Vision is a two-step process, including phototransduction initiated by a retinal-bound opsin photopigment, and subsequent photopigment regeneration wherein the inactive state is restored. Because retinal isomerization, or changing of the molecular structural conformation, drives the activation of opsin photopigments, the reverse process is a necessary part of regeneration. In some animal lineages, a second opsin functions as a photoisomerase that converts retinal back to the isomer needed for the visual opsin, but very little is known about their spectral properties. We investigated spectral variation and spatial expression of scallop retinochrome, an opsin used for light-driven regeneration of molluscan visual photopigments. We show that retinochrome is expressed in scallop retina, along with visual opsins, indicating a conserved role in supporting vision. We compared retinochrome spectral absorption across six species with different ecologies, revealing considerable functional variation possibly associated with photic environments. Finally, we characterized multiple amino acid sites contributing to spectral tuning-shifts in the specific maximally sensitive wavelength of light-of this opsin. These results reveal retinochrome as a functionally dynamic component of the scallop visual cycle that may be adapted to different photic environments across species and highlights spectral tuning in an ocular but otherwise non-visual photopigment, which has been largely overlooked in studying the evolution of visual cycles.

Key unanswered questions in biology center on the causes, consequences, and maintenance of sexual reproduction ("sex"). Genome-driven processes are central to the evolutionary and genetic mechanisms inherent to sex, making genomic resources a fundamental part of answering these questions. We present the first genome assembly for a species that is uniquely well-suited for the study of (a)sex in nature, Potamopyrgus antipodarum. This New Zealand snail is unusual in featuring multiple separate transitions from obligately sexual to obligately asexual reproduction, leading to the coexistence of phenotypically similar sexual and asexual forms, a feature that is required to directly study the maintenance of sex. These separately derived asexual lineages constitute separate evolutionary experiments, providing a powerful means of characterizing how the absence of sex affects genome evolution. Our genome assembly provides critical steps toward understanding the causes and consequences of sex in this system and important resources for the rapidly growing P. antipodarum and molluscan genomics research community. In characterizing this genome, we uncovered unexpected evidence for a recent whole-genome duplication (WGD) in P. antipodarum. This discovery sets the stage for using P. antipodarum to evaluate processes of rediploidization following WGD and to assess whether WGD might drive transitions to asexuality.

IntroductionMany marine organisms have a biphasic life cycle that transitions between a swimming larva with a more sedentary adult form. At the end of the first phase, larvae must identify suitable sites to settle and undergo a dramatic morphological change. Environmental factors, including photic and chemical cues, appear to influence settlement, but the sensory receptors involved are largely unknown. We targeted the protein receptor, opsin, which belongs to large superfamily of transmembrane receptors that detects environmental stimuli, hormones, and neurotransmitters. While opsins are well-known for light-sensing, including vision, a growing number of studies have demonstrated light-independent functions. We therefore examined opsin expression in the Pteriomorphia, a large, diverse clade of marine bivalves, that includes commercially important species, such as oysters, mussels, and scallops.MethodsGenomic annotations combined with phylogenetic analysis show great variation of opsin abundance among pteriomorphian bivalves, including surprisingly high genomic abundance in many species that are eyeless as adults, such as mussels. Therefore, we investigated the diversity of opsin expression from the perspective of larval development. We collected opsin gene expression in four families of Pteriomorphia, across three distinct larval stages, i.e., trochophore, veliger, and pediveliger, and compared those to adult tissues.ResultsWe found larvae express all opsin types in these bivalves, but opsin expression patterns are largely species-specific across development. Few opsins are expressed in the adult mantle, but many are highly expressed in adult eyes. Intriguingly, opsin genes such as retinochrome, xenopsins, and Go-opsins have higher levels of expression in the later larval stages when substrates for settlement are being tested, such as the pediveliger.ConclusionInvestigating opsin gene expression during larval development provides crucial insights into their intricate interactions with the surroundings, which may shed light on how opsin receptors of these organisms respond to various environmental cues that play a pivotal role in their settlement process.

ABSTRACTThe bivalve subclass Pteriomorphia, which includes the economically important scallops, oysters, mussels, and ark clams, exhibits extreme ecological, morphological, and behavioral diversity. Among this diversity are five morphologically distinct eye types, making Pteriomorphia an excellent setting to explore the molecular basis for the evolution of novel traits. Of pteriomorphian bivalves, Limida is the only order lacking genomic resources, greatly limiting the potential phylogenomic analyses related to eyes and phototransduction. Here, we present the first limid genome assembly, the disco clam,Ctenoides ales, which is characterized by invaginated eyes, exceptionally long tentacles, and a flashing light display. This genome assembly was constructed with PacBio long reads and Dovetail Omni-CTMproximity-ligation sequencing. The final assembly is ∼2.3Gb and over 99% of the total length is contained in 18 pseudomolecule scaffolds. We annotated 41,064 protein coding genes and report a BUSCO completeness of 91.9% for metazoa_obd10. Additionally, we report a completely annotated mitochondrial genome, also a first for Limida. The ∼20Kb mitogenome has 12 protein coding genes, 22 tRNAs, 2 rRNA genes, and a 1,589 bp duplicated sequence containing the origin of replication. TheC. alesnuclear genome size is substantially larger than other pteriomorphian genomes, mainly accounted for by transposable element sequences. We inventoried the genome for opsins, the signaling proteins that initiate phototransduction, and found that, unlike its closest eyed-relatives, the scallops,C. aleslacks duplication of the rhabdomeric Gq-protein coupled opsin that is typically used for invertebrate vision. In fact,C. aleshas uncharacteristically few opsins relative to the other pteriomorphian families, all of which have unique expansions of xenopsins, a recently discovered opsin subfamily. This chromosome-level assembly, along with the mitogenome, will be valuable resources for comparative genomics and phylogenetics in bivalves and particularly for the understudied but charismatic limids.SignificanceThis high-quality chromosome-level genome assembly forCtenoides ales, the disco clam, is the first genome sequenced from the bivalve order Limida, the only group of Pteriomorphia – a highly diverse and ecologically important clade – lacking genomic resources. The sequence and annotation of theC. alesgenome will be a useful resource for molluscan phylogenetics and comparative genomics.

Animals have evolved numerous mechanisms to perceive and interact with the environment that can be translated into different sensory modalities. However, the genomic and phenotypic features that support sensory functions remain enigmatic for many invertebrates, such as bivalves, an ecologically and economically important taxonomic group. No repertoire of sensory genes has been characterized in bivalves, representing a significant knowledge gap in molluscan sensory biology. Here, we gather multiple lines of evidence to explore the specialized sensory function of bivalve tentacles in the common jingle shell, Anomia simplex. In addition to applying microscopy techniques, we performed transcriptome sequencing of dissected tentacles using phylogenetically-informed annotation to identify candidate receptors. Our results demonstrate the expression of candidate GPCRs, including one opsin type, five small-molecule receptors, and 11 chemosensory-related receptors, supporting the involvement of sensory neurons in the organ, likely in association with the ciliated receptor cells observed along the tentacle surface. In addition, we identified seven ionotropic receptors as putative chemosensory receptors and one member of the Piezo mechanosensitive ion channel, which might be involved in touch sensation by ciliated sensory receptors. Our results provide the first evidence of putative sensory genes expressed in a bivalve sensory organ, representing an important starting point to investigate chemosensation in this class.

We have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes of Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater species Potamopyrgus antipodarum-a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness-and thus provide key evolutionary context for understanding its unusual biology. The P. estuarinus and P. kaitunuparaoa genomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses of P. antipodarum-a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate that P. kaitunuparaoa is likely the closest relative to P. antipodarum. We nevertheless show that there has been considerable introgression between P. estuarinus and P. kaitunuparaoa. That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization between P. estuarinus and P. kaitunuparaoa. Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of nonintrogression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of mitochondrial genome architecture and landscapes of recombination. We used PacBio sequencing to re-assemble mitochondrial genomes from two species of New Zealand freshwater snails, Potamopyrgus antipodarum and Potamopyrgus estuarinus . These assemblies revealed a ∼1.7 kb structure within the mitochondrial genomes of both species that was previously undetected by assembly of short sequencing reads and likely corresponding to a large non-coding region commonly present in mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy region (LSC) and a small single-copy region (SSC) separated by a pair of inverted repeats (IRa and IRb). Individual sequencing reads that spanned across the Potamopyrgus IRa–SSC–IRb structure revealed the occurrence of “flip-flop” recombination, apparently mediated by the IRs. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus , as well as extensive inter-molecular recombination between SNPs in the LSC region. Together, these observations suggest that mitochondrial inheritance is not strictly maternal in these snails. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of such architecture, whether and how recombination mediates mitochondrial genome evolution, and the role of genome architecture in driving cytoplasmic genome biology and the maintenance of cytoplasmic genomes.

AbstractWe have sequenced, assembled, and analyzed the nuclear and mitochondrial genomes and transcriptomes ofPotamopyrgus estuarinusandPotamopyrgus kaitunuparaoa, two prosobranch snail species native to New Zealand that together span the continuum from estuary to freshwater. These two species are the closest known relatives of the freshwater speciesP. antipodarum—a model for studying the evolution of sex, host-parasite coevolution, and biological invasiveness—and thus provide key evolutionary context for understanding its unusual biology. TheP. estuarinusandP. kaitunuparaoagenomes are very similar in size and overall gene content. Comparative analyses of genome content indicate that these two species harbor a near-identical set of genes involved in meiosis and sperm functions, including seven genes with meiosis-specific functions. These results are consistent with obligate sexual reproduction in these two species and provide a framework for future analyses ofP. antipodarum—a species comprising both obligately sexual and obligately asexual lineages, each separately derived from a sexual ancestor. Genome-wide multigene phylogenetic analyses indicate thatP. kaitunuparaoais likely the closest relative toP. antipodarum.We nevertheless show that there has been considerable introgression betweenP. estuarinusandP. kaitunuparaoa.That introgression does not extend to the mitochondrial genome, which appears to serve as a barrier to hybridization betweenP. estuarinusandP. kaitunuparaoa.Nuclear-encoded genes whose products function in joint mitochondrial-nuclear enzyme complexes exhibit similar patterns of non-introgression, indicating that incompatibilities between the mitochondrial and the nuclear genome may have prevented more extensive gene flow between these two species.Significance StatementNo whole-nuclear genome sequences are currently available for snails of the genusPotamopyrgus, best known forPotamopyrgus antipodarum, an invasive species of rivers and lakes worldwide, and a famous model for the study of the evolution of sex. We have sequenced and analyzed the genome of sexualP. estuarinusandP. kaitunuparaoa, the closest known relatives ofP. antipodarum. We show that 1) the genomes are very similar in gene content and size, 2)P. kaitunuparaoais the closest relative toP. antipodarum, 3) significant introgression has occurred betweenP. estuarinusandP. kaitunuparaoa; these genomes set the stage for powerful direct analyses of the genomic features,e.g., sex to asexual transitions and invasive success, that makeP. antipodarumunique.

Why sexual reproduction is so common when asexual reproduction should be much more efficient and less costly remains an open question in evolutionary biology. Comparisons between otherwise similar sexual and asexual taxa allow us to characterize the genetic architecture underlying asexuality, which can, in turn, illuminate how this reproductive mode transition occurred and the mechanisms by which it is maintained or disrupted. Here, we used transcriptome sequencing to compare patterns of ovarian gene expression between actively reproducing obligately sexual and obligately asexual females from multiple lineages of Potamopyrgus antipodarum, a freshwater New Zealand snail characterized by frequent separate transitions to asexuality and coexistence of otherwise similar sexual and asexual lineages. We also used these sequence data to evaluate whether population history accounts for variation in patterns of gene expression. We found that source population was a major source of gene expression variation, and likely more influential than reproductive mode. This outcome for these common garden-raised snails is strikingly similar to earlier results from field-collected snails. While we did not identify a likely set of candidate genes from expression profiles that could plausibly explain how transitions to asexuality occurred, we identified around 1,000 genes with evidence of differential expression between sexual and asexual reproductive modes, and 21 genes that appear to exhibit consistent expression differences between sexuals and asexuals across genetic backgrounds. This second smaller set of genes provides a good starting point for further exploration regarding a potential role in the transition to asexual reproduction. These results mark the first effort to characterize the causes of asexuality in P. antipodarum, demonstrate the apparently high heritability of gene expression patterns in this species, and hint that for P. antipodarum, transitions to asexuality might not necessarily be strongly associated with broad changes in gene expression.

The relationship between genotype and phenotype is nontrivial due to often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype (λmax). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket are responsible for fine spectral tuning, or small changes in the λmax of these light sensitive proteins Most mutations caused a blue shift regardless of the retinochrome background, with shifts ranging from a 12 nm blue shift to a 5 nm red shift from the wild-type λmax. These mutations do not show an additive effect, but rather suggests the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis. Author summary Site-directed mutagenesis determined that spectral tuning in retinochrome is not solely additive, but is influenced by intra-molecular epistasis.

Abstract How does asexual reproduction influence genome evolution? Although is it clear that genomic structural variation is common and important in natural populations, we know very little about how one of the most fundamental of eukaryotic traits—mode of genomic inheritance—influences genome structure. We address this question with the New Zealand freshwater snail Potamopyrgus antipodarum, which features multiple separately derived obligately asexual lineages that coexist and compete with otherwise similar sexual lineages. We used whole-genome sequencing reads from a diverse set of sexual and asexual individuals to analyze genomic abundance of a critically important gene family, rDNA (the genes encoding rRNAs), that is notable for dynamic and variable copy number. Our genomic survey of rDNA in P. antipodarum revealed two striking results. First, the core histone and 5S rRNA genes occur between tandem copies of the 18S–5.8S–28S gene cluster, a unique architecture for these crucial gene families. Second, asexual P. antipodarum harbor dramatically more rDNA–histone copies than sexuals, which we validated through molecular and cytogenetic analysis. The repeated expansion of this genomic region in asexual P. antipodarum lineages following distinct transitions to asexuality represents a dramatic genome structural change associated with asexual reproduction—with potential functional consequences related to the loss of sexual reproduction.