Genome-wide analyses reveal drivers of penguin diversification.

Penguins are the only extant family of flightless diving birds. They currently comprise at least 18 species, distributed from polar to tropical environments in the Southern Hemisphere. The history of their diversification and adaptation to these diverse environments remains controversial. We used 22 new genomes from 18 penguin species to reconstruct the order, timing, and location of their diversification, to track changes in their thermal niches through time, and to test for associated adaptation across the genome. Our results indicate that the penguin crown-group originated during the Miocene in New Zealand and Australia, not in Antarctica as previously thought, and that Aptenodytes is the sister group to all other extant penguin species. We show that lineage diversification in penguins was largely driven by changing climatic conditions and by the opening of the Drake Passage and associated intensification of the Antarctic Circumpolar Current (ACC). Penguin species have introgressed throughout much of their evolutionary history, following the direction of the ACC, which might have promoted dispersal and admixture. Changes in thermal niches were accompanied by adaptations in genes that govern thermoregulation and oxygen metabolism. Estimates of ancestral effective population sizes (Ne ) confirm that penguins are sensitive to climate shifts, as represented by three different demographic trajectories in deeper time, the most common (in 11 of 18 penguin species) being an increased Ne between 40 and 70 kya, followed by a precipitous decline during the Last Glacial Maximum. The latter effect is most likely a consequence of the overall decline in marine productivity following the last glaciation.

More than the eye can see: Genomic insights into the drivers of genetic differentiation in Royal/Macaroni penguins across the Southern Ocean.

The study of systematics in wide-ranging seabirds can be challenging due to the vast geographic scales involved, as well as the possible discordance between molecular, morphological and behavioral data. In the Southern Ocean, macaroni penguins (Eudyptes chrysolophus) are distributed over a circumpolar range including populations in Antarctic and sub-Antarctic areas. Macquarie Island, in its relative isolation, is home to a closely related endemic taxon - the royal penguin (Eudyptes schlegeli), which is distinguishable from E. chrysolophus mainly by facial coloration. Although these sister taxa are widely accepted as representing distinct species based on morphological grounds, the extent of their genome-wide differentiation remains uncertain. In this study, we use genome-wide Single Nucleotide Polymorphisms to test genetic differentiation between these geographically isolated taxa and evaluate the main drivers of population structure among breeding colonies of macaroni/royal penguins. Genetic similarity observed between macaroni and royal penguins suggests they constitute a single evolutionary unit. Nevertheless, royal penguins exhibited a tendency to cluster only with macaroni individuals from Kerguelen Island, suggesting that dispersal occurs mainly between these neighboring colonies. A stepping stone model of differentiation of macaroni/royal populations was further supported by a strong pattern of isolation by distance detected across its whole distribution range, possibly driven by large geographic distances between colonies as well as natal philopatry. However, we also detected intraspecific genomic differentiation between Antarctic and sub-Antarctic populations of macaroni penguins, highlighting the role of environmental factors together with geographic distance in the processes of genetic differentiation between Antarctic and sub-Antarctic waters.

Correction to: Chinstrap penguin population genetic structure: one or more populations along the Southern Ocean?

Correction to: BMC Evolutionary Biology (2018) 18:90 https://doi.org/10.1186/s12862-018-1207-0 .

Chinstrap penguin population genetic structure: one or more populations along the Southern Ocean?

BACKGROUND: Historical factors, demography, reproduction and dispersal are crucial in determining the genetic structure of seabirds. In the Antarctic marine environment, penguins are a major component of the avian biomass, dominant predators and important bioindicators of ecological change. Populations of chinstrap penguins have decreased in nearly all their breeding sites, and their range is expanding throughout the Antarctic Peninsula. Population genetic structure of this species has been studied in some colonies, but not between breeding colonies in the Antarctic Peninsula or at the species' easternmost breeding colony (Bouvetøya). RESULTS: Connectivity, sex-biased dispersal, diversity, genetic structure and demographic history were studied using 12 microsatellite loci and a mitochondrial DNA region (HVRI) in 12 breeding colonies in the South Shetland Islands (SSI) and the Western Antarctic Peninsula (WAP), and one previously unstudied sub-Antarctic island, 3600 km away from the WAP (Bouvetøya). High genetic diversity, evidence of female bias-dispersal and a sign of population expansion after the last glacial maximum around 10,000 mya were detected. Limited population genetic structure and lack of isolation by distance throughout the region were found, along with no differentiation between the WAP and Bouvetøya (overall microsatellite F ST = 0.002, p = 0.273; mtDNA F ST  = - 0.004, p = 0.766), indicating long distance dispersal. Therefore, genetic assignment tests could not assign individuals to their population(s) of origin. The most differentiated location was Georges Point, one of the southernmost breeding colonies of this species in the WAP. CONCLUSIONS: The subtle differentiation found may be explained by some combination of low natal philopatric behavior, high rates of dispersal and/or generally high mobility among colonies of chinstrap penguins compared to other Pygoscelis species.

Landscape genomics: natural selection drives the evolution of mitogenome in penguins.

BACKGROUND: Mitochondria play a key role in the balance of energy and heat production, and therefore the mitochondrial genome is under natural selection by environmental temperature and food availability, since starvation can generate more efficient coupling of energy production. However, selection over mitochondrial DNA (mtDNA) genes has usually been evaluated at the population level. We sequenced by NGS 12 mitogenomes and with four published genomes, assessed genetic variation in ten penguin species distributed from the equator to Antarctica. Signatures of selection of 13 mitochondrial protein-coding genes were evaluated by comparing among species within and among genera (Spheniscus, Pygoscelis, Eudyptula, Eudyptes and Aptenodytes). The genetic data were correlated with environmental data obtained through remote sensing (sea surface temperature [SST], chlorophyll levels [Chl] and a combination of SST and Chl [COM]) through the distribution of these species. RESULTS: We identified the complete mtDNA genomes of several penguin species, including ND6 and 8 tRNAs on the light strand and 12 protein coding genes, 14 tRNAs and two rRNAs positioned on the heavy strand. The highest diversity was found in NADH dehydrogenase genes and the lowest in COX genes. The lowest evolutionary divergence among species was between Humboldt (Spheniscus humboldti) and Galapagos (S. mendiculus) penguins (0.004), while the highest was observed between little penguin (Eudyptula minor) and Adélie penguin (Pygoscelis adeliae) (0.097). We identified a signature of purifying selection (Ka/Ks < 1) across the mitochondrial genome, which is consistent with the hypothesis that purifying selection is constraining mitogenome evolution to maintain Oxidative phosphorylation (OXPHOS) proteins and functionality. Pairwise species maximum-likelihood analyses of selection at codon sites suggest positive selection has occurred on ATP8 (Fixed-Effects Likelihood, FEL) and ND4 (Single Likelihood Ancestral Counting, SLAC) in all penguins. In contrast, COX1 had a signature of strong negative selection. ND4 Ka/Ks ratios were highly correlated with SST (Mantel, p-value: 0.0001; GLM, p-value: 0.00001) and thus may be related to climate adaptation throughout penguin speciation. CONCLUSIONS: These results identify mtDNA candidate genes under selection which could be involved in broad-scale adaptations of penguins to their environment. Such knowledge may be particularly useful for developing predictive models of how these species may respond to severe climatic changes in the future.

Marked phylogeographic structure of Gentoo penguin reveals an ongoing diversification process along the Southern Ocean.

Two main hypotheses have been debated about the biogeography of the Southern Ocean: (1) the Antarctic Polar Front (APF), acting as a barrier between Antarctic and sub-Antarctic provinces, and (2) the Antarctic Circumpolar Current (ACC), promoting gene flow among sub-Antarctic areas. The Gentoo penguin is distributed throughout these two provinces, separated by the APF. We analyzed mtDNA (HVR1) and 12 microsatellite loci of 264 Gentoo penguins, Pygoscelis papua, from 12 colonies spanning from the Western Antarctic Peninsula and the South Shetland Islands (WAP) to the sub-Antarctic Islands (SAI). While low genetic structure was detected among WAP colonies (mtDNA ФST=0.037-0.133; microsatellite FST=0.009-0.063), high differentiation was found between all SAI and WAP populations (mtDNA ФST=0.678-0.930; microsatellite FST=0.110-0.290). These results suggest that contemporary dispersal around the Southern Ocean is very limited or absent. As predicted, the APF appears to be a significant biogeographical boundary for Gentoo penguin populations; however, the ACC does not promote connectivity in this species. Our data suggest demographic expansion in the WAP during the last glacial maximum (LGM, about 20kya), but stability in SAI. Phylogenetic analyses showed a deep divergence between populations from the WAP and those from the SAI. Therefore, taxonomy should be further revised. The Crozet Islands resulted as a basal clade (3.57Mya), followed by the Kerguelen Islands (2.32Mya) as well as a more recent divergence between the Falkland/Malvinas Islands and the WAP (1.27Mya). Historical isolation, local adaptation, and past climate scenarios of those Evolutionarily Significant Units may have led to different potentials to respond to climate changes.

The niche and phylogeography of a passerine reveal the history of biological diversification between the Andean and the Atlantic forests.

The Atlantic Forest is separated from the Andean tropical forest by dry and open vegetation biomes (Chaco and Cerrado). Despite this isolation, both rainforests share closely related lineages, which suggest a past connection. This connection could have been important for forest taxa evolution. In this study, we used the Saffron-billed Sparrow (Arremon flavirostris) as a model to evaluate whether the Andean and the Atlantic forests act as a refugia system, as well as to test for a history of biogeographic connection between them. In addition, we evaluated the molecular systematic of intraspecific lineages of the studied species. We modeled the current and past distribution of A. flavirostris, performed phylogeographic analyses based on mitochondrial and nuclear genes, and used Approximate Bayesian Computation (ABC) analyses to test for biogeographic scenarios. The major phylogeographic disjunction within A. flavirostris was found between the Andean and the Atlantic forests, with a divergence that occurred during the Mid-Pleistocene. Our paleodistribution models indicated a connection between these forest domains in different periods and through both the Chaco and Cerrado. Additionally, the phylogeographic and ABC analyses supported that the Cerrado was the main route of connection between these rainforests, but without giving decisive evidence against a Chaco connection. Our study with A. flavirostris suggest that the biodiversity of the Andean and of the Atlantic forests could have been impacted (and perhaps enriched?) by cycles of connections through the Cerrado and Chaco. This recurrent cycle of connection between the Andean and the Atlantic Forest could have been important for the evolution of Neotropical forest taxa. In addition, we discussed taxonomic implications of the results and proposed to split the studied taxon into two full species.

Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing.

Microsatellites are valuable molecular markers for evolutionary and ecological studies. Next generation sequencing is responsible for the increasing number of microsatellites for non-model species. Penguins of the Pygoscelis genus are comprised of three species: Adélie (P. adeliae), Chinstrap (P. antarcticus) and Gentoo penguin (P. papua), all distributed around Antarctica and the sub-Antarctic. The species have been affected differently by climate change, and the use of microsatellite markers will be crucial to monitor population dynamics. We characterized a large set of genome-wide microsatellites and evaluated polymorphisms in all three species. SOLiD reads were generated from the libraries of each species, identifying a large amount of microsatellite loci: 33,677, 35,265 and 42,057 for P. adeliae, P. antarcticus and P. papua, respectively. A large number of dinucleotide (66,139), trinucleotide (29,490) and tetranucleotide (11,849) microsatellites are described. Microsatellite abundance, diversity and orthology were characterized in penguin genomes. We evaluated polymorphisms in 170 tetranucleotide loci, obtaining 34 polymorphic loci in at least one species and 15 polymorphic loci in all three species, which allow to perform comparative studies. Polymorphic markers presented here enable a number of ecological, population, individual identification, parentage and evolutionary studies of Pygoscelis, with potential use in other penguin species.

Uncovering population structure in the Humboldt penguin (Spheniscus humboldti) along the Pacific coast at South America.

The upwelling hypothesis has been proposed to explain reduced or lack of population structure in seabird species specialized in food resources available at cold-water upwellings. However, population genetic structure may be challenging to detect in species with large population sizes, since variation in allele frequencies are more robust under genetic drift. High gene flow among populations, that can be constant or pulses of migration in a short period, may also decrease power of algorithms to detect genetic structure. Penguin species usually have large population sizes, high migratory ability but philopatric behavior, and recent investigations debate the existence of subtle population structure for some species not detected before. Previous study on Humboldt penguins found lack of population genetic structure for colonies of Punta San Juan and from South Chile. Here, we used mtDNA and nuclear markers (10 microsatellites and RAG1 intron) to evaluate population structure for 11 main breeding colonies of Humboldt penguins, covering the whole spatial distribution of this species. Although mtDNA failed to detect population structure, microsatellite loci and nuclear intron detected population structure along its latitudinal distribution. Microsatellite showed significant Rst values between most of pairwise locations (44 of 56 locations, Rst = 0.003 to 0.081) and 86% of individuals were assigned to their sampled colony, suggesting philopatry. STRUCTURE detected three main genetic clusters according to geographical locations: i) Peru; ii) North of Chile; and iii) Central-South of Chile. The Humboldt penguin shows signal population expansion after the Last Glacial Maximum (LGM), suggesting that the genetic structure of the species is a result of population dynamics and foraging colder water upwelling that favor gene flow and phylopatric rate. Our findings thus highlight that variable markers and wide sampling along the species distribution are crucial to better understand genetic population structure in animals with high dispersal ability.

Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins.

The evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans-species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long-term survival of the species.

Molecular Epidemiology of Avian Malaria in Wild Breeding Colonies of Humboldt and Magellanic Penguins in South America.

Avian malaria is a disease caused by species of the genera Haemoproteus, Leucocytozoon, and Plasmodium. It affects hundreds of bird species, causing varied clinical signs depending on the susceptibility of the host species. Although high mortality has been reported in captive penguins, limited epidemiological studies have been conducted in wild colonies, and isolated records of avian malaria have been reported mostly from individuals referred to rehabilitation centers. For this epidemiological study, we obtained blood samples from 501 adult Humboldt and 360 adult Magellanic penguins from 13 colonies throughout South America. To identify malaria parasitaemia, we amplified the mtDNA cytochrome b for all three parasite genera. Avian malaria was absent in most of the analyzed colonies, with exception of the Punta San Juan Humboldt penguin colony, in Peru, where we detected at least two new Haemoproteus lineages in three positive samples, resulting in a prevalence of 0.6% for the species. The low prevalence of avian malaria detected in wild penguins could be due to two possible causes: A low incidence, with high morbidity and mortality in wild penguins or alternatively, penguins sampled in the chronic stage of the disease (during which parasitaemia in peripheral blood samples is unlikely) would be detected as false negatives.

Have historical climate changes affected Gentoo penguin (Pygoscelis papua) populations in Antarctica?

The West Antarctic Peninsula (WAP) has been suffering an increase in its atmospheric temperature during the last 50 years, mainly associated with global warming. This increment of temperature trend associated with changes in sea-ice dynamics has an impact on organisms, affecting their phenology, physiology and distribution range. For instance, rapid demographic changes in Pygoscelis penguins have been reported over the last 50 years in WAP, resulting in population expansion of sub-Antarctic Gentoo penguin (P. papua) and retreat of Antarctic Adelie penguin (P. adeliae). Current global warming has been mainly associated with human activities; however these climate trends are framed in a historical context of climate changes, particularly during the Pleistocene, characterized by an alternation between glacial and interglacial periods. During the last maximal glacial (LGM∼21,000 BP) the ice sheet cover reached its maximum extension on the West Antarctic Peninsula (WAP), causing local extinction of Antarctic taxa, migration to lower latitudes and/or survival in glacial refugia. We studied the HRVI of mtDNA and the nuclear intron ßfibint7 of 150 individuals of the WAP to understand the demographic history and population structure of P. papua. We found high genetic diversity, reduced population genetic structure and a signature of population expansion estimated around 13,000 BP, much before the first paleocolony fossil records (∼1,100 BP). Our results suggest that the species may have survived in peri-Antarctic refugia such as South Georgia and North Sandwich islands and recolonized the Antarctic Peninsula and South Shetland Islands after the ice sheet retreat.

Comparative genome-wide polymorphic microsatellite markers in Antarctic penguins through next generation sequencing

Abstract Microsatellites are valuable molecular markers for evolutionary and ecological studies. Next generation sequencing is responsible for the increasing number of microsatellites for non-model species. Penguins of the Pygoscelis genus are comprised of three species: Adélie (P. adeliae), Chinstrap (P. antarcticus) and Gentoo penguin (P. papua), all distributed around Antarctica and the sub-Antarctic. The species have been affected differently by climate change, and the use of microsatellite markers will be crucial to monitor population dynamics. We characterized a large set of genome-wide microsatellites and evaluated polymorphisms in all three species. SOLiD reads were generated from the libraries of each species, identifying a large amount of microsatellite loci: 33,677, 35,265 and 42,057 for P. adeliae, P. antarcticus and P. papua, respectively. A large number of dinucleotide (66,139), trinucleotide (29,490) and tetranucleotide (11,849) microsatellites are described. Microsatellite abundance, diversity and orthology were characterized in penguin genomes. We evaluated polymorphisms in 170 tetranucleotide loci, obtaining 34 polymorphic loci in at least one species and 15 polymorphic loci in all three species, which allow to perform comparative studies. Polymorphic markers presented here enable a number of ecological, population, individual identification, parentage and evolutionary studies of Pygoscelis, with potential use in other penguin species.