Convergent evolution of the sensory pits in and within flatworms.

BACKGROUND: Unlike most free-living platyhelminths, catenulids, the sister group to all remaining flatworms, do not have eyes. Instead, the most prominent sensory structures in their heads are statocysts or sensory pits. The latter, found in the family Stenostomidae, are concave depressions located laterally on the head that represent one of the taxonomically important traits of the family. In the past, the sensory pits of flatworms have been homologized with the cephalic organs of nemerteans, a clade that occupies a sister position to platyhelminths in some recent phylogenies. To test for this homology, we studied morphology and gene expression in the sensory pits of the catenulid Stenostomum brevipharyngium. RESULTS: We used confocal and electron microscopy to investigate the detailed morphology of the sensory pits, as well as their formation during regeneration and asexual reproduction. The most prevalent cell type within the organ is epidermally-derived neuron-like cells that have cell bodies embedded deeply in the brain lobes and long neurite-like processes extending to the bottom of the pit. Those elongated processes are adorned with extensive microvillar projections that fill up the cavity of the pit, but cilia are not associated with the sensory pit. We also studied the expression patterns of some of the transcription factors expressed in the nemertean cephalic organs during the development of the pits. Only a single gene, pax4/6, is expressed in both the cerebral organs of nemerteans and sensory pits of S. brevipharyngium, challenging the idea of their deep homology. CONCLUSIONS: Since there is no morphological or molecular correspondence between the sensory pits of Stenostomum and the cerebral organs of nemerteans, we reject their homology. Interestingly, the major cell type contributing to the sensory pits of stenostomids shows ultrastructural similarities to the rhabdomeric photoreceptors of other flatworms and expresses ortholog of the gene pax4/6, the pan-bilaterian master regulator of eye development. We suggest that the sensory pits of stenostomids might have evolved from the ancestral rhabdomeric photoreceptors that lost their photosensitivity and evolved secondary function. The mapping of head sensory structures on plathelminth phylogeny indicates that sensory pit-like organs evolved many times independently in flatworms.

Evolutionary dynamics of whole-body regeneration across planarian flatworms.

Regenerative abilities vary dramatically across animals. Even amongst planarian flatworms, well-known for complete regeneration from tiny body fragments, some species have restricted regeneration abilities while others are almost entirely regeneration incompetent. Here, we assemble a diverse live collection of 40 planarian species to probe the evolution of head regeneration in the group. Combining quantification of species-specific head-regeneration abilities with a comprehensive transcriptome-based phylogeny reconstruction, we show multiple independent transitions between robust whole-body regeneration and restricted regeneration in freshwater species. RNA-mediated genetic interference inhibition of canonical Wnt signalling in RNA-mediated genetic interference-sensitive species bypassed all head-regeneration defects, suggesting that the Wnt pathway is linked to the emergence of planarian regeneration defects. Our finding that Wnt signalling has multiple roles in the reproductive system of the model species Schmidtea mediterranea raises the possibility that a trade-off between egg-laying, asexual reproduction by fission/regeneration and Wnt signalling drives regenerative trait evolution. Although quantitative comparisons of Wnt signalling levels, yolk content and reproductive strategy across our species collection remained inconclusive, they revealed divergent Wnt signalling roles in the reproductive system of planarians. Altogether, our study establishes planarians as a model taxon for comparative regeneration research and presents a framework for the mechanistic evolution of regenerative abilities.

Support for a radiation of free-living flatworms in the African Great Lakes region and the description of five new Macrostomum species.

BACKGROUND: The African Great Lakes have long been recognized as an excellent location to study speciation. Most famously, cichlid fishes have radiated in Lake Tanganyika and subsequently spread into Lake Malawi and Lake Victoria, where they again radiated. Other taxa have diversified in these lakes, such as catfish, ostracods, gastropods, and Monegenean gill parasites of cichlids. However, these radiations have received less attention, and the process leading to their speciation in this unique region remains to be further explored. Here I present evidence that suggests a radiation of Macrostomum flatworms has occurred in the African Great Lakes region, offering a good opportunity for such investigations. RESULTS: Recent field work has revealed a monophyletic clade of 16 Macrostomum flatworms that have, to date, only been collected from Lake Tanganyika. Additionally, a species collected from Lake Malawi was found nested within this clade. Molecular phylogenetic analysis, largely based on transcriptome data, suggests that this clade underwent rapid speciation, possibly due to a large habitat diversity in the lake. I also observed significant differences in the sperm morphology of these flatworms compared to those of species found outside Lake Tanganyika and Lake Malawi. These included the elongation of an anterior structure, a reduction in the size of the lateral sperm bristles, and changes in relative proportions. I propose functional hypotheses for these changes in sperm design, and formally describe Macrostomum gracilistylum sp. nov from Lake Malawi and its sister species Macrostomum crassum sp. nov., Macrostomum pellitum sp. nov., Macrostomum longispermatum sp. nov., and Macrostomum schäreri sp. nov., from Lake Tanganyika. CONCLUSIONS: The available evidence is consistent with the hypothesis that Macrostomum flatworms have radiated in Lake Tanganyika and subsequently spread to Lake Malawi. However, whether this represents a bona fide adaptive radiation still needs to be determined. Therefore, the African Great Lakes are promising targets for further research into flatworm diversity and speciation.

Genome assemblies of the simultaneously hermaphroditic flatworms Macrostomum cliftonense and Macrostomum hystrix.

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.

Frequent origins of traumatic insemination involve convergent shifts in sperm and genital morphology.

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.

Mating behavior and reproductive morphology predict macroevolution of sex allocation in hermaphroditic flatworms.

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.

Large-scale phylogenomics of the genus Macrostomum (Platyhelminthes) reveals cryptic diversity and novel sexual traits.

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.

Faster Rates of Molecular Sequence Evolution in Reproduction-Related Genes and in Species with Hypodermic Sperm Morphologies.

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.

RNA-Seq of three free-living flatworm species suggests rapid evolution of reproduction-related genes.

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.