G C A T T A C G G C A T genes Article The Genetic Basis of Scale-Loss Phenotype in the Rapid Radiation of Takifugu Fishes Dong In Kim y, Wataru Kai y, Sho Hosoya y, Mana Sato, Aoi Nozawa, Miwa Kuroyanagi, Yuka Jo, Satoshi Tasumi, Hiroaki Suetake, Yuzuru Suzuki and Kiyoshi Kikuchi * Fisheries Laboratory, University of Tokyo, Maisaka, Shizuoka 431-0214, Japan; [email protected] (D.I.K.); Wataru.Kai@fluidigm.com (W.K.); [email protected] (S.H.); [email protected] (M.S.); [email protected] (A.N.); [email protected] (M.K.); [email protected] (Y.J.); tasumi@fish.kagoshima-u.ac.jp (S.T.); [email protected] (H.S.); [email protected] (Y.S.) * Correspondence: [email protected] These authors contributed equally to this work. y Received: 16 October 2019; Accepted: 3 December 2019; Published: 10 December 2019 Abstract: Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development. Keywords: QTL mapping; rapid adaptation; phenotypic divergence; pufferfish; scale morphology; convergence; developmental diversity 1. Introduction Rapid evolutionary radiation generates both phenotypic disparity and similarity. Classic examples of the rapid radiation include finch birds, silversword plants, anole lizards, threespine sticklebacks, and cichlid fishes [1–6]. Recent advances in genomics have enabled the study of the genetic basis of phenotypic evolution associated with rapid adaptation not only in the classic examples but also in many other groups of organisms [7,8]. Interestingly, this line of research also allows investigations into the predictability of evolutionary change [9–13]. However, more studies are needed for a better understanding of genetic architecture that promotes diversification, which would facilitate the stable prediction of evolutionary changes. The scale is attractive for comparative studies of phenotypic evolution since it is widely distributed among vertebrates, and shows an impressive diversity in morphology [14–16]. For example, scale loss or reduction is a recurring feature in a number of teleost lineages, such as anguilliformes (eels), Genes 2019, 10, 1027; doi:10.3390/genes10121027 www.mdpi.com/journal/genes Genes 2019, 10, 1027 2 of 18 Genes 2019, 10, x FOR PEER REVIEW 2 of 18 cypriniformes (carps), gasterosteiformes (sticklebacks), and siluriformes (catfish) [17–23]. Detailed (catfish)studies on[17–23]. the three-spine Detailed studie sticklebacks on the ( Gasterosteusthree-spine stickleback aculeatus Linnaeus) (Gasterosteus have aculeatus revealed Linnaeus) that a specific have revealedallele of thethat ectodysplasin a specific allele (Eda of )the gene, ectodysplasin fixed in most (Eda freshwater) gene, fixed populations, in most freshwater is associated populations, with the isconvergent associated loss with of the lateral convergent plates (modified loss of lateral scales) plates [17 ,(modified24]. In addition, scales) mutations[17,24]. In addition, in fibroblast mutations growth infactor fibroblast receptor growth 1a1 gene factor (Fgfr1a1 receptor) are 1a1 associated gene (Fgfr1a1 with the) are scale associated reduction with of common the scale carp reduction(Cyprinus of commoncarpio L.) andcarp zebrafish(Cyprinus (Danio carpio rerio L.) andHamilton), zebrafish whereas (Danio a rerio mutation Hamilton), in the ectodysplasinwhereas a mutation receptor in gene the ectodysplasin(Edar) leads to receptor almost complete gene (Edar loss) leads of scales to almost in medaka complete (Oryzias losslatipes of scalesTemminck in medaka & Schlegel) (Oryzias [latipes22,25]. TemminckFugu (&Takifugu Schlegel) rubripes [22,25].Temminck & Schlegel) and its closely related species, being a prominent exampleFugu of (Takifugu recent adaptive rubripes radiationTemminck in & marine Schlegel) fishes, and provide its closely an opportunityrelated species, for being understanding a prominent the examplegenetic basis of recent of phenotypic adaptive radiation evolution in of marine the scale fishes, morphology provide an for opportunity the reasons setfor forthunderstanding below. Genus the geneticTakifugu basisunderwent of phenotypic an explosive evolution radiation of the scale in the morphology last 2–5 million for the years, reasons resulting set forth in approximately below. Genus Takifugu20 extant underwent species, and an the explosive evolutionary radiation history in the has last been 2–5 studied million foryears, 15 of resulting them [26 in,27 approximately]. While most 20Takifugu extantspecies species, have and spinythe evolutio scalesnary covering history the has integument been studied (the for scale-covered 15 of them phenotype),[26,27]. While at most least Takifugufive species species show have a loss spiny or massivescales covering reduction the in integu the numberment (the of scales,scale-covered resulting phenotype), in the “scale-loss” at least fiveor “scale-uncovered” species show a loss phenotype or massive [ 28reduction] (Figure in1). the Interestingly, number of scales, previous resulting phylogenetic in the “scale-loss” studies and or “scale-uncovered”phenotypic descriptions phenotype showed [28] incongruences (Figure 1). Inte betweenrestingly, phylogenies previous derived phylogenetic from mitochondrial studies and phenotypicDNA (mtDNA) descriptions data and showed morphological incongruences data [28 ,between29] (Figure phylogenies1). These incongruences derived from implymitochondrial that the DNAscale-uncovered (mtDNA) data phenotype and morphological evolved repeatedly data [28,29] in this (Figure genus, 1). althoughThese incongruences a robust phylogeny imply that using the scale-uncoverednuclear genes of phenotype the Takifugu evolved fishes repeatedly is currently in lacking. this genus, In addition, althoughTakifugu a robustcan phylogeny produce fertileusing nuclearprogeny genes when of crossed the Takifugu with closely fishes related is currently species, lacking. which permitsIn addition, the genetic Takifugu mapping can produce of implicated fertile progenygenes [30 when–32]. crossed with closely related species, which permits the genetic mapping of implicated genes [30–32]. FigureFigure 1. 1. PhylogeneticPhylogenetic relationship relationship of of 14 14 TakifuguTakifugu andand 1 1 other other pufferfishes, pufferfishes, and and their their scale scale phenotypes. phenotypes. The phylogeny isis redrawredraw using using data data from from [29 [29]] where where mitochondrial mitochondrial DNA DNA data data of 14 ofTakifugu 14 Takifuguandother and otherrelated related species species have beenhave analyzedbeen analyzed using usin maximumg maximum likelihood likelihood method. method. Nodes Nodes supported supported by high by highbootstrap bootstrap values values ( 80%) (≥80%) are shown are shown in black in circles.black circles. Scale barScale represents bar represents 5 million 5 million years before years present.before ≥ present.Also shown Also was shown a summary was a summary of previous of previous results fromresults [28 from] on the[28] scale on the phenotypes scale phenotypes for each for species. each species.The scale-covered The scale-covered (red) or-uncovered (red) or -uncovered (blue) phenotypes (blue) phenotypes are illustrated are byillustrated colored squaresby colored on thesquares right onof eachthe right species. of each Examples species. ofExamples mineralized of mineralized scales on thescales ventral on the surface ventral of surface the body of the of body three of species, three species,T. pardalis T., pardalisT. niphobles, T. niphoblesand T. snyderi and T., were snyderi shown, were under shown the under picture the of picture each species. of each species. Here,Here, we we aim aim to to understand the the genetic and developmental basi basiss of morphological divergence/convergencedivergence/convergence associatedassociated with with rapid rapid radiation radiation of ofTakifugu Takifuguspecies species inmarine in marine ecosystems. ecosystems. To this To thisend, end, we focusedwe focused on one on one species species with with the scale-coveredthe scale-covered phenotype phenotype (T. niphobles (T. niphoblesJordan Jordan & Snyder) & Snyder) and and two species with the scale-uncovered