The multilevel organismal diversity approach deciphers difficult to distinguish nudibranch species complex.

Species identification is a key procedure for broad-scoped ecological, phylogeographic and evolutionary studies. However, to perform a taxonomic study in the molecular era is a complicated task that has many pitfalls. In the present study we use particular examples of common but difficult to distinguish European species within the genus of Polycera (Nudibranchia, Mollusca) to discuss the general issues of the "cryptic species" problem that has broad biological and interdisciplinary importance and can significantly impede ecological, evolutionary, and other biodiversity-related research. The largest dataset of molecular and morphological information for European nudibranchs ever applied encompasses a wide geographical area and shapes a robust framework in this study. Four species are recognized in the species complex, including a new one. It is shown that a lack of appropriate taxonomic analysis led recently to considerable errors in species identity assessment of this complex. Chromatic polymorphism for each species is mapped in a periodic-like framework and combined with statistical analysis of the diagnostic features that considerably facilitates identification of particular species in the complex for biologists and practitioners. The present study evidently shows that "cryptic" and "non-cryptic" components are present within the same species. Therefore, this species complex is well suited for the exploring and testing of general biological problems. One of the main conclusions of this study is that division of biological diversity into "cryptic" and "non-cryptic" components is counterproductive. We propose that the central biological phenomenon of a species can instead be universally designated as multilevel organismal diversity thereby provide a practical set of methods for its investigation.

Vulnerability of Temperate Mesophotic Ecosystems (TMEs) to environmental impacts: Rapid ecosystem changes at Lough Hyne Marine Nature Reserve, Ireland.

Temperate Mesophotic Ecosystems (TMEs) are stable habitats, usually dominated by slow-growing, long-lived sessile invertebrates and sciaphilous algae. Organisms inhabiting TMEs can form complex three-dimensional structures and support many commercially important species. However, TMEs have been poorly studied, with little known about their vulnerability to environmental impacts. Lough Hyne Marine Nature Reserve (Ireland) supports TMEs in shallower waters (12-40 m) compared with other locations (30-150+ m) as a result of the unusual hydrodynamic conditions. Here, we report changes that have occurred on the sponge-dominated cliffs at Lough Hyne between 1990 and 2019, providing insights into TME long-term stability and vulnerability to environmental impacts. Our main finding was a marked decline in most three-dimensional sponges at the internal sites of the lough. This was likely the result of one or more mass mortality events that occurred between 2010 and 2015. We also found an increase in ascidians, which might have been more tolerant and benefited from the space freed by the sponge mortality. Finally, in the most recent surveys, we found a high abundance of sponge recruits, indicating that a natural recovery may be underway. The possible factors involved in these community changes include eutrophication, increased temperature, and a toxic event due to an anomaly in the oxycline breakdown. However, the absence of comprehensive monitoring of biotic and abiotic variables makes it impossible to identify the cause with certainty. Our Lough Hyne example shows the potential vulnerability of TMEs to short-term disturbance events, highlighting the importance of monitoring these habitats globally to ensure they are appropriately conserved.

Multiple paedomorphic lineages of soft-substrate burrowing invertebrates: parallels in the origin of Xenocratena and Xenoturbella.

Paedomorphosis is an important evolutionary force. It has previously been suggested that a soft-substrate sediment-dwelling (infaunal) environment facilitates paedomorphic evolution in marine invertebrates. However, until recently this proposal was never rigorously tested with robust phylogeny and broad taxon selection. Here, for the first time, we present a molecular phylogeny for a majority of the 21 families of one of the largest nudibranch subgroups (Aeolidacea) and show that the externally highly simplified vermiform nudibranch family, Pseudovermidae, with clearly defined paedomorphic traits and inhabiting a soft-substrata environment, is a sister group to the complex nudibranch family, Cumanotidae. We also report the rediscovery of one of the most enigmatic nudibranchs-Xenocratena suecica-on the Swedish and Norwegian coasts 70 years after it was first found. Xenocratena was described from the same location and environment in the Swedish Gullmar fjord as one of the most enigmatic vermiform organisms, Xenoturbella bocki, which represents either an original simple bilaterian body plan or secondary simplification of a more complex organisation. Our results show that Xenocratena suecica reveals an onset of parallel paedomorphic evolution so we have proposed the new family, Xenocratenidae fam. n., to accommodate the molecular and morphological disparities we discovered. The paedomorphic origin of another aeolidacean family, Embletoniidae, is also demonstrated for the first time. Thus, by presenting three independent lineages from non-closely related aeolidacean families, Xenocratenidae fam. n., Cumanotidae and Embletoniidae, we confirm with phylogenetic data that a soft-substrata burrowing-related environment strongly favours paedomorphic evolution. We suggest criteria to distinguish ancestral and derived characters in the context of modifications of ontogenetic cycles. Applying an evolutionary model of the soft substrate-driven multiple paedomorphic origin of several families of nudibranch molluscs we propose that it is plausible to extend this model to other marine invertebrates and suggest that the ancestral organisation of the enigmatic metazoan, Xenoturbella, might correspond to the larval part of a complex ancestral bilaterian ontogenetic cycle with sedentary/semi-sedentary adult stages and planula-like larval stages.

Multilevel fine-scale diversity challenges the 'cryptic species' concept.

'Cryptic' species are an emerging biological problem that is broadly discussed in the present study. Recently, a cryptic species definition was suggested for those species which manifest low morphological, but considerable genetic, disparity. As a case study we present unique material from a charismatic group of nudibranch molluscs of the genus Trinchesia from European waters to reveal three new species and demonstrate that they show a dual nature: on one hand, they can be considered a 'cryptic' species complex due to their overall similarity, but on the other hand, stable morphological differences as well as molecular differences are demonstrated for every species in that complex. Thus, this species complex can equally be named 'cryptic', 'pseudocryptic' or 'non-cryptic'. We also present evidence for an extremely rapid speciation rate in this species complex and link the species problem with epigenetics. Available metazoan-wide data, which are broadly discussed in the present study, show the unsuitability of a 'cryptic' species concept because the degree of crypticity represents a continuum when a finer multilevel morphological and molecular scale is applied to uncover more narrowly defined species making the 'cryptic' addition to 'species' redundant. Morphological and molecular methods should be applied in concordance to form a fine-scale multilevel taxonomic framework, and not necessarily implying only an a posteriori transformation of exclusively molecular-based 'cryptic' species into morphologically-defined 'pseudocryptic' ones. Implications of the present study have importance for many fields, including conservation biology and fine-scale biodiversity assessments.

The formerly enigmatic Unidentiidae in the limelight again: a new species of the genus Unidentia from Thailand (Gastropoda: Nudibranchia).

An integrative molecular and morphological study is presented for the family Unidentiidae. Molecular phylogenetic analyses were conducted with the inclusion of all previous and newly obtained molecular data for the family Unidentiidae Millen Hermosillo 2012. A new species of the genus Unidentia Millen Hermosillo 2012, U. aliciae sp. nov., is described from Thailand as part of an inventory of sea slugs at Koh Tao. All up-to-date available morphological data for the species of the genus Unidentia is for the first time summarized. Morphological differences among the different species of Unidentia are clarified showing that every species has its own distinguishable morphological traits. According to the new molecular and morphological data, the family Unidentiidae is re-confirmed as a well-supported taxon of the aeolidacean nudibranchs. The taxonomy and phylogeny of the Aeolidacea in the light of the family Unidentiidae is briefly discussed and necessity of a fine-scale and narrowly-defined taxa approach instead of a ''superlumping'' one is highlighted.

The extraordinary genus Myja is not a tergipedid, but related to the Facelinidae s. str. with the addition of two new species from Japan (Mollusca, Nudibranchia).

Morphological and molecular data are presented for the first time in an integrative way for the genus Myja Bergh, 1896. In accordance with the new molecular phylogenies, the traditional Facelinidae is paraphyletic. Herein is presented the phylogenetic placement of true Facelinidae s. str., including the first molecular data for F.auriculata (Müller, 1776), type species of the genus Facelina Alder & Hancock, 1855. The taxonomic history of F.auriculata is reviewed. The genus Myja is related to the clade Facelinidae s. str., but shows disparate morphological traits. Two new species of the genus Myja, M.karin sp. n., and M.hyotan sp. n., are described from the Pacific waters of Japan (middle Honshu), and M.cf.longicornis Bergh, 1896 is investigated from Thailand. According to molecular analysis and review of available morphological information, the genus Myja contains more hidden diversity. The family-level relationship within aeolidacean nudibranchs with emphasis on the family Facelinidae is outlined. The problem of the relationship between Facelinidae Bergh, 1889 and Glaucidae Gray, 1827 is discussed. The family Glaucidae has precedence over Facelinidae and is phylogenetically related to the core group of Facelinidae s. str., but has a profoundly modified aberrant external morphology, thus making a purely molecular-based approach to the taxonomy an unsatisfactory solution. To accommodate recently discovered hidden diversity within glaucids, the genus Glaucilla Bergh, 1861 is restored. The family Facelinidae s. str. is separate from, and not closely related to, a clade containing the genera Dondice Marcus, 1958, Godiva MacNae 1954, Hermissenda Bergh, 1879, and Phyllodesmium Ehrenberg, 1831 (= Myrrhine Bergh, 1905). The oldest valid available name for the separate ex-facelinid paraphyletic clade that contains several facelinid genera is Myrrhinidae Bergh, 1905, and resurrection of this family name under provision of the ICZN article 40.1 can preliminarily solve the problem of paraphyly of the traditional Facelinidae. "Facelinidae" s. l. needs to be further divided into several separate families, pending further study.

The genus Zelentia is an amphi-boreal taxon expanded to include three new species from the North Pacific and Atlantic oceans (Gastropoda: Nudibranchia: Trinchesiidae).

The taxonomy of Zelentia Korshunova, Martynov Picton, 2017, a genus of aeolidacean nudibranchs recently separated from Trinchesia Ihering, 1879, is reviewed. Using previous and novel data, including the defining morphological characters of a supplementary gland inserted into the penis, which is also armed with stylet, it is demonstrated that the genus Zelentia is a well-established amphi-boreal taxon. A phylogenetic tree and haplotype network for species of the genus Zelentia are provided. A new species from the Northeastern Pacific, Zelentia willowsi sp. nov. and a second Northeastern Pacific species, Zelentia nepunicea sp. nov., previously thought to be "Cuthona" pustulata (Alder Hancock, 1854), are described using morphological and molecular data. A new species from the North Atlantic and sub-Arctic White Sea, Zelentia roginskae sp. nov., is also described using morphological and molecular data. The Northeastern Pacific Zelentia nepunicea sp. nov. and"Cuthona" punicea Millen, 1986, which externally share a similar reddish colouration, are shown to belong to two different families since "Cuthona" punicea possesses a supplementary gland inserted into the vas deferens and an unarmed penis, characters of the genus Cuthonella Bergh, 1884, belonging to the Cuthonellidae family. Despite belonging to different families and having significant internal differences, externally Cuthonella punicea and Zelentia nepunicea sp. nov. appear similar, thus the new sympatric species of Zelentia is given the name Z. nepunicea sp. nov. which means non punicea. To facilitate identification and avoid further confusion we provide a comparative table which encompasses diagnostic morphological data for Northeastern Pacific species of the families Cuthonidae, Cuthonellidae and Trinchesiidae which are externally similar to the described new taxa.

First true brackish-water nudibranch mollusc provides new insights for phylogeny and biogeography and reveals paedomorphosis-driven evolution.

A unique example of brackish water fjord-related diversification of a new nudibranch genus and species Bohuslania matsmichaeli gen. n., sp. n. is presented. There are only few previously known brackish-water opisthobranchs and B. matsmichaeli gen. n., sp. n. is the first ever described brackish-water nudibranch with such an extremely limited known geographical range and apparently strict adherence to salinity levels lower than 20 per mille. Up to date the new taxon has been found only in a very restricted area in the Idefjord, bordering Sweden and Norway, but not in any other apparently suitable localities along the Swedish and Norwegian coasts. We also show in this study for the first time the molecular phylogenetic sister relationship between the newly discovered genus Bohuslania and the genus Cuthona. This supports the validity of the family Cuthonidae, which was re-established recently. Furthermore, it contributes to the understanding of the evolutionary patterns and classification of the whole group Nudibranchia. Molecular and morphological data indicate that brackish water speciation was triggered by paedomorphic evolution among aeolidacean nudibranchs at least two times independently. Thus, the present discovery of this new nudibranch genus contributes to several biological fields, including integration of molecular and morphological data as well as phylogenetic and biogeographical patterns.

Polyphyly of the traditional family Flabellinidae affects a major group of Nudibranchia: aeolidacean taxonomic reassessment with descriptions of several new families, genera, and species (Mollusca, Gastropoda).

The Flabellinidae, a heterogeneous assembly of supposedly plesiomorphic to very derived sea slug groups, have not yet been addressed by integrative studies. Here novel material of rarely seen Arctic taxa as well as North Atlantic, North and South Pacific, and tropical Indo-West Pacific flabellinid species is investigated morpho-anatomically and with multi-locus markers (partial COI, 16S rDNA, 28S rDNA and H3) which were generated and analysed in a comprehensive aeolid taxon sampling. It was found that the current family Flabellinidae is polyphyletic and its phylogeny and taxonomic patterns cannot be understood without considering members from all the Aeolidacean families and, based on a robust phylogenetic hypothesis, morpho-anatomical evolution of aeolids is more complex than suspected in earlier works and requires reclassification of the taxon. Morphological diversity of Flabellinidae is corroborated by molecular divergence rates and supports establishing three new families (Apataidae fam. n., Flabellinopsidae fam. n., Samlidae fam. n.), 16 new genera, 13 new species, and two new subspecies among the former Flabellinidae. Two families, namely Coryphellidae and Paracoryphellidae, are restored and traditional Flabellinidae is considerably restricted. The distinctness of the recently described family Unidentiidae is confirmed by both morphological and molecular data. Several species complexes among all ex-"Flabellinidae" lineages are recognised using both morphological and molecular data. The present study shows that Facelinidae and Aeolidiidae, together with traditional "Tergipedidae", deeply divide traditional "Flabellinidae." Diagnoses for all aeolidacean families are therefore provided and additionally two new non-flabellinid families (Abronicidae fam. n. and Murmaniidae fam. n.) within traditional tergipedids are established to accommodate molecular and morphological disparity. To address relationships and disparity, we propose a new family system for aeolids. Here the aeolidacean species are classified into at least 102 genera and 24 families. Operational rules for integration of morphological and molecular data for taxonomy are suggested.

Corrigenda: Polyphyly of the traditional family Flabellinidae affects a major group of Nudibranchia: aeolidacean taxonomic reassessment with descriptions of several new families, genera, and species (Mollusca, Gastropoda). https://doi.org/10.3897/zookeys.717.21885.

[This corrects the article DOI: 10.3897/zookeys.717.21885.].

Diaphorodoris alba Portmann & Sandmeier, 1960 is a valid species: molecular and morphological comparison with D. luteocincta (M. Sars, 1870) (Gastropoda: Nudibranchia).

The nudibranch Diaphorodoris luteocincta (M. Sars, 1870) shows two colour morphotypes defined as D. luteocincta var. alba and D. luteocincta var. reticulata, which are easy to identify and which share an overlapping distribution in the Mediterranean Sea and the North-Eastern Atlantic Ocean. Their systematics has long been discussed by several authors until recently when a molecular study proposed the two varieties as intraspecific colour variability occurring within D. luteocincta species. In order to solve their ranking status, we have carried out a morphological study on anatomical characters and molecular analyses on the mitochondrial markers (COI and 16S rDNA) and the nuclear H3 gene. Results proved the usefulness of the integrative taxonomy approach in assessing species delimitation; in fact Diaphorodoris alba stat. nov. and D. luteocincta were revealed to be two different species. D. luteocincta var. reticulata is confirmed as synonym of D. luteocincta s.str. A hypothesis on phylogenetic relationship among most of the currently recognised species of the genus Diaphorodoris Iredale & O'Donoghue, 1923 is also here presented.

Molecular phylogenies support homoplasy of multiple morphological characters used in the taxonomy of Heteroscleromorpha (Porifera: Demospongiae).

Sponge classification has long been based mainly on morphocladistic analyses but is now being greatly challenged by more than 12 years of accumulated analyses of molecular data analyses. The current study used phylogenetic hypotheses based on sequence data from 18S rRNA, 28S rRNA, and the CO1 barcoding fragment, combined with morphology to justify the resurrection of the order Axinellida Lévi, 1953. Axinellida occupies a key position in different morphologically derived topologies. The abandonment of Axinellida and the establishment of Halichondrida Vosmaer, 1887 sensu lato to contain Halichondriidae Gray, 1867, Axinellidae Carter, 1875, Bubaridae Topsent, 1894, Heteroxyidae Dendy, 1905, and a new family Dictyonellidae van Soest et al., 1990 was based on the conclusion that an axially condensed skeleton evolved independently in separate lineages in preference to the less parsimonious assumption that asters (star-shaped spicules), acanthostyles (club-shaped spicules with spines), and sigmata (C-shaped spicules) each evolved more than once. Our new molecular trees are congruent and contrast with the earlier, morphologically based, trees. The results show that axially condensed skeletons, asters, acanthostyles, and sigmata are all homoplasious characters. The unrecognized homoplasious nature of these characters explains much of the incongruence between molecular-based and morphology-based phylogenies. We use the molecular trees presented here as a basis for re-interpreting the morphological characters within Heteroscleromorpha. The implications for the classification of Heteroscleromorpha are discussed and a new order Biemnida ord. nov. is erected.

Reconstruction of family-level phylogenetic relationships within Demospongiae (Porifera) using nuclear encoded housekeeping genes.

BACKGROUND: Demosponges are challenging for phylogenetic systematics because of their plastic and relatively simple morphologies and many deep divergences between major clades. To improve understanding of the phylogenetic relationships within Demospongiae, we sequenced and analyzed seven nuclear housekeeping genes involved in a variety of cellular functions from a diverse group of sponges. METHODOLOGY/PRINCIPAL FINDINGS: We generated data from each of the four sponge classes (i.e., Calcarea, Demospongiae, Hexactinellida, and Homoscleromorpha), but focused on family-level relationships within demosponges. With data for 21 newly sampled families, our Maximum Likelihood and Bayesian-based approaches recovered previously phylogenetically defined taxa: Keratosa(p), Myxospongiae(p), Spongillida(p), Haploscleromorpha(p) (the marine haplosclerids) and Democlavia(p). We found conflicting results concerning the relationships of Keratosa(p) and Myxospongiae(p) to the remaining demosponges, but our results strongly supported a clade of Haploscleromorpha(p)+Spongillida(p)+Democlavia(p). In contrast to hypotheses based on mitochondrial genome and ribosomal data, nuclear housekeeping gene data suggested that freshwater sponges (Spongillida(p)) are sister to Haploscleromorpha(p) rather than part of Democlavia(p). Within Keratosa(p), we found equivocal results as to the monophyly of Dictyoceratida. Within Myxospongiae(p), Chondrosida and Verongida were monophyletic. A well-supported clade within Democlavia(p), Tetractinellida(p), composed of all sampled members of Astrophorina and Spirophorina (including the only lithistid in our analysis), was consistently revealed as the sister group to all other members of Democlavia(p). Within Tetractinellida(p), we did not recover monophyletic Astrophorina or Spirophorina. Our results also reaffirmed the monophyly of order Poecilosclerida (excluding Desmacellidae and Raspailiidae), and polyphyly of Hadromerida and Halichondrida. CONCLUSIONS/SIGNIFICANCE: These results, using an independent nuclear gene set, confirmed many hypotheses based on ribosomal and/or mitochondrial genes, and they also identified clades with low statistical support or clades that conflicted with traditional morphological classification. Our results will serve as a basis for future exploration of these outstanding questions using more taxon- and gene-rich datasets.

Effects of two decades of rising sea surface temperatures on sublittoral macrobenthos communities in Northern Ireland, UK.

We examined whether two decades of rising sea surface temperatures have resulted in significant changes in the benthic community and frequency of occurrence of Northern and Southern species in three areas of Northern Ireland, using visual census data collected by SCUBA surveys undertaken during two periods: pre-1986 and post-2006. We found little evidence to suggest that rising sea surface temperatures have contributed to the changes in benthic assemblage structure between the pre-1986 and post-2006 surveys. However, there were slight but not significant declines in extreme Northern species at Rathlin Island, and increases in the mean number and frequency of occurrence of extreme Southern species in all three areas. There were also substantial declines in the spatial presence of 7 extreme Northern species and notable increases in distribution of 19 extreme Southern species. In contrast, there were no clear trends in the intermediate to Northern and intermediate to Southern species. These results suggest that rising sea surface temperatures have had significant impacts on the occurrence of rarer marine invertebrate species at the edges of their biogeographic range however the trends differed between areas in Northern Ireland.

Evidence of bacteriophage-mediated horizontal transfer of bacterial 16S rRNA genes in the viral metagenome of the marine sponge Hymeniacidon perlevis.

Marine sponges have never been directly examined with respect to the presence of viruses or their potential involvement in horizontal gene transfer. Here we demonstrate for the first time, to our knowledge, the presence of viruses in the marine sponge Hymeniacidon perlevis. Moreover, bacterial 16S rDNA was detected in DNA isolated from these viruses, indicating that phage-derived transduction appears to occur in H. perlevis. Phylogenetic analysis revealed that bacterial 16S rDNA isolated from sponge-derived viral and total DNA differed significantly, indicating that not all species are equally involved in transduction.

Phylogenetic relationships among NE Atlantic Plocamionida Topsent (1927) (Porifera, Poecilosclerida): under-estimated diversity in reef ecosystems.

BACKGROUND: Small and cryptic sponges associated with cold-water coral reefs are particularly numerous and challenging to identify, but their ecological and biochemical importance is likely to compete with megabenthic specimens. METHODOLOGY/PRINCIPAL FINDINGS: Here we use a combination of the standard M1M6 and I3M11 partitions of the COI fragment, partial rDNA 28S sequences and morphology to delineate small encrusting Plocamionida species. In total, 46 specimens were retrieved from seven shallow to deep-water coral locations, crossing 3,000 km along the European margins. Our work provides evidence that the Plocamionida ambigua f. tylotata and f. grandichelata can be considered valid species, whereas Plocamionida ambigua f. tornata corresponds to the species P. ambigua. Within the monophyletic group of Plocamionida, P. microcionides is shown as really divergent from the other taxa, and four putative new Plocamionida species are suggested. CONCLUSIONS/SIGNIFICANCE: This study shows that the use of molecular and morphological information in an integrative approach is a powerful tool for the identification of sponge species, and suggests that an under-estimated biodiversity of sponges occurs in cold-water coral reefs.