Walter R. Hoeh

MULTIPLE ORIGINS OF GENDER-ASSOCIATED MITOCHONDRIAL DNA LINEAGES IN BIVALVES (MOLLUSCA: BIVALVIA)

Previous studies have shown that marine mussels (genus Mytilus) and a freshwater mussel (Pyganodon grandis) contain two distinct gender‐associated mitotypes, which is a characteristic feature of the phenomenon of doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA). Here we present evidence for the presence of distinct male (M) and female (F) mitotypes in three other bivalve species, the mytilid Geukensia demissa, and the unionid species P. fragilis and Fusconaia flava. Nucleotide sequences of a segment of the COI gene from the M and F mitotypes from each of the three mytilid species (M. edulis, M. trossulus, G. demissa) and three unionid species (P. grandis, P. fragilis, F. flava) were used for phylogenetic analysis. The analysis suggests three independent origins of M and F mitotypes for the six species examined; one for the three unionid species, one for the two Mytilus species, and one for Geukensia. The first of these F/M divergence events, while of uncertain age, predates the divergence of the two unionid genera and is likely older than either of the two F/M divergence events in the mytilid taxa. The most parsimonious explanation of multiple F/M divergence events is that they represent independent origins of DUI. Another possibility is that, in a given taxon, an F or M mitotype assumes the role of the opposite mitotype (by virtue of a mechanism that remains to be clarified) and subsequently was fixed within its new gender. The fixation of a mtDNA lineage derived from a mitotype of switched function would reset the divergence of the gender‐associated lineages to zero, thereby mimicking a de novo split of F and M lineages from a preexisting mtDNA genome that was not gender specific. Further broad‐scale taxonomic studies of the occurrence of distinct M and F mitotypes may allow for the evaluation of the latter hypothesis.

Global diversity of large branchiopods (Crustacea: Branchiopoda) in freshwater

With about 500 known species worldwide, the large brachiopods are a relatively small group of primitive crustaceans. With few exceptions they live in temporary aquatic systems that are most abundant in arid and semi arid areas. As many regions remain unexplored and as especially the number of species in clam shrimps and tadpole shrimps is underestimated due to difficult identification, the species list will increase with future surveys. The Branchiopoda are monophyletic, but inter-ordinal relationships, as well as many evolutionary relationships at lower taxonomic levels are still unclear. Ongoing molecular studies will more accurately depict species diversity and phylogenetic patterns. With the exception of some anostracan families, most families are not restricted to the northern or southern hemisphere or specific zoogeographical regions. Large branchiopods are used for the assessment of the quality and function of temporary wetlands. Due to the reduction in number and quality of temporary wetlands, several species became endangered and are red listed by the IUCN.

Intraspecific phylogeography of Lasmigona subviridis (Bivalvia: Unionidae): conservation implications of range discontinuity

A nucleotide sequence analysis of the first internal transcribed spacer region (ITS‐1) between the 5.8S and 18S ribosomal DNA genes (640 bp) and cytochrome c oxidase subunit I (COI) of mitochondrial DNA (mtDNA) (576 bp) was conducted for the freshwater bivalve Lasmigona subviridis and three congeners to determine the utility of these regions in identifying phylogeographic and phylogenetic structure. Sequence analysis of the ITS‐1 region indicated a zone of discontinuity in the genetic population structure between a group of L. subviridis populations inhabiting the Susquehanna and Potomac Rivers and more southern populations. Moreover, haplotype patterns resulting from variation in the COI region suggested an absence of gene exchange between tributaries within two different river drainages, as well as between adjacent rivers systems. The authors recommend that the northern and southern populations, which are reproductively isolated and constitute evolutionarily significant lineages, be managed as separate conservation units. Results from the COI region suggest that, in some cases, unionid relocations should be avoided between tributaries of the same drainage because these populations may have been reproductively isolated for thousands of generations. Therefore, unionid bivalves distributed among discontinuous habitats (e.g. Atlantic slope drainages) potentially should be considered evolutionarily distinct. The DNA sequence divergences observed in the nuclear and mtDNA regions among the Lasmigona species were congruent, although the level of divergence in the COI region was up to three times greater. The genus Lasmigona, as represented by the four species surveyed in this study, may not be monophyletic.

HIGH FIDELITY OF MITOCHONDRIAL GENOME TRANSMISSION UNDER THE DOUBLY UNIPARENTAL MODE OF INHERITANCE IN FRESHWATER MUSSELS (BIVALVIA: UNIONOIDEA)

Abstract.— Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) has been demonstrated in both mytilid and unionid bivalves. Under DUI, females pass on their mtDNA to both sons and daughters, whereas males pass on their mtDNA to only sons. In mytilids, the loss of an original male (or M) mitotype, with its subsequent replacement by that lineages female (or F) mitotype, has been called a role‐reversal or, more specifically, a masculinization event. Multiple masculinization events have been inferred during the evolutionary history of mytilids but not unionids. The perceived lack of role‐reversal events in unionids may represent a significant difference in the evolutionary dynamics of DUI between the two bivalve taxa or simply a lack of sufficient taxon sampling in unionids. To evaluate these alternative hypotheses, six additional unionoidean bivalve genera were sampled for DUI including one genus from the sister taxon of the Unionidae, the Hyriidae. Phylogenetic analyses of 619 base pairs of cytochrome c oxidase I (COI) from eight genera (nine species) of unionoidean bivalves, plus the sister taxon to the Unionoida, Neotrigonia, revealed that the M and F unionoidean mitotypes were contained in gender‐specific, topologically congruent clades. This supports the hypothesis that either role‐reversal events do not occur in unionoideans or, if they do occur, their products are ephemeral in an evolutionary sense. Furthermore, the fact that the mantle‐tissue‐derived Neotrigonia mitotype is the sister mitotype to the unionoidean F mitotype clade suggests that DUI has been operating with high fidelity in unionoids for at least 200 million years. A relatively low incidence of interspecific hybridization in unionoideans and a possibly obligate role for the M mitotype in unionoidean gender determination are offered as potential explanations for the disparate evolutionary dynamics of DUI observed between mytilid and unionoidean bivalves.

Comparative Mitochondrial Genomics of Freshwater Mussels (Bivalvia: Unionoida) With Doubly Uniparental Inheritance of mtDNA: Gender-Specific Open Reading Frames and Putative Origins of Replication

Doubly uniparental inheritance (DUI) of mitochondrial DNA in marine mussels (Mytiloida), freshwater mussels (Unionoida), and marine clams (Veneroida) is the only known exception to the general rule of strict maternal transmission of mtDNA in animals. DUI is characterized by the presence of gender-associated mitochondrial DNA lineages that are inherited through males (male-transmitted or M types) or females (female-transmitted or F types), respectively. This unusual system constitutes an excellent model for studying basic aspects of mitochondrial DNA inheritance and the evolution of mtDNA genomes in general. Here we compare published mitochondrial genomes of unionoid bivalve species with DUI, with an emphasis on characterizing unassigned regions, to identify regions of the F and M mtDNA genomes that could (i) play a role in replication or transcription of the mtDNA molecule and/or (ii) determine whether a genome will be transmitted via the female or the male gamete. Our results reveal the presence of one F-specific and one M-specific open reading frames (ORFs), and we hypothesize that they play a role in the transmission and/or gender-specific adaptive functions of the M and F mtDNA genomes in unionoid bivalves. Three major unassigned regions shared among all F and M unionoid genomes have also been identified, and our results indicate that (i) two of them are potential heavy-strand control regions (OH) for regulating replication and/or transcription and that (ii) multiple and potentially bidirectional light-strand origins of replication (OL) are present in unionoid F and M mitochondrial genomes. We propose that unassigned regions are the most promising candidate sequences in which to find regulatory and/or gender-specific sequences that could determine whether a mitochondrial genome will be maternally or paternally transmitted.

Genetic diversity and divergence among freshwater mussel (Anodonta) populations in the Bonneville Basin of Utah

Populations of the freshwater mussel genus Anodonta appear to be in a state of rapid decline in western North America, following a trend that unfortunately seems to be prevalent among these animals (Mollusca: Unionoida). Here we describe the patterns of molecular divergence and diversity among Anodonta populations in the Bonneville Basin, a large sub‐basin of the Great Basin in western North America. Using amplified fragment length polymorphism (AFLP) analysis, we found a striking lack of nuclear diversity within some of these populations, along with a high degree of structuring among populations (FST = 0.61), suggesting post‐Pleistocene isolation, due either to a long‐term loss of hydrologic connectivity among populations or to more recent fish introductions. We also found evidence of recent hybridization in one of these populations, possibly mediated by fish‐stocking practices. Using mitochondrial sequence data, we compared the Bonneville Basin populations to Anodonta in several other drainages in western North America. We found a general lack of resolution in these phylogenetic reconstructions, although there was a tendency for the Bonneville Basin Anodonta (tentatively A. californiensis) to cluster with A. oregonensis from the adjacent Lahontan Basin in Nevada. We recommend further investigation of anthropogenic factors that may be contributing to the decline of western Anodonta and a broad‐scale analysis and synthesis of genetic and morphological variation among Anodonta in western North America.

On becoming cemented: evolutionary relationships among the genera in the freshwater bivalve family Etheriidae (Bivalvia: Unionoida)

Abstract A robust phylogeny for the Unionoida is emerging and presumed relationships of some major clades are being questioned. The Etheriidae or freshwater oysters has been a distinct family for over 160 years and currently contains three cemented genera: Acostaea (Columbia, South America), Pseudomulleria (India) and Etheria (Africa and Madagascar). Starobogatov (1970, Nauka, 1–372), Mansur and da Silva (1990, Amazoniana, 11(2), 147–166) and Bonetto (1997, Biociências, 5, 113–142) present conflicting testable hypotheses regarding the evolution of these taxa. Using cytochrome c oxidase subunit I DNA sequences the evolutionary relationships of these three genera has been examined, by comparing them to representatives of 30 other unionoid taxa from around the world. These analyses place Acostaea and Etheria within the Mycetopodidae while Pseudomulleria falls within the Unionidae. A monophyletic Etheriidae, composed of cemented freshwater bivalves, is not supported by the present analyses. Furthermore, the analyses indicate that cementation in the Unionida has evolved at least twice.

Characterization of a mitochondrial ORF from the gender-associated mtDNAs of Mytilus spp. (Bivalvia: Mytilidae): identification of the "missing" ATPase 8 gene.

Bivalve species are characterized by extraordinary variability in terms of mitochondrial (mt) genome size, gene arrangement and tRNA gene number. Many species are thought to lack the mitochondrial protein-coding gene atp8. Of these species, the Mytilidae appears to be the only known taxon with doubly uniparental inheritance of mtDNA that does not possess the atp8 gene. This raises the question as to whether mytilids have completely lost the ATP8 protein, whether the gene has been transferred to the nucleus or whether they possess a highly modified version of the gene/protein that has led to its lack of annotation. In the present study, we re-investigated all complete (or nearly complete) F and M mytilid mt genomes previously sequenced for the presence of conserved open reading frames (ORFs) that might code for ATP8 and/or have other functional importance in these bivalves. We also revised the annotations of all available complete mitochondrial genomes of bivalves and nematodes that are thought to lack atp8 in an attempt to detect it. Our results indicate that a novel mytilid ORF of significant length (i.e., the ORF is >85 amino acids in length), with complete start and stop codons, is a candidate for the atp8 gene: (1) it possesses a pattern of evolution expected for a protein-coding gene evolving under purifying selection (i.e., the 3rd>1st>2nd codon pattern of evolution), (2) it is actively transcribed in Mytilus species, (3) it has one predicted transmembrane helix (as do other metazoan ATP8 proteins), (4) it has conserved functional motifs and (5), comparisons of its amino acid sequence with ATP8 sequences of other molluscan or bivalve species reveal similar hydropathy profiles. Furthermore, our revised annotations also confirmed the mt presence of atp8 in almost all bivalve species and in one nematode species. Our results thus support recognizing the presence of ATPase 8 in most bivalves mt genomes (if not all) rather than the continued characterization of these genomes as lacking this gene.

A Phylogenetic Perspective on the Evolution of Morphological and Reproductive Characteristics in the Unionoida

Unionoid bivalves have a long history of classification and reclassification. Pre-Darwin classifications were deductive and artificial, reflecting character selection and weighting. More recent classifications, which should be natural and inductive to reflect phylogeny, reveal a diversity of methodologies and results.

Ancient androdioecy in the freshwater crustacean Eulimnadia

Among the variety of reproductive mechanisms exhibited by living systems, one permutation—androdioecy (mixtures of males and hermaphrodites)—is distinguished by its rarity. Models of mating system evolution predict that androdioecy should be a brief stage between hermaphroditism and dioecy (separate males and females), or vice versa. Herein we report evidence of widespread and ancient androdioecy in crustaceans in the genus Eulimnadia, based on observations of over 33 000 shrimp from 36 locations from every continent except Antarctica. Using phylogenetic, biogeographical and palaeontological evidence, we infer that androdioecy in Eulimnadia has persisted for 24–180 million years and has been maintained through multiple speciation events. These results suggest that androdioecy is a highly successful aspect of the life history of these freshwater crustaceans, and has persisted for orders of magnitude longer than predicted by current models of this rare breeding system.