Penguins (Sphenisciformes) are an iconic order of flightless, diving seabirds distributed across a large latitudinal range in the Southern Hemisphere. The extensive area over which penguins are endemic is likely to have fostered variation in pathogen pressure, which in turn will have imposed differential selective pressures on the penguin immune system. At the front line of pathogen detection and response, the Toll-like receptors (TLRs) provide insight into host evolution in the face of microbial challenge. TLRs respond to conserved pathogen-associated molecular patterns and are frequently found to be under positive selection, despite retaining specificity for defined agonist classes. We undertook a comparative immunogenetics analysis of TLRs for all penguin species and found evidence of adaptive evolution that was largely restricted to the cell surface-expressed TLRs, with evidence of positive selection at, or near, key agonist-binding sites in TLR1B, TLR4, and TLR5. Intriguingly, TLR15, which is activated by fungal products, appeared to have been pseudogenized multiple times in the Eudyptes spp., but a full-length form was present as a rare haplotype at the population level. However, in vitro analysis revealed that even the full-length form of Eudyptes TLR15 was nonfunctional, indicating an ancestral cryptic pseudogenization prior to its eventual disruption multiple times in the Eudyptes lineage. This unusual pseudogenization event could provide an insight into immune adaptation to fungal pathogens such as Aspergillus, which is responsible for significant mortality in wild and captive bird populations.

BGI Shenzhen Beishan Industrial Zone Yantian District Shenzhen China

Biology Department Reedley College Reedley CA USA

Center for Excellence in Animal Evolution and Genetics Chinese Academy of Sciences Kunming China

Departamento de Ecosistemas y Medio Ambiente Facultad de Agronomía e Ingeniería Forestal Pontificia Universidad Católica de Chile Macul Santiago Chile

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Department of Zoology University of Oxford Oxford United Kingdom

Institute of Palaeoanatomy Domestication Research and the History of Veterinary Medicine Faculty of Veterinary Medicine Ludwig Maximilian University of Munich Munich Germany

Jenner Institute University of Oxford Oxford United Kingdom

School of Biological and Chemical Sciences Fogg Building Queen Mary University of London London United Kingdom

Section for Ecology and Evolution Department of Biology University of Copenhagen Copenhagen Denmark

State Key Laboratory of Genetic Resources and Evolution Kunming Institute of Zoology Chinese Academy of Sciences Kunming China

The Palaeogenomics and Bio Archaeology Research Network Research Laboratory for Archaeology and History of Art University of Oxford Oxford United Kingdom

Wildlife Health Services Zoological Society of London London United Kingdom

Zobrazit více v PubMed

Alcaide M, Edwards SV.. 2011. Molecular evolution of the Toll-like receptor multigene family in birds. Mol Biol Evol. 28(5):1703–1715. PubMed

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ.. 1990. Basic local alignment search tool. J Mol Biol. 215(3):403–410. PubMed

Andersen-Nissen E, Smith KD, Strobe KL, Barrett SLR, Cookson BT, Logan SM, Aderem A.. 2005. Evasion of Toll-like receptor 5 by flagellated bacteria. Proc Natl Acad Sci U S A. 102(26):9247–9252. PubMed PMC

Arbour NC, Lorenz E, Schutte BC, Zabner J, Kline JN, Jones M, Frees K, Watt JL, Schwartz DA.. 2000. TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nat Genet. 25(2):187–191. PubMed

Areal H, Abrantes J, Esteves PJ.. 2011. Signatures of positive selection in Toll-like receptor (TLR) genes in mammals. BMC Evol Biol. 11:368. PubMed PMC

AZA Penguin Taxon Advisory Group. 2014. Penguin (Spheniscidae) care manual. Silver Spring (MD): Association of Zoos and Aquariums.

Bainova H, Kralova T, Bryjova A, Albrecht T, Bryja J, Vinkler M.. 2014. First evidence of independent pseudogenization of Toll-like receptor 5 in passerine birds. Dev Comp Immunol. 45(1):151–155. PubMed

Balakirev ES, Ayala FJ.. 1996. Is esterase-P encoded by a cryptic pseudogene in Drosophila melanogaster? Genetics 144(4):1511–1518. PubMed PMC

Beernaert LA, Pasmans F, Van Waeyenberghe L, Haesebrouck F, Martel A.. 2010. Aspergillus infections in birds: a review. Avian Pathol. 39(5):325–331. PubMed

Bodenhofer U, Bonatesta E, Horejš-Kainrath C, Hochreiter S.. 2015. msa: an R package for multiple sequence alignment. Bioinformatics 31(24):3997–3999. PubMed

Boyd A, Philbin VJ, Smith AL.. 2007. Conserved and distinct aspects of the avian Toll-like receptor (TLR) system: implications for transmission and control of bird-borne zoonoses. Biochem Soc Trans. 35(Pt 6):1504–1507. PubMed

Boyd AC, Peroval MY, Hammond JA, Prickett MD, Young JR, Smith AL.. 2012. TLR15 Is unique to avian and reptilian lineages and recognizes a yeast-derived agonist. J Immunol. 189(10):4930–4938. PubMed

Browning BL, Zhou Y, Browning SR.. 2018. A one-penny imputed genome from next-generation reference panels. Am J Hum Genet. 103(3):338–348. PubMed PMC

Browning SR, Browning BL.. 2007. Rapid and accurate haplotype phasing and missing-data inference for whole-genome association studies by use of localized haplotype clustering. Am J Hum Genet. 81(5):1084–1097. PubMed PMC

Brownlie R, Allan B, 2011. Avian toll-like receptors 2001. Cell Tissue Res. 343:121–130. PubMed

Carrasco L, Lima JS, Halfen DC, Salguero FJ, Sánchez-Cordon P, Becker G.. 2001. Systemic aspergillosis in an oiled magallanic penguin (Spheniscus magellanicus). J Vet Med B Infect Dis Vet Public Health 48:551–554. PubMed

Choi Y, Chan AP.. 2015. PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 31(16):2745–2747. PubMed PMC

Clements J, Schulenberg T, Iliff M, Billerman S, Fredericks T, Sullivan B, Wood C.. 2019. The eBird/Clements Checklist of Birds of the World: v2019. Available from: https://www.birds.cornell.edu/clementschecklist/download/.

Cohen JM, Sauer EL, Santiago O, Spencer S, Rohr JR.. 2020. Divergent impacts of warming weather on wildlife disease risk across climates. Science 370(6519):eabb1702. PubMed PMC

Cole TL, Dutoit L, Dussex N, Hart T, Alexander A, Younger JL, Clucas GV, Frugone MJ, Cherel Y, Cuthbert R, et al.2019a. Receding ice drove parallel expansions in Southern Ocean penguins. Proc Natl Acad Sci U S A. 116(52):26690–26696. PubMed PMC

Cole TL, Ksepka DT, Mitchell KJ, Tennyson AJD, Thomas DB, Pan H, Zhang G, Rawlence NJ, Wood JR, Bover P, et al.2019. b. Mitogenomes uncover extinct penguin taxa and reveal island formation as a key driver of speciation. Mol Biol Evol. 36(4):784–797. PubMed

Cumming GS, Guegan JF.. 2006. Food webs and disease: is pathogen diversity limited by vector diversity? Ecohealth 3(3):163–170.

Cummings SM, McMullan M, Joyce DA, van Oosterhout C.. 2010. Solutions for PCR, cloning and sequencing errors in population genetic analysis. Conserv Genet. 11(3):1095–1097.

Dalton DL, Vermaak E, Roelofse M, Kotze A.. 2016. Diversity in the toll-like receptor genes of the African Penguin (Spheniscus demersus). PLoS One 11(10):e0163331. PubMed PMC

de Zoete MR, Bouwman LI, Keestra AM, van Putten JPM.. 2011. Cleavage and activation of a Toll-like receptor by microbial proteases. Proc Natl Acad Sci U S A. 108(12):4968–4973. PubMed PMC

Delport W, Poon AFY, Frost SDW, Pond SLK.. 2010. Datamonkey 2010: a suite of phylogenetic analysis tools for evolutionary biology. Bioinformatics 26(19):2455–2457. PubMed PMC

DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, del Angel G, Rivas MA, Hanna M, et al.2011. A framework for variation discovery and genotyping using nextgeneration DNA sequencing data. Nat Genet. 43(5):491–498. PubMed PMC

Dionne M, Miller KM, Dodson JJ, Caron F, Bernatchez L.. 2007. Clinal variation in MHC diversity with temperature: evidence for the role of host-pathogen interaction on local adaptation in Atlantic salmon. Evolution 61(9):2154–2164. PubMed

Dunnett CW. 1955. A multiple comparison procedure for comparing several treatments with a control. J Am Stat Assoc. 50(272):1096–1121.

Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32(5):1792–1797. PubMed PMC

Ferwerda B, McCall MBB, Alonso S, Mouktaroudi M, Giamarellos-Bourboulis EJ, Izagirre N, Syafruddin D, Kibiki G, Cristea T, Hijmans A, et al.2007. TLR4 polymorphisms, infectious diseases, and evolutionary pressure during migration of modern humans. Proc Natl Acad Sci U S A. 104(42):16645–16650. PubMed PMC

Flach EJ, Stevenson MF, Henderson GM.. 1990. Aspergillosis in Gentoo penguins (Pygoscelis papua) at Edinburgh Zoo, 1964 to 1988. Vet Rec. 126:81–85. PubMed

Fornůsková A, Vinkler M, Pagès M, Galan M, Jousselin E, Cerqueira F, Morand S, Charbonnel N, Bryja J, Cosson J-F.. 2013. Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE). BMC Evol Biol. 13:194. PubMed PMC

Frugone MJ, López ME, Segovia NI, Cole TL, Lowther A, Pistorius P, Dantas GPM, Petry MV, Bonadonna F, Trathan P, et al.2019. More than the eye can see: genomic insights into the drivers of genetic differentiation in Royal/Macaroni penguins across the Southern Ocean. Mol Phylogenet Evol. 139:106563. PubMed

Frugone MJ, Lowther A, Noll D, Ramos B, Pistorius P, Dantas GPM, Petry MV, Bonadonna F, Steinfurth A, Polanowski A, et al.2018. Contrasting phylogeographic pattern among Eudyptes penguins around the Southern Ocean. Sci Rep. 8(1):17481. PubMed PMC

Garate JA, Oostenbrink C.. 2013. Lipid A from lipopolysaccharide recognition: structure, dynamics and cooperativity by molecular dynamics simulations. Proteins 81(4):658–674. PubMed

Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA III, Smith HO.. 2009. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6(5):343–U341. PubMed

Graczyk TK, Cockrem JF.. 1995. Aspergillus spp seropositivity in New Zealand penguins. Mycopathologia 131(3):179–184. PubMed

Grueber CE, Wallis GP, Jamieson IG.. 2014. Episodic positive selection in the evolution of avian Toll-like receptor innate immunity genes. PLoS One 9(3):e89632. PubMed PMC

Hawn TR, Verbon A, Lettinga KD, Zhao LP, Li SS, Laws RJ, Skerrett SJ, Beutler B, Schroeder L, Nachman A, et al.2003. A common dominant TLR5 stop codon polymorphism abolishes flagellin signaling and is associated with susceptibility to Legionnaires’ disease. J Exp Med. 198(10):1563–1572. PubMed PMC

Higgs R, Cormican P, Cahalane S, Allan B, Lloyd AT, Meade K, James T, Lynn DJ, Babiuk LA, O’Farrelly C.. 2006. Induction of a novel chicken Toll-like receptor following Salmonella enterica serovar Typhimurium infection. Infect Immun. 74(3):1692–1698. PubMed PMC

Hocken AG. 2000. Cause of death in blue penguins (Eudyptula m. minor) in North Otago, New Zealand. N Z J Zool. 27(4):305–309.

Huang Y, Temperley ND, Ren L, Smith J, Li N, Burt DW.. 2011. Molecular evolution of the vertebrate TLR1 gene family–a complex history of gene duplication, gene conversion, positive selection and co-evolution. BMC Evol Biol. 11:149. PubMed PMC

Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee HY, Lee JO.. 2007. Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130(6):1071–1082. PubMed

Kall L, Krogh A, Sonnhammer ELL.. 2004. A combined transmembrane topology and signal peptide prediction method. J Mol Biol. 338(5):1027–1036. PubMed

Kall L, Krogh A, Sonnhammer ELL.. 2007. Advantages of combined transmembrane topology and signal peptide prediction - the Phobius web server. Nucleic Acids Res. 35(Web Server issue):W429–W432. PubMed PMC

Kamiya T, O’Dwyer K, Nakagawa S, Poulin R.. 2014. Host diversity drives parasite diversity: metaanalytical insights into patterns and causal mechanisms. Ecography 37(7):689–697.

Keestra AM, de Zoete MR, Bouwman LI, van Putten JPM.. 2010. Chicken TLR21 is an innate CpG DNA receptor distinct from mammalian TLR9. J Immunol. 185(1):460–467. PubMed

Khan ZU, Pal M, Paliwal DK, Damodaran VN.. 1977. Aspergillosis in imported penguins. Sabouraudia 15(1):43–45. PubMed

Krol L, Moore RP, Mutlow AG, Brady SM, Dorsa D.. 2020. A retrospective analysis of mortality in captive Magellanic penguins (Spheniscus magellanicus) in the United States, 2008-2018. Zoo Biol. 39(6):405–410. PubMed

Králová T, Albrecht T, Bryja J, Hořák D, Johnsen A, Lifjeld JT, Novotný M, Sedláček O, Velová H, Vinkler M.. 2018. Signatures of diversifying selection and convergence acting on passerine Toll-like receptor 4 in an evolutionary context. Mol Ecol. 27(13):2871–2883. PubMed

Levy H, Fiddaman SR, Vianna JA, Noll D, Clucas GV, Sidhu JKH, Polito MJ, Bost CA, Phillips RA, Crofts S, et al.2020. Evidence of pathogen-induced immunogenetic selection across the large geographic range of a wild seabird. Mol Biol Evol. 37(6):1708–1726. PubMed PMC

Li C, Zhang Y, Li J, Kong L, Hu H, Pan H, Xu L, Deng Y, Li Q, Jin L, et al.2014. Two Antarctic penguin genomes reveal insights into their evolutionary history and molecular changes related to the Antarctic environment. Gigascience 3(1):27. PubMed PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R; 1000 Genome Project Data Processing Subgroup. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25(16):2078–2079. PubMed PMC

Li WH, Gojobori T, Nei M.. 1981. Pseudogenes as a paradigm of neutral evolution. Nature 292(5820):237–239. PubMed

Liu GS, Zhang HX, Zhao C, Zhang HH.. 2020. Evolutionary history of the Toll-like receptor gene family across vertebrates. Genome Biol Evol. 12(1):3615–3634. PubMed PMC

Medzhitov R, Preston-Hurlburt P, Janeway CA.. 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388(6640):394–397. PubMed

Mikami T, Miyashita H, Takatsuka S, Kuroki Y, Matsushima N.. 2012. Molecular evolution of vertebrate Toll-like receptors: evolutionary rate difference between their leucine-rich repeats and their TIR domains. Gene 503(2):235–243. PubMed

Murrell B, Moola S, Mabona A, Weighill T, Sheward D, Pond SLK, Scheffler K.. 2013. FUBAR: a fast, unconstrained Bayesian AppRoximation for inferring selection. Mol Biol Evol. 30(5):1196–1205. PubMed PMC

Murrell B, Wertheim JO, Moola S, Weighill T, Scheffler K, Pond SLK.. 2012. Detecting individual sites subject to episodic diversifying selection. PLoS Genet. 8(7):e1002764. PubMed PMC

Nakajima T, Ohtani H, Satta Y, Uno Y, Akari H, Ishida T, Kimura A.. 2008. Natural selection in the TLR-related genes in the course of primate evolution. Immunogenetics 60(12):727–735. PubMed

Ng PC, Henikoff S.. 2003. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res. 31(13):3812–3814. PubMed PMC

O'Connor EA, Hasselquist D, Nilsson J, Westerdahl H, Cornwallis CK.. 2020. Wetter climates select for higher immune gene diversity in resident, but not migratory, songbirds. Proc Biol Sci. 287(1919):20192675. PubMed PMC

Obendorf DL, McColl K.. 1980. Mortality in little penguins (Eudyptula minor) along the coast of Victoria, Australia. J Wildl Dis. 16(2):251–259. PubMed

Ohto U, Yamakawa N, Akashi-Takamura S, Miyake K, Shimizu T.. 2012. Structural analyses of human Toll-like receptor 4 polymorphisms D299G and T399I. J Biol Chem. 287(48):40611–40617. PubMed PMC

Oven I, Resman Rus K, Dušanić D, Benčina D, Keeler CL, Narat M.. 2013. Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses. Vet Res. 44:99. PubMed PMC

Pan HL, Cole TL, Bi XP, Fang MQ, Zhou CR, Yang ZT, Ksepka DT, Hart T, Bouzat JL, Argilla LS, et al.2019. High-coverage genomes to elucidate the evolution of penguins. Gigascience 8(9):giz117. PubMed PMC

Paramo T, Piggot TJ, Bryant CE, Bond PJ.. 2013. The structural basis for endotoxin-induced allosteric regulation of the Toll-like receptor 4 (TLR4) innate immune receptor. J Biol Chem. 288(51):36215–36225. PubMed PMC

Park BS, Song DH, Kim HM, Choi B-S, Lee H, Lee J-O.. 2009. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature 458(7242):1191–1195. PubMed

Pond SLK, Frost SDW.. 2005. Datamonkey: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21(10):2531–2533. PubMed

Poplin R, Ruano-Rubio V, DePristo MA, Fennell TJ, Carneiro MO, Van der Auwera GA, Kling DE, Gauthier LD, Levy-Moonshine A, Roazen D, et al.2018. Scaling accurate genetic variant discovery to tens of thousands of samples. bioRxiv 201178. Available from: 10.1101/201178. DOI

Poulin R. 2014. Parasite biodiversity revisited: frontiers and constraints. Int J Parasitol. 44(9):581–589. PubMed

Qureshi ST, Lariviere L, Leveque G, Clermont S, Moore KJ, Gros P, Malo D.. 1999. Endotoxin tolerant mice have mutations in toll-like receptor 4 (Tlr4). J Exp Med. 189(4):615–625. PubMed PMC

Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD, Hood LE, Aderem A.. 2005. The evolution of vertebrate Toll-like receptors. Proc Natl Acad Sci U S A. 102(27):9577–9582. PubMed PMC

Ropert-Coudert Y, Hindell MA, Phillips RA, Jean-Benoit C, Trudelle L, Raymond B, et al.2014. Biogeographic patterns of birds and mammals. In: De Broyer C, Koubbi P, Griffiths H, Raymond B, d’Udekem d’Acoz C, Van de Putte A, Danis B, David B, Grant S, Gutt J, editors. Biogeographic atlas of the Southern Ocean. Cambridge: The Scientific Committee on Antarctic Research. p. 364–387.

Roy A, Kucukural A, Zhang Y.. 2010. I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc. 5(4):725–738. PubMed PMC

Rozas J, Ferrer-Mata A, Sanchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sanchez-Gracia A.. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Mol Biol Evol. 34(12):3299–3302. PubMed

Sallaberry-Pincheira N, González-Acuña D, Padilla P, Dantas GPM, Luna-Jorquera G, Frere E, Valdés-Velásquez A, Vianna JA.. 2016. Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins. Ecol Evol. 6(20):7498–7510. PubMed PMC

Scior T, Lozano-Aponte J, Figueroa-Vazquez V, Yunes-Rojas JA, Zähringer U, Alexander C.. 2013. Three-dimensional mapping of differential amino acids of human, murine, canine and equine TLR4/MD-2 receptor complexes conferring endotoxic activation by lipid A, antagonism by Eritoran and species-dependent activities of Lipid IVA in the mammalian LPS sensor system. Comput Struct Biotechnol J. 7:e201305003. PubMed PMC

Sharma V, Walther F, Hecker N, Stuckas H, Hiller M.. 2020. Convergent losses of TLR5 suggest altered extracellular flagellin detection in four mammalian lineages. Mol Biol Evol. 37(7):1847–1854. PubMed

Shen T, Xu SX, Wang XH, Yu WH, Zhou KY, Yang G.. 2012. Adaptive evolution and functional constraint at TLR4 during the secondary aquatic adaptation and diversification of cetaceans. BMC Evol Biol. 12:39. PubMed PMC

Shultz AJ, Sackton TB.. 2019. Immune genes are hotspots of shared positive selection across birds and mammals. Elife 8:e41815. PubMed PMC

Świderská Z, Šmídová A, Buchtová L, Bryjová A, Fabiánová A, Munclinger P, Vinkler M.. 2018. Avian Toll-like receptor allelic diversity far exceeds human polymorphism: an insight from domestic chicken breeds. Sci Rep. 8(1):17878. PubMed PMC

Takeda K, Kaisho T, Akira S.. 2003. Toll-like receptors. Annu Rev Immunol. 21:335–376. PubMed

Temperley ND, Berlin S, Paton IR, Griffin DK, Burt DW.. 2008. Evolution of the chicken Toll-like receptor gene family: a story of gene gain and gene loss. BMC Genomics 9:62. PubMed PMC

Těšický M, Velová H, Novotný M, Kreisinger J, Beneš V, Vinkler M.. 2020. Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae). Mol Ecol. 29(16):3056–3070. PubMed

Uhart M, Vanstreels RET, Gallo L, Cook RA, Karesh WB.. 2020. Serological survey for selection infectious agents in wild magellanic penguins (Spheniscus magellanicus) in Argentina, 1994-2008. J Wildl Dis. 56(1):66–81. PubMed

Van der Auwera GA, Carneiro MO Hartl C, Poplin R, Angel Levy-Moonshine DG, Jordan A, Shakir T, Roazen K, Thibault D. J.. 2013. From FastQ data to high confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Curr Protoc Bioinformatics 43:11.10.11–11.10.33. PubMed PMC

Velova H, Gutowska-Ding MW, Burt DW, Vinkler M.. 2018. Toll-like receptor evolution in birds: gene duplication, pseudogenization, and diversifying selection. Mol Biol Evol. 35(9):2170–2184. PubMed PMC

Vianna JA, Fernandes FAN, Frugone MJ, Figueiró HV, Pertierra LR, Noll D, Bi K, Wang-Claypool CY, Lowther A, Parker P, et al.2020. Genome-wide analyses reveal drivers of penguin diversification. Proc Natl Acad Sci U S A. 117(36):22303–22310. PubMed PMC

Vinkler M, Bainova H, Bryja J.. 2014. Protein evolution of Toll-like receptors 4, 5 and 7 within Galloanserae birds. Genet Select Evol. 46(1):72. PubMed PMC

Vinkler M, Bryjova A, Albrecht T, Bryja J.. 2009. Identification of the first Toll-like receptor gene in passerine birds: TLR4 orthologue in zebra finch (Taeniopygia guttata). Tissue Antigens 74(1):32–41. PubMed

Voogdt CGP, Merchant ME, Wagenaar JA, van Putten JPM.. 2018. Evolutionary regression and species-specific codon usage of TLR15. Front Immunol. 9:2626. PubMed PMC

Weaver S, Shank SD, Spielman SJ, Li M, Muse SV, Pond SLK.. 2018. Datamonkey 2.0: a modern web application for characterizing selective and other evolutionary processes. Mol Biol Evol. 35(3):773–777. PubMed PMC

Wlasiuk G, Nachman MW.. 2010. Adaptation and constraint at Toll-like receptors in primates. Mol Biol Evol. 27(9):2172–2186. PubMed PMC

Wolfe AD, dePamphilis CW.. 1998. The effect of relaxed functional constraints on the photosynthetic gene rbcL in photosynthetic and nonphotosynthetic parasitic plants. Mol Biol Evol. 15(10):1243–1258. PubMed

Xavier MO, Soares MP, Meinerz ARM, Nobre MO, Osorio LG, da Silva RP, Meireles MCA.. 2007. Aspergillosis: a limiting factor during recovery of captive Magellanic penguins. Braz J Microbiol. 38(3):480–484.

Yang Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol. 24(8):1586–1591. PubMed

Yang ZH. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci. 13(5):555–556. PubMed

Yang ZH, Wong WSW, Nielsen R.. 2005. Bayes empirical Bayes inference of amino acid sites under positive selection. Mol Biol Evol. 22(4):1107–1118. PubMed

Yeh DW, Liu YL, Lo YC, Yuh CH, Yu GY, Lo JF, Luo YP, Xiang R, Chuang TH.. 2013. Toll-like receptor 9 and 21 have different ligand recognition profiles and cooperatively mediate activity of CpG-oligodeoxynucleotides in zebrafish. Proc Natl Acad Sci U S A. 110(51):20711–20716. PubMed PMC

Yoon SI, Kurnasov O, Natarajan V, Hong MS, Gudkov AV, Osterman AL, Wilson IA.. 2012. Structural Basis of TLR5-Flagellin Recognition and Signaling. Science. 335(6070):859–864. PubMed PMC

Zhang G, Rahbek C, Graves GR, Lei F, Jarvis ED, Gilbert MT.. 2015. Genomics: bird sequencing project takes off. Nature. 522(7554):34. PubMed

Zhang JZ. 2003. Evolution by gene duplication: an update. Trends in Ecology & Evolution. 18(6):292–298.

Zhang ZK, Ohto U, Shibata T, Krayukhina E, Taoka M, Yamauchi Y, Tanji H, Isobe T, Uchiyama S, Miyake K, et al.2016. Structural analysis reveals that Toll-like receptor 7 is a dual receptor for guanosine and single-stranded RNA. Immunity 45(4):737–748. PubMed

Understanding the evolution of immune genes in jawed vertebrates

Dynamic Evolution of Avian RNA Virus Sensors: Repeated Loss of RIG-I and RIPLET

Selection Balancing at Innate Immune Genes: Adaptive Polymorphism Maintenance in Toll-Like Receptors