DOI: 10.5061/dryad.80gb5mktn
title: Genomics of cold adaptations in the Antarctic notothenioid fish radiation

description: Numerous novel adaptations characterise the radiation of notothenioids,
 the dominant fish group in the freezing seas of the Southern Ocean. To
 improve understanding of the evolution of this iconic fish group, we
 generated and analysed new genome assemblies for 24 species covering all
 major subgroups of the radiation, including five long-read assemblies. We
 present a new estimate for the onset of the radiation at 10.7 million
 years ago, based on a time-calibrated phylogeny derived from genome-wide
 sequence data. We identify a two-fold variation in genome size, driven by
 expansion of multiple transposable element families, and use the long-read
 data to reconstruct two evolutionarily important, highly repetitive gene
 family loci. First, we present the most complete reconstruction to date of
 the antifreeze glycoprotein gene family, whose emergence enabled survival
 in sub-zero temperatures, showing the expansion of the antifreeze gene
 locus from the ancestral to the derived state. Second, we trace the loss
 of haemoglobin genes in icefishes, the only vertebrates lacking functional
 haemoglobins, through complete reconstruction of the two haemoglobin gene
 clusters across notothenioid families. Both the haemoglobin and antifreeze
 genomic loci are characterised by multiple transposon expansions that may
 have driven the evolutionary history of these genes.
description: Phylogenetic analysis was performed using single copy ortholog
 genes identified with BUSCO, for the 24 newly sequenced notothenioid
 genomes and 17 previously published genomes of seven notothenioids and ten
 further species of percomorph fishes. BUSCO (v2) was run with lineage
 “actinopterygii_odb9”, and the sequences of single- opy orthologs
 identified in each assembly and extracted for use in further
 analysis. We used MAFFT v.7.453 to align 266 selected
 BUSCO genes that were single copy in our annotated gene sets. The 266
 alignments were inspected by eye, and apparently misaligned sequence
 regions were set to missing data. A total of 1,141,524 amino acids were
 set to missing out of 6,410,688, including nine alignments that were
 excluded completely, leaving 257 alignments for further analysis. We then
 aligned nucleotide sequences of the same BUSCO genes according to the
 amino-acid alignments, ensuring that regions corresponding to the removed
 sequences were again set to missing data in the nucleotide sequence
 alignments. Sites with high entropy (entropy like score &gt; 0.5) or
 high proportion of missing data (gap rate &gt; 0.2) were removed with
 BMGE v.1.1 and alignments with more than three completely missing
 sequences, a minimum length below 500 bp, or a standard deviation of
 among-sequence GC-content variation greater than 0.03 were excluded. These
 filters were passed by 228 alignments. Each of these
 alignments was subjected to Bayesian phylogenetic analysis with BEAST 2
 v.2.6.0, with an uncorrelated lognormal relaxed clock model and a
 Markov-chain Monte Carlo chain (MCMC) length of 25 million iterations.
 “Strict” and “permissive” sets of alignments were compiled based on
 estimates of the mutation rate and its among-species variation and
 contained 140 and 200 of the alignments, respectively. For the strict set
 of 140 alignments, the permissive set of 200 alignments, and the “full”
 set of 257 alignments, we performed maximum-likelihood phylogenetic
 analyses with IQ-TREE v.1.7 after alignment concatenation, maintaining
 separate partitions with unlinked instances of the GTR+Gamma substitution
 model for each of the original alignments. Node support was assessed with
 1,000 ultrafast bootstrap replicates. Each of the three analyses was
 complemented with an estimation of gene- and site-specific concordance
 factors, and the three resulting sets of gene trees were used for separate
 species-tree analyses with ASTRAL v.5.7.3. Finally, we estimated the
 phylogeny and the divergence times of notothenioid species with BEAST 2
 from a concatenated alignment combining all alignments of the strict set.
 The original data blocks were grouped in 12 positions selected with the
 rcluster algorithm of PartitionFinder v.2.1.1, assuming linked branch
 lengths, equal weights for all model parameters, a minimum partition size
 of 5,000 bp, and the GTR+Gamma substitution model. The same substitution
 model was also assumed in the BEAST 2 analysis, together with the
 birth-death model of diversification and the uncorrelated lognormal
 relaxed clock model. Time calibration of the phylogeny was based on four
 age constraints defined according to a recent timeline of teleost
 evolution inferred from genome and fossil information, at the most recent
 common ancestors of clades: Eupercaria, around 97.47 MYA (2.5–97.5
 inter-percentile range: 91.3–104.0 MYA); the clade combining Eupercaria,
 Ovalentaria, and Anabantaria – around 101.79 MYA (95.4–109.0 MYA); the
 clade combining these four groups with Syngnatharia and Pelagiaria –
 around 104.48 MYA (97.3–112.0 MYA); and the clade combining those six
 groups with Gobiaria – around 107.08 MYA (100.0–114.0 MYA). All
 constraints were implemented as lognormal prior distributions with mean
 values as specified above and a standard deviation between 0.033 and
 0.036. Additionally, we constrained the unambiguous monophyly of the
 groups Notothenioidei, Perciformes, Ovalentaria, Anabantaria, and the
 clade combining the latter two groups. We performed six replicate BEAST 2
 analyses with 330 million MCMC iterations, and convergence among MCMC
 chains was confirmed by ESS values greater than 120 for all model
 parameters and greater than 270 for the likelihood and the prior and
 posterior probabilities. The posterior tree distribution was summarised in
 the form of a maximum-clade credibility tree with TreeAnnotator v.2.6.0.
 We attempted to repeat the BEAST 2 analyses with the permissive and full
 datasets, but these proved too computationally demanding to complete.
 Nevertheless, the preliminary results from these analyses supported the
 same tree topology as the analyses with the strict dataset.