Evolution & Development

Hard cornification is the developmental process that produces the hardened epidermal structures that characterize tetrapod diversity, including scales, feathers, hair, and claws. This process relies on keratin proteins and additional reinforcing proteins that assemble, cross-link, and strengthen tissues to become mechanically resilient. Although the molecular composition of hard cornified tissues is known to vary among tetrapods, the program is least understood in amphibians. Within frogs, hard cornification occurs in the mouthparts of most tadpoles and in the nuptial pads, hindfoot claws, and foot tubercles of some adults. We characterized transcriptomic profiles of Bombina orientalis tadpoles at three developmental stages before, during, and after mouthpart cornification. We identified 25 keratin genes orthologous to amniote hard keratins in the B. bombina genome, 18 of which are upregulated in cornifying larval mouthparts. Furthermore, two of these hard keratin paralogs and an amphibian epidermal differentiation gene exhibit distinct spatial expression patterns within the jaw sheaths and keratodonts evidenced by in situ hybridization, suggesting they have discrete functional roles. The newly discovered diversity of the hard keratin repertoire in Bombina , and the concurrent expression of many of these paralogs in tadpole mouthparts, suggests that anuran hard cornification is much more complex than adult cornified traits thus far characterized in Xenopus frogs. Our results also challenge the notion that the diversification of keratins in Amphibia is primarily associated with the transition to more terrestrial habits, and may instead be shaped, in part, by the novel feeding apparatus of aquatic tadpoles.

Aquaporin-10 (AQP10) is an aquaglyceroporin that transports small, uncharged molecules, such as water and glycerol. In some species, such as mice and cattle, the AQP10 gene (Aqp10) is pseudogenized. In rodents, pseudogenization of Aqp10 is limited to the Myomorpha suborder, while Aqp10 remains intact in other rodent lineages. The order Cetartiodactyla encompasses diverse species, including Suina, Tylopoda, Ruminantia, and Whippomorpha. The distribution of intact Aqp10 across the entire Cetartiodactyla and analyses estimating the timing of Aqp10 pseudogenization have not been sufficiently investigated. In this study, we identified species within Cetartiodactyla that retain intact Aqp10 and analyzed the detailed patterns of pseudogenization to elucidate the specifics of pseudogenization within this order. We collected Aqp10 loci by BLAST and synteny analyses of publicly available genome databases of 51 cetartiodactyl species from 17 families. Dot plot and alignment analyses revealed that seudogenization of Aqp10 occurred in 42 Cetartiodactyla species, including the Camelidae, warthog (Suidae), Antilocapridae, okapi (Giraffidae), Cervidae, Bovidae, Hippopotamidae, and Cetacea. In contrast, nine species retained an intact Aqp10: pig and babirusa (Suidae), peccary (Tayassuidae), mouse deeres (Tragulidae), giraffes (Giraffidae), and musk deers (Moschidae). The timing of pseudogenization was estimated by comparing patterns of exon deletions, frameshifts, and nonsense mutations in Aqp10 pseudogenes. RT-PCR analysis of cattle and sheep tissues revealed that these species express Aqp3 and Aqp7 in their intestines, but not Aqp10. Collectively, these findings indicate that Aqp10 pseudogenization occurred independently in multiple cetartiodactyl lineages and provide new insights into the evolutionary history of Aqp10 loss within this order.

DNA methylation, an epigenetic mechanism, has been proposed as a basis for Lamarckian inheritance of acquired characteristics. This hypothesis raises questions about environmental induction of DNA methylation, its persistence across generations, and associations with gene expression. We investigated these processes in the California oak, Quercus lobata. Based on 40 range-wide adult trees and their progeny grown in two common gardens, we tested four predictions: (i) DNA methylation is environmentally induced; (ii) methylation shows parent-offspring correspondence; (iii) environmentally induced patterns remain stable after transmission; and (iv) DNA methylation is associated with gene expression, varying by sequence context and TE proximity. Using methylomes and transcriptomes, we found support for all predictions except the third: CHH methylation was environmentally induced, but rarely showed parent/offspring correspondence, whereas CG methylation showed the opposite pattern. DNA methylation was associated with gene expression, depending on the sequence context and proximity to TEs. We identified one stress-associated gene with environmentally responsive CHH methylation that showed parent offspring correspondence in two common gardens in both gardens. Still, overall evidence for Lamarckian like evolution is not compelling. Nevertheless, DNA methylation was environmentally responsive, showed parent offspring correspondence, and was associated with gene expression, highlighting its potential importance for the ecological and evolutionary responses of long-lived trees to environmental change.

The repeated evolution of similar phenotypes in response to shared ecological pressures provides striking examples of natural selection and raises broader questions about the predictability of evolution. Evolutionary constraint may shape this predictability, while the apparent extent of parallelism may also depend on the biological scale at which it is measured. When are adaptive solutions channeled along the same route? We addressed this question using three sympatric benthic-limnetic ecotype pairs of threespine stickleback (Gasterosteus aculeatus), characterizing gene expression in gill and liver from wild-caught fish. We tested whether proxies of pleiotropic and coding-sequence constraint predicted the repeatability of transcriptional divergence across lakes. At the gene level, differential expression was strongly biased toward parallel rather than antiparallel responses. At the functional level, numerous pathways showed parallel enrichment, but the conservation of their underlying member-gene responses varied substantially, indicating that similar functional outcomes can arise with differing degrees of gene-level concordance. Most importantly, several proxies of evolutionary constraint predicted transcriptional repeatability, particularly in gill: broadly expressed, highly connected, and more slowly evolving genes showed more stable expression responses across lakes, with similar relationships evident for repeated use of genes within functionally parallel pathways. Together, these results show that both gene-level constraint and biological scale contribute to variation in molecular repeatability and suggest that the predictability of repeated adaptation may reflect an interaction between genetic architecture, population-genetic context, and the level of biological organization at which parallelism emerges.

Premise:: Conifers comprise one-sixth of global forests and landscape genomic studies are urgently needed to understand their biology, yet relatively few conifer species have reference genomes. Targeted exon-capture is a practical alternative to whole-genome sequencing, but selecting loci without a reference genome remains a barrier. We developed a multi-tissue reference transcriptome for pi non pine (Pinus edulis) to ask how multi-tissue sampling improves probe design, and what is lost under a conventional single-tissue approach. Methods: We assembled a reference transcriptome from four diploid tissues (needle, phloem, root, vegetative bud) and one haploid tissue (megagametophyte), using the latter as a pseudo-guide. We designed a target-capture probe-set by prioritizing highly expressed P. edulis transcripts and transcripts matching a conifer exon database. Results: We recovered 76% of conserved plant genes with few duplicated transcripts. Filtering yielded 15,664 transcripts totaling 31.4 Mb, ~0.1% of the genome. Tissue-specific expression was substantial: 27.4% of transcripts occurred exclusively in non-needle tissues. Functional annotation confirmed complementary contributions, including seed-desiccation and germination genes from megagametophytes, and defense- and stress-associated genes from roots and phloem. Discussion: We built a transcriptome and efficient probe set using a roadmap suitable for other non-model plants with large genomes; keys to success were prioritizing complementary tissues and using expression-based filtering.

Metabolic rate governs the pace of life through the allocation and expenditure of energy, yet metabolic rate data are unavailable for most species. External morphological traits, such as gill slits, have been used to infer respiratory physiology and metabolism in sharks, yet, whether that pattern is generalizable across rays is unknown. In rays (Batoidea), spiracle length seems adaptive to oxygen uptake capacity, environmental oxygen availability, and activity level. Thus, we evaluate the allometry of external respiratory morphology (spiracles and gill slits) in rays using 550 museum specimens representing 88 species. We test whether spiracle length and gill slit height grow isometrically with body size (total length or disc width) within and across species. Overall, we find spiracles and gill slits are morphologically diverse, varying widely in shape within and between families. Spiracle length and gill slit height scale isometrically and are a consistent proportion of body size, despite such high morphological variability. While that proportion is consistent through ontogeny, each species proportion differs (e.g. gill slit height ranges from 0.93% of total length in Spotted Legskate to 2.93% in Pacific Electric Ray). Of the three swimming styles, pectoral oscillating species (Mobulid and Eagle Rays) may have steeper scaling of spiracle length with gill slit height - consistent with greater respiratory capacity in species with more active lifestyles. Our findings are consistent with literature hypothesizing external respiratory morphology (spiracles and gill slits) is tightly related to metabolic rate and activity level, and demonstrate the value of natural history collections in ecological research. Importantly, the isometry of both respiratory traits means that a single measure of any individual, irrespective of size, will be representative of the species thus providing a basis for expanding our understanding of metabolic morphology, enabling future work on life history, extinction risk, and ecological function.

A longstanding question in evolution is if similar morphologies shared by distantly related organisms facing similar environmental pressures belie similar functions. The carnivorous pitcher form has evolved in three distinct plant lineages and encompasses a suite of convergent morphological traits, providing a natural experiment to test hypothesized links between morphology and function. Multiple pitcher plant species possess fenestrations (i.e., small windows) that let light pass through the pitcher leaf. While research on one species, Nepenthes aristolochioides, demonstrates that fenestrations manipulate light to create false exits and increase prey capture, whether this convergent morphology serves this convergent function in other pitcher species remains unresolved. We explored the function of fenestrations in two additional species of pitcher plants, Nepenthes klossii and Cephalotus follicularis, where the morphology and function of fenestration were previously unexplored. We inspected micromorphology of pitcher structure, conducted an insect bioassay with live plants to test for links between morphology and function, and quantified traits associated with light manipulation from herbarium specimens. Both species exhibited fenestration and related pitcher morphology similar to N. aristolochioides. Yet our experiments did not reveal a significant effect of light passing through fenestrations on fly capture for these two species. Key differences between these species and the previously investigated species may be the presence of wax crystals and the absence of viscoelastic fluid. While future studies may uncover weak to moderate light manipulation function not detected in our study, our data do not support convergent function in these distantly related, morphologically similar species.

Primordial germ cells (PGCs) are the stem cell lineage responsible for transmitting genetic information across generations and are therefore central to studies of germline development, reproduction, and evolution. However, a lack of molecular tools in jawless vertebrates such as lampreys and hagfish has limited comparative insights into the evolutionary origins of vertebrate germline cells. Here, we report the development of the first germline-specific reporter (GSR) in the sea lamprey (Petromyzon marinus), Tg(piwil1: egfp-UTRnanos1), a piwil1-based germline reporter that is driven by the sea lamprey piwil1 regulatory region that directs Green Fluorescent Protein (GFP) expression in early PGCs. Promoter activity was validated by robust GFP expression in PGCs of zebrafish embryos co-injected with Tol2 transposase mRNA. Injection of the construct into sea lamprey embryos resulted in early, persistent, and gonad-localized GFP-positive cells. These results demonstrate stable germline-specific transgene expression in a jawless vertebrate and suggest deep evolutionary conservation of germline regulatory mechanisms between jawless and jawed vertebrates. Furthermore, the piwil1-based germline reporter line enabled lifetime visualization of zebrafish germline cells, revealing that increased PGC abundance during embryogenesis biases toward female sexual differentiation. Together, this study establishes the first GSR system in a jawless vertebrate and provides a useful platform for investigating germline biology and vertebrate germline evolution.

BackgroundN6-Methyladenosine (m6A) regulates RNA fate in flowering plants, but the repertoire and expression patterns of m6A-related genes remain incompletely characterized in liverworts. Here, we characterized these gene families in the liverwort Marchantia polymorpha within a comparative framework spanning diverse plant lineages. ResultsA comparative survey of 119 plant genomes using sequence similarity and conserved-domain criteria identified 18 candidate m6A-related loci in M. polymorpha, comprising seven writer-related genes, five AlkB homolog genes, and six YTH- or CPSF30-related genes. Across the surveyed genomes, canonical writer components generally had low copy numbers, whereas AlkB homolog and reader-related families showed greater copy-number variation. Phylogenetic and domain analyses supported the family assignments of the M. polymorpha candidates. Eight of the 18 candidate loci showed detectable collinearity with Marchantia quadrata. Putative promoter analysis identified motifs annotated as hormone-, light-, and stress-responsive, with hormone-responsive motifs accounting for the largest share of predicted sites. Public transcriptome analysis revealed variation in candidate gene expression across developmental stages and tissues. Quantitative reverse-transcription PCR showed higher transcript abundance for all six YTH- or CPSF30-related genes in thalli than in gemmae or gemma cups. Cold, heat, osmotic, salt, and dark treatments elicited gene-specific expression responses. Darkness increased transcript abundance of several candidates, including MpMTA, MpALKBH1A, and MpECT2. A predicted protein-association network suggested links between subsets of the candidates and RNA-processing proteins. ConclusionsThese findings define a candidate m6A-related gene repertoire in M. polymorpha and identify tissue- and stress-responsive genes for targeted functional studies of RNA methylation in this liverwort.

Social aggregation is widespread in vertebrate larvae, yet how genetic relatedness shapes group cohesion remains poorly understood. In particular, the association between genetic similarity and social aggregation may depend on the scale at which genetic relatedness is evaluated. Here, we quantitatively examined aggregation behaviour in tadpoles from three lineages of Japanese toads, Bufo japonicus, B. formosus, and B. gargarizans miyakonis . We experimentally combined tadpole cohorts differing in relatedness and lineage and quantified aggregation using spatial dispersion analyses. We then examined relationships between aggregation behaviour and genetic variation using mitochondrial DNA (mtDNA), major histocompatibility complex (MHC) class II variation, and microsatellite diversity. Overall degree of aggregation did not differ significantly between sibling and non-sibling cohorts, and Morisitas index provided no evidence that tadpoles formed separate clumps according to relatedness. However, the correlation between genetic variation and aggregation differed across scales of genetic relatedness: among non-sibling cohorts, greater mtDNA pairwise distance was associated with weaker aggregation, whereas among sibling cohorts, greater MHC amino-acid difference was associated with weaker aggregation. These results indicate that the degree of aggregation is related to genetic similarity across different scales of relatedness, rather than being explained by a simple sibling/non-sibling distinction. Our findings suggest that tadpole group cohesion is related to genetic similarity in a scale-dependent manner, highlighting the importance of considering the scale of genetic relatedness when investigating social discrimination in vertebrates, while the sensory and molecular mechanisms underlying these relationships remain to be determined.

The turquoise killifish (Nothobranchius furzeri) is an emerging vertebrate model for development and ageing due to its exceptionally rapid maturation and short lifespan. However, how microRNA (miRNA) regulation coordinates gene expression across its compressed developmental trajectory remains poorly understood. Here, we integrated miRNA and mRNA expression from the same unexposed animals across three developmental stages: newly hatched fry (4 weeks total age), juveniles (~8 weeks total age), and aged adults (~8 months of age). Developmental stage was the dominant source of variation in both datasets, with miRNA and mRNA profiles independently resolving a consistent progression from newly hatched fry to juveniles and adults. Transcriptomic changes reflected ongoing tissue maturation, including increased expression of crystallin genes associated with lens development, while more than 2,000 genes were differentially expressed during the juvenile transition. Integration of 398 developmentally dynamic miRNAs with predicted mRNA targets identified specific miRNAs whose developmental changes were significantly enriched for opposing changes in their target genes. These included conserved developmental regulators from the miR-200/141, miR-29, let-7, and miR-34 families, which increased toward adulthood as their predicted target cohorts decreased. Stage-controlled analyses further identified miR-34a, miR-29a, miR-29b, miR-15a, and a novel candidate whose target relationships persisted independently of the overall developmental trajectory. Notably, miR-29 showed particularly strong induction during maturation, consistent with its conserved roles in extracellular-matrix remodeling, differentiation, and vertebrate ageing. Together, these findings reveal coordinated remodeling of conserved miRNA-mRNA regulatory programs across the rapid maturation of N. furzeri and establish a framework for investigating post-transcriptional regulation during vertebrate development in a species with limited miRNA annotation.

Multifunctional anatomical structures must accommodate potentially competing performance demands, leading to functional tradeoffs. The mammal rostrum (snout) is involved in multiple functions, including feeding, respiratory conditioning, olfaction, and, in most bats, echolocation. Therefore, it serves as an ideal system to investigate whether and how functional tradeoffs promote or constrain morphological diversification. While the external morphology of the rostrum has been linked with mechanical demands of feeding in bats, the factors and processes influencing its internal morphology, including the turbinals in the nasal cavity, remain comparatively unexplored. Here we investigate how internal rostral morphology reflects specialization and tradeoffs among respiratory air conditioning, echolocation, and olfaction by quantifying turbinal morphological diversity in a phylogenetically and ecologically diverse sample of bat species. We find that echolocation mode predominantly influences both the ethmoturbinal complex (ETc) and maxilloturbinal (MT) systems, with nasal-emitting bats having reduced and less complex turbinals compared to oral and non-laryngeally echolocating bat species. MT complexity and surface area are not associated with bioclimatic variables representing thermal variability and aridity. However, the ETc exhibits higher complexity in plantivorous bats than faunivorous bats, suggesting higher olfactory reliance. While MT morphological disparity was predominantly explained by echolocation mode, ETc morphological disparity was associated with both echolocation mode and diet, suggesting potentially opposing demands favoring turbinal reduction for sound transmission and elaboration for olfaction. Among nasal emitters, which must accommodate both demands within the nasal cavity, ETc complexity exhibited higher relative disparity compared to MT complexity. These patterns suggest that competing functional demands may promote or maintain morphological diversity, possibly through ecological specialization and/or many-to-one mapping between morphology and performance.

The teleost whole-genome duplication (TGD) contributed to functional diversification of opsins. Some TGD paralogs, including those of parapinopsin (PP), Vertebrate Ancient (VA) opsin, and long wavelength-sensitive (LWS) opsin, show different absorption spectra and/or expression patterns. However, our knowledge of detailed evolutionary processes and mechanisms by which TGD contributed to opsin diversification is still limited. Here, we report that TGD paralogs of parietopsin (PT1 and PT2) are retained in extant species. While most species, including the zebrafish, have only PT1, several characins, including the red piranha (Pygocentrus nattereri), retain both PT1 and PT2. The Mexican tetra (Astyanax mexicanus) and catfishes have only PT2. To assess the degree of functional diversification between PT1 and PT2, we characterized spectral properties and expression patterns. Maximum absorption spectra differ slightly among PTs. Those of red piranha PT1, PT2, Mexican tetra PT2, and Japanese catfish (Silurus asotus) PT2 were located at 517 nm, 528 nm, 517 nm, and ~535 nm, respectively. Fluorescence in situ hybridization showed that (1) piranha PT1 and PT2 are expressed in the same pineal cells, and (2) they are also co-expressed with PP1. Based on these findings, functional diversification of opsins after gene duplication is discussed.

Two rounds of whole-genome duplication (2R-WGD) in the vertebrate lineage are thought to have driven gene diversification. Mammals have four genes encoding five nebulin superfamily proteins (LASP1, LASP2/NEBL, NEB, and NRAP) hypothesised to have arisen from invertebrate LASP via WGD. Here, we examined the evolutionary fate of the nebulin superfamily after an additional WGD, teleost-specific genome duplication (TGD). Genome and transcriptome analysis of zebrafish (Danio rerio) and medaka (Oryzias latipes), of which the complete genomes were recently determined, showed only lasp family genes retained duplicate copies. Zebrafish retained two LASP2/NEBL-derived ohnologs; one (LASP2L) had been misidentified as a nebulette gene in the Zebrafish Information Network (ZFIN) despite lacking nebulette-specific exons. Another (LASP2/NEBL) is newly identified in this study because it lies in a newly determined region of the latest genome assembly. Contrastingly, medaka retained two LASP1 ohnologs and one LASP2/NEBL. The duplicated lasp family proteins showed overlapping and specific tissue expression patterns. We confirmed all three zebrafish lasp proteins bound actin and co-localized with actin-rich structures when ectopically expressed in cultured fibroblasts. This supports that TGD-derived lasp duplicates underwent lineage-specific retention and expression divergence in teleosts whilst conserving core actin-binding properties.

Parasitoid wasps develop in or on other arthropods and have evolved a wide range of strategies for exploiting their hosts. Ascogaster reticulata is a solitary egg to larval endoparasitoid wasp belonging to the subfamily Cheloninae of Braconidae. We constructed a genome assembly using long-read sequence data. The assembled genome was 280.5 Mbp, consisting of 209 contigs. The N50 value and GC content ratio were 13.9 Mbp and 30.43%, respectively. The percentage of transposable elements and repetitive regions was 52.43%. The predicted number of genes is 27,261. Merqury revealed that the QV value and completeness of the genome were 61.79 and 99.3917%, respectively. BUSCO analysis detected approximately 99% of the core genes. These results suggest that the genome data were of sufficient quality for use as a reference genome. Therefore, these data can be utilized as a genomic resource for comparative studies within Braconidae and for future investigations into the biology and evolution of Cheloninae parasitoid wasps.

Prospero-related homeobox (Prox) genes encode a family of transcription factors that play essential roles in the development of several organs and systems, including the central nervous system, lymphatic endothelium, musculature, and liver. Despite their developmental importance, the evolutionary history of the vertebrate Prox gene family remains poorly understood. In this study we combined phylogenetic and synteny analysis to characterise the evolution of the Prox family in vertebrates. Our results reveal that two to three Prox subfamilies were already present in the last common ancestor of jawed vertebrates. We clarify the identity and evolutionary relationships of well-studied members of this family and identify multiple independent losses of Prox2 and Prox3 genes in specific vertebrate lineages. Furthermore, we uncover evidence for the existence of a fourth Prox gene in the ancestral vertebrate genome, which was subsequently lost. Overall, this study provides the first comprehensive analysis of the evolutionary history of the vertebrate Prox gene family and establishes a foundations for future studies on the functional roles of these genes.

Ecdysozoans is one of the most abundant and diverse groups of metazoans and grow through moulting the rigid exoskeleton that provided protection. But the moulting process is often dangerous, resulting in malformations or death. Injuries have been well documented in the calcitic exoskeleton of trilobites, some of which have been attributed to moulting complications when long spines are deformed. Injuries in trilobites are often found in the trunk rather than the cephalon, and abnormal genal spines are rarely documented. Here we examine injuries in eleven specimens of Olenoides serratus from the Burgess Shale (Cambrian, Wuluian) and demonstrate an increased frequency of genal spine malformations compared to other trilobites. The unusual pattern of malformations may be attributed to moulting injuries of the long and delicate genal spines in O. serratus. To moult, trilobites curved ventrally to press the anterior margin of their cephalon into the substrate and open the facial sutures. This process exerted force on the librigenae. To exit the exuvia, the genal spines were forced to bend as the old librigenae dipped ventrally along the anterior edge, resulting in the injured genal spines in O. serratus. A similar process of moulting resulting in injuries may be seen in other early Euarthropods.

Premise of the study: Doryopteris J.Sm. is a genus of dry-adapted ferns found widely across the southern hemisphere and the tropics. In the Hawaiian Islands, the genus is represented by four endemic species and one putative hybrid. Two of the endemic species (D. decora and D. decipiens) are widespread across all major islands, while two (D. angelica and D. takeuchii) are federally and state-listed endangered single-island endemics.We aimed to resolve the evolutionary relationships and biogeographic history of Hawaiian Doryopteris. Methods: We used a target-capture approach and the GoFlag 408 flagellate land plant probe set to sequence more than 400 nuclear loci for 40 Hawaiian and 29 non-Hawaiian individuals in Doryopteris and the related genera Lytoneuron and Ormopteris. After processing the sequences into alignments and single nucleotide polymorphism sites, we performed phylogenetic inference and population-level principal component analyses. Key results: We infer that Hawaiian Doryopteris are monophyletic and are nested within the pantropical species D. concolor. Within Hawai'i, clades and clusters of individuals correspond to geographical, geological, and climatological factors, more so than species identifications, with a few exceptions. We posit that Hawaiian Doryopteris represents an incipient radiation at the very earliest stages of divergence, and that budding speciation has led to non-monophyly of several taxa. Conclusions Our inference using genome-wide sequencing data sheds light on the complex evolutionary history of Hawaiian Doryopteris. Overall, the phylogeny of Hawaiian Doryopteris shows strong links with geographical, geological, and climatological processes and little with morphological evolution.

Hydractiniidae is a diverse family of colonial hydrozoans exhibiting substantial variation in colony organization, reproduction and life-cycle strategies, including species with and without a medusa stage. Several hydractiniids are also established model organisms, yet species boundaries and distributions remain incompletely resolved, limiting comparisons of reproductive and developmental traits across taxa. Here, we combine morphological and molecular data to reassess hydractiniid diversity, species identities, and life-cycle variation in material from the North Atlantic and Arctic Ocean. We integrate detailed observations of colonies and reproductive structures with DNA barcoding and mitochondrial genomes to evaluate species boundaries and provide molecular support for taxonomic identifications. Our results confirm 10 species in the study area, occurring from shallow waters to 2600 m depth and from coastal fjords to deep-sea hydrothermal vents. We further show that some historical species identifications and distribution records require reassessment. We establish a new genus for Arctic hydractiniid species (Bonneviactinia gen. nov.) and reassign two species to Schuchertinia. Mapping reproductive strategies onto the phylogeny reveals a broad pattern of increasing retention of medusa characters, from fixed sporosacs through reduced and fixed medusoids and non-liberated eumedusoids to fully developed, free-swimming feeding medusae, suggesting a progressive evolutionary trajectory in life-cycle complexity across Hydractiniidae. Furthermore, we clarify the identity of Podocoryna carnea and, through rearing experiments from polyp stage to medusae, characterize its morphological variation and developmental stages. By linking morphologically documented specimens and vouchers to genetic data, we establish reference resources that will facilitate future taxonomic, phylogenetic and biodiversity studies, including molecular surveys of marine communities. Together, these results demonstrate the value of integrative taxonomy for resolving hydractiniid diversity and provide a comparative foundation for investigating the evolution of reproductive strategies and life-cycle complexity in Medusozoa.

Background Comparisons of single-cell and single-nucleus RNA sequencing (scRNA-seq and snRNA-seq) data have been described in some mammalian tissues and, subsequently, in Drosophila, but remain unexplored in most invertebrate lineages. The xenacoelomorphs occupy key phylogenetic positions, yet they differ anatomically from mammals. They have a reduced extracellular matrix, high-salt body fluid, and no circulatory system. Despite these differences, they possess a well-developed nervous system. One of the xenacoelomorphs, the photosymbiotic acoel (Praesagittifera naikaiensis) also harbours symbiotic Tetraselmis algae, whose RNA can be co-captured with host RNA. Results We compared scRNA-seq and snRNA-seq data from whole P. naikaiensis specimens. Both methods yielded high-quality data with comparable gene detection but a larger share of scRNA-seq reads derived from symbiotic algae. Gene-level analyses revealed that neural genes were enriched in snRNA-seq relative to non-neural genes. Cross-method label transfer and integration-based validation identified six snRNA-seq clusters, including some neural populations, that lacked a clear scRNA-seq counterpart. In contrast, three cell populations, including muscle and metabolically active clusters, were reciprocally validated as captured by both methods. Conclusions Our results show that key snRNA-seq advantages, particularly the enhanced recovery of neural transcripts, are recapitulated in our dataset, which is consistent with previous reports in mammals. Furthermore, snRNA-seq reduces symbiont-derived reads and recovers several cell populations underrepresented in scRNA-seq. These findings provide practical guidance for cell atlas construction in non-model, symbiotic invertebrates.