A global phylogeny of the deep-sea gastropod family Scaphandridae (Heterobranchia: Cephalaspidea): Redefinition and generic classification.

We present the most comprehensive phylogeny of a globally distributed deep-sea group of gastropods published to date including over 80% of the recognized diversity of the family Scaphandridae. The definition and taxonomic composition of the Scaphandridae has been hampered by the lack of a sound phylogenetic framework and definition of synapomorphic traits. We used a combination of molecular phylogenetics (Bayesian Inference and Maximum Likelihood) based on five gene markers (cytochrome c oxidase subunit I, 12S rRNA, 16S rRNA, 18S rRNA, and 28S rRNA) and morpho-anatomical characters to redefine the Scaphandridae and its genera. A new classification is proposed with the three genera Nipponoscaphander, Sabatia, and Scaphander. Main differences between genera lie on the shells (shape, parietal callus, spire) and male reproductive system (prostate). The species Hamineobulla kawamurai is reassigned to the closely related family Eoscaphandridae, currently defined mostly based on pleisiomorphic traits. Biogeographically the genus Nipponoscaphander is restricted to the Indo-West Pacific; Sabatia is mostly circumscribed to the Indo-West Pacific, but has one lineage present in the north Atlantic Ocean. Polyphyly across ocean realms prevails in the specious and globally distributed genus Scaphander with multiple speciation events between Indo-Pacific and Atlantic lineages but also with several episodes of cladogenesis within realms. Two rare cases of species with a broad distribution spanning the Indo-West Pacific and Atlantic realms are confirmed (S. meridionalis and S. nobilis).

A molecular phylogeny of Thuridilla Bergh, 1872 sea slugs (Gastropoda, Sacoglossa) reveals a case of flamboyant and cryptic radiation in the marine realm.

The genus Thuridilla Bergh, 1872 comprises mostly tropical sap-sucking sea slugs species with flamboyantly coloured forms. However, the potential for cryptic or pseudocryptic species masked by convergent or polymorphic colour patterns has not been tested using molecular characters. In this study, we sampled 20 of the 23 recognized worldwide species and performed the most comprehensive molecular phylogenetic analysis of the genus to date using a multi-locus approach combining two mitochondrial (cytochrome c oxidase subunit I, 16S rRNA) and two nuclear (Histone H3, 28S rRNA) genes using maximum likelihood, maximum-parsimony and Bayesian criteria. Three molecular species delimitation methods (ABGD, GMYC, bPTP) and the morphology of radular teeth were additionally used to aid in species delimitation. Our analyses supported 35 species within Thuridilla, of which more than one-third (13) are part of a single radiation here named the Thuridilla gracilis (Risbec, 1928) species-complex. This complex includes T. gracilis, T. splendens (Baba, 1949), T. bayeri (Er. Marcus, 1965), and T. ratna (Er. Marcus, 1965), plus nine additional undescribed species. All 13 species are distinguishable by radular characters, external morphology and their DNA. The detection of this radiation led diversity of Thuridilla to be underestimated by about 25% and provides a new comparative system for studying the role of colour patterns in marine diversification.

A molecular phylogeny of the Indo-West Pacific species of Haloa sensu lato gastropods (Cephalaspidea: Haminoeidae): Tethyan vicariance, generic diversity, and ecological specialization.

The cephalaspidean gastropod genus Haminoea has been considered a worldwide radiation with species living in intertidal and shallow areas with algae, seagrass sandy-mud, mangroves, and coral reefs. Recently this monophyletic status was questioned and it was suggested that Haminoea proper only included Atlantic plus eastern Pacific species, whereas the Indo-West Pacific species were a separate radiation belonging to the genus Haloa. In this work we used an extended dataset of Haminoea sensu lato including to our best knowledge representatives of all Indo-West Pacific species, plus a large representation of Atlantic and eastern Pacific species. Bayesian and maximum likelihood molecular phylogenetic analyses using three mitochondrial (COI, 12S rRNA, 16S rRNA) and two nuclear genes (28S rRNA and Histone-3) were employed to study relationships and diversification including also the closely related genus Smaragdinella. Our results support a monophyletic Atlantic + eastern Pacific Haminoea clade and a monophyletic Indo-West Pacific radiation with five genera all defined by unique morphological characters and ecological requirements, namely Haloa proper with tropical and sub-tropical species spread across the Indo-West Pacific, Lamprohaminoea including only colourful species of tropical and sub-tropical affinity, Bakawan gen. nov. with species associated with mangrove habitats across the tropical eastern Indian Ocean and western Pacific, Papawera gen. nov. restricted to temperate waters of Australasia, and Smaragdinella the only cephalaspidean genus inhabiting intertidal hard substrates across sub-tropical and tropical regions of the Indo-West Pacific. This result suggests the role of the closure of the Tethys seaway structuring the phylogeny of worldwide "Haminoea" snails and of ecology driving the phylogenetic structure of the Indo-West Pacific radiation.

Unmasking Antarctic mollusc lineages: novel evidence from philinoid snails (Gastropoda: Cephalaspidea).

Since its introduction, the genus Philine has epitomized numerous mollusc snails with strong morphological convergence. Recently, a molecular analysis including a wide taxon sampling split this group into four non-sister families. Although they are especially diverse in cold and deep waters, no comprehensive studies are available for the Antarctic counterparts. Here, our morpho-anatomical and molecular data suggest major changes in the systematics of the group. From the eight known species, two are synonymized, Antarctophiline amoena with A. alata, and A. gouldi with A. gibba, and two are transferred to the genus Antarctophiline, namely A. apertissima comb.n. and A. falklandica comb.n. Two new species are described, A. easmithi sp.n. and A. amundseni sp.n. from different depths in the eastern Weddell Sea. The elusive P. antarctica from the Ross Sea was found in the Weddell Sea and Waegelea gen.n. is erected to place this species. Both phylogenetic and morphological data support the erection of Antarctophilinidae fam.n. to embrace most of the Philinoidea species described in the Southern Ocean. Only two species of Philinidae are found in Antarctic waters, Spiraphiline bathyalis gen. et sp.n. from bathyal depths in the Weddell Sea and S. kerguelensis comb.n. from the Kerguelen Islands. In light of the new data provided for all described species and the phylogenetic framework proposed herein, we briefly discuss the diversification and biogeographical patterns of Antarctic philinoid snails. Overall, antarctophilinid species seem to have restricted and grossly nonoverlapping distributions suggesting allopatric speciation connected possibly to geographical or bathymetric isolation.

A test of color-based taxonomy in nudibranchs: Molecular phylogeny and species delimitation of the Felimida clenchi (Mollusca: Chromodorididae) species complex.

Traditionally, species identification in nudibranch gastropods relies heavily on body color pattern. The Felimida clenchi species complex, a group of brightly colored Atlantic and Mediterranean species in the family Chromodorididae, has a history of exceptional controversy and discussion among taxonomists. The most widely accepted hypothesis is that the complex includes four species (Felimida clenchi, F. neona, F. binza and F. britoi), each with a characteristic body color pattern. In this study, we investigated the taxonomic value of coloration in the Felimida clenchi complex, using molecular phylogenetics, species-delimitation analyses (ABGD, GMYC, PTP), haplotype-network methods, and the anatomy of the reproductive system. None of our analyses recovered the traditional separation into four species. Our results indicated the existence of three species, a result inconsistent with previous taxonomic hypotheses. We distinguished an undescribed species of Felimida and redefined the concepts of F. clenchi and F. binza, both highly polychromatic species. For the first time, molecular data support the existence of extreme color polymorphism in chromatic nudibranch species, with direct implications for the taxonomy of the group and its diversity. The polychromatism observed in the F. clenchi complex apparently correlates with the regional occurrence of similar color patterns in congeneric species, suggesting different mimicry circles. This may represent a parallel in the marine environment to the mechanisms that play a major role in the diversification of color in terrestrial and fresh-water chromatic groups, such as heliconian butterflies.

A new phylogeny of the Cephalaspidea (Gastropoda: Heterobranchia) based on expanded taxon sampling and gene markers.

The Cephalaspidea is a diverse marine clade of euthyneuran gastropods with many groups still known largely from shells or scant anatomical data. The definition of the group and the relationships between members has been hampered by the difficulty of establishing sound synapomorphies, but the advent of molecular phylogenetics is helping to change significantly this situation. Yet, because of limited taxon sampling and few genetic markers employed in previous studies, many questions about the sister relationships and monophyletic status of several families remained open. In this study 109 species of Cephalaspidea were included covering 100% of traditional family-level diversity (12 families) and 50% of all genera (33 genera). Bayesian and maximum likelihood phylogenetics analyses based on two mitochondrial (COI, 16S rRNA) and two nuclear gene markers (28S rRNA and Histone-3) were used to infer the relationships of Cephalaspidea. The monophyly of the Cephalaspidea was confirmed. The families Cylichnidae, Diaphanidae, Haminoeidae, Philinidae, and Retusidae were found non-monophyletic. This result suggests that the family level taxonomy of the Cephalaspidea warrants a profound revision and several new family and genus names are required to reflect the new phylogenetic hypothesis presented here. We propose a new classification of the Cephalaspidea including five new families (Alacuppidae, Colinatydidae, Colpodaspididae, Mnestiidae, Philinorbidae) and one new genus (Alacuppa). Two family names (Acteocinidae, Laonidae) and two genera (Laona, Philinorbis) are reinstated as valid. An additional lineage with family rank (Philinidae "Clade 4") was unravelled, but no genus and species names are available to reflect the phylogeny and formal description will take place elsewhere.

The family Diaphanidae (Gastropoda: Heterobranchia: Cephalaspidea) in Europe, with a redescription of the enigmatic species Colobocephalus costellatus M. Sars, 1870.

Diaphanidae has traditionally been considered the basal family of the heterobranch gastropod clade Cephalaspidea with 14 species present in Europe ascribed to five genera. The monophyletic status of the family is controversial because of lack of sound synapomorphies. In this paper we present a review of the diversity of Diaphanidae in Europe and give new data on the morphology and anatomy of 50% of the species together with a synopsis of the relevant systematic characters to distinguish between all European species and genera. The rare species Colobocephalus costellatus is redescribed and shown alive for the first time.Further evidence supporting the non-monophyly of Diaphanidae is given; no synapomorphies were found to unite traditional Diaphanidae taxa, but three groups can be recognized: (1) Diaphaninae with genus Diaphana is united by a globose external, thin, and umbilicate shell and radulae with a bilobed rachidian tooth; (2) Toledoniinae, with genera Toledonia and Bogasonia share the presence of a shell with elevated spire and radulae with a unicuspid rachidian tooth; and (3) an unnamed group, with Colobocephalus and Colpodaspis which are the only genera with internal shells, sculptured and globose with short but protruding spires, and radulae lacking rachidian tooth and with smooth hook-shaped lateral teeth.

Diversity and systematics of Haminoeidae gastropods (Heterobranchia: Cephalaspidea) in the tropical West Pacific Ocean: new data on the genera Aliculastrum, Atys, Diniatys and Liloa.

Four genera of Haminoeidae gastropods inhabiting the tropical West Pacific (Aliculastrum, Atys, Diniatys, and Liloa) were studied based on the combined analyses of live animals, external morphology, anatomy and shells aided by scanning electron microscopy and molecular sequencing. Eleven species are described including two new to Science, namely Atys pittmani sp. nov. and Atys ukulele sp. nov.. The putative synapomorphies of the genera Aliculastrum, Diniatys, and Liloa are discussed. High levels of morpho-anatomical variability were found among representatives of the genus Atys, which hampered the recognition of synapomorphies. 

Assessment of mitochondrial genomes for heterobranch gastropod phylogenetics.

BACKGROUND: Heterobranchia is a diverse clade of marine, freshwater, and terrestrial gastropod molluscs. It includes such disparate taxa as nudibranchs, sea hares, bubble snails, pulmonate land snails and slugs, and a number of (mostly small-bodied) poorly known snails and slugs collectively referred to as the "lower heterobranchs". Evolutionary relationships within Heterobranchia have been challenging to resolve and the group has been subject to frequent and significant taxonomic revision. Mitochondrial (mt) genomes can be a useful molecular marker for phylogenetics but, to date, sequences have been available for only a relatively small subset of Heterobranchia. RESULTS: To assess the utility of mitochondrial genomes for resolving evolutionary relationships within this clade, eleven new mt genomes were sequenced including representatives of several groups of "lower heterobranchs". Maximum likelihood analyses of concatenated matrices of the thirteen protein coding genes found weak support for most higher-level relationships even after several taxa with extremely high rates of evolution were excluded. Bayesian inference with the CAT + GTR model resulted in a reconstruction that is much more consistent with the current understanding of heterobranch phylogeny. Notably, this analysis recovered Valvatoidea and Orbitestelloidea in a polytomy with a clade including all other heterobranchs, highlighting these taxa as important to understanding early heterobranch evolution. Also, dramatic gene rearrangements were detected within and between multiple clades. However, a single gene order is conserved across the majority of heterobranch clades. CONCLUSIONS: Analysis of mitochondrial genomes in a Bayesian framework with the site heterogeneous CAT + GTR model resulted in a topology largely consistent with the current understanding of heterobranch phylogeny. However, mitochondrial genomes appear to be too variable to serve as good phylogenetic markers for robustly resolving a number of deeper splits within this clade.

Histological, histochemical, and ultrastructural investigation of the male copulatory apparatus of Haminoea navicula (Gastropoda, Cephalaspidea).

Due to its biological and systematic importance, the morphology and function of the male copulatory apparatus of Haminoea navicula, a Cephalaspidea gastropod mollusk, was investigated by light and electron microscopy. These systems are poorly understood in haminoids, but are often used in the taxonomy of the genus. In H. navicula, the male copulatory apparatus comprises the penis within a penial sheath, a seminal duct and the prostate with two lobes. The penis is a muscular structure with a tip covered by spikes formed by muscular cells. The penial sheath consists of muscular tissue folds lined by an epithelium. Below this epithelium, polysaccharide-secreting cells and pigment cells were observed. A large number of vacuolar cells were found below the ciliated epithelium of the seminal duct. The proximal lobe of the prostate was formed by tubules that could be divided in basal, middle and apical zones, containing cells that secrete polysaccharides and proteins. The tubules of the prostate distal lobe contained a single type of secretory cells with vesicles that were stained by histochemical techniques for detection of polysaccharides and proteins. Ciliated cells were present along the tubules in both lobes of the prostate. This study revealed a complex prostate with five types of secretory cells, which secrete substances that should be involved in the maintenance and eventually also in the maturation of spermatozoa. A comparison with previous publications, shows that the male copulatory apparatus can differ substantially among cephalaspideans, even between H. navicula and non-European species attributed to this genus.

Molecular and morphological systematics of Dolabrifera Gray, 1847 (Mollusca: Gastropoda: Heterobranchia: Aplysiomorpha).

Molecular and morphological data from newly collected specimens and a review of the literature and type material indicate that the widespread tropical sea hare Dolabrifera dolabrifera is a species complex of five genetically distinct taxa. The name Dolabrifera dolabrifera is retained for a widespread species in the Indo-Pacific tropics. Dolabrifera nicaraguana is endemic to the eastern Pacific. Dolabrifera ascifera, D. virens and a new species described herein are restricted to the tropical Atlantic, with partially overlapping ranges in the Caribbean region and St. Helena. The temperate Pacific species Dolabrifera brazieri is also distinct and endemic to temperate southeastern Australia and New Zealand. These species of Dolabrifera constitute highly divergent lineages and most contain unique internal anatomical characteristics, particularly in the male reproductive morphology and shell shape, making them relatively easy to identify upon dissection. However, externally all these species are extremely variable in colour pattern and morphology and are virtually indistinguishable. This is particularly problematic for identification in the Atlantic Ocean where three species co-occur in the Caribbean region.

Ecosystem Alterations and Species Range Shifts: An Atlantic-Mediterranean Cephalaspidean Gastropod in an Inland Egyptian Lake.

The eastern Atlantic and Mediterranean marine Cephalaspidea gastropod Haminoea orbignyana was collected from Lake Qarun (Fayoum, Egypt), a landlocked lake that has undergone a shift from freshwater to estuarine conditions in the past 100 years. Species identity was confirmed by both morphological (anatomical dissection and scanning electron microscopy) and molecular methods (COI gene phylogeny). Observations suggested a robust population of H. orbignyana in the lake with a density of ca. 64 individuals/m2 and ca. 105 egg masses/m2 during surveys conducted in the summer of 2013. The vast majority of snails and egg masses were found under rocks. Observations of egg masses in the lab showed a gradual change from whitish to yellow-green as the eggs matured and the release of veliger larvae alone after about a week. Although adult cephalaspideans readily consumed filamentous red and green algae, and cyanobacteria, laboratory trials showed that they consumed significantly more of the red alga Ceramium sp., than of the green alga Cladophora glomerata, with consumption of Oscillatoria margaritifera being similar to those on the two algae. When grown on these resources for 16 days, H. orbignyana maintained their mass on the rhodophyte and cyanobacterium, but not in starvation controls. No cephalaspideans grew over the course of this experiment. Lake Qarun has been periodically restocked with Mediterranean fishes and prawns since the 1920s to maintain local fisheries, which represents a possible route of colonization for H. orbignyana. Yet, based on literature records, it seems more likely that invasion of the lake by this gastropod species has occurred only within the last 20 years. As human activities redistribute species through direct and indirect means, the structure of the community of this inland lake has become unpredictable and the long-term effects of these recent introductions are unknown.

Speciation in the dark: diversification and biogeography of the deep-sea gastropod genus Scaphander in the Atlantic Ocean.

AIM: The aim of this work was to improve understanding about the mode, geography and tempo of diversification in deep-sea organisms, using a time-calibrated molecular phylogeny of the heterobranch gastropod genus Scaphander. LOCATION: Atlantic and Indo-West Pacific (IWP) oceans. METHODS: Two mitochondrial gene markers (COI and 16S) and one nuclear ribosomal gene (28S) from six Atlantic species of Scaphander, and four IWP species were used to generate a multilocus phylogenetic hypothesis using uncorrelated relaxed-clock Bayesian methods implemented in beast and calibrated with the first occurrence of Scaphander in the fossil record (58.7-55.8 Ma). RESULTS: Two main clades were supported: clade A, with sister relationships between species and subclades from the Atlantic and IWP; and clade B, with two western Atlantic sister species. Our estimates indicate that the two earliest divergences in clade A occurred between the middle Eocene and late Miocene and the most recent speciation occurred within the middle Miocene to Pleistocene. The divergence between the two western Atlantic species in clade B was estimated at late Oligocene-Pliocene. MAIN CONCLUSIONS: The prevailing mode of speciation in Scaphander was allopatric, but one possible case of sympatric speciation was detected between two western Atlantic species. Sister relationships between IWP and Atlantic lineages suggest the occurrence both of vicariance events caused by the closure of the Tethyan Seaway and of dispersal between the two ocean basins, probably around South Africa during episodic disruptions of the deep-sea regional current system caused by glacial-interglacial cycles. Cladogenetic estimates do not support comparatively older diversification of deep-sea faunas, but corroborate the hypothesis of a pulse of diversification centred in the Oligocene and Miocene epochs. Amphi-Atlantic species were found to occur at deeper depths (bathyal-abyssal) and we hypothesize that trans-Atlantic connectivity is maintained by dispersal between neighbouring reproductive populations inhabiting the abyssal sea floor and by dispersal across the shelf and slope of Arctic and sub-Arctic regions.

The opisthobranch gastropods (Mollusca: Heterobranchia) from Venezuela: an annotated and illustrated inventory of species.

The Caribbean waters of Venezuela are composed by a large variety of habitats, with over 2800 km of coastline, islands, and islets. This area is a transitional zone between two main biogeographic provinces, the Caribbean and the Brazilian, separated by the fresh water outflows of the Orinoco and Amazon rivers, and is therefore expected to be an area of high species diversity. However, concerning the study of molluscs, Venezuela is probably the poorest known region in the Caribbean. The best compilation of opisthobranch species known in Venezuela was produced almost a decade ago, mentioning the occurrence of 57 species, plus seven determined only to genus level. In this work, 134 species are reported for Venezuela (71 are illustrated), representing about 40 % of the entire diversity of opisthobranchs known in the Caribbean. Among the species occurring in Venezuela, 49 have here the southern limit of their distribution range and only one the northern limit. Forty-six species are recorded for the first time to the country and one is a new record for the Caribbean Sea, namely Placida cremoniana. In addition, the distribution and ecology of the species are given based in literature and new data.