Selected Lark Mitochondrial Genomes Provide Insights into the Evolution of Second Control Region with Tandem Repeats in Alaudidae (Aves, Passeriformes).

The control region (CR) regulates the replication and transcription of the mitochondrial genome (mitogenome). Some avian mitogenomes possess two CRs, and the second control region (CR2) may enhance replication and transcription; however, the CR2 in lark mitogenome appears to be undergoing loss and is accompanied by tandem repeats. Here, we characterized six lark mitogenomes from Alaudala cheleensis, Eremophila alpestris, Alauda razae, and Calandrella cinerea and reconstructed the phylogeny of Passerida. Through further comparative analysis among larks, we traced the evolutionary process of CR2. The mitochondrial gene orders were conserved in all published lark mitogenomes, with Cytb-trnT-CR1-trnP-ND6-trnE-remnant CR2 with tandem repeat-trnF-rrnS. Phylogenetic analysis revealed Alaudidae and Panuridae are sister groups at the base of Sylvioidea, and sporadic losses of CR2 may occur in their common ancestor. CR sequence and phylogeny analysis indicated CR2 tandem repeats were generated within CR2, originating in the ancestor of all larks, rather than inherited from CR1. The secondary structure comparison of tandem repeat units within and between species suggested slipped-strand mispairing and DNA turnover as suitable models for explaining the origin and evolution of these repeats. This study reveals the evolutionary process of the CR2 containing tandem repeat in Alaudidae, providing reference for understanding the evolutionary characteristics and dynamics of tandem repeats.

Plumage Balances Camouflage and Thermoregulation in Horned Larks (Eremophila alpestris).

AbstractAnimal coloration serves many biological functions and must therefore balance potentially competing selective pressures. For example, many animals have camouflage in which coloration matches the visual background that predators scan for prey. However, different colors reflect different amounts of solar radiation and may therefore have thermoregulatory implications as well. In this study, we examined geographic variation in dorsal patterning, coloration, and solar reflectance among horned larks (Eremophila alpestris) of the western United States. We found that plumage brightness was positively associated with soil granularity, aridity, and temperature. Plumage redness-both in terms of saturation (i.e., chroma) and hue-was positively associated with soil redness and temperature, while plumage patterning was positively associated with soil granularity. Together, these plumage-environment associations support both background matching and Gloger's rule, a widespread ecogeographic pattern in animal coloration. We also constructed thermoregulatory models that estimated cooling benefits provided by solar reflectance profiles of the dorsal plumage of each specimen based on the collection site. We found increased cooling benefits in hotter, more arid environments. Finally, cooling benefits were positively associated with residual brightness, such that individuals that were brighter than expected based on environmental conditions also had higher cooling benefits, suggesting a trade-off between camouflage and thermoregulation. Together, these data suggest that natural selection has balanced camouflage and thermoregulation in horned larks, and they illustrate how multiple competing evolutionary pressures may interact to shape geographic variation in adaptive phenotypes.

Cloacal microbiota are biogeographically structured in larks from desert, tropical and temperate areas.

BACKGROUND: In contrast with macroorganisms, that show well-documented biogeographical patterns in distribution associated with local adaptation of physiology, behavior and life history, strong biogeographical patterns have not been found for microorganisms, raising questions about what determines their biogeography. Thus far, large-scale biogeographical studies have focused on free-living microbes, paying little attention to host-associated microbes, which play essential roles in physiology, behavior and life history of their hosts. Investigating cloacal gut microbiota of closely-related, ecologically similar free-living songbird species (Alaudidae, larks) inhabiting desert, temperate and tropical regions, we explored influences of geographical location and host species on α-diversity, co-occurrence of amplicon sequence variants (ASVs) and genera, differentially abundant and dominant bacterial taxa, and community composition. We also investigated how geographical distance explained differences in gut microbial community composition among larks. RESULTS: Geographic location did not explain variation in richness and Shannon diversity of cloacal microbiota in larks. Out of 3798 ASVs and 799 bacterial genera identified, 17 ASVs (< 0.5%) and 43 genera (5%) were shared by larks from all locations. Desert larks held fewer unique ASVs (25%) than temperate zone (31%) and tropical larks (34%). Five out of 33 detected bacterial phyla dominated lark cloacal gut microbiomes. In tropical larks three bacterial classes were overrepresented. Highlighting the distinctiveness of desert lark microbiota, the relative abundances of 52 ASVs differed among locations, which classified within three dominant and 11 low-abundance phyla. Clear and significant phylogenetic clustering in cloacal microbiota community composition (unweighted UniFrac) showed segregation with geography and host species, where microbiota of desert larks were distinct from those of tropical and temperate regions. Geographic distance was nonlinearly associated with pairwise unweighted UniFrac distances. CONCLUSIONS: We conclude that host-associated microbiota are geographically structured in a group of widespread but closely-related bird species, following large-scale macro-ecological patterns and contrasting with previous findings for free-living microbes. Future work should further explore if and to what extent geographic variation in host-associated microbiota can be explained as result of co-evolution between gut microbes and host adaptive traits, and if and how acquisition from the environmental pool of bacteria contributes to explaining host-associated communities.

No seasonal curtailment of the Eurasian Skylark's (Alauda arvensis) breeding season in German heterogeneous farmland.

The lack of suitable nesting sites is one key driver behind the farmland bird crisis in Europe. Winter cereals become impenetrable for ground-breeding birds like the Eurasian Skylark (Alauda arvensis), curtailing breeding time. Stable Skylark populations depend on multiple breeding attempts per year; thus, the widespread cultivation of winter cereals has strongly contributed to their tremendous decline. Crop diversification is thought to be a potential measure to counteract this development. Therefore, we explored how individual Skylarks respond to the decreasing suitability of winter cereals as nesting habitats in heterogeneous but otherwise conventionally managed farmland. Our study focused on: (i) the degree to which Skylarks prematurely cease nesting activity, switch nesting habitats, or breed on linear structures like tramlines. Additionally, we analyzed: (ii) if nest success decreases throughout the breeding season and (iii) how often Skylarks make a successful breeding attempt per year. We radio-tagged 28 adults in a German population during April 2018 and 2019, tracked half of them for more than 3 months, and measured their breeding success. Additionally, we monitored nests of untagged pairs, resulting in 96 nests found. None, except one tagged individual, stopped breeding activity before July 1st. Home ranges were mainly stable, but Skylarks switched nesting habitats away from winter cereals to crops like sugar beet or set-aside. High-risk nesting sites like corn and linear structures played a minor role in breeding. Overall, Mayfield logistic regressions revealed no seasonal decrease in nest success, and tagged Skylarks had sufficient time to make 1.5-1.8 breeding attempts, of which 0.8 were successful. We suggest that heterogeneous farmland in our study area, which enabled diversely composed home ranges, prevented a curtailment of the breeding season. Thus, our study reinforces the need for crop diversification which gives Skylarks a chance to survive in modern farmland.

Neo-sex chromosome evolution and phenotypic differentiation across an elevational gradient in horned larks (Eremophila alpestris).

Genetic structure and phenotypic variation among populations are affected by both geographic distance and environmental variation across species' distributions. Understanding the relative contributions of isolation by distance (IBD) and isolation by environment (IBE) is important for elucidating population dynamics across habitats and ecological gradients. In this study, we compared phenotypic and genetic variation among Horned Lark (Eremophila alpestris) populations from 10 sites encompassing an elevational gradient from low-elevation desert scrub in Death Valley (285 a.s.l.) to high-elevation meadows in the White Mountains of the Sierra Nevada of California (greater than 3000 m a.s.l.). Using a ddRAD data set of 28,474 SNPs aligned to a high-quality reference genome, we compared genetic structure with elevational, environmental, and spatial distance to quantify how different aspects of the landscape drive genomic and phenotypic differentiation in Horned Larks. We found larger-bodied birds were associated with sites that had less seasonality and higher annual precipitation, and longer spurs occurred in soils with more clay and silt content, less sand, and finer fragments. Larks have large neo-sex chromosomes, and we found that associations with elevation and environmental variation were much stronger among neo-sex chromosomes compared to autosomes. Furthermore, we found that putative chromosomal translocations, fusions, and inversions were associated with elevation and may underlie local adaptation across an elevational gradient in Horned Larks. Our results suggest that genetic variation in Horned Larks is affected more by IBD than IBE, but specific phenotypes and genomic regions-particually on neo-sex chromosomes-bear stronger associations with the environment.

Mitogenome of emAlaudala/em emcheleensis/em (Passeriformes: Alaudidae) and comparative analyses of Sylvioidea mitogenomes.

To gain a better understanding of mitogenome features and phylogenetic relationships in Sylvioidea, a superfamily of Passerida, suborder Passeri, Passeriformes, the whole mitogenome of Alaudala cheleensis Swinhoe (Alaudidae) was sequenced, a comparative mitogenomic analysis of 18 Sylvioidea species was carried out, and finally, a phylogeny was reconstructed based on the mitochondrial dataset. Gene order of the A. cheleensis mitogenome was similar to that of other Sylvioidea species, including the gene rearrangement of cytb-trnT-CR1-trnP-nad6-trnE-remnant CR2-trnF-rrnS. There was slightly higher A+T content than that of G+C in the mitogenome, with an obvious C skew. The ATG codon initiated all protein-coding genes, while six terminating codons were used. The secondary structure of rrnS contained three domains and 47 helices, whereas rrnL included six domains and 60 helices. All tRNAs could be folded into a classic clover-leaf secondary structure except for trnS (AGY). The CR1 could be divided into three domains, including several conserved boxes (C-string, F, E, D, C and B-box, Bird similarity box, CSB1). Comparative analyses within Sylvioidea mitogenomes showed that most mitochondrial features were consistent with that of the A. cheleensis mitogenome. The basal position of the Alaudidae within the Sylvioidea in our phylogenetic analyses is consistent with other recent studies.

De Novo Assembly of a High-Quality Reference Genome for the Horned Lark (Eremophila alpestris).

The Horned Lark (Eremophila alpestris) is a small songbird that exhibits remarkable geographic variation in appearance and habitat across an expansive distribution. While E. alpestris has been the focus of many ecological and evolutionary studies, we still lack a highly contiguous genome assembly for the Horned Lark and related taxa (Alaudidae). Here, we present CLO_EAlp_1.0, a highly contiguous assembly for E. alpestris generated from a blood sample of a wild, male bird captured in the Altiplano Cundiboyacense of Colombia. By combining short-insert and mate-pair libraries with the ALLPATHS-LG genome assembly pipeline, we generated a 1.04 Gb assembly comprised of 2713 scaffolds, with a largest scaffold size of 31.81 Mb, a scaffold N50 of 9.42 Mb, and a scaffold L50 of 30. These scaffolds were assembled from 23685 contigs, with a largest contig size of 1.69 Mb, a contig N50 of 193.81 kb, and a contig L50 of 1429. Our assembly pipeline also produced a single mitochondrial DNA contig of 14.00 kb. After polishing the genome, we identified 94.5% of single-copy gene orthologs from an Aves data set and 97.7% of single-copy gene orthologs from a vertebrata data set, which further demonstrates the high quality of our assembly. We anticipate that this genomic resource will be useful to the broader ornithological community and those interested in studying the evolutionary history and ecological interactions of larks, which comprise a widespread, yet understudied lineage of songbirds.

Thermal physiology of a range-restricted desert lark.

Much recent work on avian physiological adaptation to desert environments has focused on larks (Passeriformes: Alaudidae). We tested the prediction that the threatened red lark (Calendulauda burra), a species restricted to very arid parts of South Africa and which is not known to drink, exhibits highly efficient evaporative cooling and makes pronounced use of facultative hyperthermia when exposed to high air temperatures (Ta). We also predicted that C. burra possesses similarly low basal metabolic rate (BMR) and total evaporative water loss (EWL) at moderate Ta as reported for species from the deserts of the Middle East. Rest-phase thermoregulation in C. burra was characterized by an unusually low lower critical limit of thermoneutrality at Ta = ~ 21 °C and a BMR of 0.317 ± 0.047 W, the lowest BMR relative to allometrically-expected values yet reported in any lark. During the diurnal active phase, red larks were able to tolerate Ta up to 50 °C, with the onset of panting occurring at Ta = 38 °C. Maximum EWL was 1.475 ± 0.107 g h- 1 at Ta = 50 °C, equivalent to 620% of minimum EWL at thermoneutrality. The maximum ratio of evaporative heat dissipation to metabolic heat production was 1.58, a value towards the lower end of the range reported for passerines. Our data support the prediction that C. burra shows metabolic traits similar to those of other larks inhabiting extremely arid climates, but not the notion that evaporative cooling at high Ta in this species is more efficient than in most passerines.

Complete mitochondrial genome of the Mongolian lark, Melanocorypha mongolica (Aves: Passeriformes).

We sequenced the entire mitochondrial genome of Melanocorypha mongolica for the first time. The mitogenome is 17,358 bp in length, which includes 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA (rRNA) genes, the control region (CR1), and the control region 2 (CR2). Gene order follows a pattern similar to those of Eurasian Skylark. Using mitochondrial genomes of Melanocorypha mongolica and other seven reference birds in Sylvioidea, we preformed Bayesian analysis based on concatenated protein-coding genes. The results reveal that Alaudidae and Acroccphalidae are clustered together, which is sister to the branches included Sylviidae and Leiothrichidae. Further sequencing of mitochondrial genomes in Alaudidae is useful to advance phylogenetic relationship of species in the family.