Identification of Novel Bacterial Microproteins Encoded by Small Open Reading Frames Using a Computational Proteogenomics Workflow.

Genome annotation has historically ignored small open reading frames (smORFs), which encode a class of proteins shorter than 100 amino acids, collectively referred to as microproteins. This cutoff was established to avoid thousands of false positives due to limitations of pure genomics pipelines. Proteogenomics, a computational approach that combines genomics, transcriptomics, and proteomics, makes it possible to accurately identify these short sequences by overlaying different levels of omics evidence. In this chapter, we showcase the use of µProteInS, a bioinformatics pipeline developed for the identification of unannotated microproteins encoded by smORFs in bacteria. The workflow covers all the steps from quality control and transcriptome assembly to the scoring and post-processing of mass spectrometry data. Additionally, we provide an example on how to apply the pipeline's machine learning method to identify high-confidence spectra and pinpoint the most reliable identifications from large datasets.

Genome- and Toxicology-Based Safety Assessment of Probiotic Akkermansia muciniphila ONE Isolated from Humans.

In this study, the genome of Akkermansia muciniphila ONE (designated AKK ONE) was sequenced, assembled, and analyzed. In addition, the safety of this strain was further evaluated by toxicological studies. The results showed that the AKK ONE genome is contained on a single chromosome with a total length of 2,817,524 bp and an average GC content of 55.48%. In total, 2411, 1131, 1168, 1745, and 1402 genes were annotated to the NR, GO, KEGG, COG, and SwissProt database, respectively. Potential resistance genes, adeF, tetW, ANT(3″)-IIa, and aadA1 were detected. AKK ONE was sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, and chloramphenicol and resistant to moxifloxacin. No potential virulence-related genes were detected. The PathogenFinder database analysis showed that AKK ONE was a non-potential human pathogen. This strain had good gastroenteric fluid tolerance and a weak ability to colonize the gut. No test item-related adverse effects were observed in the acute and subchronic toxicity test. AKK ONE did not display mutagenic activity either. This strain did not change the hematological and clinical biochemical parameters of mice. The weights of the organs were not affected by AKK ONE treatment. These results support that AKK ONE is safe for use as a probiotic at a dose of 8.28 × 109 CFU/kg bw/day.

A data-driven genome annotation approach for cassava.

Genome annotation files play a critical role in dictating the quality of downstream analyses by providing essential predictions for gene positions and structures. These files are pivotal in decoding the complex information encoded within DNA sequences. Here, we generated experimental data resolving RNA 5'- and 3'-ends as well as full-length RNAs for cassava TME12 sticklings in ambient temperature and cold. We used these data to generate genome annotation files using the TranscriptomeReconstructoR (TR) tool. A careful comparison to high-quality genome annotations suggests that our new TR genome annotations identified additional genes, resolved the transcript boundaries more accurately and identified additional RNA isoforms. We enhanced existing cassava genome annotation files with the information from TR that maintained the different transcript models as RNA isoforms. The resultant merged annotation was subsequently utilized for comprehensive analysis. To examine the effects of genome annotation files on gene expression studies, we compared the detection of differentially expressed genes during cold using the same RNA-seq data but alternative genome annotation files. We found that our merged genome annotation that included cold-specific TR gene models identified about twice as many cold-induced genes. These data indicate that environmentally induced genes may be missing in off-the-shelf genome annotation files. In conclusion, TR offers the opportunity to enhance crop genome annotations with implications for the discovery of differentially expressed candidate genes during plant-environment interactions.

Evidence-based unification of potato gene models with the UniTato collaborative genome browser.

Potato (Solanum tuberosum) is the most popular tuber crop and a model organism. A variety of gene models for potato exist, and despite frequent updates, they are not unified. This hinders the comparison of gene models across versions, limits the ability to reuse experimental data without significant re-analysis, and leads to missing or wrongly annotated genes. Here, we unify the recent potato double monoploid v4 and v6 gene models by developing an automated merging protocol, resulting in a Unified poTato genome model (UniTato). We subsequently established an Apollo genome browser (unitato.nib.si) that enables public access to UniTato and further community-based curation. We demonstrate how the UniTato resource can help resolve problems with missing or misplaced genes and can be used to update or consolidate a wider set of gene models or genome information. The automated protocol, genome annotation files, and a comprehensive translation table are provided at github.com/NIB-SI/unitato.

Genome-wide mining and characterization of MATE transporters in Coriandrum sativum L.

Multidrug and Toxic Compound Extrusion (MATE) proteins are responsible for the transport of a wide range of metabolites out of plant cells. This helps to protect the cells from toxins and other harmful compounds. MATE proteins also play a role in plant development, by regulating the transport of hormones and other signalling molecules. They transport a wide variety of substances, including organic acids, plant hormones, flavonoids, alkaloids, terpenes and other secondary metabolites. MATE proteins are thought to play similar roles in Coriander, in addition to stress responses. The MATE genes in the coriander genome have been identified and characterized. Detailed genome homology search and domain identification analysis have identified 91 MATE proteins in the genome assembly of coriander. A phylogenetic analysis of the identified proteins divided them into five major clades. The functions of the transporters in each cluster were predicted based on the clustering pattern of the functionally characterized proteins. The amino acid sequences, exon-intron structures and motif details of all the 91 proteins are identified and described. This is the first work on the MATE transporters in coriander and the results deliver clues for the molecular mechanisms behind the stress responses and secondary metabolite transport in coriander.

Draft assembly and annotation of the Cuban crocodile (Crocodylus rhombifer) genome.

OBJECTIVES: The new data provide an important genomic resource for the Critically Endangered Cuban crocodile (Crocodylus rhombifer). Cuban crocodiles are restricted to the Zapata Swamp in southern Matanzas Province, Cuba, and readily hybridize with the widespread American crocodile (Crocodylus acutus) in areas of sympatry. The reported de novo assembly will contribute to studies of crocodylian evolutionary history and provide a resource for informing Cuban crocodile conservation. DATA DESCRIPTION: The final 2.2 Gb draft genome for C. rhombifer consists of 41,387 scaffolds (contigs: N50 = 104.67 Kb; scaffold: N50-518.55 Kb). Benchmarking Universal Single-Copy Orthologs (BUSCO) identified 92.3% of the 3,354 genes in the vertebrata_odb10 database. Approximately 42% of the genome (960Mbp) comprises repeat elements. We predicted 30,138 unique protein-coding sequences (17,737 unique genes) in the genome assembly. Functional annotation found the top Gene Ontology annotations for Biological Processes, Molecular Function, and Cellular Component were regulation, protein, and intracellular, respectively. This assembly will support future macroevolutionary, conservation, and molecular studies of the Cuban crocodile.

Genomic exploration of the fermented meat isolate Staphylococcus shinii IMDO-S216 with a focus on competitiveness-enhancing secondary metabolites.

BACKGROUND: Staphylococcus shinii appears as an umbrella species encompassing several strains of Staphylococcus pseudoxylosus and Staphylococcus xylosus. Given its phylogenetic closeness to S. xylosus, S. shinii can be found in similar ecological niches, including the microbiota of fermented meats where the species may contribute to colour and flavour development. In addition to these conventional functionalities, a biopreservation potential based on the production of antagonistic compounds may be available. Such potential, however, remains largely unexplored in contrast to the large body of research that is available on the biopreservative properties of lactic acid bacteria. The present study outlines the exploration of the genetic basis of competitiveness and antimicrobial activity of a fermented meat isolate, S. shinii IMDO-S216. To this end, its genome was sequenced, de novo assembled, and annotated. RESULTS: The genome contained a single circular chromosome and eight plasmid replicons. Focus of the genomic exploration was on secondary metabolite biosynthetic gene clusters coding for ribosomally synthesized and posttranslationally modified peptides. One complete cluster was coding for a bacteriocin, namely lactococcin 972; the genes coding for the pre-bacteriocin, the ATP-binding cassette transporter, and the immunity protein were also identified. Five other complete clusters were identified, possibly functioning as competitiveness factors. These clusters were found to be involved in various responses such as membrane fluidity, iron intake from the medium, a quorum sensing system, and decreased sensitivity to antimicrobial peptides and competing microorganisms. The presence of these clusters was equally studied among a selection of multiple Staphylococcus species to assess their prevalence in closely-related organisms. CONCLUSIONS: Such factors possibly translate in an improved adaptation and competitiveness of S. shinii IMDO-S216 which are, in turn, likely to improve its fitness in a fermented meat matrix.

Exploring crop genomes: assembly features, gene prediction accuracy, and implications for proteomics studies.

Plant genomics plays a pivotal role in enhancing global food security and sustainability by offering innovative solutions for improving crop yield, disease resistance, and stress tolerance. As the number of sequenced genomes grows and the accuracy and contiguity of genome assemblies improve, structural annotation of plant genomes continues to be a significant challenge due to their large size, polyploidy, and rich repeat content. In this paper, we present an overview of the current landscape in crop genomics research, highlighting the diversity of genomic characteristics across various crop species. We also assessed the accuracy of popular gene prediction tools in identifying genes within crop genomes and examined the factors that impact their performance. Our findings highlight the strengths and limitations of BRAKER2 and Helixer as leading structural genome annotation tools and underscore the impact of genome complexity, fragmentation, and repeat content on their performance. Furthermore, we evaluated the suitability of the predicted proteins as a reliable search space in proteomics studies using mass spectrometry data. Our results provide valuable insights for future efforts to refine and advance the field of structural genome annotation.

Advancing microbial production through artificial intelligence-aided biology.

Microbial cell factories (MCFs) have been leveraged to construct sustainable platforms for value-added compound production. To optimize metabolism and reach optimal productivity, synthetic biology has developed various genetic devices to engineer microbial systems by gene editing, high-throughput protein engineering, and dynamic regulation. However, current synthetic biology methodologies still rely heavily on manual design, laborious testing, and exhaustive analysis. The emerging interdisciplinary field of artificial intelligence (AI) and biology has become pivotal in addressing the remaining challenges. AI-aided microbial production harnesses the power of processing, learning, and predicting vast amounts of biological data within seconds, providing outputs with high probability. With well-trained AI models, the conventional Design-Build-Test (DBT) cycle has been transformed into a multidimensional Design-Build-Test-Learn-Predict (DBTLP) workflow, leading to significantly improved operational efficiency and reduced labor consumption. Here, we comprehensively review the main components and recent advances in AI-aided microbial production, focusing on genome annotation, AI-aided protein engineering, artificial functional protein design, and AI-enabled pathway prediction. Finally, we discuss the challenges of integrating novel AI techniques into biology and propose the potential of large language models (LLMs) in advancing microbial production.

A haplotype-resolved reference genome of a long-distance migratory bat, Pipistrellus nathusii (Keyserling & Blasius, 1839).

We present a complete, chromosome-scale reference genome for the long-distance migratory bat Pipistrellus nathusii. The genome encompasses both haplotypic sets of autosomes and the separation of both sex chromosomes by utilizing highly accurate long-reads and preserving long-range phasing information through the use of three-dimensional chromatin conformation capture sequencing (Hi-C). This genome, accompanied by a comprehensive protein-coding sequence annotation, provides a valuable genomic resource for future investigations into the genomic bases of long-distance migratory flight in bats as well as uncovering the genetic architecture, population structure and evolutionary history of Pipistrellus nathusii. The reference-quality genome presented here gives a fundamental resource to further our understanding of bat genetics and evolution, adding to the growing number of high-quality genetic resources in this field. Here, we demonstrate its use in the phylogenetic reconstruction of the order Chiroptera, and in particular, we present the resources to allow detailed investigations into the genetic drivers and adaptations related to long-distance migration.

Denitrificimonas halotolerans sp. nov., a novel species isolated from UASB sludge treating landfill leachate.

A Gram-stain-negative, facultative anaerobe, rod-shaped strain JX-1T was isolated from UASB sludge treating landfill leachate in Wuhan, China. The isolate is capable of growing under conditions of pH 6.0-11.0 (optimum, pH 7.0-8.0), temperature 4-42 â„ƒ (optimum, 20-30 â„ƒ), 0-8.0% (w/v) NaCl (optimum, 5.0%), and ammonia nitrogen concentration of 200-5000 mg/L (optimum, 500 mg/L) on LB plates. The microorganism can utilize malic acid, D-galactose, L-rhamnose, inosine, and L-glutamic acid as carbon sources, but does not reduce nitrates and nitrites. The major fatty acids are C18:1ω7c/C18:1ω6c, iso-C15:0, and anteiso-C15:0. The respiratory quinones are Q9 (91.92%) and Q8 (8.08%). Polar lipids include aminolipid, aminophospholipid, diphosphatidylglycerol, glycolipid, phosphatidylethanolamine, phosphatidylglycerol, and phospholipid. Compared with other strains, strain JX-1T and Denitrificimonas caeni HY-14T have the highest values in terms of 16S rRNA gene sequence similarity (96.79%), average nucleotide identity (ANI; 76.06%), and average amino acid identity (AAI; 78.89%). Its digital DNA-DNA hybridization (dDDH) result is 20.3%. The genome of strain JX-1T, with a size of 2.78 Mb and 46.12 mol% G + C content, lacks genes for denitrification and dissimilatory nitrate reduction to ammonium (DNRA), but contains genes for ectoine synthesis as a secondary metabolite. The results of this polyphasic study allow genotypic and phenotypic differentiation of the analysed strain from the closest related species and confirm that the strain represents a novel species within the genus Denitrificimonas, for which the name Denitrificimonas halotolerans sp. nov. is proposed with JX-1T (= MCCC 1K08958T = KCTC 8395T) as the type strain.

Chromosome-level genome assembly and annotation of the black sea urchin Arbacia lixula (Linnaeus, 1758).

The black sea urchin (Arbacia lixula) is a keystone species inhabiting the coastal shallow waters of the Mediterranean Sea, which is a key driver of littoral communities' structure. Here, we present the first genome assembly and annotation of this species, standing as the first Arbacioida genome, including both nuclear and mitochondrial genomes. To obtain a chromosome-level assembly, we used a combination of PacBio high fidelity (HiFi) reads and chromatin capture reads (Omni-C). In addition, we generated a high-quality nuclear annotation of both coding and non-coding genes, by using published RNA-Seq data from several individuals of A. lixula and gene models from closely related species. The nuclear genome assembly has a total span of 607.91 Mb, being consistent with its experimentally estimated genome size. The assembly contains 22 chromosome-scale scaffolds (96.52% of the total length), which coincides with its known karyotype. A total of 72,767 transcripts were predicted from the nuclear genome, 24,171 coding, and 48,596 non-coding that included lncRNA, snoRNA, and tRNAs. The circularized mitochondrial genome had 15740 bp comprising 13 protein-coding genes, 2 rRNA, and 22 tRNA. This reference genome will enhance ongoing A. lixula studies and benefit the wider sea urchin scientific community.

Draft genome sequence data of methicillin-resistant Staphylococcus aureus, strain 4233.

Staphylococcus aureus is a conditionally pathogenic microorganism and one of the main causative agents of antibiotic resistant nosocomial infections. In immunocompromised people, S. aureus infection can cause folliculitis, furuncles, impetigo, osteomyelitis, septic arthritis, sepsis, endocarditis, pneumonia and meningitis. In the presented work, sequencing of a methicillin-resistant S. aureus, strain 4233, was performed on the Illumina MiSeq platform, followed by bioinformatics processing and gene annotation using SPAdes, RAST and CARD programs and databases. The submitted genome is a total of 2,790,390 bp long and contains 2759 genes, including 82 RNA genes. 33 % of the genes are functionally significant and represent 25 functional groups. Fourteen genes encoding resistance factors to 14 different types of antibacterial drugs were predicted. The information provided on the genome of S. aureus, strain 4233 will be of value in investigating the evolution and formation of antibiotic-resistant forms of S. aureus.

First draft reference genome and annotation of the alternative oil species Physaria fendleri.

In the wake of increasing demand for renewable energy sources, plant-based sources including alternative oilseeds have come to the forefront of interest. Hydroxy fatty acids (HFAs), produced in a few oilseed species, are important chemical feedstocks for industrial applications. An integrated approach was taken to assemble the first draft genome of the alternative HFA producer Physaria fendleri (n = 6), an outcrossing species with high heterozygosity. Both de novo transcriptome assemblies and genome assemblies were produced with public and generated sequencing reads. Resulting intermediate assemblies were then scaffolded and patched with multiple data sources, followed by super-scaffolding onto a masked genome of Camelina laxa (n = 6). Despite a current lack of available resources for the physical mapping of genomic scaffolds of Physaria fendleri, topography of the genome with respect to repeat and gene content was preserved at the scaffold level and not significantly lost via super-scaffolding. Read representation, gene and genome completion statistics, and annotation results illustrated the creation of a functional draft genome and a tool for future research on alternative oil species.

Unraveling a Small Secreted Peptide SUBPEP3 That Positively Regulates Salt-Stress Tolerance in Pyrus betulifolia.

Small secreted peptides (SSPs) play important roles in regulating plants' growth and development in response to external stimulus, but the genes and functions of SSPs in many species are still unknown. Therefore, it is particularly significant to characterize and annotate SSP genes in plant genomes. As a widely used stock of pears, Pyrus betulifolia has strong resistance to biotic and abiotic stresses. In this study, we analyzed the SSPs genes in the genome of P. betulifolia according to their characteristics and homology. A total of 1195 SSP genes were identified, and most of them are signaling molecules. Among these, we identified a new SSP, subtilase peptide 3 (SUBPEP3), which derived from the PA region of preSUBPEP3, increasing the expression level under salt stress. Both adding synthetic peptide SUBPEP3 to the culture medium of pears and the overexpression of SUBPEP3 in tobacco can improve the salt tolerance of plants. In summary, we annotated the SSP genes in the P. betulifolia genome and identified a small secreted peptide SUBPEP3 that regulates the salt tolerance of P. betulifolia, which provides an important theoretical basis for further revealing the function of SSPs.

Isolation, Characterization, Genome Annotation, and Evaluation of Hyaluronidase Inhibitory Activity in Secondary Metabolites of Brevibacillus sp. JNUCC 41: A Comprehensive Analysis through Molecular Docking and Molecular Dynamics Simulation.

Brevibacillus sp. JNUCC 41, characterized as a plant-growth-promoting rhizobacterium (PGPR), actively participates in lipid metabolism and biocontrol based on gene analysis. This study aimed to investigate the crucial secondary metabolites in biological metabolism; fermentation, extraction, and isolation were performed, revealing that methyl indole-3-acetate showed the best hyaluronidase (HAase) inhibitory activity (IC50: 343.9 µM). Molecular docking results further revealed that the compound forms hydrogen bonds with the residues Tyr-75 and Tyr-247 of HAase (binding energy: -6.4 kcal/mol). Molecular dynamics (MD) simulations demonstrated that the compound predominantly binds to HAase via hydrogen bonding (MM-PBSA binding energy: -24.9 kcal/mol) and exhibits good stability. The residues Tyr-247 and Tyr-202, pivotal for binding in docking, were also confirmed via MD simulations. This study suggests that methyl indole-3-acetate holds potential applications in anti-inflammatory and anti-aging treatments.

Genome divergence and reproductive incompatibility among populations of Ganaspis near brasiliensis.

During the last decade, the spotted wing drosophila, Drosophila suzukii, has spread from eastern Asia to the Americas, Europe, and Africa. This fly attacks many species of cultivated and wild fruits with soft, thin skins, where its serrated ovipositor allows it to lay eggs in undamaged fruit. Parasitoids from the native range of D. suzukii may provide sustainable management of this polyphagous pest. Among these parasitoids, host-specificity testing has revealed a lineage of Ganaspis near brasiliensis, referred to in this paper as G1, that appears to be a cryptic species more host-specific to D. suzukii than other parasitoids. Differentiation among cryptic species is critical for introduction and subsequent evaluation of their impact on D. suzukii. Here, we present results on divergence in genomic sequences and architecture and reproductive isolation between lineages of Ganaspis near brasiliensis that appear to be cryptic species. We studied five populations, two from China, two from Japan, and one from Canada, identified as the G1 vs G3 lineages based on differences in cytochrome oxidase l sequences. We assembled and annotated the genomes of these populations and analyzed divergences in sequence and genome architecture between them. We also report results from crosses to test reproductive compatibility between the G3 lineage from China and the G1 lineage from Japan. The combined results on sequence divergence, differences in genome architectures, ortholog divergence, reproductive incompatibility, differences in host ranges and microhabitat preferences, and differences in morphology show that these lineages are different species. Thus, the decision to evaluate the lineages separately and only import and introduce the more host-specific lineage to North America and Europe was appropriate.

Phylogenetic Analysis and Comparative Genomics of Brucella abortus and Brucella melitensis Strains in Egypt.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

PPanG: a precision pangenome browser enabling nucleotide-level analysis of genomic variations in individual genomes and their graph-based pangenome.

Graph-based pangenome is gaining more popularity than linear pangenome because it stores more comprehensive information of variations. However, traditional linear genome browser has its own advantages, especially the tremendous resources accumulated historically. With the fast-growing number of individual genomes and their annotations available, the demand for a genome browser to visualize genome annotation for many individuals together with a graph-based pangenome is getting higher and higher. Here we report a new pangenome browser PPanG, a precise pangenome browser enabling nucleotide-level comparison of individual genome annotations together with a graph-based pangenome. Nine rice genomes with annotations were provided by default as potential references, and any individual genome can be selected as the reference. Our pangenome browser provides unprecedented insights on genome variations at different levels from base to gene, and reveals how the structures of a gene could differ for individuals. PPanG can be applied to any species with multiple individual genomes available and it is available at https://cgm.sjtu.edu.cn/PPanG .

SMA20/PMIS2 Is a Rapidly Evolving Sperm Membrane Alloantigen with Possible Species-Divergent Function in Fertilization.

Immunodominant alloantigens in pig sperm membranes include 15 known gene products and a previously undiscovered Mr 20,000 sperm membrane-specific protein (SMA20). Here we characterize SMA20 and identify it as the unannotated pig ortholog of PMIS2. A composite SMA20 cDNA encoded a 126 amino acid polypeptide comprising two predicted transmembrane segments and an N-terminal alanine- and proline (AP)-rich region with no apparent signal peptide. The Northern blots showed that the composite SMA20 cDNA was derived from a 1.1 kb testis-specific transcript. A BLASTp search retrieved no SMA20 match from the pig genome, but it did retrieve a 99% match to the Pmis2 gene product in warthog. Sequence identity to predicted PMIS2 orthologs from other placental mammals ranged from no more than 80% overall in Cetartiodactyla to less than 60% in Primates, with the AP-rich region showing the highest divergence, including, in the extreme, its absence in most rodents, including the mouse. SMA20 immunoreactivity localized to the acrosome/apical head of methanol-fixed boar spermatozoa but not live, motile cells. Ultrastructurally, the SMA20 AP-rich domain immunolocalized to the inner leaflet of the plasma membrane, the outer acrosomal membrane, and the acrosomal contents of ejaculated spermatozoa. Gene name search failed to retrieve annotated Pmis2 from most mammalian genomes. Nevertheless, individual pairwise interrogation of loci spanning Atp4a-Haus5 identified Pmis2 in all placental mammals, but not in marsupials or monotremes. We conclude that the gene encoding sperm-specific SMA20/PMIS2 arose de novo in Eutheria after divergence from Metatheria, whereupon rapid molecular evolution likely drove the acquisition of a species-divergent function unique to fertilization in placental mammals.