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During development of the spermatid, two free flagella are transported distally from the main spermatid mass by elongation of the shaft. They rotate back towards the shaft and lie parallel with it prior to fusion in a distal-proximal direction. This process conforms to that found in other free-living platyhelminths that have fused or adjoined axonemes in their sperm (Acoela, Kalyptorhynchia, Polycladida). It is basically different from the process of fusion in the majority of neodermatan species (major groups of parasitic platyhelminths), where the attachment of axonemes remains near the main spermatid body, a median process grows out between them, the nucleus and mitochondria migrate into this process and axonemes then fuse with it in a proximal-distal manner. Pterastericola astropectinis also differs from the Neodermata in the presence of dense bodies in the sperm. The ultrastructure of the sperm, spermiogenesis and protonephridia does not support the view of a close affinity between Pterastericolidae and Neodermata. [ABSTRACT FROM AUTHOR]

Xenacoelomorpha are mostly microscopic, morphologically simple worms, lacking many structures typical of other bilaterians. Xenacoelomorphs -which include three main groups: Acoela, Nemertodermatida, and Xenoturbella- have been proposed to be an early diverging Bilateria, sister to protostomes and deuterostomes, but other phylogenomic analyses have recovered this clade nested within the deuterostomes, as sister to Ambulacraria. The position of Xenacoelomorpha within the metazoan tree has understandably attracted a lot of attention, overshadowing the study of phylogenetic relationships within this group. Given that Xenoturbella includes only six species whose relationships are well understood, we decided to focus on the most speciose Acoelomorpha (Acoela + Nemertodermatida). Here, we have sequenced 29 transcriptomes, doubling the number of sequenced species, to infer a backbone tree for Acoelomorpha based on genomic data. The recovered topology is mostly congruent with previous studies. The most important difference is the recovery of Paratomella as the first off-shoot within Acoela, dramatically changing the reconstruction of the ancestral acoel. Besides, we have detected incongruence between the gene trees and the species tree, likely linked to incomplete lineage sorting, and some signal of introgression between the families Dakuidae and Mecynostomidae, which hampers inferring the correct placement of this family and, particularly, of the genus Notocelis. We have also used this dataset to infer for the first time diversification times within Acoelomorpha, which coincide with known bilaterian diversification and extinction events. Given the importance of morphological data in acoelomorph phylogenetics, we tested several partitions and models. Although morphological data failed to recover a robust phylogeny, phylogenetic placement has proven to be a suitable alternative when a reference phylogeny is available., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems., (© 2024 The Author(s). Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Background: Xenacoelomorpha is a marine clade of microscopic worms that is an important model system for understanding the evolution of key bilaterian novelties, such as the excretory system. Nevertheless, Xenacoelomorpha genomics has been restricted to a few species that either can be cultured in the lab or are centimetres long. Thus far, no genomes are available for Nemertodermatida, one of the group's main clades and whose origin has been dated more than 400 million years ago. Methods: DNA was extracted from a single specimen and sequenced with HiFi following the PacBio Ultra-Low DNA Input protocol. After genome assembly, decontamination, and annotation, the genome quality was benchmarked using two acoel genomes and one Illumina genome as reference. The gene content of three cnidarians, three acoelomorphs, four deuterostomes, and eight protostomes was clustered in orthogroups to make inferences of gene content evolution. Finally, we focused on the genes related to the ultrafiltration excretory system to compare patterns of presence/absence and gene architecture among these clades. Results: We present the first nemertodermatid genome sequenced from a single specimen of Nemertoderma westbladi . Although genome contiguity remains challenging (N50: 60 kb), it is very complete (BUSCO: 80.2%, Metazoa; 88.6%, Eukaryota) and the quality of the annotation allows fine-detail analyses of genome evolution. Acoelomorph genomes seem to be relatively conserved in terms of the percentage of repeats, number of genes, number of exons per gene and intron size. In addition, a high fraction of genes present in both protostomes and deuterostomes are absent in Acoelomorpha. Interestingly, we show that all genes related to the excretory system are present in Xenacoelomorpha except Osr , a key element in the development of these organs and whose acquisition seems to be interconnected with the origin of the specialised excretory system. Conclusion: Overall, these analyses highlight the potential of the Ultra-Low Input DNA protocol and HiFi to generate high-quality genomes from single animals, even for relatively large genomes, making it a feasible option for sequencing challenging taxa, which will be an exciting resource for comparative genomics analyses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Abalde, Tellgren-Roth, Heintz, Vinnere Pettersson and Jondelius.)

Eight new species of Isodiametridae (Acoela) are presented and phylogenetic relationships of the family using 18S and COI sequence data reconstructed. Four species of Baltalimania, two species of Praeaphanostoma and two species of Pseudoposthia are described that all can be distinguished both morphologically and through differences in the DNA sequences as supported by mPTP analysis. The addition of eight new species increases the number of nominal species of Isodiametridae worldwide by 10% and within Sweden by 31%, but also highlights several taxonomic problems. Several species are now confirmed as isodiametrids that are otherwise incongruent with the familys diagnostic characters. Generic keys are presented for Baltalimania and Praeaphanostoma.

The meiofauna is an important part of the marine ecosystem, but its composition and distribution patterns are relatively unexplored. Here we assessed the biodiversity and community structure of meiofauna from five locations on the Swedish western and southern coasts using a high-throughput DNA sequencing (metabarcoding) approach. The mitochondrial cytochrome oxidase 1 (COI) mini-barcode and nuclear 18S small ribosomal subunit (18S) V1-V2 region were amplified and sequenced using Illumina MiSeq technology. Our analyses revealed a higher number of species than previously found in other areas: thirteen samples comprising 6.5 dm 3 sediment revealed 708 COI and 1,639 18S metazoan OTUs. Across all sites, the majority of the metazoan biodiversity was assigned to Arthropoda, Nematoda and Platyhelminthes. Alpha and beta diversity measurements showed that community composition differed significantly amongst sites. OTUs initially assigned to Acoela, Gastrotricha and the two Platyhelminthes sub-groups Macrostomorpha and Rhabdocoela were further investigated and assigned to species using a phylogeny-based taxonomy approach. Our results demonstrate that there is great potential for discovery of new meiofauna species even in some of the most extensively studied locations., (Sarah Atherton, Ulf Jondelius.)

Whereas most work to understand impacts of humans on biodiversity on coastal areas has focused on large, conspicuous organisms, we highlight effects of tourist access on the diversity of microscopic marine animals (meiofauna). We used a DNA metabarcoding approach with an iterative and phylogeny-based approach for the taxonomic assignment of meiofauna and relate diversity patterns to the numbers of tourists accessing sandy beaches on an otherwise un-impacted island National Park. Tourist frequentation, independently of differences in sediment granulometry, beach length, and other potential confounding factors, affected meiofaunal diversity in the shallow "swash" zone right at the mean water mark; the impacts declined with water depth (up to 2 m). The indicated negative effect on meiofauna may have a consequence on all the biota including the higher trophic levels. Thus, we claim that it is important to consider restricting access to beaches in touristic areas, in order to preserve biodiversity.

Microstomidae (Platyhelminthes: Macrostomorpha) diversity has been almost entirely ignored within recent years, likely due to inconsistent and often old taxonomic literature and a general rarity of sexually mature collected specimens. Herein, we reconstruct the phylogenetic relationships of the group using both previously published and new 18S and CO1 gene sequences. We present some taxonomic revisions of Microstomidae and further describe 8 new species of Microstomum based on both molecular and morphological evidence. Finally, we briefly review the morphological taxonomy of each species and provide a key to aid in future research and identification that is not dependent on reproductive morphology. Our goal is to clarify the taxonomy and facilitate future research into an otherwise very understudied group of tiny (but important) flatworms., Competing Interests: The authors have declared that no competing interests exist.

[This corrects the article DOI: 10.1038/s42003-018-0119-2.].

Accurate assessments of biodiversity are crucial to advising ecosystem-monitoring programs and understanding ecosystem function. Nevertheless, a standard operating procedure to assess biodiversity accurately and consistently has not been established. This is especially true for meiofauna, a diverse community (>20 phyla) of small benthic invertebrates that have fundamental ecological roles. Recent studies show that metabarcoding is a cost-effective and time-effective method to estimate meiofauna biodiversity, in contrast to morphological-based taxonomy. Here, we compare biodiversity assessments of a diverse meiofaunal community derived by applying multiple taxonomic methods based on comparative morphology, molecular phylogenetic analysis, DNA barcoding of individual specimens, and metabarcoding of environmental DNA. We show that biodiversity estimates are strongly biased across taxonomic methods and phyla. Such biases affect understanding of community structures and ecological interpretations. This study supports the urgency of improving aspects of environmental high-throughput sequencing and the value of taxonomists in correctly understanding biodiversity estimates., Competing Interests: The authors declare no competing interests.

It has been hypothesized that a condensed nervous system with a medial ventral nerve cord is an ancestral character of Bilateria. The presence of similar dorsoventral molecular patterns along the nerve cords of vertebrates, flies, and an annelid has been interpreted as support for this scenario. Whether these similarities are generally found across the diversity of bilaterian neuroanatomies is unclear, and thus the evolutionary history of the nervous system is still contentious. Here we study representatives of Xenacoelomorpha, Rotifera, Nemertea, Brachiopoda, and Annelida to assess the conservation of the dorsoventral nerve cord patterning. None of the studied species show a conserved dorsoventral molecular regionalization of their nerve cords, not even the annelid Owenia fusiformis, whose trunk neuroanatomy parallels that of vertebrates and flies. Our findings restrict the use of molecular patterns to explain nervous system evolution, and suggest that the similarities in dorsoventral patterning and trunk neuroanatomies evolved independently in Bilateria.

Precision and reliability of barcode-based biodiversity assessment can be affected at several steps during acquisition and analysis of data. Identification of operational taxonomic units (OTUs) is one of the crucial steps in the process and can be accomplished using several different approaches, namely, alignment-based, probabilistic, tree-based and phylogeny-based. The number of identified sequences in the reference databases affects the precision of identification. This paper compares the identification of marine nematode OTUs using alignment-based, tree-based and phylogeny-based approaches. Because the nematode reference dataset is limited in its taxonomic scope, OTUs can only be assigned to higher taxonomic categories, families. The phylogeny-based approach using the evolutionary placement algorithm provided the largest number of positively assigned OTUs and was least affected by erroneous sequences and limitations of reference data, compared to alignment-based and tree-based approaches., Competing Interests: We declare we have no competing interests.

The position of Xenacoelomorpha in the tree of life remains a major unresolved question in the study of deep animal relationships. Xenacoelomorpha, comprising Acoela, Nemertodermatida, and Xenoturbella, are bilaterally symmetrical marine worms that lack several features common to most other bilaterians, for example an anus, nephridia, and a circulatory system. Two conflicting hypotheses are under debate: Xenacoelomorpha is the sister group to all remaining Bilateria (= Nephrozoa, namely protostomes and deuterostomes) or is a clade inside Deuterostomia. Thus, determining the phylogenetic position of this clade is pivotal for understanding the early evolution of bilaterian features, or as a case of drastic secondary loss of complexity. Here we show robust phylogenomic support for Xenacoelomorpha as the sister taxon of Nephrozoa. Our phylogenetic analyses, based on 11 novel xenacoelomorph transcriptomes and using different models of evolution under maximum likelihood and Bayesian inference analyses, strongly corroborate this result. Rigorous testing of 25 experimental data sets designed to exclude data partitions and taxa potentially prone to reconstruction biases indicates that long-branch attraction, saturation, and missing data do not influence these results. The sister group relationship between Nephrozoa and Xenacoelomorpha supported by our phylogenomic analyses implies that the last common ancestor of bilaterians was probably a benthic, ciliated acoelomate worm with a single opening into an epithelial gut, and that excretory organs, coelomic cavities, and nerve cords evolved after xenacoelomorphs separated from the stem lineage of Nephrozoa.

Nemertodermatida are microscopically small, benthic marine worms. Specimens of two nominal species, Sterreria psammicola and Nemertinoides elongatus from 33 locations worldwide were sequenced for three molecular markers. Species delimitation and validation was done using gene trees, haplotype networks and multilocus Bayesian analysis. We found 20 supported species of which nine: Nemertinoides glandulosum n.sp., N. wolfgangi n.sp., Sterreria boucheti n.sp., S. lundini n.sp., S. martindalei n.sp., S. monolithes n.sp., S. papuensis n.sp., S. variabilis n.sp. and S. ylvae n.sp., are described including nucleotide-based diagnoses. The distribution patterns indicate transoceanic dispersal in some of the species. Sympatric species were found in many cases. The high level of cryptic diversity in this meiofauna group implies that marine diversity may be higher than previously estimated.

Some taxonomic groups are less amenable to mitochondrial DNA barcoding than others. Due to the paucity of molecular information of understudied groups and the huge molecular diversity within flatworms, primer design has been hampered. Indeed, all attempts to develop universal flatworm-specific COI markers have failed so far. We demonstrate how high molecular variability and contamination problems limit the possibilities for barcoding using standard COI-based protocols in flatworms. As a consequence, molecular identification methods often rely on other widely applicable markers. In the case of Monogenea, a very diverse group of platyhelminth parasites, and Rhabdocoela, representing one-fourth of all free-living flatworm taxa, this has led to a relatively high availability of nuclear ITS and 18S/28S rDNA sequences on GenBank. In a comparison of the effectiveness in species assignment we conclude that mitochondrial and nuclear ribosomal markers perform equally well. In case intraspecific information is needed, rDNA sequences can guide the selection of the appropriate (i.e. taxon-specific) COI primers if available.

Acoels are with few exceptions marine worms and a common component of the interstitial meiofauna. In this study we present new species to science belonging to Isodiametridae and Solenofilomorphidae. The new species, Isodiametra finkei n. sp., Postaphanostoma nilssoni n. sp., Pseudaphanostoma hyalinorhabdoida n. sp. and Solenofilomorpha pellucida n. sp. were all collected in Chile during March 2012. Nucleotide sequences for the ribosomal genes 18S rDNA and 28S rDNA as well as COI mtDNA have been determined for the new species and used in a maximum likelihood analysis to further support their classification.

In this study we elaborate the phylogeny of Dalytyphloplanida based on complete 18S rDNA (156 sequences) and partial 28S rDNA (125 sequences), using a Maximum Likelihood and a Bayesian Inference approach, in order to investigate the origin of a limnic or limnoterrestrial and of a symbiotic lifestyle in this large group of rhabditophoran flatworms. The results of our phylogenetic analyses and ancestral state reconstructions indicate that dalytyphloplanids have their origin in the marine environment and that there was one highly successful invasion of the freshwater environment, leading to a large radiation of limnic and limnoterrestrial dalytyphloplanids. This monophyletic freshwater clade, Limnotyphloplanida, comprises the taxa Dalyelliidae, Temnocephalida, and most Typhloplanidae. Temnocephalida can be considered ectosymbiotic Dalyelliidae as they are embedded within this group. Secondary returns to brackish water and marine environments occurred relatively frequently in several dalyeliid and typhloplanid taxa. Our phylogenies also show that, apart from the Limnotyphloplanida, there have been only few independent invasions of the limnic environment, and apparently these were not followed by spectacular speciation events. The distinct phylogenetic positions of the symbiotic taxa also suggest multiple origins of commensal and parasitic life strategies within Dalytyphloplanida. The previously established higher-level dalytyphloplanid clades are confirmed in our topologies, but many of the traditional families are not monophyletic. Alternative hypothesis testing constraining the monophyly of these families in the topologies and using the approximately unbiased test, also statistically rejects their monophyly.

Background: Within an evolutionary framework of Gastrotricha Marinellina flagellata and Redudasys fornerise bear special interest, as they are the only Macrodasyida that inhabit freshwater ecosystems. Notwithstanding, these rare animals are poorly known; found only once (Austria and Brazil), they are currently systematised as incertae sedis. Here we report on the rediscovery of Redudasys fornerise, provide an account on morphological novelties and present a hypothesis on its phylogenetic relationship based on molecular data., Methodology/principal Findings: Specimens were surveyed using DIC microscopy and SEM, and used to obtain the 18 S rRNA gene sequence; molecular data was analyzed cladistically in conjunction with data from 42 additional species belonging to the near complete Macrodasyida taxonomic spectrum. Morphological analysis, while providing new information on taxonomically relevant traits (adhesive tubes, protonephridia and sensorial bristles), failed to detect elements of the male system, thus stressing the parthenogenetic nature of the Brazilian species. Phylogenetic analysis, carried out with ML, MP and Bayesian approaches, yielded topologies with strong nodal support and highly congruent with each other. Among the supported groups is the previously undocumented clade showing the alliance between Redudasys fornerise and Dactylopodola agadasys; other strongly sustained clades include the densely sampled families Thaumastodermatidae and Turbanellidae and most genera., Conclusions/significance: A reconsideration of the morphological traits of Dactylopodola agadasys in light of the new information on Redudasys fornerise makes the alliance between these two taxa very likely. As a result, we create Anandrodasys gen. nov. to contain members of the previously described D. agadasys and erect Redudasyidae fam. nov. to reflect this novel relationship between Anandrodasys and Redudasys. From an ecological perspective, the derived position of Redudasys, which is deeply nested within the Macrodasyida clade, unequivocally demonstrates that invasion of freshwater by gastrotrichs has taken place at least twice, in contrast with the single event hypothesis recently put forward.

Background: Biogeographical and macroecological principles are derived from patterns of distribution in large organisms, whereas microscopic ones have often been considered uninteresting, because of their supposed wide distribution. Here, after reporting the results of an intensive faunistic survey of marine microscopic animals (meiofauna) in Northern Sardinia, we test for the effect of body size, dispersal ability, and habitat features on the patterns of distribution of several groups., Methodology/principal Findings: As a dataset we use the results of a workshop held at La Maddalena (Sardinia, Italy) in September 2010, aimed at studying selected taxa of soft-bodied meiofauna (Acoela, Annelida, Gastrotricha, Nemertodermatida, Platyhelminthes and Rotifera), in conjunction with data on the same taxa obtained during a previous workshop hosted at Tjärnö (Western Sweden) in September 2007. Using linear mixed effects models and model averaging while accounting for sampling bias and potential pseudoreplication, we found evidence that: (1) meiofaunal groups with more restricted distribution are the ones with low dispersal potential; (2) meiofaunal groups with higher probability of finding new species for science are the ones with low dispersal potential; (3) the proportion of the global species pool of each meiofaunal group present in each area at the regional scale is negatively related to body size, and positively related to their occurrence in the endobenthic habitat., Conclusion/significance: Our macroecological analysis of meiofauna, in the framework of the ubiquity hypothesis for microscopic organisms, indicates that not only body size but mostly dispersal ability and also occurrence in the endobenthic habitat are important correlates of diversity for these understudied animals, with different importance at different spatial scales. Furthermore, since the Western Mediterranean is one of the best-studied areas in the world, the large number of undescribed species (37%) highlights that the census of marine meiofauna is still very far from being complete.

Acoela are marine microscopic worms currently thought to be the sister taxon of all other bilaterians. Acoels have long been used as models in evolutionary scenarios, and generalized conclusions about acoel and bilaterian ancestral features are frequently drawn from studies of single acoel species. There is no extensive phylogenetic study of Acoela and the taxonomy of the 380 species is chaotic. Here we use two nuclear ribosomal genes and one mitochondrial gene in combination with 37 morphological characters in an analysis of 126 acoel terminals (about one-third of the described species) to estimate the phylogeny and character evolution of Acoela. We present an estimate of posterior probabilities for ancestral character states at 31 control nodes in the phylogeny. The overall reconstruction signal based on the shape of the posterior distribution of character states was computed for all morphological characters and control nodes to assess how well these were reconstructed. The body-wall musculature appears more clearly reconstructed than the reproductive organs. Posterior similarity to the root was calculated by averaging the divergence between the posterior distributions at the nodes and the root over all morphological characters. Diopisthoporidae is the sister group to all other acoels and has the highest posterior similarity to the root. Convolutidae, including several "model" acoels, is most divergent. Finally, we present a phylogenetic classification of Acoela down to the family level where six previous family level taxa are synonymized.

Background: Acoels are simply organized unsegmented worms, lacking hindgut and anus. Several publications over recent years challenge the long-held view that acoels are early offshoots of the flatworms. Instead a basal position as sister group to all other bilaterian animals was suggested, mainly based on molecular evidence. This led to the view that features of acoels might reflect those of the last common ancestor of Bilateria, and resulted in several evo-devo studies trying to interpret bilaterian evolution using acoels as a proxy model for the "Urbilateria"., Results: We describe the first complete mitochondrial genome sequence of a member of the Acoela, Symsagittifera roscoffensis. Gene content and circular organization of the mitochondrial genome does not significantly differ from other bilaterian animals. However, gene order shows no similarity to any other mitochondrial genome within the Metazoa. Phylogenetic analyses of concatenated alignments of amino acid sequences from protein coding genes support a position of Acoela and Nemertodermatida as the sister group to all other Bilateria. Our data provided no support for a sister group relationship between Xenoturbellida and Acoela or Acoelomorpha. The phylogenetic position of Xenoturbella bocki as sister group to or part of the deuterostomes was also unstable., Conclusions: Our phylogenetic analysis supports the view that acoels and nemertodermatids are the earliest divergent extant lineage of Bilateria. As such they remain a valid source for seeking primitive characters present in the last common ancestor of Bilateria. Gene order of mitochondrial genomes seems to be very variable among Acoela and Nemertodermatida and the groundplan for the metazoan mitochondrial genome remains elusive. More data are needed to interpret mitochondrial genome evolution at the base of Bilateria.

A clear picture of animal relationships is a prerequisite to understand how the morphological and ecological diversity of animals evolved over time. Among others, the placement of the acoelomorph flatworms, Acoela and Nemertodermatida, has fundamental implications for the origin and evolution of various animal organ systems. Their position, however, has been inconsistent in phylogenetic studies using one or several genes. Furthermore, Acoela has been among the least stable taxa in recent animal phylogenomic analyses, which simultaneously examine many genes from many species, while Nemertodermatida has not been sampled in any phylogenomic study. New sequence data are presented here from organisms targeted for their instability or lack of representation in prior analyses, and are analysed in combination with other publicly available data. We also designed new automated explicit methods for identifying and selecting common genes across different species, and developed highly optimized supercomputing tools to reconstruct relationships from gene sequences. The results of the work corroborate several recently established findings about animal relationships and provide new support for the placement of other groups. These new data and methods strongly uphold previous suggestions that Acoelomorpha is sister clade to all other bilaterian animals, find diminishing evidence for the placement of the enigmatic Xenoturbella within Deuterostomia, and place Cycliophora with Entoprocta and Ectoprocta. The work highlights the implications that these arrangements have for metazoan evolution and permits a clearer picture of ancestral morphologies and life histories in the deep past.

One of the main characters used in acoel taxonomy is the male copulatory organ. Despite this, ultrastructural studies of this structure are scarce. We studied the ultrastructure of the copulatory organ in eight species of acoels belonging to the taxon Childia. Members of Childia possess a well-developed conical or cylindrical stylet-like structure composed of needles. Immunogold cytochemistry of tubulin was used to determine the composition of the needles. Stylet-like structures of Childia species at the ultrastructural level are basically similar. Stylet needles show intracellular differentiations. As shown both by ultrastructural and immunocytochemical methods, the stylet needles, in all species studied, are composed of long, parallel microtubules, either tightly packed or polymerized. We report unusual polymerization of microtubules, resulting in formation of a honeycomb-like structure in cross section. Variations of ultrastructure among Childia species include numbers and arrangement of stylet needles, shape of needles, needle compactness, microtubule polymerization, direction of stylet growth, and presence/absence of different types of granules. The stylet-like structures are homologous within Childia, but are likely to prove nonhomologous with the other needle-like structures found in acoel copulatory organs. Stylets in Platyhelminthes are not homologous with stylet-like structures in acoels.