Positively selected genes in the hoary bat (Lasiurus cinereus) lineage: prominence of thymus expression, immune and metabolic function, and regions of ancient synteny

Abstract

Background: Bats of the genus Lasiurus occur throughout the Americas and have diversified into at least 20 species among three subgenera. The hoary bat (Lasiurus cinereus) is highly migratory and ranges farther across North America than any other wild mammal. Despite the ecological importance of this species as a major insect predator, and the particular susceptibility of lasiurine bats to wind turbine strikes, our understanding of hoary bat ecology, physiology, and behavior remains poor.

Methods: To better understand adaptive evolution in this lineage, we used whole-genome sequencing to identify protein-coding sequence and explore signatures of positive selection. Gene models were predicted with Maker and compared to seven well-annotated and phylogenetically representative species. Evolutionary rate analysis was performed with PAML.

Results: Of 9,447 single-copy orthologous groups that met evaluation criteria, 150 genes had a significant excess of nonsynonymous substitutions along the L. cinereus branch (P < 0.001 after manual review of alignments). Selected genes as a group had biased expression, most strongly in thymus tissue. We identified 23 selected genes with reported immune functions as well as a divergent paralog of Steep1 within suborder Yangochiroptera. Seventeen genes had roles in lipid and glucose metabolic pathways, partially overlapping with 15 mitochondrion-associated genes; these adaptations may reflect the metabolic challenges of hibernation, long-distance migration, and seasonal variation in prey abundance. The genomic distribution of positively selected genes differed significantly from background expectation by discrete Kolmogorov-Smirnov test (P < 0.001). Remarkably, the top three physical clusters all coincided with islands of conserved synteny predating Mammalia, the largest of which shares synteny with the human cat-eye critical region (CECR) on 22q11. This observation coupled with the expansion of a novel Tbx1-like gene family may indicate evolutionary innovation during pharyngeal arch development: both the CECR and Tbx1 cause dosage-dependent congenital abnormalities in thymus, heart, and head, and craniodysmorphy is associated with human orthologs of other positively selected genes as well.

Keywords: Adaptation; Cat-eye critical region; Chrna9; Conserved synteny; Hoary bat; Immunogenetics; Positive selection; Tbx gene family; Thymus.

Figure 1. Genes positively selected in the Lasiurus cinereus lineage have biased expression in the mouse thymus.

(A) Proportion of mouse orthologs of selected genes that have peak relative expression in each tissue. The proportion is significantly higher in thymus for positively selected genes than for other genes when all other tissues are binned together (see text for details). (B) The mean relative expression of positively selected genes is highest in thymus. The difference in means is significant at P < 0.05 (Student’s t-test) for both thymus and heart after Benjamini–Hochberg correction for multiple tests.

Figure 2. Positively selected genes are clustered in the Lasiurus cinereus genome.

(A) The cumulative distribution of significant genes along scaffolds differs significantly from that of all tested orthogroups by discrete Kolmogorov–Smirnov test. (B) Distribution of the counts of significant genes in consecutive 5-Mb windows. The scaffold locations are shown for the three windows with three or more significant genes. (C) Schematic of the distribution of significant genes (orange marks) and nonsignificant genes (purple marks) by coordinate position on the three scaffolds marked in (B). Red boxes indicate the selected genes within 5-Mb windows marked in panel (B).

Figure 3. Positively selected genes clustered on scaffold 533 are syntenic with the human cat-eye critical region (CECR).

(A) Protein-based alignment between human chromosome 22 in the vicinity of the CECR (gray-shaded region) and six other species. Red and blue dots indicate homology in the same and reverse orientations, respectively. (B) Positively selected genes and structural variation in the syntenic region in Lasiurus cinereus.

Figure 4. A derived Steep1 paralog is positively selected within Yangochiroptera.

(A) Neighbor-joining dendrogram of the Steep1 ortholog together with the Steep1 paralog (shaded in green), with distances based on nonsynonymous amino-acid substitutions only. The evolutionary rate parameter ω computed under PAML Model 0 is shown for each group of orthologous sequences. The likelihoods of PAML Model 2 were computed with and without branch labels denoting the Steep1 paralog as the foreground branch. The labeled model was significantly more likely than the unlabeled model or Model 1. (B) A protein alignment of bat Steep1 orthologs and the novel paralogous sequences in Yangochiroptera. Four-letter species codes for bats are: EpFu = Eptesicus fuscus, MyLu = Myotis lucifugus, HiAr = Hipposideros armiger, RhFe = Rhinolophus ferrumequinum, RoAe = Rousettus aegyptiacus, PtVA = Pteropus vampyrus, PhDi = Phyllostomus discolor [Steep 1 paralog clade] MoMo = Molossus molussus, MyLu = M. lucifugus, Laci = Lasiurus cinereus, PiKu = Pipistrellus kuhlii, PhDi = P. discolor, ArJa = Artibeus jamaicensis, StHo = Sturnira hondurensis.

Figure 5. Protein substitutions unique to the L. cinereus Chrna9 gene relative to protein structure and functional sites.

Black squares denote bat sequences included in the selection test, with additional bat sequences and the human sequence also included for comparison. Alignment coordinates are shaded orange, pink, or blue according to the secondary structure predicted by TMHMM (Krogh et al., 2001). Gaps occur at each end of the alignment because the tested orthogroup alignment was shorter than the full human protein sequence, for which glycosylation sites (red stars) are annotated in the corresponding NCBI accession record. The second glycosylation site has been abolished by an N–>K amino acid change in the L. cinereus sequence. See text for additional details.