First chromosome-level genome assembly of a ribbon worm from the Hoplonemertea clade, Emplectonema gracile, and its structural annotation.

Genome-wide information has so far been unavailable for ribbon worms of the clade Hoplonemertea, the most species-rich class within the phylum Nemertea. While species within Pilidiophora, the sister clade of Hoplonemertea, possess a pilidium larval stage and lack stylets on their proboscis, Hoplonemertea species have a planuliform larva and are armed with stylets employed for the injection of toxins into their prey. To further compare these developmental, physiological, and behavioral differences from a genomic perspective, the availability of a reference genome for a Hoplonemertea species is crucial. Such data will be highly useful for future investigations towards a better understanding of molecular ecology, venom evolution, and regeneration not only in Nemertea, but also in other marine invertebrate phyla. To this end, we herein present the annotated chromosome-level genome assembly for Emplectonema gracile (Nemertea; Hoplonemertea; Monostilifera; Emplectonematidae), an easily collected nemertean well-suited for laboratory experimentation. The genome has an assembly size of 157.9 Mbp. Hi-C scaffolding yielded chromosome level scaffolds, with a scaffold N50 of 10.0 Mbp and a score of 95.1% for complete BUSCO genes found as a single copy. Annotation predicted 20,684 protein-coding genes. The high-quality reference genome reaches an Earth BioGenome standard level of 7.C.Q50.

Multiple paths toward repeated phenotypic evolution in the spiny-leg adaptive radiation (Tetragnatha; Hawai'i).

The repeated evolution of phenotypes provides clear evidence for the role of natural selection in driving evolutionary change. However, the evolutionary origin of repeated phenotypes can be difficult to disentangle as it can arise from a combination of factors such as gene flow, shared ancestral polymorphisms or mutation. Here, we investigate the presence of these evolutionary processes in the Hawaiian spiny-leg Tetragnatha adaptive radiation, which includes four microhabitat-specialists or ecomorphs, with different body pigmentation and size (Green, Large Brown, Maroon, and Small Brown). We investigated the evolutionary history of this radiation using 76 newly generated low-coverage, whole-genome resequenced samples, along with phylogenetic and population genomic tools. Considering the Green ecomorph as the ancestral state, our results suggest that the Green ecomorph likely re-evolved once, the Large Brown and Maroon ecomorphs evolved twice and the Small Brown evolved three times. We found that the evolution of the Maroon and Small Brown ecomorphs likely involved ancestral hybridization events, while the Green and Large Brown ecomorphs likely evolved through novel mutations, despite a high rate of incomplete lineage sorting in the dataset. Our findings demonstrate that the repeated evolution of ecomorphs in the Hawaiian spiny-leg Tetragnatha is influenced by multiple evolutionary processes.

Identifying and addressing methodological incongruence in phylogenomics: A review.

The availability of phylogenetic data has greatly expanded in recent years. As a result, a new era in phylogenetic analysis is dawning-one in which the methods we use to analyse and assess our data are the bottleneck to producing valuable phylogenetic hypotheses, rather than the need to acquire more data. This makes the ability to accurately appraise and evaluate new methods of phylogenetic analysis and phylogenetic artefact identification more important than ever. Incongruence in phylogenetic reconstructions based on different datasets may be due to two major sources: biological and methodological. Biological sources comprise processes like horizontal gene transfer, hybridization and incomplete lineage sorting, while methodological ones contain falsely assigned data or violations of the assumptions of the underlying model. While the former provides interesting insights into the evolutionary history of the investigated groups, the latter should be avoided or minimized as best as possible. However, errors introduced by methodology must first be excluded or minimized to be able to conclude that biological sources are the cause. Fortunately, a variety of useful tools exist to help detect such misassignments and model violations and to apply ameliorating measurements. Still, the number of methods and their theoretical underpinning can be overwhelming and opaque. Here, we present a practical and comprehensive review of recent developments in techniques to detect artefacts arising from model violations and poorly assigned data. The advantages and disadvantages of the different methods to detect such misleading signals in phylogenetic reconstructions are also discussed. As there is no one-size-fits-all solution, this review can serve as a guide in choosing the most appropriate detection methods depending on both the actual dataset and the computational power available to the researcher. Ultimately, this informed selection will have a positive impact on the broader field, allowing us to better understand the evolutionary history of the group of interest.

nRCFV: a new, dataset-size-independent metric to quantify compositional heterogeneity in nucleotide and amino acid datasets.

MOTIVATION: Compositional heterogeneity-when the proportions of nucleotides and amino acids are not broadly similar across the dataset-is a cause of a great number of phylogenetic artefacts. Whilst a variety of methods can identify it post-hoc, few metrics exist to quantify compositional heterogeneity prior to the computationally intensive task of phylogenetic tree reconstruction. Here we assess the efficacy of one such existing, widely used, metric: Relative Composition Frequency Variability (RCFV), using both real and simulated data. RESULTS: Our results show that RCFV can be biased by sequence length, the number of taxa, and the number of possible character states within the dataset. However, we also find that missing data does not appear to have an appreciable effect on RCFV. We discuss the theory behind this, the consequences of this for the future of the usage of the RCFV value and propose a new metric, nRCFV, which accounts for these biases. Alongside this, we present a new software that calculates both RCFV and nRCFV, called nRCFV_Reader. AVAILABILITY AND IMPLEMENTATION: nRCFV has been implemented in RCFV_Reader, available at: https://github.com/JFFleming/RCFV_Reader . Both our simulation and real data are available at Datadryad: https://doi.org/10.5061/dryad.wpzgmsbpn .

Mitochondrial Genome Evolution in Annelida-A Systematic Study on Conservative and Variable Gene Orders and the Factors Influencing its Evolution.

The mitochondrial genomes of Bilateria are relatively conserved in their protein-coding, rRNA, and tRNA gene complement, but the order of these genes can range from very conserved to very variable depending on the taxon. The supposedly conserved gene order of Annelida has been used to support the placement of some taxa within Annelida. Recently, authors have cast doubts on the conserved nature of the annelid gene order. Various factors may influence gene order variability including, among others, increased substitution rates, base composition differences, structure of noncoding regions, parasitism, living in extreme habitats, short generation times, and biomineralization. However, these analyses were neither done systematically nor based on well-established reference trees. Several focused on only a few of these factors and biological factors were usually explored ad-hoc without rigorous testing or correlation analyses. Herein, we investigated the variability and evolution of the annelid gene order and the factors that potentially influenced its evolution, using a comprehensive and systematic approach. The analyses were based on 170 genomes, including 33 previously unrepresented species. Our analyses included 706 different molecular properties, 20 life-history and ecological traits, and a reference tree corresponding to recent improvements concerning the annelid tree. The results showed that the gene order with and without tRNAs is generally conserved. However, individual taxa exhibit higher degrees of variability. None of the analyzed life-history and ecological traits explained the observed variability across mitochondrial gene orders. In contrast, the combination and interaction of the best-predicting factors for substitution rate and base composition explained up to 30% of the observed variability. Accordingly, correlation analyses of different molecular properties of the mitochondrial genomes showed an intricate network of direct and indirect correlations between the different molecular factors. Hence, gene order evolution seems to be driven by molecular evolutionary aspects rather than by life history or ecology. On the other hand, variability of the gene order does not predict if a taxon is difficult to place in molecular phylogenetic reconstructions using sequence data or not. We also discuss the molecular properties of annelid mitochondrial genomes considering canonical views on gene evolution and potential reasons why the canonical views do not always fit to the observed patterns without making some adjustments. [Annelida; compositional biases; ecology; gene order; life history; macroevolution; mitochondrial genomes; substitution rates.].

Three new species of the genus Perinereis (Annelida, Nereididae) from Egyptian coasts.

Despite being one of the most common groups of polychaetes on intertidal shores, the genus Perinereis (Nereididae) is comparatively poorly known taxonomically, with confusion still existing due to the lack of comprehensive systematic studies. The systematics of Perinereis species from the intertidal Egyptian coasts of the Red Sea, Gulf of Suez and Suez Canal have been investigated using morphology and the mitochondrial barcoding marker cytochrome oxidase subunit I (COI). New sequence data was obtained for 102 Perinereis specimens and analysis included all publicly available COI data from other Perinereis species. The COI data indicate that monophyly of the P.nuntia species group is doubtful, as specimens identified in this species group from south-eastern Asia and Australia form a monophyletic group exclusive of the three new species described in this study from the Red Sea region. A morphometric character set (26 characters) was used to identify and characterize each specimen in the study. Three distinct morphospecies belonging to the P.nuntia species group were found, each differentiated by the number and type of paragnaths on pharyngeal areas V and VI, relative sizes of parapodial lobes, type of notochaetae and neurochaetae, and form of the neurochaetal falciger blades. The three morphospecies were well supported by COI data: two of the three new species, Perinereissuezensis sp. nov. and Perinereisfayedensis sp. nov., are closely similar to P.nuntia sensu stricto, while the other, Perinereisdamietta sp. nov., is similar to P.heterodonta. The new species are described and illustrated, and bring the number of species in Perinereis to 97. The new species are compared and contrasted to the closely similar P.heterodonta, P.nuntia and other congeners from the region.

Incomplete lineage sorting and ancient admixture, and speciation without morphological change in ghost-worm cryptic species.

Morphologically similar species, that is cryptic species, may be similar or quasi-similar owing to the deceleration of morphological evolution and stasis. While the factors underlying the deceleration of morphological evolution or stasis in cryptic species remain unknown, decades of research in the field of paleontology on punctuated equilibrium have originated clear hypotheses. Species are expected to remain morphologically identical in scenarios of shared genetic variation, such as hybridization and incomplete lineage sorting, or in scenarios where bottlenecks reduce genetic variation and constrain the evolution of morphology. Here, focusing on three morphologically similar Stygocapitella species, we employ a whole-genome amplification method (WGA) coupled with double-digestion restriction-site associated DNA sequencing (ddRAD) to reconstruct the evolutionary history of the species complex. We explore population structure, use population-level statistics to determine the degree of connectivity between populations and species, and determine the most likely demographic scenarios which generally reject for recent hybridization. We find that the combination of WGA and ddRAD allowed us to obtain genomic-level data from microscopic eukaryotes (∼1 millimetre) opening up opportunities for those working with population genomics and phylogenomics in such taxa. The three species share genetic variance, likely from incomplete lineage sorting and ancient admixture. We speculate that the degree of shared variation might underlie morphological similarity in the Atlantic species complex.

A review of the worldwide distribution of Marenzelleria viridis, with new records for M. viridis, M. neglecta and Marenzelleria sp. (Annelida: Spionidae).

Marenzelleria Mesnil, 1896 is a small group of spionid polychaetes comprising five valid species, all of which appear similar to each other. The identification of worms based on morphological features is often confusing, and thus molecular data have been suggested as providing crucial additional diagnostic characters. Here we summarize and map available records of M. viridis (Verrill, 1873) worldwide, and, based on the analysis of fragment sequences of COI, 16S, 18S, 28S and Histone 3, report this species for the first time from Norway. We also summarize and map the records of Marenzelleria from North America, distinguishing those based on morphology and molecular data. We report new records for Marenzelleria sp. from Baffin Is., Nunavut, Canada, and for M. neglecta Sikorski Bick, 2004 from Washington, USA.

A new species of the Spirobranchus kraussii-complex (Annelida, Serpulidae) from the Persian Gulf and Gulf of Oman.

A recent study (Simon et al. 2019) confirmed that Spirobranchus kraussii is neither a widely distributed tropical species of Indo-Pacific origin nor a Lessepsian migrant to the Mediterranean, but a large complex of species, some of which might be indeed invasive. Thus, a common intertidal gregarious serpulid, previously attributed to S. kraussii in the Persian Gulf and Gulf of Oman, is described herein as Spirobranchus sinuspersicus sp. nov., using a combination of morphological and molecular data. The new species differs from S. kraussii by smaller size, fewer abdominal chaetigers, arrangement of abdominal chaetae and shape of opercular endplate talon. Results of phylogenetic analyses of a dataset combining 18S nucleotide and Cyt-b amino-acid sequences of S. sinuspersicus sp. nov. and Spirobranchus spp. available from GenBank supported monophyly of S. kraussii complex (including S. cariniferus) nested within Spirobranchus and thus, provided molecular support for synonymy of Pomatoleios with Spirobranchus proposed based on morphological criteria. The new species forms a well-supported clade with (S. kraussii (sp. 2 Hawaii + sp. 3 Australia)) clade, which in turn forms a clade with Spirobranchus sp.1 from temperate Japan, while S. cariniferus from New Zealand forms a basal grade. Evidence of substitution saturation of Cyt-b nucleotide sequences suggests that using translated amino-acid sequences to exclude non-informative substitutions should provide a better phylogenetic resolution for the genus Spirobranchus. Further studies are required to determine the invasive status of S. sinuspersicus sp. nov. as well as taxonomic and invasive status of S. cf. kraussii populations from the Mediterranean Sea, Suez Bay, Pakistan, India, Sri Lanka, Philippines, Singapore, and Panama.

Delimitation of cryptic species drastically reduces the geographical ranges of marine interstitial ghost-worms (Stygocapitella; Annelida, Sedentaria).

The recognition of cryptic species concealed in traditionally established species may reveal new biogeographical patterns and alter the understanding of how biodiversity is geographically distributed. This is particularly relevant for marine ecosystems where the incidence of cryptic species is high and where species distribution data are often challenging to collect and interpret. Here, we studied specimens of the 'cosmopolitan' interstitial meiofaunal annelid Stygocapitella subterranea Knöllner, 1934 (Parergodrilidae, Orbiniida), obtaining data from four coastlines in the Northern hemisphere. Using phylogenetic tools and several species-delimitation methods (haplotype networks, GMYC, bPTP, maximum likelihood, posterior probability and morphology) we describe eight new Stygocapitella species. With one exception, all species are present along a single coastline, ultimately challenging the idea that Stygocapitella subterranea has a cosmopolitan distribution. We found evidence for several oceanic transitions having occurred in the past as well as a recent translocation, potentially due to human activity. No diagnostic characters were found, and qualitative and quantitative morphological data do not allow an unequivocal differentiation of the identified cryptic species. This suggests that (i) neither traditional diagnostic features nor quantitative morphology suffice to recognise species boundaries in cryptic species complexes, such as the Stygocapitella species complex; and that (ii) the recognition and description of cryptic species is of seminal importance for biodiversity assessments, biogeography and evolutionary biology.

Deceleration of morphological evolution in a cryptic species complex and its link to paleontological stasis.

Morphological stasis or the absence of morphological change is a well-known phenomenon in the paleontological record, yet it is poorly integrated with neontological evidence. Recent evidence suggests that cryptic species complexes may remain morphologically identical due to morphological stasis. Here, we describe a case of long-term stasis in the Stygocapitella cryptic species complex (Parergodrilidae, Orbiniida, Annelida). Using phylogenetic methods and morphological data, we find that rates of morphological evolution in Stygocapitella are significantly slower than in closely related taxa (Nerillidae, Orbiniidae). Assessment of quantitative and qualitative morphology revealed the presence of four morphotypes with only subtle differences, whereas molecular data supports 10 reproductively isolated clades. Notably, estimates for the time of Stygocapitella species divergence range from ∼275 million years to ∼18 million years, including one case of two morphologically similar species that have diverged about 140 million years ago. These findings provide evidence for morphological deceleration and long-term morphological stasis in Stygocapitella, and that speciation is not necessarily accompanied by morphological changes. The deceleration of morphological divergence in Stygocapitella can be potentially linked to niche conservatism and tracking, coupled with the fluctuating dynamics of the interstitial environment, or genetic constraints due to progenetic evolution. Finally, we conclude that failing to integrate speciation without morphological evolution in paleontology may bias estimates of rates of speciation and morphological evolution.

Convergent evolution of the ladder-like ventral nerve cord in Annelida.

BACKGROUND: A median, segmented, annelid nerve cord has repeatedly been compared to the arthropod and vertebrate nerve cords and became the most used textbook representation of the annelid nervous system. Recent phylogenomic analyses, however, challenge the hypothesis that a subepidermal rope-ladder-like ventral nerve cord (VNC) composed of a paired serial chain of ganglia and somata-free connectives represents either a plesiomorphic or a typical condition in annelids. RESULTS: Using a comparative approach by combining phylogenomic analyses with morphological methods (immunohistochemistry and CLSM, histology and TEM), we compiled a comprehensive dataset to reconstruct the evolution of the annelid VNC. Our phylogenomic analyses generally support previous topologies. However, the so far hard-to-place Apistobranchidae and Psammodrilidae are now incorporated among the basally branching annelids with high support. Based on this topology we reconstruct an intraepidermal VNC as the ancestral state in Annelida. Thus, a subepidermal ladder-like nerve cord clearly represents a derived condition. CONCLUSIONS: Based on the presented data, a ladder-like appearance of the ventral nerve cord evolved repeatedly, and independently of the transition from an intraepidermal to a subepidermal cord during annelid evolution. Our investigations thereby propose an alternative set of neuroanatomical characteristics for the last common ancestor of Annelida or perhaps even Spiralia.

Molecular phylogeny, morphology, and distribution of Polygordius (Polychaeta: Polygordiidae) in the Atlantic and Mediterranean.

Low morphological diversity among interstitial taxa makes it difficult to delimit species and their geographic boundaries based solely on morphology and molecular data often reveal cryptic species. Polygordius (Annelida, Polygordiidae) have low morphological diversity, but are unusual among interstitial species in their comparatively large size due to their elongated form, high fecundity, and potential for long-distance dispersal via a planktotrophic larval stage. Polygordius species collected from 14 localities in the Northwest Atlantic, Mediterranean Sea, and Southwest Atlantic including several of the respective type localities were analysed. This study presents the first phylogeny of the genus Polygordius and combines molecular data, sequences of COI, 16S and ITS1/2 genes, and morphological data for a systematic re-evaluation focusing on Atlantic species, with an emphasis on populations from European waters. Phylogenetic analyses recovered six valid species (P. appendiculatus, P. lacteus, P. neapolitanus, P. triestinus, P. jouinae, and P. eschaturus) and their distinctness is confirmed by haplotype network analyses. Thus, molecular data supported the validity of the previously recognized morpho-species and no new species were present. P. erythrophthalmus and P. villoti are invalid species being synonymous with P. lacteus. Subtle differences in head and pygidial morphology and larval type (endolarva vs. exolarva), were useful characters for discrimination. Yet seemingly significant variation in characters among individuals in some species was not diagnostic (e.g., number of pygidial cirri). Highly similar species based on adult morphology were shown to be sister taxa occurring in allopatry. Present day distribution patterns of species are summarized in light of this study.

Finding Evolutionary Processes Hidden in Cryptic Species.

Cryptic species could represent a substantial fraction of biodiversity. However, inconsistent definitions and taxonomic treatment of cryptic species prevent informed estimates of their contribution to biodiversity and impede our understanding of their evolutionary and ecological significance. We propose a conceptual framework that recognizes cryptic species based on their low levels of phenotypic (morphological) disparity relative to their degree of genetic differentiation and divergence times as compared with non-cryptic species. We discuss how application of a more rigorous definition of cryptic species in taxonomic practice will lead to more accurate estimates of their prevalence in nature, better understanding of their distribution patterns on the tree of life, and increased abilities to resolve the processes underlying their evolution.

Phylogenomics of Lophotrochozoa with Consideration of Systematic Error.

Phylogenomic studies have improved understanding of deep metazoan phylogeny and show promise for resolving incongruences among analyses based on limited numbers of loci. One region of the animal tree that has been especially difficult to resolve, even with phylogenomic approaches, is relationships within Lophotrochozoa (the animal clade that includes molluscs, annelids, and flatworms among others). Lack of resolution in phylogenomic analyses could be due to insufficient phylogenetic signal, limitations in taxon and/or gene sampling, or systematic error. Here, we investigated why lophotrochozoan phylogeny has been such a difficult question to answer by identifying and reducing sources of systematic error. We supplemented existing data with 32 new transcriptomes spanning the diversity of Lophotrochozoa and constructed a new set of Lophotrochozoa-specific core orthologs. Of these, 638 orthologous groups (OGs) passed strict screening for paralogy using a tree-based approach. In order to reduce possible sources of systematic error, we calculated branch-length heterogeneity, evolutionary rate, percent missing data, compositional bias, and saturation for each OG and analyzed increasingly stricter subsets of only the most stringent (best) OGs for these five variables. Principal component analysis of the values for each factor examined for each OG revealed that compositional heterogeneity and average patristic distance contributed most to the variance observed along the first principal component while branch-length heterogeneity and, to a lesser extent, saturation contributed most to the variance observed along the second. Missing data did not strongly contribute to either. Additional sensitivity analyses examined effects of removing taxa with heterogeneous branch lengths, large amounts of missing data, and compositional heterogeneity. Although our analyses do not unambiguously resolve lophotrochozoan phylogeny, we advance the field by reducing the list of viable hypotheses. Moreover, our systematic approach for dissection of phylogenomic data can be applied to explore sources of incongruence and poor support in any phylogenomic data set. [Annelida; Brachiopoda; Bryozoa; Entoprocta; Mollusca; Nemertea; Phoronida; Platyzoa; Polyzoa; Spiralia; Trochozoa.].

Syllidae mitochondrial gene order is unusually variable for Annelida.

Complete mitochondrial genomes of five syllids (Streptosyllis sp., Eusyllis blomstrandi, Myrianida brachycephala, Typosyllis antoni and Typosyllis sp.) have been obtained using Illumina sequencing. Together with two previous studied taxa (Ramisyllis multicaudata and Trypanobia cryptica), the analysed sequences represent most of the main lineages within the family Syllidae (Anoplosyllinae, Eusyllinae, Autolytinae and Syllinae). The genomic features, gene order and phylogenetic relationships are examined. Unusual for annelids, syllid mitochondrial genomes are highly variable in their gene order. Considering genomic features, such as length, skewness, gene content, and codon bias, most similar to the rest of annelids are the genomes of E. blomstrandi and M. brachycephala, while Streptosyllis sp. and the analysed sylline taxa (R. multicaudata, T. cryptica, T. antoni and Typosyllis sp.) are the most dissimilar. Two methionine tRNA's (trnM) have been found in T. antoni and Typosyllis sp. The mt genomes of these latter taxa are the longest with numerous non-coding regions. The 13 protein coding genes, as well as the rRNA's are used to perform phylogenetic analyses that recovered the relationships within the family explored before by previous authors. The gene order in Syllidae shows very different patterns. E. blomstrandi and M. prolifera show a similar pattern to the one found in Pleistoannelida; however this might have changed at least twice within Syllidae: in Streptosyllis sp. and within Syllinae. All analysed Syllinae show different gene orders, thereby illustrating more variability as all other pleistoannelids analysed so far. The information provided herein allows a more accurate reconstruction of the possible evolutionary scenarios in Syllidae.

Evolution of mitochondrial gene order in Annelida.

Annelida is a highly diverse animal group with over 21,000 described species. As part of Lophotrochozoa, the vast majority of annelids are currently classified into two groups: Errantia and Sedentaria, together forming Pleistoannelida. Besides these taxa, Sipuncula, Amphinomidae, Chaetopteridae, Oweniidae and Magelonidae can be found branching at the base of the tree. Comparisons of mitochondrial genomes have been used to investigate phylogenetic relationship within animal taxa. Complete annelid mitochondrial genomes are available for some Sedentaria and Errantia and in most cases exhibit a highly conserved gene order. Only two complete genomes have been published from the basal branching lineages and these are restricted to Sipuncula. We describe the first complete mitochondrial genome sequences for all other basal branching annelid families: Owenia fusiformis (Oweniidae), Magelona mirabilis (Magelonidae), Eurythoe complanata (Amphinomidae), Chaetopterus variopedatus and Phyllochaetopterus sp. (Chaetopteridae). The mitochondrial gene order of all these taxa is substantially different from the pattern found in Pleistoannelida. Additionally, we report the first mitochondrial genomes in Annelida that encode genes on both strands. Our findings demonstrate that the supposedly highly conserved mitochondrial gene order suggested for Annelida is restricted to Pleistoannelida, representing the ground pattern of this group. All investigated basal branching annelid taxa show a completely different arrangement of genes than observed in Pleistoannelida. The gene order of protein coding and ribosomal genes in Magelona mirabilis differs only in two transposition events from a putative lophotrochozoan ground pattern and might be the closest to an ancestral annelid pattern. The mitochondrial genomes of Myzostomida show the conserved pattern of Pleistoannelida, thereby supporting their inclusion in this taxon.

Phylogeny of Syndermata (syn. Rotifera): Mitochondrial gene order verifies epizoic Seisonidea as sister to endoparasitic Acanthocephala within monophyletic Hemirotifera.

A monophyletic origin of endoparasitic thorny-headed worms (Acanthocephala) and wheel-animals (Rotifera) is widely accepted. However, the phylogeny inside the clade, be it called Syndermata or Rotifera, has lacked validation by mitochondrial (mt) data. Herein, we present the first mt genome of the key taxon Seison and report conflicting results of phylogenetic analyses: while mt sequence-based topologies showed monophyletic Lemniscea (Bdelloidea+Acanthocephala), gene order analyses supported monophyly of Pararotatoria (Seisonidea+Acanthocephala) and Hemirotifera (Bdelloidea+Pararotatoria). Sequence-based analyses obviously suffered from substitution saturation, compositional bias, and branch length heterogeneity; however, we observed no compromising effects in gene order analyses. Moreover, gene order-based topologies were robust to changes in coding (genes vs. gene pairs, two-state vs. multistate, aligned vs. non-aligned), tree reconstruction methods, and the treatment of the two monogonont mt genomes. Thus, mt gene order verifies seisonids as sister to acanthocephalans within monophyletic Hemirotifera, while deviating results of sequence-based analyses reflect artificial signal. This conclusion implies that the complex life cycle of extant acanthocephalans evolved from a free-living state, as retained by most monogononts and bdelloids, via an epizoic state with a simple life cycle, as shown by seisonids. Hence, Acanthocephala represent a rare example where ancestral transitional stages have counterparts amongst the closest relatives.

Elucidating the phylogenetic position of Gnathostomulida and first mitochondrial genomes of Gnathostomulida, Gastrotricha and Polycladida (Platyhelminthes).

Gnathostomulida is a taxon of small marine worms, which exclusively inhabit the interstitium. The evolution of Gnathostomulida has been discussed for decades. Originally regarded as primitive animals with affinities to flatworms, the phylogenetic position of Gnathostomulida has been debated. Given the lack of an anus a close relationship to Platyhelminthes has been maintained (i.e., Plathelminthomorpha hypothesis). Alternative hypotheses proposed Gnathostomulida as being close to Gastrotricha due to the presence of a monociliary epidermis (i.e., Monokonta/Neotrichozoa hypothesis) or to Syndermata based on the complicated jaw apparatus (i.e., Gnathifera hypothesis). Molecular analyses using only few genes were inconclusive. Recent phylogenomic studies brought some progress by placing Gnathostomulida as sister to Syndermata, but support for this relationship was low and depended on the analytical strategy. Herein we present the first data of complete or nearly complete mitochondrial genomes for two gnathostomulids (Gnathostomula paradoxa &G. armata), one gastrotrich (Lepidodermella squamata) and one polyclad flatworm (Stylochoplana maculata) to address the uncertain phylogenetic affinity of Gnathostomulida. Our analyses found Gnathostomulida as sister to Syndermata (Gnathifera hypothesis). Thorough sensitivity analyses addressing taxon instability, branch length heterogeneity (also known as long branch attraction) and base composition heterogeneity showed that the position of Gnathostomulida is consistent across the different analyses and, hence, independent of potential misleading biases. Moreover, by ameliorating these different biases nodal support values could be increased to maximum values. Thus, our data support the hypothesis that the different jaw apparatuses of Syndermata and Gnathostomulida are indeed homologous structures as proposed by the Gnathifera hypothesis.