 93 (or 82%) of the 113 currently accepted bivalve families. This study was designed to populate the bivalve Tree of Life at the family level and to place many genera into a clear phylogenetic context, but also pointing to several major clades where taxonomic work is sorely needed. Despite not recovering monophyly of Bivalvia or Protobranchia-as in most previous Sanger-based approaches to bivalve phylogeny-our study provides increased resolution in many higher-level clades, and supports the monophyly of Autobranchia, Pteriomorphia, Heteroconchia, Palaeoheterodonta, Heterodonta, Archiheterodonta, Euheterodonta, Anomalodesmata, Imparidentia, and Neoheterodontei, in addition to many other lower clades. However, deep nodes within some of these clades, especially Pteriomorphia and Imparidentia, could not be resolved with confidence. In addition, many families are not supported, and several are supported as non-monophyletic, including Malletiidae, Nuculanidae, Yoldiidae, Malleidae, Pteriidae, Arcidae, Propeamussiidae, Iridinidae, Carditidae, Myochamidae, Lyonsiidae, Pandoridae, Montacutidae, Galeommatidae, Tellinidae, Semelidae, Psammobiidae, Donacidae, Mactridae, and Cyrenidae; Veneridae is paraphyletic with respect to Chamidae, although this result appears to be an artifact. The denser sampling however allowed testing specific placement of species, showing, for example, that the unusual Australian Plebidonax deltoides is not a member of Donacidae and instead nests within Psammobiidae, suggesting that major revision of Tellinoidea may be required. We also showed that Cleidothaerus is sister group to the cementing member of Myochamidae, suggesting that Cleidothaeridae may not be a valid family and that cementation in Cleidothaerus and Myochama may have had a single origin. These results highlight the need for an integrative approach including as many genera as possible, and that the monophyly and relationships of many families require detailed reassessment. NGS approaches may be able to resolve the most recalcitrant nodes in the near future.

Sperm ultrastructure in Glauconome plankta and its relevance to the affinities of the Glauconomidae (Bivalvia: Heterodonta)

Summary Using transmission electron microscopy, sperm morphology was investigated for the first time in a representative of the heterodont bivalve family Glauconomidae. Spermatozoa of Glauconome plankta (Iredale, 1936) consist of a head region (acrosomal complex + nucleus), short midpiece and a flagellum. The membrane-bound acrosomal vesicle is attenuate-conical (length 1.1 ± 0.08 μm) with a moderately deep basal invagination extending for most of the vesicles length. Contents of the vesicle are differentiated into a highly electron-dense basal ring (cylindrical) sheathed by less dense material that also occupies the anterior two-thirds of the vesicle. Finely granular subacrosomal material fills the vesicle invagination and forms a layer between the nuclear apex and the vesicle, but no axial rod is discernible. The nucleus is rodshaped and straight, 4.4 ± 0.2 μm long and tapers slightly towards the apex. Nuclear contents are highly electron-dense, with the exception of occasional, electron-lucent lacunae. The midpiece consists of a pair of triplet-microtubular centrioles surrounded by four, approximately spherical mitochondria. Highly electron-dense granules of putative glycogen were observed between the mitochondria and around the distal centriole. The distal centriole is anchored to the plasma membrane via satellite fibres and is continuous with the flagellum (the latter 46 ± 3.0 μm long and exhibiting a conventional 9+2 axoneme). Comparison of sperm results does not suggest any especially close relationship between Glauconomidae (as exemplified by G. plankta) and other member families of the superfamily Veneroidea. Elsewhere within the Heterodonta no close relationship with the Corbiculoidea, Myoidea, Mactroidea, Tellinoidea or the Crassatelloidea is indicated by sperm morphology. Most promising seems a possible connection with Arctica (Arcticidae, Arcticoidea), which shows similar acrosomal and nuclear features. Supporting morphological and molecular evidence for this relationship is inconclusive but suggests further study.

Patterns of digenean parasitism of bivalves from the Great Barrier Reef and associated waters

Digenean parasites of marine bivalves are relatively poorly known, particularly in Australia. We surveyed 2256 bivalve individuals ( 47 species, 17 families) from Queensland marine waters incorporating south-east Queensland, Heron Island ( southern Great Barrier Reef) and Lizard Island ( northern Great Barrier Reef). Infections of trematode species from three families, Bucephalidae, Gorgoderidae and Monorchiidae, were found. Overall prevalence of infection was 2.3%. The Bucephalidae was the most commonly found family; 11 species were found in Tellinidae, Ostreidae, Isognomonidae and Spondylidae - the latter two previously unknown as hosts for bucephalids. A single gorgoderid infection was found in a venerid, Lioconcha castrensis. Five species of monorchiids were found from Tellinidae and Lucinidae. All infections are new host/parasite records. No infections were found in 35 of the 47 bivalve species sampled. The generally low prevalence of infection by digeneans of bivalves suggests that it is unlikely that any of the species reported here are seriously damaging to bivalve populations in these waters. We deduce that, at best, we have some life-cycle information but no actual identifications for 10% of the species of trematodes that infect bivalves of Queensland marine waters.

Sperm Ultrastructure of the Protobranchia: Comparison with Other Bivalve Mollusks and Potential Taxonomic and Phylogenetic Significance

Abstract Sperm ultrastructure of nine species of protobranch bivalves, representing three of four extant orders (Solemyida, Nuculida, Nuculanida), is discussed. Greatest diversity occurs in Solemyida (acrosomal vesicle low-conical, tall-conical, or very elongate, with radial plates; nucleus rod-shaped, teardrop-shaped, or very elongate; four, five, or six mitochondria) and the least in Nuculida (acrosomal vesicle low- to tall-conical; lacking radial plates; nucleus rod-shaped, five or six mitochondria) followed by Nuculanida (short, conical acrosomal vesicle with radial plates; spheroidal nucleus; four or five mitochondria). The wide variety of shapes in Solemyidae suggests taxonomic potential, especially in resolution and/or recognition of supraspecific taxa, but no diagnostic family characters were identified. Taxonomic potential exists for Nuculida (acrosomal shape) and Nuculanida (mitochondrial number). Protobranch sperm is highly diverse, and no defining character of the whole group was found. Support was found for the Nuculida and Nuculanida as natural groups but not for their close relationship. Nuculanida and Solemyida exhibit radial plates in the acrosomal vesicle but otherwise share no derived characters. The striking similarity of most sperm features of Nuculanida with certain pteriomorphians, especially Pectinoidea, a relationship also suggested by some mitochondrial DNA sequence data, poses interesting questions concerning their relationships and/or shared functional constraints.