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The free‐living flatworm Macrostomum lignano is used as a model in a range of research fields—including aging, bioadhesion, stem cells, and sexual selection—culminating in the establishment of genome assemblies and transgenics. However, the Macrostomum community has run into a roadblock following the discovery of an unusual genome organization in M. lignano, which could now impair the development of additional resources and tools. Briefly, M. lignano has undergone a whole‐genome duplication, followed by rediploidization into a 2n = 8 karyotype (distinct from the canonical 2n = 6 karyotype in the genus). Although this karyotype appears visually diploid, it is in fact a hidden tetraploid (with rarer 2n = 9 and 2n = 10 individuals being pentaploid and hexaploid, respectively). Here, we report on a phylogenetically informed search for close relatives of M. lignano, aimed at uncovering alternative Macrostomum models with the canonical karyotype and a simple genome organization. We taxonomically describe three new species: the first, Macrostomum janickei n. sp., is the closest known relative of M. lignano and shares its derived genome organization; the second, Macrostomum mirumnovem n. sp., has an even more unusual genome organization, with a highly variable karyotype based on a 2n = 9 base pattern; and the third, Macrostomum cliftonensis n. sp., does not only show the canonical 2n = 6 karyotype, but also performs well under standard laboratory culture conditions and fulfills many other requirements. M. cliftonensis is a viable candidate for replacing M. lignano as the primary Macrostomum model, being outcrossing and having an estimated haploid genome size of only 231 Mbp.

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.

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 malefemale 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.

Sexual selection is expected to drive the evolution of many striking behaviours and morphologies, leaving signatures of selection at loci underlying these phenotypes. However, relatively few studies have contrasted molecular sequence evolution at such loci across lineages that differ in their sexual selection context. Our comparative genomics study of Macrostomum, a large genus of free-living simultaneously hermaphroditic flatworms, takes advantage of functional annotations from the model species, M. lignano, and transcriptome assemblies of 97 congeners. We compare molecular sequence evolution in species with contrasting sperm morphologies, which are strongly associated with multiple convergent shifts in the mating strategy and thus reflect the sexual selection context in Macrostomum. The sperm of most reciprocally mating species carry lateral bristles, likely functioning as anchoring mechanisms against post-copulatory sperm removal. Hypodermically mating species lack these bristles, potentially as adaptations to a different environment experienced by hypodermic sperm. We document faster molecular sequence evolution in reproduction-related, compared to ubiquitously-expressed, genes across all sperm morphologies, consistent with more intense selection acting on the former. Furthermore, we observed faster molecular sequence evolution in species with hypodermic sperm morphologies, in both reproduction-related and ubiquitously-expressed genes. These genome-wide patterns suggest that shifts to hypodermic mating reduce the efficiency of selection, possibly due to higher selfing rates in hypodermically mating species. Moreover, we find little evidence for convergent amino acid changes across species. We provide the first comprehensive comparative analysis of molecular sequence evolution in a group of simultaneously hermaphroditic animals, across well-replicated contrasts of lineages with divergent sperm morphologies.

Sexual selection drives the evolution of many striking behaviours and morphologies, and should leave signatures of selection at loci underlying these phenotypes. However, while 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, M. 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.

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 possible 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.

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.