Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids.

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

Genomic basis of Y-linked dwarfism in cichlids pursuing alternative reproductive tactics.

Sexually antagonistic selection, which favours different optima in males and females, is predicted to play an important role in the evolution of sex chromosomes. Body size is a sexually antagonistic trait in the shell-brooding cichlid fish Lamprologous callipterus, as "bourgeois" males must be large enough to carry empty snail shells to build nests whereas females must be small enough to fit into shells for breeding. In this species, there is also a second male morph: smaller "dwarf" males employ an alternative reproductive strategy by wriggling past spawning females into shells to fertilize eggs. L. callipterus male morphology is passed strictly from father to son, suggesting Y-linkage. However, sex chromosomes had not been previously identified in this species, and the genomic basis of size dimorphism was unknown. Here we used whole-genome sequencing to identify a 2.4-Mb sex-linked region on scaffold_23 with reduced coverage and single nucleotide polymorphism density in both male morphs compared to females. Within this sex region, distinct Y-haplotypes delineate the two male morphs, and candidate genes for body size (GHRHR, a known dwarfism gene) and sex determination (ADCYAP1R1) are in high linkage disequilibrium. Because differences in body size between females and males are under strong selection in L. callipterus, we hypothesize that sexual antagonism over body size initiated early events in sex chromosome evolution, followed by Y divergence to give rise to bourgeois and dwarf male reproductive strategies. Our results are consistent with the hypothesis that sexually antagonistic traits should be linked to young sex chromosomes.

Phylogenomics of trophically diverse cichlids disentangles processes driving adaptive radiation and repeated trophic transitions.

Cichlid fishes of the tribe Tropheini are a striking case of adaptive radiation, exemplifying multiple trophic transitions between herbivory and carnivory occurring in sympatry with other established cichlid lineages. Tropheini evolved highly specialized eco-morphologies to exploit similar trophic niches in different ways repeatedly and rapidly. To better understand the evolutionary history and trophic adaptations of this lineage, we generated a dataset of 532 targeted loci from 21 out of the 22 described Tropheini species. We resolved the Tropheini into seven monophyletic genera and discovered one to be polyphyletic. The polyphyletic genus, Petrochromis, represents three convergent origins of the algae grazing trophic specialization. This repeated evolution of grazing may have been facilitated by adaptive introgression as we found evidence for gene flow among algae grazing genera. We also found evidence of gene flow among algae browsing genera, but gene flow was restricted between herbivorous and carnivorous genera. Furthermore, we observed no evidence supporting a hybrid origin of this radiation. Our molecular evolutionary analyses suggest that opsin genes likely evolved in response to selection pressures associated with trophic ecology in the Tropheini. We found surprisingly little evidence of positive selection in coding regions of jaw-shaping genes in this trophically diverse lineage. This suggests low degrees of freedom for further change in these genes, and possibly a larger role for regulatory variation in driving jaw adaptations. Our study emphasizes Tropheini cichlids as an important model for studying the evolution of trophic specialization and its role in speciation.

A comprehensive DNA barcode inventory of Austria's fish species.

Austria is inhabited by more than 80 species of native and non-native freshwater fishes. Despite considerable knowledge about Austrian fish species, the latest Red List of threatened species dates back 15 years and a systematic genetic inventory of Austria's fish species does not exist. To fulfill this deficit, we employed DNA barcoding to generate an up-to-date and comprehensive genetic reference database for Austrian fish species. In total, 639 newly generated cytochrome c oxidase subunit 1 (COI) sequences were added to the 377 existing records from the BOLD data base, to compile a near complete reference dataset. Standard sequence similarity analyses resulted in 83 distinct clusters almost perfectly reflecting the expected number of species in Austria. Mean intraspecific distances of 0.22% were significantly lower than distances to closest relatives, resulting in a pronounced barcoding gap and unique Barcode Index Numbers (BINs) for most of the species. Four cases of BIN sharing were detected, pointing to hybridization and/or recent divergence, whereas in Phoxinus spp., Gobio spp. and Barbatula barbatula intraspecific splits, multiple BINs and consequently cryptic diversity were observed. The overall high identification success and clear genetic separation of most of the species confirms the applicability and accuracy of genetic methods for bio-surveillance. Furthermore, the new DNA barcoding data pinpoints cases of taxonomic uncertainty, which need to be addressed in further detail, to more precisely assort genetic lineages and their local distribution ranges in a new National Red-List.

Cumulative SARS-CoV-2 mutations and corresponding changes in immunity in an immunocompromised patient indicate viral evolution within the host.

Different scenarios explaining the emergence of novel variants of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including their evolution in scarcely monitored populations, in animals as alternative hosts, or in immunocompromised individuals. Here we report SARS-CoV-2 immune escape mutations over a period of seven months in an immunocompromised patient with prolonged viral shedding. Signs of infection, viral shedding and mutation events are periodically analyzed using RT-PCR and next-generation sequencing based on naso-pharyngeal swabs, with the results complemented by immunological diagnostics to determine humoral and T cell immune responses. Throughout the infection course, 17 non-synonymous intra-host mutations are noted, with 15 (88.2%) having been previously described as prominent immune escape mutations (S:E484K, S:D950N, S:P681H, S:N501Y, S:del(9), N:S235F and S:H655Y) in VOCs. The high frequency of these non-synonymous mutations is consistent with multiple events of convergent evolution. Thus, our results suggest that specific mutations in the SARS-CoV-2 genome may represent positions with a fitness advantage, and may serve as targets in future vaccine and therapeutics development for COVID-19.

Somewhere I belong: phylogeny and morphological evolution in a species-rich lineage of ectoparasitic flatworms infecting cichlid fishes.

A substantial portion of biodiversity has evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multimarker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58 of 137 species and characterize major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data for 136 of 137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters not only can mask phylogenetic signals, but also are key for characterizing species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterizations.

Expression variations in ectodysplasin-A gene (eda) may contribute to morphological divergence of scales in haplochromine cichlids.

BACKGROUND: Elasmoid scales are one of the most common dermal appendages and can be found in almost all species of bony fish differing greatly in their shape. Whilst the genetic underpinnings behind elasmoid scale development have been investigated, not much is known about the mechanisms involved in moulding of scales. To investigate the links between gene expression differences and morphological divergence, we inferred shape variation of scales from two different areas of the body (anterior and posterior) stemming from ten haplochromine cichlid species from different origins (Lake Tanganyika, Lake Malawi, Lake Victoria and riverine). Additionally, we investigated transcriptional differences of a set of genes known to be involved in scale development and morphogenesis in fish. RESULTS: We found that scales from the anterior and posterior part of the body strongly differ in their overall shape, and a separate look on scales from each body part revealed similar trajectories of shape differences considering the lake origin of single investigated species. Above all, nine as well as 11 out of 16 target genes showed expression differences between the lakes for the anterior and posterior dataset, respectively. Whereas in posterior scales four genes (dlx5, eda, rankl and shh) revealed significant correlations between expression and morphological differentiation, in anterior scales only one gene (eda) showed such a correlation. Furthermore, eda displayed the most significant expression difference between species of Lake Tanganyika and species of the other two younger lakes. Finally, we found genetic differences in downstream regions of eda gene (e.g., in the eda-tnfsf13b inter-genic region) that are associated with observed expression differences. This is reminiscent of a genetic difference in the eda-tnfsf13b inter-genic region which leads to gain or loss of armour plates in stickleback. CONCLUSION: These findings provide evidence for cross-species transcriptional differences of an important morphogenetic factor, eda, which is involved in formation of ectodermal appendages. These expression differences appeared to be associated with morphological differences observed in the scales of haplochromine cichlids indicating potential role of eda mediated signal in divergent scale morphogenesis in fish.

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro.

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.

Maintenance of neutralizing antibodies over ten months in convalescent SARS-CoV-2 afflicted patients.

Knowledge of the level and duration of protective immunity against SARS-CoV-2 after primary infection is of crucial importance for preventive approaches. Currently, there is a lack of evidence on the persistence of specific antibodies. We investigated the generation and maintenance of neutralizing antibodies of convalescent SARS-CoV-2-afflicted patients over a ten-month period post-primary infection using an immunofluorescence assay, a commercial chemiluminescent immunoassay and an in-house enzyme-linked neutralization assay. We present the successful application of an improved version of the plaque-reduction neutralization assay which can be analysed optometrically to simplify data interpretation. Based on the results of the enzyme-linked neutralization assay, neutralizing antibodies were maintained in 77.4% of convalescent individuals without relevant decay over ten months. Furthermore, a positive correlation between severity of infection and antibody titre was observed. In conclusion, SARS-CoV-2-afflicted individuals have been proven to be able to develop and maintain neutralizing antibodies over a period of ten months after primary infection. Findings suggest long-lasting presumably protective humoral immune responses after wild-type infection.

Microevolutionary change in viscerocranial bones under congeneric sympatry in the Lake Tanganyikan cichlid genus Tropheus.

The endemic Lake Tanganyika cichlid genus Tropheus lives at rocky shores all around the lake and comprises six species which are subdivided into about 120 morphologically similar but color-wise distinct populations. Typically, they live without a second Tropheus species, but there are some regions where two or even three sister species live in sympatry. We previously showed that there are morphological differences concerning head shape, eye size and insertion of fins among populations living alone compared to those living in sympatry with a second Tropheus. This study goes one step further to test if sympatry affects the shape of viscerocranial bones. By means of geometric morphometrics, we compare the shape of four bones among thirteen Tropheus populations, some of which in sympatry and some living alone. We quantify patterns of shape variation and estimate morphological disparity among the four bony elements in the study species and populations. We found consistent differences in the shape of one bony element among non-sympatric and sympatric populations, besides an extensive variation in the shape of viscerocranial bones within and among species. Furthermore, sexual dimorphism in Tropheus is clearly evident in the viscerocranial bones analyzed. We suggest that the relatively subtle morphological signal in sympatric vs. non-sympatric Tropheus populations is owed to the fact that the depth segregation does not yet represent a full shift in the trophic niche, albeit our data confirm that differences in ecologically relevant traits, such as bones of the preorbital region, play an important role in the process of niche separation and in the context of explosive diversification of cichlid fishes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10750-021-04536-7.

Transcriptomics unravels molecular players shaping dorsal lip hypertrophy in the vacuum cleaner cichlid, Gnathochromis permaxillaris.

BACKGROUND: Teleosts display a spectacular diversity of craniofacial adaptations that often mediates ecological specializations. A considerable amount of research has revealed molecular players underlying skeletal craniofacial morphologies, but less is known about soft craniofacial phenotypes. Here we focus on an example of lip hypertrophy in the benthivorous Lake Tangnayika cichlid, Gnathochromis permaxillaris, considered to be a morphological adaptation to extract invertebrates out of the uppermost layer of mud bottom. We investigate the molecular and regulatory basis of lip hypertrophy in G. permaxillaris using a comparative transcriptomic approach. RESULTS: We identified a gene regulatory network involved in tissue overgrowth and cellular hypertrophy, potentially associated with the formation of a locally restricted hypertrophic lip in a teleost fish species. Of particular interest were the increased expression level of apoda and fhl2, as well as reduced expression of cyp1a, gimap8, lama5 and rasal3, in the hypertrophic lip region which have been implicated in lip formation in other vertebrates. Among the predicted upstream transcription factors, we found reduced expression of foxp1 in the hypertrophic lip region, which is known to act as repressor of cell growth and proliferation, and its function has been associated with hypertrophy of upper lip in human. CONCLUSION: Our results provide a genetic foundation for future studies of molecular players shaping soft and exaggerated, but locally restricted, craniofacial morphological changes in fish and perhaps across vertebrates. In the future, we advocate integrating gene regulatory networks of various craniofacial phenotypes to understand how they collectively govern trophic and behavioural adaptations.

Gene coexpression networks reveal molecular interactions underlying cichlid jaw modularity.

BACKGROUND: The oral and pharyngeal jaw of cichlid fishes are a classic example of evolutionary modularity as their functional decoupling boosted trophic diversification and contributed to the success of cichlid adaptive radiations. Most studies until now have focused on the functional, morphological, or genetic aspects of cichlid jaw modularity. Here we extend this concept to include transcriptional modularity by sequencing whole transcriptomes of the two jaws and comparing their gene coexpression networks. RESULTS: We show that transcriptional decoupling of gene expression underlies the functional decoupling of cichlid oral and pharyngeal jaw apparatus and the two units are evolving independently in recently diverged cichlid species from Lake Tanganyika. Oral and pharyngeal jaw coexpression networks reflect the common origin of the jaw regulatory program as there is high preservation of gene coexpression modules between the two sets of jaws. However, there is substantial rewiring of genetic architecture within those modules. We define a global jaw coexpression network and highlight jaw-specific and species-specific modules within it. Furthermore, we annotate a comprehensive in silico gene regulatory network linking the Wnt and AHR signalling pathways to jaw morphogenesis and response to environmental cues, respectively. Components of these pathways are significantly differentially expressed between the oral and pharyngeal jaw apparatus. CONCLUSION: This study describes the concerted expression of many genes in cichlid oral and pharyngeal jaw apparatus at the onset of the independent life of cichlid fishes. Our findings suggest that - on the basis of an ancestral gill arch network-transcriptional rewiring may have driven the modular evolution of the oral and pharyngeal jaws, highlighting the evolutionary significance of gene network reuse. The gene coexpression and in silico regulatory networks presented here are intended as resource for future studies on the genetics of vertebrate jaw morphogenesis and trophic adaptation.

An in vitro model for assessment of SARS-CoV-2 infectivity by defining the correlation between virus isolation and quantitative PCR value: isolation success of SARS-CoV-2 from oropharyngeal swabs correlates negatively with Cq value.

BACKGROUND: At the beginning of the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), little was known about its actual rate of infectivity and any COVID-19 patient positive in laboratory testing was supposed to be highly infective and a public health risk factor. METHODS: One hundred oropharyngeal samples were obtained during routine work flow of testing symptomatic persons by quantitative polymerase chain reaction (qPCR) and were inoculated onto cell culture of VeroB4 cells to study the degree of infectivity of SARS-CoV-2 in vitro. Quantification by virus titration and an external standard using synthetic RNA gave the breaking point of infectivity in SARS-CoV-2 in vitro. RESULTS: A clear negative correlation (r = - 0.76; p < 0.05) could be asserted between the viral load in quantitative polymerase chain reaction (qPCR) and the probability of a successful isolation in serial isolation experiments of specific oropharyngeal samples positive in qPCR. Quantification by virus titration and an external standard using synthetic RNA indicate a Cq between 27 and 30 in E-gene screening PCR as a breaking point in vitro, where infectivity decreases significantly and isolations become less probable. CONCLUSIONS: This study showed that only the 21% of samples with the highest viral load were infectious enough to transmit the virus in vitro and determined that the dispersion rate in vitro is surprisingly close to those calculated in large retrospective epidemiological studies for SARS-CoV-2. This raises the question of whether this simple in vitro model is suitable to give first insights in dispersion characters of novel or neglected viral pathogens. The statement that SARS-CoV-2 needs at least 40,000 copies to reliably induce infection in vitro is an indication of its transmissibility in Public Health decisions. Applying quantitative PCR systems in diagnosis of SARS-CoV2 can distinguish between patients providing a high risk of transmission and those, where the risk of transmission is probably limited to close and long-lasting contacts.

Discriminating larvae of two syntopic Cychramus species (Coleoptera, Nitidulidae) by means of bar-HRM analysis.

Molecular genetic methods are increasingly used to supplement or substitute classical morphology-based species identification. Here, we employ a COI mini-barcode coupled high-resolution melting analysis to quickly, cost-efficiently and reliably determine larvae of two closely related Cychramus (Coleoptera, Nitidulidae) species. Euclidean distance comparison (p < 0.01) and a Welch t-test of the melting point temperatures (p < 0.01) provide highly significant statistical evidence for species specific differences in melting and fluorescence curves, thus allowing the assignment of larvae to either of the two species. This protocol serves as a fast, low-cost and low-tech method to discriminate between pairs or groups of closely related species and can be adapted and applied to various ecological research questions.

Appetite regulating genes may contribute to herbivory versus carnivory trophic divergence in haplochromine cichlids.

Feeding is a complex behaviour comprised of satiety control, foraging, ingestion and subsequent digestion. Cichlids from the East African Great Lakes are renowned for their diverse trophic specializations, largely predicated on highly variable jaw morphologies. Thus, most research has focused on dissecting the genetic, morphological and regulatory basis of jaw and teeth development in these species. Here for the first time we explore another aspect of feeding, the regulation of appetite related genes that are expressed in the brain and control satiety in cichlid fishes. Using qPCR analysis, we first validate stably expressed reference genes in the brain of six haplochromine cichlid species at the end of larval development prior to foraging. We next evaluate the expression of 16 appetite related genes in herbivorous and carnivorous species from the parallel radiations of Lake Tanganyika, Malawi and Victoria. Interestingly, we find increased expression of two appetite-regulating genes (anorexigenic genes), cart and npy2r, in the brain of carnivorous species in all the three lakes. This supports the notion that appetite gene regulation might play a part in determining trophic niche specialization in divergent cichlid species, already prior to exposure to different diets. Our study contributes to the limited body of knowledge on the neurological circuitry that controls feeding transitions and adaptations in cichlids and other teleosts.

Divergence in larval jaw gene expression reflects differential trophic adaptation in haplochromine cichlids prior to foraging.

BACKGROUND: Understanding how variation in gene expression contributes to morphological diversity is a major goal in evolutionary biology. Cichlid fishes from the East African Great lakes exhibit striking diversity in trophic adaptations predicated on the functional modularity of their two sets of jaws (oral and pharyngeal). However, the transcriptional basis of this modularity is not so well understood, as no studies thus far have directly compared the expression of genes in the oral and pharyngeal jaws. Nor is it well understood how gene expression may have contributed to the parallel evolution of trophic morphologies across the replicate cichlid adaptive radiations in Lake Tanganyika, Malawi and Victoria. RESULTS: We set out to investigate the role of gene expression divergence in cichlid fishes from these three lakes adapted to herbivorous and carnivorous trophic niches. We focused on the development stage prior to the onset of exogenous feeding that is critical for understanding patterns of gene expression after oral and pharyngeal jaw skeletogenesis, anticipating environmental cues. This framework permitted us for the first time to test for signatures of gene expression underlying jaw modularity in convergent eco-morphologies across three independent adaptive radiations. We validated a set of reference genes, with stable expression between the two jaw types and across species, which can be important for future studies of gene expression in cichlid jaws. Next we found evidence of modular and non-modular gene expression between the two jaws, across different trophic niches and lakes. For instance, prdm1a, a skeletogenic gene with modular anterior-posterior expression, displayed higher pharyngeal jaw expression and modular expression pattern only in carnivorous species. Furthermore, we found the expression of genes in cichlids jaws from the youngest Lake Victoria to exhibit low modularity compared to the older lakes. CONCLUSION: Overall, our results provide cross-species transcriptional comparisons of modularly-regulated skeletogenic genes in the two jaw types, implicating expression differences which might contribute to the formation of divergent trophic morphologies at the stage of larval independence prior to foraging.

Only true pelagics mix: comparative phylogeography of deepwater bathybatine cichlids from Lake Tanganyika.

In the absence of dispersal barriers, species with great dispersal ability are expected to show little, if at all, phylogeographic structure. The East African Great Lakes and their diverse fish faunas provide opportunities to test this hypothesis in pelagic fishes, which are presumed to be highly mobile and unrestricted in their movement by physical barriers. Here, we address the link between panmixis and pelagic habitat use by comparing the phylogeographic structure among four deepwater cichlid species of the tribe Bathybatini from Lake Tanganyika. We show that the mitochondrial genealogies (based on the most variable part or the control region) of the four species are very shallow (0.8-4% intraspecific divergence across entire distribution ranges) and that all species experienced recent population growth. A lack of phylogeographic structure in the two eupelagic species, Bathybates fasciatus and B. leo, was consistent with expectations and with findings in other pelagic cichlid species. Contrary to expectations, a clear phylogeographic structure was detected in the two benthopelagic species, B. graueri and Hemibates stenosoma. Differences in genetic diversity between eupelagic and benthopelagic species may be due to differences in their dispersal propensity, mediated by their respective predatory niches, rather than precipitated by external barriers to dispersal.

Molecular mechanisms underlying nuchal hump formation in dolphin cichlid, Cyrtocara moorii.

East African cichlid fishes represent a model to tackle adaptive changes and their connection to rapid speciation and ecological distinction. In comparison to bony craniofacial tissues, adaptive morphogenesis of soft tissues has been rarely addressed, particularly at the molecular level. The nuchal hump in cichlids fishes is one such soft-tissue and exaggerated trait that is hypothesized to play an innovative role in the adaptive radiation of cichlids fishes. It has also evolved in parallel across lakes in East Africa and Central America. Using gene expression profiling, we identified and validated a set of genes involved in nuchal hump formation in the Lake Malawi dolphin cichlid, Cyrtocara moorii. In particular, we found genes differentially expressed in the nuchal hump, which are involved in controlling cell proliferation (btg3, fosl1a and pdgfrb), cell growth (dlk1), craniofacial morphogenesis (dlx5a, mycn and tcf12), as well as regulators of growth-related signals (dpt, pappa and socs2). This is the first study to identify the set of genes associated with nuchal hump formation in cichlids. Given that the hump is a trait that evolved repeatedly in several African and American cichlid lineages, it would be interesting to see if the molecular pathways and genes triggering hump formation follow a common genetic track or if the trait evolved in parallel, with distinct mechanisms, in other cichlid adaptive radiations and even in other teleost fishes.

Maternal mRNA input of growth and stress-response-related genes in cichlids in relation to egg size and trophic specialization.

BACKGROUND: Egg size represents an important form of maternal effect determined by a complex interplay of long-term adaptation and short-term plasticity balancing egg size with brood size. Haplochromine cichlids are maternal mouthbrooders showing differential parental investment in different species, manifested in great variation in egg size, brood size and duration of maternal care. Little is known about maternally determined molecular characters of eggs in fishes and their relation to egg size and trophic specialization. Here we investigate maternal mRNA inputs of selected growth- and stress-related genes in eggs of mouthbrooding cichlid fishes adapted to different trophic niches from Lake Tanganyika, Lake Malawi, Lake Victoria and compare them to their riverine allies. RESULTS: We first identified two reference genes, atf7ip and mid1ip1, to be suitable for cross-species quantification of mRNA abundance via qRT-PCR in the cichlid eggs. Using these reference genes, we found substantial variation in maternal mRNA input for a set of candidate genes related to growth and stress response across species and lakes. We observed negative correlation of mRNA abundance between two of growth hormone receptor paralogs (ghr1 and ghr2) across all haplochromine cichlid species which also differentiate the species in the two younger lakes, Malawi and Lake Victoria, from those in Lake Tanganyika and ancestral riverine species. Furthermore, we found correlations between egg size and maternal mRNA abundance of two growth-related genes igf2 and ghr2 across the haplochromine cichlids as well as distinct clustering of the species based on their trophic specialization using maternal mRNA abundance of five genes (ghr1, ghr2, igf2, gr and sgk1). CONCLUSIONS: These findings indicate that variations in egg size in closely related cichlid species can be linked to differences in maternal RNA deposition of key growth-related genes. In addition, the cichlid species with contrasting trophic specialization deposit different levels of maternal mRNAs in their eggs for particular growth-related genes; however, it is unclear whether such differences contribute to differential morphogenesis at later stages of development. Our results provide first insights into this aspect of gene activation, as a basis for future studies targeting their role during ecomorphological specialization and adaptive radiation.