DOI: 10.5061/dryad.qm364n4
title: Data from: Convergent evolution of cytochrome P450s underlies independent origins of keto-carotenoid pigmentation in animals

description: Keto-carotenoids contribute to many important traits in animals, including
 vision and coloration. In a great number of animal species,
 keto-carotenoids are endogenously produced from carotenoids by carotenoid
 ketolases. Despite the ubiquity and functional importance of
 keto-carotenoids in animals, the underlying genetic architectures of their
 production have remained enigmatic. The body and eye colorations of spider
 mites (Arthropoda: Chelicerata) are determined by β-carotene and
 keto-carotenoid derivatives. Here, we focus on a carotenoid pigment mutant
 of the spider mite Tetranychus kanzawai that, as shown by chromatography,
 lost the ability to produce keto-carotenoids. We employed bulked segregant
 analysis and linked the causal locus to a single narrow genomic interval.
 The causal mutation was fine-mapped to a minimal candidate region that
 held only one complete gene, the cytochrome P450 monooxygenase CYP384A1,
 of the CYP3 clan. Using a number of genomic approaches, we revealed that
 an inactivating deletion in the fourth exon of CYP384A1 caused the
 aberrant pigmentation. Phylogenetic analysis indicated that CYP384A1 is
 orthologous across mite species of the ancient Trombidiformes order where
 carotenoids typify eye and body coloration, suggesting a deeply conserved
 function of CYP384A1 as a carotenoid ketolase. Previously, CYP2J19, a
 cytochrome P450 of the CYP2 clan, has been identified as a carotenoid
 ketolase in birds and turtles. Our study shows that selection for
 endogenous production of keto-carotenoids led to convergent evolution
 whereby cytochrome P450s were independently co-opted in vertebrate and
 invertebrate animal lineages.
description: De novo genome
 assemblies of lemon selected and wild-type selected mites from a
 segregating T. kanzawai population and its lemon and wild-type
 parent.The segregating mite
 population was generated by crossing Jp-inbred-lemon to Jp2-WT and was
 used to genetically map the lemon phenotype in Tetranychus kanzawai. After
 approximately 10-12 generations, a total of three replicates were
 collected that consisted of 1100, 900 and 500 adult lemon females. In
 parallel, 1500 wild-type females were selected and pooled into a single
 sample. From these four populations and the two parental strains, genomic
 DNA was extracted and assembled using the CLC Genomics Workbench 9.0.1
 (https://www.qiagenbioinformatics.com/).de_novo_Tkanzawai_genome_assemblies.rarLocalized de novo transcriptome assemblies of CYP384A1 of a T. kanzawai population that segregates for lemon pigmentation.RNA was extracted from 110 lemon and wild-type adult females in two replicates using an RNeasy Minikit (Qiagen). RNA reads that aligned to CYP384A1 were assembled into contigs using Trinity 2.5.1.localized_de_novo_Tkanzawai_transcriptome_assemblies.rarβ-carotene and astaxanthin levels detected by HPLC in wild-type and lemon T. kanzawai.For each treatment 30 females were collected in three replicates and homogenized in 1 ml of acetone. The homogenate was filtrated using a glass syringe with a membrane possessing a pore size of 0.45 μm. The filtrate was dried under a nitrogen gas flow and dissolved in 300 μl of methanol. Five μl of the solution was used for HPLC analysis. Carotenoids were quantified by monitoring the absorbance at 450 nm. External calibration curves were constructed with authentic standards.carotenoid_levels_Tkanzawai_wild-type_lemon.txt