Origin and significance of two pairs of head tentacles in the radiation of euthyneuran sea slugs and land snails

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.

www.nature.com/scientificreports/ head tentacles. We found that inclusion of these taxa in a phylogenetic analysis greatly improves resolution of relationships within Heterobranchia and provides a novel scenario on the evolution of the head tentacles and nervous system of Euthyneura.

Results and discussion
Mesoneura, a new clade sister to euthyneuran Heterobranchia. Our molecular phylogenetic reconstruction (Fig. 2, Supplementary Fig. S1; Table 1) recovered Tjaernoeia sp. from off Japan (Fig. 3a), Parvaplustrum tenerum Powell, 1951  With this topology, we reclassify the genus Parvaplustrum into a new monotypic family of bubble snails, Parvaplustridae nov., which is sister to Tjaernoeiidae. A new superfamily Tjaernoeioidea is also erected to contain the families Tjaernoeiidae and Parvaplustridae nov. Surprisingly, this new superfamily formed a strongly supported sister (BP: 96%, TBE: 93%, PP: 1; Fig. 2f-h) to the recently recognized Allomorpha, which contains morphologically divergent rhodopid slugs and murchisonellid snails 39 . Allomorpha was confirmed in the present study as a monophyletic group with strong support (96%, 98%, 1; Fig. 2i, j). The clade Tjaernoeioidea nov. + Allomorpha, here named as a new infraclass Mesoneura, was identified as a robustly supported sister to the over 30,000 species of the infraclass Euthyneura (97%, 96%, 1; Fig. 2k-y). We here propose the name Tetratentaculata nov. for the clade of Mesoneura nov. and Euthyneura. Mesoneura presents a novel taxon in a phylogenetic position between the species-rich clade Euthyneura and a less-diverse grade of 'lower' heterobranchs ( Fig. 2a-e). The present tree topology otherwise conformed to previous studies using either comparable marker and taxon sets 33,35,40 , mitogenomics 44,45 , or phylogenomics 21,46 in retrieving the paraphyletic 'lower' heterobranchs on the one hand and the monophyletic Acteonacea (or Acteonimorpha), Ringipleura, Euopisthobranchia and Panpulmonata as constituting Euthyneura on the other (some previous authors classified Acteonacea as lower heterobranchs, e.g. 33 ). Internal support values of derived clades (i.e., euopisthobranchs and panpulmonates) were lower than in comparable studies with more expansive taxon sampling of these groups 33,47 . Interestingly, careful BLAST searches for the present and previous Genbank sequences, followed by the removal of ambiguous or misidentified data (see Supplementary Table S2), resolved the topology of the lower heterobranchs differently from previous studies (Fig. 2a-e; 39,40 ): Orbitestellidae with discoidal shells were recovered as the sister (77%, 87%, 1) to high-spired Cimidae (Cima and Larochella; full support), instead of having these taxa separate in a large grade 40 or in a polytomy 39 .
This topology was stable to sensitivity analyses where taxa were selectively added or removed (Table 1, Supplementary Fig. S2). Inclusion of the only remaining lower heterobranch clades Architectonicoidea (presumed here to encompass Mathildidae based on morphology) and Ammonicera (currently classified as 'Omalogyroidea') into separate ML analyses found these to form a monophyletic taxon (BP: 100%, TBE: 100%) as sister to Valvatoidea but with low support (45%, 72%) and an extremely long branch ( Supplementary Fig. S2). Inclusion of this long-branched clade resulted in the masking of more numerous alignment-ambiguous sites and thereby lowered nodal support over the tree, yet without significantly affecting the topology. Exclusion of either Rhodopidae or Murchisonellidae also resulted in the same topology with the monophyletic Mesoneura (BP: 78-83%, TBE: 64-93%) and Tetratentaculata (63-89%, 81-92%) ( Table 1; Supplementary Fig. S2).  27 . Parvaplustrum species on the other hand have a fragile, oval and inflated 'bubble' shell with a sunken spire and without an umbilicus, resembling those of many euthyneuran groups (e.g. Hydatina and Haminoea, see Fig. 2q) 28,[41][42][43] . Based on our study of aligned serial histological sections and three-dimensional reconstructions of the body shape and internal organs, we here show aspects of microanatomy for the type species of each genus, namely the North Atlantic Tjaernoeia exquisita (Jeffreys, 1883) (Fig. 3b,c) and South Atlantic P. tenerum (Fig. 3d-f). Particular focus is here placed on the head morphology and configuration of the central www.nature.com/scientificreports/ nervous system (CNS) in comparison with other gastropods including newly studied Ebala (Mesoneura: Allomorpha: Murchisonellidae) and Rissoella (Euthyneura: Rissoellidae) (see Supplementary Fig. S3). The CNS is of particular interest due to its conservativeness during gastropod evolution, with neuronal connections inside its ganglia conserved for hundreds of millions of years 13,14 .
The herein reconstructed, preserved animal of T. exquisita was reproductively mature, yet only 550-µm long (Fig. 3b) and much smaller than P. tenerum (ca. 2 mm; Fig. 3d). Although with different sizes and shell shapes, the two species are very similar to each other in head-foot morphology. They both resemble lower heterobranchs including the Cimidae and many of Valvatoidea (see e.g. Fig. 2a,b,d) in having a slender and anteriorly bifurcated foot and a short snout 27 . This snout bears a short, finger-shaped tentacle on either side of the tip (TS), as seen in some species of Valvatoidea 18 . More posteriorly, the head of T. exquisita and P. tenerum has two conspicuous pairs of long, deeply bifurcated and slightly flattened head tentacles of which the anterior branch is about 30-40% shorter than the posterior one (TA and TP in Fig. 3b,c,f). The surface of these tentacles is uniformly smooth, with interspersed gland cells and with ciliation mainly on the inner side. Eyes are lacking in both species, and the body is colourless (see also 27 ). The posterior side of the foot lacks an operculum in both Tjaernoeia and Parvaplustrum, as in the Rhodopidae and the majority of Euthyneura, whereas the Murchisonellidae and lower heterobranchs are all operculate 48,49 .
The general similarity of their head-foot is also reflected in the nervous system of Tjaernoeia and Parvaplustrum. The CNS, shown here in the larger-bodied P. tenerum (Fig. 3e,f), contains a cerebral nerve ring with four ganglia (paired cerebropleural and pedal ganglia: GCP and GP) and two buccal ganglia (GB) below the pharynx. The asymmetric, twisted visceral nerve loop (VL) encircles the oesophagus and bears dorsal (G2) and posteroventral ganglia (G1). Parvaplustrum tenerum differs from T. exquisita in having small additional ganglia of unknown function joined to the sides of the cerebropleural ganglia (GR). Moreover, only P. tenerum has a cluster of accessory ganglia (GA) at the base of the external copulatory organ (P) on the right side of the head behind the bifid tentacles; this copulatory organ in P. tenerum is larger than that of T. exquisita and bears a chitinous stylet (not shown). In both species, several paired nerves emanate from the cerebropleural ganglia. The snout is innervated by two pairs (in P. tenerum) or a single pair (in T. exquisita) of nerves. The bifurcated head tentacles are each innervated by two independent nerves that emerge directly adjacent to each other (NTA, NTP); these nerves do not bear obvious lateral ramifications except two small branches near the nerve tip in Parvaplustrum.
In addition, certain ganglia of P. tenerum (GP, GB, G1, G2 and left GCP) have several large neurons (orange circles in Fig. 3f) that are histologically and topologically identifiable with the 'giant' neurons of the neurobiological model organism Aplysia (Figs. 1d, 2f) and many other euthyneurans (Gillette, 1991: p. 235 8 ). These cells could not be identified in T. exquisita with its smaller body size, as was previously the case in Rhodopidae 48 and Murchisonellidae 49 . The two species resemble lower heterobranchs in lacking specialized epidermal sensory areas innervated by smaller branches of the tentacle nerves, such as the so-called Hancock's organs (which are present at the base of posterior head tentacles of most aquatic euthyneurans; 14 ; see below). The osphradium, another chemosensory organ in the molluscan pallial cavity, is present in both T. exquisita and P. tenerum as a small ciliated patch of epidermis on the anterior roof of the mantle (O in Fig. 3e,f).
The different shell shapes of T. exquisita and P. tenerum are also reflected in the different organization of their mantle. Specifically, P. tenerum has its plicate gill, glands and kidney all located on the posterior right of the mantle (a condition called 'detorted' in Euthyneura). On the other hand, in T. exquisita the gill is a more medially lying, microscopic leaf without folds, glands are spread along the anterior margin of the mantle, and the kidney lies centrally, reflecting a more ancestral condition typical of non-euthyneuran heterobranchs (e.g. 10,18 ). Opposing ciliary strips in the mantle cavity, regarded as an apomorphy of Heterobranchia 10,29 , could not be reliably identified in Tjaernoeia and Parvaplustrum and among the Mesoneura such structures are so far only confirmed for some Murchisonellidae 50 . Tjaernoeia and Parvaplustrum are similar in their complex hermaphroditic reproductive system (Fig. 3b) and in the simple digestive tract with a very narrow cuticular radula that was shown by other authors to bear only leaf-shaped lateral teeth 27,41,43 .
Morphological diagnoses of the newly proposed taxa can be summarized as follows: Tjaernoeioidea, a new superfamily for Tjaernoeia (Tjaernoeiidae) and Parvaplustrum (Parvaplustridae nov.): Small to minute heterobranch snails with a single pair of deeply bifid head tentacles (or, depending on perspective, two pairs of basally joined tentacles) and a slender snout with a lateral projection on either side; eyes lacking and skin unpigmented; external copulatory organ on the right side of the head-foot; shell fragile, globose to oval, smooth or with sculpture of small dimples, protoconch hyperstrophic; foot without an operculum. Parvaplustridae, a new monotypic family for Parvaplustrum [ZooBank registration (LSID): urn:lsid:zoobank.org:act:A7416D49-113A-4E60-86A7-6AE46818B20A]: Shell inflated, oval, with a large aperture and an involute spire; shell surface smooth or with minute, irregularly scattered pits; mantle detorted with the gill, kidney and large glands all located on the posterior right; copulatory organ with a tubular chitinous stylet. We regard these differences in external morphology to warrant separate family status. Mesoneura, a new infraclass for Tjaernoeioidea + Allomorpha: Named after its nervous system showing a mix of plesiomorphic and apomorphic conditions in their nervous system, with tentacle nerves that innervate independent areas of the head but otherwise largely lack ramifications; Hancock's organ and median lip absent; visceral loop with torsion yet long; radula (if present) narrow without a rachidian tooth and with only one slender lateral tooth on either side of a transverse row. Tetratentaculata, a new clade name for Mesoneura + Euthyneura: Head with four individual sensory areas corresponding to four tentacles or two deeply bifurcated tentacles, although tentacles per se may be reduced secondarily; giant neurons present in the pedal, buccal, dorsal, posteroventral, and left cerebropleural ganglia (see below); visceral nerve cord at least slightly detorted or completely detorted. Accordingly, Euthyneura can be newly diagnosed as tetratentaculate heterobranchs distinguished from Mesoneura by having (1) labial tentacles that are medially fused to form an upper lip or velum and (2) tentacle nerves bearing many small ramifications that innervate sensory cells including those of the Hancock's organ (see discussion below). www.nature.com/scientificreports/ Origin of euthyneuran head tentacles. The herein recovered phylogenetic position of the Tjaernoeioidea corroborates a recent morphology-based hypothesis 13,20 that the two pairs of specialized euthyneuran head tentacles might have originated through bifurcation of an ancestral single pair of tentacles, where each tentacle was already innervated by two nerve cords, as now seen in the Caenogastropoda and lower Heterobranchia 3 . It was previously assumed that only the anterior pair (labial tentacles) of the Euthyneura was homologous to the plesiomorphic head tentacles of the Gastropoda (see 13,24,51 ). The posterior pair (rhinophores or ommatophores or eyestalks) was regarded as secondarily acquired or even repeatedly acquired (e.g. 25 ). However, convincing evidence for the homology of rhinophores across the Euthyneura came from the use of axonal backfilling techniques 12,14,15 that reliably identified and correlated individual tentacle nerves across a broad set of taxa. Furthermore, Staubach 13 identified highly conserved neuron clusters associated with the tentacle nerves in the cerebral ganglia of the periwinkle (Caenogastropoda: Littorina) and giant African snail (Heterobranchia: Stylommatophora: Achatina). This conservatism in details of neuronal architecture of tentacle innervation further supports homology of tentacles across the Apogastropoda (see Fig. 1, Supplementary Fig. S3).
In combination with these results, our new findings lead to an evolutionary scenario of two steps. First, at the origin of Tetratentaculata, the single ancestral tentacle with double nerves split into two tentacles, each with one of the two ancestral nerve cords. Second, at the origin of Euthyneura, the two tentacles became specialized into the anterior and posterior tentacles, with different shapes and with more elaborate sensory areas such as the rhinophores and Hancock's organs. The first of these evolutionary steps might still be visible in the extant Tjaernoeioidea, which have a pair of deeply bifurcated yet basally joined tentacles. With only few exceptions, the two pairs of ramified tentacle nerves-acquired in the second step-persist across the Euthyneura including groups with atypical tentacles (Fig. 4, Supplementary Fig. S3, see 3,11,13,14,24,35 ).
The Allomorpha, although being sister to the Tjaernoeioidea, do not share the long and bifid head tentacles and thus might question the evolutionary scenario proposed above. However, allomorph snails and slugs may have modified or lost the bifid tentacles in relation to their sediment-dwelling or even infaunal lifestyles 39,48,49 as have many euthyneurans 12,13,29,35 . Murchisonellids bear a pair of posterior, oftentimes broad, tentacles that are innervated with two pairs of essentially unbranched nerve cords (see 49 , and Supplementary Fig. S3 for reexamination of Ebala). Rhodopids have entirely lost the head tentacles per se but there remain the two pairs of the tentacle nerves in the head 39,48 . After a confused taxonomic history (see 39 ), the Allomorpha are now found to resemble their previously unrecognized sistergroup Tjaernoeioidea more than other heterobranchs in having four separate tentacle nerves, but lacking unequivocal Hancock's organs, distal ramifications of tentacle nerves, and a typical medially-fused upper lip 49 . Absence of the Hancock's organ and upper lip also distinguishes the Tjaernoeioidea from the shallow-water, herbivorous euthyneuran snails of the superfamily Rissoelloidea (Supplementary Fig. S3), regardless of the short, bifid head tentacles of Rissoella that externally might recall those of tjaernoeioids ( Fig. 2k; 52,53 ). The speciose radiation of Euthyneura (node 7), leading also to diverse shell-shapes and instances of shell loss ( Fig. 4: circles at branch terminals), was estimated to have started in the Carboniferous-Permian time (296 Mya, 345-248). This predates the oldest known euthyneuran fossils, the diverse Cylindrobullinoidea occurring since the Early Triassic (245 Mya) (see 54,55 for discussion). Cylindrobullinoids have been suspected to contain paraphyletic or polyphyletic members of already diverged euthyneuran lineages (see 55 for review), some of which may lead to the extant Acteonimorpha, Ringipleura, Euopisthobranchia and Panpulmonata. The early Euthyneura are suggested to have a characteristic bubble shell with a large body whorl-a morphology found in several lineages of the extant Euthyneura (Fig. 4: blue circles at terminals)-as well as a hypertrophied foot and headshield for an oftentimes infaunal mode of life 35 . The morphological variability found in Mesoneura does not allow unequivocal reconstruction, but they display bubble-shell and slug morphotypes (Parvaplustridae and Rhodopidae) in parallel with the Euthyneura. Fossils of putatively ancestral murchisonellids (as Donaldinidae and Streptacididae, see 56 ) come from the strata of 350-260 Mya, which is much older than the first occurrences of the fossil Cylindrobullinoidea and closely fit the herein proposed age of divergence (node 3 in Fig. 4, see above). Some of those Palaeozoic taxa [57][58][59] are indeed fairly similar to the modern Murchisonella 60,61 in teleoconch and protoconch morphology. On the other hand, certain early-Triassic fossils have a murchisonellid-like protoconch and a cylindrobullinoid-like teleoconch and were therefore interpreted as a phylogenetic link between the Streptacididae and Cylindrobullinoidea 56 .

Shell shapes and radiation of
The Euthyneura comprise almost half of all molluscan species richness -why could they have radiated into so many ecological niches and diversified into so many species? Kano et al. 35 hypothesized that early euthyneurans were freed from the strict connection of the shell and mantle margin, thereby releasing the mantle from morphological constraints and allowing the creation of evolutionary novelty. We here add that the modification of the head, although not evident in the fossil record, seems to present another overlooked key event in their evolutionary history. The diverse nature of head tentacles is considered important for the taxonomy of euthyneuran subgroups, particularly those of sea slugs 14 www.nature.com/scientificreports/ as a whole has been overshadowed by a focus on shell loss and coinciding chemical defence. The diverse rhinophores, eyestalks, and Hancock's organs of Euthyneura play crucial roles in directional chemosensing 11,13,14,25,62 and are much more specialized than head tentacles in other gastropods (shown in Fig. 4 at right; Supplementary  Fig. S3). For the other (aquatic) gastropods, the osphradium in the mantle cavity is the primary chemosensory organ 3 , but it is generally simplified in euthyneuran and non-euthyneuran heterobranchs 63,64 . This suggests that, at least in Euthyneura, sensory capacity of the head has not only become more pronounced relative to other Gastropoda but it has also functionally replaced much of mantle-based chemosensing, as implied by previous authors (see Morton, 1972: p. 337 65 ; Gosliner, 1994: p. 346 25 ). We here suggest that the acquisition of the more elaborate head sensors has determined the observed shift of euthyneuran ecology towards more motile and predatory lifestyles, often with specialized prey items and habitats 66,67 . Furthermore, reduced reliance on the osphradium for chemosensing may have removed constraints to reorganization of the mantle, allowing additional evolutionary plasticity and innovations in the morphology of the posterior body and shell. The acquisition www.nature.com/scientificreports/ of the enhanced head sensors may therefore be linked to the shell loss and also to the explosive radiation and speciation of Euthyneura. This study highlights that inclusion of rare, microscopic taxa in an integrated analysis has the potential to greatly improve the resolution of phylogenetic relationships and provides a novel scenario on a large-scale evolutionary process, in the present case within heterobranch and euthyneuran gastropods.

Methods
Sampling and preparation of specimens. Living snails of the following seven heterobranch species were collected from coastal to bathyal waters using various methods (see below; Supplementary Tables S1, S2 and Supplementary Fig. S3 for additional data). Specimens were preserved either (1) directly in 95-99% ethanol, or (2) fixed in a 10% formalin-seawater solution after anesthetization in isotonic magnesium chloride solution and then transferred to 80% ethanol. All sectioned specimens and DNA extracts are deposited at the Mollusca section of the Bavarian State Collection of Zoology, Munich, Germany (ZSM) or at the Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Japan (AORI).
Helminthope cf. DNA extraction, PCR amplification and sequencing. Full genomic DNA was extracted from clipped foot or mantle tissue using DNeasy Blood and Tissue Kit (Qiagen) or Macherey-Nagel Blood and Tissue Set and following the manufacturers' instructions. Partial sequences of nuclear (18S and 28S rRNA) and mitochondrial (16 s rRNA and COI) markers were amplified using primers shown in Supplementary Table S1; see 69 and 35 for amplification conditions and other details. Amplicons were purified with ExoSAP-IT (Affymetrix) and then sequenced with Big Dye Terminator Cycle Sequence Kit 3.1 (Applied Biosystems) and amplification and sequencing primers (Supplementary Table S1). The reaction mixtures were analyzed on an ABI PRISM 3130xl sequencer (at AORI) or an ABI 3730 sequencer (at the Department of Biology Genomic Service Unit of the Ludwig-Maximilians-University Munich) after purification with Big Dye XTerminator Purification Kit (ABI). New DNA sequences have been deposited in the DDBJ⁄EMBL⁄GenBank with accession numbers LC631476-LC631487 (Supplementary Table S2).
Phylogenetic reconstruction. For molecular phylogenetic analyses we selected 49 heterobranch species including one Tjaernoeia and two Parvaplustrum (Supplementary Table S2). The selection was made on the basis of covering the phylogenetic diversity of Heterobranchia and consistent evolutionary rates of all four targeted genes. The lower heterobranch genera Architectonica and Ammonicera were excluded from the main analyses due to their extremely long branches in a preliminarily tree. Dubious sequences, identified by BLAST searches and by careful comparison in the context of larger alignments, were excluded from succeeding analyses (see Supplementary Table S2 for notes on excluded sequences). Three species of Caenogastropoda were included in the dataset for outgroup comparison, resulting in a total of 52 taxa. The sequences of the four genes were aligned individually with MAFFT 7.182 using the L-INS-i strategy 70 ; COI sequences were aligned as amino acids. Each aligned dataset was masked to remove alignment ambiguous sites on Gblocks Server 0.91b with all three options for a less stringent selection 71  www.nature.com/scientificreports/ Phylogenetic trees were reconstructed from single-gene and concatenated multi-gene datasets using the Maximum-Likelihood (ML) method implemented in raxmlGUI 2.0 (RAxML-HPC and RAxML-NG; [72][73][74]. Each gene and codon position was allowed to have different parameters, resulting in six partitions for the four-gene dataset. The RAxML-HPC analyses were performed using following commands: a rapid bootstrap analysis with 1000 replicates and search for the best-scoring ML tree in a single program run under the default GTR + G model, following the software manual. The RAxML-NG runs were carried out with the same setting to calculate transfer bootstrap expectation (TBE) 75 values with 1000 replicates. The concatenated four-gene dataset was also analysed under Bayesian inference using MrBayes 3.1.2 76 . Substitution models used (estimated with jModeltest 2.1.10; 77 ) were GTR + G for the 3rd codon of COI and GTR + I + G for all other partitions. Two parallel runs were made for 10 M generations with a sample frequency of 1000, using the default value of four Markov chains. The first 5000 trees for each run were discarded to make sure the four chains reached stationarity by referring to the average standard deviation of split frequencies 76 . The consensus tree and posterior probabilities (PP) were computed from the remaining 10,000 trees (5000 trees, two runs). Bootstrap proportion (BP) and TBE of ≥ 80% and PP of ≥ 0.99 were considered significant support.
The stability of clades was further tested in sensitivity analyses where taxa were selectively added or removed. The sequences of individual genes were aligned and masked by adding the long-branched clade of Architectonica and Ammonicera (55 taxa), or excluding rhodopid slugs or murchisonellid snails (50 taxa each), to generate three additional sets of four-gene matrices. These datasets were analysed in raxmlGUI2 with 1000 replicates to obtain BP and TBE values.
Divergence times were calculated from the four-gene dataset used in the main analyses (52 taxa) with the relaxed molecular clock model implemented in BEAST 1.5.4 78 . The tree was calibrated by setting ages for three nodes with reliable fossil records: (1) the split of Heterobranchia and Caenogastropoda by Early Devonian time (Gamma distribution, Shape: 1, Offset: 400, Scale: 13.34), (2) the first split in Euopisthobranchia by Early Jurassic (Offset: 190, Scale: 6.33), and (3) first split in Ellobioidea by Late Jurassic (Offset: 152, Scale: 5.07). See 35 for the details of the calibration points and other settings for BEAST analysis.