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1.  Chromosomal rearrangements, phenotypic variation and modularity: a case study from a contact zone between house mouse Robertsonian races in Central Italy 
Ecology and Evolution  2016;10.1002/ece3.1912.
The Western European house mouse, Mus musculus domesticus, is well‐known for the high frequency of Robertsonian fusions that have rapidly produced more than 50 karyotipic races, making it an ideal model for studying the mechanisms of chromosomal speciation. The mouse mandible is one of the traits studied most intensively to investigate the effect of Robertsonian fusions on phenotypic variation within and between populations. This complex bone structure has also been widely used to study the level of integration between different morphogenetic units. Here, with the aim of testing the effect of different karyotypic assets on the morphology of the mouse mandible and on its level of modularity, we performed morphometric analyses of mice from a contact area between two highly metacentric races in Central Italy. We found no difference in size, while the mandible shape was found to be different between the two Robertsonian races, even after accounting for the genetic relationships among individuals and geographic proximity. Our results support the existence of two modules that indicate a certain degree of evolutionary independence, but no difference in the strength of modularity between chromosomal races. Moreover, the ascending ramus showed more pronounced interpopulation/race phenotypic differences than the alveolar region, an effect that could be associated to their different polygenic architecture. This study suggests that chromosomal rearrangements play a role in the house mouse phenotypic divergence, and that the two modules of the mouse mandible are differentially affected by environmental factors and genetic makeup.
PMCID: PMC4733104  PMID: 26855768
Chromosomal speciation; geometric morphometrics; house mouse; modularity; phenotypic variation; Robertsonian fusions
2.  Intrastrand triplex DNA repeats in bacteria: a source of genomic instability 
Nucleic Acids Research  2015;43(21):10126-10142.
Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.
PMCID: PMC4666352  PMID: 26450966
3.  Sympatric ecological divergence associated with a color polymorphism 
BMC Biology  2015;13:82.
Color polymorphisms are a conspicuous feature of many species and a way to address broad ecological and evolutionary questions. Three potential major evolutionary fates of color polymorphisms are conceivable over time: maintenance, loss, or speciation. However, the understanding of color polymorphisms and their evolutionary implications is frequently impaired by sex-linkage of coloration, unknown inheritance patterns, difficulties in phenotypic characterization, and a lack of evolutionary replicates. Hence, the role of color polymorphisms in promoting ecological and evolutionary diversification remains poorly understood. In this context, we assessed the ecological and evolutionary consequences of a color polymorphic study system that is not hampered by these restrictions: the repeated adaptive radiations of the gold/dark Midas cichlid fishes (the Amphilophus citrinellus species complex) from the great lakes and crater lakes of Nicaragua, Central America.
We conducted multi-trait morphological and ecological analyses from ten populations of this young adaptive radiation (<6,000 years old), which revealed sympatric ecological differentiation associated with the conspicuous binary (gold/dark) color polymorphism. Varying degrees of intraspecific ecological divergence were observed across the ten color morph pairs, but most pairs exhibited a consistently parallel ecological and evolutionary trajectory across populations. Specifically, gold Midas cichlids are frequently deeper-bodied, have more robust pharyngeal jaws, and feed at a lower trophic level compared to conspecific, sympatric dark individuals. A common garden experiment suggests there is a genetic correlation of color and eco-morphological traits.
We demonstrate unprecedented ecological and evolutionary consequences of color polymorphism in this adaptive radiation. Across the species complex, sympatric conspecific individuals differed in eco-morphology depending on color morph (gold/dark) and the axis of differentiation tended to be consistent across replicates. The consistent divergence across wild populations and the common garden experiment suggests that color is genetically correlated to ecology. Because Midas cichlids are known to mate color assortatively, the putative genetic correlation of this color polymorphism with an eco-morphological divergence suggests an innate potential to promote ecological and evolutionary divergence across this species complex. However, there are to date no examples of speciation based on color in this radiation, suggesting long-term maintenance of this color polymorphism.
Electronic supplementary material
The online version of this article (doi:10.1186/s12915-015-0192-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4594650  PMID: 26437665
Adaptive radiation; Ecological diversification; Evolutionary ecology; Parallel evolution; Color polymorphism; Stable isotope analysis; Crater lake; Cichlids; Genetic correlation; Pleiotropy; Genetic linkage
4.  Genetic and environmental effects on the morphological asymmetry in the scale‐eating cichlid fish, Perissodus microlepis  
Ecology and Evolution  2015;5(19):4277-4286.
The scale‐eating cichlid fish, Perissodus microlepis, from Lake Tanganyika are a well‐known example of an asymmetry dimorphism because the mouth/head is either left‐bending or right‐bending. However, how strongly its pronounced morphological laterality is affected by genetic and environmental factors remains unclear. Using quantitative assessments of mouth asymmetry, we investigated its origin by estimating narrow‐sense heritability (h 2) using midparent–offspring regression. The heritability estimates [field estimate: h 2 = 0.22 ± 0.06, P = 0.013; laboratory estimate: h 2 = 0.18 ± 0.05, P = 0.004] suggest that although variation in laterality has some additive genetic component, it is strongly environmentally influenced. Family‐level association analyses of a putative microsatellite marker that was claimed to be linked to gene(s) for laterality revealed no association of this locus with laterality. Moreover, the observed phenotype frequencies in offspring from parents of different phenotype combinations were not consistent with a previously suggested single‐locus two‐allele model, but they neither were able to reject with confidence a random asymmetry model. These results reconcile the disputed mechanisms for this textbook case of mouth asymmetry where both genetic and environmental factors contribute to this remarkable case of morphological asymmetry.
PMCID: PMC4667837  PMID: 26664678
Adaptation; behavioral laterality; mouth asymmetry; narrow‐sense heritability; negative frequency‐dependent selection; phenotypic plasticity
5.  The phantoms of a high-seven - or - why do our thumbs stick out? 
Frontiers in Zoology  2015;12:23.
The earliest tetrapods had hands and feet with up to eight digits but this number was subsequently reduced during evolution. It was assumed that lineages with more than five digits no longer exist but investigations of clawed-frogs now indicate that they posses a rudimentary or atavistic sixth digit in their hindlimb. A recent reevaluation of the stem tetrapod Ichthyostega predicts that its seven digits evolved from two different types of ancestral fin radials, pre-axial and post-axial. In this context we now ask the question, should we consider a pre-axial origin of the thumb as reason for its unique genetic signature?
PMCID: PMC4570229  PMID: 26379756
Thumb; Pentadactyly; Tetrapod; Fin; Limb; Digit; Pre-axial; Post-axial
6.  The genomic substrate for adaptive radiation in African cichlid fish 
Brawand, David | Wagner, Catherine E. | Li, Yang I. | Malinsky, Milan | Keller, Irene | Fan, Shaohua | Simakov, Oleg | Ng, Alvin Y. | Lim, Zhi Wei | Bezault, Etienne | Turner-Maier, Jason | Johnson, Jeremy | Alcazar, Rosa | Noh, Hyun Ji | Russell, Pamela | Aken, Bronwen | Alföldi, Jessica | Amemiya, Chris | Azzouzi, Naoual | Baroiller, Jean-François | Barloy-Hubler, Frederique | Berlin, Aaron | Bloomquist, Ryan | Carleton, Karen L. | Conte, Matthew A. | D'Cotta, Helena | Eshel, Orly | Gaffney, Leslie | Galibert, Francis | Gante, Hugo F. | Gnerre, Sante | Greuter, Lucie | Guyon, Richard | Haddad, Natalie S. | Haerty, Wilfried | Harris, Rayna M. | Hofmann, Hans A. | Hourlier, Thibaut | Hulata, Gideon | Jaffe, David B. | Lara, Marcia | Lee, Alison P. | MacCallum, Iain | Mwaiko, Salome | Nikaido, Masato | Nishihara, Hidenori | Ozouf-Costaz, Catherine | Penman, David J. | Przybylski, Dariusz | Rakotomanga, Michaelle | Renn, Suzy C. P. | Ribeiro, Filipe J. | Ron, Micha | Salzburger, Walter | Sanchez-Pulido, Luis | Santos, M. Emilia | Searle, Steve | Sharpe, Ted | Swofford, Ross | Tan, Frederick J. | Williams, Louise | Young, Sarah | Yin, Shuangye | Okada, Norihiro | Kocher, Thomas D. | Miska, Eric A. | Lander, Eric S. | Venkatesh, Byrappa | Fernald, Russell D. | Meyer, Axel | Ponting, Chris P. | Streelman, J. Todd | Lindblad-Toh, Kerstin | Seehausen, Ole | Di Palma, Federica
Nature  2014;513(7518):375-381.
Cichlid fishes are famous for large, diverse and replicated adaptive radiations in the Great Lakes of East Africa. To understand the molecular mechanisms underlying cichlid phenotypic diversity, we sequenced the genomes and transcriptomes of five lineages of African cichlids: the Nile tilapia (Oreochromis niloticus), an ancestral lineage with low diversity; and four members of the East African lineage: Neolamprologus brichardi/pulcher (older radiation, Lake Tanganyika), Metriaclima zebra (recent radiation, Lake Malawi), Pundamilia nyererei (very recent radiation, Lake Victoria), and Astatotilapia burtoni (riverine species around Lake Tanganyika). We found an excess of gene duplications in the East African lineage compared to tilapia and other teleosts, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel microRNAs. In addition, we analysed sequence data from sixty individuals representing six closely related species from Lake Victoria, and show genome-wide diversifying selection on coding and regulatory variants, some of which were recruited from ancient polymorphisms. We conclude that a number of molecular mechanisms shaped East African cichlid genomes, and that amassing of standing variation during periods of relaxed purifying selection may have been important in facilitating subsequent evolutionary diversification.
PMCID: PMC4353498  PMID: 25186727
7.  Embryonic and larval development in the Midas cichlid fish species flock (Amphilophus spp.): a new evo-devo model for the investigation of adaptive novelties and species differences 
Central American crater lake cichlid fish of the Midas species complex (Amphilophus spp.) are a model system for sympatric speciation and fast ecological diversification and specialization. Midas cichlids have been intensively analyzed from an ecological and morphological perspective. Genomic resources such as transcriptomic and genomic data sets, and a high-quality draft genome are available now. Many ecologically relevant species-specific traits and differences such as pigmentation and cranial morphology arise during development. Detailed descriptions of the early development of the Midas cichlid in particular, will help to investigate the ontogeny of species differences and adaptations.
We describe the embryonic and larval development of the crater lake cichlid, Amphilophus xiloaensis, until seven days after fertilization. Similar to previous studies on teleost development, we describe six periods of embryogenesis - the zygote, cleavage, blastula, gastrula, segmentation, and post-hatching period. Furthermore, we define homologous stages to well-described teleost models such as medaka and zebrafish, as well as other cichlid species such as the Nile tilapia and the South American cichlid Cichlasoma dimerus. Key morphological differences between the embryos of Midas cichlids and other teleosts are highlighted and discussed, including the presence of adhesive glands and different early chromatophore patterns, as well as variation in developmental timing.
The developmental staging of the Midas cichlid will aid researchers in the comparative investigation of teleost ontogenies. It will facilitate comparative developmental biological studies of Neotropical and African cichlid fish in particular. In the past, the species flocks of the African Great Lakes have received the most attention from researchers, but some lineages of the 300–400 species of Central American lakes are fascinating model systems for adaptive radiation and rapid phenotypic evolution. The availability of genetic resources, their status as a model system for evolutionary research, and the possibility to perform functional experiments including transgenesis makes the Midas cichlid complex a very attractive model for evolutionary-developmental research.
PMCID: PMC4352272  PMID: 25887993
Teleostei; Ontogeny; Parallel evolution; Phenotypic diversification; Cichlidae; Pigmentation; Melanophore; Xanthophore; Amphilophus citrinellus; Amphilophus xiloaensis
8.  Parallel evolution in Ugandan crater lakes: repeated evolution of limnetic body shapes in haplochromine cichlid fish 
BMC Evolutionary Biology  2015;15(1):9.
The enormous diversity found in East African cichlid fishes in terms of morphology, coloration, and behavior have made them a model for the study of speciation and adaptive evolution. In particular, haplochromine cichlids, by far the most species-rich lineage of cichlids, are a well-known textbook example for parallel evolution. Southwestern Uganda is an area of high tectonic activity, and is home to numerous crater lakes. Many Ugandan crater lakes were colonized, apparently independently, by a single lineage of haplochromine cichlids. Thereby, this system could be considered a natural experiment in which one can study the interaction between geographical isolation and natural selection promoting phenotypic diversification.
We sampled 13 crater lakes and six potentially-ancestral older lakes and, using both mitochondrial and microsatellite markers, discovered strong genetic and morphological differentiation whereby (a) geographically close lakes tend to be genetically more similar and (b) three different geographic areas seem to have been colonized by three independent waves of colonization from the same source population. Using a geometric morphometric approach, we found that body shape elongation (i.e. a limnetic morphology) evolved repeatedly from the ancestral deeper-bodied benthic morphology in the clear and deep crater lake habitats.
A pattern of strong genetic and morphological differentiation was observed in the Ugandan crater lakes. Our data suggest that body shape changes have repeatedly evolved into a more limnetic-like form in several Ugandan crater lakes after independent waves of colonization from the same source population. The observed morphological changes in crater lake cichlids are likely to result from a common selective regime.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-015-0287-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4322459  PMID: 25648727
Parallel evolution; Benthic-limnetic; Speciation
9.  Transcriptomics of two evolutionary novelties: how to make a sperm-transfer organ out of an anal fin and a sexually selected “sword” out of a caudal fin 
Ecology and Evolution  2015;5(4):848-864.
Swords are exaggerated male ornaments of swordtail fishes that have been of great interest to evolutionary biologists ever since Darwin described them in the Descent of Man (1871). They are a novel sexually selected trait derived from modified ventral caudal fin rays and are only found in the genus Xiphophorus. Another phylogenetically more widespread and older male trait is the gonopodium, an intromittent organ found in all poeciliid fishes, that is derived from a modified anal fin. Despite many evolutionary and behavioral studies on both traits, little is known so far about the molecular mechanisms underlying their development. By investigating transcriptomic changes (utilizing a RNA-Seq approach) in response to testosterone treatment in the swordtail fish, Xiphophorus hellerii, we aimed to better understand the architecture of the gene regulatory networks underpinning the development of these two evolutionary novelties. Large numbers of genes with tissue-specific expression patterns were identified. Among the “sword genes” those involved in embryonic organ development, sexual character development and coloration were highly expressed, while in the gonopodium rather more morphogenesis-related genes were found. Interestingly, many genes and genetic pathways are shared between both developing novel traits derived from median fins: the sword and the gonopodium. Our analyses show that a larger set of gene networks was co-opted during the development and evolution of the “older” gonopodium than in the “younger,” and morphologically less complex trait, the sword. We provide a catalog of candidate genes for future efforts to dissect the development of those sexually selected exaggerated male traits in swordtails.
PMCID: PMC4338968  PMID: 25750712
Co-option; gonopodium; key innovation; male-specific traits; RNA-Seq; swordtails; Xiphophorus
10.  Evolution of the Vertebrate Pax4/6 Class of Genes with Focus on Its Novel Member, the Pax10 Gene 
Genome Biology and Evolution  2014;6(7):1635-1651.
The members of the paired box (Pax) family regulate key developmental pathways in many metazoans as tissue-specific transcription factors. Vertebrate genomes typically possess nine Pax genes (Pax1–9), which are derived from four proto-Pax genes in the vertebrate ancestor that were later expanded through the so-called two-round (2R) whole-genome duplication. A recent study proposed that pax6a genes of a subset of teleost fishes (namely, acanthopterygians) are remnants of a paralog generated in the 2R genome duplication, to be renamed pax6.3, and reported one more group of vertebrate Pax genes (Pax6.2), most closely related to the Pax4/6 class. We propose to designate this new member Pax10 instead and reconstruct the evolutionary history of the Pax4/6/10 class with solid phylogenetic evidence. Our synteny analysis showed that Pax4, -6, and -10 originated in the 2R genome duplications early in vertebrate evolution. The phylogenetic analyses of relationships between teleost pax6a and other Pax4, -6, and -10 genes, however, do not support the proposed hypothesis of an ancient origin of the acanthopterygian pax6a genes in the 2R genome duplication. Instead, we confirmed the traditional scenario that the acanthopterygian pax6a is derived from the more recent teleost-specific genome duplication. Notably, Pax6 is present in all vertebrates surveyed to date, whereas Pax4 and -10 were lost multiple times in independent vertebrate lineages, likely because of their restricted expression patterns: Among Pax6-positive domains, Pax10 has retained expression in the adult retina alone, which we documented through in situ hybridization and quantitative reverse transcription polymerase chain reaction experiments on zebrafish, Xenopus, and anole lizard.
PMCID: PMC4122933  PMID: 24951566
Pax6; Pax4; Pax10; gene loss; conserved synteny; gene duplication
11.  Evolution of genomic structural variation and genomic architecture in the adaptive radiations of African cichlid fishes 
Frontiers in Genetics  2014;5:163.
African cichlid fishes are an ideal system for studying explosive rates of speciation and the origin of diversity in adaptive radiation. Within the last few million years, more than 2000 species have evolved in the Great Lakes of East Africa, the largest adaptive radiation in vertebrates. These young species show spectacular diversity in their coloration, morphology and behavior. However, little is known about the genomic basis of this astonishing diversity. Recently, five African cichlid genomes were sequenced, including that of the Nile Tilapia (Oreochromis niloticus), a basal and only relatively moderately diversified lineage, and the genomes of four representative endemic species of the adaptive radiations, Neolamprologus brichardi, Astatotilapia burtoni, Metriaclima zebra, and Pundamila nyererei. Using the Tilapia genome as a reference genome, we generated a high-resolution genomic variation map, consisting of single nucleotide polymorphisms (SNPs), short insertions and deletions (indels), inversions and deletions. In total, around 18.8, 17.7, 17.0, and 17.0 million SNPs, 2.3, 2.2, 1.4, and 1.9 million indels, 262, 306, 162, and 154 inversions, and 3509, 2705, 2710, and 2634 deletions were inferred to have evolved in N. brichardi, A. burtoni, P. nyererei, and M. zebra, respectively. Many of these variations affected the annotated gene regions in the genome. Different patterns of genetic variation were detected during the adaptive radiation of African cichlid fishes. For SNPs, the highest rate of evolution was detected in the common ancestor of N. brichardi, A. burtoni, P. nyererei, and M. zebra. However, for the evolution of inversions and deletions, we found that the rates at the terminal taxa are substantially higher than the rates at the ancestral lineages. The high-resolution map provides an ideal opportunity to understand the genomic bases of the adaptive radiation of African cichlid fishes.
PMCID: PMC4042683  PMID: 24917883
SNPs; insertions; deletions; inversions; adaptive radiation; structural variation
12.  The Gut Microbial Community of Midas Cichlid Fish in Repeatedly Evolved Limnetic-Benthic Species Pairs 
PLoS ONE  2014;9(4):e95027.
Gut bacterial communities are now known to influence a range of fitness related aspects of organisms. But how different the microbial community is in closely related species, and if these differences can be interpreted as adaptive is still unclear. In this study we compared microbial communities in two sets of closely related sympatric crater lake cichlid fish species pairs that show similar adaptations along the limnetic-benthic axis. The gut microbial community composition differs in the species pair inhabiting the older of two crater lakes. One major difference, relative to other fish, is that in these cichlids that live in hypersaline crater lakes, the microbial community is largely made up of Oceanospirillales (52.28%) which are halotolerant or halophilic bacteria. This analysis opens up further avenues to identify candidate symbiotic or co-evolved bacteria playing a role in adaptation to similar diets and life-styles or even have a role in speciation. Future functional and phylosymbiotic analyses might help to address these issues.
PMCID: PMC3986361  PMID: 24733403
13.  Crater lake cichlids individually specialize along the benthic–limnetic axis 
Ecology and Evolution  2014;4(7):1127-1139.
A common pattern of adaptive diversification in freshwater fishes is the repeated evolution of elongated open water (limnetic) species and high-bodied shore (benthic) species from generalist ancestors. Studies on phenotype-diet correlations have suggested that population-wide individual specialization occurs at an early evolutionary and ecological stage of divergence and niche partitioning. This variable restricted niche use across individuals can provide the raw material for earliest stages of sympatric divergence. We investigated variation in morphology and diet as well as their correlations along the benthic-limnetic axis in an extremely young Midas cichlid species, Amphilophus tolteca, endemic to the Nicaraguan crater lake Asososca Managua. We found that A. tolteca varied continuously in ecologically relevant traits such as body shape and lower pharyngeal jaw morphology. The correlation of these phenotypes with niche suggested that individuals are specialized along the benthic-limnetic axis. No genetic differentiation within the crater lake was detected based on genotypes from 13 microsatellite loci. Overall, we found that individual specialization in this young crater lake species encompasses the limnetic-as well as the benthic macro-habitat. Yet there is no evidence for any diversification within the species, making this a candidate system for studying what might be the early stages preceding sympatric divergence.
A common pattern of adaptive diversification in freshwater fishes is the repeated evolution of open water (limnetic) species and of shore (benthic) species. Individual specialization can reflect earliest stages of evolutionary and ecological divergence. We here demonstrate individual specialization along the benthic–limnetic axis in a young adaptive radiation of crater lake cichlid fishes.
PMCID: PMC3997327  PMID: 24772288
Benthic–limnetic axis; divergent selection; ecological opportunity; individual specialization; parallel evolution; phenotype-diet correlation
14.  Introgressive hybridization and latitudinal admixture clines in North Atlantic eels 
Hybridization, the interbreeding of diagnosably divergent species, is a major focus in evolutionary studies. Eels, both from North America and Europe migrate through the Atlantic to mate in a vast, overlapping area in the Sargasso Sea. Due to the lack of direct observation, it is unknown how these species remain reproductively isolated. The detection of inter-species hybrids in Iceland suggests on-going gene flow, but few studies to date have addressed the influence of introgression on genetic differentiation in North Atlantic eels.
Here, we show that while mitochondrial lineages remain completely distinct on both sides of the Atlantic, limited hybridization is detectable with nuclear DNA markers. The nuclear hybridization signal peaks in the northern areas and decreases towards the southern range limits on both continents according to Bayesian assignment analyses. By simulating increasing proportions of both F1 hybrids and admixed individuals from the southern to the northern-most locations, we were able to generate highly significant isolation-by-distance patterns in both cases, reminiscent of previously published data for the European eel. Finally, fitting an isolation-with-migration model to our data supports the hypothesis of recent asymmetric introgression and refutes the alternative hypothesis of ancient polymorphism.
Fluctuating degrees of introgressive hybridization between Atlantic eel species are sufficient to explain temporally varying correlations of geographic and genetic distances reported for populations of the European eel.
PMCID: PMC3986858  PMID: 24674242
Anguilla anguilla; Anguilla rostrata; Gene flow; Isolation-by-distance; Simulation; Migration barriers
15.  Revisiting de Beer’s textbook example of heterochrony and jaw elongation in fish: calmodulin expression reflects heterochronic growth, and underlies morphological innovation in the jaws of belonoid fishes 
EvoDevo  2014;5:8.
Heterochronic shifts during ontogeny can result in adaptively important innovations and might be initiated by simple developmental switches. Understanding the nature of these developmental events can provide insights into fundamental molecular mechanisms of evolutionary change. Fishes from the Suborder Belonoidei display a vast array of extreme craniofacial morphologies that appear to have arisen through a series of heterochronic shifts. We performed a molecular heterochrony study, comparing postembryonic jaw development in representatives of the Suborder Belonoidei, the halfbeak Dermogenys pusilla (where the lower jaw is considerably elongated compared to the upper jaw) and the needlefish Belone belone (where both jaws are elongated), to a representative of their sister group the Suborder Adrianichthyoidei, the medaka Oryzias latipes, which has retained the ancestral morphology.
Early in development, the lower jaw displays accelerated growth both in needlefish and halfbeak compared to medaka, and secondary acceleration of the upper jaw is seen in needlefish later in their development, representing a case of mosaic heterochrony. We identified toothless extensions of the dentaries as innovations of Belonoid fishes and the source of heterochronic growth. The molecular basis of growth heterochronies in the Belonoidei was examined through comparing expression of skeletogenic genes during development of halfbeak and medaka. The calmodulin paralogue calm1 was identified as a potential regulator of jaw length in halfbeak as its expression gradually increases in the lower jaw, but not the upper jaw, in a pattern that matches its outgrowth. Moreover, medaka displays equal expression of calm1 in the upper and lower jaws, consistent with the lack of jaw outgrowth in this species.
Heterochronic shifts in jaw growth have occurred repeatedly during the evolution of Belonoid fishes and we identify toothless extensions of the dentaries as an important innovation of this group. Our results suggest that calm1 contributes to jaw heterochrony in halfbeak, potentially driving further heterochronic shifts in jaw growth across the Suborder Belonoidei, such as the upper jaw acceleration observed in needlefish.
PMCID: PMC3927394  PMID: 24499543
Heterochrony; Morphological innovation; Jaw development; Beloniformes; Gavin de Beer; Dermogenys; Belone; Medaka; Calmodulin
16.  The Imprinted NPAP1 Gene in the Prader–Willi Syndrome Region Belongs to a POM121-Related Family of Retrogenes 
Genome Biology and Evolution  2014;6(2):344-351.
We have recently shown that the human Nuclear pore-associated protein (NPAP1)/C15orf2 gene encodes a nuclear pore-associated protein. This gene is one of several paternally expressed imprinted genes in the genomic region 15q11q13. Because the Prader–Willi syndrome is known to be caused by the loss of function of paternally expressed genes in 15q11q13, a phenotypic contribution of NPAP1 cannot be excluded. NPAP1 appears to be under strong positive Darwinian selection in primates, suggesting an important function in primate biology. Interestingly, however, in contrast to all other protein-coding genes in 15q11q13, NPAP1 has no ortholog in the mouse. Our investigation of the evolutionary origin of NPAP1 showed that the gene is specific to primate species and absent from the 15q11q13-orthologous regions in all nonprimate mammals. However, we identified a group of paralogous genes, which we call NPAP1L, in all placental mammals except rodents. Phylogenetic analysis revealed that NPAP1, NPAP1L, and another group of genes (UPF0607), which is also restricted to primates, are closely related to the vertebrate transmembrane nucleoporin gene POM121, although they lack the transmembrane domain. These three newly identified groups of genes all lack conserved introns, and hence, are likely retrogenes. We hypothesize that, in the common ancestor of placentals, the POM121 gene retrotransposed and gave rise to an NPAP1-ancestral retrogene NPAP1L/NPAP1/UPF0607. Our results suggest that the nuclear pore-associated gene NPAP1 originates from the vertebrate nucleoporin gene POM121 and—after several steps of retrotransposition and duplication—has been subjected to genomic imprinting and positive selection after integration into the imprinted SNRPN-UBE3A chromosomal domain.
PMCID: PMC3942032  PMID: 24482533
C15orf2; NPAP1L; UPF0607; imprinting; nucleoporin; nuclear pore complex
17.  Mouth asymmetry in the textbook example of scale-eating cichlid fish is not a discrete dimorphism after all 
Individuals of the scale-eating cichlid fish, Perissodus microlepis, from Lake Tanganyika tend to have remarkably asymmetric heads that are either left-bending or right-bending. The ‘left’ morph opens its mouth markedly towards the left and preferentially feeds on the scales from the right-hand side of its victim fish, and the ‘right’ morph bites scales from the victims’ left-hand side. This striking dimorphism made these fish a textbook example of their astonishing degree of ecological specialization and as one of the few known incidences of negative frequency-dependent selection acting on an asymmetric morphological trait, where left and right forms are equally frequent within a species. We investigated the degree and the shape of the frequency distribution of head asymmetry in P. microlepis to test whether the variation conforms to a discrete dimorphism, as generally assumed. In both adult and juvenile fish, mouth asymmetry appeared to be continuously and unimodally distributed with no clear evidence for a discrete dimorphism. Mixture analyses did not reveal evidence of a discrete or even strong dimorphism. These results raise doubts about previous claims, as reported in textbooks, that head variation in P. microlepis represents a discrete dimorphism of left- and right-bending forms. Based on extensive field sampling that excluded ambiguous (i.e. symmetric or weakly asymmetric) individual adults, we found that left and right morphs occur in equal abundance in five populations. Moreover, mate pairing for 51 wild-caught pairs was random with regard to head laterality, calling into question reports that this laterality is maintained through disassortative mating.
PMCID: PMC3497103  PMID: 23055070
behavioural laterality; Lake Tanganyika; mixture analysis; negative frequency-dependent selection; Perissodus microlepis; random mating
18.  Retinoic Acid Is Involved in the Metamorphosis of the Anal Fin into an Intromittent Organ, the Gonopodium, in the Green Swordtail (Xiphophorus hellerii) 
PLoS ONE  2013;8(10):e77580.
In poeciliid fish the male anal fin has been transformed into a gonopodium, an intromittent organ required for internal fertilization. Elevated testosterone levels induce metamorphosis of a subset of anal fin rays to grow and form the specialized terminal structures of the gonopodium. The molecular mechanisms underlying these processes are largely unknown. Here, we investigated whether retinoic acid (RA) signaling is involved in gonopodium development in the swordtail Xiphophorus hellerii. We showed that aldh1a2, a RA synthesizing enzyme, and the RA receptors, rar-ga and rar-gb, are expressed in anal fins during metamorphosis. aldh1a2 expression is regulated by testosterone in a concentration-dependent manner and is up-regulated in both hormone-induced and naturally developing gonopodia. Androgen receptor (ar), a putative regulator of gonopodial development, is co-expressed with aldh1a2 and the RA receptors in gonopodial rays. Importantly, experimental increase of RA signaling promoted growth of the gonopodium and increased the number of new segments. Based on gene expression analyses and pharmacological manipulation of gonopodium development, we show that the RA signaling pathway is activated in response to androgen signaling and promotes fin ray growth and development during the metamorphosis of the anal fin into the gonopodium.
PMCID: PMC3808415  PMID: 24204880
19.  Analysis of the African coelacanth genome sheds light on tetrapod evolution 
Amemiya, Chris T. | Alföldi, Jessica | Lee, Alison P. | Fan, Shaohua | Philippe, Hervé | MacCallum, Iain | Braasch, Ingo | Manousaki, Tereza | Schneider, Igor | Rohner, Nicolas | Organ, Chris | Chalopin, Domitille | Smith, Jeramiah J. | Robinson, Mark | Dorrington, Rosemary A. | Gerdol, Marco | Aken, Bronwen | Biscotti, Maria Assunta | Barucca, Marco | Baurain, Denis | Berlin, Aaron M. | Blatch, Gregory L. | Buonocore, Francesco | Burmester, Thorsten | Campbell, Michael S. | Canapa, Adriana | Cannon, John P. | Christoffels, Alan | De Moro, Gianluca | Edkins, Adrienne L. | Fan, Lin | Fausto, Anna Maria | Feiner, Nathalie | Forconi, Mariko | Gamieldien, Junaid | Gnerre, Sante | Gnirke, Andreas | Goldstone, Jared V. | Haerty, Wilfried | Hahn, Mark E. | Hesse, Uljana | Hoffmann, Steve | Johnson, Jeremy | Karchner, Sibel I. | Kuraku, Shigehiro | Lara, Marcia | Levin, Joshua Z. | Litman, Gary W. | Mauceli, Evan | Miyake, Tsutomu | Mueller, M. Gail | Nelson, David R. | Nitsche, Anne | Olmo, Ettore | Ota, Tatsuya | Pallavicini, Alberto | Panji, Sumir | Picone, Barbara | Ponting, Chris P. | Prohaska, Sonja J. | Przybylski, Dariusz | Saha, Nil Ratan | Ravi, Vydianathan | Ribeiro, Filipe J. | Sauka-Spengler, Tatjana | Scapigliati, Giuseppe | Searle, Stephen M. J. | Sharpe, Ted | Simakov, Oleg | Stadler, Peter F. | Stegeman, John J. | Sumiyama, Kenta | Tabbaa, Diana | Tafer, Hakim | Turner-Maier, Jason | van Heusden, Peter | White, Simon | Williams, Louise | Yandell, Mark | Brinkmann, Henner | Volff, Jean-Nicolas | Tabin, Clifford J. | Shubin, Neil | Schartl, Manfred | Jaffe, David | Postlethwait, John H. | Venkatesh, Byrappa | Di Palma, Federica | Lander, Eric S. | Meyer, Axel | Lindblad-Toh, Kerstin
Nature  2013;496(7445):311-316.
It was a zoological sensation when a living specimen of the coelacanth was first discovered in 1938, as this lineage of lobe-finned fish was thought to have gone extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features . Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain, and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues demonstrate the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.
PMCID: PMC3633110  PMID: 23598338
20.  Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution 
Nature genetics  2013;45(4):415-421e2.
Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.
PMCID: PMC3709584  PMID: 23435085
21.  Resampling-Based Approaches to Study Variation in Morphological Modularity 
PLoS ONE  2013;8(7):e69376.
Modularity has been suggested to be connected to evolvability because a higher degree of independence among parts allows them to evolve as separate units. Recently, the Escoufier RV coefficient has been proposed as a measure of the degree of integration between modules in multivariate morphometric datasets. However, it has been shown, using randomly simulated datasets, that the value of the RV coefficient depends on sample size. Also, so far there is no statistical test for the difference in the RV coefficient between a priori defined groups of observations. Here, we (1), using a rarefaction analysis, show that the value of the RV coefficient depends on sample size also in real geometric morphometric datasets; (2) propose a permutation procedure to test for the difference in the RV coefficient between a priori defined groups of observations; (3) show, through simulations, that such a permutation procedure has an appropriate Type I error; (4) suggest that a rarefaction procedure could be used to obtain sample-size-corrected values of the RV coefficient; and (5) propose a nearest-neighbor procedure that could be used when studying the variation of modularity in geographic space. The approaches outlined here, readily extendable to non-morphometric datasets, allow study of the variation in the degree of integration between a priori defined modules. A Java application – that will allow performance of the proposed test using a software with graphical user interface – has also been developed and is available at the Morphometrics at Stony Brook Web page (
PMCID: PMC3712944  PMID: 23874956
22.  Saltatory Evolution of the Ectodermal Neural Cortex Gene Family at the Vertebrate Origin 
Genome Biology and Evolution  2013;5(8):1485-1502.
The ectodermal neural cortex (ENC) gene family, whose members are implicated in neurogenesis, is part of the kelch repeat superfamily. To date, ENC genes have been identified only in osteichthyans, although other kelch repeat-containing genes are prevalent throughout bilaterians. The lack of elaborate molecular phylogenetic analysis with exhaustive taxon sampling has obscured the possible link of the establishment of this gene family with vertebrate novelties. In this study, we identified ENC homologs in diverse vertebrates by means of database mining and polymerase chain reaction screens. Our analysis revealed that the ENC3 ortholog was lost in the basal eutherian lineage through single-gene deletion and that the triplication between ENC1, -2, and -3 occurred early in vertebrate evolution. Including our original data on the catshark and the zebrafish, our comparison revealed high conservation of the pleiotropic expression pattern of ENC1 and shuffling of expression domains between ENC1, -2, and -3. Compared with many other gene families including developmental key regulators, the ENC gene family is unique in that conventional molecular phylogenetic inference could identify no obvious invertebrate ortholog. This suggests a composite nature of the vertebrate-specific gene repertoire, consisting not only of de novo genes introduced at the vertebrate origin but also of long-standing genes with no apparent invertebrate orthologs. Some of the latter, including the ENC gene family, may be too rapidly evolving to provide sufficient phylogenetic signals marking orthology to their invertebrate counterparts. Such gene families that experienced saltatory evolution likely remain to be explored and might also have contributed to phenotypic evolution of vertebrates.
PMCID: PMC3762194  PMID: 23843192
vertebrate novelty; saltation; gene loss; conserved synteny; whole genome duplication
23.  Revealing Less Derived Nature of Cartilaginous Fish Genomes with Their Evolutionary Time Scale Inferred with Nuclear Genes 
PLoS ONE  2013;8(6):e66400.
Cartilaginous fishes, divided into Holocephali (chimaeras) and Elasmoblanchii (sharks, rays and skates), occupy a key phylogenetic position among extant vertebrates in reconstructing their evolutionary processes. Their accurate evolutionary time scale is indispensable for better understanding of the relationship between phenotypic and molecular evolution of cartilaginous fishes. However, our current knowledge on the time scale of cartilaginous fish evolution largely relies on estimates using mitochondrial DNA sequences. In this study, making the best use of the still partial, but large-scale sequencing data of cartilaginous fish species, we estimate the divergence times between the major cartilaginous fish lineages employing nuclear genes. By rigorous orthology assessment based on available genomic and transcriptomic sequence resources for cartilaginous fishes, we selected 20 protein-coding genes in the nuclear genome, spanning 2973 amino acid residues. Our analysis based on the Bayesian inference resulted in the mean divergence time of 421 Ma, the late Silurian, for the Holocephali-Elasmobranchii split, and 306 Ma, the late Carboniferous, for the split between sharks and rays/skates. By applying these results and other documented divergence times, we measured the relative evolutionary rate of the Hox A cluster sequences in the cartilaginous fish lineages, which resulted in a lower substitution rate with a factor of at least 2.4 in comparison to tetrapod lineages. The obtained time scale enables mapping phenotypic and molecular changes in a quantitative framework. It is of great interest to corroborate the less derived nature of cartilaginous fish at the molecular level as a genome-wide phenomenon.
PMCID: PMC3692497  PMID: 23825540
24.  Transcriptomics of morphological color change in polychromatic Midas cichlids 
BMC Genomics  2013;14:171.
Animal pigmentation has received much attention in evolutionary biology research due to its strong implications for adaptation and speciation. However, apart from a few cases the genetic changes associated with these evolutionary processes remain largely unknown. The Midas cichlid fish from Central America are an ideal model system for investigating pigmentation traits that may also play a role in speciation. Most Midas cichlids maintain their melanophores and exhibit a grayish (normal) color pattern throughout their lives. A minority of individuals, however, undergo color change and exhibit a distinctive gold or even white coloration in adulthood. The ontogenetic color change in the Midas cichlids may also shed light on the molecular mechanisms underlying pigmentation disorders in humans.
Here we use next-generation sequencing (Illumina) RNAseq analyses to compare skin transcriptome-wide expression levels in three distinct stages of color transformation in Midas cichlids. cDNA libraries of scale tissue, for six biological replicates of each group, were generated and sequenced using Illumina technology. Using a combination of three differential expression (DE) analyses we identified 46 candidate genes that showed DE between the color morphs. We find evidence for two key DE patterns: a) genes involved in melanosomal pathways are up-regulated in normally pigmented fish; and b) immediate early and inflammatory response genes were up-regulated in transitional fish, a response that parallels some human skin disorders such as melanoma formation and psoriasis. One of the DE genes segregates with the gold phenotype in a genetic cross and might be associated with incipient speciation in this highly “species-rich” lineage of cichlids.
Using transcriptomic analyses we successfully identified key expression differences between different color morphs of Midas cichlid fish. These differentially expressed genes have important implications for our understanding of the molecular mechanisms underlying speciation in this lineage of extremely young species since they mate strongly assortatively, and new species may arise by sexual selection due to this color polymorphism. Some of the human orthologues of the genes identified here may also be involved in pigmentation differences and diseases and therefore provide genetic markers for the detection of human pigmentation disorders.
PMCID: PMC3623868  PMID: 23497064
RNAseq; Color change; Melanophore; Differential expression; Tyrosinase genes; Midas cichlids
25.  Genomics of adaptation and speciation in cichlid fishes: recent advances and analyses in African and Neotropical lineages 
Cichlid fishes are remarkably phenotypically diverse and species-rich. Therefore, they provide an exciting opportunity for the study of the genetics of adaptation and speciation by natural and sexual selection. Here, we review advances in the genomics and transcriptomics of cichlids, particularly regarding ecologically relevant differences in body shape, trophic apparatus, coloration and patterning, and sex determination. Research conducted so far has focused almost exclusively on African cichlids. To analyse genomic diversity and selection in a Neotropical radiation, we conducted a comparative transcriptomic analysis between sympatric, ecologically divergent crater-lake Midas cichlids (Lake Xiloá Amphilophus amarillo and Amphilophus sagittae). We pyrosequenced (Roche 454) expressed sequence tag (EST) libraries and generated more than 178 000 000 ESTs and identified nine ESTs under positive selection between these sister species (Ka/Ks > 1). None of these ESTs were found to be under selection in African cichlids. Of 11 candidate genes for ecomorphological differentiation in African cichlids, none showed signs of selection between A. amarillo and A. sagittae. Although more population-level studies are now needed to thoroughly document patterns of divergence during speciation of cichlids, available information so far suggests that adaptive phenotypic diversification in Neotropical and African cichlids may be evolving through non-parallel genetic bases.
PMCID: PMC3233715  PMID: 22201168
next-generation sequencing; cichlid fish; ecological speciation; coloration; body shape; expressed sequence tags

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