Dynamics of sex chromosome evolution in a rapid radiation of cichlid fishes

[Figure: see text]

Gene expression remodelling and immune response during adaptive divergence in an African cichlid fish

Variation in gene expression contributes to ecological speciation by facilitating population persistence in novel environments. Likewise, immune responses can be of relevance in speciation driven by adaptation to different environments. Previous studies examining gene expression differences between recently diverged ecotypes have 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 abilities 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 set-up. In all investigated population pairs, lake fish were more heavily parasitized than river fish, in terms of both parasite taxon composition and infection abundance. The innate immune response in the wild was higher in lake than in river populations and was elevated in a river population exposed to lake parasites in the pond set-up. Environmental differences between lake and river habitat and their distinct parasite communities have 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 set-up were based on a plastic response. Finally, gene expression and bacterial communities of wild-caught individuals and individuals acclimatized to lake-like pond conditions showed shifts underlying adaptive phenotypic plasticity

Drivers and dynamics of a massive adaptive radiation in cichlid fishes

Adaptive radiation is the likely source of much of the ecological and morphological diversity of life; 1-4; . How adaptive radiations proceed and what determines their extent remains unclear in most cases; 1,4; . Here we report the in-depth examination of the spectacular adaptive radiation of cichlid fishes in Lake Tanganyika. On the basis of whole-genome phylogenetic analyses, multivariate morphological measurements of three ecologically relevant trait complexes (body shape, upper oral jaw morphology and lower pharyngeal jaw shape), scoring of pigmentation patterns and approximations of the ecology of nearly all of the approximately 240 cichlid species endemic to Lake Tanganyika, we show that the radiation occurred within the confines of the lake and that morphological diversification proceeded in consecutive trait-specific pulses of rapid morphospace expansion. We provide empirical support for two theoretical predictions of how adaptive radiations proceed, the 'early-burst' scenario; 1,5; (for body shape) and the stages model; 1,6,7; (for all traits investigated). Through the analysis of two genomes per species and by taking advantage of the uneven distribution of species in subclades of the radiation, we further show that species richness scales positively with per-individual heterozygosity, but is not correlated with transposable element content, number of gene duplications or genome-wide levels of selection in coding sequences

Unraveling transcriptome dynamics in Lake Tanganyika cichlid fishes

During the last decade, understanding the causes of phenotypic diversification has become one of the most puzzling topics of today’s biology (Romero et al., 2012). A major motivation of current evolutionary biology is to identify the genetic basis of interspecific phenotypic variation. This obviously includes the study of the pronounced phenotypic shifts that have accumulated in humans since our lineage diverged from the common ancestor with chimpanzees (Khaitovich et al., 2006) five to seven million years ago (Glazko and Nei, 2003). Over the past century, evolutionary geneticists have thus focused on identifying the genetic basis of this famous phenotypic diversification event. Surprisingly, it appeared that the genetic distance between humans and chimpanzees is possibly too small to account for their substantial organismal differences (King and Wilson, 1975) and it has therefore been suggested that evolutionary changes in anatomy and behaviour might have been triggered through regulatory evolution rather than through variations in coding sequences (King and Wilson, 1975). Large-scale studies of gene expression evolution over longer evolutionary distances are now possible as a result of the increasing number of available sequenced transcriptomes. Extensive comparative surveys demonstrate that variation in gene expression patterns play a key role in the evolution of morphological differences (Brawand et al., 2011; Necsulea and Kaessmann, 2014; Romero et al., 2012). Nevertheless, the evolutionary dynamics of gene expression has so far mainly been studied between species that diverged long ago, whereas comparatively little is known about how gene expression evolves during rapid organismal diversification. Adaptive radiation, which is defined as the rapid proliferation of eco-morphological diversity within an organismal lineage on the basis of accelerated adaptation to distinct ecological niches (Gavrilets and Losos, 2009; Schluter, 2000), can be responsible for extremely rapid morphological diversification (Berner and Salzburger, 2015). One of the most striking examples of adaptive radiation is found in East African Lake Tanganyika, where hundreds of cichlid fish species have evolved within a few million years only (Berner and Salzburger, 2015; Salzburger, 2018; Salzburger and Meyer, 2004). Cichlids occupy a great variety of ecological niches (Berner and Salzburger, 2015; Ronco et al., 2019; Salzburger, 2018) and are drastically diverse in many phenotypic traits (Berner and Salzburger, 2015; Salzburger, 2018). The cichlids’ impressive ability to rapidly diversify and adapt to new environments has been documented multiple times and in different geographic regions (Crispo and Chapman, 2010; Härer et al., 2017; Muschick et al., 2011) and extensive efforts have been made to identify and explain the genetic basis underlying and allowing such remarkable events (Brawand et al., 2014). Several molecular mechanisms (e.g. excess of gene duplication, expression divergence associated with transposable element insertions, accelerated coding sequence evolution) have been associated with rapid diversification in cichlids (Brawand et al., 2014). A particularly promising candidate factor is gene expression regulation (Brawand et al., 2014). It has been shown at several occasions that gene expression variations account for important phenotypic shifts observed among cichlids (Colombo et al., 2013; Henning et al., 2013; Hofmann et al., 2009; Santos et al., 2016, 2014), including the differential expression of opsin genes for adaptation of the senses to different light environments (Carleton and Kocher, 2001; Hofmann et al., 2009; Seehausen et al., 2008). And yet, it is not fully understood how intrinsic factors and external events interplay to promote this exceptional phenotypic diversity (Losos, 2010; Salzburger, 2018, 2009; Seehausen, 2007; Yoder et al., 2010). The main goal of this thesis is to retrace the evolutionary history of the Tanganyikan cichlids from different perspectives (genomic, transcriptomic and ecology) and extend the current knowledge of how selection acts on an entire biological system. It is an integrative approach in which I combined bioinformatic and experimental methods to understand the inception of such an impressive evolutionary process at the level of the transcriptome. This thesis is developed along two axes. The first part of the thesis (Chapter 1-6) is dedicated to an in-depth exploration of the entire Lake Tanganyikan cichlid radiation (~250 species). By focusing on the species flock as a whole, this survey provides a unique opportunity to gain insights into the causes and triggers, the progression and the resultant communities of an adaptive radiation. The first chapter entitled “The evolution of gene expression levels during rapid organismal diversification” focuses on understanding to what extend gene expression evolution has contributed to the rapid evolution of the astonishing diversity of cichlids in Lake Tanganyika. For a comprehensive analysis, we sequenced the transcriptomes of six organs from 73 emblematic cichlid species representing all the different Tanganyikan cichlid lineages (Ronco et al., 2019). We selected six organs involved in ecological (lowerpharyngeal jaw (LPJ), liver, gills) and behavioural (brain, testis, ovary) important traits known to be very diverse in cichlids (Fig.1) (Baldo et al., 2011; Böhne et al., 2013; Schneider et al., 2014). By comparing gene expression patterns across the species tree, we investigated the rate of gene expression evolution among organs, lineage and transcriptome parts (coding vs. non-coding) and studied the difference in the degree of expression specificity among organs and lineages. The second chapter entitled “Tempo and mode of sex chromosome turnovers in an adaptive radiation” focuses on another component of speciation, namely the evolution of sex chromosome systems. In several large animal clades, sex chromosomes are conserved and shared across many species, whereas other clades undergo frequent sex chromosome turnovers leading to an uneven distribution of sex chromosome diversity (Bachtrog et al., 2014). In particular, many fish families feature young sex chromosomes and experience rapid turnovers (Kitano and Peichel, 2012). East African cichlids recently emerged as a model to study the evolutionary dynamics of sex determination (Gammerdinger et al., 2018). As we collected male and female specimens for each species of cichlids (Chapter 1 and chapter 4), we use expression (Chapter 1) and genomic (Chapter 4) data to investigate sex chromosome evolution in the Tanganyikan cichlids. We aimed at identifying new sex chromosome systems and at providing a better understanding of when and how new sex chromosomes evolve in a short evolutionary framework by focusing on a case of explosive speciation accompanied by spectacular phenotypic diversity. In the third chapter which is entitled “The transcriptional basis of adaptive diversification in the lower pharyngeal jaw bone of cichlid fishes”, we focused on the association between gene expression and the morphology of an important trophic trait of cichlid fishes, the lower pharyngeal jaw (LPJ) (Gunter and Meyer, 2014; Muschick et al., 2012, 2011; Schneider et al., 2014). To gain new insights on how gene expression modulates LPJ morphology at a higher taxonomic level, we correlated gene expression levels (see Chapter 1 for more details) of 71 cichlid species – covering the entire spectrum of the eco-morphological and genetic diversity of the Lake Tanganyika cichlid adaptive radiation – with their LPJ morphology (see Chapter 4 for more details). With this approach, we aimed at identifying genes and functional categories involved in the LPJ morphological divergence observed across the Lake Tanganyika cichlid adaptive radiation but also at measuring how much gene expression levels support those important morphological divergence. Chapter 4 is entitled “Drivers, dynamics and progression of a massive adaptive radiation in African cichlid fish” and focuses on the in-depth investigation of nearly the entire taxonomic diversity of the Tanganyika cichlid adaptive radiation. Based on whole genome sequencing, multivariate morphological measurements of several important trophic traits (e.g. body shape and LPJ) and ecological indicators (e.g. stable isotopes), this chapter focused on bringing new insights on how genetic change is associated with phenotypic and ecological diversity and trace back patterns of eco-morphological evolution through the phylogenetic history of the radiation. Chapter 5 is entitled “Community assembly patterns and niche evolution in the species-flock of cichlid fishes from East African Lake Tanganyika” and focuses on characterizing the community structure and the niche evolution of the adaptive radiation of Lake Tanganyika cichlids. To study underwater communities, point-combination transect (PCT) (Widmer et al., 2019) was used allowing the investigation of habitat differentiation and co-occurrence across almost the entire radiation. Chapter 6 “Does eDNA within sediments reflect local cichlid assemblages in Lake Tanganyika?” explores the applicability of environmental DNA (eDNA) being used to asses the cichlid fish diversity at various sites at Lake Tanganyika (Klymus et al., 2017; Lobo et al., 2017; Thomsen and Willerslev, 2015). Compared with PCT data (Chapter 5), chapter 6 compiled an ideal data set for proof of concept of PCT and discusses the validity of eDNA as a tool for future community assessments in cichlids. The second part of this thesis focuses on a very central concept of adaptive radiation: adaptation to new ecological niches (Gavrilets and Losos, 2009; Schluter, 2000). When encountering ecological opportunities, species during an adaptive radiation rapidly adapt and diversify to fill available niches (Gavrilets and Losos, 2009; Schluter, 2000). This part of the thesis investigates the transcriptomic basis of adaptation to different environments in one particular cichlid species, the East African haplochromine Astatotilapia burtoni (Chapter 7-8). This fish has adapted to lake and river environments and different pairs of lake-stream populations, representing different stages of the “speciation continuum”, can be found around Lake Tanganyika (Pauquet et al., 2018; Theis et al., 2014). Over the past decades, A. burtoni has been established as a cichlid model system to study many key questions in ecology and evolutionary biology (behaviour (Theis et al., 2012), neuronal processes (Huffman et al., 2015), sex determination (Böhne et al., 2016; Göppert et al., 2016; Heule et al., 2014; Roberts et al., 2016), pigmentation (Santos et al., 2014), genomics and speciation (Brawand et al., 2014; Pauquet et al., 2018; Salzburger, 2018). As a consequence, A. burtoni is also an emerging system in developmental biology (Heule and Salzburger, 2011; Woltering et al., 2018), which is greatly facilitated by the availability of a reference genome (Brawand et al., 2014). This genome, however, remains fragmented (scaffold level assembly) and with poorer annotations as compared to the most widely used cichlid reference genome, the one of the Nile tilapia Oreochromis niloticus, which has been assembled to the chromosome level (Brawand et al., 2014; Conte et al., 2017). Chapter 7 “Time matters! Developmental shift in gene expression between the head and the trunk region of the cichlid fish Astatotilapia burtoni” investigates gene expression throughout development in A. burtoni. Combining RNA-sequencing from different developmental time points as well as integrating adult gene expression data, we aimed at creating new genome annotations for this established cichlid model system. Using the newly constructed comprehensive reference transcriptome, we investigated differential gene expression and gene expression dynamics through time and across different body parts. In the last chapter of the thesis, which is entitled “From river to lake: Gene expression remodelling and immune response during adaptation to new environments” we focus on adaptive phenotypic plasticity to different environments in A. burtoni. We aimed at characterizing this plasticity by describing gene expression and microbiota communities of lake and stream ecotypes in the wild, and describing how they change when exposed to environmental changes. The seven chapters of this thesis are followed by an overall discussion, which quotes all chapters’ results and brings into perspective how such an integrative project allows addressing key questions in evolutionary biology. I would like to note that all the chapters presented in this thesis are the results of valuable collaborations. My personal contribution to each chapter is described in the “Author Contributions” section of each chapter

Time matters! Developmental shift in gene expression between the head and the trunk region of the cichlid fish Astatotilapia burtoni

Abstract Background Differential gene expression can be translated into differing phenotypic traits. Especially during embryogenesis, specific gene expression networks regulate the development of different body structures. Cichlid fishes, with their impressive phenotypic diversity and propensity to radiate, are an emerging model system in the genomics era. Here we set out to investigate gene expression throughout development in the well-studied cichlid fish Astatotilapia burtoni, native to Lake Tanganyika and its affluent rivers. Results Combining RNA-sequencing from different developmental time points as well as integrating adult gene expression data, we constructed a new genome annotation for A. burtoni comprising 103,253 transcripts (stemming from 52,584 genomic loci) as well as a new reference transcriptome set. We compared our transcriptome to the available reference genome, redefining transcripts and adding new annotations. We show that about half of these transcripts have coding potential. We also characterize transcripts that are not present in the genome assembly. Next, using our newly constructed comprehensive reference transcriptome, we characterized differential gene expression through time and showed that gene expression is shifted between different body parts. We constructed a gene expression network that identified connected genes responsible for particular phenotypes and made use of it to focus on genes under potential positive selection in A. burtoni, which were implicated in fin development and vision. Conclusions We provide new genomic resources for the cichlid fish Astatotilapia burtoni, which will contribute to its further establishment as a model system. Tracing gene expression through time, we identified gene networks underlying particular functions, which will help to understand the genetic basis of phenotypic diversity in cichlids

Gene expression dynamics during rapid organismal diversification in African cichlid fishes

Changes in gene expression play a fundamental role in phenotypic evolution. Transcriptome evolutionary dynamics have so far mainly been compared among distantly related species and remain largely unexplored during rapid organismal diversification, in which gene regulatory changes have been suggested as particularly effective drivers of phenotypic divergence. Here we studied gene expression evolution in a model system of adaptive radiation, the cichlid fishes of African Lake Tanganyika. By comparing gene expression profiles of 6 different organs in 74 cichlid species representing all subclades of this radiation, we demonstrate that the rate of gene expression evolution varies among organs, transcriptome parts and the subclades of the radiation, indicating different strengths of selection. We found that the noncoding part of the transcriptome evolved more rapidly than the coding part, and that the gonadal transcriptomes evolved more rapidly than the somatic ones, with the exception of liver. We further show that the rate of gene expression change was not constant over the course of the radiation but accelerated at its later phase. Finally, we show that-at the per-gene level-the evolution of expression patterns is dominated by stabilizing selection

Drivers and dynamics of a massive adaptive radiation in cichlid fishes

Adaptive radiation is the likely source of much of the ecological and morphological diversity of life1–4. How adaptive radiations proceed and what determines their extent remains unclear in most cases1,4. Here we report the in-depth examination of the spectacular adaptive radiation of cichlid fishes in Lake Tanganyika. On the basis of whole-genome phylogenetic analyses, multivariate morphological measurements of three ecologically relevant trait complexes (body shape, upper oral jaw morphology and lower pharyngeal jaw shape), scoring of pigmentation patterns and approximations of the ecology of nearly all of the approximately 240 cichlid species endemic to Lake Tanganyika, we show that the radiation occurred within the confines of the lake and that morphological diversification proceeded in consecutive trait-specific pulses of rapid morphospace expansion. We provide empirical support for two theoretical predictions of how adaptive radiations proceed, the ‘early-burst’ scenario1,5 (for body shape) and the stages model1,6,7 (for all traits investigated). Through the analysis of two genomes per species and by taking advantage of the uneven distribution of species in subclades of the radiation, we further show that species richness scales positively with per-individual heterozygosity, but is not correlated with transposable element content, number of gene duplications or genome-wide levels of selection in coding sequences

Overview of denitrification capacity of <i>P</i>. <i>veronii</i> 1YdBTEX2.

<p>(A) Overnight growth of <i>P</i>. <i>veronii</i> 1YdBTEX2 wild type (WT) and the Δ<i>nar</i> mutant in presence (+O₂, left) or absence of air but with 15 mM nitrate supplemented medium (+NO₃,–O₂, right panel) conditions. Note the gas formation in the right panel of the WT incubation. (B) Gene regions predicted for denitrification in the <i>P</i>. <i>veronii</i> 1YdBTEX2 chromosome 1 with trivial gene names indicated. Black bar represents the deleted region in <i>P</i>. <i>veronii</i> Δ<i>nar</i>.</p

Circular maps of the replicons encompassing the <i>P</i>. <i>veronii</i> 1YdBTEX2 genome.

<p>(A) Chromosome 1 (chr1) with indication of possible genomic islands (GEI) and prophages (pf). The outermost circles show the location and orientation of predicted coding regions (blue and cyan), followed by tRNA (olive green) and rRNA genes (black), predicted regions of genome plasticity (blue-green-brown) islands and prophages (grey). The inner circles represent BLASTN comparisons with the close relatives <i>P</i>. <i>fluorescens</i> SBW25 (red, Acc. No. AM181176.4), <i>P</i>. <i>trivialis</i> strain IHBB745 (deep pink, CP011507.1), <i>P</i>. <i>syringae</i> pv. syringae B728a (dark purple, CP000075.1), <i>P</i>. <i>putida</i> KT2440 (light purple, AE015451.1) and <i>P</i>. <i>knackmussii</i> B13 (persian green, HG322950). GC skew (dark magenta and yellow green) is shown in the most central circle. (B) As A, but for the chromosome 2 replicon (chr2). Inner circles, from outwards to inwards, predicted transposons (dark purple) and <i>tra</i> genes (green), regions of genome plasticity (blue-green-brown) and prophages (grey), followed by BLASTN comparisons to <i>P</i>. <i>fluorescens</i> SBW25 plasmid pQB103 (red, AM235768.1, NC_009444.1), <i>Pseudomonas stutzeri</i> strain 19SMN4 plasmid pLIB119 (deep pink, CP007510.1), <i>Pseudomonas mandelii</i> JR-1 plasmid (dark purple, CP005961.1) and <i>Pseudomonas resinovorans</i> NBRC 106553 plasmid pCAR1.3 (Persian green, AP013069.1). (C) As B, but for the plasmid replicon. The inner circles represent the BLASTN comparisons with <i>P</i>. <i>putida</i> S12 plasmid pTTS12 (red, CP009975.1), and <i>Pseudomonas</i> sp. VLB120 plasmid pSTY (purple, CP003962.1). Plots generated with DNAPlotter [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165850#pone.0165850.ref046" target="_blank">46</a>].</p