Giant petrels (Macronectes
spp.) belonging to the family Procellariidae of the order Procellariiformes are pelagic birds distributed throughout the Southern Ocean and Antarctic region. Members of the genus Macronectes
were initially considered to be single polymorphic species until Bourne and Warham [1
] separated these into two sibling species based on morphological and behavioral differences: The southern giant petrel Macronectes giganteus
and the northern giant petrel Macronectes halli
. Nonetheless, as hybridization and back-crossing have been reported in some habitats where both species breed sympatrically, their reproductive isolation would appear to be incomplete [2
]. Both species are categorized as species of “Least Concern” on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species 2018 [3
With a wingspan of 150–210 cm and a body mass of 3.8–5.0 kg, the southern giant petrel is the largest species in the order Procellariiformes, [4
], and two representative color morphs, characterized by grayish-brown and white with scattered blackish plumage, are recognized. Moreover, adults display sexual size dimorphism, with the males having larger bills and wing lengths and being heavier than females [5
]. Hunter [6
] suggested that sexual dimorphism may represent an adaptation associated with differences in foraging behavior with respect to preferred prey items, namely, krill and fish versus penguin carcasses foraged by females and males, respectively. Unlike other piscivorous petrels, the southern giant petrel feeds on animal carcasses [7
], garbage from fishing vessels, and marine organisms such as fish, krill, and cephalopods [3
]. These petrels can convert their high-fat diets into stomach oil comprising wax esters and triglycerides to feed their chicks, and which can be used as an energy source for long-distance flights [8
]. Moreover, given that this stomach oil is not only sticky, but also gives off a sickening odor, the birds use the oil as a defensive weapon against intruders approaching their nest by spraying this from their bills. The bills of these birds are also characterized by long nasal tubes connected to salt glands located on the upper mandible, which play a role in excreting salt from the body [8
Technical advances in the past decade have improved access to sequencing data, with lower costs leading to an explosion in the number of species. Genomes from many diverse organisms have been sequenced of high-quality references using hybrid approaches that combine complementary technologies, e.g., PacBio, 10x Genomics, and Hi-C sequencing technologies. Despite the increasing availability of genetic resources with research and economic value, a fully annotated genome is currently limited for Antarctic avian. Genomic resources has proven itself invaluable, not only for informing the understanding of the environmental adaptations, but for illuminating evolutionary mechanisms and forces. In Antarctic birds, large-scale genome analysis in 18 penguin species has shown that changing climatic conditions, i.e., changes in thermal niches, are accompanied by adaptations in genes that govern thermoregulation and oxygen metabolism, largely leading to the lineage diversification of the species [9
]. Herein, we report the genome of M. giganteus
assembled by integrating Pacific Biosciences single-molecule real-time sequencing and the Chromium system developed by 10x Genomics. We subsequently estimated the phylogenetic position of M. giganteus
relative to that of other avian species and examined its historical effective population size. The genome of the southern giant petrel will facilitate more effective genetic monitoring of threatened species, thereby enabling us to conserve species based on a more comprehensive understanding of their evolutionary mechanisms. Moreover, it will provide a basis for gene functional studies and further comparative genomic studies on the life history and ecological traits of specific avian species.
Recently, reference-mapped genome assembly of the southern giant petrel using short-read Illumina data have been reported [9
], though the genome was fragmented into many scaffolds due to low coverage and sequencing by synthesis technology. With the development of third-generation single-molecule sequencing technology, long-read sequences can be precisely assembled into genome and discovery features of DNA areas that have previously unavailable DNA regions. The genome of M. giganteus
was assembled using the PacBio long reads and 10x Genomics Chromium platform, achieving a final scaffold assembly of 1.247 Gb (94% of the predicted 1.328 Gb genome size). The final scaffolding assembly resulted in a significantly improved assembly, the longest scaffold length was 120 Mb, and the scaffold N50 value was 27 Mb, which were 6- and 4.5-fold greater than the corresponding draft assembly values, respectively (Table 1
). Thirty-six superscaffolds were greater than 10 Mb in size, and 74 scaffolds were over 1 Mb in size. The total assembly length of over 1 Mb was 1.191 Gb.
We compared our data to those pertaining to the B. regulorum
genome to confirm the chromosomal stability of the constructed M. giganteus
genome. Chromosome 1 of B. regulorum
was found to be highly contiguous with the Mgig_0035, Mgig_0020, Mgig_0004, Mgig_0055, Mgig_0008, Mgig_0045, and Mgig_0043 scaffolds of M. giganteus
, with small portions of syntenic inversion. However, these seven M. giganteus
scaffolds mapped two chromosomes (chromosomes 1 and 4) of Tauraco erythrolophus
). Although the southern giant petrel genome sequence does not fully assemble at the chromosome level, lineage-specific chromosome rearrangements are evident, as confirmed by analysis of the chromosomes of other species, despite the paucity of translocations during evolution.
The repeat sequences of M. giganteus
genome contains 11.06% repeat sequences, of which 8.61% comprise transposable elements (TEs). In order to estimate the “relative age” and transposition history of TEs [46
], Kimura distances (K-values) were calculated for all TE copies of each element. Copy divergence is correlated with the age of activity, with very similar copies (low K-values) being indicative of rather recent activity, whereas divergent copies (high K-values) are assumed to have been generated by more ancient transposition events. Kimura substitution levels indicated significant interspecific differences in profiles (Figure S4
), with the M. giganteus
genome being dominated by relatively recent copies (K-values <5) and strongly shaped by LINE and LTR transposons, which can be taken to be indicative of recent bursts of transposition in M. giganteus
. TE expansion may have facilitated gene duplication and other genomic evolutionary events during particular periods of evolutionary history [47
], and thus may have contributed to adaptation to the specifics of Antarctic environments.
A total of 14,993 protein-coding genes were annotated in the M. giganteus genome. The predicted genes were initially annotated by alignment with the NCBI nr database, and subsequently using the InterProScan, EggNOG, and Pfam databases. Consequently, 14,822 genes were annotated in one or more databases, which represents 98.86% of the total genes.
The estimated history of effective population size (Ne) using PSMC analysis revealed evidence of population expansion from 200 to 300 thousand years ago, followed by a subsequent contraction in size (Figure 3
). The Ne curve shown in Figure 3
indicates a reduction during the last glacial maximum and population growth during the last glacial period. Historically, global climate change has substantially influenced the distribution and abundance of biodiversity, including that of birds. In particular, during unfavorable glacial periods, many species experienced range contractions, followed by subsequent expansions during interglacial periods [48
]. A number of the species currently included in the IUCN Red List of Threatened Species have shown a long-term reduction in population size, predating recent declines [48
], among which, the southern giant petrel experienced a long-term reduction in Ne to 40,000 individuals around 0.5 Mya.
Phylogenetic relationships of M. giganteus and other avian species to examine evolutionary relationships showed that clustering in the constructed phylogenetic tree indicates that the southern giant petrel and northern fulmar (Fulmarus glacialis) form a sister group, and that the order Sphenisciformes (including the Adélie penguin and emperor penguin) have a close genetic relationship. On the basis of phylogeny, we calculated species divergence times according to the molecular clock with respect to TimeTree, and accordingly estimated that M. giganteus and F. glacialis diverged 19.97 Mya.
The southern giant petrel has 562 significantly expanded gene families, and the vast majority of expanded pathways are associated with protein catabolism, including proteolysis involved in the cellular protein catabolic process (GO:0051603), the modification-dependent protein catabolic process (GO:0019941), and the ubiquitin-dependent protein catabolic process (GO:0006511). The southern giant petrel is circumpolar, with a distribution that encompasses the sub-Antarctic Islands and the Antarctic Peninsula [49
]. Maintenance of energy homeostasis is essential for survival and effective functioning in cold environments, and intracellular energy homeostasis is closely related to protein degradation and synthesis, including the functioning of the ubiquitous-dependent protein and autophagy systems for protein decomposition and synthesis as energy-saving processes. Consequently, the expansion of these genes may be required to maintain efficient energy homeostasis in cold environments. Among the KEGG metabolic pathway maps we obtained, the MAPK signaling pathway of signal transduction was activated in response to almost any change in the extracellular or intracellular environment that affects the metabolism of cells, organs, or entire organisms required for physiological metabolic adaptation shown to be expanded. Genes associated with carbohydrate and lipid metabolism were also frequently found to be expanded (Figures S5 and S6
The breeding populations of southern giant petrels are distributed on several sub-Antarctic islands, the Antarctic Peninsula, southern Chile, the Malvinas (Falkland) Islands, and Patagonia, Argentina [50
]. Although the trends show a general decline in the total breeding population of M. giganteus
, whereas some colonies have decreased in size over the past few decades, others have increased [49
]. Such population declines are attributed to the detrimental effects of habitat destruction, human disturbance, introduced predators, and fisheries. In this regard, a sufficient genetic diversity is essential to enable adaptation to changing environmental conditions, and is recognized as a key component of biodiversity. M. giganteus
and M. halli
are the only members of the genus Macronectes
. Although M. giganteus
breed both further north and further south than M. halli
, these two species breed sympatrically across five islands: South Georgia, the Prince Edward Islands, Îles Crozet, Îles Kerguelen, and Macquarie Island. The proportion of crossbred species pairs has been reported to be 0.4–2.4% annually for South Georgia island [1
]. We identified a total of 24,887 SSR loci within the southern giant petrel genome. These microsatellite markers will provide useful information for future analyses of the genetic diversity within and among populations, and also these data can be used to identify hybrids between giant petrel species. The SSR data will, in turn, enable us to draw conclusions regarding genome-wide diversity patterns pertinent to conservation of the southern giant petrel, and potentially contribute to research on the population trends of other listed threatened species.