Description: Disentangling the processes and mechanisms underlying adaptive diversification is facilitated by the comparative study of replicate population pairs that have diverged along a similar environmental gradient. Such a setting is realized in a cichlid fish from southern Lake Tanganyika, Astatotilapia burtoni, which occurs within the lake proper as well as in various affluent rivers. Previously, we demonstrated that independent lake and stream populations show similar adaptations to the two habitat regimes. However, little is known about the evolutionary and demographic history of the A. burtoni populations in question and the patterns of genome divergence among them. Here, we apply restriction site-associated DNA sequencing (RADseq) to examine the evolutionary history, the population structure and genomic differentiation of lake and stream populations in A. burtoni. A phylogenetic reconstruction based on genome-wide molecular data largely resolved the evolutionary relationships among populations, allowing us to re-evaluate the independence of replicate lake-stream population clusters. Further, we detected a strong pattern of isolation by distance, with baseline genomic divergence increasing with geographic distance and decreasing with the level of gene flow between lake and stream populations. Genome divergence patterns were heterogeneous and inconsistent among lake-stream population clusters, which is explained by differences in divergence times, levels of gene flow and local selection regimes. In line with the latter, we only detected consistent outlier loci when the most divergent lake-stream population pair was excluded. Several of the thus identified candidate genes have inferred functions in immune and neuronal systems and show differences in gene expression between lake and stream populations.

Description: The origin of novel phenotypic characters is a key component in organismal diversification; yet, the mechanisms underlying the emergence of such evolutionary novelties are largely unknown. Here we examine the origin of egg-spots, an evolutionary innovation of the most species-rich group of cichlids, the haplochromines, where these conspicuous male fin colour markings are involved in mating. Applying a combination of RNAseq, comparative genomics and functional experiments, we identify two novel pigmentation genes, fhl2a and fhl2b, and show that especially the more rapidly evolving b-paralog is associated with egg-spot formation. We further find that egg-spot bearing haplochromines, but not other cichlids, feature a transposable element in the cis-regulatory region of fhl2b. Using transgenic zebrafish, we finally demonstrate that this region shows specific enhancer activities in iridophores, a type of pigment cells found in egg-spots, suggesting that a cis-regulatory change is causally linked to the gain of expression in egg-spot bearing haplochromines

Description: Highlights: • Pelvic brooding induces tissue-specific changes in gene expression • Inflammatory signaling characterizes transcriptome of the egg-anchoring plug • Similar to embryo implantation, the plug likely evolved from an inflammatory response • Mammalian placenta genes were independently co-opted into the plug Summary: The evolution of pregnancy exposes parental tissues to new, potentially stressful conditions, which can trigger inflammation.1 Inflammation is costly2,3 and can induce embryo rejection, which constrains the evolution of pregnancy.1 In contrast, inflammation can also promote morphological innovation at the maternal-embryonic interface as exemplified by co-option of pro-inflammatory signaling for eutherian embryo implantation.1,4,5 Given its dual function, inflammation could be a key process explaining how innovations such as pregnancy and placentation evolved many times convergently. Pelvic brooding ricefishes evolved a novel “plug” tissue,6,7 which forms inside the female gonoduct after spawning, anchors egg-attaching filaments, and enables pelvic brooders to carry eggs externally until hatching.6,8 Compared to pregnancy, i.e., internal bearing of embryos, external bearing should alleviate constraints on inflammation in the reproductive tract. We thus hypothesized that an ancestral inflammation triggered by the retention of attaching filaments gave rise to pathways orchestrating plug formation. In line with our hypothesis, histological sections of the developing plug revealed signs of gonoduct injuries by egg-attaching filaments in the pelvic brooding ricefish Oryzias eversi. Tissue-specific transcriptomes showed that inflammatory signaling dominates the plug transcriptome and inflammation-induced genes controlling vital processes for plug development such as tissue growth and angiogenesis were overexpressed in the plug. Finally, mammalian placenta genes were enriched in the plug transcriptome, indicating convergent gene co-option for building, attaching, and sustaining a transient tissue in the female reproductive tract. This study highlights the role of gene co-option and suggests that recruiting inflammatory signaling into physiological processes provides a fast-track to evolutionary innovation.

Description: The unique male pregnancy in pipefishes and seahorses ranges from basic attachment (pouch-less species: Nerophinae) of maternal eggs to specialized internal gestation in pouched species (e.g. Syngnathus and Hippocampus) with many transitions in between. Due to this diversity, male pregnancy offers a unique platform for assessing physiological and molecular adaptations in pregnancy evolution. These insights will contribute to answering long-standing questions of why and how pregnancy evolved convergently in so many vertebrate systems. To understand the molecular congruencies and disparities in male pregnancy evolution, we compared transcriptome-wide differentially expressed genes in four syngnathid species, at four pregnancy stages (nonpregnant, early, late and parturition). Across all species and pregnancy forms, metabolic processes and immune dynamics defined pregnancy stages, especially pouched species shared expression features akin to female pregnancy. The observed downregulation of adaptive immune genes in early-stage pregnancy and its reversed upregulation during late/parturition in pouched species, most notably in Hippocampus, combined with directionless expression in the pouch-less species, suggests immune modulation to be restricted to pouched species that evolved placenta-like systems. We propose that increased foeto-paternal intimacy in pouched syngnathids commands immune suppression processes in early gestation, and that the elevated immune response during parturition coincides with pouch opening and reduced progeny reliance. Immune response regulation in pouched species supports the recently described functional MHC II pathway loss as critical in male pregnancy evolution. The independent co-option of similar genes and pathways both in male and female pregnancy highlights immune modulation as crucial for the evolutionary establishment of pregnancy.

Description: Variation in gene expression contributes to ecological speciation by facilitating population persistence in novel environments. Likewise, immune response can be a relevant factor in speciation driven by adaptation to different environments. Previous studies examining gene expression differences between recently diverged ecotypes often relied on only one pair of populations, targeted the expression of only a subset of genes, or used wild caught‐individuals. Here, we investigated the contribution of habitat‐specific parasites and symbionts and the underlying immunological capabilities of ecotype hosts to adaptive divergence in lake‐river population pairs of the cichlid fish Astatotilapia burtoni. To shed light on the role of phenotypic plasticity in adaptive divergence, we compared parasite and microbiota communities, immune response, and gene expression patterns of fish from natural habitats and a lake‐like pond setup. In all investigated population pairs, lake fish were more heavily parasitized than river fish, both in terms of parasite taxa composition and infection abundance. Innate immune response in the wild was higher in lake than in river populations and elevated in a river population exposed to lake parasites in the pond setup. Environmental differences between lake and river habitat and their distinct parasite communities shaped differential gene expression, involving genes functioning in osmoregulation and immune response. Most changes in gene expression between lake and river samples in the wild and in the pond setup were based on a plastic response. Finally, gene expression and bacterial communities of wild‐caught individuals and individuals acclimated to lake‐like pond conditions showed shifts underlying adaptive phenotypic plasticity.