Received: 3 October 2018 | Revised: 15 February 2019 | Accepted: 22 February 2019 DOI: 10.1111/jzs.12286 ORIGINAL ARTICLE Pseudogenization of Mc1r gene associated with transcriptional changes related to melanogenesis explains leucistic phenotypes in Oreonectes cavefish (Cypriniformes, Nemacheilidae) Zhijin Liu1,2* | Huamei Wen1* | Frank Hailer3* | Fang Dong2,4* | Zuomin Yang5 | Tao Liu1 | Ling Han1 | Fanglei Shi2 | Yibo Hu2 | Jiang Zhou1,2 1State Key Laboratory Cultivation for Karst Mountain Ecology Environment of Guizhou Abstract Province, Guizhou Normal University, Organisms that have colonized underground caves encounter vastly different selec- Guiyang, Guizhou, China tive pressures than their relatives in above- ground habitats. While disruption of cer- 2CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, tain pigmentation genes has been documented in various cave- dwelling taxa, little is Chaoyang District, Beijing, China known about wider impacts across pigmentation and other gene pathways. We here 3School of Biosciences, Cardiff University, Cardiff, UK study the timeframe and transcriptional landscape of a leucistic and blind cypriniform 4Institute of Physical Science and fish (Oreonectes daqikongensis, Nemacheilidae) that inhabits karst caves in Guizhou, Information Technology, Anhui University, China. Based on data from the mitochondrial ND4, ND5, and Cytb genes, we show Hefei, Anhui, China that the divergence between O. daqikongensis and its most closely related pigmented 5School of Life Sciences, Qufu Normal University, Qufu, Shandong, China species occurred ca. 6.82 million years ago (95% HPD, 5.12–9.01), providing ample time for widespread phenotypic change. Indeed, we found that the DNA sequence of Correspondence Zhijin Liu, Yibo Hu and Jiang Zhou, CAS Mc1r (melanocortin- 1 receptor), a key gene regulating the biosynthesis of melanin in Key Laboratory of Animal Ecology and most vertebrates, is pseudogenized in O. daqikongensis, caused by a 29 bp deletion in Conservation Biology, Institute of Zoology, Chaoyang District, Beijing, China. the protein- coding region. Furthermore, 99,305 unigenes were annotated based on Emails: [email protected] (J. Z.); huyb@ the transcriptome of skin tissue of Oreonectes fish. Among the differentially ex- ioz.ac.cn (Y. H.); [email protected] (Z. L.) pressed unigenes, 7,326 (7.4% of the total unigenes) had decreased expression and Funding information 2,530 (2.5% of the total unigenes) had increased expression in O. daqikongensis skin. The Key Project of Science and Technology Program of Guizhou Province, Grant/Award As predicted, the expression of Mc1r and 18 additional genes associated with melanin Number: 3052 2015; Provincial Governor biosynthesis was significantly downregulated in the skin tissue of O. daqikongensis, Fund of Guizhou (Qian-Sheng-Zhuan-He-Zi (2010) 14); Youth Innovation Promotion but not in its congener. Our results, integrating with other studies on cavefishes, sug- Association of Chinese Academy of gest that loss of pigmentation was caused by coding region loss- of- function muta- Sciences, Grant/Award Number: 2011077; The Ministry of Science and Technology of tions along with widespread transcriptional changes, resulting from extended the People's Republic of China, Grant/Award evolutionary time as a cave- dwelling form. Number: 2005DKA21402 Contributing authors: Huamei Wen KEYWORDS ([email protected]); Frank Hailer (HailerF@ cavefish, frameshift mutation, leucism, Mc1r, Oreonectes daqikongensis, transcriptome cardiff.ac.uk); Fang Dong ([email protected]); Zuomin Yang ([email protected]); Tao Liu ([email protected]); Ling Han (ling_ling_han@ sina.com); Fanglei Shi ([email protected]) *These authors Contributed equally to this work. 900 | © 2019 Blackwell Verlag GmbH wileyonlinelibrary.com/journal/jzs J Zool Syst Evol Res. 2019;57:900–909. LIU et aL. | 901 1 | INTRODUCTION polymorphism in lesser snow geese (Anser caerulescens) and arctic skuas (Stercorarius parasiticus) is tightly associated with Uncovering the speciation process and genetic basis of phenotypic sequence variation within the Mc1r gene (Mundy et al., 2004). adaptations of animals to a specific environment is a key goal in evo- In fish, two different Mc1r alleles (963 and 969 bp long) were lutionary and comparative zoology. Underground rivers and caves found in wild populations of guppies (Poecilia reticulata) and in- are a globally widespread ecosystem that have been independently dividuals with the 963 bp Mc1r allele tended to show less black colonized by numerous animals (Bilandžija, Ma, Parkhurst, & Jeffeary, pigmentation than those with the 969 bp allele (Ayumi, Hiroaki, 2013; McGaugh et al., 2014). Since light is lacking year- round, many Jun, van Cock, & Masakado, 2011). animals inhabiting such habitats show degeneration of skin pigmen- The genus Oreonectes (Cypriniformes, Nemacheilidae) was first tation (Gross & Wilkens, 2013). However, the timeframes on which described by Günther (1868), with O. platycephalus as the type cave dwellers have evolved their specialized phenotypes, and the species, and a total of 18 species of Oreonectes are currently con- molecular mechanisms underlying such phenotypes, remain largely sidered as valid (Deng, Wen, Xiao, & Zhou, 2016; Günther, 1868). unknown. Besides O. platycephalus, Oreonectes fish are semi- cave- dwelling The gene coding for the melanocortin- 1 receptor (MC1R) plays a or cave- restricted species, rendering the genus an interesting key role in the regulation of melanin biosynthesis (Majerus & Mundy, model to study evolutionary consequences colonization of cave 2003). The MC1R receptor protein encoded by Mc1r gene belongs habitats (Deng et al., 2016). One particularly specialized species to the G- protein coupled receptors family, which has seven trans- is O. daqikongensis, which was only recently described (Deng et al., membrane α- helices. α- MSH (α- Melanocyte- Stimulating hormone) 2016). The species exhibit numerous typical characteristics of cave binds to and activates MC1R receptor at the membrane of melano- dwellers (e.g., lack of eyes), but is the only leucistic Oreonectes fish phore cells and promotes the biosynthesis of cAMP (cyclic adenos- which lack pigmentation (Figure 1a,b) (Deng et al., 2016). We here ine monophosphate), followed by the synthesis of melanin (Barsh & ask the following evolutionary questions: What is the timeframe Cotsarelis, 2007). High levels of basal Mc1r signaling cause increased on which O. daqikongensis evolved the leucistic phenotype? Is the expression of the microphthalmia- associated transcription factor Mc1r gene involved in the occurrence of leucism? More broadly, (MITF), tyrosinase (TYR), tyrosinase- related protein 1 (TYRP1), and how is the translational landscape of the melanogenesis pathway other melanogenesis- related genes, which leads to increased eumel- changed in this species? To answer these questions, we first re- anin synthesis (Nishimura, 2011). constructed a dated phylogeny of five Oreonectes species based Mc1r has been identified as the key candidate gene for ani- on mitochondrial Cytb, ND4, and ND5 gene sequences. To de- mal pigmentation, and several Mc1r mutations have been shown termine whether the Mc1r gene is functional in O. daqikongensis, to explain the presence of leucistic/light- colored phenotypes the locus was sequenced in O. daqikongensis and another three (Majerus & Mundy, 2003). Leucism describes a partial loss of Oreonectes species. Finally, to illuminate the evolutionary impacts pigmentation, with phenotypes such as white or patchily white- of long- term cave- dwelling on Oreonectes fishes, determine the colored skin, hair, or feathers, but pigmentation still being par- function of other melanogenesis- related genes in O. daqikongensis, tially present, for example, the pigment cells in the eyes which we further collected skin tissue samples of O. daqikongensis and are not affected by the leucistic condition. Albinism, on the its close- related species with pigmentation and performed whole- other hand, is a phenotype with complete absence of melanin, transcriptome sequencing. resulting in albinistic skin, feathers/hair, and eyes. Vertebrates with albinism are not only white (or sometimes pale yellowish) 2 | MATERIALS AND METHODS in exterior coloration, but also possess pale eyes, often pink or red in color due to blood vessels showing through. In mam- 2.1 | Sample collection mals, the leucistic phenotype of Ursus americanus kermodei is caused by the substitution of tyrosine by cysteine at the 298th Fifteen individuals of O. daqikongensis, 16 O. jiarongensis individuals, amino acid (Y298C) of MC1R (Ritland, Newton, & Marshall, six O. dongliangensis individuals, and ten O. shuilongensis individu- 2001). The substitution of arginine by cysteine at 18th amino als were collected in Libo and Sando county of Guizhou Province acid and arginine by tryptophan at the 109th amino acid (R18C (Figure 1). These samples have been deposited in the Museum of and R109W) of MC1R also results in the light- coated phenotype Guizhou Normal University (for catalog numbers, see Table 1). of Chaetodipus intermedius (Nachman, Hoekstra, & D'Agostino, 2003). In reptiles, the light- colored skins are also associated 2.2 | Phylogenetic analyses and divergence with mutations of Mc1r in lizards and geckos (Machado et al., time estimation 2018; Mendes, Salvi, Harris, Els, & Carranza, 2018). In birds, the leucistic phenotype of the plumage in Falco rusticolus