Paula M. Mikkelsen

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.

Ultrastructure of Sperm Development and Mature Sperm Morphology in Three Species of Commensal Bivalves (Mollusca: Galeommatoidea)

Ultrastructural features of the ovotestes, spermatogenesis, and the mature sperm are described for three galeommatid bivalves, Divariscintilla yoyo, Divariscintilla troglodytes, and Scintilla sp., from stomatopod burrows in eastern Florida. All three species yielded similar results except with respect to mature sperm dimensions. The ovotestis contains three types of somatic cells within the testicular portion: flattened myoepithelial cells defining the outer acinal wall; underlying pleomorphic follicle cells containing abundant glycogen deposits; and scattered, amoeboid cells containing lysosomal‐like inclusions which are closely associated with developing sperm. Early spermatogenesis is typical of that reported from other bivalves. In contrast, the late stages of spermiogenesis involve the migration and gradual rotation of the acrosomal vesicle, resulting in a mature acrosome tilted about 70° from the long axis of the cell. The mature sperm possesses an elongated, slightly curved nucleus; a subterminal, concave acrosome with a nipple‐like central projection; five spherical mitochondria and two centnoles in the middlepiece; and a long flagellum. The rotational asymmetry and the presence of perimitochondrial glycogen deposits in these sperm are unusual in the Bivalvia and may be associated with fertilization specializations and larval brooding common among galeommatoideans.

A phylogenetic backbone for Bivalvia: an RNA-seq approach.

Bivalves are an ancient and ubiquitous group of aquatic invertebrates with an estimated 10 000–20 000 living species. They are economically significant as a human food source, and ecologically important given their biomass and effects on communities. Their phylogenetic relationships have been studied for decades, and their unparalleled fossil record extends from the Cambrian to the Recent. Nevertheless, a robustly supported phylogeny of the deepest nodes, needed to fully exploit the bivalves as a model for testing macroevolutionary theories, is lacking. Here, we present the first phylogenomic approach for this important group of molluscs, including novel transcriptomic data for 31 bivalves obtained through an RNA-seq approach, and analyse these data with published genomes and transcriptomes of other bivalves plus outgroups. Our results provide a well-resolved, robust phylogenetic backbone for Bivalvia with all major lineages delineated, addressing long-standing questions about the monophyly of Protobranchia and Heterodonta, and resolving the position of particular groups such as Palaeoheterodonta, Archiheterodonta and Anomalodesmata. This now fully resolved backbone demonstrates that genomic approaches using hundreds of genes are feasible for resolving phylogenetic questions in bivalves and other animals.

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.

Speciation in Modern Marine Bivalves (Mollusca: Bivalvia): Insights from the Published Record*

Abstract: What can living marine bivalves tell us about speciation in the marine environment? Three sets of literature data on Recent marine bivalves are analyzed for insight into the mechanisms behind bivalve speciation processes. (1) A dataset of all marine bivalves described as new to science during the years 2000–2009 (381 species in 135 published papers) reveals that malacologists are still describing undiscovered biodiversity, based largely upon newly collected expedition material. New species include those of both large and small body size (0.86-500 mm, mean 28 mm), from 51 bivalve families, all oceanic basins, and a wide range of water depths (intertidal to 7,333 m, mean 444 m). External shell characters dominate the diagnoses but are increasingly supplemented by anatomical, molecular, and phylogenetic evidence. Endemism is low (2.6%) when stated as such although another 57% of species were described as (thus far) restricted to a particular geographic region, habitat, or both. High percentages of deep-water and otherwise (geographically or ecologically) restricted species, plus several case studies, suggest that physiological specialization, in the form of bathymetrie limits, unique dietary adaptations, or host/symbiont associations, plays an important role in setting up barriers to gene flow in marine bivalves. (2) Bivalve species complexes [i.e., closely related, cryptic (possibly sibling) species with obscure morphological boundaries, or highly variable single species] also imply factors involved in ongoing speciation. Seven recently studied marine taxa are presented in which species complexes are either revealed or resolved by molecular data. Apparent barriers to gene flow are in most cases physiological (sympatric), or (in two cases) physical (allopatric), and in one case can be readily broken down by anthropogenic transport. (3) Two recent published phylogenetic analyses are discussed that show (a) disparately sized sister taxa with their synapomorphies (glochidia in Unionoidea; chemosymbiotic bacteria and mucus-tube feeding in Lucinoidea; cruciform muscle and long siphons in Tellinoidea; aortic bulb in Veneroidea) as recognized innovations that facilitated radiation of the more species-rich sister, and (b) polytomies in Lucinoidea that suggest rapid ongoing evolutionary change in several clades. Together these three sets of published data defeat the concept of a Marine Speciation Paradox in bivalves—speciation clues are merely subtler in marine bivalves and most often act at the physiological, rather than physical, level.

Marine bivalves of the Florida Keys: discovered biodiversity

Abstract A survey of marine bivalve biodiversity in the Florida Keys, an island archipelago off southern Florida, was compiled from original collecting, museum specimens and the literature. Assembly of over 6000 records resulted in 325 species, 47% of which can be considered common to abundant in the Keys. This represents a 100% increase over the previously known fauna, largely attributable to critical review of museum specimens. Capture of species occurrences from the literature, especially when non-traditional sources (newsletters, agency reports) are excluded, is shown to be least effective, producing only 44% of the total. Bivalve distributions within the Keys show that the fauna is tropical. One-third of the species are wide ranging along the island chain; however, a latitudinal cline in faunal similarity exists from the Upper Keys southwestwards to Dry Tortugas. The fauna of Florida Bay is the most divergent within the study region and also compared to other, ecologically complex, western Atlantic tropical-subtropical regions. Limited historical records indicate little species turnover in the Keys, although population reductions along the main highway and habitat shifts (from natural to artificial substrata) are evident. These results have implications for biodiversity survey methods and, more locally, for management of the Florida Keys National Marine Sanctuary.

Bivalvia—A Discussion of Known Unknowns*

Abstract. Bivalves share many of the “deeper” questions with the other molluscan groups - issues such as their origin and sister-group relationships within the Mollusca, and their suitability to explore molecular data in a “known” fossil framework. Other questions are more specific to bivalves, a group that radiated so successfully and nowadays predominantly specializes as infaunal and sessile epifaunal suspension feeders. This paper highlights and explores unanswered questions, from the seemingly trivial and mundane (e.g., how many species are actually out there?), to exploring enigmatic clades about which we know extremely little besides their shells, to macroevolutionary questions that could best be addressed by bivalve-based data. Fast-developing molecular approaches, including the first genome-level and transcriptomic data, a resurgence of detailed morphological and soft-anatomical research, and a renewed focus on Bivalvia by biological and paleontological workers provide us with an opportunity to address such issues. Coordination of efforts - and reciprocal illumination - across traditional disciplinary boundaries will be key factors in such endeavors.

Molecular phylogenetics and historical biogeography amid shifting continents in the cockles and giant clams (Bivalvia: Cardiidae).

Reconstructing historical biogeography of the marine realm is complicated by indistinct barriers and, over deeper time scales, a dynamic landscape shaped by plate tectonics. Here we present the most extensive examination of model-based historical biogeography among marine invertebrates to date. We conducted the largest phylogenetic and molecular clock analyses to date for the bivalve family Cardiidae (cockles and giant clams) with three unlinked loci for 110 species representing 37 of the 50 genera. Ancestral ranges were reconstructed using the dispersal-extinction-cladogenesis (DEC) method with a time-stratified paleogeographic model wherein dispersal rates varied with shifting tectonics. Results were compared to previous classifications and the extensive paleontological record. Six of the eight prior subfamily groupings were found to be para- or polyphyletic. Cardiidae originated and subsequently diversified in the tropical Indo-Pacific starting in the Late Triassic. Eastern Atlantic species were mainly derived from the tropical Indo-Mediterranean region via the Tethys Sea. In contrast, the western Atlantic fauna was derived from Indo-Pacific clades. Our phylogenetic results demonstrated greater concordance with geography than did previous phylogenies based on morphology. Time-stratifying the DEC reconstruction improved the fit and was highly consistent with paleo-ocean currents and paleogeography. Lastly, combining molecular phylogenetics with a rich and well-documented fossil record allowed us to test the accuracy and precision of biogeographic range reconstructions.

In Memoriam: William K. Emerson (1925–2016), with a List of His Publications and Taxa

82 On October 19, 2016, malacology lost one of its most respected senior members. William “Bill” Emerson died in New York City at the age of 91, following a 50-year career mainly spent at the American Museum of Natural History. It was there that he built a legacy of research, curation, and public service, including supervising world-class exhibitions and serving as President of most of this country’s major malacological societies. His accolades include honorary life memberships and other tributes in recognition of his career achievements. In memoriam: William K. Emerson (1925–2016), with a list of his publications and taxa