DOI: 10.5061/dryad.6djh9w15t
title: Species delimitation beyond phylogenomics: integrative approaches reveal gentoo penguin speciation

description: Isolation and adaptation to new environments are important steps for
 reproductive isolation and consequently speciation. Seabirds have low
 phenotypic variation along their ranges in the absence of clear geographic
 or environmental barriers to dispersal. Despite the lacking visible
 phenotypic differences, the number of taxa for the gentoo penguin
 (Pygoscelis papua, Forster 1781) in the Southern Ocean has been under
 debate for the last decade, ranging from one to six different taxa. Here,
 we provide several lines of evidence from genomics, ecology, morphological
 data, and a complete systematic review that supports four distinctive
 gentoo penguin species, including the description of a new species. We
 also provide future niche projections for each of these species. Gentoo
 penguin genomes (n = 64) recover four main lineages: the northern gentoo
 (from South America), the southern gentoo (Antarctic Peninsula and
 maritime Antarctica, south of the Antarctic Polar Front, APF), the
 southeastern gentoo (from Kerguelen Islands), and the eastern gentoo
 (colonies located at lower latitudes north of the APF). Our analysis of
 selection across the genome recovered between 42 and 101 genes under
 selection for each of the four species, demonstrating that the four
 species are experiencing differing selective pressures that have caused
 them to diverge adaptively. The function of these genes affects traits
 that include reproduction, thermoregulation, osmoregulation, feed
 efficiency, and morphological variation. Morphological data were taken
 from museum individuals of all lineages, including from South Georgia
 gentoos, which have previously been considered a distinct taxon.
 Multivariate morphological comparisons of all pairs of lineages showed
 that the northern, southern, southeastern, and South Georgia gentoo
 penguins are morphologically distinct from each other (p &lt; 0.05 for
 all pairwise comparisons), while the eastern lineage is intermediate in
 size and overlaps in morphospace with other lineages. This result also
 suggests that body size across latitudes is in direct contrast to
 Bergmann’s rule. Here, we describe the southeastern gentoo penguin from
 Kerguelen Island and confirm the taxonomic rank of gentoos from Macquarie
 Island and South Georgia Island as subspecies. Species distribution
 modelling suggests that climate change will expand the favourable space
 for the southern range expansion of the southern gentoo penguin but would
 result in a net loss of suitable habitats for compensatory niche shift
 relocation for the northern and southeastern gentoos. Despite this,
 amongst the three subantarctic species, the northern and southeastern
 gentoos possess high neutral and adaptive genetic diversity, including
 genes related to cold and heat response. This may represent a higher
 potential to evolve under environmental changes compared with the eastern
 gentoo penguin; therefore, the future resilience of each species remains
 uncertain. This study reinforces the urgent need for explicit recognition
 and protection of the four regional gentoo species based on their genetic,
 morphological, and ecological distinctiveness.
description: The whole genomes of gentoo penguins were resequenced
 from 60 individuals and four GenBank genomes (Vianna et al. 2020) were
 included to complement the number of samples per breeding colony. The
 genome data set contains individuals distributed within the four main
 evolutionary lineages previously identified. This study contains several
 lines of evidence, from genomics, ecology, morphology, and population
 trends and future niche projections that support the existence of four
 distinctive gentoo penguin species around the Southern Ocean.
 The
 bioinformatic processing of genomic data includes cleaning of low-quality
 reads and remotion of adapters. Fastq trimmed reads were mapped to the
 reference genome (GenBank access GCA_010090195.1) and BAM files were
 generated using BWA-mem2 and processed using GATK. Variant calling was
 performed using bcftools mpileup/call and after SNPs processing, a fasta
 consensus file was generated using bcftools consensus. From the fasta
 files, CDS were extracted with gffread using genomic coordinates from the
 reference genome. In addition, UCE were extracted using PHYLUCE. Each
 genomic marker was aligned using MAFFT and gene/species trees were
 constructed using the RAxML-ASTRAL III pipeline. Additionally, a subset of
 CDS was used to infer the divergence time using StarBEAST2. For this, a
 subset of genes was extracted using the approach described by Koch (2021),
 which calculates multiple gene properties for a range of phylogenomic
 datasets and predicts their patterns of covariance, enabling the ordering
 of loci by their putative rate of evolution and their relative
 phylogenetic informativeness.  Differences in morphology were explored from
 museum individuals from different locations around the Southern Ocean,
 covering all the lineages and subpopulations, and were performed using
 lineal morphology information. Based on previous studies of niche modelling of
 gentoo penguin habitats, we used occurrence data and relevant bioclimatic
 variables defined by Pertierra et al. (2020) to perform future niche
 projections for each species.
description: Beast2, Figtree