Evolution of the Immune System Influences Speciation Rates in Teleost Fishes
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ARTICLES OPEN Evolution of the immune system influences speciation rates in teleost fishes Martin Malmstrøm1,7, Michael Matschiner1,7, Ole K Tørresen1, Bastiaan Star1, Lars G Snipen2, Thomas F Hansen1, Helle T Baalsrud1, Alexander J Nederbragt1, Reinhold Hanel3, Walter Salzburger1,4, Nils C Stenseth1,5,6, Kjetill S Jakobsen1 & Sissel Jentoft1,6 Teleost fishes constitute the most species-rich vertebrate clade and exhibit extensive genetic and phenotypic variation, including diverse immune defense strategies. The genomic basis of a particularly aberrant strategy is exemplified by Atlantic cod, in which a loss of major histocompatibility complex (MHC) II functionality coincides with a marked expansion of MHC I genes. Through low-coverage genome sequencing (9–39×), assembly and comparative analyses for 66 teleost species, we show here that MHC II is missing in the entire Gadiformes lineage and thus was lost once in their common ancestor. In contrast, we find that MHC I gene expansions have occurred multiple times, both inside and outside this clade. Moreover, we identify an association between high MHC I copy number and elevated speciation rates using trait-dependent diversification models. Our results extend current understanding of the plasticity of the adaptive immune system and suggest an important role for immune-related genes in animal diversification. With over 32,000 extant species1, teleost fishes comprise the majority in an immune system that can detect a large number of pathogens at of vertebrate species. Their taxonomic diversity is matched by exten- the expense of being less efficient in removing them. The evolution sive genetic and phenotypic variation, including novel immunological of MHC copy numbers is therefore likely driven toward intermediate strategies. Although the functionality of the adaptive immune optima determined by a tradeoff between detection and elimination of Nature America, Inc. All rights reserved. America, Inc. Nature system has been considered to be conserved since its emergence in pathogens—as suggested by selection for 5–10 copies inferred in case 6 the ancestor of all jawed vertebrates2,3, fundamental modifications studies of fish17,18 and birds19. Because pathogen load and the associ- of the immune gene repertoire have recently been reported in tel- ated selective pressures vary between habitats, the optimal number © 201 eosts4–7. One of the most dramatic changes has occurred in Atlantic of MHC copies depends on the environment20–22. As a result, inter- cod (Gadus morhua), involving complete loss of the MHC II pathway breeding between different locally adapted populations is expected to that is otherwise responsible for the detection of bacterial pathogens produce hybrids with excess (above optimal) MHC diversity that are npg in vertebrates4. Moreover, this loss is accompanied by a substan- characterized by T cell deprivation and low fitness. This process would tially enlarged repertoire of MHC I genes, which normally encode introduce postzygotic reproductive isolation and promote reinforce- molecules for protection against viral pathogens. It has thus been ment of premating isolation between the populations. Consequently, hypothesized that the expanded MHC I repertoire of cod evolved as MHC genes have been suggested to have an important role in spe- a compensatory mechanism, whereby broader MHC I functional- ciation22,23, but, to our knowledge, this role has never been tested ity makes up for the initial loss of MHC II (refs. 4,6). However, the comparatively in a macroevolutionary context. questions of how and when MHC II was lost relative to the MHC I Here we report comparative analyses of 76 teleost species, of expansion, and whether these genomic modifications are causally which 66 were sequenced to produce partial draft genome assem- related, have so far remained unresolved. blies, including 27 representatives of cod-like fishes within the order As key components of the vertebrate adaptive immune system, the Gadiformes. First, we use phylogenomic analysis to resolve stand- complex MHC pathways and their functionality are now well charac- ing controversy regarding early-teleost divergences and to firmly terized8–10, but less is known about the causes of MHC copy number establish the relationships of gadiform fishes. Second, by analyzing variation, which poses an immunological tradeoff11,12. Although an the presence and absence of key genes in the vertebrate adaptive increase in the number of MHC genes facilitates pathogen detection, immune system, we place the loss of MHC II functionality in the it will also decrease the number of circulating T cells13–16, resulting common ancestor of all Gadiformes; by time calibrating the teleost 1Department of Biosciences, University of Oslo, Oslo, Norway. 2Department of Chemistry, Biotechnology and Food Sciences, Norwegian University of Life Sciences, Ås, Norway. 3Institute of Fisheries Ecology, Federal Research Institute for Rural Areas, Forestry and Fisheries, Hamburg, Germany. 4Zoological Institute, University of Basel, Basel, Switzerland. 5Institute of Marine Research, Flødevigen Marine Research Station, His, Norway. 6Department of Natural Sciences, University of Agder, Kristiansand, Norway. 7These authors contributed equally to this work. Correspondence should be addressed to K.S.J. ([email protected]) or S.J. ([email protected]). Received 8 April; accepted 15 July; published online 22 August 2016; doi:10.1038/ng.3645 1204 VOLUME 48 | NUMBER 10 | OCTOBER 2016 NATURE GENETICS ARTICLES phylogeny on the basis of fossil sampling rate estimation, we fur- The resulting time tree (Fig. 1) supports a monophyletic clade ther show that this loss occurred 105–85 million years ago. Third, including the orders Gadiformes, Stylephoriformes, Zeiformes, we demonstrate that expansions of the MHC I gene repertoire are Polymixiiformes and Percopsiformes, thus corroborating the not limited to Gadiformes but are also found in three other clades, Paracanthopterygii sensu Miya et al.30. We further confirm the including the exceptionally species-rich group of percomorph fishes placement of Stylephorus chordatus as the closest extant relative (Percomorphaceae). By applying phylogenetic comparative meth- of the Gadiformes30,31 and estimate the crown age of this order at ods and ancestral state reconstruction, we trace the evolutionary approximately 85 million years ago. Within Gadiformes, we find sup- history of MHC I gene expansions and infer distinct copy number port for a sister group relationship between Bregmacerotidae and all optima in each of them. Finally, using trait-dependent diversifica- other gadiform families, which began to radiate around 70 million tion models, we identify a positive association between MHC I gene years ago. copy number and speciation rate. Our results highlight the plasticity of the vertebrate adaptive immune system and support the role of Loss of MHC II pathway genes MHC genes as ‘speciation genes’, promoting rapid diversification To assess the origin and extent of the MHC II pathway gene loss in teleost fishes. observed in Atlantic cod, we investigated the immune gene repertoires of the teleost genomes through a comparative gene mining pipeline RESULTS comprising BLAST searches, prediction of ORFs and annotation Sequencing and draft assembly of 66 teleost genomes (Supplementary Note). We explicitly investigated the presence of To investigate the evolution of the immune gene repertoire of tel- 27 genes chosen for their central role in MHC class I, MHC class II eost fishes, we selected representatives of all major lineages in the and cross-presentation pathways8,10. In addition, three highly con- Neoteleostei24 for low-coverage genome sequencing. As both loss and served genes were included as a control (Fig. 2). As query sequences, expansion of MHC genes have been reported in the Gadiformes4, we used orthologs from zebrafish (Danio rerio), medaka (Oryzias we sampled this order more densely with 27 species representing latipes), spotted green pufferfish (Tetraodon nigroviridis), fugu most gadiform families and subfamilies (Supplementary Table 1)25. (Takifugu rubripes), three-spined stickleback (Gasterosteus aculeatus), The sequencing strategy for de novo assembly of the 66 genomes Nile tilapia (Oreochromis niloticus), Amazon molly (Poecilia formosa), was designed on the basis of gene space completeness and assem- platyfish (Xiphophorus maculatus), cavefish (Astyanax mexicanus) bly statistics of simulated genomes (Supplementary Note). For each and Atlantic cod (G. morhua) (Supplementary Table 6). species, a single paired-end library was sequenced to 9–39× cover- The genome-based phylogeny allowed us to place the immune age on the Illumina HiSeq 2000 platform (Supplementary Table 2 gene characterization into an evolutionary perspective. Whereas the and Supplementary Note). The genome sequences were assembled three control genes were identified in all species, we found that genes with the Celera Assembler26, resulting in N50 scaffold sizes between associated with the MHC II pathway were consistently missing in 3.2 and 71 kb (Supplementary Table 3 and Supplementary Note). all inspected Gadiformes draft genome assemblies, as no orthologs For most species, we recovered more than 75% of the conserved could be identified for invariant chain (also known as cd74), cd4 and eukaryotic genes included in the CEGMA27 analysis, and on average the MHC II α and β chains (Fig. 2a and Supplementary Table 7). we recovered 74% of the conserved