Symbiotic Algae of Acoel Species in the Seto Inland Sea and Symbiont Selectivity in the Hosts.

Praesagittifera naikaiensis is an acoel flatworm that inhabits the sandy beaches in the intertidal zone of the Seto Inland Sea. This species carries Tetraselmis sp., a green unicellular chlorophyte, as a symbiont in its body, and depends on algal photosynthetic products to survive. However, the eggs of P. naikaiensis contain no symbiotic algae, and juvenile P. naikaiensis acquire symbionts from the surrounding environment through horizontal transfer after hatching, thereby establishing new symbiotic relationships in each generation. Other acoel species, Symsagittifera spp., also inhabit the Seto Inland Sea shores and acquire symbiotic green algae via horizontal transfers. To characterize their symbionts, these acoels were collected from a wide area of the Seto Inland Sea and partial nucleotide sequences of the chloroplast ribulose diphosphate carboxylase large subunit (rbcL) of the symbiotic algae were determined and used for molecular phylogenetic analysis. Symbionts of both P. naikaiensis and Symsagittifera spp. belonged to the genus Tetraselmis but were phylogenetically distant, and both species established symbiotic relationships with different symbionts even when they were sympatric. To test whether each species selects specific algae in the environment for symbiosis, we established algal strains from P. naikaiensis and Symsagittifera sp. symbionts and conducted uptake experiments on aposymbiotic juveniles of P. naikaiensis. The results suggest that symbiotic algae from Symsagittifera could be taken up by P. naikaiensis juveniles, but were unable to establish a normal symbiotic relationship with the juveniles.

Genome assembly of the acoel flatworm Symsagittifera roscoffensis, a model for research on body plan evolution and photosymbiosis.

Symsagittifera roscoffensis is a well-known member of the order Acoela that lives in symbiosis with the algae Tetraselmis convolutae during its adult stage. Its natural habitat is the eastern coast of the Atlantic, where at specific locations thousands of individuals can be found, mostly, lying in large pools on the surface of sand at low tide. As a member of the Acoela it has been thought as a proxy for ancestral bilaterian animals; however, its phylogenetic position remains still debated. In order to understand the basic structural characteristics of the acoel genome, we sequenced and assembled the genome of aposymbiotic species S. roscoffensis. The size of this genome was measured to be in the range of 910-940 Mb. Sequencing of the genome was performed using PacBio Hi-Fi technology. Hi-C and RNA-seq data were also generated to scaffold and annotate it. The resulting assembly is 1.1 Gb large (covering 118% of the estimated genome size) and highly continuous, with N50 scaffold size of 1.04 Mb. The repetitive fraction of the genome is 61%, of which 85% (half of the genome) are LTR retrotransposons. Genome-guided transcriptome assembly identified 34,493 genes, of which 29,351 are protein coding (BUSCO score 97.6%), and 30.2% of genes are spliced leader trans-spliced. The completeness of this genome suggests that it can be used extensively to characterize gene families and conduct accurate phylogenomic reconstructions.

Studying Xenacoelomorpha WBR Using Isodiametra pulchra.

Xenacoelomorpha are a phylogenetically and biologically interesting, but severely understudied group of worm-like animals. Among them, the acoel Isodiametra pulchra has been shown to be amenable to experimental work, including the study of stem cells and regeneration. The animal is capable of regenerating the posterior part of the body, but not its head. Here, methods such as nucleic acid extractions, in situ hybridisation, RNA interference, antibody and cytochemical stainings, and the general handling of the animals are presented.

Observing Phylum-Level Metazoan Diversity by Environmental DNA Analysis at the Ushimado Area in the Seto Inland Sea.

The dynamics of microscopic marine plankton in coastal areas is a fundamental theme in marine biodiversity research, but studies have been limited because the only available methodology was collection of plankton using plankton-nets and microscopic observation. In recent years, environmental DNA (eDNA) analysis has exhibited potential for conducting comprehensive surveys of marine plankton diversity in water at fixed points and depths in the ocean. However, few studies have examined how eDNA analysis reflects the actual distribution and dynamics of organisms in the field, and further investigation is needed to determine whether it can detect distinct differences in plankton density in the field. To address this, we analyzed eDNA in seawater samples collected at 1 km intervals at three depths over a linear distance of approximately 3.0 km in the Seto Inland Sea. The survey area included a location with a high density of Acoela (Praesagittifera naikaiensis). However, the eDNA signal for this was little to none, and its presence would not have been noticed if we did not have this information beforehand. Meanwhile, eDNA analysis enabled us to confirm the presence of a species of Placozoa that was previously undiscovered in the area. In summary, our results suggest that the number of sequence reads generated from eDNA samples in our project was not sufficient to predict the density of a particular species. However, eDNA can be useful for detecting organisms that have been overlooked using other methods.

Geographical Distribution and Genetic Diversity of Praesagittifera naikaiensis (Acoelomorpha) in the Seto Inland Sea, Japan.

Acoel flatworms are simple bilaterians that lack digestive lumens and coelomic cavities. Although they are a significant taxon for evaluating the evolution of metazoans, suitable species for biological experiments are not available in Japan. We recently focused on Praesagittifera naikaiensis, which inhabits the sandy shores of intertidal zones in the Seto Inland Sea in Japan, as a candidate for a representative acoel species to be used in experiments. However, reports on its distribution range remain limited. Here, we surveyed the habitats of P. naikaiensis on 108 beaches along the Seto Inland Sea. Praesagittifera naikaiensis is reported here from 37 sites (six previously known and 31 newly discovered sites) spread over a wide area of the Seto Inland Sea, from Awaji Island in Hyogo Prefecture to Fukuoka Prefecture (364 km direct distance). Based on the mitochondrial cytochrome oxidase subunit I (COI) gene haplotypes, we evaluated the genetic diversity of 145 individuals collected from 33 sites. Out of 42 COI haplotypes, 13 haplotypes were shared by multiple individuals. The most frequent haplotype was observed in 67 individuals collected from 31 sites. Eight other haplotypes were detected at geographically distant locations (maximum of 299 km direct distance). Multiple haplotypes were found at 32 sites. These results demonstrate that sufficient genetic flow exists among P. naikaiensis populations throughout the Seto Inland Sea. Molecular phylogenetic analysis of the COI haplotypes of P. naikaiensis revealed that all specimens were grouped into one clade. The genetic homogeneity of the animals in this area favors their use as an experimental animal.

Biodiversity between sand grains: Meiofauna composition across southern and western Sweden assessed by metabarcoding.

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 dm3 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.

Immunostaining and In Situ Hybridization of the Developing Acoel Nervous System.

The study of acoel morphologies has been recently stimulated by the knowledge that this group of animals represents an early offshoot of the Bilateria. Understanding how organ systems and tissues develop and the molecular underpinnings of the processes involved has become an area of new research. The microscopic anatomy of these organisms is best understood through the systematic use of immunochemistry and in situ hybridization procedures. These methods allow us to map, in precise detail, the expression patterns of genes and proteins, in space and time. With the additional use of genomic resources, they provide us with insights on how a group of "early" bilaterians have diversified over time. As these animals are new to the world of molecular studies, the protocols have involved a lot of new and specific adaptations to their specific anatomical-histological characteristics. Here we explain some of these protocols in detail, with the aim that should prove useful in our much-needed understanding of the origins of bilaterian animals. An anatomical sketch is provided at the beginning as a necessary guide for those not familiar with the Acoela.

The complete mitochondrial DNA sequence of Heterochaerus australis (Acoela, Convolutidae).

One complete mitochondrial genomes (mitogenomes) was determined for Heterochaerus australis (Acoela, Convolutidae). Its mitochondrial genome size was 13,885 bp. The sequence contains 2 ribosomal RNA genes (rrnL and rrnS), 20 tRNA genes, and 12 protein-coding genes (PCGs). The A + T content of the complete mitochondrial genome sequence was 70.8%. The base composition showed a tendency of high AT. The resulted maximum likelihood (ML) tree supported that Acoela had a distant relationship with other orders of Turbellaria and the Xenacoelomorpha.

Morphological and phylogenetic diversity of Waminoa and similar flatworms (Acoelomorpha) in the western Pacific Ocean.

The genus Waminoa currently contains two described species, which each contains two types of endosymbiotic algae. Waminoa individuals are basically brown in body color, derived from these algal symbionts, and their body shape has been described as "discoid to obcordate". They have been found as associates of various anthozoans (Cnidaria) in the Indo-Pacific Ocean and the Red Sea. In order to reveal the diversity of the genus Waminoa and their hosts, we conducted phylogenetic and morphological analyses on acoelomate flatworms specimens collected from Japan, Palau and Indonesia. At least 18 Waminoa morphotypes were found on at least 20 anthozoan host species, and two specimens were found on species of two sea stars. Overall, there were two main body shapes of specimens; obcordate, as seen in W. litus and W. brickneri, and the other molar-like with an elongated body. These two body shapes each represented a separate clade in 18S rDNA and mitochondrial cytochrome c oxidase subunit 1 (COI) phylogenetic trees, with W. brickneri included in the obcordate subclade. Automatic Barcode Gap Discovery (ABGD) analyses on COI sequences of our specimens revealed the presence of at least five operational taxonomic units (OTUs). These five OTUs consisted of one large group of all obcordate animals, three OTUs consisting of one specimen each within the molar-like clade, and one large group of the remaining molar-like specimens. Both clades contain numerous morphotypes and were associated with a variety of hosts. Finally, based on genetic distances, the molar-like specimens are considered as an unnamed genus group separate from Waminoa, which needs to be clarified in future studies.

The digestive system of xenacoelomorphs.

Interest in the study of Xenacoelomorpha has recently been revived due to realization of its key phylogenetic position as the putative sister group of the remaining Bilateria. Phylogenomic studies have attracted the attention of researchers interested in the evolution of animals and the origin of novelties. However, it is clear that a proper understanding of novelties can only be gained in the context of thorough descriptions of the anatomy of the different members of this phylum. A considerable literature, based mainly on conventional histological techniques, describes different aspects of xenacoelomorphs' tissue architecture. However, the focus has been somewhat uneven; some tissues, such as the neuro-muscular system, are relatively well described in most groups, whereas others, including the digestive system, are only poorly understood. Our lack of knowledge of the xenacoelomorph digestive system is exacerbated by the assumption that, at least in Acoela, which possess a syncytial gut, the digestive system is a derived and specialized tissue with little bearing on what is observed in other bilaterian animals. Here, we try to remedy this lack of attention by revisiting the different studies of the xenacoelomorph digestive system, and we discuss the diversity present in the light of new evolutionary knowledge.

New replacement name for Anaperus Graff, 1911 (Acoelomorpha: Acoela: Convolutidae).

The acoel genus Anaperus was established by Graff in 1911 for Amphiscolops gardineri Graff, 1910, making Anaperus gardineri (Graff, 1910) its type species. Since then, six more valid species were described: A. tvaerminnensis (Luther, 1912); A. sulcatus Beklemischev, 1914; A. rubellus Westblad, 1945; A. biaculeatus Boguta, 1970; A. ornatus Beltagi, 2001; A. singularis Hooge Smith, 2004. A seventh species, A. australis Westblad, 1952, is incertae sedis (Dörjes Karling, 1975). The genus was placed to family Convolutidae Graff, 1905, until Dörjes (1968) erected the family Anaperidae on the basis of a distinctive male copulatory apparatus. Jondelius et al. (2011) returned it to Convolutidae on the basis of molecular-sequence data.

Characterization of the bHLH family of transcriptional regulators in the acoel S. roscoffensis and their putative role in neurogenesis.

BACKGROUND: The basic helix-loop-helix (bHLH) family of transcription factors is one of the largest superfamilies of regulatory transcription factors and is widely used in eukaryotic organisms. They play an essential role in a range of metabolic, physiological, and developmental processes, including the development of the nervous system (NS). These transcription factors have been studied in many metazoans, especially in vertebrates but also in early branching metazoan clades such as the cnidarians and sponges. However, currently very little is known about their expression in the most basally branching bilaterian group, the xenacoelomorphs. Recently, our laboratory has characterized the full complement of bHLH in the genome of two members of the Xenacoelomorpha, the xenoturbellid Xenoturbella bocki and the acoel Symsagittifera roscoffensis. Understanding the patterns of bHLH gene expression in members of this phylum (in space and time) provides critical new insights into the conserved roles of the bHLH and their putative specificities in this group. Our focus is on deciphering the specific roles that these genes have in the process of neurogenesis. RESULTS: Here, we analyze the developmental expression of the whole complement of bHLH genes identified in the acoel S. roscoffensis. Based on their expression patterns, several members of bHLH class A appear to have specific conserved roles in neurogenesis, while other class A genes (as well as members of other classes) have likely taken on more generalized functions. All gene expression patterns are described in embryos and early juveniles. CONCLUSION: Our results suggest that the main roles of the bHLH genes of S. roscoffensis are evolutionarily conserved, with a specific subset dedicated to patterning the nervous system: SrAscA, SrAscB, SrHes/Hey, SrNscl, SrSrebp, SrE12/E47 and SrOlig.

Xenacoelomorpha: a case of independent nervous system centralization?

Centralized nervous systems (NSs) and complex brains are among the most important innovations in the history of life on our planet. In this context, two related questions have been formulated: How did complex NSs arise in evolution, and how many times did this occur? As a step towards finding an answer, we describe the NS of several representatives of the Xenacoelomorpha, a clade whose members show different degrees of NS complexity. This enigmatic clade is composed of three major taxa: acoels, nemertodermatids and xenoturbellids. Interestingly, while the xenoturbellids seem to have a rather 'simple' NS (a nerve net), members of the most derived group of acoel worms clearly have ganglionic brains. This interesting diversity of NS architectures (with different degrees of compaction) provides a unique system with which to address outstanding questions regarding the evolution of brains and centralized NSs. The recent sequencing of xenacoelomorph genomes gives us a privileged vantage point from which to analyse neural evolution, especially through the study of key gene families involved in neurogenesis and NS function, such as G protein-coupled receptors, helix-loop-helix transcription factors and Wnts. We finish our manuscript proposing an adaptive scenario for the origin of centralized NSs (brains).

A Flatworm from the Genus Waminoa (Acoela: Convolutidae) Associated with Bleached Corals in Western Australia.

A flatworm isolated from bleached colonies of the coral Coscinaraea marshae at Rottnest Island, Western Australia, is described using a combination of morphological and molecular systematics. This flatworm shares morphological features characteristic of the genus Waminoa (Acoelomorpha: Acoela), including the presence of two algal symbionts, but appears to have genital regions different from those of other described species of Waminoa. The design of new oligonucleotide primers enabled the amplification of partial 18S rDNA of the Rottnest Island acoel specimens, and phylogenetic analysis positioned them within Waminoa, confirming their placement in the genus. Furthermore, Waminoa specimens from Rottnest Island grouped into a sister clade to Waminoa brickneri, indicating that the morphological and genetic differences observed are most likely intraspecific and due to geographic variation. As such, we name these Rottnest Island specimens W. cf. brickneri, but highlight that key differences warrant further exploration before assignment to this species can be confirmed. This is the first acoel flatworm described from Western Australia and contributes to our understanding of the diversity and evolutionary relationship of the Acoela.

The nervous system of Xenacoelomorpha: a genomic perspective.

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of 'cephalized' (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of 'progressive' cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper.