Ctenostome bryozoans pose ongoing challenges for taxonomists and systematists, owing to their lack of character-rich mineralized skeletons. We present the largest (super)family-level phylogeny of Ctenostomatida to date. We resolved the three major clades (A–C) that were recognized previously. Clade A included endolithic taxa Penetrantiidae, Immergentiidae, and Terebripora ramosa, the latter two being sister taxa. Other taxa in this clade were Nolella, Arachnidium, Pottsiella, and Paludicella. Clade B was composed of Alcyonidioidea and Multiporata. The genus Alcyonidium was revealed as non-monophyletic, with the two recovered clades likely to be characterized by contrasting reproductive modes (brooding and zygote spawning). Members of the presumptive brooding clade had insertions of possible exogenous origin in their 18S and 28S ribosomal RNA genes, which might be the result of horizontal gene transfer. Clade C was sister to the Cheilostomatida and was composed of Vesicularioidea, Hislopioidea, Walkerioidea, and three clades of Victorellidae. Anguinella and Hypophorella were consistently sister taxa, but their position was unstable. Results are discussed in light of morphology, reproduction, and previous phylogenetic hypotheses. A revised classification is proposed. Timwoodiellina natans is transferred to Hislopia, Vesicularia spinosa to Amathia, and Monobryozoon ambulans to Alcyonidium. Members of the putative zygote-spawning Alcyonidium clade are transferred to Alcyonidioides in the family Alcyonidioididae.

While marine bryozoans were shown to be a promising source of bioactive compounds with the potential to be developed into drugs, their freshwater counterparts remain understudied. Considering that bioactive compounds isolated from bryozoans may originate from bacterial communities associated with the hosts, we explored the bacterial community of the freshwater bryozoan Cristatella mucedo using genomics and metabolomics. 16 S rRNA gene amplicon sequencing of the bacterial community associated with C. mucedo showed a considerable overlap with communities from surrounding water and sediment. Using different isolation approaches we retrieved a diverse collection of bacterial strains representing 26 genera, including a potentially new one. Genome sequencing and analyses of representative isolates of each genus revealed considerable potential for secondary metabolite biosynthesis. The secondary metabolomes of both mono- and co-cultures of selected isolates and enriched bryozoan-derived communities were investigated, allowing the identification of several known and potentially novel secondary metabolites. This work provides important information regarding the composition of the bacterial community associated with C. mucedo and sets the stage for its further exploration and exploitation for drug discovery.

Colonial invertebrates consist of iterative semi-autonomous modules (usually termed zooids) whose lifespan is significantly shorter than that of the entire colony. Typically, module development begins with budding and ends with degeneration. Most studies on the developmental biology of colonial invertebrates have focused on blastogenesis, whereas the changes occurring throughout the entire zooidal life were examined only for a few tunicates. Here we provide the first description of transcriptomic changes during polypide development in the freshwater bryozoan Cristatella mucedo. For the first time for Bryozoa, we performed bulk RNA sequencing of six polypide stages in C. mucedo (buds, juvenile polypides, three mature stages, and degeneration stage) and generated a high-quality de novo reference transcriptome. Based on these data, we analyzed clusters of differentially expressed genes for enriched pathways and biological processes that may be involved in polypide budding, growth, active functioning, and degradation. Although stem cells have never been described in Bryozoa, our analysis revealed the expression of conservative "stemness" markers in developing buds and juvenile polypides. Our data also indicate that polypide degeneration is a complex regulated process involving autophagy and other types of programmed cell death. We hypothesize that the mTOR signaling pathway plays an important role in regulating the polypide lifespan.

AimQuantify the contribution of environmental factors (water temperature, salinity and depth) and evolutionary history to varied skeletal mineralogy in calcifying marine organisms.LocationGlobal Ocean.Time periodPresent.Major taxa studiedOrder: Cheilostomatida; Phylum: Bryozoa.MethodsWe employed X-ray diffraction (XRD) to analyse the skeletal mineral composition of 872 individual colonies, representing 437 bryozoan species, in terms of calcite/aragonite ratios. We integrated these data with equivalent published data, thus reaching 981 species, and applied linear models (LMs), generalized linear models (GLMs) and phylogenetic generalized least squares models (PGLSs) to investigate the influences of temperature, salinity, depth and phylogenetic history on the mineralogy of nearly 1000 cheilostome bryozoan species.ResultsCheilostome bryozoans vary considerably in their skeletal mineral composition: in our dataset 65% of the species possess purely calcite skeletons, 15% exclusively employ aragonite and 20% exhibit mixed (i.e. calcite and aragonite) mineralogies. Temperature is the predominant measured environmental factor influencing bryozoan skeletal mineralogy, accounting for 20% of its variability across species, when phylogenetic relatedness is unaccounted for. Bryozoans in lower latitudes, characterized by higher seawater temperatures, have higher aragonite concentrations. By accounting for phylogenetic structure using a subset of 87 species for which we have topological information, 40% of the observed mineralogical variability could be attributed to present-day temperature. In contrast, depth and salinity played minor roles, explaining less than 1% of the mineralogical variation each.Main conclusionsThis study emphasizes the influence of evolutionary history on the mineralogical variability of calcifying organisms, even when it can be shown that a single environmental factor (temperature) explains a substantial amount of this variability. When confronted with changing temperature, calcifiers such as bryozoans are likely to respond in diverse ways, depending on the species, given their phylogenetic relatedness and the external conditions they meet.

The medium-sized lophotrochozoan phylum Bryozoa demonstrates a surprisingly wide range of larval forms. Few zygoparous species from the class Gymnolaemata possess long-lived planktotrophic larva (cyphonautes and paracyphonautes). The rest of gymnolaemates, and all species from classes Stenolaemata and Phylactolamata, incubate their embryos, whose development relies on egg’s yolk, extraembryonic nutrition (matrotrophy) or both, and have a brief free-swimming larval stage. Comparative morpho-functional analysis indicates that in bryozoans, similar to many other marine invertebrates, transitions from planktotrophic to endotrophic larvae were multiple and, obviously, were based on changes in oogenesis. Besides, the acquisition of a new larval type has always occurred in association with the evolution of embryonic incubation in Bryozoa. In myolaemates, the main trends in the evolution of endotrophy were reduction of the larval gut, loss of the larval protective cuticle/shell, invagination of the pallial epithelium of the episphere, and increase of the corona. Furthermore, larvae of stenolaemates lost their aboral and pyriform organs. Although being planktotrophic, the cyphonautes is a highly modified larval form, and cannot be considered as an ancestral type of bryozoan larvae. Phylactolaemates have a highly derived heterochronous development with a free-swimming stage that is, in fact, a chimera—either an ancestrula or a juvenile colony having a larval ciliary covering.

Parental care is considered crucial for the enhanced survival of offspring and evolutionary success of many metazoan groups. Most bryozoans incubate their young in brood chambers or intracoelomically. Based on the drastic morphological differences in incubation chambers across members of the order Cheilostomatida (class Gymnolaemata), multiple origins of incubation were predicted in this group. This hypothesis was tested by constructing a molecular phylogeny based on mitogenome data and nuclear rRNA genes 18S and 28S with the most complete sampling of taxa with various incubation devices to date. Ancestral character estimation suggested that distinct types of brood chambers evolved at least 10 times in Cheilostomatida. In Eucratea loricata and Aetea spp. brooding evolved unambiguously from a zygote-spawning ancestral state, as it probably did in Tendra zostericola, Neocheilostomata, and 'Carbasea' indivisa. In two further instances, brooders with different incubation chamber types, skeletal and non-skeletal, formed clades (Scruparia spp., Leiosalpinx australis) and (Catenicula corbulifera (Steginoporella spp. (Labioporella spp., Thalamoporella californica))), each also probably evolved from a zygote-spawning ancestral state. The modular nature of bryozoans probably contributed to the evolution of such a diverse array of embryonic incubation chambers, which included complex constructions made of polymorphic heterozooids, and maternal zooidal invaginations and outgrowths.

PHYLOGENIES All_genes_alignment.nex The concatenated mixed alignment consisting of, 13 mitochondrial protein-coding genes as amino acids, mitochondrial ribosomal RNA genes 12S+16S, and nuclear 18S+28S rRNA genes. Gene boundaries and excludes sites are indicated. Fig_2.nex Topology of the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. The analysis was performed in MrBayes5D v. 3.2.6 under the GTR+G model of nucleotide evolution (nucleotides) and the MTZOA+G model (amino acids). The analysis was run for 2.4 million generations; 1.5 million generations were discarded as burn-in. Fig_S3 Topology of the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes (PCGs) as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. The analysis was performed in p4 under the GTR+G model of nucleotide evolution (nucleotides) and the MTZOA+G+F model (amino acids). The +F model component accommodates empirical composition in the amino acid model. The analysis used three separate runs for 300,000 generations; 200,000 generations were discarded as burn-in. Fig_S4 Topology of the maximum likelihood phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. The analysis was performed in RAxML HPC-PTHREADS-SSE3 v. 8.2.12 under the GTR+G (nucleotides) and the MTZOA+G+F models (amino acids). Fig_S5 Topology of the Bayesian phylogenetic analysis of the 12S+16S rRNA gene partition constructed using MrBayes v. 3.2.6 under the GTR + G model. The analysis was run for 20 million generations; 10 million generations were discarded as burn-in. Fig_S6 Topology of the maximum likelihood phylogenetic analysis of the 12S+16S rRNA gene partition constructed using RAxML HPC-PTHREADS-SSE3 v. 8.2.12 under the GTRCAT model. Fig_S7 Topology of the Bayesian phylogenetic analysis of the 18S+28S rRNA gene partition constructed using MrBayes v. 3.2.6 under the GTR + G model. The analysis was run for 20 million generations; 10 million generations were discarded as burn-in. Fig_S8 Topology of the maximum likelihood phylogenetic analysis of the 18S+28S rRNA gene partition constructed using RAxML HPC-PTHREADS-SSE3 v. 8.2.12 under the GTRCAT model. Fig_S9 Topology of the Bayesian phylogenetic analysis of 13 mitochondrial protein-coding genes as amino acids constructed using MrBayes5D v. 3.2.6 under the MTZOA+G model. The analysis was run for 3.7 million generations; 2.5 million generations were discarded as burn-in. Fig_S10 Topology of the maximum likelihood phylogenetic analysis of 13 mitochondrial protein-coding genes as amino acids constructed using RAxML HPC-PTHREADS-SSE3 v. 8.2.12 under the PROTGAMMAMTZOA model. Fig_S11 Topology of the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes (PCGs) as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. The analysis was performed in p4 under the NDCH-C2 model. The analysis used four separate runs for 300,000 generations; 200,000 generations were discarded as burn-in. The NDCH model accommodates compositional tree-heterogeneity and was used because there was a large amount of compositional heterogeneity over the sequences, especially in the PCGs and 12S+16S rRNA data partitions. This is an NDCH model with two composition vectors on each of the three data partitions. Fig_S12 Topology of the Bayesian phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. This analysis excluded all terminals for which less than half of mitogenome genes were available, or which only had one of the two nuclear rRNA genes. The analysis was performed in MrBayes5D v. 3.2.6 under the GTR+G model of nucleotide evolution (nucleotides) and the MTZOA+G model (amino acids). The analysis was run for 350,000 generations; 125,000 generations were discarded as burn-in. Fig_S13 Topology of the maximum likelihood phylogenetic analysis of the mixed concatenated alignment consisting of three partitions: (i) 13 mitochondrial protein-coding genes as amino acids, (ii) mitochondrial ribosomal RNA genes 12S+16S, (iii) nuclear 18S+28S rRNA genes. This analysis excluded all terminals for which less than half of mitogenome genes were available, or which only had one of the two nuclear rRNA genes. The analysis was performed in RAxML HPC-PTHREADS-SSE3 v. 8.2.12 under the GTR+G (nucleotides) and the MTZOA+G+F models (amino acids). ANCESTRAL CHARACTER ESTIMATION: ACE.R R script of the ancestral character estimation carried out in phytools. Reproductive_strategy_numbers.csv Data input file for ACE analysis (reproductive strategies coded as numbers) Reproductive_strategies.xlsx List of reproductive strategies per taxon with the corresponding numerical codes used in the file 'Reproductive_stategies_numbers.csv'. Tree.tre Input tree for ACE analysis.

Symbiotic associations are dynamic systems influenced by both intrinsic and extrinsic factors. Here we describe for the first time the developmental and seasonal changes of the funicular bodies in the bryozoan Dendrobeania fruticosa, which are unique temporary organs of cheilostome bryozoans containing prokaryotic symbionts. Histological and ultrastructural studies showed that these organs undergo strong seasonal modification in the White Sea during the ice-free period. Initially (in June) they play a trophic function and support the development of a large population of bacteria. From June to September, both funicular bodies and bacteria show signs of degradation accompanied by development of presumed virus-like particles (VLPs); these self-organize to hollow spheres inside bacteria and are also detected outside of them. Although the destruction of bacteria coincides with the development of VLPs and spheres, the general picture differs considerably from the known instances of bacteriophagy in bryozoans. We broadly discuss potential routes of bacterial infection in Bryozoa and question the hypothesis of vertical transfer, which, although widely accepted in the literature, is contradicted by molecular, morphological and ecological evidence.

A large body of research is dedicated to marine sponges, filter-feeding animals harboring rich bacterial microbiomes believed to play an important role in protecting the host from predators and infections. Freshwater sponges have received so far much less attention with respect to their microbiomes, members of which may produce bioactive secondary metabolites with potential to be developed into drugs to treat a variety of diseases.

Capitate hydrozoans are a morphologically and ecologically diverse hydrozoan suborder, currently including about 200 species. Being grouped in two clades, Corynida and Zancleida, these hydrozoans still show a number of taxonomic uncertainties at the species, genus and family levels. Many Capitata species established symbiotic relationships with other benthic organisms, including bryozoans, other cnidarians, molluscs and poriferans, as well as with planktonic dinoflagellates for mixotrophic relationships and with bacteria for thiotrophic ectosymbioses. Our study aimed at providing an updated and comprehensive phylogeny reconstruction of the suborder, at modelling the evolution of selected morphological and ecological characters, and at testing evolutionary relationships between the symbiotic lifestyle and the other characters, by integrating taxonomic, ecological and evolutionary data. The phylogenetic hypotheses here presented shed light on the evolutionary relationships within Capitata, with most families and genera being recovered as monophyletic. The genus Zanclea and family Zancleidae, however, were divided into four divergent clades, requiring the establishment of the new genus Apatizanclea and the new combinations for species in Zanclea and Halocoryne genera. The ancestral state reconstructions revealed that symbiosis arose multiple times in the evolutionary history of the Capitata, and that homoplasy is a common phenomenon in the group. Correlations were found between the evolution of symbiosis and morphological characters, such as the perisarc. Overall, our results highlighted that the use of genetic data and a complete knowledge of the life cycles are strongly needed to disentangle taxonomic and systematic issues in capitate hydrozoans. Finally, the colonization of tropical habitat appears to have influenced the evolution of a symbiotic lifestyle, playing important roles in the evolution of the group.

The life of sedentary organisms faces strong competition for space against neighboring epibionts, and the impact of predators. The emergence of various adaptations to cope these problems includes establishing various interactions with other members of benthic communities. Various symbiotic (commensal, mutualistic, and even parasitic) relationships presented in multiple variations allow not only to succeed in the competition for free space, but also provide other equally important advantages for survival. Being one of the most abundant groups of colonial invertebrates in marine benthic ecosystems, bryozoans are not an exception. This group demonstrates both common and unique symbiotic associations. This article provides an overview of all known forms of symbiosis in Bryozoa, fossil and modern, and discusses the consequences of such relationships.

Morpho-functional polymorphism of modules, also known as “division of labor”, is a widespread phenomenon independently evolving multiple times in the colonies of many aquatic invertebrates and invertebrate chordates. Polymorphic zooids are especially diverse among Cheilostomata, the evolutionarily most successful clade in the phylum Bryozoa. The most diverse among cheilostome polymorphs are avicularia and vibracularia, acting as defensive, repelling, cleaning and locomotory “colonial organs”. While their skeletal characters were intensively studied, the soft tissues have largely been neglected. This hampers evolutionary interpretations. In this study, we compared the muscular system in five contrasting types of these polymorphs from 10 species of eight related as well as distant families. In contrast with the prevailing view, we found that the structural and functional changes affecting the muscular system during evolutionary transition from autozooid to polymorph were considerably more diverse and complex than mere “vestigialization”. These changes included muscle loss, hypertrophy, rearrangement (regrouping and relocation), fusion, and acquisition of asymmetry and muscle striation. Asynchronous contraction of originally synchronously working muscles was presumably evolved in advanced vibracularia. Some of these modifications were recorded in all avicularian types, whereas others were characteristic only for particular taxa or polymorphic categories. Our study showed that not only skeletal, but also soft parts of the polymorphs were evolutionarily very flexible and modified to various degrees and in various directions.

Bryozoans are small benthic suspension-feeding colonial animals. Among this phylum, there are representatives showing a lesser or greater degree of polymorphism, and the most common type of polymorphic zooids is the avicularium. Here we present a detailed description of the bird's-head shaped avicularium in Dendrobeania fruticosa. The body cavity of the avicularium demonstrates an acoelomate condition: along the cystid walls, there is neither the layer of extracellular matrix toward the epidermis, nor coelomic lining. However, a layer of extracellular matrix and epithelialized cells lie under the epidermis of the tentacle sheath. Probably, such construction helps the tentacle sheath to acquire some rigidity-it is the only region of the body wall without an ectocyst. We did not find typical funicular strands in the avicularium, but there is a delicate mesh composed of stellate cells with thin and long projections, which sometimes isolate the spaces filled with a heterogeneous matrix. The proximal ends of the adductors, abductors, and polypide retractors are attached to the body wall via typical epidermal tendon cells, which possess numerous bundles of tonofilaments. The distal ends of the abductors and adductors attach to the frontal membrane or upper vestibular membrane, respectively. The inner organic layer of the ectocyst in these regions forms large protrusions, from which numerous thin outgrowths branch off. We suggest them to be a functional analogue of apodemes and apodemal filaments in arthropods. "Apodemal" tendon cells have long and thin projections that line the outgrowths of the ectocyst and surround the distal ends of the muscle cells. At these sites, "apodemal" tendon cells possess numerous tonofilaments. The vestigial polypide includes the tentacle sheath, rudimentary lophophore, cerebral ganglion, and polypide retractors. The sensory part of 5HT-positive cells of the frontal membrane is dendrite-shaped and embedded in the inner organic layer of the ectocyst.

The outer epithelium of free-swimming larvae of marine invertebrates is composed of several cell types and performs a variety of vital functions.In bryozoan larvae, early anatomical studies revealed a rather complex organization of this epithelium.The present contribution re-examines the available morphological descriptions, as well as investigates anatomically and ultrastructurally the epispheral (aboral) part of the outer pallial epithelium in contrasting larval types of six marine bryozoans from three orders -Cheilostomatida, Ctenostomatida, and Cyclostomatida.A total of 11 cell types were identified -three of them are typical for most of the studied larvae, the rest are unique to specific larval types.Based on the data obtained, we conducted a comparative analysis and assigned potential functions to particular types of cells during larval life.We proposed the hypothesis that immersion/invagination of the larval pallial epithelium (which was accompanied by cell enlargement in ciliary locomotory corona) occurred independently in different bryozoan lineages and was associated with acquisition of incubation.We also consider the presence of the cuticle above the episphere and the absence of cilia in most principal pallial epithelial cells as plesiomorphic traits of bryozoan larvae.

Twenty-four Recent species of the boreal-Arctic and Pacific cheilostome bryozoan genus Rhamphostomella are described. The species R. tatarica and R. pacifica are transferred to Rhamphostomella from Posterula and Porella, respectively. Eight species are new: R. aleutica n. sp., R. aspera n. sp., R. commandorica n. sp., R. echinata n. sp., R. microavicularia n. sp., R. morozovi n. sp., R. multirostrata n. sp. and R. obliqua n. sp. Neotypes are selected for six species, and lectotypes for eight species. Mixtoscutella n. gen. is established for several Rhamphostomella-like species, including M. androsovae [formerly Smittina androsovae Gontar], M. cancellata [formerly Escharella porifera forma cancellata Smitt], M. harmsworthi [formerly Schizoporella harmsworthi Waters], M. ovata [formerly Cellepora ovata (Smitt)], and M. ussowi [formerly Schizoporella ussowi (Kluge)]. In addition to taxonomic revision, the morphology (frontal shields, ovicells and multiporous septula), ecology and zoogeography of these cheilostomes are discussed, and identification keys are presented. Most species of Rhamphostomella have broad bathymetric distributions. Some have long protuberances on their basal walls that allow them to grow elevated above allelopathically active substrates such as sponges. The diversity of Rhamphostomella peaks in the northwestern Pacific.

Phylogenetic relationships and the timing of evolutionary events are essential for understanding evolution on longer time scales. Cheilostome bryozoans are a group of ubiquitous, species-rich, marine colonial organisms with an excellent fossil record but lack phylogenetic relationships inferred from molecular data. We present genome-skimmed data for 395 cheilostomes and combine these with 315 published sequences to infer relationships and the timing of key events among c. 500 cheilostome species. We find that named cheilostome genera and species are phylogenetically coherent, rendering fossil or contemporary specimens readily delimited using only skeletal morphology. Our phylogeny shows that parental care in the form of brooding evolved several times independently but was never lost in cheilostomes. Our fossil calibration, robust to varied assumptions, indicates that the cheilostome lineage and parental care therein could have Paleozoic origins, much older than the first known fossil record of cheilostomes in the Late Jurassic.

Aquatic and, in particular, marine invertebrates demonstrate a wide range of sexual reproduction traits, which is unsurprising given their enormous morphological and ecological diversity. The descriptive data published on their reproduction are massive. Providing an integrated picture of their evolution, however, remains difficult due to the large number, variability, and numerous combinations of reproductive traits, as well as due to the incomplete data on the reproduction of many groups. Nonetheless, the known diversity reveals the main directions and stages in the evolution of sexual reproduction within particular clades, an important step in outlining the general patterns in marine invertebrates. The present brief overview analyzes this issue based on our current level of knowledge.