ARTICLE Received 3 Mar 2015 | Accepted 12 Jun 2015 | Published 17 Jul 2015 DOI: 10.1038/ncomms8814 OPEN Insect glycerol transporters evolved by functional co-option and gene replacement Roderick Nigel Finn1,2, Franc¸ois Chauvigne´1,3, Jon Anders Stavang1, Xavier Belles4 & Joan Cerda`3 Transmembrane glycerol transport is typically facilitated by aquaglyceroporins in Prokaryota and Eukaryota. In holometabolan insects however, aquaglyceroporins are absent, yet several species possess polyol permeable aquaporins. It thus remains unknown how glycerol transport evolved in the Holometabola. By combining phylogenetic and functional studies, here we show that a more efficient form of glycerol transporter related to the water-selective channel AQP4 specifically evolved and multiplied in the insect lineage, resulting in the replacement of the ancestral branch of aquaglyceroporins in holometabolan insects. To recapitulate this evolutionary process, we generate specific mutants in distantly related insect aquaporins and human AQP4 and show that a single mutation in the selectivity filter con- verted a water-selective channel into a glycerol transporter at the root of the crown clade of hexapod insects. Integration of phanerozoic climate models suggests that these events were associated with the emergence of complete metamorphosis and the unparalleled radiation of insects. 1 Department of Biology, Bergen High Technology Center, University of Bergen, PO Box 7803, N-5020 Bergen, Norway. 2 Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway. 3 Institut de Recerca i Tecnologia Agroalimenta`ries (IRTA)-Institut de Cie`ncies del Mar, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Passeig Marı´tim 37-49, 08003 Barcelona, Spain. 4 Institute of Evolutionary Biology (CSIC-Universitat Pompeu Fabra), Passeig Marı´tim 37-49, 08003 Barcelona, Spain. Correspondence and requests for materials should be addressed to R.N.F. (email: nigel.fi[email protected]) or to J.C. (email: [email protected]). NATURE COMMUNICATIONS | 6:7814 | DOI: 10.1038/ncomms8814 | www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8814 stablished models suggest that evolutionary novelty typically observed in engineered immotile strains of bacteria that can arises from gene duplication followed by gradual neo- recover their lost flagella in just a few mutational steps3. Whether Efunctionalization or subfunctionalization1,2. Due to the real world examples of such functional co-option and gene modular and interrelated nature of multigene families, however, replacement have occurred through natural selection remains to an alternative non-linear pathway of gene evolution could occur if be established. a chance mutation in a duplicated gene lead to the gain-of- In terms of numbers of species, the holometabolan insects function of a distantly related member of the superfamily. The (Holometabola; or superorder Endopterygota) that undergo potential for such a rewired evolutionary pathway has been complete metamorphosis mediated by a unique pupal stage are the most successful clade of organisms in the history of life, with current estimates accounting for more than half of the world’s 12 Diptera: Brachycera - Bib 4 5 Diptera: Nematocera - Bib eukaryotic biodiversity . As in other insects, holometabolan 2 Lepidoptera - Bib species can accumulate high levels of colligative polyols, such as > 95% 0.4 10 Hymenoptera - Bib > 90% Coleoptera - Bib glycerol or sorbitol, as an adaptive response to dessication and > 80% Psocodea - Bib Bib 5–7 > 70% Hemiptera - Bib freezing temperatures . While maximum glycerol accumulation > 50% Blattodea - Bib 8–10 Malacostraca - Bib and cold tolerance predominantly occurs in the pupal stage , 2 Ixodida - Bib recent phylogenetic studies of insect aquaporins have suggested Branchiopoda - Bib 2 Lepidoptera - Aqp that members of the aquaporin gene superfamily known as aquaglyceroporin (Glp) channels, which typically transport water 14 Diptera: Brachycera - Drip 11–13 5 Diptera: Nematocera - Drip and glycerol, are absent in holometabolan insects . This Hemiptera: Sternorrhyncha - Drip notion nevertheless contrasts with experimental studies showing Psocodea - Drip that some aquaporins from different insect species can transport Blattodea - Drip 14–19 Isoptera - Drip Drip glycerol . Such opposing observations represent a paradox Hemiptera: Euhemiptera - Drip 10 Lepidoptera - Drip since it has been established that the aquaporin superfamily split 11 Hymenoptera - Drip into two major phyletic branches before the evolution of 2 Coleoptera - Drip Collembola - Drip Eukaryota, one that includes multifunctional Glps and the other 13,20,21 14 Diptera: Brachycera - Prip water-selective aquaporins . The structural basis for the 6 Diptera: Nematocera - Prip P. vanderplanki - Prip selective aquaporin permeation properties was resolved through 6 Lepidoptera - Prip crystallographic studies, which determined that conserved central 10 Hymenoptera - Prip Prip Hemiptera: Euhemiptera - Prip Asn-Pro-Ala (NPA) motifs and a quartet of aromatic arginine Orthoptera - Prip (ar/R) residues in the outer channel vestibule form the major B.germanica - Prip 22,23 3 Coleoptera - Prip selectivity filters . Experimental studies have also shown that Psocodea - Prip Archeognatha - Prip at least two ar/R point mutations are required to convert a typical Collembola - Prip Malacostraca - Prip water-selective channel, such as mammalian aquaporin-1 Branchiopoda - Prip 24 2 Maxillipoda - Prip-like (AQP1), into a glycerol transporter , yet no such substitutions have been observed in the vertically transferred aquaporin 13 Diptera: Brachycera - Eglp 4 13 Diptera: Brachycera - Eglp 3 repertoires of extant biota, despite billions of years of 10 25 10 Diptera: Brachycera - Eglp 2 evolution . We therefore phylogenetically investigated the interrelationships of insect aquaporins and glycerol transporters 4 in relation to the genomic repertoires of other arthropods and Diptera: Nematocera - Eglp 2 P.vanderplanki - Eglp used mutagenesis studies to experimentally probe the origin and Psocodea - Eglp 2 functional evolution of the insect glycerol transporters. Our data 8 Lepidoptera - Eglp 1–4 Hemiptera: Euhemiptera - Eglp 2 Unclassified reveal that mutated water-selective channels specifically co-opted 15 Diptera: Brachycera - Eglp 1 = Eglp the glycerol transport function of the ancestral Glps in the oldest 4 Diptera: Nematocera - Eglp 1 Hemiptera: Euhemiptera - Eglp 1 lineages of hexapod insects. Uniquely, these new, more efficient 4 Coleoptera - Eglp 1–4 forms of glycerol transporters were positively selected in the 9 Eglp 3 8 Eglp 2 Hymenoptera megadiverse holometabolan insects resulting in the replacement 8 Eglp 1 of the Glps as the major vehicles of polyol conductance. B. germanica - Eglp Isoptera - Eglp Psocodea - Eglp 1 3 Hemiptera: Sternorryncha - Eglp Results and discussion Bacteria 3 AqpZ Insects genomes encode non-classical glycerol transporters.To resolve the glycerol paradox in holometabolan insects, we con- 9 Chelicerata - Glp 10 Hexapoda- Glp ducted an indepth phylogenetic analysis of 269 non-redundant 5 Branchiopoda - Glp Archaea - AqpM arthropod aquaporins assembled from available whole-genome Glp 4 and transcriptome shotgun sequences. These data show that the Bacteria - GlpF arthropod orthodox aquaporin superfamily consists of five major subfamilies, including classical Glps, water and urea-transporting Figure 1 | Molecular phylogeny of arthropod orthodox aquaporins. channels termed Pyrocoelia rufa integral proteins (Prip)26,27, The tree was inferred through maximum likelihood (3,002,760 heuristic water-specific channels termed Drosophila intrinsic proteins rearrangements) and Bayesian analysis (15 million MCMC generations) of (Drip)28–32, the cation permeating big brain channels (Bib)33,34, 219,583 nucleotide sites in a codon alignment of 269 non-redundant and a previously unclassified clade (Fig. 1). A sixth group of aquaporins, and was rooted with aqpM. The number of taxa in collapsed unorthodox aquaporins related to the vertebrate Aqp12 channels (triangular) nodes are indicated, with coloured circles at each node is also identified in all insects and other protostome animals indicating posterior probabilites as defined by the key. Scale bar represents (Supplementary Table 1). Although we identify glp genes in the rate of nucleotide substitution per site. Dotted lines and insect images approximately half of the extant orders of insects (see below), illustrate the conducted experiments. BLAST searches using nucleotides or peptides corresponding to 2 NATURE COMMUNICATIONS | 6:7814 | DOI: 10.1038/ncomms8814 | www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms8814 ARTICLE the exons as queries did not uncover classical glps in 46 of the 47 Prip-like proteins in the four extant lineages of arthropods, species of holometabolan insect studied. Only an N-terminal Chelicerata (scorpions, spiders and ticks), Myriapoda fragment corresponding to the first of seven exons of the human (centipedes), Crustacea (copepods and water fleas) and body louse (Pediculus humanus) glp was detected in the Hexapoda (insects). The results provide robust statistical neuropteran Ant lion (Euroleon nostras). To support these evidence that the neofunctionalized glycerol-transporting genes observations, we conducted an alignview synteny analysis