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Sexual traits may be selected during multiple consecutive episodes of selection, occurring before, during, or after copulation. The overall strength and form of selection acting on traits may thus be determined by how selection (co-)varies along different episodes. However, it is challenging to measure pre- and postcopulatory phenotypic traits alongside variation in fitness components at each different episode. Here, we used a transgenic line of the transparent flatworm Macrostomum lignano expressing green fluorescent protein (GFP) in all cell types, including sperm cells, enabling in vivo sperm tracking. We assessed the mating success, sperm-transfer efficiency, and sperm fertilizing efficiency of GFP(+) focal worms in which we measured 13 morphological traits. We found linear selection on sperm production rate arising from pre- and postcopulatory components and on copulatory organ shape arising from sperm fertilizing efficiency. We further found nonlinear (mostly concave) selection on combinations of copulatory organ and sperm morphology traits arising mostly from sperm-transfer efficiency and sperm fertilizing efficiency. Our study provides a fine-scale quantification of sexual selection, showing that both the form and strength of selection can change across fitness components. Quantifying how sexual selection builds up along episodes of selection allows us to better understand the evolution of sexually selected traits., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE).)

The free-living, simultaneously hermaphroditic flatworms of the genus Macrostomum are increasingly used as model systems in various contexts. In particular, Macrostomum lignano, the only species of this group with a published genome assembly, has emerged as a model for the study of regeneration, reproduction, and stem-cell function. However, challenges have emerged due to M. lignano being a hidden polyploid, having recently undergone whole-genome duplication and chromosome fusion events. This complex genome architecture presents a significant roadblock to the application of many modern genetic tools. Hence, additional genomic resources for this genus are needed. Here, we present such resources for Macrostomum cliftonense and Macrostomum hystrix, which represent the contrasting mating behaviors of reciprocal copulation and hypodermic insemination found in the genus. We use a combination of PacBio long-read sequencing and Illumina shot-gun sequencing, along with several RNA-Seq data sets, to assemble and annotate highly contiguous genomes for both species. The assemblies span ∼227 and ∼220 Mb and are represented by 399 and 42 contigs for M. cliftonense and M. hystrix, respectively. Furthermore, high BUSCO completeness (∼84-85%), low BUSCO duplication rates (8.3-6.2%), and low k-mer multiplicity indicate that these assemblies do not suffer from the same assembly ambiguities of the M. lignano genome assembly, which can be attributed to the complex karyology of this species. We also show that these resources, in combination with the prior resources from M. lignano, offer an excellent foundation for comparative genomic research in this group of organisms., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Anisogamy-the size dimorphism of gametes-is the defining difference between the male and female sexual strategies. Game-theoretic thinking led to the first convincing explanation for the evolutionary origins of anisogamy in the 1970s. Since then, formal game-theoretic models have continued to refine our understanding of when and why anisogamy should evolve. Such models typically presume that the earliest anisogamous organisms had separate sexes. However, in most taxa, there is no empirical evidence to support this assumption. Here, we present a model of the coevolution of gamete size and sex allocation, which allows for anisogamy to emerge alongside either hermaphroditism or separate sexes. We show that hermaphroditic anisogamy can evolve directly from isogamous ancestors when the average size of spawning groups is small and fertilization is relatively efficient. Sex allocation under hermaphroditism becomes increasingly female-biased as group size decreases and the degree of anisogamy increases. When spawning groups are very small, our model also predicts the existence of complex isogamous organisms in which individuals allocate resources equally to two large gamete types. We discuss common, but potentially unwarranted, assumptions in the literature that could be relaxed in future models. This article is part of the theme issue 'Half a century of evolutionary games: a synthesis of theory, application and future directions'.

Gene repertoire turnover is a characteristic of genome evolution. However, we lack well-replicated analyses of presence/absence patterns associated with different selection contexts. Here, we study ∼100 transcriptome assemblies across Macrostomum, a genus of simultaneously hermaphroditic flatworms exhibiting multiple convergent shifts in mating strategy and associated reproductive morphologies. Many species mate reciprocally, with partners donating and receiving sperm at the same time. Other species convergently evolved to mate by hypodermic injection of sperm into the partner. We find that for orthologous transcripts annotated as expressed in the body region containing the testes, sequences from hypodermically inseminating species diverge more rapidly from the model species, Macrostomum lignano, and have a lower probability of being observed in other species. For other annotation categories, simpler models with a constant rate of similarity decay with increasing genetic distance from M. lignano match the observed patterns well. Thus, faster rates of sequence evolution for hypodermically inseminating species in testis-region genes result in higher rates of homology detection failure, yielding a signal of rapid evolution in sequence presence/absence patterns. Our results highlight the utility of considering appropriate null models for unobserved genes, as well as associating patterns of gene presence/absence with replicated evolutionary events in a phylogenetic context., (© 2022 The Authors. Evolution published by Wiley Periodicals LLC on behalf of The Society for the Study of Evolution.)

Sex allocation theory in simultaneous hermaphrodites predicts that optimal sex allocation is influenced by local sperm competition, which occurs when related sperm compete to fertilize a given set of eggs. Different factors, including the mating strategy and the ability to self-fertilize, are predicted to affect local sperm competition and hence the optimal SA. Moreover, since the local sperm competition experienced by an individual can vary temporally and spatially, this can favour the evolution of sex allocation plasticity. Here, using seven species of the free-living flatworm genus Macrostomum, we document interspecific variation in sex allocation, but neither their mating strategy nor their ability to self-fertilize significantly predicted sex allocation among these species. Since we also found interspecific variation in sex allocation plasticity, we further estimated standardized effect sizes for plasticity in response to (i) the presence of mating partners (i.e. in isolation vs. with partners) and (ii) the strength of local sperm competition (i.e. in small vs. large groups). We found that self-fertilization predicted sex allocation plasticity with respect to the presence of mating partners, with plasticity being lower for self-fertilizing species. Finally, we showed that interspecific variation in sex allocation is higher than intraspecific variation due to sex allocation plasticity. Our study suggests that both sex allocation and sex allocation plasticity are evolutionarily labile, with self-fertilization predicting the latter in Macrostomum., (© 2022 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Mitochondria - the cell's power stations - are inherited uniparentally via eggs, not sperm. In hermaphroditic plants, they sometimes prevent their hosts from making pollen (and sperm), causing cytoplasmic male sterility. New evidence from a hermaphroditic freshwater snail now documents cytoplasmic male sterility in animals., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Sex allocation is the distribution of resources to male or female reproduction. In hermaphrodites, this concerns an individual's resource allocation to, for example, the production of male or female gametes. Macroevolutionary studies across hermaphroditic plants have revealed that the self-pollination rate and the pollination mode are strong predictors of sex allocation. Consequently, we expect similar factors such as the selfing rate and aspects of the reproductive biology, like the mating behaviour and the intensity of postcopulatory sexual selection, to predict sex allocation in hermaphroditic animals. However, comparative work on hermaphroditic animals is limited. Here, we study sex allocation in 120 species of the hermaphroditic free-living flatworm genus Macrostomum. We ask how hypodermic insemination, a convergently evolved mating behaviour where sperm are traumatically injected through the partner's epidermis, affects the evolution of sex allocation. We also test the commonly-made assumption that investment into male and female reproduction should trade-off. Finally, we ask if morphological indicators of the intensity of postcopulatory sexual selection (female genital complexity, male copulatory organ length, and sperm length) can predict sex allocation., Results: We find that the repeated evolution of hypodermic insemination predicts a more female-biased sex allocation (i.e., a relative shift towards female allocation). Moreover, transcriptome-based estimates of heterozygosity reveal reduced heterozygosity in hypodermically mating species, indicating that this mating behavior is linked to increased selfing or biparental inbreeding. Therefore, hypodermic insemination could represent a selfing syndrome. Furthermore, across the genus, allocation to male and female gametes is negatively related, and larger species have a more female-biased sex allocation. Finally, increased female genital complexity, longer sperm, and a longer male copulatory organ predict a more male-biased sex allocation., Conclusions: Selfing syndromes have repeatedly originated in plants. Remarkably, this macroevolutionary pattern is replicated in Macrostomum flatworms and linked to repeated shifts in reproductive behavior. We also find a trade-off between male and female reproduction, a fundamental assumption of most theories of sex allocation. Beyond that, no theory predicts a more female-biased allocation in larger species, suggesting avenues for future work. Finally, morphological indicators of more intense postcopulatory sexual selection appear to predict more intense sperm competition., (© 2022. The Author(s).)

Free-living flatworms of the genus Macrostomum are small and transparent animals, representing attractive study organisms for a broad range of topics in evolutionary, developmental, and molecular biology. The genus includes the model organism M. lignano for which extensive molecular resources are available, and recently there is a growing interest in extending work to additional species in the genus. These endeavours are currently hindered because, even though >200 Macrostomum species have been taxonomically described, molecular phylogenetic information and geographic sampling remain limited. We report on a global sampling campaign aimed at increasing taxon sampling and geographic representation of the genus. Specifically, we use extensive transcriptome and single-locus data to generate phylogenomic hypotheses including 145 species. Across different phylogenetic methods and alignments used, we identify several consistent clades, while their exact grouping is less clear, possibly due to a radiation early in Macrostomum evolution. Moreover, we uncover a large undescribed diversity, with 94 of the studied species likely being new to science, and we identify multiple novel morphological traits. Furthermore, we identify cryptic speciation in a taxonomically challenging assemblage of species, suggesting that the use of molecular markers is a prerequisite for future work, and we describe the distribution of putative synapomorphies and suggest taxonomic revisions based on our finding. Our large-scale phylogenomic dataset now provides a robust foundation for comparative analyses of morphological, behavioural and molecular evolution in this genus., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Traumatic insemination is a mating behavior during which the (sperm) donor uses a traumatic intromittent organ to inject an ejaculate through the epidermis of the (sperm) recipient, thereby frequently circumventing the female genitalia. Traumatic insemination occurs widely across animals, but the frequency of its evolution, the intermediate stages via which it originates, and the morphological changes that such shifts involve remain poorly understood. Based on observations in 145 species of the free-living flatworm genus Macrostomum , we identify at least nine independent evolutionary origins of traumatic insemination from reciprocal copulation, but no clear indication of reversals. These origins involve convergent shifts in multivariate morphospace of male and female reproductive traits, suggesting that traumatic insemination has a canalizing effect on morphology. We also observed sperm in both the sperm receiving organ and within the body tissue of two species. These species had intermediate trait values indicating that traumatic insemination evolves through initial internal wounding during copulation. Finally, signatures of male-female coevolution of genitalia across the genus indicate that sexual selection and sexual conflict drive the evolution of traumatic insemination, because it allows donors to bypass postcopulatory control mechanisms of recipients., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Evolution Letters published by Wiley Periodicals LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).)

Sexual selection drives the evolution of many striking behaviors and morphologies and should leave signatures of selection at loci underlying these phenotypes. However, although loci thought to be under sexual selection often evolve rapidly, few studies have contrasted rates of molecular sequence evolution at such loci across lineages with different sexual selection contexts. Furthermore, work has focused on separate sexed animals, neglecting alternative sexual systems. We investigate rates of molecular sequence evolution in hermaphroditic flatworms of the genus Macrostomum. Specifically, we compare species that exhibit contrasting sperm morphologies, strongly associated with multiple convergent shifts in the mating strategy, reflecting different sexual selection contexts. Species donating and receiving sperm in every mating have sperm with bristles, likely to prevent sperm removal. Meanwhile, species that hypodermically inject sperm lack bristles, potentially as an adaptation to the environment experienced by hypodermic sperm. Combining functional annotations from the model, Macrostomum lignano, with transcriptomes from 93 congeners, we find genus-wide faster sequence evolution in reproduction-related versus ubiquitously expressed genes, consistent with stronger sexual selection on the former. Additionally, species with hypodermic sperm morphologies had elevated molecular sequence evolution, regardless of a gene's functional annotation. These genome-wide patterns suggest reduced selection efficiency following shifts to hypodermic mating, possibly due to higher selfing rates in these species. Moreover, we find little evidence for convergent amino acid changes across species. Our work not only shows that reproduction-related genes evolve rapidly also in hermaphroditic animals, but also that well-replicated contrasts of different sexual selection contexts can reveal underappreciated genome-wide effects., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Bradynectes ensifer is a new species of Macrostomorpha from shallow subtidal sediment in Bogue Inlet, NC, USA. It differs primarily from all other named members of the genus in the radically different shape of its copulatory stylet. We also consider historical records of currently unnamed specimens with similar stylet morphology collected from the NC coast by the late Dr. Reinhard M. Rieger and his students.

In species with multiple mating, intense sexual selection may occur both before and after copulation. However, comparing the strength of pre- and postcopulatory selection is challenging, because (i) postcopulatory processes are generally difficult to observe and (ii) the often-used opportunity for selection ( I ) metric contains both deterministic and stochastic components. Here, we quantified pre- and postcopulatory male fitness components of the simultaneously hermaphroditic flatworm, Macrostomum lignano . We did this by tracking fluorescent sperm-using transgenics-through the transparent body of sperm recipients, enabling to observe postcopulatory processes in vivo . Moreover, we sequentially exposed focal worms to three independent mating groups, and in each assessed their mating success, sperm-transfer efficiency, sperm fertilizing efficiency, and partner fecundity. Based on these multiple measures, we could, for each fitness component, combine the variance ( I ) with the repeatability ( R ) in individual success to assess the amount of repeatable variance in individual success-a measure we call the repeatable opportunity for selection ( I R ). We found higher repeatable opportunity for selection in sperm-transfer efficiency and sperm fertilizing efficiency compared to mating success, which clearly suggests that postcopulatory selection is stronger than precopulatory selection. Our study demonstrates that the opportunity for selection contains a repeatable deterministic component, which can be assessed and disentangled from the often large stochastic component, to provide a better estimate of the strength of selection., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Evolution Letters published by Wiley Periodicals, LLC on behalf of Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEB).)

Matrical differentiation is the distinctive feature of pilomatricoma and other purely matrical adnexal neoplasms; additionally, foci of matrical differentiation have been also described in hybrid cysts of Gardner syndrome, as well as in a wide variety of benign and malignant cutaneous tumors, including basal cell carcinoma. We report an exceptional case of Bowen disease exhibiting multiple foci of matrical differentiation, as confirmed by means of immunohistochemical studies. Several types of divergent, non-squamous differentiation have been exceptionally reported in cutaneous squamous cell carcinoma in situ (cSCCIS), including sebaceous, mucinous/glandular, poroid, tricholemmal, and neuroendocrine differentiation; matrical differentiation may be added to this list. Our findings further emphasize the undifferentiated nature of neoplastic cells in cSCCIS., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Reproductive traits are some of the fastest diverging characters and can serve as reproductive barriers. The free-living flatworm Macrostomum lignano, and its congener M. janickei are closely related, but differ substantially in their male intromittent organ (stylet) morphology. Here, we examine whether these morphological differences are accompanied by differences in behavioural traits, and whether these could represent barriers to successful mating and hybridization between the two species. Our data shows that the two species differ in many aspects of their mating behaviour. Despite these differences, the species mate readily with each other in heterospecific pairings. Although both species have similar fecundity in conspecific pairings, the heterospecific pairings revealed clear postmating barriers, as few heterospecific pairings produced F1 hybrids. These hybrids had a stylet morphology that was intermediate between that of the parental species, and they were fertile. Finally, using a mate choice experiment, we show that the nearly two-fold higher mating rate of M. lignano caused it to mate more with conspecifics, leading to assortative mating, while M. janickei ended up mating more with heterospecifics. Thus, while the two species can hybridize, the mating rate differences could possibly lead to higher fitness costs for M. janickei compared to M. lignano.

Background: The genus Macrostomum consists of small free-living flatworms and contains Macrostomum lignano, which has been used in investigations of ageing, stem cell biology, bioadhesion, karyology, and sexual selection in hermaphrodites. Two types of mating behaviour occur within this genus. Some species, including M. lignano, mate via reciprocal copulation, where, in a single mating, both partners insert their male copulatory organ into the female storage organ and simultaneously donate and receive sperm. Other species mate via hypodermic insemination, where worms use a needle-like copulatory organ to inject sperm into the tissue of the partner. These contrasting mating behaviours are associated with striking differences in sperm and copulatory organ morphology. Here we expand the genomic resources within the genus to representatives of both behaviour types and investigate whether genes vary in their rate of evolution depending on their putative function., Results: We present de novo assembled transcriptomes of three Macrostomum species, namely M. hystrix, a close relative of M. lignano that mates via hypodermic insemination, M. spirale, a more distantly related species that mates via reciprocal copulation, and finally M. pusillum, which represents a clade that is only distantly related to the other three species and also mates via hypodermic insemination. We infer 23,764 sets of homologous genes and annotate them using experimental evidence from M. lignano. Across the genus, we identify 521 gene families with conserved patterns of differential expression between juvenile vs. adult worms and 185 gene families with a putative expression in the testes that are restricted to the two reciprocally mating species. Further, we show that homologs of putative reproduction-related genes have a higher protein divergence across the four species than genes lacking such annotations and that they are more difficult to identify across the four species, indicating that these genes evolve more rapidly, while genes involved in neoblast function are more conserved., Conclusions: This study improves the genus Macrostomum as a model system, by providing resources for the targeted investigation of gene function in a broad range of species. And we, for the first time, show that reproduction-related genes evolve at an accelerated rate in flatworms.

Macrostomum lignano is a free-living flatworm that is emerging as an attractive experimental animal for research on a broad range of biological questions. One feature setting it apart from other flatworms is the successful establishment of transgenesis methods, facilitated by a steady supply of eggs in the form of single-cell zygotes that can be readily manipulated. This, in combination with the transparency of the animal and its small size, creates practical advantages for imaging and fluorescence-activated cell sorting in studies related to stem cell biology and regeneration. M. lignano can regenerate most of its body parts, including the germline, thanks to the neoblasts, which represent the flatworm stem cell system. Interestingly, neoblasts seem to have a high capacity of cellular maintenance, as M. lignano can survive up to 210 Gy of γ-irradiation, and partially offset the negative consequence of ageing. As a non-self-fertilizing simultaneous hermaphrodite that reproduces in a sexual manner, M. lignano is also used to study sexual selection and other evolutionary aspects of sexual reproduction. Work over the past several years has led to the development of molecular resources and tools, including high-quality genome and transcriptome assemblies, transcriptional profiling of the germline and somatic neoblasts, gene knockdown, and in situ hybridization. The increasingly detailed characterization of this animal has also resulted in novel research questions, such as bio-adhesion based on its adhesion-release glands and genome evolution due to its recent whole-genome duplication., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2 n = 8) and its sibling species M. janickei (2 n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis , and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2 n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2 n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2 n = 9 karyotype discovered in M. mirumnovem , we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei , our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD.

Abstract: There is an ongoing debate about whether simultaneous hermaphrodites capable of selfing should prefer selfing over outcrossing or vice versa. While many theoretical models predict a transmission advantage for alleles that favour selfing, empirical studies often reveal low selfing rates. Despite these considerations, the underlying mechanisms that determine reproductive strategies in simultaneously hermaphroditic animals are poorly understood. In our study on the facultatively selfing free-living flatworm, Macrostomum hystrix , we ask whether the initiation of selfing, as inferred from the differential spatial distribution of received sperm, is linked to an individual's female or male reproductive function. Specifically, the initiation of selfing could (i) be linked to the male function, when an individual is unable to donate sperm to others and hence donates sperm to self, or it could (ii) be linked to the female function, when an individual fails to receive sperm from others-and hence is unable to fertilize its eggs via outcrossing-thus inducing it to self-fertilize. We experimentally created a social environment that allowed focals to outcross via sperm donation, but simultaneously prevented them from receiving sperm-by pairing them with a partner lacking the male copulatory organ-so that fertilization of the focal's eggs was restricted to selfing. Our results suggest that such focals generally do not initiate selfing, while we readily observe selfing in isolated worms. This suggests that in isolated M. hystrix , it is the male function that is linked to the initiation of selfing, likely due to a lack of opportunities for sperm donation., Significance Statement: A variety of simultaneously hermaphroditic animals are capable of reproducing via both selfing and outcrossing. While the reproductive choices of such animals can be modelled by the joint action of genetic (e.g. inbreeding depression) and ecological factors (e.g. partner availability), experimental evaluation of theoretical results is often lacking. By manipulating the social environment of focal individuals, we here provide evidence that explores the respective role that the co-occurring male and female sex functions have on the initiation of selfing in a simultaneously hermaphroditic flatworm species. Specifically, our results suggest that the initiation of selfing is linked to the worm's male function. Insights about which function is linked to the initiation of selfing may ultimately help to better understand reproductive decisions in simultaneous hermaphrodites., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

Background: Impairments in psychosocial functioning have been demonstrated in 30-60% of adults with bipolar disorder (BD). However, the majority of studies investigating the effect of comorbid mental health disorders and age at onset outcomes in BD have focused on traditional outcome measures such as mood symptoms, mortality and treatment response. Therefore, this project aimed to investigate the impact of comorbid mental health disorders and age at onset on longitudinal psychosocial outcome in participants with BD., Method: Mixed effects modelling was conducted using data from the Stanley Foundation Bipolar Network. Baseline factors were entered into a model, with Global Assessment of Functioning (GAF) score as the longitudinal outcome measure. Relative model fits were calculated using Akaike's Information Criterion., Results: No individual comorbidities predicted lower GAF scores, however an interaction effect was demonstrated between attention deficit hyperactivity disorder (ADHD) and any anxiety disorder (t = 2.180, p = 0.030). Participants with BD I vs BD II (t = 2.023, p = 0.044) and those in the lowest vs. highest income class (t = 2.266, p = 0.024) predicted lower GAF scores. Age at onset (t = 1.672, p = 0.095) did not significantly predict GAF scores., Conclusions: This is the first study to demonstrate the negative psychosocial effects of comorbid anxiety disorders and ADHD in BD. This study adds to the growing database suggesting that comorbid mental health disorders are a significant factor hindering psychosocial recovery.

Sex allocation (SA) theory for simultaneous hermaphrodites predicts an influence of group size on SA. Since group size can vary within an individual's lifetime, this can favor the evolution of phenotypically plastic SA. In an emerging comparative context, we here report on SA plasticity in three closely related Macrostomum flatworm species, namely Macrostomum janickei , Macrostomum cliftonensis , and Macrostomum mirumnovem . For each species, we experimentally raised worms in three group sizes (isolated, pairs, and octets) and two enclosure sizes (small and large) in all factorial combinations and studied the effects of these factors on different estimates of SA. In addition, we also evaluated whether isolated worms engage in self-fertilization. We found that all species have plastic SA, with M. cliftonensis being more plastic than the other two species, as assessed by comparing standardized effect sizes of (a) the presence/absence of mating partners and (b) the strength of sexual competition. Moreover, we found that sperm production rate-but not sperm morphology-is plastic in M. cliftonensis , and that only M. mirumnovem self-fertilized during our observation period. Our study suggests that both SA and SA plasticity can diverge even between closely related species., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Phenotypic plasticity can enable organisms to produce optimal phenotypes in multiple environments. A crucial life history trait that is often highly plastic is sex allocation, which in simultaneous hermaphrodites describes the relative investment into the male versus female sex functions. Theory predicts-and morphological evidence supports-that greater investment into the male function is favoured with increasing group size, due to the increasing importance of sperm competition for male reproductive success. Here, we performed a genome-wide gene expression assay to test for such sex allocation plasticity in a model simultaneous hermaphrodite, the free-living flatworm Macrostomum lignano. Based on RNA-Seq data from 16 biological replicates spanning four different group size treatments, we demonstrate that at least 10% of the >75,000 investigated transcripts in M. lignano are differentially expressed according to the social environment, rising to >30% of putative gonad-specific transcripts (spermatogenesis and oogenesis candidates) and tail-specific transcripts (seminal fluid candidates). This transcriptional response closely corresponds to the expected shift away from female and towards male reproductive investment with increasing sperm competition level. Using whole-mount in situ hybridization, we then confirm that many plastic transcripts exhibit the expected organ-specific expression, and RNA interference of selected testis- and ovary-specific candidates establishes that these indeed function in gametogenesis pathways. We conclude that a large proportion of sex-specific transcripts in M. lignano are differentially expressed according to the prevailing ecological conditions and that these are functionally relevant to key reproductive phenotypes. Our study thus begins to bridge organismal and molecular perspectives on sex allocation plasticity., (© 2019 John Wiley & Sons Ltd.)

Background: Along with sperm, in many taxa ejaculates also contain large numbers of seminal fluid proteins (SFPs). SFPs and sperm are transferred to the mating partner, where they are thought to play key roles in mediating post-mating sexual selection. They modulate the partner's behavior and physiology in ways that influence the reproductive success of both partners, thus potentially leading to sexual conflict. Despite the presumed general functional and evolutionary significance of SFPs, their identification and characterization has to date focused on just a few animal groups, predominantly insects and mammals. Moreover, until now seminal fluid profiling has mainly focused on species with separate sexes. Here we report a comprehensive screen for putative SFPs in the simultaneously hermaphroditic flatworm Macrostomum lignano., Results: Based on existing transcriptomic data, we selected 150 transcripts known to be (a) predominantly expressed in the tail region of the worms, where the seminal fluid-producing prostate gland cells are located, and (b) differentially expressed in social environments differing in sperm competition level, strongly implying that they represent a phenotypically plastic aspect of male reproductive allocation in this species. For these SFP candidates, we then performed whole-mount in situ hybridization (ISH) experiments to characterize tissue-specific expression. In total, we identified 98 transcripts that exhibited prostate-specific expression, 76 of which we found to be expressed exclusively in the prostate gland cells; additional sites of expression for the remaining 22 included the testis or other gland cells. Bioinformatics analyses of the prostate-limited candidates revealed that at least 64 are predicted to be secretory proteins, making these especially strong candidates to be SFPs that are transferred during copulation., Conclusions: Our study represents a first comprehensive analysis using a combination of transcriptomic and ISH screen data to identify SFPs based on transcript expression in seminal fluid-producing tissues. We thereby extend the range of taxa for which seminal fluid has been characterized to a flatworm species with a sequenced genome and for which several methods such as antibody staining, transgenesis and RNA interference have been established. Our data provide a basis for testing the functional and evolutionary significance of SFPs.

Hermaphroditic animals face the fundamental evolutionary optimization problem of allocating their resources to their male vs. female reproductive function (e.g. testes and sperm vs. ovaries and eggs), and this optimal sex allocation can be affected by both pre- and post-copulatory sexual selection. For example, local sperm competition (LSC) - the competition between related sperm for the fertilization of a partner's ova - occurs in small mating groups and can favour a female-biased sex allocation, because, under LSC, investment into sperm production is predicted to show diminishing fitness returns. Here, we test whether higher testis investment increases an individual's paternity success under sperm competition, and whether the strength of this effect diminishes when LSC is stronger, as predicted by sex allocation theory. We created two subsets of individuals of the simultaneously hermaphroditic flatworm Macrostomum lignano - by sampling worms from either the highest or lowest quartile of the testis investment distribution - and estimated their paternity success in group sizes of either three (strong LSC) or eight individuals (weak LSC). Specifically, using transgenic focal individuals expressing a dominant green-fluorescent protein marker, we showed that worms with high testis investment sired 22% more offspring relative to those with low investment, corroborating previous findings in M. lignano and other species. However, the strength of this effect was not significantly modulated by the experienced group size, contrasting theoretical expectations of more strongly diminishing fitness returns under strong LSC. We discuss the possible implications for the evolutionary maintenance of hermaphroditism in M. lignano., (© 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.)

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.

The free-living flatworm Macrostomum lignano is a model organism for evolutionary and developmental biology studies. Recently, an unusual karyotypic diversity was revealed in this species. Specifically, worms are either 'normal' 2n = 8, or they are aneuploid with one or two additional large chromosome(s) (i.e. 2n = 9 or 2n = 10, respectively). Aneuploid worms did not show visible behavioral or morphological abnormalities and were successful in reproduction. In this study, we generated microdissected DNA probes from chromosome 1 (further called MLI1), chromosome 2 (MLI2), and a pair of similar-sized smaller chromosomes (MLI3, MLI4). FISH using these probes revealed that MLI1 consists of contiguous regions homologous to MLI2-MLI4, suggesting that MLI1 arose due to the whole genome duplication and subsequent fusion of one full chromosome set into one large metacentric chromosome. Therefore, one presumably full haploid genome was packed into MLI1, leading to hidden tetraploidy in the M. lignano genome. The study of Macrostomum sp. 8 - a sibling species of M. lignano - revealed that it usually has one additional pair of large chromosomes (2n = 10) showing a high homology to MLI1, thus suggesting hidden hexaploidy in its genome. Possible evolutionary scenarios for the emergence of the M. lignano and Macrostomum sp. 8 genomes are discussed.

The expression of an individual's phenotypic traits can be influenced by genes expressed in its social partners. Theoretical models predict that such indirect genetic effects (IGEs) on reproductive traits should play an important role in determining the evolutionary outcome of sexual conflict. However, empirical tests of (i) whether reproductive IGEs exist, (ii) how they vary among genotypes, and (iii) whether they are uniform for different types of reproductive traits are largely lacking. We addressed this in a series of experiments in the simultaneously hermaphroditic flatworm Macrostomum lignano. We found strong evidence for IGEs on both morphological and behavioral reproductive traits. Partner genotype had a significant impact on the testis size of focal individuals-varying up to 2.4-fold-suggesting that IGEs could mediate sexual conflicts that target the male sex function. We also found that time to first copulation was affected by a genotype × genotype interaction between mating partners, and that partner genotype affected the propensity to copulate and perform the postcopulatory suck behavior, which may mediate conflicts over the fate of received ejaculate components. These findings provide clear empirical evidence for IGEs on multiple behavioral and morphological reproductive traits, which suggests that the evolutionary dynamics of these traits could be altered by genes contained in the social environment., (© 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.)