A tale of two genomes: What drives mitonuclear discordance in asexual lineages of a freshwater snail?

We use genomic information to tell us stories of evolutionary origins. But what does it mean when different genomes report wildly different accounts of lineage history? This genomic "discordance" can be a consequence of a fascinating suite of natural history and evolutionary phenomena, from the different inheritance mechanisms of nuclear versus cytoplasmic (mitochondrial and plastid) genomes to hybridization and introgression to horizontal transfer. Here, we explore how we can use these distinct genomic stories to provide new insights into the maintenance of sexual reproduction, one of the most important unanswered questions in biology. We focus on the strikingly distinct nuclear versus mitochondrial versions of the story surrounding the origin and maintenance of asexual lineages in Potamopyrgus antipodarum, a New Zealand freshwater snail. While key questions remain unresolved, these data inspire multiple testable hypotheses that can be powerfully applied across a broad range of taxa toward a deeper understanding of the causes and consequences of mitonuclear discordance, the maintenance of sex, and the origin of new asexual lineages.

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.

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.

Sticky steps and the gender gap: how thoughtful practices could help keep caregivers in science.

Many fewer women than men hold senior academic positions, a widely recognized and increasing problem. Our goal is to identify effective and feasible solutions. We begin by providing an in-depth assessment of the drivers of this gender inequity. In our synthesis of existing data, we provide many lines of evidence highlighting caregiving as a primary main factor. This is not a 'new' insight per se, but a point worth repeating that we back up by a strong and synthetic body of recent data. We also believe that our analysis provides a step forward in tackling a complex issue. We then develop a more detailed understanding of the challenges academic caregivers face and discuss whether and why it is important to keep caregivers in science. We find that the attrition due to caregiving should not be seen as a factor but rather as a process with multiple 'sticky steps' that eventually drive caregivers out of science-which, as we argue, is partly also good news. Indeed, it is here that we believe actions could be taken that would have a real impact: for example, one could effectively increase and expand upon current funding practices that focus on caregiver career advancement.

Life-history trait variation in native versus invasive asexual New Zealand mud snails.

Potamopyrgus antipodarum is a New Zealand freshwater snail that is invasive worldwide. While native P. antipodarum populations are characterized by frequent coexistence between obligately sexual and obligately asexual individuals, only the asexual snails are known to invade other ecosystems. Despite low genetic diversity and the absence of sex, invasive asexual P. antipodarum are highly successful. Here, we quantified variation in three key life-history traits across invasive P. antipodarum lineages and compared this variation to already documented variation in these same traits in asexual native lineages to provide a deeper understanding of why some lineages become invasive. In particular, we evaluated if invasive lineages of P. antipodarum could be successful because they represent life-history variation from native ancestors that could facilitate invasion. We found that invasive snails displayed a non-representative sample of native diversity, with invasive snails growing more slowly and maturing more rapidly than their native counterparts. These results are consistent with expectations of a scenario where invasive lineages represent a subset of native variation that is beneficial in the setting of invasion. Together, these results help illuminate the mechanisms driving the worldwide expansion of invasive populations of these snails.

Development, implementation and impact of a new preprint solicitation process at Proceedings B.

Preprints are manuscripts posted on a public server that do not yet have formal certification of peer review from a scholarly journal. The increasingly prominent online repositories for these preprints provide a means of rapidly making scientific results accessible to all with an Internet connection. We here describe the catalysis and subsequent development of a successful new process to solicit preprints for consideration for publication in Proceedings B. We present preliminary comparisons between the focal topics and geographic origin of submitting authors of papers submitted in the traditional (non-solicited) route versus solicited preprints. This analysis suggests that the solicitation process seems to be achieving one of the primary goals of the preprint solicitation endeavour: broadening the scope of the papers featured in Proceedings B. We also use an informal survey of the early-career scientists that are or have been involved with the Preprint Editorial Team to find that these scientists view their participation positively with respect to career development and knowledge in their field. The inclusion of early-career researchers from across the world in the preprint solicitation process could also translate into social justice benefits by providing a career-building opportunity and a window into the publishing process for young scientists.

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.

A layover in Europe: Reconstructing the invasion route of asexual lineages of a New Zealand snail to North America.

Non-native invasive species are threatening ecosystems and biodiversity worldwide. High genetic variation is thought to be a critical factor for invasion success. Accordingly, the global invasion of a few clonal lineages of the gastropod Potamopyrgus antipodarum is thus both puzzling and has the potential to help illuminate why some invasions succeed while others fail. Here, we used SNP markers and a geographically broad sampling scheme (N = 1617) including native New Zealand populations and invasive North American and European populations to provide the first widescale population genetic assessment of the relationships between and among native and invasive P. antipodarum. We used a combination of traditional and Bayesian molecular analyses to demonstrate that New Zealand populations harbour very high diversity relative to the invasive populations and are the source of the two main European genetic lineages. One of these two European lineages was in turn the source of at least one of the two main North American genetic clusters of invasive P. antipodarum, located in Lake Ontario. The other widespread North American group had a more complex origin that included the other European lineage and two New Zealand clusters. Altogether, our analyses suggest that just a small handful of clonal lineages of P. antipodarum were responsible for invasion across continents. Our findings provide critical information for prevention of additional invasions and control of existing invasive populations and are of broader relevance towards understanding the establishment and evolution of asexual populations and the forces driving biological invasion.

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.

Science policies: How should science funding be allocated? An evolutionary biologists' perspective.

In an ideal world, funding agencies could identify the best scientists and projects and provide them with the resources to undertake these projects. Most scientists would agree that in practice, how funding for scientific research is allocated is far from ideal and likely compromises research quality. We, nine evolutionary biologists from different countries and career stages, provide a comparative summary of our impressions on funding strategies for evolutionary biology across eleven different funding agencies. We also assess whether and how funding effectiveness might be improved. We focused this assessment on 14 elements within four broad categories: (a) topical shaping of science, (b) distribution of funds, (c) application and review procedures, and (d) incentives for mobility and diversity. These comparisons revealed striking among-country variation in those elements, including wide variation in funding rates, the effort and burden required for grant applications, and the extent of emphasis on societal relevance and individual mobility. We use these observations to provide constructive suggestions for the future and urge the need to further gather informed considerations from scientists on the effects of funding policies on science across countries and research fields.

Genomic evidence for population-specific responses to co-evolving parasites in a New Zealand freshwater snail.

Reciprocal co-evolving interactions between hosts and parasites are a primary source of strong selection that can promote rapid and often population- or genotype-specific evolutionary change. These host-parasite interactions are also a major source of disease. Despite their importance, very little is known about the genomic basis of co-evolving host-parasite interactions in natural populations, especially in animals. Here, we use gene expression and sequence evolution approaches to take critical steps towards characterizing the genomic basis of interactions between the freshwater snail Potamopyrgus antipodarum and its co-evolving sterilizing trematode parasite, Microphallus sp., a textbook example of natural coevolution. We found that Microphallus-infected P. antipodarum exhibit systematic downregulation of genes relative to uninfected P. antipodarum. The specific genes involved in parasite response differ markedly across lakes, consistent with a scenario where population-level co-evolution is leading to population-specific host-parasite interactions and evolutionary trajectories. We also used an FST -based approach to identify a set of loci that represent promising candidates for targets of parasite-mediated selection across lakes as well as within each lake population. These results constitute the first genomic evidence for population-specific responses to co-evolving infection in the P. antipodarum-Microphallus interaction and provide new insights into the genomic basis of co-evolutionary interactions in nature.

Phosphorus availability in the source population influences response to dietary phosphorus quantity in a New Zealand freshwater snail.

We investigated whether previously documented variation among populations in availability of dietary phosphorus (P) is linked to heterogeneity in growth rate of the New Zealand freshwater snail Potamopyrgus antipodarum on a P-limited diet. We chose this system because P. antipodarum inhabits water bodies that vary in P availability and because P. antipodarum growth rate varies considerably in response to low P. We quantified specific growth rate and alkaline phosphatase (AP) expression in a diverse array of juvenile P. antipodarum fed high vs. low-P diets. We found strong associations between P content of epilithon in the source lake and P. antipodarum growth rate on high vs. low-P diets, with snails collected from lakes with relatively low-P epilithon showing the greatest increase in growth rate on the high-P relative to low-P diet. We also found substantial intraspecific variation in growth response to P limitation. Expression of AP also varied among lineages and was negatively associated with C: P of lake epilithon but did not explain the relationship between C: P in the lake of origin and sensitivity to P limitation. Together, our results demonstrate a strong signature of the P environment in the lake of origin on how this snail responds to P limitation as well as preliminary evidence for intraspecific variation of AP expression in animals.

Genetic Variation for Mitochondrial Function in the New Zealand Freshwater Snail Potamopyrgus antipodarum.

The proteins responsible for mitochondrial function are encoded by 2 different genomes with distinct inheritance regimes, rendering rigorous inference of genotype-phenotype connections intractable for all but a few model systems. Asexual organisms provide a powerful means to address these challenges because offspring produced without recombination inherit both nuclear and mitochondrial genomes from a single parent. As such, these offspring inherit mitonuclear genotypes that are identical to the mitonuclear genotypes of their parents and siblings but different from those of other asexual lineages. Here, we compared mitochondrial function across distinct asexual lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail model for understanding the evolutionary consequences of asexuality. Our analyses revealed substantial phenotypic variation across asexual lineages at 3 levels of biological organization: mitogenomic, organellar, and organismal. These data demonstrate that different asexual lineages have different mitochondrial function phenotypes, likely reflecting heritable variation (i.e., the raw material for evolution) for mitochondrial function in P. antipodarum. The discovery of this variation combined with the methods developed here sets the stage to use P. antipodarum to study central evolutionary questions involving mitochondrial function, including whether mitochondrial mutation accumulation influences the maintenance of sexual reproduction in natural populations.

Validation of the OECD reproduction test guideline with the New Zealand mudsnail Potamopyrgus antipodarum using trenbolone and prochloraz.

The Organisation for Economic Cooperation and Development (OECD) provides several standard test methods for the environmental hazard assessment of chemicals, mainly based on primary producers, arthropods, and fish. In April 2016, two new test guidelines with two mollusc species representing different reproductive strategies were approved by OECD member countries. One test guideline describes a 28-day reproduction test with the parthenogenetic New Zealand mudsnail Potamopyrgus antipodarum. The main endpoint of the test is reproduction, reflected by the embryo number in the brood pouch per female. The development of a new OECD test guideline involves several phases including inter-laboratory validation studies to demonstrate the robustness of the proposed test design and the reproducibility of the test results. Therefore, a ring test of the reproduction test with P. antipodarum was conducted including eight laboratories with the test substances trenbolone and prochloraz and results are presented here. Most laboratories could meet test validity criteria, thus demonstrating the robustness of the proposed test protocol. Trenbolone did not have an effect on the reproduction of the snails at the tested concentration range (nominal: 10-1000 ng/L). For prochloraz, laboratories produced similar EC10 and NOEC values, showing the inter-laboratory reproducibility of results. The average EC10 and NOEC values for reproduction (with coefficient of variation) were 26.2 µg/L (61.7%) and 29.7 µg/L (32.9%), respectively. This ring test shows that the mudsnail reproduction test is a well-suited tool for use in the chronic aquatic hazard and risk assessment of chemicals.

Sensitivity to dietary phosphorus limitation in native vs. invasive lineages of a New Zealand freshwater snail.

Why some species and lineages are more likely to be invasive than others is one of the most important unanswered questions in basic and applied biology. In particular, the relative contributions to the invasion process of factors like pre-adaptation to invasiveness in the native range, evolution post-colonization, and random vs. non-random sampling of colonist lineages remain unclear. We use a powerful common garden approach to address the potential for a role for sensitivity to nutrient limitation in determining the invasiveness of particular lineages of Potamopyrgus antipodarum, a New Zealand freshwater snail that has become globally invasive. We quantified specific growth rate (SGR), an important fitness-related trait in this species, under high phosphorus (P) vs. low-P conditions for a diverse set of native and invasive P. antipodarum. This experiment revealed that native-range P. antipodarum experience a more severe decline in SGR in low-P conditions relative to SGR in high-P conditions than their invasive range counterparts. Although these results suggest resilience to P limitation in invasive lineages, the absence of significant absolute differences in SGR between native and invasive lineages indicates that a straightforward connection between response to P limitation and invasiveness in P. antipodarum is unlikely. Regardless, our data demonstrate that invasive vs. native lineages of P. antipodarum exhibit consistently different responses to an important environmental variable that is rarely studied in the context of invasion success. Further studies directed at exploring and disentangling the roles of sampling effects, selection on preexisting variation, and evolution after colonization will be required to provide a comprehensive picture of the role (or lack thereof) of nutrient limitation in the global invasion of P. antipodarum, as well for as other invasive taxa.

Invasive freshwater snails are less sensitive to population density than native conspecifics.

Understanding how and why some species or lineages become invasive is critically important for effectively predicting and mitigating biological invasions. Here, we address an important unanswered question in invasion biology: do key life-history traits of invasive versus native lineages within a species differ in response to key environmental stressors? We focus on the environmental factor of population density, which is a fundamental characteristic of all populations, and investigate how changes in density affect native versus invasive Potamopyrgus antipodarum (New Zealand mudsnail). P. antipodarum has invaded 39 countries and detrimentally affects invaded environments. Previous studies of native and invasive populations and from laboratory experiments have demonstrated that growth and reproduction of P. antipodarum is sensitive to population density, though whether and how this sensitivity varies across native versus invasive lineages remains uncharacterized. We quantified individual growth rate and reproduction in P. antipodarum from multiple distinct native and invasive lineages across three different population density treatments. The growth of native but not invasive lineages decreased as density increased. There was no differential effect of density treatment on embryo production of invasive versus native snails, but a significantly higher proportion of snails were reproductive in high density compared to intermediate density for invasive lineages. In native lineages, there were no significant differences in the relative frequency of reproductive snails across density treatments. While the extent to which these results from our laboratory study can be extrapolated to the more complex natural world remain unclear, our findings are consistent with a scenario where differential sensitivity to population density could help explain why some lineages become successful invaders. Our findings also align with previous studies that show that invasive P. antipodarum lineages exhibit a relatively wide range of tolerance to environmental stressors.

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.

Discordance between nuclear and mitochondrial genomes in sexual and asexual lineages of the freshwater snail Potamopyrgus antipodarum.

The presence and extent of mitonuclear discordance in coexisting sexual and asexual lineages provides insight into 1) how and when asexual lineages emerged, and 2) the spatial and temporal scales at which the ecological and evolutionary processes influencing the evolution of sexual and asexual reproduction occur. Here, we used nuclear single-nucleotide polymorphism (SNP) markers and a mitochondrial gene to characterize phylogeographic structure and the extent of mitonuclear discordance in Potamopyrgus antipodarum. This New Zealand freshwater snail is often used to study the evolution and maintenance of sex because obligately sexual and obligately asexual individuals often coexist. While our data indicate that sexual and asexual P. antipodarum sampled from the same lake population are often genetically similar, suggesting recent origin of these asexuals from sympatric sexual P. antipodarum, we also found significantly more population structure in sexuals vs. asexuals. This latter result suggests that some asexual lineages originated in other lakes and/or in the relatively distant past. When comparing mitochondrial and nuclear population genetic structure, we discovered that one mitochondrial haplotype ('1A') was rare in sexuals, but common and widespread in asexuals. Haplotype 1A frequency and nuclear genetic diversity were not associated, suggesting that the commonness of this haplotype cannot be attributed entirely to genetic drift and pointing instead to a role for selection.

Higher rate of tissue regeneration in polyploid asexual versus diploid sexual freshwater snails.

Characterizing phenotypic differences between sexual and asexual organisms is a critical step towards understanding why sexual reproduction is so common. Because asexuals are often polyploid, understanding how ploidy influences phenotype is directly relevant to the study of sex and will provide key insights into the evolution of ploidy-level variation. The well-established association between genome size and cell cycle duration, evidence for a link between genome size and tissue regeneration rate and the growing body of research showing that ploidy influences growth rate and gene expression led us to hypothesize that healing and tissue regeneration might be affected by ploidy-level variation. We evaluated this hypothesis by measuring the rate of regeneration of antenna tissue of Potamopyrgus antipodarum, a New Zealand snail characterized by frequent coexistence between diploid sexuals and polyploid asexuals. Antennae of triploid and presumptive tetraploid asexuals regenerated more rapidly than the antennae of diploid sexuals, but regeneration rate did not differ between triploids and tetraploids. These results suggest either that ploidy elevation has nonlinear positive effects on tissue regeneration and/or that factors associated directly with reproductive mode affect regeneration rate more than ploidy level. The results of this study also indicate that the lower ploidy of sexual P. antipodarum is unlikely to confer advantages associated with more rapid regeneration.