Conservation and divergence in the frog immunome: pyrosequencing and de novo assembly of immune tissue transcriptomes Anna E. Savage a,b, ⁎, Karen M. Kiemnec-Tyburczy b , Amy R. Ellison b , Robert C. Fleischer a , Kelly R. Zamudio b a Center for Conservation and Evolutionary Genetics, Smithsonian Institution, Washington, DC 20013, United States b Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, United States abstract article info Article history: Received 1 November 2013 Received in revised form 6 March 2014 Accepted 26 March 2014 Available online 27 March 2014 Keywords: Anura Neobatrachia RNA-seq Immune system Functional genomics Background: Frogs are a diverse group of vertebrates for which limited genomic resources are available. Natural frog populations face a multitude of threats, including habitat degradation, infectious disease, and environmental change. Characterizing the functional genomics of anuran tissues in general – and the immune system in particular – will enhance our knowledge of genetic and epigenetic responses to environmental threats and inform conservation and recovery efforts. Results: To increase the number of species with genomic datasets and characterize gene expression in immune- related tissues, we sequenced the transcriptomes of three tissues from two frogs (Espadarana prosoblepon and Lithobates yavapaiensis) on the Roche 454 GS FLX platform. Our sequencing produced 8881 E. prosoblepon and 5428 L. yavapaiensis annotated gene products after de novo assembly and Gene Ontology classification. Transcripts of the innate and acquired immune system were expressed in all three tissues. Inflammatory response and acquired immunity transcripts were significantly more diverged between E. prosoblepon and L. yavapaiensis compared to innate immunity and immune system development transcripts. Immune-related transcripts did not show an overall elevated rate of functional evolution, with the exception of glycosyl proteases, which include lysozymes, central bacterial and fungal-killing enzymes of the innate immune system. Conclusions: The three frog transcriptomes provide more than 600 Mbp of new genomic data, and will serve as a valuable framework for future comparative studies of non-model anurans. Additionally, we show that immune gene divergence varies by functional group and that transcriptome studies can be useful in comparing rates of evolutionary change across gene families. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Amphibians are a diverse group of vertebrates for which limited ge- nomic resources are available (Calboli et al., 2011). Although over 7200 species of amphibians are recognized (Amphibiaweb 2014), genome- scale sequencing projects are limited by the intrinsic complexity of many amphibian genomes. For example, approximately 43 frog species across 12 families are known to be polyploid, as are numerous salaman- ders in the Ambystomatidae and Sirenidae families (Mable et al., 2011). Regardless of ploidy levels, amphibian genomes also rank among the largest within animal taxa (Gregory, 2005), thus they require considerably more sequencing to achieve the coverage needed for whole-genome assembly projects. Because of these limitations, most amphibian genomic data – including EST databases, transcriptomes, and sequenced genomes – come from two model species: the Western clawed frog, Silurana tropicalis (Hellsten et al., 2010) and the African clawed frog, Xenopus laevis (e.g., Morin et al., 2006). Recently, a draft transcriptome assembly generated using both Sanger and high- throughput sequencing was released for a salamander, the axolotl Ambystoma mexicanum (http://www.ambystoma.org/). Although these newly developed resources are extremely useful for comparative genomics (e.g., Voss et al., 2011), amphibians are an ancient and diverse vertebrate lineage which has diverged over 350 million years ago (Roelants et al., 2007). Consequently, the genomic architecture and expression profiles of A. mexicanum, S. tropicalis, and X. laevis are likely not representative of amphibians as a whole. S. tropicalis and X. laevis are members of the family Pipidae, an early diverging group of anurans (Pyron and Wiens,' 2011), yet 95% of extant frog species belong to the clade Neobatrachia, a much more recent radiation approximately 300 million years diverged from Pipidae (Igawa et al., 2008) that includes most of the evolutionary diversity within this group (Cannatella et al., 2008). Thus, genomic data from multiple species better representing diversity in the Anuran phylogenetic tree are neces- sary for comparative inferences about patterns and processes shaping amphibian genomes. Gene 542 (2014) 98–108 Abbreviations: aa, amino acid(s); Asn (N), asparagine; Asp (D), aspartic acid; Bd, Batrachochytrium dendrobatidis; BLAST, Basic Local Alignment Search Tool; Espr, Espadarana prosoblepon; gi, GenBank identification number; Glu (E), glutamic acid; Liya, Lithobates yavapaiensis; Lys (K), lysine; MHC, major histocompatibility complex. ⁎ Corresponding author at: Center for Conservation and Evolutionary Genetics, Smithsonian Institution, Washington, DC 20013, United States. E-mail addresses: savagea@si.edu (A.E. Savage), kmk877@humboldt.edu (K.M. Kiemnec-Tyburczy), are35@cornell.edu (A.R. Ellison), fleischerr@si.edu (R.C. Fleischer), kelly.zamudio@cornell.edu (K.R. Zamudio). http://dx.doi.org/10.1016/j.gene.2014.03.051 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene