Ancestral role of Pax2/5/8 in molluscan brain and multimodal sensory system development
© Wollesen et al. 2015
Received: 20 April 2015
Accepted: 1 October 2015
Published: 28 October 2015
Mollusks represent the largest lophotrochozoan phylum and exhibit highly diverse body plans. Previous studies have demonstrated that transcription factors such as Pax genes play important roles during their development. Accordingly, in ecdysozoan and vertebrate model organisms, orthologs of Pax2/5/8 are among others involved in the formation of the midbrain/hindbrain boundary, the auditory/geosensory organ systems, and the excretory system.
Pax2/5/8 expression was investigated by in situ hybridization during the development of representatives of the two major molluscan subclades, Aculifera and Conchifera.
Compared to the investigated polyplacophoran and bivalve species that lack larval statocysts as geosensory organs and elaborate central nervous systems (CNS), cephalopods possess highly centralized brains and statocysts. Pax2/5/8 is expressed in regions where sensory cells develop subsequently during ontogenesis. Expression domains include esthetes and the ampullary system in polyplacophorans as well as the eyes of cephalopods. No Pax2/5/8 expression was observed in the less centralized CNS of bivalve, polyplacophoran, and gastropod embryos, thus arguing for a loss of Pax2/5/8 involvement in CNS development in these lineages. In contrast, Pax2/5/8 is expressed among others in brain lobes along the trajectory of the esophagus that divides the cephalopod brain.
Our results, along with those on Otx- and Hox-gene expression, demonstrate that the cephalopod condition is similar to that in mouse and fruit fly, with Otx being expressed in the anterior-most brain region (except for the vertical lobe) and a Pax2/5/8 expression domain separating the Otx-domain from a Hox-gene-expressing posterior brain region. Thus, Pax2/5/8 appears to have been recruited independently into regionalization of non-homologous complex brains of organisms as different as squid, fruit fly, and mouse. In addition, Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans as well as the eyes of cephalopods. Pax2/5/8-expressing cells are present in regions where the future sensory cells such as the polyplacophoran esthetes are situated and hence Pax2/5/8 expression probably predates sensory cell development during ontogeny. In mollusks, Pax2/5/8 is only expressed in derivatives of the ectoderm and hence an ancestral role in molluscan ectoderm differentiation is inferred.
KeywordsBivalvia Cephalopod Complexity Evolution Homeobox Hox Lophotrochozoa Neurogenesis Sensory cell Polyplacophora
Members of the paired box (Pax) gene family encode transcription factors that are involved in a variety of processes such as the development of excretory organs, the differentiation of the musculature, neuroectoderm specification, and the development of auditory/geosensory systems [10, 11]. In bilaterians, nine orthologous groups of Pax proteins are known that can be distinguished on the basis of the presence or absence of highly conserved structural domains . Members of the Pax2/5/8 group exhibit a conserved N-terminal paired domain, an octapeptide, a partial homeodomain, and a C-terminal transactivation domain . The N-terminal domain and the homeodomain serve as DNA binding sites, the octapeptide is the site for activation or repression of the gene, and the C-terminal domain is the protein/protein interaction domain . In vertebrates and ecdysozoans, Pax2/5/8 is involved among others in the genesis of the auditory/geosensory system as well as in the formation of the midbrain/hindbrain boundary [13, 14]. For lophotrochozoans, so far only one study has been published on the expression domains and the putative function of Pax2/5/8 during development. In this study, Pax2/5/8 expression was found in the statocysts, i.e. the geosensory organ, of the gastropod veliger larva .
In order to elucidate putative roles of Pax2/5/8 during molluscan development, we investigated the expression of its orthologs in representatives of both major molluscan lineages, the Aculifera and the Conchifera [15, 16] (Fig. 1). As a polyplacophoran, Acanthochitona crinita has been shown to exhibit putative ancestral features of the Aculifera , while the conchiferan Nucula tumidula is a protobranch that holds a basal position among the bivalves and possesses a unique pericalymma-type larva [4, 18]. A. crinita and N. tumidula do not possess statocysts in their larvae and exhibit a less centralized CNS. In contrast, the conchiferan cephalopod Idiosepius notoides has a highly centralized CNS and statocysts. This study will test whether Pax2/5/8 is expressed in ectodermal, endodermal, or mesodermal derivatives during the ontogeny of all three molluscan species and it will shed light on the shared Pax2/5/8 expression among bilaterian representatives.
Pax2/5/8 gene orthologs and phylogenetic analysis
Pax2/5/8 expression in the polyplacophoran Acanthochitona crinita
An additional Acr-Pax2/5/8 expression domain is present close to the lateral borders of the foot (Fig. 5, group 5 in Fig. 6h, i). Both groups 5 are interconnected via a thin band of Acr-Pax2/5/8-expressing cells that runs along the abapical side of the mouth (Figs. 5 and 6h, i). Few Acr-Pax2/5/8-expressing cell are present in the posterior mantle fold (Figs. 5 and 6k), while no Acr-Pax2/5/8-expressing cells are located in the region of the apical organ (dashed circle in Fig. 6k).
Settled animals (130 hpf) commence to reduce their episphere and the foot becomes more pronounced. Metamorphosis is largely completed with the shedding of the prototroch, although the eighth shell plate is not formed until probably several weeks after settlement. Early settled animals solely possess few faintly stained Acr-Pax2/5/8-expressing cells in the region of the shell fields (Fig. 6l) and only non-specific staining was observed in the perinotum that surrounds the plates (Fig. 6l).
Pax2/5/8 expression in the bivalve Nucula tumidula
Pax2/5/8 expression in the cephalopod Idiosepius notoides
As a typical cephalopod, I. notoides is a direct developer with discoidal cleavage. Hatching of the juvenile occurs within 9–10 days after oviposition at 25 °C. In the present study, we adhere to the common designation of embryological orientation in cephalopods, i.e., the mantle apex is considered dorsal and the cephalic region including the brachial crown that surrounds the mouth is considered ventral. The funnel is located posteriorly, while the opposite side including the supraesophageal mass is considered anterior.
Evolution of Pax2/5/8 proteins
Our phylogenetic analysis confirms the identities of all three molluscan Pax2/5/8 proteins presented herein. The sequences of Acanthochitona crinita, Idiosepius notoides, and Nucula tumidula exhibit a paired domain, an octapeptide, the lysine-arginine rich region, as well as the partial homeodomain (not shown) diagnostic for Pax2/5/8.
Pax2/5/8 is expressed in the development of multimodal sensory systems of mollusks
Among bilaterians, Pax2/5/8 expression is particularly well-investigated in the Deuterostomia and Ecdysozoa [13, 20]. Studies on various deuterostomes revealed that orthologs of Pax2/5/8 play a role during development of auditory/ geosensory organs [21–23]. Interestingly, the phylogenetically distantly related gastropod mollusk Haliotis asinina also expresses Pax2/5/8 in its geosensory organ, the statocysts . This present study demonstrated that homeobox genes may be recruited into the development of organs with similar function but of different evolutionary origin (i.e., in non-homologous systems). It also poses the question as to how Pax2/5/8 is recruited during the ontogeny of organisms without auditory and equilibrial sense.
In our study, we included the chiton Acanthochitona crinita and the bivalve Nucula tumidula, both lophotrochozoan representatives without larval statocysts, in order to assess the role of Pax2/5/8 during their development. Pax2/5/8 is expressed in several groups of ectodermal cells in the episphere and the hyposphere of the polyplacophoran trochophore. Although A. crinita and N. tumidula possess larval apical (sensory) organs, no Pax2/5/8 expression was detected in components of this organ. In A. crinita cells of groups 2–3 are located in a similar location as cells of the larval ampullary sensory system that is an autapomorphy of Polyplacophora (Figs. 5 and 6 in present study; ). Cells of the ampullary system are flask-shaped and probably chemosensory rather than mechanosensory . Pax2/5/8 expression and FMRFamide-like immunoreactivity may be co-localized in ectodermal cells in the dorsal episphere adjacent to the prototroch (group 2, c.f. Figs. 5 and 6j). Further Pax2/5/8-expressing ectodermal cells (group 4) are located in the ventral episphere and might correspond to the four serotonin-like immunoreactive flask-shaped cells (c.f. group 4 in Figs. 4h, 5, 6b). The immunochemical and molecular phenotype, their flask-shaped somata, and their subepithelial location argue for a sensory role of the above-mentioned cells . This might also be true for the Pax2/5/8-expressing groups 5 that are located on the ventral side along the borders of the foot of advanced trochophore larvae (Fig. 5). Both groups are connected to each other via a small band along the abapical side of the mouth/ apical side of the foot (Fig. 5). This expression domain also includes a region where the “Schwabe organ”, a putative chemosensory organ of a polyplacophoran ingroup, is located . Although the chitonid A. crinita has not been studied with respect to the presence of this organ, it is likely that it is not present in A. crinita since other congeneric species of the Chitonida lack this sensory structure. Pax2/5/8-expressing cells are also located in the posterior mantle fold that exhibits the chemosensory osphradia of adult chitons (arrowheads in Fig. 6k; see also . In both described expression domains, however, no Pax2/5/8 expression is found in postmetamorphic chitons.
A prominent group of Pax2/5/8-expressing cells is present in the shell fields of A. crinita (arrowheads in Figs. 4j, k, 5 and 6a). The majority of these cells is located in central regions of each shell field and exhibits processes that penetrate the epidermis and the nascent calcareous layers of the shell plates (inset in Fig. 6j). These processes exhibit FMRFamide-like immunoreactivity in earlier developmental stages than the associated cell somata (c.f. Figs. 4j, 5 and 6a, j). Judging by their location and morphology, these Pax2/5/8-expressing and FMRFamide-like immunoreactive cells are probably esthetes. Esthetes are primarily photoreceptive organs that constitute a polyplacophoran autoapomorphy and penetrate the surface of the shell plates of adult specimens [25–27]. During ontogeny, esthetes may first be identified by the narrow holes of their canals in shell plates of metamorphosed specimens .
In the pygmy squid, Pax2/5/8 is expressed in various organs that are known to be equipped with sensory cells. During a short developmental time frame (stages 24–25), Ino-Pax2/5/8 is expressed in the eyes (Figs. 5 and 10b, c), resembling the situation of the adult gastropod Haliotis asinina . The brachial crown, i.e. all five arm pairs, expresses Pax2/5/8 in epidermal cells until stage 24 (Fig. 10a, b). In stage 25 individuals, Pax2/5/8 expression is restricted to the proximal part of each arm/tentacle. Expression fades in subsequent developmental stages.
Cephalopod arms and tentacles house a plethora of sensory cells. Although cell proliferation experiments during cephalopod arm growth are still lacking, it is tempting to speculate that Pax2/5/8 is expressed in ectodermal sensory cells in regions of high proliferation activity, such as the proximal portion of the brachial crown in stage 25 specimens. The funnel shows a similar pattern as the brachial crown. During early development the entire funnel expresses Pax2/5/8, while only the proximal portion expresses Pax2/5/8 in subsequent stages until it fades entirely in late prehatching stages. Here, Pax2/5/8 appears to be expressed in the epidermis as it has also been reported for vertebrates . Along the lateral sides of the foot the future gills develop and Pax2/5/8 expression predates their development.
Expression of Pax2/5/8 in the molluscan mantle
Pax2/5/8 is expressed in different regions of the developing mantle of the aculiferan polyplacophoran Acanthochitona crinita, the basal bivalve Nucula tumidula, and the cephalopod Idiosepius notoides. This corroborates data on the veliger larva of Haliotis asinina, where Pax2/5/8 is expressed in the anterior region of the mantle that is known to be richly equipped with sensory cells . In contrast to the esthetes as distinct expression domain of A. crinita, Pax2/5/8 is rather broadly expressed in the mantle of N. tumidula and I. notoides. During earlier development of N. tumidula, Pax2/5/8 expression is, however, first restricted to the dorsal mantle epithelium, including the shell gland. In contrast, the shell gland of H. asinina does not exhibit Pax2/5/8 expression (present study and ). Shortly before metamorphosis, Pax2/5/8 expression shifts to the ventral side of the mantle and disappears in the dorsal region after metamorphosis. This may be explained by the onset of sensory cell development on the ventral side of the mantle that is exposed to water currents in the postmetamorphic, settled animal. Other ectodermal expression domains comprise the cephalopedal epidermis of stage 24 individuals of I. notoides (Fig. 10a).
Pax2/5/8 plays a role in cephalopod brain regionalization
Otx is expressed in the supraesophageal mass of the cuttlefish Sepia officinalis and the pygmy squid I. notoides (Fig. 11; ; Wollesen, unpublished data). Compared to Pax2/5/8 it is, however, expressed in more anterior regions of the supraesophageal mass; i.e., in components of the vertical lobe complex (i.e. the superior frontal and subpedunculate lobe) and the median basal lobe of the supraesophageal mass. In contrast to Pax2/5/8 expression, no Otx expression was found in the subesophageal mass (present study, , Wollesen, unpublished data). In cephalopods, Hox genes are expressed in the CNS with exception of the cerebral ganglia/supraesophageal mass . Although no collinear spatial expression is reported from cephalopods, the Otx- and Pax2/5/8 expression patterns adhere to the overall pattern reported for Drosophila and mouse . Data on other bilaterians suggest a Six3 expression domain in the anterior-most region of the cephalopod brain, i.e. the vertical lobe complex (white box in Fig. 11; ). Surprisingly, Pax2/5/8 is not expressed in the developing nervous system of the polyplacophorans, bivalves, or gastropods investigated so far (present study; ). In contrast to cephalopods, the fruit fly, or mouse none of the latter mollusks possess a highly centralized brain.
Pax2/5/8 expression is restricted to the ectoderm in mollusks
The present study shows that Pax2/5/8 is expressed in multimodal sensory systems in mollusks such as the esthetes and the ampullary system of polyplacophorans, the eyes of squids, but not in the larval apical organ of A. crinita and N. tumidula. Pax2/5/8 expression probably predates sensory cell development during ontogenesis, since Pax2/5/8-expressing cells are present in regions where the future sensory cells are situated. Compared to other bilaterians, Pax2/5/8 is not expressed in the development of the less centralized nervous system of bivalves, gastropods, and polyplacophorans, and hence it most likely lost its role in brain development in these mollusks. Interestingly, Pax2/5/8 is expressed along the trajectory of the esophagus that divides the cephalopod brain into a supraesophageal and a subesophageal mass. Together with Otx and Hox genes Pax2/5/8 might have been recruited into brain regionalization, thus representing an extreme case of convergent evolution of gene function to the situation found in vertebrates (mouse) and insects (fruit fly). Since Pax2/5/8 is largely expressed in ectodermal domains throughout the Bilateria, its ancestral role was most likely in the differentiation of this outer most germ layer of bilaterians.
Collection and culture of animals
Adults of the polyplacophoran Acanthochitona crinita were collected in the intertidal zone close to the Station Biologique Roscoff in Roscoff, France, during the summers of 2013 and 2014. Adults were kept at 20 °C in running seawater. Prior to spawning, adults were isolated and maintained separately in glass dishes. Spawning was induced by exposing adults to sunlight for 3–4 h or thrice each for 15 min to alternating water temperatures, i.e. 25–30 °C and 10–15 °C. Released sperm and oocytes were separated and the latter rinsed several times in Millipore-filtered seawater (MFSW) and fertilized immediately. Early cleavage stages were kept at 20 °C in running seawater, while post-gastrulation stages were kept in MFSW with the addition of antibiotics against bacterial and fungal growth (50 mg streptomycin sulfate (Sigma-Aldrich) and 60 mg penicillin (Sigma-Aldrich) per liter MFSW). Water was changed every other day and settlement of metamorphic competent larvae was induced by the addition of substrate on which adult animals had been found.
Adults of the protobranch bivalve Nucula tumidula were collected from sediment that was sampled with a hyperbenthic sled at 180–220 m depth on muddy seafloor in Hauglandsosen (Bergen, Norway) during the winters 2012 and 2013. Adult individuals were kept at 6.5 °C in MFSW (UV-treated) at the marine living animal facilities of the Department of Biology, University of Bergen. N. tumidula is dioecious and spawning of gametes was induced by keeping adults in seawater of alternating temperatures. Accordingly, adults were exposed thrice to water of 10–15 and 2 °C for 10 min each. Males that released sperm were separated immediately to avoid polyspermy and the oocytes were rinsed several times in MFSW. Oocytes were fertilized and developmental stages were cultured at 6.5 °C in MFSW in glass bowls with water changes every other day.
Adults of the pygmy squid Idiosepius notoides were dip-netted in the sea grass beds of Moreton Bay, Queensland, Australia. Adults were kept in closed aquaria facilities at the School of Biological Sciences of the University of Queensland in Brisbane and fed with various crustaceans. Fertilization is internal and females attached egg clutches to sea grass or to the glass surface of the aquaria. Embryos were cultured and staged as described previously .
RNA extraction and fixation of animals
Several hundred individuals of early cleavage stages, larvae, metamorphic competent individuals to early juveniles of Acanthochitona crinita and Nucula tumidula were collected and stored in RNAlater (Lifetechnologies, Vienna, Austria) at −20 to −80 °C. RNA was extracted with a RNA extraction kit (Qiagen, Roermond, Netherlands) and stored at −80 °C. For I. notoides, egg jelly and chorion of embryos were removed and RNA from approximately 300 specimens including freshly laid zygotes (stage 1) to hatchlings (stage 30) was extracted using TriReagent according to the manufacturer’s instructions (Astral Scientific Pty. Ltd., Caringbah, Australia; see also ). Several hundred individuals of representative developmental stages of all three species were fixed for in situ hybridization experiments as previously described [36, 37]. Specimens were either stored in 75 % EtOH or in 100 % Methanol at −20 °C.
RNAseq and transcriptome assembly
Pooled total RNA of all above-mentioned developmental stages was used for the preparation of amplified short insert cDNA libraries (150–250 bp insert size) for Nucula tumidula and Acanthochitona crinita (Eurofins, Ebersberg, Germany). Both libraries (Kit version TruSeq SBS Kit v3) were sequenced together with another two bar-coded libraries in 1 channel of HiSeq 2000 with Illumina chemistry v3.0. Sequences were demultiplexed according to the 6 bp index code with 0 mismatch allowed. In both cases a PhiX library was added before sequencing to estimate the error rate of the sequences. Sequencing resulted in a total amount of 8.160 Mbp for N. tumidula and 7.147 Mbp for A. crinita. Paired-end reads with an average read length of 100 bp were obtained. These reads were subsequently filtered (rRNA removal) and adapter and low quality sequences were trimmed, normalized, and assembled de novo into contigs with the assembler Trinity . The transcriptomes of A. crinita and N. tumidula comprised 166,556 contigs and 224,633 contigs, respectively. The sequencing strategy for developmental stages of I. notoides was described previously . Briefly, RNA of developmental stages was sequenced by 454 technology by Eurofins (Ebersberg, Germany). After filtering, adapter and low quality read trimming, the reads were normalized and assembled de novo by Eurofins.
Alignment and phylogenetic analysis
GenBank accession numbers of genes used for the phylogenetic analysis
Gene name (Abbreviation)
Molecular isolation of Pax2/5/8 sequence orthologs
RNA pooled from different developmental stages of A. crinita, N. tumidula, and I. notoides, respectively, was used for first-strand cDNA synthesis by reverse transcription using the First strand cDNA Synthesis Kit for rt-PCR (Roche Diagnostics GmbH, Mannheim, Germany). Gene-specific primers were designed from identified Pax2/5/8 orthologs of A. crinita, N. tumidula, and I. notoides and transcripts were amplified via standard PCR. PCR products were size-fractioned by gel electrophoresis and gel bands of the expected length were excised and cleaned up using a QIAquick Gel Extraction Kit (Qiagen). Subsequently, cleaned-up products were cloned by insertion into pGEM-T Easy Vectors (Promega, Mannheim, Germany). Plasmid minipreps were grown overnight, cleaned-up, and sent for sequencing. All Pax2/5/8-gene orthologs were identified using the BLASTx algorithm screening the database of the National Center for Biotechnology Information (NCBI). All sequences and phylogenetic data that have been obtained in this study have been deposited in appropriate data bases.
Probe syntheses and whole-mount in situ hybridization
From the miniprepped plasmids the probe template was amplified via standard PCR using M13 forward and reverse primers, and in vitro transcription reactions were performed with these templates, digoxigenin-UTP (DIG RNA Labeling Kit, Roche Diagnostics GmbH), and SP6/ T7 polymerase (Roche Diagnostics GmbH) for the syntheses of antisense riboprobes, according to the manufacturer’s instructions. Whole-mount in situ hybridization experiments were carried out as described previously [36, 37]. Briefly, specimens were rehydrated into PBT (PBS + 0.1 % Tween-20). They were treated with Proteinase-K (50-60 μg/ml for Acanthochitona crinita, 10 μg/ml for Nucula tumidula, and 25 μg/ml for Idiosepius notoides) in PBT at 37 °C for 15 min and prehybridized in hybridization buffer for 4 h or overnight at 65 °C for A. crinita and I. notoides, and at 56 °C for N. tumidula. Hybridization with a probe concentration of 0.5 to 1 μg/ml was carried out overnight at the same temperatures as those for prehybridization. For I. notoides, a minimum of 20 individuals per stage was investigated and for A. crinita and N. tumidula approximately 50 individuals per stage. In addition, negative controls were carried out with sense probes for all genes and developmental stages. The majority of whole-mount preparations were cleared in a 3:1 solution of benzyl-benzoate and benzyl alcohol, mounted on objective slides, and analyzed. The results were documented with an Olympus BX53 microscope (Olympus, Hamburg, Germany) and polyplacophoran and bivalve developmental stages were additionally scanned with a Leica confocal SP5 II microscope (Leica Microsystems,Wetzlar, Germany) using bright-field and autofluorescence scans as well as the reflection mode . If necessary, images were processed with Adobe Photoshop 9.0.2 software (Adobe Systems, San Jose, USA) to adjust contrast and brightness. Sketch drawings were done with Adobe Illustrator CS5 software (Adobe Systems).
Specimens stored in 75 % EtOH or 100 % MetOH at −20 °C were rehydrated and washed several times in PBS. Samples were rinsed thrice for 20 min each in PBS with 2 % Triton X-100 (PBT) to increase tissue permeability and were then blocked for 4 h in PBT with 3 % normal swine serum (NSS; Jackson ImmunoResearch, West Grove, USA) at RT. Specimens were incubated for 12 to 24 h at RT in a cocktail of primary antibodies directed against serotonin (raised in rabbit, polyclonal, Immunostar, Hudson, USA) or FMRFamide-related peptides (raised in rabbit, polyclonal; Biotrend, Cologne, Germany), and a primary antibody against acetylated α-tubulin (raised in mouse, monoclonal; Sigma Aldrich), all diluted 1:800 in PBT with 2 % NGS. After rinsing specimens five times for 20 min each in PBS at RT, secondary fluorochrome–coupled antibodies were applied in a 1:1.000 dilution over night at RT. These cocktails contained PBT with 1 % NGS and Alexa Fluor 488 (anti-rabbit; Invitrogen) and Alexa Fluor 633 (anti-mouse, Invitrogen), as well as 1 % Hoechst 33342 ((2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole trihydrochloride trihydrate); Thermo Scientific) for cell nuclei staining. Samples were rinsed in PBS five times for 20 min each at RT and subsequently mounted on glass slides in Fluoromount G (Southern Biotech, Birmingham, Alabama, USA). Glass slides were stored in the dark at 4 °C until the mounting medium had hardened. Specimens were investigated and results analyzed as described above. Isosurfaces of FMRFamide-like reactive and serotonin-like immunoreactive elements were created by surface rendering algorithms using the 3D reconstruction imaging software IMARIS (Bitplane, Zurich, Switzerland). Specificity of the FMRFamide-like and serotonin-like antibodies was tested by omission of the primary antibodies and rendered no signal.
Statement of ethical approval
Animals were collected, anesthetized, and fixed according to internationally recognized standards (University of Queensland Animal Welfare Permit No. 158/09 “The cultivation of Idiosepius (pygmy squid) for studies in developmental biology” to BMD). Field work permission was granted by the ethics committee for every collection site of I. notoides used in this study.
Thomas Rattei und Thomas Eder are thanked for advice with transcriptome assembly. Dan Jackson (Göttingen), Martin Fritsch (Vienna), and Oliver Vöcking (Bergen) are thanked for technical advice. Emanuel Redl (Vienna) is thanked for collecting some of the postmetamorphic N. tumidula. TW thanks the staff of the research vessel Neomys (SBR in Roscoff) for collecting adult scaphopods and Henrik Glenner (Department of Biology, University of Bergen) for providing lab space and boat time. We are grateful to the crew of the RV Hans Brattström (University of Bergen) for assistance with the collection of adults of N. tumidula. TW was supported by ASSEMBLE (Association of European Marine Biological Laboratories) during his stay in at the SBR in Roscoff (France) and thanks the Faculty of Life Sciences, University of Vienna, for generous support. This work was support by an Australia Research Council grant to BMD and a grant by the Austrian Science Fund (FWF; grant number P24276-B22) on molluscan EvoDevo to AW.
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