Heterobranchia is a subclass of Gastropoda for which phylogenetic analyses based on mitochondrial gene sequences have not been entirely successful. Here, we report a complete mitochondrial genome of an ellobiid snail, Marinula pepita (Euthyneura: Eupulmonata: Ellobioidea), and its wholesale gene rearrangements from the presumed ancestral order for Heterobranchia. Our phylogenetic reconstruction based on this new genome and the available mitogenomic sequences recovered two sister clades in the superfamily Ellobioidea. Six genera with the ancestral gene order and short branches formed one of the clades (Trimusculus, Carychium, Ovatella, Ellobium, Leucophytia and Auriculastra). Four ellobiid genera with rearranged gene orders and long branches formed the other clade (Marinula, Pedipes, Melampus and Myosotella), regardless of their subfamilial assignments, thereby strongly questioning this topology [long branch attraction (LBA)]. A separate phylogenetic tree inferred from near-complete sequences of the nuclear 18S and 28S rRNA genes, on the other hand, was largely consistent with the conventional, morphology-based classification although suggesting the need of a new subfamily for Marinula. Mitochondrial sequences can be useful for inferring shallow divergences among certain ellobioid genera and species, provided that particular attention is paid to avoid LBA artefacts.

Recent studies have revealed significant hidden diversity and a high incidence of cryptic speciation in Antarctic marine gastropods. Originally, philinoid cephalaspideans in the Southern Ocean were classified within the genus Philine . However, molecular and morphological studies have shown that three genera encompass all known diversity instead. These are Antarctophiline , Waegelea and Spiraphiline , the first two belonging to the recently erected family Antarctophilinidae. In this study, 55 specimens were collected from the South Shetland Islands, across the South Atlantic Antarctic Ridge to Bouvet Island, and from the South Sandwich Islands and Bransfield Strait, between 134 and 4548 m depth. We conducted morpho-anatomical and phylogenetic analyses to describe two new Antarctophiline species: Antarctophiline abyssalis sp. nov. and Antarctophiline malaquiasi sp. nov. Molecular results support the validity of the two distinct species, consistent with observed morpho-anatomical differences in the digestive system (i.e. the shape of gizzard plates and salivary glands), shell shape and other external characters. Additionally, we evaluate the morphological affinities of the most common Antarctic species, Antarctophiline alata , throughout its distribution range. Overall biogeographical distributions are discussed in a systematic context. Our study is yet another example of how Antarctica keeps revealing itself as a cornerstone of gastropod diversity.

The repeated colonisation of non-marine environments in panpulmonate molluscs represents a major evolutionary transition, yet many lineages remain poorly understood. Among gastropods, Acochlidimorpha stands out for its ecological and morphological diversity, originating in marine interstitial habitats before independently invading freshwater and terrestrial ecosystems. Here, we present the most complete phylogeny of Acochlidimorpha to date based on a global taxon sampling collected over several decades. We integrate ultra-conserved element (UCE) data from 58 museum-preserved specimens-comprising over 1,700 nuclear loci-with all publicly available Sanger-sequencing markers, totalling more than 150 specimens. This pioneering approach sets a new benchmark for phylogenomic studies in minute molluscan lineages. Our results resolve long-standing systematic ambiguities, reinstating Pontohedylidae stat. rest. and Strubelliidae stat. rest. and establishing Helicohedylidae fam. nov. Within Acochlidioidea, Strubelliidae is recovered as the sister to Pseudunelidae + (Tantulidae + Acochlidiidae). At the species level, we clarify the position of key taxa, including Asperspina loricata and two unidentified species of Hedylopsis. Ancestral habitat reconstruction analyses reveal at least four independent transitions to freshwater and terrestrial environments, highlighting the anatomical innovations facilitating these shifts, and further reinforcing the Indo-West Pacific as the centre of origin and diversification of most extant acochlids. Altogether, our study provides a robust evolutionary framework for Acochlidimorpha, offering new insights into the processes underlying terrestrialisation, morphological evolution, and ecological diversification in a lineage characterised by both extreme miniaturisation and evolutionary dynamism. Zoobank: urn:lsid:zoobank.org:pub:264EB1DD-A0B1-4D30-94B3-8AEA2D41C34B.

Recent molecular phylogenetic studies have tested the monophyly of Chromodorididae nudibranchs. Although polyphyletic, Felimida was revalidated to include species from the Eastern Pacific, Atlantic, and Mediterranean Sea formerly assigned to Chromodoris and Glossodoris. One clade within Felimida includes Felimida purpurea and other similar Atlantic species. In this study, we employ an integrative approach combining molecular phylogenetics, species delimitation analyses (ASAP, ABGD, GMYC, PTP), and morphological analysis (1) to investigate the systematics of the Felimida purpurea clade and (2) to assess the wide range of color morphs in F. paulomarcioi and its relationship to F. grahami. Our analyses confirm that the F. purpurea clade constitutes a distinct new genus, Rudmania gen. nov., characterized by white to purple species, the absence of rachidian teeth , and a thin, very long, and coiled vagina. All color morphs of F. paulomarcioi and F. grahami represent a single species, Rudmania grahami comb. nov., a variable species distributed throughout the tropical western Atlantic. The new genus includes Rudmania fentoni comb. nov. from Florida, Rudmania atlantica comb. nov. from Ascension Island, Rudmania purpurea comb. nov. and Rudmania krohni comb. nov. from the Eastern Atlantic and Mediterranean Sea, and Rudmania kpone comb. nov. from Ghana. A careful analysis of morphological traits proved effective in identifying diagnostic characters of a Chromodorididae clade revealed by molecular data.

The genus Doto Oken, 1815 is, taxonomically, one of the most complex genera of nudibranchs due to the cryptic nature of its species, their small body size, and the homogeneity among their internal and external features. Here, an extensive molecular analysis of Mediterranean, Northeastern Atlantic, and South American specimens sheds light on the species-level taxonomy of the group. Our multilocus analyses include 171 specimens, 59 of which are newly sequenced, corresponding to 20 species. Ten species are included in a molecular phylogeny for the first time, two being new species. Here, we provide detailed morphological and ecological descriptions of three Atlantic species, 11 Mediterranean species, and one from the Pacific, complemented by live photographs evidencing their chromatic variation. The phylogenetic and species delimitation analyses suggest that coloration and external morphology are often unreliable for differentiating among species. Molecular evidence corroborates and expands the geographical distribution of Doto species, some of which have never been included in molecular studies. The Mediterranean species currently recognized as D. coronata (Gmelin, 1791) and D. dunnei Lemche, 1976 correspond to D. millbayana Lemche, 1976. Consequently, we suggest the junior synonymy with D. dunnei syn. nov. Nevertheless, we found evidence for a restricted distribution of D. coronata in the western Mediterranean, coexisting in sympatry with D. cavernicola sp. nov. An additional new species from the Chilean Patagonia is also described as D. vrenifossorum sp. nov. Considering the present phylogenetic scenario, there is a highlighted need for further new morphological and genetic evidence, expanding the number of taxa and species to further unravel the taxonomy of Doto.

Recent molecular phylogenies have shown the polyphyly of widespread genera within the Chromodorididae. In this context, the genus Felimare was resurrected to include Eastern Pacific, Atlantic, and Mediterranean species previously included in Hypselodoris, plus Mexichromis porterae (Cockerell, 1901) and Mexichromis kempfi (Ev. Marcus, 1971). The somewhat controversial genus Felimare currently includes species with different external, radular, and jaw shapes as well as different reproductive morphologies, resulting in the absence of a clear diagnosis for the genus. Here, we advance the systematics of Chromodorididae with emphasis on a group of small blue species of Felimare from the Eastern Pacific and Atlantic Ocean. Based on consistent morphological and molecular characteristics we describe a new genus, Neptunazurea gen. nov., that includes small chromodorids with mole-paw shaped jaw rodlets, a small radula compared to the body length, and a short, undifferentiated prostate. A taxonomic review of Neptunazurea kempfi (Ev. Marcus, 1971) comb. nov. is presented. Comprehensive morphological studies are crucial for the study of chromodoridids, enabling the thorough evaluation of diagnostic traits and the effective integration of morphological data with molecular information.

With the advances in high-throughput sequencing and bioinformatic pipelines, mitochondrial genomes have become increasingly popular for phylogenetic analyses across different clades of invertebrates. Despite the vast rise in available mitogenomic datasets of molluscs, one class of aplacophoran molluscs – Solenogastres (or Neomeniomorpha) – is still neglected. Here, we present six new mitochondrial genomes from five families of Solenogastres (Amphimeniidae, Gymnomeniidae, Proneomeniidae, Pruvotinidae, Simrothiellidae), including the first complete mitogenomes, thereby now representing three of the four traditional orders. Solenogaster mitogenomes are variable in size (ranging from approximately 15,000 bp to over 17,000 bp). The gene order of the 13 protein coding genes and two rRNA genes is conserved in three blocks, but considerable variation occurs in the order of the 22 tRNA genes. Based on phylogenetic analyses and reconstruction of ancestral mitochondrial genomes of Aculifera, the position of (1) trnD gene between atp8 and atp6, (2) trnT and P genes between atp6 and nad5, and (3) trnL1 gene between G and E, resulting in a ‘MCYWQGL1E’-block of tRNA genes, are all three considered synapomorphies for Solenogastres. The tRNA gene block ‘KARNI’ present in Polyplacophora and several conchiferan taxa is dissolved in Solenogastres. Our study shows that mitogenomes are suitable to resolve the phylogenetic relationships among Aculifera and within Solenogastres, thus presenting a cost and time efficient compromise to approach evolutionary history in these clades.

Stylommatophoran pulmonate land slugs and snails successfully completed the water-to-land transition from an aquatic ancestor and flourished on land. Of the 30,000 estimated species, very few genomes have so far been published. Here, we assembled and characterized a chromosome-level genome of the “Spanish” slug, Arion vulgaris Moquin-Tandon, 1855, a notorious pest land slug in Europe. Using this reference genome, we conclude that a whole-genome duplication event occurred approximately 93–109 Mya at the base of Stylommatophora and might have promoted land invasion and adaptive radiation. Comparative genomic analyses reveal that genes related to the development of kidney, blood vessels, muscle, and nervous systems had expanded in the last common ancestor of land pulmonates, likely an evolutionary response to the terrestrial challenges of gravity and water loss. Analyses of A. vulgaris gene families and positively selected genes show the slug has evolved a stronger ability to counteract the greater threats of external damage, radiation, and water loss lacking a protective shell. Furthermore, a recent burst of long interspersed elements in the genome of A. vulgaris might affect gene regulation and contribute to rapid phenotype changes in A. vulgaris , which might be conducive to its rapid adaptation and invasiveness.

Tritoniella belli is the only valid species of a nudibranch genus endemic to the Southern Ocean. Recent exhaustive sampling and molecular analyses led to the discovery of several new lineages. A total of 69 specimens were collected from 25 sites across the Weddell and Scotia Seas, from 5 to 751 m depth. In this study, we provide morphological and anatomical characters to describe five new Tritoniella species, namely T. gnocchi n. sp., T. prinzess n. sp., T. gnathodentata n. sp., T. schoriesi n. sp., T. heideae n. sp. Detailed descriptions of colouration, external morphology, digestive and reproductive organs, distribution, and ecology are presented in a systematic context. These are compared to the type material from the Ross Sea of T. belli and its synonym T. sinuata, whose status requires additional sampling to be solved. Discrete differences in external characters, including the shape of dorsal notum ridge and mantle edges, support the species hypotheses delimited by Moles, Berning et al. (2021). Moreover, detailed scanning electron microscopy images of the masticatory border of the jaws, radula teeth, and penial papilla were provided and their differences discussed. The gut content of all species revealed sclerites of Primnoidae gorgonians as their preferred prey. Pseudo-cryptic radiations along the Scotia Arc, explained by the combination of distribution reduction due to glacial cycles and the existence of refugia, and enhanced by their direct development, could explain the allopatric speciation events in Tritoniella species.

The gastropod infraclass Euthyneura comprises at least 30,000 species of snails and slugs, including nudibranch sea slugs, sea hares and garden snails, that flourish in various environments on earth. A unique morphological feature of Euthyneura is the presence of two pairs of sensory head tentacles with different shapes and functions: the anterior labial tentacles and the posterior rhinophores or eyestalks. Here we combine molecular phylogenetic and microanatomical evidence that suggests the two pairs of head tentacles have originated by splitting of the original single tentacle pair (with two parallel nerve cords in each tentacle) as seen in many other gastropods. Minute deep-sea snails of Tjaernoeia and Parvaplustrum, which in our phylogeny belonged to the euthyneurans' sister group (new infraclass Mesoneura), have tentacles that are split along much of their lengths but associated nerves and epidermal sense organs are not as specialized as in Euthyneura. We suggest that further elaboration of cephalic sense organs in Euthyneura closely coincided with their ecological radiation and drastic modification of body plans. The monotypic family Parvaplustridae nov., superfamily Tjaernoeioidea nov. (Tjaernoeiidae + Parvaplustridae), and new major clade Tetratentaculata nov. (Mesoneura nov. + Euthyneura) are also proposed based on their phylogenetic relationships and shared morphological traits.

Sampling impediments and paucity of suitable material for molecular analyses have precluded the study of speciation and radiation of deep-sea species in Antarctica. We analyzed barcodes together with genome-wide single nucleotide polymorphisms obtained from double digestion restriction site-associated DNA sequencing (ddRADseq) for species in the family Antarctophilinidae. We also reevaluated the fossil record associated with this taxon to provide further insights into the origin of the group. Novel approaches to identify distinctive genetic lineages, including unsupervised machine learning variational autoencoder plots, were used to establish species hypothesis frameworks. In this sense, three undescribed species and a complex of cryptic species were identified, suggesting allopatric speciation connected to geographic or bathymetric isolation. We further observed that the shallow waters around the Scotia Arc and on the continental shelf in the Weddell Sea present high endemism and diversity. In contrast, likely due to the glacial pressure during the Cenozoic, a deep-sea group with fewer species emerged expanding over great areas in the South-Atlantic Antarctic Ridge. Our study agrees on how diachronic paleoclimatic and current environmental factors shaped Antarctic communities both at the shallow and deep-sea levels, promoting Antarctica as the center of origin for numerous taxa such as gastropod mollusks.

The Tritoniidae provides one of the most famous model species for neurophysiology and behaviour, yet a well-developed phylogenetic framework for this family is still incomplete. In this study, we explored the species-level taxonomy, phylogenetic relationships, and geographic distributions of the tritoniid nudibranchs. During numerous expeditions, specimens from southern South America, Sub-Antarctic Islands, and Antarctica were collected, documented alive, and fixed for anatomical descriptions and genetic sequencing. DNA from 167 specimens were extracted and sequenced for mitochondrial (COI, 16S) and nuclear (H3) markers. An additional 109 sequences of all available tritoniids plus additional outgroups were downloaded from GenBank for comparative purposes. Maximum Likelihood under the GHOST model of evolution and Bayesian inference using the GTR + GAMMA model produced congruent topologies from concatenated alignments. The results of ABGD, GMYC, bPTP, and mPTP species delimitation analyses suggest many separately evolving units that do not coincide with traditionally recognized species limits. Southern Ocean Tritoniella and Tritonia species split into several previously unrecognized species. This result is in accordance with the limited dispersal abilities of some southern tritoniids. Along with the most complete phylogeny of Tritoniidae to date, we also provided many taxonomic notes at the species and genus level. Tritoniidae species are yet another example of under-recognized diversity in the Southern Ocean.

Stylommatophora is one of the most speciose orders of Gastropoda, including terrestrial snails and slugs, some of which are economically important as human food, agricultural pests, vectors of parasites or due to invasiveness. Despite their great diversity and relevance, the internal phylogeny of Stylommatophora has been debated. To date, only 34 stylommatophoran mitogenomes were sequenced. Here, the complete mitogenome of an invasive pest slug, Arion vulgaris Moquin-Tandon, 1855 (Stylommatophora: Arionidae), was sequenced using next generation sequencing, analysed and compared with other stylommatophorans. The mitogenome of A. vulgaris measures 14,547 bp and contains 13 protein-coding, two rRNA, 22 tRNA genes, and one control region, with an A + T content of 70.20%. All protein coding genes (PCGs) are initiated with ATN codons except for COX1, ND5 and ATP8 and all are ended with TAR or T-stop codons. All tRNAs were folded into a clover-leaf secondary structure except for trnC and trnS1 (AGN). Phylogenetic analyses confirmed the position of A. vulgaris within the superfamily Arionoidea, recovered a sister group relationship between Arionoidea and Orthalicoidea, and supported monophyly of all currently recognized superfamilies within Stylommatophora except for the superfamily Helicoidea. Initial diversification time of the Stylommatophora was estimated as 138.55 million years ago corresponding to Early Cretaceous. The divergence time of A. vulgaris and Arion rufus (Linnaeus, 1758) was estimated as 15.24 million years ago corresponding to one of Earth’s most recent, global warming events, the Mid-Miocene Climatic Optimum. Furthermore, selection analyses were performed to investigate the role of different selective forces shaping stylommatophoran mitogenomes. Although purifying selection is the predominant selective force shaping stylommatophoran mitogenomes, six genes ( ATP8 , COX1 , COX3 , ND3 , ND4 and ND6 ) detected by the branch-specific aBSREL approach and three genes ( ATP8 , CYTB and ND4L ) detected by codon-based BEB, FUBAR and MEME approaches were exposed to diversifying selection. The positively selected substitutions at the mitochondrial PCGs of stylommatophoran species seems to be adaptive to environmental conditions and affecting mitochondrial ATP production or protection from reactive oxygen species effects. Comparative analysis of stylommatophoran mitogenome rearrangements using MLGO revealed conservatism in Stylommatophora; exceptions refer to potential apomorphies for several clades including rearranged orders of trnW-trnY and of trnE-trnQ-rrnS-trnM-trnL2-ATP8-trnN-ATP6-trnR clusters for the genus Arion . Generally, tRNA genes tend to be rearranged and tandem duplication random loss, transitions and inversions are the most basic mechanisms shaping stylommatophoran mitogenomes.

Stylommatophora is one of the most speciose orders of Gastropoda, including terrestrial snails and slugs, some of which are economically important as human food, agricultural pests, vectors of parasites or due to invasiveness. Despite their great diversity and relevance, the internal phylogeny of Stylommatophora has been debated. To date, only 34 stylommatophoran mitogenomes were sequenced. Here, the complete mitogenome of an invasive pest slug, Arion vulgaris Moquin-Tandon, 1855 (Stylommatophora: Arionidae), was sequenced using next generation sequencing, analysed and compared with other stylommatophorans. The mitogenome of A. vulgaris measures 14,547 bp and contains 13 protein-coding, two rRNA, 22 tRNA genes, and one control region, with an A + T content of 70.20%. All protein coding genes (PCGs) are initiated with ATN codons except for COX1, ND5 and ATP8 and all are ended with TAR or T-stop codons. All tRNAs were folded into a clover-leaf secondary structure except for trnC and trnS1 (AGN). Phylogenetic analyses confirmed the position of A. vulgaris within the superfamily Arionoidea, recovered a sister group relationship between Arionoidea and Orthalicoidea, and supported monophyly of all currently recognized superfamilies within Stylommatophora except for the superfamily Helicoidea. Initial diversification time of the Stylommatophora was estimated as 138.55 million years ago corresponding to Early Cretaceous. The divergence time of A. vulgaris and Arion rufus (Linnaeus, 1758) was estimated as 15.24 million years ago corresponding to one of Earth’s most recent, global warming events, the Mid-Miocene Climatic Optimum. Furthermore, selection analyses were performed to investigate the role of different selective forces shaping stylommatophoran mitogenomes. Although purifying selection is the predominant selective force shaping stylommatophoran mitogenomes, six genes (ATP8, COX1, COX3, ND3, ND4 and ND6) detected by the branch-specific aBSREL approach and three genes (ATP8, CYTB and ND4L) detected by codon-based BEB, FUBAR and MEME approaches were exposed to diversifying selection. The positively selected substitutions at the mitochondrial PCGs of stylommatophoran species seems to be adaptive to environmental conditions and affecting mitochondrial ATP production or protection from reactive oxygen species effects. Comparative analysis of stylommatophoran mitogenome rearrangements using MLGO revealed conservatism in Stylommatophora; exceptions refer to potential apomorphies for several clades including rearranged orders of trnW-trnY and of trnE-trnQ-rrnS-trnM-trnL2-ATP8-trnN-ATP6-trnR clusters for the genus Arion. Generally, tRNA genes tend to be rearranged and tandem duplication random loss, transitions and inversions are the most basic mechanisms shaping stylommatophoran mitogenomes.

AbstractBackgroundThe “Spanish” slug,Arion vulgarisMoquin-Tandon, 1855, is considered to be among the 100 worst pest species in Europe. It is common and invasive to at least northern and eastern parts of Europe, probably benefitting from climate change and the modern human lifestyle. The origin and expansion of this species, the mechanisms behind its outstanding adaptive success and ability to outcompete other land slugs are worth to be explored on a genomic level. However, a high-quality chromosome-level genome is still lacking.FindingsThe final assembly ofA. vulgariswas obtained by combining short reads, linked reads, Nanopore long reads, and Hi-C data. The genome assembly size is 1.54 Gb with a contig N50 length of 8.6 Mb. We found a recent expansion of transposable elements (TEs) which results in repetitive sequences accounting for more than 75% of theA. vulgarisgenome, which is the highest among all known gastropod species. We identified 32,518 protein coding genes, and 2,763 species specific genes were functionally enriched in response to stimuli, nervous system and reproduction. With 1,237 single-copy orthologs fromA. vulgarisand other related mollusks with whole-genome data available, we reconstructed the phylogenetic relationships of gastropods and estimated the divergence time of stylommatophoran land snails (Achatina) andArionslugs at around 126 million years ago, and confirmed the whole genome duplication event shared by them.ConclusionsTo our knowledge, theA. vulgarisgenome is the first land slug genome assembly published to date. The high-quality genomic data will provide valuable genetic resources for further phylogeographic studies ofA. vulgarisorigin and expansion, invasiveness, as well as molluscan aquatic-land transition and shell formation.

Background The “Spanish” slug, Arion vulgaris Moquin-Tandon, 1855, is considered to be among the 100 worst pest species in Europe. It is common and invasive to at least northern and eastern parts of Europe, probably benefitting from climate change and the modern human lifestyle. The origin and expansion of this species, the mechanisms behind its outstanding adaptive success and ability to outcompete other land slugs are worth to be explored on a genomic level. However, a high-quality chromosome-level genome is still lacking.Findings The final assembly of A. vulgaris was obtained by combining short reads, linked reads, Nanopore long reads, and Hi-C data. The genome assembly size is 1.54 Gb with a contig N50 length of 8.6 Mb. We found a recent expansion of transposable elements (TEs) which results in repetitive sequences accounting for more than 75% of the A. vulgaris genome, which is the highest among all known gastropod species. We identified 32,518 protein coding genes, and 2,763 species specific genes were functionally enriched in response to stimuli, nervous system and reproduction. With 1,237 single-copy orthologs from A. vulgaris and other related mollusks with whole-genome data available, we reconstructed the phylogenetic relationships of gastropods and estimated the divergence time of stylommatophoran land snails ( Achatina ) and Arion slugs at around 126 million years ago, and confirmed the whole genome duplication event shared by them.Conclusions To our knowledge, the A. vulgaris genome is the first land slug genome assembly published to date. The high-quality genomic data will provide valuable genetic resources for further phylogeographic studies of A. vulgaris origin and expansion, invasiveness, as well as molluscan aquatic-land transition and shell formation.### Competing Interest StatementThe authors have declared no competing interest.* BLAST : Basic Local Alignment Search Tool BUSCO : Benchmarking Universal Single-Copy Orthologs BWA : Burrows-Wheeler Aligner CDS : coding DNA sequence CTAB : Cetyltrimethylammonium-bromide DAISIE : Delivering Alien invasive species inventoried for Europe DNAs : DNA transposons EVM : EvidenceModeller GATK : Genome Analysis Toolkit GO : gene ontology KAT : the K-mer Analysis Toolkit KEGG : Kyoto Encyclopedia of Genes and Genomes LINEs : Long Interspersed Nuclear Elements LTR : Long Terminal Repeat MY : million years NCBI : National Center for Biotechnology Information PASA : Program to Assemble Spliced Alignments RC : Rolling Circle SAM : Sequence Alignment Map SINEs : Short Interspersed Nuclear Elements SNPs : single nucleotide polymorphisms TEs : transposable elements TrEMBL : Translation of European Molecular Biology Laboratory TRF : Tandem Repeat Finder WGD : whole-genome duplication

Molecular and morphological evidence support the view that the widely distributed species Berthella stellata (Risso, 1826) is a species complex of at least eight different species. The closely related species Berthella plumula (Montagu, 1803), examined for comparison, is also a complex of two species; the name B. plumula is retained for the Atlantic species and the name Berthella perforata (Philippi, 1844) is proposed for the Mediterranean species. The B. stellata species complex forms a monophyletic group when the Eastern Pacific species Berthella strongi (MacFarland, 1966) is included. Based on a critical review of the literature, the name Berthella stellata is retained for the Eastern Atlantic and Mediterranean species, and the name Berthella pellucida (Pease, 1860) is resurrected for a species found in the Hawaiian Islands. Two new species from the Caribbean region ( Berthella nebula sp. nov., Berthella vialactea sp. nov.) and one from the Eastern Pacific ( Berthella andromeda sp. nov.) are described herein, but the status of the Brazilian species B. tupala Er. Marcus, 1957 remains uncertain. Two possible new species from the Eastern Pacific, represented by one specimen each, were recovered in the phylogenetic analyses but not formally described. It is hypothesized that additional species of this complex may occur in other parts of the Indo-Pacific tropics, particularly in the Indian Ocean.

Stylommatophora is one of the most speciose orders of Gastropoda, including terrestrial snails and slugs, some of which are economically important as human food, agricultural pests, vectors of parasites or due to invasiveness. Despite their great diversity and relevance, the internal phylogeny of Stylommatophora has been debated. To date, only 34 stylommatophoran mitogenomes were sequenced. Here, the complete mitogenome of an invasive pest slug,Arion vulgarisMoquin-Tandon, 1855 (Stylommatophora: Arionidae), was sequenced using next generation sequencing, analysed and compared with other stylommatophorans. The mitogenome ofA. vulgarismeasures 14,547 bp and contains 13 protein-coding, two rRNA, 22 tRNA genes, and one control region, with an A + T content of 70.20%. All protein coding genes (PCGs) are initiated with ATN codons except forCOX1, ND5andATP8and all are ended with TAR or T-stop codons. All tRNAs were folded into a clover-leaf secondary structure except fortrnCandtrnS1(AGN). Phylogenetic analyses confirmed the position ofA. vulgariswithin the superfamily Arionoidea, recovered a sister group relationship between Arionoidea and Orthalicoidea, and supported monophyly of all currently recognized superfamilies within Stylommatophora except for the superfamily Helicoidea. Initial diversification time of the Stylommatophora was estimated as 138.55 million years ago corresponding to Early Cretaceous. The divergence time ofA. vulgarisandArion rufus(Linnaeus, 1758) was estimated as 15.24 million years ago corresponding to one of Earth’s most recent, global warming events, the Mid-Miocene Climatic Optimum. Furthermore, selection analyses were performed to investigate the role of different selective forces shaping stylommatophoran mitogenomes. Although purifying selection is the predominant selective force shaping stylommatophoran mitogenomes, six genes (ATP8,COX1,COX3,ND3,ND4andND6) detected by the branch-specific aBSREL approach and three genes (ATP8,CYTBandND4L) detected by codon-based BEB, FUBAR and MEME approaches were exposed to diversifying selection. The positively selected substitutions at the mitochondrial PCGs of stylommatophoran species seems to be adaptive to environmental conditions and affecting mitochondrial ATP production or protection from reactive oxygen species effects. Comparative analysis of stylommatophoran mitogenome rearrangements using MLGO revealed conservatism in Stylommatophora; exceptions refer to potential apomorphies for several clades including rearranged orders oftrnW-trnYand oftrnE-trnQ-rrnS-trnM-trnL2-ATP8-trnN-ATP6-trnRclusters for the genusArion. Generally, tRNA genes tend to be rearranged and tandem duplication random loss, transitions and inversions are the most basic mechanisms shaping stylommatophoran mitogenomes.

Knowledge about the Splanchnotrophidae, a family of endoparasitic copepods infesting opisthobranch sea slugs, currently is restricted to the external morphology. In contrast, their internal anatomy is still largely unknown and many questions concerning life-history traits remain unanswered. Therefore, the microanatomy of both sexes of Ismaila aliena Haumayr & Schrödl, 2003, a splanchnotrophid infesting the nudibranch Thecacera darwini Pruvot-Fol, 1950 in Chile, was studied using computer-based 3D-reconstruction methods on serial semithin histological sections. The body musculature comprises three paired longitudinal strands. Regarding the cephalic and thoracic appendages, besides the antennae only the first pair of male thoracopods is supplied with strong musculature. The digestive system consists of an oesophagus and a voluminous, sack-like midgut, while hindgut and anus are lacking. Structural, functional and observational evidences suggest that I. aliena and at least some other splanchnotrophids are body fluid rather than – eponymous – tissue feeders. The gonad of I. aliena is large in both sexes and neither antrum nor seminal receptacle was detected in the female. Compared to ectoparasitic copepods, the central nervous system of I. aliena is modified, especially in males. Microanatomical results of the present study are compared with available literature results on I. belciki Ho, 1987 (as I. monstrosa Bergh) and discussed regarding potential functions. Within an emerging functional and evolutionary framework we provide some new insights in the life history of the splanchnotrophid parasites.