Daniel L. Graf

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.

Palearctic freshwater mussel (Mollusca: Bivalvia: Unionoida) diversity and the Comparatory Method as a species concept

ABSTRACT The current taxonomy of freshwater mussels (Mollusca: Bivalvia: Unionoida) in the Palearctic (western and northern Eurasia, from Western Europe to Eastern Russia, Korea and Japan) is confused by two competing species concepts: the Biological Species Concept (BSC) and the Comparatory Method (CM). The CM uses the “frontal contour” of the shell as the primary/sole character to delimit bivalve species. Based upon review of the literature, 45 Biological species in 16 genera are recognized in the Palearctic vs. 156 Comparatory species in 34 genera. I argue that the Comparatory Method is typological and that the “species” recognized have no evolutionary or biological basis. The traditional, Biological species are regarded as better representative of actual species diversity, but further revision is required. The problematic legacy of the Nouvelle École on Palearctic freshwater mollusk systematics is discussed.

Molecular phylogenetic analysis of tropical freshwater mussels (Mollusca: Bivalvia: Unionoida) resolves the position of Coelatura and supports a monophyletic Unionidae

In previous molecular phylogenetic analyses of the freshwater mussel family Unionidae (Bivalvia: Unionoida), the Afrotropical genus Coelatura had been recovered in various positions, generally indicating a paraphyletic Unionidae. However that result was typically poorly supported and in conflict with morphology-based analyses. We set out to test the phylogenetic position of Coelatura by sampling tropical lineages omitted from previous studies. Forty-one partial 28S nuclear rDNA and partial COI mtDNA sequences (1130 total aligned nucleotides) were analyzed separately and in combination under both maximum parsimony and likelihood, as well as Bayesian inference. There was significant phylogenetic incongruence between the character sets (partition homogeneity test, p<0.01), but a novel heuristic for comparing bootstrap values among character sets analyzed separately and in combination illustrated that the observed conflict was due to homoplasy rather than separate gene histories. Phylogenetic analyses robustly supported a monophyletic Unionidae, with Coelatura recovered as part of a well-supported Africa-India clade (=Parreysiinae). The implications of this result are discussed in the context of Afrotropical freshwater mussel evolution and the classification of the family Unionidae.

Patterns of Freshwater Bivalve Global Diversity and the State of Phylogenetic Studies on the Unionoida, Sphaeriidae, and Cyrenidae *

Abstract. The objective of this paper is to review the current state of our knowledge of freshwater bivalve diversity and evolution in order to identify some of the “Great Unanswered Questions” in the field. Twenty-one bivalve families have been attributed to fresh waters, but only 16 actually live and reproduce in inland waterways. Of 1209 species of freshwater bivalves, 1178 (97%) belong to eight primary freshwater families: Unionidae, Margaritiferidae, Hyriidae, Mycetopodidae, Iridinidae, and Etheriidae (all Unionoida), Sphaeriidae, and Cyrenidae (both Veneroida). The remaining 31 species represent secondary freshwater lineages of predominantly brackish/marine bivalve families. The global geographical patterns of freshwater bivalve richness are discussed, and an appendix detailing the families, genera, and species of freshwater bivalves is provided. Although the primary freshwater bivalve families represent at least three independent radiations from marine into inland aquatic habitats, these lineages have converged on similar adaptations to life in a flowing hypoosmotic medium. For example, all have abandoned broadcast spawning and planktonic veligers. Phylogenetic studies of the Unionoida, Sphaeriidae, and Cyrenidae have also converged on a suite of common challenges: outgroup issues, biased ingroup taxon and character sampling, and atypical modes of genetic inheritance that uncouple gene trees from species trees. The recent phylogenetic literature on the three primary radiations is reviewed, emphasizing areas in need of research. Ample opportunities exist in freshwater bivalve research but progress is hindered by the limited numbers of researchers and students in the field.

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.

Molecular phylogenetic analysis supports a Gondwanan origin of the Hyriidae (Mollusca: Bivalvia: Unionida) and the paraphyly of Australasian taxa

The freshwater mussel family Hyriidae (Mollusca: Bivalvia: Unionida) has a disjunct trans-Pacific distribution in Australasia and South America. Previous phylogenetic analyses have estimated the evolutionary relationships of the family and the major infra-familial taxa (Velesunioninae and Hyriinae: Hyridellini in Australia; Hyriinae: Hyriini, Castaliini, and Rhipidodontini in South America), but taxon and character sampling have been too incomplete to support a predictive classification or allow testing of biogeographical hypotheses. We sampled 30 freshwater mussel individuals representing the aforementioned hyriid taxa, as well as outgroup species representing the five other freshwater mussel families and their marine sister group (order Trigoniida). Our ingroup included representatives of all Australian genera. Phylogenetic relationships were estimated from three gene fragments (nuclear 28S, COI and 16S mtDNA) using maximum parsimony, maximum likelihood, and Bayesian inference, and we applied a Bayesian relaxed clock model calibrated with fossil dates to estimate node ages. Our analyses found good support for monophyly of the Hyriidae and the subfamilies and tribes, as well as the paraphyly of the Australasian taxa (Velesunioninae, (Hyridellini, (Rhipidodontini, (Castaliini, Hyriini)))). The Hyriidae was recovered as sister to a clade comprised of all other Recent freshwater mussel families. Our molecular date estimation supported Cretaceous origins of the major hyriid clades, pre-dating the Tertiary isolation of South America from Antarctica/Australia. We hypothesize that early diversification of the Hyriidae was driven by terrestrial barriers on Gondwana rather than marine barriers following disintegration of the super-continent.

Freshwater mussel (Mollusca: Bivalvia: Unionoida) richness and endemism in the ecoregions of Africa and Madagascar based on comprehensive museum sampling

The objective of this study was to assess freshwater mussel (Mollusca: Bivalvia: Unionoida) species distributions among the freshwater ecoregions of Africa and Madagascar to discover areas of high richness and endemism. These are among the top criteria for identifying biodiversity hotspots and establishing conservation priorities. Distributions were determined from museum specimens in 17 collections. In total, 5,612 records for 87 unionoid species could each be assigned to one of 90 freshwater ecoregions. The majority of species (55%) are known from only one (34 spp.) or two (14) ecoregions. Only three are known from more than 20 ecoregions: Etheria elliptica (38 ecoregions), Chambardia wahlbergi (25), and Mutela rostrata (21). The most species-rich ecoregions are Lake Victoria Basin (17 spp.), Upper Nile (16), Upper Congo (14), Senegal–Gambia (13), and Sudanic Congo–Oubangi (13). Those with the most endemic species are Lake Tanganyika (8 spp.), Lake Victoria Basin (6), Bangweulu–Mweru (4), and Lake Malawi (3). Twenty-five ecoregions have no known freshwater mussels. These patterns are significantly correlated with fish and general freshwater mollusk richness. Unionoid richness also varies significantly among major habitat types. These patterns are relevant to biogeography and conservation and indicate areas in need of further research. We argue that freshwater mussels are valuable as focal species for conservation assessments, and they themselves merit management consideration for their ecosystem functions and distributions in imperiled habitats. It is recommended that field surveys be conducted to determine the current status of species in all areas of Africa and Madagascar.

Freshwater mussels (Mollusca: Bivalvia: Unionoida) of Angola, with description of a new species, Mutela wistarmorrisi

ABSTRACT The aquatic fauna of the Angola region in southwestern Africa, including the Cuanza (= Kwanza), Cunene, Okavango, upper Zambezi, Cassai (= Kasai) and lower Congo basins, is poorly known. The freshwater mussels (Mollusca: Unionoida) of the region have received little attention, and then usually as fringes to the Congo and Zambezi-Okavango systems. All available freshwater mussel specimens from thirteen major mollusk collections were examined and, along with a literature review, form the basis for this checklist. Twenty-three species in three families (Etheriidae, Iridinidae and Unionidae) are recognized from the Angola region, eight of which are newly recognized in the region or had not been treated as valid in recent revisions (Mandahl-Barth, 1988; Daget, 1998). One of these species, Mutela wistarmorrisi, is new to science and described herein. Chambardia welwitschi (Morelet), a widely recognized subspecies, is elevated to species-level status, and four more names are resurrected to valid status from synonymy: Mutela langi Pilsbry & Bequaert, Chambardia moutai (Dartevelle), Coelatura stagnorum (Dautzenberg) and C. rotula Pilsbry & Bequaert. Mutela legumen (Rochebrune) is recognized as the senior synonym for the species formerly referred to as Mutela carrei (Putzeys), and the known range of Aspatharia subreniformis (Sowerby) is expanded to include the Cunene, Okavango and Zambezi basins. Figures of each species are provided, and known distributions and persistent taxonomic issues are discussed. An appendix lists geocodes for localities associated with specimen records.

Actual and Alleged Freshwater Mussels (Mollusca: Bivalvia: Unionoida) from Madagascar and the Mascarenes, with Description of a New Genus, Germainaia

ABSTRACT. Madagascar is widely recognized as a biodiversity hotspot, but the freshwater bivalves have received only limited recent attention. Based upon examination of records from 15 major museums and a literature review, at least nine species of freshwater mussels (Mollusca: Bivalvia: Unionoida) have been reported from either Madagascar or the Mascarene Islands (specifically, Reunion or Mauritius) in the Indian Ocean, east of Africa. The quality of the data, however, is generally poor. Seven of those species records are regarded as either erroneous reports of taxa known from other regions or as nomina dubia. No records from the Mascarenes are considered to be valid. The two remaining species, Etheria elliptica Lamarck 1807 and Unio geayi Germain 1911 (= Coelatura geayi) are both from Madagascar, and the latter is discussed in the context of two alternative hypotheses for the origin of the family Unionidae, nicknamed “Out of Africa” and “Into Africa.” Germainaia gen. nov. is introduced for Unio geayi to emphasize the distinction of that species from other Afrotropical freshwater mussel lineages. The possibility that Germainaia may represent the Hyriidae in Madagascar is discussed, but the new genus is left incertae sedis at the family-level in the absence of complete data. New taxon: Germainaia Graf and Cummings

Funeral for the Nouvelle École -iana Generic Names Introduced for Freshwater Mussels (Mollusca: Bivalvia: Unionoida)

ABSTRACT. Bourguignat, Locard, Servain, and others of the French Nouvelle École of the late 18th century introduced a number of subgeneric names for the freshwater mussel (Bivalvia: Unionoida) species of western Europe. Based largely upon latinizations of species-group names, most of these generic names end in the characteristic suffix -iana. For the most part, these taxa have not been regarded as valid and, in fact, they have been largely ignored since the early 20th century. However, most of these genus-group level names are available and need to be accounted for as the malacological community moves forward to update the taxonomy of the European fauna. A survey of the relevant primary and secondary literature revealed 131 available -iana generic nomina, only three of which are currently regarded as valid. The type species of each is reported (or designated) herein, and these are used to place the subgenera into the current European system of genera: Unio, Anodonta, Pseudanodonta, Potomida, and Margaritifera.