Chemical communication underpins virtually all aspects of vertebrate social life, yet remains poorly understood because of its highly complex mechanistic basis. We therefore used chemical fingerprinting of skin swabs and genetic analysis to explore the chemical cues that may underlie mother-offspring recognition in colonially breeding Antarctic fur seals. By sampling mother-offspring pairs from two different colonies, using a variety of statistical approaches and genotyping a large panel of microsatellite loci, we show that colony membership, mother-offspring similarity, heterozygosity, and genetic relatedness are all chemically encoded. Moreover, chemical similarity between mothers and offspring reflects a combination of genetic and environmental influences, the former partly encoded by substances resembling known pheromones. Our findings reveal the diversity of information contained within chemical fingerprints and have implications for understanding mother-offspring communication, kin recognition, and mate choice. Olfaction in particular is fundamental to animal communication, mediating social interactions as varied as territorial behavior, kin recognition, and mate choice (1). Metabolomic tools, such as gas chromatography-mass spectrometry (GC-MS) have made it possible to generate individual-specific chemical "fingerprints." By separating compounds and quantifying their relative abundances, these fingerprints provide a wealth of information, even though not all compounds can necessarily be identified. Both volatile and contact cues are potentially hidden within the extreme complexity of chemical profiles, which is why a mechanistic understanding of chemical communication is still lacking in natural vertebrate populations (2).In particular, "surprisingly little progress" has been made in understanding the link between vertebrate chemical fingerprints and genotype (2). Experimental studies have shown that females of several species are capable of discriminating potential partners based on olfactory cues (3-5). However, very few studies have demonstrated a convincing link between the molecular composition of chemical fingerprints and genetic traits, such as heterozygosity (a measure of genetic quality) and relatedness (6-9). These studies were almost exclusively conducted on a captive population of lemurs, a species known for its conspicuous use of scent marking.A functional understanding of how genotype is chemically encoded also requires knowledge of how many and which types of substances are involved. This is challenging because, especially in natural populations, an individual's mixture of surface chemicals is not only the product of its genotype but may also be mediated by hormones, the microbial flora, body condition, and environmental factors (2). Thus, analyses based on overall chemical fingerprints may overlook subtle genetic signatures and make little if any headway toward identifying the specific substances involved. A second less-appreciated problem is that the modest panels of around 10-15 microsat...

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BackgroundSperm competition between rival ejaculates over the fertilization of ova typically selects for the production of large numbers of sperm. An obvious way to increase sperm production is to increase testis size, and most empirical work has focussed on this parameter. Adaptive plasticity in sperm production rate could also arise due to variation in the speed with which each spermatozoon is produced, but whether animals can respond to relevant environmental conditions by modulating the kinetics of spermatogenesis in this way has not been experimentally investigated.ResultsHere we demonstrate that the simultaneously hermaphroditic flatworm Macrostomum lignano exhibits substantial plasticity in the speed of spermatogenesis, depending on the social context: worms raised under higher levels of sperm competition produce sperm faster.ConclusionsOur findings overturn the prevailing view that the speed of spermatogenesis is a static property of a genotype, and demonstrate the profound impact that social environmental conditions can exert upon a key developmental process. We thus identify, to our knowledge, a novel mechanism through which sperm production rate is maximised under sperm competition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0629-9) contains supplementary material, which is available to authorized users.

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.

The free-living flatworm genus Macrostomum is an emerging model system for studying the links between sex allocation, sexual selection and mating system evolution, as well as the underlying developmental and physiological mechanisms responsible for wide intra-and inter-specific variability in reproductive phenotypes. Despite compelling comparative morphological evidence of sexual diversity, detailed experimental work on reproductive behaviour and physiology in Macrostomum has so far been largely limited to just two species, M. lignano and M. hystrix, an obligate and a preferential outcrosser, respectively. In this study, we establish that a third species, M. pusillum, exhibits a combination of reproductive traits strikingly different from both of its congeners. Unlike M. lignano, we demonstrate that M. pusillum does not adjust sex allocation or the speed of spermatogenesis to the prevailing social group size. Macrostomum pusillum's relatively simple sperm morphology likely explains the short spermatogenesis duration we report, and is linked to a hypodermically inseminating mode of fertilization, which we show also means that these worms are capable of self-fertilization. Surprisingly, and unlike M. hystrix, selfing in isolated worms commences after only a short (if any) delay compared with the onset of reproduction in grouped individuals, with little evidence of differential inbreeding depression in 'isolated' progeny. These combined results suggest that, in nature, M. pusillum may be regularly selfing, in contrast to the congeners studied to date. Our findings highlight the rapid and correlated evolution of reproductive traits, and reinforce the utility of the genus Macrostomum for understanding the evolutionary and developmental mechanisms responsible for this diversity.

As a class, seminal fluid proteins are expected to exert strong effects on mating partners due to the selection pressures of sperm competition and sexual conflict. But because of the complexity of this secretion, linking specific proteins to downstream effects on own fitness—via manipulating the reproductive behavior, physiology, and ultimately the sperm utilization of mating partners—is not straightforward. Here, we adopted a systematic gene knockdown approach to screen for seminal fluid‐mediated fitness effects in the simultaneously hermaphroditic flatworm Macrostomum lignano. We focused on 18 transcripts in M. lignano seminal fluid, testing how their RNA interference‐induced knockdown impacted on three aspects of donor (male) reproductive success: (a) fertility (offspring production of the partner); (b) defensive sperm competitive ability, P1; and (c) offensive sperm competitive ability, P2. In general, the knockdown of most individual transcripts appeared to have only a minor impact on male reproductive success, though we found evidence that the knockdown of up to five different transcripts impacted on fertility; the knockdown of two other transcripts resulted in reduced P2; and knockdown of a further transcript actually increased P2. We thus identify a number of candidate seminal fluid transcripts that appear to modulate offspring production and sperm competitiveness in M. lignano. That only a minority of transcripts exhibit such a pattern likely reflects both the difficulty of accurately estimating sperm competitiveness and the functional redundancy of seminal fluid.

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