Genome Evolution and Introgression in the New Zealand mud Snails Potamopyrgus estuarinus and Potamopyrgus kaitunuparaoa.

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.

Single-molecule Sequencing of an Animal Mitochondrial Genome Reveals Chloroplast-like Architecture and Repeat-mediated Recombination.

Recent advances in long-read sequencing technology have allowed for single-molecule sequencing of entire mitochondrial genomes, opening the door for direct investigation of the mitochondrial genome architecture and recombination. We used PacBio sequencing to reassemble 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 an assembly of short reads and likely corresponding to a large noncoding region commonly present in the mitochondrial genomes. The overall architecture of these Potamopyrgus mitochondrial genomes is reminiscent of the chloroplast genomes of land plants, harboring a large single-copy (LSC) region and a small single-copy (SSC) region 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 a "flip-flop" recombination. We also detected evidence for two distinct IR haplotypes and recombination between them in wild-caught P. estuarinus, as well as extensive intermolecular recombination between single-nucleotide polymorphisms in the LSC region. The chloroplast-like architecture and repeat-mediated mitochondrial recombination we describe here raise fundamental questions regarding the origins and commonness of inverted repeats in cytoplasmic genomes and their role in mitochondrial genome evolution.

Evidence for stronger discrimination between conspecific and heterospecific mating partners in sexual vs. asexual female freshwater snails.

Once-useful traits that no longer contribute to fitness tend to decay over time. Here, we address whether the expression of mating-related traits that increase the fitness of sexually reproducing individuals but are likely less useful or even costly to asexual counterparts seems to exhibit decay in the latter. Potamopyrgus antipodarum is a New Zealand freshwater snail characterized by repeated transitions from sexual to asexual reproduction. The frequent coexistence of sexual and asexual lineages makes P. antipodarum an excellent model for the study of mating-related trait loss. Under the presumption (inherent in the Biological Species Concept) that failure to discriminate between conspecific and heterospecific mating partners represents a poor mate choice, we used a mating choice assay including sexual and asexual P. antipodarum females and conspecific (presumed better choice) vs. heterospecific (presumed worse choice) males to evaluate the loss of behavioral traits related to sexual reproduction. We found that sexual females engaged in mating behaviors with conspecific mating partners more frequently and for a greater duration than with heterospecific mating partners. By contrast, asexual females mated at similar frequency and duration as sexual females, but did not mate more often or for longer duration with conspecific vs. heterospecific males. While further confirmation will require inclusion of a more diverse array of sexual and asexual lineages, these results are consistent with a scenario where selection acting to maintain effective mate discrimination in asexual P. antipodarum is weak or ineffective relative to sexual females and, thus, where asexual reproduction is associated with the evolutionary decay of mating-related traits in this system.

Asexuality Associated with Marked Genomic Expansion of Tandemly Repeated rRNA and Histone Genes.

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.

Intraspecific genetic variation for anesthesia success in a New Zealand freshwater snail.

Intraspecific genetic variation can drive phenotypic variation even across very closely related individuals. Here, we demonstrate that genetic differences between snails are a major contributor to wide variation in menthol anesthesia success in an important freshwater snail model system, Potamopyrgus antipodarum. Anesthesia is used to immobilize organisms for experiments and surgical procedures and to humanely mitigate pain. This is the first example of which we are aware of a role for genetic variation in anesthesia success in a mollusk. These findings highlight the fact that using only one strain or lineage for many experiments will not provide a full picture of phenotypic variation, demonstrate the importance of optimizing biomedically relevant techniques and protocols across a variety of genetic backgrounds, illuminate a potential mechanism underlying previously documented challenges in molluscan anesthesia, and set the stage for powerful and humane manipulative experiments in P. antipodarum.

Male phenotypes in a female framework: Evidence for degeneration in sperm produced by male snails from asexual lineages.

How changes in selective regimes affect trait evolution is an important open biological question. We take advantage of naturally occurring and repeated transitions from sexual to asexual reproduction in a New Zealand freshwater snail species, Potamopyrgus antipodarum, to address how evolution in an asexual context-including the potential for relaxed selection on male-specific traits-influences sperm morphology. The occasional production of male offspring by the otherwise all-female asexual P. antipodarum lineages affords a unique and powerful opportunity to assess the fate of sperm traits in a context where males are exceedingly rare. These comparisons revealed that the sperm produced by 'asexual' males are markedly distinct from sexual counterparts. We also found that the asexual male sperm harboured markedly higher phenotypic variation and was much more likely to be morphologically abnormal. Together, these data suggest that transitions to asexual reproduction might be irreversible, at least in part because male function is likely to be compromised. These results are also consistent with a scenario where relaxed selection and/or mutation accumulation in the absence of sex translates into rapid trait degeneration.