Emergence of a Chimeric Globin Pseudogene and Increased Hemoglobin Oxygen Affinity Underlie the Evolution of Aquatic Specializations in Sirenia

Abstract

As limits on O2 availability during submergence impose severe constraints on aerobic respiration, the oxygen binding globin proteins of marine mammals are expected to have evolved under strong evolutionary pressures during their land-to-sea transition. Here, we address this question for the order Sirenia by retrieving, annotating, and performing detailed selection analyses on the globin repertoire of the extinct Steller's sea cow (Hydrodamalis gigas), dugong (Dugong dugon), and Florida manatee (Trichechus manatus latirostris) in relation to their closest living terrestrial relatives (elephants and hyraxes). These analyses indicate most loci experienced elevated nucleotide substitution rates during their transition to a fully aquatic lifestyle. While most of these genes evolved under neutrality or strong purifying selection, the rate of nonsynonymous/synonymous replacements increased in two genes (Hbz-T1 and Hba-T1) that encode the α-type chains of hemoglobin (Hb) during each stage of life. Notably, the relaxed evolution of Hba-T1 is temporally coupled with the emergence of a chimeric pseudogene (Hba-T2/Hbq-ps) that contributed to the tandemly linked Hba-T1 of stem sirenians via interparalog gene conversion. Functional tests on recombinant Hb proteins from extant and ancestral sirenians further revealed that the molecular remodeling of Hba-T1 coincided with increased Hb-O2 affinity in early sirenians. Available evidence suggests that this trait evolved to maximize O2 extraction from finite lung stores and suppress tissue O2 offloading, thereby facilitating the low metabolic intensities of extant sirenians. In contrast, the derived reduction in Hb-O2 affinity in (sub)Arctic Steller's sea cows is consistent with fueling increased thermogenesis by these once colossal marine herbivores.

Keywords: ancient DNA; aquatic adaptation; cytoglobin; gene conversion; hemoglobin; molecular evolution; myoglobin; neuroglobin; oxygen affinity; pseudogene.

Fig. 1.

(A) Organization and synteny analysis of rock hyrax (Procavia capensis), African elephant (Loxodonta africana), and Florida manatee (Trichechus manatus) α-globin gene clusters. Red and grey boxes represent exonic and intronic sequences, respectively. The “ps” suffix denotes pseudogenes, whereas “-T1” and “T2” suffices indicate the 5′–3′ linkage order of tandemly duplicated genes. Thick black lines represent intergenic sequences. Gaps in the contiguous sequence denote missing data. (B) Organization and synteny analysis of rock hyrax (P. capensis), African elephant (L. africana), and Florida manatee (T. manatus) β-globin gene clusters. Red and grey boxes represent exonic and intronic sequences, respectively. The “ps” suffix denotes pseudogenes. Thick black lines represent intergenic sequences. Gaps in the contiguous sequence denote missing data.

Fig. 2.

Maximum likelihood phylogenies depicting relationships between (A) the first exon and intron (bp 1–353) and (B) the second intron and third exon (bp 593–848) of paenungulate Hba- and Hbq-type genes. Values adjacent to each node represent maximum likelihood bootstrap support. The “ps” suffix denotes pseudogenes, whereas “-T1” and “-T2” suffices indicate the 5′–3′ linkage order of tandemly duplicated genes. Boxes highlight noteworthy gene groupings (see text for details).

Fig. 3.

Time-calibrated phylogeny modified from Springer et al. (2015) and Rohland et al. (2007) (top panel). Column plots representing the rate of nucleotide substitutions (substitutions per nucleotide site per million years ± SE) among the eight functional globin genes of paenungulate mammals (bottom panel). Black and grey regions of each column represent the contribution of nonsynonymous and synonymous substitutions to the overall substitution rate, respectively. Column numbers correspond to specific branches in the above time-calibrated phylogeny. Asterisks denote sirenian branches that have a rate of nucleotide substitution significantly higher than the locus average (denoted by grey dotted lines). Columns marked “N” denote negative selection.

Fig. 4.

Rates of nucleotide substitution (substitutions per nucleotide site) for ancestral sirenian and ancestral elephant Hba genes. Gene wide substitution rate (total substitutions/423 nt) is represented by black dotted lines. Vertical grey bars represent the substitution rate within a 40 bp sliding window. The blue shaded area denotes the location of the gene conversion event (identified by GENECONV; table 1) that took place between Hba-T1 and Hba-T2/Hbq-ps genes in the sirenian ancestor and the green arrows denote exon boundaries.

Fig. 5.

Oxygen tensions at half saturation (P₅₀) for representative paenungulate hemoglobins (0.0625 mM Hb₄) in the presence of both 0.1 M KCl and 0.125 mM 2, 3-diphosphoglycerate at 37 °C, pH 7.2. Values are shown as P₅₀ ± SE. The phylogenetic tree is colored according to P₅₀ value. The P₅₀ at branches without in vitro measurements were estimated using the phytools package in R.