Cryptic species in tropic sands--interactive 3D anatomy, molecular phylogeny and evolution of meiofaunal Pseudunelidae (Gastropoda, Acochlidia)

Towards realistic estimations of the diversity of marine animals, tiny meiofaunal species usually are underrepresented. Since the biological species concept is hardly applicable on exotic and elusive animals, it is even more important to apply a morphospecies concept on the best level of information possible, using accurate and efficient methodology such as 3D modelling from histological sections. Molecular approaches such as sequence analyses may reveal further, cryptic species. This is the first case study on meiofaunal gastropods to test diversity estimations from traditional taxonomy against results from modern microanatomical methodology and molecular systematics

How to describe a cryptic species? Practical challenges of molecular taxonomy

Background: Molecular methods of species delineation are rapidly developing and widely considered as fast and efficient means to discover species and face the `taxonomic impediment' in times of biodiversity crisis. So far, however, this form of DNA taxonomy frequently remains incomplete, lacking the final step of formal species description, thus enhancing rather than reducing impediments in taxonomy. DNA sequence information contributes valuable diagnostic characters and - at least for cryptic species-could even serve as the backbone of a taxonomic description. To this end solutions for a number of practical problems must be found, including a way in which molecular data can be presented to fulfill the formal requirements every description must meet. Multi-gene barcoding and a combined molecular species delineation approach recently revealed a radiation of at least 12 more or less cryptic species in the marine meiofaunal slug genus Pontohedyle (Acochlidia, Heterobranchia). All identified candidate species are well delimited by a consensus across different methods based on mitochondrial and nuclear markers. Results: The detailed microanatomical redescription of Pontohedyle verrucosa provided in the present paper does not reveal reliable characters for diagnosing even the two major clades identified within the genus on molecular data. We thus characterize three previously valid Pontohedyle species based on four genetic markers (mitochondrial cytochrome c oxidase subunit I, 16S rRNA, nuclear 28S and 18S rRNA) and formally describe nine cryptic new species (P. kepii sp. nov., P. joni sp. nov., P. neridae sp. nov., P. liliae sp. nov., P. wiggi sp. nov., P. wenzli sp. nov., P. peteryalli sp. nov., P. martynovi sp. nov., P. yurihookeri sp. nov.) applying molecular taxonomy, based on diagnostic nucleotides in DNA sequences of the four markers. Due to the minute size of the animals, entire specimens were used for extraction, consequently the holotype is a voucher of extracted DNA ('DNA-type'). We used the Character Attribute Organization System (CAOS) to determine diagnostic nucleotides, explore the dependence on input data and data processing, and aim for maximum traceability in our diagnoses for future research. Challenges, pitfalls and necessary considerations for applied DNA taxonomy are critically evaluated. Conclusions: To describe cryptic species traditional lines of evidence in taxonomy need to be modified. DNA sequence information, for example, could even serve as the backbone of a taxonomic description. The present contribution demonstrates that few adaptations are needed to integrate into traditional taxonomy novel diagnoses based on molecular data. The taxonomic community is encouraged to join the discussion and develop a quality standard for molecular taxonomy, ideally in the form of an automated final step in molecular species delineation procedures

Undersized and underestimated: 3D visualization of the Mediterranean interstitial acochlidian gastropod Pontohedyle milaschewitchii (Kowalevsky, 1901)

AbstractPontohedyle milaschewitchii (Kowalevsky, 1901) is one of the most common mesopsammic opisthobranchs in the Mediterranean and Black Seas and has been considered as a comparably well-described acochlidian species. However, data on its complex internal anatomy were fragmentary and little detailed due to inadequate methodology available, and contradictory between different sources. The present study redescribes all major organ systems of P. milaschewitchii in full detail by three-dimensional reconstruction from serial semithin sections using AMIRA software. The prepharyngeal central nervous system (cns) of P. milaschewitchii is highly concentrated and shows a euthyneurous and epiathroid condition. Contrary to earlier reports, the cerebral and pleural ganglia are not fused. Aggregations of precerebral accessory ganglia can be grouped into three complexes supplied by distinct cerebral nerves. Rhinophoral ganglia with thin, double cerebro-rhinophoral connectives are described for the first time in acochlidians. A Hancock's organ is present in the form of a conspicuous, curved fold in the epidermis posterior to the oral tentacles. Cerebral nervous features and sensory structures are discussed comparatively. Our study confirms P. milaschewitchii as having the male genital opening in an unusual position above the mouth. Homology of the ciliated vas deferens of the gonochoristic and aphallic P. milaschewitchii with that of hermaphroditic acochlidian species with cephalic male genitals is discussed. The radula formula of P. milaschewitchii is 41–54×1-1-1, i.e. the single lateral teeth are broad and, contrary to previous descriptions, undivided. SEM examination of the body wall of entire specimens revealed a special and constant ciliary pattern. Providing a novel additional set of characters for taxonomic and phylogenetic purposes, external SEM examination is suggested as the standard method for describing acochlidian species in the future

On the origin of Acochlidia and other enigmatic euthyneuran gastropods, with implications for the systematics of Heterobranchia

<p>Abstract</p> <p>Background</p> <p>A robust phylogenetic hypothesis of euthyneuran gastropods, as a basis to reconstructing their evolutionary history, is still hindered by several groups of aberrant, more or less worm-like slugs with unclear phylogenetic relationships. As a traditional "order" in the Opisthobranchia, the Acochlidia have a long history of controversial placements, among others influenced by convergent adaptation to the mainly meiofaunal habitats. The present study includes six out of seven acochlidian families in a comprehensive euthyneuran taxon sampling with special focus on minute, aberrant slugs. Since there is no fossil record of tiny, shell-less gastropods, a molecular clock was used to estimate divergence times within Euthyneura.</p> <p>Results</p> <p>Our multi-locus molecular study confirms Acochlidia in a pulmonate relationship, as sister to Eupulmonata. Previous hypotheses of opisthobranch relations, or of a common origin with other meiofaunal Euthyneura, are clearly rejected. The enigmatic amphibious and insectivorous Aitengidae <it>incerta sedis </it>clusters within Acochlidia, as sister to meiofaunal and brackish Pseudunelidae and limnic Acochlidiidae. Euthyneura, Opisthobranchia and Pulmonata as traditionally defined are non-monophyletic. A relaxed molecular clock approach indicates a late Palaeozoic diversification of Euthyneura and a Mesozoic origin of the major euthyneuran diversity, including Acochlidia.</p> <p>Conclusions</p> <p>The present study shows that the inclusion of small, enigmatic groups is necessary to solve deep-level phylogenetic relationships, and underlines that "pulmonate" and "opisthobranch" phylogeny, respectively, cannot be solved independently from each other. Our phylogenetic hypothesis requires reinvestigation of the traditional classification of Euthyneura: morphological synapomorphies of the traditionally defined Pulmonata and Opisthobranchia are evaluated in light of the presented phylogeny, and a redefinition of major groups is proposed. It is demonstrated that the invasion of the meiofaunal habitat has occurred several times independently in various euthyneuran taxa, leading to convergent adaptations previously misinterpreted as synapomorphies. The inclusion of Acochlidia extends the structural and biological diversity in pulmonates, presenting a remarkable flexibility concerning habitat choice.</p

The unique deep sea-land connection: interactive 3D visualization and molecular phylogeny of Bathyhedyle boucheti n. sp (Bathyhedylidae n. fam.)-the first panpulmonate slug from bathyal zones

The deep sea comprises vast unexplored areas and is expected to conceal significant undescribed invertebrate species diversity. Deep waters may act as a refuge for many relictual groups, including elusive and enigmatic higher taxa, but the evolutionary pathways by which colonization of the deep sea has occurred have scarcely been investigated. Sister group relationships between shallow water and deep sea taxa have been documented in several invertebrate groups, but are unknown between amphibious/terrestrial and deep-sea species. Here we describe in full and interactive 3D morphoanatomical detail the new sea slug species Bathyhedyle boucheti n. sp., dredged from the continental slope off Mozambique. Molecular and morphological analyses reveal that it represents a novel heterobranch gastropod lineage which we establish as the new family Bathyhedylidae. The family is robustly supported as sister to the recently discovered panpulmonate acochlidian family Aitengidae, which comprises amphibious species living along the sea shore as well as fully terrestrial species. This is the first marine-epibenthic representative among hedylopsacean Acochlidiida, the first record of an acochlidian from deep waters and the first documented panpulmonate deep-sea slug. Considering a marine mesopsammic ancestor, the external morphological features of Bathyhedyle n. gen. may be interpreted as independent adaptations to a benthic life style in the deep sea, including the large body size, broad foot and propodial tentacles. Alternatively, the common ancestor of Bathyhedylidae and Aitengidae may have been a macroscopic amphibious or even terrestrial species. We hypothesize that oophagy in the common ancestor of Aitengidae and Bathyhedylidae might explain the impressive ecological and evolutionary flexibility in habitat choice in the Acochlidiida

3D-microanatomy of the mesopsammic Pseudovermis salamandrops Marcus, 1953 from Brazil (Nudibranchia, Gastropoda)

Species of the nudibranch Pseudovermidae Thiele, 1931 are rare but conspicuous inhabitants of the marine mesopsammon. Their characteristic vermiform body with reduced\ud cerata and acorn-shaped head lacking appendages is well adapted to life in the interstices of sand grains. Traditionally, species descriptions are based mainly on external morphology\ud and radula characteristics; knowledge on their anatomy is scarce. Here we provide the first microanatomical redescription of a member of Pseudovermidae based on 3D-reconstruction\ud from histological semi-thin section series. The present study on Pseudovermis salamandrops Marcus, 1953 reveals several discrepancies to the original description especially within the complex triaulic genital system (i.e., absence of a connection between vas deferens and kidney, presence of a receptaculum seminis and a large muscular penial sheath gland).We also add microanatomical details such as the presence of gastroesophageal ganglia in the central nervous system, described for the\ud first time in Pseudovermidae. Concluding from the nematocysts found in the cnidosacs of P. salamandrops, this species is a cnidarivore which likely preys on various meiofaunal cnidarians. We show that microanatomical redescriptions of poorly known Pseudovermidae are needed to gather comparative data as a backbone to place these neglected meiofaunal slugs in a phylogeny and trace their evolutionary pathway into the mesopsammon. Traditional characters used for species delineation are insufficient to diagnose Pseudovermis and an integrative approach is needed to reliably address pseudovermid diversity in the future

Potential contribution of surface-dwelling Sargassum algae to deep-sea ecosystems in the southern North Atlantic

Deep-sea ecosystems, limited by their inability to use primary production as a source of carbon, rely on other sources to maintain life. Sedimentation of organic carbon into the deep sea has been previously studied, however, the high biomass of sedimented Sargassum algae discovered during the VEMA Transit expedition in 2014/2015 to the southern North Atlantic, and its potential as a regular carbon input, has been an underestimated phenomenon. To determine the potential for this carbon flux, a literature survey of previous studies that estimated the abundance of surface water Sargassum was conducted. We compared these estimates with quantitative analyses of sedimented Sargassum appearing on photos taken with an autonomous underwater vehicle (AUV) directly above the abyssal sediment during the expedition. Organismal communities associated to Sargassum fluitans from surface waters were investigated and Sargassum samples collected from surface waters and the deep sea were biochemically analyzed (fatty acids, stable isotopes, C:N ratios) to determine degradation potential and the trophic significance within deep-sea communities. The estimated Sargassum biomass (fresh weight) in the deep sea (0.07 − 3.75 g/m2) was several times higher than that estimated from surface waters in the North Atlantic (0.024 – 0.84 g/m2). Biochemical analysis showed degradation of Sargassum occurring during sedimentation or in the deep sea, however, fatty acid and stable isotope analysis did not indicate direct trophic interactions between the algae and benthic organisms. Thus, it is assumed that components of the deep-sea microbial food web form an important link between the macroalgae and larger benthic organisms. Evaluation of the epifauna showed a diverse nano- micro-, meio, and macrofauna on surface Sargassum and maybe transported across the Atlantic, but we had no evidence for a vertical exchange of fauna components. The large-scale sedimentation of Sargassum forms an important trophic link between surface and benthic production and has to be further considered in the future as a regular carbon input to the deep-sea floor in the North Atlantic

Abyssal Solenogastres (Mollusca, Aplacophora) from the Northwest Pacific: Scratching the Surface of Deep-Sea Diversity Using Integrative Taxonomy

Solenogastres (Aplacophora) is a small clade of marine, shell-less worm-molluscs with close to 300 valid species. Their distribution ranges across all oceans, and whereas the vast majority of species has been collected and described from the continental shelf and slope, only few species are known from depths below 4,000 m. Following traditional taxonomy, identification of specimens to species level is complex and time-consuming and requires detailed investigations of morphology and anatomy—often resulting in the exclusion of the clade in biodiversity or biogeographic studies. During the KuramBio expedition (Kuril-Kamchatka Biodiversity Studies) to the abyssal plain of the Northwest Pacific and the Kuril-Kamchatka Trench, 33 solenogaster specimens were sampled from 4,830 m to 5,397 m. Within this study we present an efficient workflow to address solenogaster diversity, even when confronted with a high degree of singletons and minute body sizes, hampering the use of single individuals for multiple morphological and molecular approaches. We combine analyses of external characters and scleritome with molecular barcoding based on a self-designed solenogaster specific set of mitochondrial primers. Overall we were able to delineate at least 19 solenogaster lineages and identify 15 species to family level and beyond. Based on our approach we identified three key lineages from the two regionally most species-rich families (Acanthomeniidae and Pruvotinidae) for deeper taxonomic investigations and describe the novel abyssal species Amboherpia abyssokurilensis sp. nov. (Cavibelonia, Acanthomeniidae) using microanatomical 3D-reconstructions. Our study more than doubles the previous records of solenogaster species from the Northwest Pacific and its marginal seas. Almost all lineages are reported for the first time from the region of the (Northwest) Pacific, vastly expanding distribution ranges of the respective clades. Moreover it doubles the number of Solenogastres collected from abyssal depths on a global scale and underlines the lack of exploratory α-diversity work in the abyssal zone for reliable species estimates in marine biodiversity