Abstract
The mammalian skin exhibits a rich spectrum of evolutionary adaptations. The pilosebaceous unit, composed of the hair shaft, follicle, and the sebaceous gland, is the most striking synapomorphy. The evolutionary diversification of mammals across different ecological niches was paralleled by the appearance of an ample variety of skin modifications. Pangolins, order Pholidota, exhibit keratin-derived scales, one of the most iconic skin appendages. This formidable armor is intended to serve as a deterrent against predators. Surprisingly, while pangolins have hair on their abdomens, the occurrence of sebaceous and sweat glands is contentious. Here, we explore various molecular modules of skin physiology in four pangolin genomes, including that of sebum production. We show that genes driving wax monoester formation, Awat1/2, show patterns of inactivation in the stem pangolin branch, while the triacylglycerol synthesis gene Dgat2l6 seems independently eroded in the African and Asian clades. In contrast, Elovl3 implicated in the formation of specific neutral lipids required for skin barrier function is intact and expressed in the pangolin skin. An extended comparative analysis shows that genes involved in skin pathogen defense and structural integrity of keratinocyte layers also show inactivating mutations: associated with both ancestral and independent pseudogenization events. Finally, we deduce that the suggested absence of sweat glands is not paralleled by the inactivation of the ATP-binding cassette transporter Abcc11, as previously described in Cetacea. Our findings reveal the sophisticated and complex history of gene retention and loss as key mechanisms in the evolution of the highly modified mammalian skin phenotypes.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
The evolution of mammals entailed some tantalizing lifestyle variations. Ecological transitions such as subterranean burrowing, powered flight, or obligate aquatic regimes, elaborated from prominent eco-physiological adaptations, notably in the skin (Themudo et al. 2020; Wu et al. 2022; Christmas et al. 2023). Some of these skin-phenotypic shifts were quite radical, as illustrated by the complete absence of glands and pelage in Cetacea skin (Fig. 1). The molecular foundations underscoring the skin phenotype of Cetacea is contingent on gene repertoire variations (Nery et al. 2014; Springer and Gatesy 2018; Lopes-Marques et al. 2019a, b; Springer et al. 2021; Themudo et al. 2020; Kowalczyk et al. 2022; Holthaus et al. 2021; Fuchs et al. 2022), which translate into a thick and smooth skin, to counterbalance the mechanical and thermal stress associated with an obligatory aquatic lifestyle (Spearman 1972; Reeb et al. 2007). In other mammalian lineages, the morphological co-occurrence of hair and associated glands (i.e., the pilosebaceous unit) has been more challenging to ascertain. In manatees (Trichechus manatus latirostris), for instance, complete sebaceous gland regression is still disputed, while in the semi-aquatic hippopotamus (Hippopotamus amphibius) hair is sparsely present yet, sebaceous and sweat glands were so far undetected (Fig. 1) (Sokolov 1982; Graham 2005; Springer et al. 2021). In agreement, the collection of key molecular modules participating in sebum production in aquatic or semi-aquatic mammals was shown to mirror such mosaic of skin morphologies: being fully absent in cetaceans but partially eroded in manatees and hippopotamuses (Lopes-Marques et al. 2019a, b; Themudo et al. 2020; Springer et al. 2021). Extreme skin modifications are not restricted to aquatic species and include the naked mole rat and pangolins (Menon et al. 2019; Li et al. 2020; Savina et al. 2022). Species from the order Pholidota display a formidable keratin-scale armor probably serving as a key deterrent against predators and infections (Fig. 1) (Meyer et al. 2013; Choo et al. 2016; Li et al. 2020). Interestingly, while pangolins have scattered hair on their abdomens, the presence of exocrine glands is contentious, since sweat and sebaceous glands were not detected in their dermis (Liumsiricharoen et al. 2008; Li et al. 2020). Consistently, the reported gene sequence decay of the melanocortin 5 receptor (Mc5r) in pangolins (Springer and Gatesy 2018; Liu et al. 2022), a gene with abundant expression in exocrine glands and centrally involved in sebogenesis (Eisinger et al. 2011; Xu et al. 2020; Shintani et al. 2021), is suggestive of a radical shift in skin exocrine function. Overall, the comparative molecular architecture governing skin physiology in particular that associated with the glandular exocrine and eccrine compartment in Pholidota is largely unknown. Here, we provide an exhaustive comparative genomic analysis of the molecular modules governing mammalian skin homeostasis in four species of pangolins. Our findings provide an insight into the richness of evolutionary routes and processes responsible for the skin physiology of extant mammalian lineages.
Methods
Genome Resources
The genome regions of the target genes were retrieved from the NCBI (National Center for Biotechnology Information) database. Two of the studied species have high-quality genome annotations (M. javanica (GCA_014570535.1) and M. pentadactyla (GCA_014570555.1)) (Table 1). Thus, target genes and corresponding genomic regions were retrieved using gene symbols and manually verified. We additionally used two recently released genome assemblies (non-annotated) from M. javanica (GCA_024605085.1) and M. pentadactyla (GCA_024244205.1) (Wang et al. 2022; Yan et al. 2023). For non-annotated genomes (Table 1), we used BLAST (Basic Local Alignment Search Tool), using as a query the target and two flanking genes from species with annotated genomes, to extract the full scaffold sequence. Gene selection involved an exhaustive literature review to define key genetic pathways involved in skin physiology (e.g., Themudo et al. 2020). This initial screening was complemented with the investigation of the skin “enriched genes” list from the Human Protein Atlas resource (Uhlén et al. 2015). Additionally, we used String to explore protein–protein networks of skin-specific gene families (Szklarczyk et al. 2021).
Sequence Alignment Analysis
Each gene sequence was aligned against a coding and curated reference sequence for each gene (Homo sapiens) using PseudoChecker (Alves et al. 2020), a program that uses the MACSE alignment software (Ranwez et al. 2018). PseudoChecker attributes a value ranging from 0 to 5, the PseudoIndex, with respect to the status of the genomic sequence in comparison to the reference: a value of 0 representing a fully functional protein coding sequence and a value of 5 suggesting pseudogenization mutations (Table 1). For the genomic regions scoring 3 or higher on PseudoIndex, a second alignment was performed, against the same reference, using Geneious Prime 2021.2.2 (https://www.geneious.com), for manual curation and evaluation.
Mutational Validation via SRAs
Identified mutations were validated using Sequence Read Archives (SRAs) (Online Resource 1). Two independent projects were used per species when possible and aligned with our genomic sequence using Geneious Prime 2021.2.2. Exceptions include P. tricuspis, for which only one SRA is available and M. crassicaudata for which no SRA project exists. Cross-species conserved non-synonymous mutations were selected for further validation and when conservation was authenticated, mutations located in the mid-section of the gene sequence were selected, due to the increased stability of gene structure and lower susceptibility to alignment artifacts.
Gene Expression Analysis of Elovl3 and Abcc11 in Pangolin Tissues
RNA-seq data for the tissues of M. javanica from previously published bioprojects were downloaded from NCBI’s database (Online Resource 2). In order to map the SRAs to the reference genome, we indexed the genome using Hisat2 v.2.2.0 (Kim et al. 2015, 2019; Zhang et al. 2021) to enable the mapping of both forward and reverse sequences (within the SRA archives) to the species’ reference genome (YNU_ManJav_2.0). In this mapping, the option for downstream transcriptome assembly was triggered to reduce computational and memory consumption with transcript assembly. The generated SAM files were then converted and sorted to BAM files using samtools and submitted to featureCounts, to quantify the raw reads that were mapped to the transcriptomic components (Liao et al. 2014); the raw read quantification was then transformed to transcripts per million (TPM) using an in-house script.
Results and Discussion
In the present work, we set to analyze the status of skin-specific genes, mostly involved in sebaceous gland function, across four Pholidota species: M. javanica, M. pentadactyla, M. crassicaudata, and P. tricuspis. Pangolins diversified approximately 38 million years ago and have since colonized two continents, Africa and Asia (Fig. 2) (Gaubert et al. 2018). We first identified the gene-containing scaffold in each of the investigated genome assemblies, using PseudoIndex to classify the coding condition of the studied genes in pangolin genomes (Alves et al. 2020) (Online Resource 3). This classification system varies in a discrete scale from 0 to 5, with 0 suggesting full functionality of the candidate gene and a value of 5 indicating a complete inactivation (Table 1). Genes with PseudoIndex above 2 were further validated and the retrieved mutations carefully annotated.
Sebum-Producing Genes Show Signs of Erosion
Sebum comprises a complex lipid mixture. The biosynthesis of sebum components involves the action of various key modules: i.e., monoacylglycerol O-acyltransferases—Mogat2 and Mogat3; diacyl-glycerol O-acyltransferases—Dgat2 and Dgat2l6; and wax alcohol acyltransferases—Awat1 and Awat2 (Bell and Coleman 1980; Turkish et al. 2005; Holmes 2010; Kawelke and Feussner 2015). Besides fatty acid esterification to produce triglycerides or waxes, these modules also encompass fatty acid elongation (i.e., elongases—Elovl3), as well as trafficking and signaling (i.e., fatty acid-binding protein—Fabp9), required for the upstream regulation of sebum production via fatty acid-responsive transcription factors (i.e., Peroxisome proliferator-activated receptors—PPARs) (Fig. 2) (Trivedi et al. 2006; Kobayashi and Fujimori 2012).
Our comparative analysis showed that numerous disruptive mutations are present in the sebum production-related genes in pangolins. Regarding Awat1, using the reference genome we identified and validated, with independent SRA data, a conserved premature stop codon in the sixth exon in M. javanica, M. pentadactyla, and P. tricuspis (Fig. 2; Online Resources 3 and 4); in the M. crassicaudata genome, no Awat1-containing scaffold was found yet, a possible problem in the genome assembly cannot be discarded (not shown). Other mutations, including nucleotide deletions and premature stop codons, were also retrieved, notably a set of mutations conserved within the Manis genus (Online Resources 3 and 4). Curiously, no Awat1 was found in the novel non-annotated genome of M. javanica (GCA_024605085.1), whereas for M. pentadactyla the additional genome largely confirmed previous observations (GCA_024244205.1) (Online Resource 5). Similarly to Awat1, Awat2 displays a mutational pattern concurrent with the inactivation of the gene in the stem of the pangolin clade, exhibiting a conserved loss of a canonical splice site in exon 6 and lack of terminal stop codon in all examined species. Additionally, other disruptive mutations were mapped and validated; notably, a four-nucleotide insertion in the fourth exon found conserved within the Manis genus or a 2-nucleotide deletion retrieved in the exon 4 of P. tricuspis (Fig. 2; Online Resources 3 and 4). Dgat2l6 orthologues also displayed several disruptive mutations in pangolins (Fig. 2; Online Resources 3 and 4); yet, unlike Awat1 and Awat2, none was shared across all pangolin species. Still, mutations were found to be conserved within the Manis genus (i.e., premature stops codons, nucleotide insertions, and losses of splice sites in multiple different exons), notably a two-nucleotide insertion in the second exon, leading to a premature stop codon, which was further validated by SRA analysis (Fig. 2; Online Resources 3 and 4). In P. tricuspis, a set of nucleotide deletions, premature stop codons, and a validated single-nucleotide insertion were identified (Online Resources 3 and 4). Additionally, exon 1 of P. tricuspis and exon 5 of M. pentadactyla were not found (not shown). Such mutational patterns indicate an independent Dgat2l6 erosion among pangolin lineages.
We next investigated Fabp9 (Fatty Acid-Binding Protein 9) and Aadacl3 (Arylacetamide Deacetylase-Like 3). Fabp9 is typically expressed in the testis (Selvaraj et al. 2010), but previous findings have also suggested a role in skin homeostasis in Artiodactyls (Jiang et al. 2014). Aadacl3, although poorly studied, appears to be related with epidermal fat deposition (Lu et al. 2020; Sweet-Jones et al. 2021). Additionally, analysis of the Human Protein Atlas (www.proteinatlas.org) shows a very specific pattern with expression noted only in skin, breast, and placenta (not shown). Importantly, both these genes have been found to be inactivated on the stem Cetacea branch (Huelsmann et al. 2019; Lopes-Marques et al. 2019a, b; Springer et al. 2021). Aadacl3 is also eroded in the African elephant (Huelsmann et al. 2019), a lineage where the presence of sebaceous glands has been contentious (Spearman 1970; Lopes-Marques et al. 2019a, b). Sequence analysis allowed the identification of a conserved single-nucleotide deletion in the third exon of Aadacl3, found in M. javanica, M. pentadactyla, and P. tricuspis, leading to the emergence of a premature stop codon (Fig. 2; Online Resources 3 and 4), as well as a conserved two-nucleotide insertion in the same exon (Fig. 2; Online Resources 3 and 4). No gene ORF was found for M. crassicaudata possibly due to low-quality genome assembly (not shown). Regarding Fabp9, a transversal canonical splice site loss was found in exon 3 for M. javanica, M. pentadactyla, and P. tricuspis. Additional lineage-specific nucleotide deletions were also retrieved and validated (Fig. 2; Online Resource 3 and 4). In M. crassicaudata the first exon was not found (Online Resources 3). Finally, for Mogat3, no conserved mutation was detected, although the genomic sequences display several species-specific mutations across the four species, including a premature stop codon on the sixth exon of M. pentadactyla, a single-nucleotide deletion in the fourth exon of M. crassicaudata and numerous insertions in the second exon of P. tricuspis (Fig. 2; Online Resources 3 and 4). During our analysis, we came across a possible case of gene duplication, followed by erosion of both copies of the gene (not shown), similarly to a previous duplication found in the hippopotamus (Springer et al. 2021). Altogether, our analyses show a comprehensive mutational landscape in sebum-related genes and highlight distinct evolutionary routes, with genes inactivated in the stem of the pangolin clade (i.e., Awat1) and genes apparently lost independently in both Asian and African lineages (i.e., Dgat2l6).
Elovl3 is Functional and Expressed in Pangolin Skin Tissue Compartments
Fatty acid elongation is a critical pathway for skin lipid homeostasis. A skin-specific elongase, Elovl3, has been shown to participate in the formation of specific and essential neutral lipids (Westerberg et al. 2004). In effect, the removal of Elovl3 in mice leads to a phenotype of sparse hair coat, hyperplastic pilosebaceous unit, and perturbation of hair lipid contents (Westerberg et al. 2004). In Cetacea, Elovl3 was previously shown to display inactivating mutations (Lopes-Marques et al. 2019a, b; Springer et al. 2021). The identified and examined pangolin Elovl3 orthologues (Online resource 6) all classified with a PseudoIndex of 0, an indication of sequence functionally (Table 1). Yet, we could not exclude deleterious mutations in the regulatory region of the gene that could hamper gene expression and function. Thus, we next examined the expression of Elovl3 in a comprehensive panel of tissues (Fig. 3; Online Resource 2). Of the examined tissues, the Pholidota Elovl3 is uniquely expressed in the skin components, including hair follicles (Fig. 3).
Hair Growth and Robustness are Shaped by Gene Loss Episodes
We next expanded our analysis to a group of genes that play a pivotal role in hair follicle homeostasis: Ectodysplasin A2 Receptor (Eda2r) and Trichohyalin-Like 1 (Tchhl1). Eda2r is a membrane receptor which participates in the regulation of the hair follicle growth cycle (Kwack et al. 2019; Lan et al. 2020; Cai et al. 2021; Font-Porterias et al. 2022). Within the Manis genus, we detected a single-nucleotide deletion in exon 2 and a premature stop codon in exon 4 of Eda2r (Fig. 2; Online Resources 3 and 4); the latter was validated using independent SRAs (Online Resource 4). For P. tricuspis, a 2-nucleotide insertion in exon 3 and a premature stop codon in exon 5 were retrieved, denoting an independent Eda2r loss across Asian and African pangolin lineage. Additionally, multiple non-conserved alterations of the nucleotide sequence were identified in all four species, including insertions, deletions, premature stop codons, loss of splicing sites, and missing exons (Online Resource 3).
We next investigated Tchhl1, a gene responsible for providing mechanical strength to the hair follicle inner root sheath through keratin intermediate filaments (Makino et al. 2020). Numerous disruptive mutations were retrieved and found conserved among members of the Manis genus; a conserved two-nucleotide deletion in the second, and last exon of the gene, was selected for further scrutiny using independent SRAs (Fig. 2; Online Resources 3 and 4). For P. tricuspis, no gene remnant was found. Curiously, Tchhl1, which is chiefly expressed in the stratum basale of the epidermal layer, is also inactivated in cetaceans and hippopotamuses (Springer et al. 2021).
Erosion of the Gasdermin Gene Repertoire
Gasdermins comprise a protein family involved in membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death leading to the release of intracellular contents (Broz et al. 2019; de Schutter et al. 2021). Importantly, members of this gene family are expressed in the skin (Tamura et al. 2007). Gsdma is specifically expressed in human epidermis, hair follicles, and sebaceous glands and was recently shown to be required for epidermal cornification and skin regeneration (Lachner et al. 2017; Huang et al. 2023); in mice, Gsdma3 mutation leads to alopecia (Runkel et al. 2004). Gsdmb and Gsdmc, expressed in human keratinocytes, were also suggested to contribute to keratinocyte differentiation and cornification (Lachner et al. 2017). In addition to skin build-up and maintenance, gasdermins, notably Gsdmb and Gsdmd, were shown to affect T-cell differentiation and function, as well as macrophage infiltration, protecting the skin against bacterial infections and contributing to the pathophysiology of skin diseases, such as psoriasis or skin fibrosis (Liu et al. 2021; Yang et al 2022; Ji et al 2023). Our study of the gasdermin family (Gsdma, Gsdmb, Gsdmc, and Gsdmd) revealed some very different scenarios regarding the conservation status of these genes. For Gsdma, preliminary PseudoIndex analysis suggested pseudogenization for M. crassicaudata and M. javanica (PseudoIndex, 5), while P. tricuspis scored 0, suggestive of an intact gene (Table 1). Further analysis highlighted a missing exon in M. crassicaudata (exon 6), out of the eleven that compose the gene (Online Resource 3). Regarding M. javanica, the available genomes displayed a single (exon 6) or two missing exons (exons 6 and 7; Online Resource 3). M. pentadactyla genomes, however, yielded contradicting results, with the PseudoIndex analysis scoring 0 for the gene region extracted from the annotated genome (GCA_014570555.1) and scoring 5 with the novel non-annotated genome (GCA_024244205.1) (Table 1 and Online Resource 3). Manual curation of the putatively pseudogenized Gsdma in M. pentadactyla unraveled two missing exons, as observed in M. javanica, in addition to the loss of a splicing site and a single-nucleotide deletion, both in exon 8, confirming the predicted coding status (Online Resource 3). Gsdmb, on the other hand, displayed various mutations conserved across the Manis lineage, including a five-nucleotide deletion in the third exon, which was independently validated by SRAs (Online Resources 3 and 4). Other non-conserved mutations were identified and several exons could not be found through our analysis, especially in P. tricuspis, for which only two out of the nine exons were retrieved, possibly due to a low-quality assembly (Fig. 2). Yet, the P. tricuspis sequence also harbored a disruptive premature stop codon in exon 1, which was further validated (Online Resources 3 and 4). Regarding Gsdmc, no gene was found for M. crassicaudata and P. tricuspis but an assembly artifact cannot be discarded (Online Resource 4). Curiously, for M. javanica and M. pentadactyla, two Gsdmc copies were found in the previous available reference genomes, yet the duplication was not corroborated by the novel genome assemblies (GCA_024244205.1; GCA_024605085.1). Nonetheless, all retrieved copies displayed PseudoIndex scores of 5 (Table 1 and Online Resource 7). Further analysis highlighted the transversal absence of exons 6 and 7 (Online Resources 3 and 4). Numerous inactivating mutations were also retrieved, including mutations conserved between the novel genome assemblies of M. javanica and M. pentadactyla, such as a two-nucleotide deletion in exon 3, as well as two losses of splicing sites in exons 3 and 4 (Online Resource 3). For Gsdmd, M. crassicaudata and P. tricuspis yielded PseudoIndex values above 2, suggesting gene sequence erosion (Table 1). While P. tricuspis revealed a case of exon loss in the sixth exon from a total of ten coding exons, in M. crassicaudata the alignment similarity of the sixth exon was particularly low, hindering further conclusions (Online Resources 3 and 4). Regarding M. javanica and M. pentadactyla, PseudoIndex scores differed between genome assemblies, yielding a value of 0, indicating a high level of conservation of the genomic sequence, for the reference genomes (Table 1), and a value of 5, suggestive of pseudogenization, for the non-annotated genomes (GCA_024244205.1; GCA_024605085.1). Regarding the latter, further scrutiny highlighted the loss of exon 6, as observed for the remaining species, and a conserved premature stop codon in the last exon of both species. Finally, although our results support several gene erosion events within this gene family, further clarifications may be required to fully ascertain the gasdermin repertoire in pangolin species, given the observed discrepancies between current genome assemblies.
Skin-Layer Integrity Genes Display ORF-Disruption Mutations
Next, we investigated a gene related to the integrity of the skin layers—Secreted LY6/PLAUR Domain Containing 1 (Slurp1)—responsible for the stabilization of epithelial cell junctions (Campbell et al. 2019; Okamoto et al. 2020) and found to be eroded in Cetacea (Themudo et al. 2020). Mutations in this gene underlie a rare palmoplantar keratoderma exhibiting increased keratinocyte proliferation, lipid accumulation, and water barrier deficiency (Fischer et al. 2001). We were unable to identify the Slurp1-containing scaffold in the M. crassicaudata genome (not shown). The remaining species showed solid evidence of pseudogenization: missing exons, in M. javanica and M. pentadactyla, loss of splicing sites in M. pentadactyla, or a deletion in the first exon of the gene in P. tricuspis (Fig. 2). Validation of the mutations using independent SRAs was only possible for P. tricuspis (Online Resource 3 and 4).
Sweat Gland Gene Marker are Functional in Pangolins
Water evapotranspiration from the skin is fundamental for thermoregulation. This physiological process is dependent on the action of a subset of skin elements and the sweat glands: eccrine, opening directly into the skin surface, and apocrine, opening into the pilosebaceous unit (Kobielak et al. 2015). Similarly to sebaceous glands, the presence of sweat glands in pangolins was so far unreported (Liumsiricharoen et al. 2008). Here, we investigated the expression status of Abcc1, a gene maker of apocrine sweat glands (Martin et al. 2010). Abcc1 gene was previously shown to be eroded in Cetacea, paralleling sweat gland loss in this lineage (Oh et al. 2015). Expression analysis revealed a marked gene expression in the skin (Fig. 3). Thus, despite the apparent absence of sweat glands, Abcc1 was found intact in pangolins.
Gene Loss and the Uniqueness of the Skin Phenotype in Pangolins
Comparative genomics is a powerful tool to decipher the origin and loss of phenotypic variations (Huelsmann et al. 2019; Zoonomia Consortium 2020; Alves et al. 2021; Fuchs et al. 2022; Zheng et al. 2022). Specifically, gene loss-aware research is reverberating, highlighting the role of secondary losses in the emergence of diverse biological features, including the simplification of body plans (i.e., urochordates), the deconstruction of the vertebrate organs (i.e., stomach, pineal gland), the modulation of sensory acuity (i.e., vision, taste), or even behavior and locomotion (Castro et al. 2014; Zhao et al. 2015; Lopes-Marques et al. 2019a, b; Valente et al. 2021a, b; Carneiro et al. 2021; Ferrández-Roldán et al. 2021; Indrischek et al. 2022).
Pholidota skin is unique among mammals (Yan et al. 2023). This group evolved from a common ancestral skin phenotype to armored keratinous-scale appendices (Meyer et al. 2013). The main function of the keratinous scales is to serve as protection for the soft skin underneath, making it a natural shield against predators and external harm, such as UV radiation, as well as pathogenic agents (Wang et al. 2016). Keratins are the key components of pangolin scales (Ehrlich et al. 2019). In effect, a recent genome analysis identified a unique expansion in the number of high glycine-tyrosine keratin-associated proteins (HGT-KRTAPs) specifically associated with the phenotype of the pangolin scale (Yan et al. 2023). Interestingly, the presence and functional status of sebaceous and sweat glands in Pholidota are so far unclear (Liumsiricharoen et al. 2008). Previous works emphasized gene loss landscapes in other mammalian lineages with divergent skin phenotypes, such as cetaceans, and large African mammals, such as the African elephant (Loxodonta Africana) or the white rhinoceros (Ceratotherium sinum) (Fig. 4) (Plochocki et al. 2017; Springer and Gatesy 2018; Lopes-Marques et al. 2019a, b; Springer et al. 2021); this prompted us to address whether similar genomic variations underlined the emergence of the distinctive skin phenotype in pangolins. Our comparative analysis led to the annotation and validation of numerous disruptive mutations in target genes—related with sebum production, skin layer development and maintenance, and hair growth—across four studied species spanning the two pangolin lineages. These results support the role of gene pseudogenization episodes as drivers of the extant skin phenotype in Pholidota. Ancestral (i.e., Awat1, Awat2, Aadacl3) and independent (i.e., Dgat2l6) pseudogenization events in genes associated with epidermal lipids and sebum production strongly suggest a progressive impairment of the sebum-producing molecular machinery in pangolin lineages. Similar genomic signatures were previously proposed for mammalian lineages with derived skin phenotypes, particularly visible in the fully aquatic Cetacea (Sokolov 1982; Springer and Gatesy 2018; Lopes-Marques et al. 2019a, b). Sebum, a mammalian synapomorphy, is mainly produced to serve as a protective layer against UV radiation, bacteria, and skin dehydration (Lobitz 1957; Pappas 2009; Niemann and Horsley 2012). These functions, although vital when considering exposed skin, may have diminished relevance in an armor-like skin, as observed in Pholidota (Fig. 4). A similar hypothesis can be drawn regarding genes related to skin protection against external dangers (i.e., microbial infection)—the gasdermin family, responsible for host defense and cell death (Tamura and Shiroishi 2015). In effect, pangolins display an innate immune gene repertoire that is strikingly variable as compared to other mammalian lineages (Haley 2022), and events of pseudogenization have been described for various genes (e.g., viral DNA sensors cGAS and STING; Fisher et al. 2020).
Whereas sebaceous gland dismantling is supported by our data, the current results do not unequivocally clarify the fate of sweat glands in pangolins. Abcc1 gene, a marker of apocrine sweat glands (Martin et al. 2010), generally associated with the hair follicle, was shown to be intact and expressed in pangolins. Yet, unlike their back and tail, the abdomen of pangolins yields an exposed skin with sparse hair and thicker stratum corneum (Meyer et al. 2013). In agreement, evidence of erosion was found in genes related with keratinocyte proliferation and stabilization (i.e., Slurp1) or hair development and mechanical strength (i.e., Edar2, Tchhl1) (Campbell et al. 2019; Lan et al. 2020; Cai et al. 2021). Conversely, other genomic components were found intact. Among these, we find Alox15A and Alox3, epidermal lipoxygenases participating in the maintenance of the cornified layer (Krieg et al. 2013) (not shown) or Elovl3. Such mosaic gene retention has been previously proposed for mammalian species with derived skin phenotypes (i.e., elephant, rhinoceros, hippopotamuses) (Lopes-Marques et al. 2019a, b; Springer et al. 2021).
Conclusion
Our findings show that species of the order Pholidota display numerous skin-related gene pseudogenization events paralleled by the dismantling of the sebaceous gland in this mammalian group and the emergence of their idiosyncratic skin phenotype. Importantly, the present work reinforces the role of gene loss as a powerful evolutionary driver, notably in transitional scenarios or radical phenotypic adaptation, as reported for other mammalian groups such as the fully aquatic Cetacea and Sirenia.
References
Alves LQ, Ruivo R, Fonseca MM, Lopes-Marques MO, Ribeiro P, Castro LFC (2020) PseudoChecker: an integrated online platform for gene inactivation inference. Nucleic Acids Res 48:321–331. https://doi.org/10.1093/nar/gkaa408
Alves LQ, Ruivo R, Valente R, Fonseca MM, Machado AM, Plön S, Monteiro N, García-Parraga D, Ruiz-Díaz S, Sánchez-Calabuig MJ, Gutiérrez-Adán A, Castro LFC (2021) A drastic shift in the energetic landscape of toothed whale sperm cells. Curr Biol 31:3648–3655. https://doi.org/10.1016/j.cub.2021.05.062
Bell RM, Coleman RA (1980) Enzymes of glycerolipid synthesis in eukaryotes. Ann Rev Biochem 49:459–487
Broz P, Pelegrín P (2019) Shao F (2019) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20:3. https://doi.org/10.1038/s41577-019-0228-2
Cai Z, Deng X, Jia J, Wang D, Yuan G (2021) Ectodysplasin A/ectodysplasin a receptor system and their roles in multiple diseases. Front Physiol. https://doi.org/10.3389/fphys.2021.788411
Campbell G, Swamynathan S, Tiwari A, Swamynathan SK (2019) The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production. Biochem Biophys Res Commun 517:729–734. https://doi.org/10.1016/j.bbrc.2019.07.123
Carneiro M, Vieillard J, Andrade P, Boucher S, Afonso S, Blanco-Aguiar JA, Santos N, Branco J, Esteves PJ, Ferrand N, Kullander K, Andersson L (2021) A loss-of-function mutation in RORB disrupts saltatorial locomotion in rabbits. PLoS Genet. https://doi.org/10.1371/journal.pgen.1009429
Castro LFC, Gonçalves O, Mazan S, Tay BH, Venkatesh B, Wilson JM (2014) Recurrent gene loss correlates with the evolution of stomach phenotypes in gnathostome history. Proc R Soc B. https://doi.org/10.1098/rspb.2013.2669
Choo SW, Rayko M, Tan TK, Hari R, Komissarov A, Wee WY, Yurchenko AA, Kliver S, Tamazian G, Antunes A, Wilson RK, Warren WC, Koepfli KP, Minx P, Krasheninnikova K, Kotze A, Dalton DL, Vermaak E, Paterson IC, Dobrynin P, Sitam FT, Rovie-Ryan JJ, Johnson WE, Yusoff AM, Luo SJ, Karuppannan KV, Fang G, Zheng D, Gerstein MB, Lipovich L, O’Brien SJ, Wong GJ (2016) Pangolin genomes and the evolution of mammalian scales and immunity. Genome Res 26(1312):1322. https://doi.org/10.1101/gr.203521.115
Christmas MJ, Kaplow IM, Genereux DP, Dong MX, Hughes GM, Li X, Sullivan PF, Hindle AG, Andrews G, Armstrong JC, Bianchi M, Breit AM, Diekhans M, Fanter C, Foley NM, Goodman DB, Goodman L, Keough KC, Kirilenko B, Kowalczyk A, Lawless C, Lind AL, Meadows JRS, Moreira LR, Redlich RW, Ryan L, Swofford R, Valenzuela A, Wagner F, Wallerman O, Brown AR, Damas J, Fan K, Gatesy J, Grimshaw J, Johnson J, Kozyrev SV, Lawler AJ, Marinescu VD, Morrill KM, Osmanski A, Paulat NS, Phan BN, Reilly SK, Schäffer DE, Steiner C, Supple MA, Wilder AP, Wirthlin ME, Xue JR; Zoonomia Consortium§; Birren BW, Gazal S, Hubley RM, Koepfli KP, Marques-Bonet T, Meyer WK, Nweeia M, Sabeti PC, Shapiro B, Smit AFA, Springer MS, Teeling EC, Weng Z, Hiller M, Levesque DL, Lewin HA, Murphy WJ, Navarro A, Paten B, Pollard KS, Ray DA, Ruf I, Ryder OA, Pfenning AR, Lindblad-Toh K, Karlsson EK, Andrews G, Armstrong JC, Bianchi M, Birren BW, Bredemeyer KR, Breit AM, Christmas MJ, Clawson H, Damas J, Di Palma F, Diekhans M, Dong MX, Eizirik E, Fan K, Fanter C, Foley NM, Forsberg-Nilsson K, Garcia CJ, Gatesy J, Gazal S, Genereux DP, Goodman L, Grimshaw J, Halsey MK, Harris AJ, Hickey G, Hiller M, Hindle AG, Hubley RM, Hughes GM, Johnson J, Juan D, Kaplow IM, Karlsson EK, Keough KC, Kirilenko B, Koepfli KP, Korstian JM, Kowalczyk A, Kozyrev SV, Lawler AJ, Lawless C, Lehmann T, Levesque DL, Lewin HA, Li X, Lind A, Lindblad-Toh K, Mackay-Smith A, Marinescu VD, Marques-Bonet T, Mason VC, Meadows JRS, Meyer WK, Moore JE, Moreira LR, Moreno-Santillan DD, Morrill KM, Muntané G, Murphy WJ, Navarro A, Nweeia M, Ortmann S, Osmanski A, Paten B, Paulat NS, Pfenning AR, Phan BN, Pollard KS, Pratt HE, Ray DA, Reilly SK, Rosen JR, Ruf I, Ryan L, Ryder OA, Sabeti PC, Schäffer DE, Serres A, Shapiro B, Smit AFA, Springer M, Srinivasan C, Steiner C, Storer JM, Sullivan KAM, Sullivan PF, Sundström E, Supple MA, Swofford R, Talbot JE, Teeling E, Turner-Maier J, Valenzuela A, Wagner F, Wallerman O, Wang C, Wang J, Weng Z, Wilder AP, Wirthlin ME, Xue JR, Zhang X (2023) Evolutionary constraint and innovation across hundreds of placental mammals Science 380:3943 https://doi.org/10.1126/science.abn3943
De Schutter E, Roelandt R, Riquet FB, van Camp G, Wullaert A, Vandenabeele P (2021) Punching holes in cellular membranes: biology and evolution of gasdermins. Trends Cell Biol 31:500–513. https://doi.org/10.1016/j.tcb.2021.03.004
Ehrlich F, Laggner M, Langbein L, Burger P, Pollreisz A, Tschachler E, Eckhart L (2019) Comparative genomics suggests loss of keratin K24 in three evolutionary lineages of mammals. Sci Rep 9:10924. https://doi.org/10.1038/s41598-019-47422-y
Eisinger M, Li WH, Anthonavage M, Pappas A, Zhang L, Rossetti D, Huang QL, Seiberg M (2011) A melanocortin receptor 1 and 5 antagonist inhibits sebaceous gland differentiation and the production of sebum-specific lipids. J Dermatol Sci 63:23–32. https://doi.org/10.1016/j.jdermsci.2011.04.001
Ferrández-Roldán A, Fabregà-Torrus M, Sánchez-Serna G, Duran-Bello E, Joaquín-Lluís M, Bujosa P, Plana-Carmona M, Garcia-Fernàndez J, Albalat R, Cañestro C (2021) Cardiopharyngeal deconstruction and ancestral tunicate sessility. Nature 599:431–435. https://doi.org/10.1038/s41586-021-04041-w
Fischer J, Bouadjar B, Heilig R, Huber M, Lefèvre C, Jobard F, Macari F, Bakija-Konsuo A, Ait-Belkacem F, Weissenbach J, Lathrop M, Hohl D, Prud’homme JF, (2001) Mutations in the gene encoding SLURP-1 in Mal de Meleda. Hum Mol Genet 10:875–880. https://doi.org/10.1093/hmg/10.8.875
Fischer H, Tschachler E, Eckhart L (2020) Cytosolic DNA sensing through cGAS and STING is inactivated by gene mutations in pangolins. Apoptosis 25:474–480. https://doi.org/10.1007/s10495-020-01614-4
Font-Porterias N, McNelis MG, Comas D, Hlusko LJ (2022) Evidence of selection in the ectodysplasin pathway among endangered aquatic mammals. Integrative Organismal Biology. https://doi.org/10.1093/iob/obac018
Fuchs P, Drexler C, Ratajczyk S, Eckhart L (2022) Comparative genomics reveals evolutionary loss of epiplakin in cetaceans. Sci Rep 12:1–9. https://doi.org/10.1038/s41598-022-05087-0
Gaubert P, Antunes A, Meng H, Miao L, Peigné S, Justy F, Njiokou F, Dufour S, Danquah E, Alahakoon J, Verheyen E, Stanley WT, O’Brien SJ, Johnson WE, Luo S (2018) The complete phylogeny of pangolins: scaling up resources for the molecular tracing of the most trafficked mammals on earth. J Hered 109:347–359. https://doi.org/10.1093/jhered/esx097
Graham AR (2005) Histological examination of the florida manatee (Trichecus manatus latirostris) INTEGUMENT. Gainesville, Florida
Haley PJ (2022) From bats to pangolins: new insights into species differences in the structure and function of the immune system. Innate Immun 28:107–121. https://doi.org/10.1177/17534259221093120
Holmes RS (2010) Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT). Comp Biochem Physiol Part D Genomics Proteomics 5:45–54. https://doi.org/10.1016/j.cbd.2009.09.004
Holthaus KB, Lachner J, Ebner B, Tschachler E, Eckhart L (2021) Gene duplications and gene loss in the epidermal differentiation complex during the evolutionary land-to-water transition of cetaceans. Sci Rep 11:1–9. https://doi.org/10.1038/s41598-021-91863-3
Huang LY, Li ST, Lin SC, Kao CH, Hong CH, Lee CH, Yang LT (2023) Gasdermin a is required for epidermal cornification during skin barrier regeneration and in an atopic dermatitis-like model. J Invest Dermatol. https://doi.org/10.1016/j.jid.2023.03.1657
Huelsmann M, Hecker N, Springer MS, Gatesy J, Sharma V, Hiller M (2019) Genes lost during the transition from land to water in cetaceans highlight genomic changes associated with aquatic adaptations. Sci Adv. https://doi.org/10.1126/sciadv.aaw6671
Indrischek H, Hammer J, Machate A, Hecker N, Kirilenko B, Roscito J, Hans S, Norden C, Brand M, Hiller M (2022) Vision-related convergent gene losses reveal SERPINE3’s unknown role in the eye. Elife. https://doi.org/10.7554/eLife.77999
Ji X, Chen H, Xie L, Chen S, Huang S, Tan Q, Yang H, Yang T, Ye X, Zeng Z, Wan C, Li L (2023) The study of GSDMB in pathogenesis of psoriasis vulgaris. PLoS ONE. https://doi.org/10.1371/journal.pone.0279908
Jiang Y, Xie M, Chen W, Talbot R, Maddox JF, Faraut T, Wu C, Muzny DM, Li Y, Zhang W, Stanton JA, Brauning R, Barris WC, Hourlier T, Aken BL, Searle SMJ, Adelson DL, Bian C, Cam GR, Chen Y, Cheng S, DeSilva U, Dixen K, Dong Y, Fan G, Franklin IR, Fu S, Guan R, Highland MA, Holder ME, Huang G, Ingham AB, Jhangiani SN, Kalra D, Kovar CL, Lee SL, Liu W, Liu X, Lu C, Lv T, Mathew T, McWilliam S, Menzies M, Pan S, Robelin D, Servin B, Townley D, Wang W, Wei B, White SN, Yang X, Ye C, Yue Y, Zeng P, Zhou Q, Hansen JB, Kristensen K, Gibbs RA, Flicek P, Warkup CC, Jones HE, Oddy VH, Nicholas FW, McEwan JC, Kijas J, Wang J, Worley KC, Archibald AL, Cockett N, Xu X, Wang W, Dalrymple BP (2014) The sheep genome illuminates biology of the rumen and lipid metabolism. Science 344:1168–1173. https://doi.org/10.1126/science.1252806
Kawelke S, Feussner I (2015) Two predicted transmembrane domains exclude very long chain fatty acyl-CoAs from the active site of mouse wax synthase. PLoS ONE. https://doi.org/10.1371/journal.pone.0145797
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360. https://doi.org/10.1038/nmeth.3317
Kim D, Paggi JM, Park C, Bennett C, Salzberg SL (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37:907–915. https://doi.org/10.1038/s41587-019-0201-4
Kobayashi T, Fujimori K (2012) Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPARγ in 3T3-L1 cells. Am J Physiol Endocrinol Metab. https://doi.org/10.1152/ajpendo.00623.2011
Kobielak K, Kandyba E, Leung Y (2015) Skin and skin appendage regeneration. In: Translational regenerative medicine. Elsevier. pp. 269–292
Kowalczyk A, Chikina M, Clark NL (2022) Complementary evolution of coding and noncoding sequence underlies mammalian hairlessness. Elife 11:e76911. https://doi.org/10.7554/eLife.76911
Krieg P, Rosenberger S, de Juanes S, Latzko S, Hou J, Dick A, Kloz U, van der Hoeven F, Hausser I, Esposito I, Rauh M, Schneider H (2013) Aloxe3 knockout mice reveal a function of epidermal lipoxygenase-3 as hepoxilin synthase and its pivotal role in barrier formation. J Invest Dermatol 133:172–180. https://doi.org/10.1038/jid.2012.250
Kwack MH, Kim JC, Kim MK (2019) Ectodysplasin-A2 induces apoptosis in cultured human hair follicle cells and promotes regression of hair follicles in mice. Biochem Biophys Res Commun 520(2):428–433. https://doi.org/10.1016/j.bbrc.2019.10.031
Lachner J, Mlitz V, Tschachler E, Eckhart L (2017) Epidermal cornification is preceded by the expression of a keratinocyte-specific set of pyroptosis-related genes. Sci Rep 7(1):1–11. https://doi.org/10.1038/s41598-017-17782-4
Lan X, Kumar V, Jha A, Aslam R, Wang H, Chen K, Yu Y, He W, Chen F, Luo H, Malhotra A, Singhal PC (2020) EDA2R mediates podocyte injury in high glucose milieu. Biochimie 174:74–83. https://doi.org/10.1016/j.biochi.2020.04.003
Li HM, Liu P, Zhang XJ, Li LM, Jiang HY, Yan H, Hou FH, Chen JP (2020) Combined proteomics and transcriptomics reveal the genetic basis underlying the differentiation of skin appendages and immunity in pangolin. Sci Rep 10:1–13. https://doi.org/10.1038/s41598-020-71513-w
Liao Y, Smyth GK, Shi W (2014) featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930. https://doi.org/10.1093/bioinformatics/btt656
Liu J, Shu M, Liu S, Xue J, Chen H, Li W, Zhou J, Amanullah A, Guan M, Bao J, Pu D, Deng C (2022) Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment. Dev Genes Evol 232:81–87. https://doi.org/10.1007/s00427-022-00688-1
Liumsiricharoen M, Prapong T, Chungsamarnyart N, Thiangtum K, Pongket P, Ruengsuphaphichat P, Suprasert A (2008) Macroscopic and Microscopic Study of the Integument and Accessory Organs of Malayan Pangolin (Manis javanica). Kasetsart Veterinarians
Lobitz WC (1957) The structure and function of the sebaceous glands. AMA Arch Derm. https://doi.org/10.1001/archderm.1957.01550200006002
Lopes-Marques M, Machado AM, Alves LQ, Fonseca MM, Barbosa S, Sinding MHS, Rasmussen MH, Iversen MR, Bertelsen MF, Campos PF, Fonseca R, Ruivo R, Castro LFC (2019a) Complete inactivation of sebum-producing genes parallels the loss of sebaceous glands in Cetacea. Mol Biol Evol 36:1270–1280. https://doi.org/10.1093/molbev/msz068
Lopes-Marques M, Ruivo R, Alves LQ, Sousa N, Machado AM, Castro LFC (2019b) The singularity of cetacea behavior parallels the complete inactivation of melatonin gene modules. Genes (basel). https://doi.org/10.3390/genes10020121
Lu Z, Yue Y, Yuan C, Liu J, Chen Z, Niu C, Sun X, Zhu S, Zhao H, Guo T, Yang B (2020) Genome-wide association study of body weight traits in chinese fine-wool sheep. Animals. https://doi.org/10.3390/ani10010170
Lui ZZ, Yang YJ, Zhou FH, Ma K, Lin XQ, Yan SQ, Gao Y, Chen W (2021) GSDMD contributes to host defense against Staphylococcus aureus skin infection by supressing the Cxcl1-Cxcr2 axis. Vet Res 52(1):1–13. https://doi.org/10.1186/s13567-021-00937-7
Makino T, Mizawa M, Yoshihisa Y, Yamamoto S, Tabuchi Y, Miyai M, Hibino T, Sasahara M, Shimizu T (2020) Trichohyalin-like 1 protein plays a crucial role in proliferation and anti-apoptosis of normal human keratinocytes and squamous cell carcinoma cells. Cell Death Discov. https://doi.org/10.1038/s41420-020-00344-5
Martin A, Saathoff M, Kuhn F, Max H, Terstegen L, Natsch A (2010) A functional ABCC11 allele is essential in the biochemical formation of human axillary odor. JID 130:529–540. https://doi.org/10.1038/jid.2009.254
Menon GK, Catania KC, Crumrine D, Bradley C, Mauldin EA (2019) Unique features of the skin barrier in naked mole rats reflect adaptations to their fossorial habitat. J Morphol 280:1871–1880. https://doi.org/10.1002/jmor.21072
Meyer W, Liumsiricharoen M, Suprasert A, Fleischer LG, Hewicker-Trautwein M (2013) Immunohistochemical demonstration of keratins in the epidermal layers of the Malayan pangolin (Manis javanica), with remarks on the evolution of the integumental scale armour. Eur J Histochem 57:172–177. https://doi.org/10.4081/ejh.2013.e27
Nery MF, Arroyo JI, Opazo JC (2014) Increased rate of hair keratin gene loss in the cetacean lineage. BMC Genomics 15:1–9. https://doi.org/10.1186/1471-2164-15-869
Niemann C, Horsley V (2012) Development and homeostasis of the sebaceous gland. Semin Cell Dev Biol 23:928–936. https://doi.org/10.1016/j.semcdb.2012.08.010
Oh JW, Chung O, Cho YS, Macgregor GR, Plikus MV (2015) Gene loss in keratinization programs accompanies adaptation of cetacean skin to aquatic lifestyle. Exp Dermatol 24:572. https://doi.org/10.1111/exd.12756
Okamoto R, Goto I, Nishimura Y, Kobayashi I, Hashizume R, Yoshida Y, Ito R, Kobayashi Y, Nishikawa M, Ali Y, Saito S, Tanaka T, Sawa Y, Ito M, Dohi K (2020) Gap junction protein beta 4 plays an important role in cardiac function in humans, rodents, and zebrafish. PLoS ONE. https://doi.org/10.1371/journal.pone.0240129
Pappas A (2009) Epidermal Surface Lipids. Dermatoendocrinol 1:72–76. https://doi.org/10.4161/derm.1.2.7811
Plochocki JH, Ruiz S, Rodriguez-Sosa JR, Hall MI (2017) Histological study of white rhinoceros integument. PLoS ONE. https://doi.org/10.1371/journal.pone.0176327
Ranwez V, Douzery EJP, Cambon C, Chantret N, Delsuc F (2018) MACSE v2: Toolkit for the alignment of coding sequences accounting for frameshifts and stop codons. Mol Biol Evol 35:2582–2584. https://doi.org/10.1093/molbev/msy159
Reeb D, Best PB, Kidson SH (2007) Structure of the integument of southern right whales, Eubalaena australis. Anat Rec Adv Integr Anat Evol Biol 290:596–613. https://doi.org/10.1002/ar.20535
Runkel F, Marquardt A, Stoeger C, Kochmann E, Simon D, Kohnke B, Korthaus D, Wattler F, Fuchs H, Hrabé de Angelis M, Stumm G, Nehls M, Wattler S, Franz T, Augustin M (2004) The dominant alopecia phenotypes Bareskin, Rex-denuded, and Reduced Coat 2 are caused by mutations in gasdermin 3. Genomics 84(5):824–835. https://doi.org/10.1016/j.ygeno.2004.07.003
Savina A, Jaffredo T, Saldmann F, Faulkes CG, Moguelet P, Leroy C, del Marmol D, Codogno P, Foucher L, Zalc A, Viltard M, Friedlander G, Aractingi S, Fontaine RH (2022) Single-cell transcriptomics reveals age-resistant maintenance of cell identities, stem cell compartments and differentiation trajectories in long-lived naked mole-rats skin. Aging 14:3728–3756. https://doi.org/10.18632/aging.204054
Selvaraj V, Asano A, Page JL, Nelson JL, Kothapalli KSD, Foster JA, Brenna JT, Weiss RS, Travis AJ (2010) Mice lacking FABP9/PERF15 develop sperm head abnormalities but are fertile. Dev Biol 348:177. https://doi.org/10.1016/j.ydbio.2010.09.019
Shintani A, Sakata-Haga H, Moriguchi K, Tomosugi M, Sakai D, Tsukada T, Taniguchi M, Asano M, Shimada H, Otani H, Shoji H, Hatta J, Mochizuki T, Hatta T (2021) MC5R contributes to sensitivity to UVB waves and barrier function in mouse epidermis. JID Innov 1:100024. https://doi.org/10.1016/j.xjidi.2021.100024
Sokolov VE (1982) Mammal skin. University of California Press, London
Spearman RI (1970) The epidermis and its keratinisation in the African elephant (Loxodonta Africana). Zoologica Africana 5:327–338
Spearman RI (1972) The epidermal stratum corneum of the whale. J Anat 113:373–381
Springer MS, Gatesy J (2018) Evolution of the MC5R gene in placental mammals with evidence for its inactivation in multiple lineages that lack sebaceous glands. Mol Phylogenet Evol 120:364–374. https://doi.org/10.1016/j.ympev.2017.12.010
Springer MS, Guerrero-Juarez CF, Huelsmann M, Collin MA, Danil K, McGowen MR, Oh JW, Ramos R, Hiller M, Plikus MV, Gatesy J (2021) Genomic and anatomical comparisons of skin support independent adaptation to life in water by cetaceans and hippos. Curr Biol 31:2124–2139. https://doi.org/10.1016/j.cub.2021.02.057
Sweet-Jones J, Yurchenko AA, Igoshin AV, Yudin NS, Swain MT, Larkin DM (2021) Resequencing and signatures of selection scan in two Siberian native sheep breeds point to candidate genetic variants for adaptation and economically important traits. Anim Genet 52:126–131. https://doi.org/10.1111/age.13015
Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R, Pyysalo S, Doncheva NT, Legeay M, Fang T, Bork P, Jensen LJ, von Mering C (2021) The STRING database in 2021: customizable protein–protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res 49:605. https://doi.org/10.1093/nar/gkaa1074
Tamura M, Shiroishi T (2015) GSDM family genes meet autophagy. Biochem J 469:5–7. https://doi.org/10.1042/BJ20150558
Tamura M, Tanaka S, Fujii T, Aoki A, Komiyama H, Ezawa K, Sumiyama K, Sagai T, Shiroishi T (2007) Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner. Genomics 89:618–629. https://doi.org/10.1016/j.ygeno.2007.01.003
Themudo G, Alves LQ, Machado AM, Lopes-Marques M, da Fonseca RR, Fonseca M, Ruivo R, Castro LFC (2020) Losing genes: the evolutionary remodeling of cetacea skin. Front Mar Sci. https://doi.org/10.3389/fmars.2020.5923
Trivedi NR, Cong Z, Nelson AM, Albert AJ, Rosamilia LL, Sivarajah S, Gilliland KL, Liu W, Mauger DT, Gabbay RA, Thiboutot DM (2006) Peroxisome proliferator-activated receptors increase human sebum production. J Invest Dermatol 126:2002–2009. https://doi.org/10.1038/sj.jid.5700336
Turkish AR, Henneberry AL, Cromley D, Padamsee M, Oelkers P, Bazzi H, Christiano AM, Billheimer JT, Sturley SL (2005) Identification of two novel human acyl-CoA wax alcohol acyltransferases: members of the diacylglycerol acyltransferase 2 (DGAT2) gene superfamily. J Biol Chem 280(15):14755–14764. https://doi.org/10.1074/jbc.M500025200
Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, Olsson I, Lundberg E, Navani S, Szigyarto CA, Odeberg J, Djureinovic D, Takanen JO, Hober S, Alm T, Edqvist P, Berling H, Tegel H, Mulder J, Rockberg J, Nilsson P, Schwenk JM, Hamsten M, Feilitzen KV, Forsberg M, Persson L, Johansson F, Zwahlen M, von Heijne G, Nielsen J, Pontén F (2015) Tissue-based map of the human proteome. Science. https://doi.org/10.1126/science.1260419
Valente R, Alves F, Sousa-Pinto I, Ruivo R, Castro LFC (2021a) Functional or Vestigial? The Genomics of the pineal gland in xenarthra. J Mol Evol 89:565–575. https://doi.org/10.1007/s00239-021-10025-1
Valente R, Alves LQ, Nabais M, Alves F, Sousa-Pinto I, Ruivo R, Castro LFC (2021b) Convergent Cortistatin losses parallel modifications in circadian rhythmicity and energy homeostasis in Cetacea and other mammalian lineages. Genomics 113:1064–1070. https://doi.org/10.1016/j.ygeno.2020.11.002
Wang B, Yang W, Sherman VR, Meyers MA (2016) Pangolin armor: Overlapping, structure, and mechanical properties of the keratinous scales. Acta Biomater 41:60–74. https://doi.org/10.1016/j.actbio.2016.05.028
Wang Q, Lan T, Li H, Sahu S, Shi M, Zhu Y, Han L, Yang S, Li Q, Zhang L, Deng Z, Liu H, Hua Y (2022) Whole-genome resequencing of Chinese pangolins reveals a population structure and provides insights into their conservation. Commun Biol 5:821. https://doi.org/10.1038/s42003-022-03757-3
Westerberg R, Tvrdik P, Undén AB, Månsson JE, Norlén L, Jakobsson A, Holleran WH, Elias PM, Asadi A, Flodby P, Toftgård R, Capecchi MR, Jacobsson A (2004) Role for ELOVL3 and fatty acid chain length in development of hair and skin function. J Biol Chem 279:5621–5629. https://doi.org/10.1074/jbc.M310529200
Wu T, Deme L, Zhang Z, Huang X, Xu S, Yang G (2022) Decay of TRPV3 as the genomic trace of epidermal structure changes in the land-to-sea transition of mammals. Ecol Evol https://doi.org/10.1002/ece3.8731
Xu Y, Guan X, Zhou R, Gong R (2020) Melanocortin 5 receptor signaling pathway in health and disease. Cell Mol Life Sci 77:3831–3840. https://doi.org/10.1007/s00018-020-03511-0
Yan D, Luo X, Tang J, Xu S, Huang K, Wang X, Feng T, Que T, Jia M, Guo X, Rehman S, Li Z, Yang Y, Li K, Cui K, Ruan J, Liu Q (2023) High-quality genomes of pangolins: insights into the molecular basis of scale formation and adaption to myrmecophagous diet. Mol Biol Evol https://doi.org/10.1093/molbev/msac262
Yang H, Shi Y, Liu LF, Qiu B, Feng Q, Wang Y, Yang B (2022) Pyroptosis executor gasdermin D plays a key role in scleroderma and bleomycin-induced skin fibrosis. Cell Death Discovery 8(1):183. https://doi.org/10.1038/s41420-022-00970-1
Zhang Y, Park C, Bennett C, Thornton M, Kim D (2021) Rapid and accurate alignment of nucleotide conversion sequencing reads with HISAT-3N. Genome Res 31:1290–1295. https://doi.org/10.1101/gr.275193.120
Zhao H, Li J, Zhang J (2015) Molecular evidence for the loss of three basic tastes in penguins. Curr Biol 25:141–142. https://doi.org/10.1016/j.cub.2015.01.026
Zheng Z, Hua R, Xu G, Yang H, Shi P (2022) Gene losses may contribute to subterranean adaptations in naked mole-rat and blind mole-rat. BMC Biol 20:1–17. https://doi.org/10.1186/s12915-022-01243-0
Zoonomia Consortium (2020) A comparative genomics multitool for scientific discovery and conservation. Nature 587:240–245. https://doi.org/10.1038/s41586-020-2876-6
Funding
Open access funding provided by FCT|FCCN (b-on). The funding was provided by NORTE 2020, NORTE-01-0145-FEDER-000040 (ATLANTIDA) to Filipe Castro, and FCT, SFRH/BD/144786/2019, to Raul Valente.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Handling editor: Konstantinos Voskarides.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Pinto, B., Valente, R., Caramelo, F. et al. Decay of Skin-Specific Gene Modules in Pangolins. J Mol Evol 91, 458–470 (2023). https://doi.org/10.1007/s00239-023-10118-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00239-023-10118-z