Abstract Molecular data have revolutionised taxonomic and ecological research on the hyperdiverse communities of aquatic benthic microinvertebrates known as meiofauna. However, reference sequence databases remain highly incomplete, with variable barcode genes or fragments studied from taxon to taxon. Furthermore, there is a typical tradeoff between universality of primers and phylogenetic resolution, with rRNA markers being robustly recoverable but failing to resolve species-level divergences, and mitochondrial markers showing the reverse trend. Here, we introduce O xford Nanopore r RNA and C OI a mplicon sequencing (OrCa-seq), a rapid, low-cost protocol for parallel long-range PCR amplification and multiplexed sequencing of four amplicons, spanning the nearly-complete rRNA cistron (∼7-8 kb) and the widely studied Folmer region of COI (represented as overlapping 313 and 658 bp amplicons). This protocol, with its associated bioinformatic workflow, was designed for conducting biodiversity inventories of meiofauna and can be easily carried out in field research and educational contexts, with data available from 96-well plates of specimens within a day of lysis. To validate the method, we processed six plates of student-isolated freshwater and limno-terrestrial meiofauna, characterising the recovery of target genes and taxa with both automated and human-curated BLAST database comparisons. These data demonstrate the universal applicability of OrCa-seq across effectively all meiofauna, including the very smallest species. Nonetheless, recovery efficiency for each amplicon shows variation by taxon, with the full-length Folmer COI amplicon standing out as the most challenging. We present exemplar phylogenetic trees integrating reference sequences, demonstrating the utility of these data in confirming morphological determinations and in identifying anonymous specimens in a reverse taxonomy context. While developed in a specific educational context for use on meiofauna, the OrCa-seq approach should be readily scalable to larger research datasets, adaptable to many specimen types, and to any combination of taxon-or target-specific primers. As such, it represents a compelling multi-locus extension to the ever-growing repertoire of nanopore DNA barcoding protocols.

Linckia laevigata is a tropical sea star commonly found throughout the Indian and Pacific Oceans, and is one of the top ten-most collected invertebrates, often encountered in the aquarium trade. It has been the subject of investigations into population structure, biodiversity and ecology, particularly regarding gene flow among populations throughout its range, the status of different colour morphs and its relationship with its putative sister species, L. multifora . Here we present and describe a high-quality genome assembly for L. laevigata. Our assembly is 585.97 Mb in length, with a scaffold N50 of 3 Mb. The genome has a typical repeat (36.99%) and GC content (41.33%), when compared with other echinoderm datasets. Our genome annotation recovers 16,178 genes, with high (89.4%) recovery of the metazoan BUSCO set. This novel resource will provide a model organism for studying the biogeography of the tropical Indo-West Pacific region, and more specifically facilitate the investigation of a range of sea star traits at the genomic level. Significance Sea stars remain poorly characterised at a genomic level. Data from the charismatic tropical sea star Linckia laevigata will enable future studies into the biology, population structure and evolution of these ecologically important species. ### Competing Interest Statement The authors have declared no competing interest. Royal Society of New Zealand, https://ror.org/04tajb587, 23-UOO-003-CSG Royal Society, URF\R1\221744 National Geographic Society, NGS-52123R-19

Although platyhelminths are common members of marine interstitial communities, the number of meiofaunal marine triclads known at present is rather limited. Here, we describe two new monotypic genera of interstitial marine triclads from Panama. In order to shed light on their phylogenetic and taxonomic position, we have followed an integrative approach that includes not only comparative analysis of their morphology but molecular phylogenetic placement with small and large rDNA sequences. Our phylogenies were broadly congruent with recently published molecular phylogenies of the marine triclads. The two new genera appeared not to be closely related, but, nevertheless, do share several morphological features, such as the presence of (1) a secondary gonopore, (2) a glandular region traversed by many septa in a large portion of the penis, (3) a copulatory bursa connected with the secondary gonopore, and (4) two retinal cells per eyecup. However, they do also differ considerably from each other. The following combination of factors made it impossible to assign the two new genera to any of the higher categories within the suborder of the marine triclads: (a) a unique combination of features present in each of the genera that does not fit into any of the current family diagnoses, (b) their present position in the phylogenetic tree, (c) the mismatch between the topologies of the molecular phylogenetic trees and the current taxonomic classification within the suborder. Therefore, the two taxa are classified as genera incertae sedis, pending a broader revision of the taxonomy of Maricola.

Cephalochordates (also known as amphioxus or lancelets) are a group of marine invertebrates occupying a critical phylogenetic position as the sister group to the remaining members of the chordate phylum (vertebrates and tunicates). As such, amphioxus have been key to help understanding the origin and evolution of vertebrate genomes, the evolution of their development and the diversification of their anatomy. However, out of the three genera of amphioxus currently known ( Branchiostoma, Asymmetron and Epigonichthys ), most studies have focused exclusively on Branchiostoma species. Indeed, all amphioxus genomes published to date belong to the Branchiostoma genus. This implies that our understanding of cephalochordate biology is limited to findings from Branchiostoma alone. It is consequently difficult to infer from the currently available Branchiostoma genomes, what aspects of their gene composition and structure are genus-specific or ancestral to cephalochordates. Here, we provide new high-quality genome assemblies and annotations from representatives of both Asymmetron and Epigonichthys . Genome-wide phylogenetic analyses reveal that, in contrast to previous mitogenomic studies, Branchiostoma is the sister group to Asymmetron and Epigonichthys . We further uncover discordant gene histories between these three genera, with notable asymmetry in the distribution of gene trees with minority topologies. Lastly, we find that extensive gene births and duplications preceded the origin of modern cephalochordates. ### Competing Interest Statement The authors have declared no competing interest. CRUK, C9545/A29580 Marie Curie, FP7-PEOPLE-EIPOD COFUND 229597 Assemble, 227799

The latest animal phylum to be discovered, Micrognathozoa, constitutes a rare group of limnic meiofauna. These microscopic 'jaw animals' are among the smallest metazoans yet possess highly complex jaw structures. The single species of Micrognathozoa, Limnognathia maerski Kristensen and Funch, 2000, was first described from Greenland, later reported from a remote Subantarctic island and more recently discovered in the Pyrenees on the European continent. Successful collections of these three known populations facilitated investigations of the intraphylum relationships and species limits within Limnognathia for the first time. Through detailed anatomical comparisons, we substantiate the lack of morphological differences between the three geographically disjunct populations. With transcriptomic data from single specimens, we conducted the first intraphylum phylogenetic analyses and extensively tested species hypotheses using standard approaches and novel machine learning methods. Analyses clearly delimited the Subantarctic population, here described as Limnognathia desmeti sp. nov., the second species of Micrognathozoa, but did not definitively split the Greenland and Pyrenees populations as separate species. Divergence dating analysis suggests the disjunct distribution of Micrognathozoa is not human mediated but the result of long-distance dispersal raising questions about their dispersal capabilities and potential undiscovered populations.

We report the results of a faunistic survey focused on freshwater and limno-terrestrial meiofauna to improve biodiversity knowledge in a protected area in the Eastern part of the French Pyrénées: the Massane Forest Reserve (336 Ha). The survey provided 1187 occurrence records from 315 taxa (most resoved at species-level), uploaded as a shared online dataset. The highest number of occurrences and distinguishable morpho-taxon belong to the group Nematoda (775 occurrences, 172 taxa), followed by Rotifera (219 occurrences, 67 taxa), Platyhelminthes (85 occurrences, 32 taxa), Tardigrada (69 occurrences, 25 taxa), and Gastrotricha (39 occurrences, 19 taxa). A diversity of meiofaunal organisms was found, in large numbers, in all the samples screened: from stream biofilms and sediments, to forest floor soils, mosses, and litter, to a broad range of tree-related micro-habitats associated with beech-like epixylic mosses and lichens, tree cavities, woodpecker breeding holes, bark pockets and fruiting bodies of saproxylic fungi. This survey makes the Massane forest one of the few protected areas of the world with a taxa-inclusive meiofauna dataset, which could serve as a standard inventory to further consider micro-invertebrates in forest conservation.

BACKGROUND:The advancement of sequencing technologies results in the rapid release of hundreds of new genome assemblies a year providing unprecedented resources for the study of genome evolution. Within this context, the significance of in-depth analyses of repetitive elements, transposable elements (TEs) in particular, is increasingly recognized in understanding genome evolution. Despite the plethora of available bioinformatic tools for identifying and annotating TEs, the phylogenetic distance of the target species from a curated and classified database of repetitive element sequences constrains any automated annotation effort. Moreover, manual curation of raw repeat libraries is deemed essential due to the frequent incompleteness of automatically generated consensus sequences.RESULTS:Here, we present an example of a crowd-sourcing effort aimed at curating and annotating TE libraries of two non-model species built around a collaborative, peer-reviewed teaching process. Manual curation and classification are time-consuming processes that offer limited short-term academic rewards and are typically confined to a few research groups where methods are taught through hands-on experience. Crowd-sourcing efforts could therefore offer a significant opportunity to bridge the gap between learning the methods of curation effectively and empowering the scientific community with high-quality, reusable repeat libraries.CONCLUSIONS:The collaborative manual curation of TEs from two tardigrade species, for which there were no TE libraries available, resulted in the successful characterization of hundreds of new and diverse TEs in a reasonable time frame. Our crowd-sourcing setting can be used as a teaching reference guide for similar projects: A hidden treasure awaits discovery within non-model organisms.

Meiofauna—a collective term to define microscopic animals—represent a numerically important component of biodiversity in most of Earth’s ecosystems and play a crucial role in biogeochemical cycles. Meiofauna have also been used as models to understand fundamental adaptive processes, have contributed to a better understanding of the animal’s Tree of Life, and are believed to be a treasure trove for future genomic studies. To celebrate the diversity of research topics brought to us by the term “meiofauna”, we gathered a multidisciplinary team of 42 ecologists, taxonomists, morphologists, biogeographers, molecular biologists, and scientific disseminators to list 194 fundamental questions in meiofaunal research. Then, through an online survey, 251 scientists, administrators, students, and stakeholders assisted us in reducing this list to 50 top-priority questions. Applied topics related to anthropogenic impact and climate change received the highest scores, whereas questions related to areas in development such as genomics or adaptations, received less attention. Whereas we might not be exploiting meiofauna’s full potential yet, more and more integrative approaches and technological developments will create opportunities to employ these fascinating organisms to answer broad and important questions, despite of their impediments related to their small body size. Meiofauna research agenda should balance amongst investigating general questions, addressing more specialized research topics, and generating primary data on distribution, taxonomy, traits, and DNA sequences. The geographical and taxonomic biases that have historically affected meiofaunal research can be alleviated by promoting international cooperation, open data sharing, and an increase effort in education, taxonomic training, as well as scientific communication. We hope that this will get both researchers and the general public intrigued by those small critters that constantly lurk unseen in front of us.

Transposable elements (TEs) regulate diverse biological processes, from early development to cancer. Expression of young TEs is difficult to measure with next-generation, single-cell sequencing technologies because their highly repetitive nature means that short complementary DNA reads cannot be unambiguously mapped to a specific locus. Single CELl LOng-read RNA-sequencing (CELLO-seq) combines long-read single cell RNA-sequencing with computational analyses to measure TE expression at unique loci. We used CELLO-seq to assess the widespread expression of TEs in two-cell mouse blastomeres as well as in human induced pluripotent stem cells. Across both species, old and young TEs showed evidence of locus-specific expression with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to the reference with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed large heterogeneity, with TEs within a class showing different patterns of correlation and suggesting distinct regulatory mechanisms.

The role of Transposable Elements (TEs) in regulating diverse biological processes, from early development to cancer, is becoming increasing appreciated. However, unlike other biological processes, next generation single-cell sequencing technologies are ill-suited for assaying TE expression: in particular, their highly repetitive nature means that short cDNA reads cannot be unambiguously mapped to a specific locus. Consequently, it is extremely challenging to understand the mechanisms by which TE expression is regulated and how they might themselves regulate other protein coding genes. To resolve this, we introduce CELLO-seq, a novel method and computational framework for performing long-read RNA sequencing at single cell resolution. CELLO-seq allows for full-length RNA sequencing and enables measurement of allelic, isoform and TE expression at unique loci. We use CELLO-seq to assess the widespread expression of TEs in 2-cell mouse blastomeres as well as human induced pluripotent stem cells (hiPSCs). Across both species, old and young TEs showed evidence of locus-specific expression, with simulations demonstrating that only a small number of very young elements in the mouse could not be mapped back to with high confidence. Exploring the relationship between the expression of individual elements and putative regulators revealed surprising heterogeneity, with TEs within a class showing different patterns of correlation, suggesting distinct regulatory mechanisms. ### Competing Interest Statement The authors have declared no competing interest. * (CELLO-seq) : CELl LOng read RNA sequencing (PacBio) : Pacific Biosciences (MaLRs) : Mammalian apparent LTR-retrotransposons (SNP) : single nucleotide polymorphism (sc) : single cell (TE) : Transposable element (ERV) : endogenous retrovirus (LINE) : Long interspersed element (SINE) : Short interspersed element (RNAseq) : RNA sequencing (hiPSCs) : Human induced pluripotent stem cells (RT) : Reverse Transcription (LTRs) : long terminal repeat elements (NGS) : next generation sequencing (ONT) : Oxford Nanopore technologies (UMIs) : unique molecular identifiers (TSO) : template switch oligo (ZNFs) : Zinc finger nucleases (nt) : Nucleotides (bp) : Base pairs (TSS) : Transcription start site (TES) : Transcription end site (ERCC) : External RNA Controls Consortium

The paper provides a taxonomic description of a new genus and species of terrestrial cavernicolan triclad flatworm from Barro Colorado Island, Panama. Molecular sequences of this species were already deposited in GenBank several years ago under the provisional taxon name Cavernicola sp. Animals have the following characteristics: absence of pigmentation and eyes; mouth opening located halfway in the pharyngeal cavity; few, ventral testes extending from a position far posterior to the ovaries to directly behind the pharyngeal cavity; strongly recurving sperm ducts, thus forming a loop; horizontally oriented cone-shaped penis papilla; common oviduct oriented perpendicular to the bursal canal and opening into the latter shortly before the canal communicates with the copulatory bursa; sac-shaped copulatory bursa lacking a distinct, single lumen and filled with a mass of syncytial cells, with interspersed nuclei.

Proper biological interpretation of a phylogeny can sometimes hinge on the placement of key taxa-or fail when such key taxa are not sampled. In this light, we here present the first attempt to investigate (though not conclusively resolve) animal relationships using genome-scale data from all phyla. Results from the site-heterogeneous CAT + GTR model recapitulate many established major clades, and strongly confirm some recent discoveries, such as a monophyletic Lophophorata, and a sister group relationship between Gnathifera and Chaetognatha, raising continued questions on the nature of the spiralian ancestor. We also explore matrix construction with an eye towards testing specific relationships; this approach uniquely recovers support for Panarthropoda, and shows that Lophotrochozoa (a subclade of Spiralia) can be constructed in strongly conflicting ways using different taxon- and/or orthologue sets. Dayhoff-6 recoding sacrifices information, but can also reveal surprising outcomes, e.g. full support for a clade of Lophophorata and Entoprocta + Cycliophora, a clade of Placozoa + Cnidaria, and raising support for Ctenophora as sister group to the remaining Metazoa, in a manner dependent on the gene and/or taxon sampling of the matrix in question. Future work should test the hypothesis that the few remaining uncertainties in animal phylogeny might reflect violations of the various stationarity assumptions used in contemporary inference methods.

The phylogenetic placement of the morphologically simple placozoans is crucial to understanding the evolution of complex animal traits. Here, we examine the influence of adding new genomes from placozoans to a large dataset designed to study the deepest splits in the animal phylogeny. Using site-heterogeneous substitution models, we show that it is possible to obtain strong support, in both amino acid and reduced-alphabet matrices, for either a sister-group relationship between Cnidaria and Placozoa, or for Cnidaria and Bilateria (=Planulozoa), also seen in most published work to date, depending on the orthologues selected to construct the matrix. We demonstrate that a majority of genes show evidence of compositional heterogeneity, and that the support for Planulozoa can be assigned to this source of systematic error. In interpreting this placozoan-cnidarian clade, we caution against a peremptory reading of placozoans as secondarily reduced forms of little relevance to broader discussions of early animal evolution.

The phylogenetic placement of the morphologically simple placozoans is crucial to understanding the evolution of complex animal traits. Here, we examine the influence of adding new genomes from placozoans to a large dataset designed to study the deepest splits in the animal phylogeny. Using site-heterogeneous substitution models, we show that it is possible to obtain strong support, in both amino acid and reduced-alphabet matrices, for either a sister-group relationship between Cnidaria and Placozoa, or for Cnidaria and Bilateria as seen in most published work to date, depending on the orthologues selected to construct the matrix. We demonstrate that a majority of genes show evidence of compositional heterogeneity, and that support for the Cnidaria + Bilateria clade can be assigned to this source of systematic error. In interpreting these results, we caution against a peremptory reading of placozoans as secondarily reduced forms of little relevance to broader discussions of early animal evolution.

Contemporary phylogeneticists enjoy an embarrassment of riches, not only in the volumes of data now available, but also in the diversity of bioinformatic tools for handling these data. Here, I discuss a subset of these tools I consider well-suited to the task of inferring ancient relationships with coding sequence data in particular, encompassing data generation, orthology assignment, alignment and gene tree inference, supermatrix construction, and analysis under the best-fitting models applicable to large-scale datasets. Throughout, I compare and critique methods, considering both their theoretical principles and the details of their implementation, and offering practical tips on usage where appropriate. I also entertain different motivations for analyzing what are almost always originally DNA sequence data as codons, amino acids, and higher-order recodings. Although presented in a linear order, I see value in using the diversity of tools available to us to assess the sensitivity of clades of biological interest to different gene and taxon sets and analytical modes, which can be an indication of the presence of systematic error, of which a few forms remain poorly controlled by even the best available inference methods.

The existence of the platyhelminth clade Adiaphanida-an assemblage comprising the well-studied order Tricladida as well as two lesser known taxa, Prolecithophora and the obligate parasitic Fecampiida-is among the more surprising results of flatworm molecular systematics. Each of these three clades is itself largely well-defined from a morphological point of view, although Adiaphanida at large, despite its strong support in molecular phylogenetic analyses, lacks known morphological synapomorphies. However, one taxon, the genus Genostoma, a parasite of the leptostracan crustacean Nebalia, rests uneasily within its current classification within the fecampiid family Genostomatidae; ultrastructural investigations on this taxon have uncovered a spermatogenesis reminiscent of Kalyptorhynchia, and a dorsal syncytium resembling the neodermatan tegument. Here, we provide molecular sequence data (nearly complete 18S and 28S rRNA) from a representative of Genostoma, with which we test hypotheses on the phylogenetic position of this taxon within Platyhelminthes, expanding upon a recently published phylum-wide analysis, and applying novel alignment algorithms and substitution models. These analyses unequivocally position Genostoma as the sister group of Prolecithophora. However, even in taxon-rich analyses, support for the position of the root of Adiaphanida is lacking, highlighting the need for new data types to study the phylogeny of this clade. Interestingly, our analyses also do not recover the monophyly of several taxa previously proposed, notably Continenticola within Tricladida and Protomonotresidae within Prolecithophora. In light of this phylogeny and the distinctive morphology (especially, spermatogenesis) of Genostoma, we advocate for a redefinition of the family Genostomatidae, outside of both Fecampiida and Prolecithophora, to encompass the members of this unique genus of parasites. Within Fecampiida, the family Piscinquilinidae fam. nov. is erected to accommodate the vertebrate-parasitic Piscinquilinus, formerly Genostomatidae.

Bdelloura candida (Platyhelminthes, Tricladida, Maricola) is an ectocommensal symbiont on the American horseshoe crab Limulus polyphemus, living on the book gills and appendages, where it spends its entire life. Given its limited dispersal capabilities and its inability to live outside of the host, we hypothesized a genetic structure that parallels that of its host. We obtained 84 planarian individuals from 19 horseshoe crabs collected from 10 sites from Massachusetts to Florida. We amplified the mitochondrial 16S rRNA and the nuclear internal transcribed spacer 2 and conducted phylogeographic and population genetic analyses, which show a clear and strong genetic break between the populations in the Atlantic and the Gulf coasts. Among the Atlantic populations, two additional, weaker barriers located along Cape Hatteras and Cape Cod restrict gene flow. Even though previous studies have suggested that the populations of the host may be in decline, those of B. candida remain stable, and some even shows signatures of expansion. Our results indicate that the phylogeography of these marine ectocommensal triclads closely mirrors that of its Limulus host, and highlight the challenges to both host and symbiont to genetically connect populations across their distribution.