Next Article in Journal
Relationship Between Groundwater Level and Rodent Community Structure Mediated by Nutrient Composition of Plants in Dongting Lake, China
Previous Article in Journal
Farmland Biodiversity Monitoring Using DNA Metabarcoding
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Diversity
Volume 17
Issue 8
10.3390/d17080586
diversity-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Molecular Updates on the ‘Warty Dorid’ Doris verrucosa Linnaeus, 1758 (Mollusca, Nudibranchia) from the Mediterranean Sea

by
Giulia Furfaro
1,2,*,
Michele Solca
1,2,
Enric Madrenas
3,
Francesco Tiralongo
4,5,6 and
Egidio Trainito
7
1
Department of Biological and Environmental Sciences and Technologies-DiSTeBA, University of Salento, Via Prov. le Lecce-Monteroni, 73100 Lecce, Italy
2
NBFC—National Biodiversity Future Center, Piazza Marina 61, 90133 Palermo, Italy
3
VIMAR (Vida Marina), C/Rocafort 246, 08029 Barcelona, Spain
4
Department of Biological, Geological and Environmental Sciences, University of Catania, 95124 Catania, Italy
5
Institute for Biological Resources and Marine Biotechnologies, National Research Council, 60125 Ancona, Italy
6
Ente Fauna Marina Mediterranea, Scientific Organization for Research and Conservation of Marine Biodiversity, 96012 Avola, Italy
7
Genoa Marine Centre Stazione Zoologica Anton Dohrn, Piazza del Principe 4, 16126 Genoa, Italy
*
Author to whom correspondence should be addressed.
Diversity 2025, 17(8), 586; https://doi.org/10.3390/d17080586
Submission received: 26 June 2025 / Revised: 28 July 2025 / Accepted: 13 August 2025 / Published: 20 August 2025
(This article belongs to the Section Marine Diversity)
Browse Figures
Versions Notes

Abstract

Basic and applied research reveals the importance of sea slugs as a source of new bioactive molecules or of still little-known intra/intercellular processes, mainly linked to the highly specialised defensive strategies typical of this group of shell-less molluscs. In this context, the nudibranch Doris verrucosa (Gastropoda, Mollusca), commonly known as ‘warty dorid’, is particularly interesting due to its ability to produce de novo biochemical compounds with pharmacological properties and being the type species of the genus Doris, one of the oldest and richest in species, currently characterised by a troubled systematics. Despite its wide distribution across the Eastern Atlantic Ocean and the Mediterranean Sea, this species has not yet been characterised from a genetic point of view. Considering the importance of assessing species identity to correctly investigate the systematics and to properly unravel potentially useful applications, results from a molecular assessment of such interesting species are provided. Genetic analysis involved species delimitation, phylogeny and haplotype network methods carried out on specimens of D. verrucosa collected from highly anthropised areas of Southern Italy (central Mediterranean Sea). Furthermore, in situ observations allowed us to fill some gaps in knowledge on the ecology and the morphological variability of this species that could be useful for future comparisons.

Graphical Abstract

1. Introduction

The study of Mediterranean marine Heterobranchia is an intriguing field of research that has gained renewed attention in recent decades due to the increased attention to conservation of neglected biodiversity and marine environments. Marine Heterobranchia molluscs are known for their unique adaptations and highly specialised defensive strategies, being model species in different fields of applied research such as neuroscience, biomedicine, and pharmaceutic [1,2,3,4,5]. In fact, some of the marine natural products currently tested as drugs were originally identified and isolated from sea slug molluscs [3] that can obtain, accumulate, and sometimes modify chemical compounds from their prey or directly biosynthesise them de novo [6]. Potential anticancer activity is currently known for compounds such as ‘kahalalide F’ obtained from sacoglossans, and ‘aplyronines’ and ‘dolastatins’ from anaspideans and from several different compounds in the Nudibranchia order and especially in Doridoidea [2,7,8,9,10,11]. However, the presence of biochemically active compounds is only one of the specialised traits evolved in response to the reduction and/or loss (in the adult stage) of the defensive shell [12]. Symbiotic relationships with different taxa [13,14,15], extraordinary regeneration ability and autotomy [16,17,18], and kleptopredation behaviour [19,20,21] are just a few examples of the great potential of these extremely specialised gastropods.
In this context, a nudibranch belonging to the Dorididae family, Doris verrucosa Linnaeus, 1758, has gained great scientific relevance, as it produces metabolites in the mantle, namely verrucosins (Verrucosins 1–9, Verrucosin A, and other diterpene glycerides) with ichthyotoxic and pharmacological properties [11,22,23,24]. This species biosynthesises these molecules de novo following a pathway not yet totally depicted [25] and can be considered an emergent model species. Doris verrucosa is a benthic nudibranch inhabiting rocky substrates around 0–12 m depth but also found in tide pool habitats where it may feed upon sponges of the genera Halicondria Fleming, 1828 and Hymeniacidon Bowerbank, 1858 [26,27,28]. Even if this species is one of the oldest known nudibranchs, originally described by Linnaeus in 1758 in the Systema Naturae, it was the object of a very complicated taxonomy that was solved only after the designation of a neotype from one specimen collected in Castropol, Asturias (Atlantic coast of Spain) that is currently deposited in the Museum National d’Histoire Naturelle in Paris (MNHN) [29]. To date, D. verrucosa is the type species of the genus Doris Linnaeus, 1758, one of the oldest and richest nudibranch genera with about 56 currently accepted species [30]. Doris verrucosa is characterised by some morphological diagnostic features, like the dorsum covered with simple, rounded tubercles, two tubercles around the rhinophoral cavity, and the presence of eight tubercles around the gills [31]. This species has also lateral, triangle-shaped extensions with a ventral furrow around the mouth, approximately 13 rhinophoral lamellae, tubercles covering the dorsum more elongated than rounded and distant from each other, a foot width that does not reach the edge of the notum, and with the same width/length of the anterior–posterior proportion [31,32]. Several authors have considered D. verrucosa a widespread species, with a geographic range including the western and eastern Atlantic Ocean; however, recent comparative anatomical studies have demonstrated that populations from the western Atlantic belong to a different species, namely Doris januarii (Bergh, 1878), while D. verrucosa is restricted to the Atlantic coast of Europe and the Mediterranean Sea [29,31,32]. The eastern Atlantic area of occurrence is wide since it ranges from the North Sea [33] to the Canary Islands [34,35,36] and probably to Ghana [37] and South Africa [38]. Within the Mediterranean Sea, D. verrucosa has been detected from the Spanish coast, with two specimens in the Barcelona Forum bathing area [27] and in the Catalan coast [35]; along the French coast, in Thau lagoon [39]; from Malta [40]; and in the Adriatic basin in Croatia [41] and in the Sicily Channel from the Tunis harbour, Tunisia [42]. In Italy it was collected in the central Tyrrhenian basin, from the Gulf of Naples, where it has been frequently sampled [23,24,25] and in the southern Tyrrhenian area from Ustica Island (reported as Doris cf. verrucosa) [43] and from the Faro Lake in north-eastern Sicily Island [44]. It was registered in the Ionian Sea along the Salento Peninsula, from the Mar Piccolo of Taranto, Santa Maria di Leuca [39,45], and close to Gallipoli (Lecce), where its spawning was observed in August [26].
Despite being a common species reported throughout the Mediterranean basin, noteworthy in applied research, and the type species of a representative nudibranch genus, it has been explored very little from a molecular point of view, with only two individuals, for the whole range of distribution, partially analysed so far [24,46]. Considering the importance of a correct identification of this species to properly interpret and discuss its potential application in other fields of research, a molecular study was conducted on specimens observed and collected from highly anthropised areas of Southern Italy. The main goals of the present study are to (i) characterise D. verrucosa from a molecular point of view, filling the gap of knowledge still existing on the genetics of this important species, (ii) provide molecular data on the nuclear H3 (histone H3) and the two mitochondrial COI (Cytochrome Oxidase subunit I) and 16S markers (the three most commonly used molecular markers in nudibranchs) that could be useful for future systematic studies, and (iii) add some data on the diagnostic characters, like the external colour variability and the shape of the egg masses that could be useful for a more effective species detection and identification in the future.

2. Materials and Methods

2.1. Field and Laboratory Sampling

A preliminary bibliographic study was conducted to define the geographic range of the known distribution of this species. Monitoring took place in two different sectors of the Italian seas (according to [47]), namely the north-eastern part of Sicily Island (Strait of Messina) and the Salento peninsula in southern Apulia (Ionian Sea). The studied areas consisted of semi-closed basins characterised by a high level of anthropogenic impact and pollution and by fish and mussel farms [44,48,49].
The coastal Faro Lake (North-Eastern Sicily) was studied using a visual census followed by the collection, cataloguing, and storing of some individuals for future molecular analyses. The Sicilian specimens were observed through snorkelling and SCUBA diving activities, and the pictures were taken in situ or in the laboratory.
In the framework of a project of the marine zoology group of the University of Salento, the Heterobranchia fauna from the Mar Piccolo of Taranto (Ionian Sea) was investigated between 2020 and 2024. A monthly SCUBA diving activity allowed us to observe and photograph the specimens in situ and in the laboratory and to collect and store them in EtOH 96% for further morphological and molecular analyses. All the collected specimens were catalogued (vouchers ‘RM3’) and stored in the Heterobranchia collection deposited at the Department of Biological and Environmental Sciences and Technologies (DiSTeBA) of the Salento University.
The photographic in situ documentation was obtained using a Nikon D500 camera (Tokyo, Japan) with a Tokina 10/17 lens (Tokyo, Japan), in an Isotta underwater housing with an 8” dome by Isotecnic (Castelnuovo del Garda, Italy) and two Sea&Sea YS-D3 underwater flashes (Tokyo, Japan), and post-produced with Camera Raw V7.0 and Photoshop CS6. Observation in the laboratory at a high magnification level was carried out under the stereomicroscope Nikon SMZ800N equipped with the Nikon Digital Sight 1000 camera.

2.2. Molecular Analysis

Total genomic DNA was extracted from a small piece of tissue of five collected specimens by using the ‘salting out’ procedure [50]. The primer pairs LCO1490 and HCO2198 [51], 16Sar-L and 16Sbr-H [52], and H3AD-F and H3BD-R [53] were used for the amplification of the two mitochondrial markers, cytochrome oxidase subunit I (COI) and 16S and the nuclear histone H3, respectively. The PCR reaction mix and the temperature profile used to amplify the three molecular markers were the same already reported in [54]. The amplified products were sequenced at the European Division of Macrogen Inc. (Milan, Italy). The newly obtained sequences were edited with Staden Package 2.0.0b9 [55] and checked using the BLASTN V2.17.0 [56] to exclude contaminations and to confirm the identity of the sequenced fragments. Newly obtained sequences were deposited in GenBank (available at https://www.ncbi.nlm.nih.gov/). Consensus sequences were aligned together with sequences already available in GenBank using the Muscle algorithm implemented in MEGA version 11 [57]. Four different alignments were generated: three single-gene datasets (COI, 16S, and H3) and one with the three genes concatenated and partitioned (ConcDNA). The program Gblocks 0.91b with less stringent options [58,59] was used to eliminate poorly aligned positions or hyper-divergent regions of the multiple sequence alignment of the 16S rDNA. The best-fitting evolutionary model for each of the four datasets (three single genes and one concatenated and partitioned) was determined using JModelTest version 2.1.10 under the BIC model [60]. To generate the concatenated and partitioned dataset, the program DnaSP 6.12.03 [61] was used. Analysis of the different haplotypes was carried out to reconstruct the COI network by using the program PopArt (Population Analysis with Reticulate Trees) (available at https://popart.maths.otago.ac.nz/, accessed on 4 May 2024) with the TCS inference method [62]. Prior to conducting phylogenetic analyses using COI sequence data, we evaluated substitution saturation in the COI alignment using the entropy-based index of substitution saturation developed by Xia et al. [63], as implemented in DAMBE v7.3.32 [64]. The proportion of invariant sites (Pinv) was set to 0.1468 based on the estimate obtained in DAMBE. Bayesian inference and maximum likelihood phylogenetic analyses were carried out to investigate the evolutionary relationships. The Bayesian inference analysis (BI) was performed using the program MrBayes (v. 3.2.6) [65], applying a Bayesian posterior likelihood methodology. Each of the four runs was conducted with four MCMC (Markov chain Monte Carlo) for five million generations, a sample frequency of one tree per 1000 generations and a burn-in of 25%. Maximum likelihood analysis was performed using raxmlGUI 1.5b2 [66], a graphical front-end for RAxML 8.2.1 [67], with 100 independent ML searches and 1000 bootstrap replicates. The species Goniobranchus vibratus (Pease, 1860) was selected as the outgroup species for both analyses.

3. Results

The results from the bibliographic study on the geographic range of distribution of this species and the on-field investigations allowed us to reveal the presence of many individuals of Doris verrucosa from the two marine sectors investigated here (Figure 1).
Four specimens were photographed from the Faro Lake, three of them collected, and many were observed from the Mar Piccolo of Taranto: six were photographed in situ and collected (Table 1).
In situ observations allowed us to obtain and document (see Table 1) useful information on the bathymetric range, preferred season, and water temperature of D. verrucosa, along with images of mating and egg-laying behaviours (Figure 2).
The body colour pattern is variable, going from a brown morphotype, through an orange one, and to the more common morphotype with bright yellow and pale creamy colours (Figure 3). The general shape of the body and the diagnostic features perfectly match those reported in the original and subsequent descriptions of this species (see the Introduction Section); however, it is noteworthy that a lighter area extends centrally along the entire dorsum, flanked by darker lateral areas. This pattern is present regardless of the colour of the different morphotypes (Figure 3).
All the D. verrucosa individuals were found in transitional water systems used as aquaculture sites, which are very complex ecotones characterised by high spatial and temporal variability of physico-chemical characteristics and productivity patterns [68]. The Sicilian specimens were found in an environment characterised by a high turnover in benthic communities and with sediments at the bottom that are in part anthropogenized, being reworked by the mollusc farmers, and very rich in remnants of shells of cultivated bivalves [68,69]. The Apulian specimens were found in a rhodolite bed habitat, extending over a sandy bottom without any significant cover of algae or phanerogams, down to a depth of approximately 3 m [70].

Molecular Analysis

A total of 15 sequences, one for each of the three molecular markers, were obtained from five specimens of D. verrucosa collected in the Tyrrhenian (Sicily) and Ionian Seas (Apulia) (Table 2). These newly obtained sequences were compared with those already present in GenBank and belonging to the same species or to congeneric and/or closely related taxa for a final dataset consisting of 67 total sequences (Table 2).
The COI single dataset included 29 sequences, and the alignment was 647 bp long. The proportion of invariant sites (Pinv) was set to 0.1468 based on the estimate obtained in DAMBE. The calculated Iss value was 0.3301, which is significantly lower (p = 0.0000) than the critical Iss.c value of 0.7289 (assuming a symmetrical tree topology), indicating limited substitution saturation. These results suggest that the COI sequences are informative and appropriate for phylogenetic inference. The 16S single dataset contained 23 sequences with a final alignment of 359 bp after GBlock processing. The histone H3 single gene dataset had 16 sequences and was 328 bp long. The concatenated (COI + 16S + H3) and partitioned dataset (ConcDNA) consisted of 20 taxa, and the alignment was 1334 bp. The best evolutionary models selected for the COI, 16S, and H3 were, respectively, TIM3 + I + G, TPM3uf + G, and K80 + G for the single gene datasets and TIM3 + I + G, TPM3uf + G, and TPM2 + G for each partition of the ConcDNA dataset. All the resulting trees were congruent with each other but showed a different ability to resolve phylogenetic relationships at different taxonomic levels. In fact, the COI single gene analysis proved powerful to investigate at the species level (Figure 4), while the concatenated analysis was the best to investigate at deeper phylogenetic relationships (Figure 5). The single gene analyses carried out using the 16S and the H3 molecular markers were congruent with COI and the concatenated and partitioned analysis, but, as expected [71,72,73], with low statistical support; for this reason, these analyses are not shown here. Values of posterior probability (from the Bayesian analysis) higher than 0.90 and of bootstrap (from the maximum likelihood) higher than 70% were considered, while values lower than 0.50 and 50, respectively, were not reported in the final topologies. The COI single-gene dataset was used to investigate at the species taxonomic level, with all the species showing high support values except for the D. berghi (Vayssière, 1901) clade, which was supported only by Bayesian analysis and not the maximum likelihood inference. The monophyletic clade consisted, with high support (BPP = 1, BP = 99), of all the D. verrucosa individuals (Figure 4). Furthermore, an additional specimen belonging to the Doris marmorata Risso, 1818 species helped us to clarify a case of misidentifications that had occurred in a previous study focused on this species and the closely related Doris bertheloti A. d’Orbigny, 1839 [46]. In fact, the addition of the newly collected specimen (voucher RM3_3419) in the present molecular study revealed that the individual with voucher ZSMMol20210045 (COI accession = OR286429) previously identified as ‘Doris bertheloti’ indeed belongs to D. marmorata (Figure 4) as well as the specimen with voucher ZSM20240263 (COI accession = OR286430) wrongly assessed to D. marmorata is indeed D. bertheloti (Figure 4). The TCS haplotype network analysis was performed on the COI single-gene alignment and revealed each five investigated D. verrucosa being characterised by their own unique haplotypes, suggesting a high genetic variability within this species and a reduced genetic flow between different populations (Figure 4).
The Bayesian and maximum likelihood analyses carried out on the concatenated and partitioned dataset (ConcDNA) gave congruent topologies and were useful to unravel phylogenetic relationships at higher taxonomic levels (Figure 5). In fact, the results suggest that D. ocelligera (Bergh, 1881) is sister to D. verrucosa (BPP = 0.9, BP = 66), evidence that was not revealed in the recent systematic study carried out on this group [46], and with D. berghi complex as sister to them (BPP = 1, BP = 100). Doris marmorata and D. bertheloti confirmed as sister taxa (BPP = 1, BP = 100) and grouped (with no statistical support) (BPP = 0.68, BP = <50) with a clade non supported (BPP = 0.76, BP = <50) that includes the conspecific D. montereyensis J.G. Cooper, 1863 (BPP = 1, BP = 100) and the outgroups Avaldesia albomacula (J. M. Chan & Gosliner, 2007) and Halgerda meringuecitrea Tibiriçá, Pola & Cervera, 2018 (BPP = 0.71, BP = <50) (Figure 5).

4. Discussion

Ten specimens of Doris verrucosa were studied from two localities of Southern Italy, consisting of areas highly impacted by human activities, like coastal lakes and semi-closed basins.
In this context, D. verrucosa shows a wide distribution across the whole Mediterranean basin; however, evidence from the present study suggests that the area of coverage is the western and central Mediterranean Sea, but with an important contribution coming from localities used as aquaculture sites, with high human impact. In fact, D. verrucosa is an euryhaline species as it lives from brackish [44] to marine waters and prefers shallower basins characterised by low hydrodynamics and eutrophic water. The ability shown by this species to live in a polluted environment with highly variable biotic and abiotic factors is noteworthy and becomes even more appealing if considering the biochemical compounds that are synthesised by D. verrucosa and that are characterised by antimicrobial and antiviral activities [74], abilities that could be particularly useful for the well-being of this species in such difficult environments. The variability observed in the body colour pattern of D. verrucosa (Figure 3) is in line with what is reported for other nudibranchs that can have different background body colours according to the prey they feed on [75,76]. In these regards, detailed images of the wide body colour variability (Figure 3) and of the shape of the egg masses with details of the veliger larvae (Figure 2c–f) are documented here and shown for the first time. These characters are important to define the ranges of intraspecific variability and could also be significant at higher taxonomic levels. In fact, the shape of the egg mass was revealed as a powerful diagnostic character also at the genus and family taxonomic levels [77]. For this reason, images of the egg masses of D. verrucosa, the type species of the genus Doris, are even more important.
Molecular investigations carried out in this study using the molecular markers mostly used in nudibranchs (i.e., COI, 16S, and H3) revealed a complicated phylogenetic history that is far from being fully understood yet (Figure 4 and Figure 5). In fact, future analyses including a more representative dataset are required to better understand the systematics of the Doris genus, especially considering that it is one of the first genera to be described and in which many species with unknown taxonomy were included, causing a consequent general confusion. However, the present study allowed us to add some important insights, like the possible sister relationship between D. verrucosa and D. ocelligera (Figure 5) that was not revealed from the recent phylogenetic study carried out on the family [46]. Finally, it is noteworthy to mention that D. berghi is a possible case of species complex since the molecular results show a high genetic intraspecific diversity at the COI barcoding marker (going from 0.5% to 13.1% of minimum and maximum uncorrected p-distances, respectively) with values that fall within the range of interspecific variability commonly accepted for nudibranchs [71,78,79]. Moreover, the D. marmorata specimen (voucher RM3_3419) observed and collected in Girona (Spain) and included in the present study (Figure 6) helped us to clarify the incorrect identifications made in the past between D. marmorata and D. bertheloti that have generated confusion in the phylogenetic analyses (and in the relative table) reported in Renau and collaborators [46].
Some speculations can be proposed based on the results of the present study and particularly regarding the importance of monitoring highly variable marine environments such as transitional waters and coastal areas with high human impact. In fact, these overlooked habitats, like ports, coastal lakes and brackish environments, have recently been revealed to be important to unravel rare or unknown diversity [80], to detect non-indigenous species early [81], and to study Heterobranchia species that are adapted to live under polluted and stressed conditions.
Finally, monitoring the future changes in the distribution range, as well as in other ecological traits of D. verrucosa, can highlight possible shifts in the conditions of natural environments and the presence of hidden environmental stressors. In this regard, it is interesting to note that we observed a potential anticipation in egg laying compared to what was previously observed for D. verrucosa. In fact, the specimens from Mar Piccolo of Taranto laid their eggs in June, while Perrone [26] observed individuals from nearby Gallipoli (about 80 Km south of the Mar Piccolo of Taranto) with eggs in August. This anticipation from late summer to early summer could be a mirror of the increasing sea water temperature due to global warming, confirming the Heterobranchia as useful bioindicators of the environmental status [82].

Author Contributions

Conceptualization, G.F. and E.T.; methodology, G.F. and E.T.; software, G.F.; validation, G.F., M.S., F.T. and E.T.; formal analysis, G.F.; investigation, G.F., M.S., E.M., F.T. and E.T.; resources, G.F.; data curation, G.F., M.S. and E.T.; writing—original draft preparation, G.F.; writing—review and editing, G.F., M.S., F.T. and E.T.; supervision, G.F.; funding acquisition, G.F. All authors have read and agreed to the published version of the manuscript.

Funding

G.F. and M.S. were supported by the project funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4—Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union—NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP D33C22000960007, Project title ‘National Biodiversity Future Center—NBFC’.

Data Availability Statement

Newly produced DNA sequences will be submitted to GenBank (at https://www.ncbi.nlm.nih.gov/) after paper acceptance.

Acknowledgments

The authors are grateful to Daniele Salvi for his valuable advice on molecular analyses, to Paolo Mariottini and Salvatore Giacobbe for their help during Sicilian sampling activities and to Sabrina Lo Brutto and Alex Carnevale for their kind support. All the authors wish to thank the two anonymous reviewers who helped in improving the quality of the present manuscript.

Conflicts of Interest

Author Enric Madrenas was employed by the company VIMAR (Vida Marina). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
NISNon-Indigenous Species
COICytochrome Oxidase Subunit I
H3Histone H3

References

  1. Cimino, G.; Ghiselin, M.T. Chemical defense and evolutionary trends in biosynthetic capacity among dorid nudibranchs (Mollusca: Gastropoda: Opisthobranchia). Chemoecology 1999, 9, 187–207. [Google Scholar] [CrossRef]
  2. Dean, L.J.; Prinsep, M.R. The chemistry and chemical ecology of nudibranchs. Nat. Prod. Rep. 2017, 34, 1359–1390. [Google Scholar] [CrossRef]
  3. Gavagnin, M.; Carbone, M.; Ciavatta, M.L.; Mollo, E. Natural products from marine heterobranchs: An overview of recent results. Chem. J. Mold. 2019, 14, 9–31. [Google Scholar] [CrossRef]
  4. Katz, P.S.; Quinlan, P.D. The importance of identified neurons in gastropod molluscs to neuroscience. Curr. Opin. Neurobiol. 2019, 56, 1–7. [Google Scholar] [CrossRef]
  5. Zhuo, J.; Gill, J.P.; Jansen, E.D.; Jenkins, M.W.; Chiel, H.J. Use of an invertebrate animal model (Aplysia californica) to develop novel neural interfaces for neuromodulation. Front. Neurosci. 2022, 16, 1080027. [Google Scholar] [CrossRef]
  6. Avila, C. Terpenoids in marine heterobranch molluscs. Mar. Drugs 2020, 18, 162. [Google Scholar] [CrossRef] [PubMed]
  7. Faircloth, G.; Cuevas, C. Kahalalide F and ES285: Potent anticancer agents from marine molluscs. In Molluscs: From Chemo-ecological Study to Biotechnological Applications; Cimino, G., Gavagnin, M., Eds.; Springer: Berlin, Germany, 2006; pp. 363–379. [Google Scholar]
  8. Molinski, T.F.; Dalisay, D.S.; Lievens, S.L.; Saludes, J.P. Drug development from marine natural products. Nat. Rev. Drug Disc. 2009, 8, 69–85. [Google Scholar] [CrossRef]
  9. Newman, D.J.; Cragg, G.M. Marine-sourced anti-cancer and cancer pain control agents in clinical and late preclinical development. Mar. Drugs 2014, 12, 255–278. [Google Scholar] [CrossRef]
  10. Kigoshi, H.; Kita, M. Antitumor effects of sea hare-derived compounds in cancer. In Handbook of Anticancer Drugs from Marine Origin; Kim, S.K., Ed.; Springer: Cham, Switzerland, 2015; pp. 701–739. [Google Scholar]
  11. Avila, C.; Núñez-Pons, L.; Renau, J. From the Tropics to the Poles: Chemical defense strategies in sea slugs (Mollusca: Heterobranchia). In Chemical Ecology; CRC Press: Boca Raton, FL, USA, 2018; pp. 71–163. [Google Scholar]
  12. Wägele, H.; Klussmann-Kolb, A. Opisthobranchia (Mollusca, Gastropoda)—More than just slimy slugs. Shell reduction and its implications on defence and foraging. Front. Zool. 2005, 2, 1–18. [Google Scholar] [CrossRef]
  13. Pelletreau, K.N.; Worful, J.M.; Sarver, K.E.; Rumpho, M.E. Laboratory culturing of Elysia chlorotica reveals a shift from transient to permanent kleptoplasty. Symbiosis 2012, 58, 221–232. [Google Scholar] [CrossRef]
  14. Chan, C.X.; Vaysberg, P.; Price, D.C.; Pelletreau, K.N.; Rumpho, M.E.; Bhattacharya, D. Active host response to algal symbionts in the sea slug Elysia chlorotica. Mol. Biol. Evol. 2018, 35, 1706–1711. [Google Scholar] [CrossRef] [PubMed]
  15. Furfaro, G.; Solca, M.; Mariottini, P. Crustaceans and marine Heterobranchia: A new symbiotic relationship in the Mediterranean Sea. Diversity 2021, 13, 613. [Google Scholar] [CrossRef]
  16. Mitoh, S.; Yusa, Y. Extreme autotomy and whole-body regeneration in photosynthetic sea slugs. Curr. Biol. 2021, 31, 233–234. [Google Scholar] [CrossRef]
  17. Furfaro, G.; Mariottini, P. Looking at the nudibranch family Myrrhinidae (Gastropoda, Heterobranchia) from a mitochondrial ‘2D folding structure’ point of view. Life 2021, 11, 583. [Google Scholar] [CrossRef]
  18. Gutierrez-Andrade, D.; Middlebrooks, M.L. Ceratal autotomy as a defensive mechanism of the sacoglossan sea slug Placida kingstoni against a generalist crustacean predator. J. Molluscan Stud. 2023, 89, eyad013. [Google Scholar] [CrossRef]
  19. Willis, T.J.; Berglöf, K.T.; McGill, R.A.; Musco, L.; Piraino, S.; Rumsey, C.M.; Fernández, T.V.; Badalamenti, F. Kleptopredation: A mechanism to facilitate planktivory in a benthic mollusc. Biol. Lett. 2017, 13, 20170447. [Google Scholar] [CrossRef] [PubMed]
  20. Goodheart, J.A.; Bleidißel, S.; Schillo, D.; Strong, E.E.; Ayres, D.L.; Preisfeld, A.; Cummings, M.P.; Wägele, H. Comparative morphology and evolution of the cnidosac in Cladobranchia (Gastropoda: Heterobranchia: Nudibranchia). Front. Zool. 2018, 15, 1–18. [Google Scholar] [CrossRef]
  21. Maggioni, D.; Furfaro, G.; Solca, M.; Seveso, D.; Galli, P.; Montano, S. Being safe, but not too safe: A nudibranch feeding on a bryozoan-associated hydrozoan. Diversity 2023, 15, 484. [Google Scholar] [CrossRef]
  22. Avila, C.; Ballesteros, M.; Cimino, G.; Crispino, A.; Gavagnin, M.; Sodano, G. Biosynthetic origin and anatomical distribution of the main secondary metabolites in the nudibranch mollusc Doris verrucosa. Comp. Biochem. Physiol. Part B Comp. Biochem. 1990, 97, 363–368. [Google Scholar] [CrossRef]
  23. Gavagnin, M.; Ungur, N.; Castelluccio, F.; Cimino, G. Novel Verrucosins from the Skin of the Mediterranean Nudibranch Doris verrucosa. Tetrahedron 1997, 53, 1491–1504. [Google Scholar] [CrossRef]
  24. De Masi, L.; Adelfi, M.G.; Pignone, D.; Laratta, B. Identification of Doris verrucosa mollusc via mitochondrial 16S rDNA. Biochem. Syst. Ecol. 2015, 58, 21–29. [Google Scholar] [CrossRef]
  25. Fontana, A.; Tramice, A.; Cutignano, A.; d’Ippolito, G.; Renzulli, L.; Cimino, G. Studies of the biogenesis of verrucosins, toxic diterpenoid glycerides of the Mediterranean mollusc Doris verrucosa. Eur. J. Org. Chem. 2003, 16, 3104–3108. [Google Scholar] [CrossRef]
  26. Perrone, A. Opistobranchi (Aplysiomorpha, Pleurobrancomorpha, Sacoglossa, Nudibranchia) del litorale salentino (Mar Jonio) (Elenco–Contributo primo). Thalass. Salentina 1983, 12, 118–144. [Google Scholar]
  27. Parera, A.; Pontes, M.; Salvador, X.; Ballesteros, M. Sea-slugs (Mollusca, Gastropoda, Heterobranchia): The other inhabitants of the city of Barcelona (Spain). Butlletí De La Inst. Catalana D’histōria Nat. 2020, 84, 75–100. [Google Scholar]
  28. López-Acosta, M.; Potel, C.; Gallinari, M.; Pérez, F.F.; Leynaert, A. Predation data of the sponge-feeding nudibranch Doris verrucosa on the sponge Hymeniacidon perlevis. Digital CSIC 2022. [Google Scholar] [CrossRef]
  29. Bouchet, P.; Valdés, A. Case 3088. Doris verrucosa Linnaeus; 1758 (Mollusca, Gastropoda): A proposed conservation of the generic and specific names by designation of a neotype. Bull. Zool. Nomencl. 2000, 57, 74–80. [Google Scholar]
  30. MolluscaBase. MolluscaBase. Doris Linnaeus, 1758. World Register of Marine Species. Available online: https://www.marinespecies.org/aphia.php?p=taxdetails&id=137914 (accessed on 6 August 2024).
  31. Valdés, A. A phylogenetic analysis and systematic revision of the cryptobranch dorids (Mollusca, Nudibranchia, Anthobranchia). Zool. J. Linn. Soc. 2002, 136, 535–636. [Google Scholar] [CrossRef]
  32. Lima, P.O.; Simone, L.R. Anatomical review of Doris verrucosa and redescription of Doris januarii (Gastropoda, Nudibranchia) based on comparative morphology. J. Mar. Biol. Assoc. UK 2015, 95, 1203–1220. [Google Scholar] [CrossRef]
  33. Høisæter, T. An annotated check-list of marine molluscs of the Norwegian coast and adjacent waters. Sarsia 1986, 71, 73–175. [Google Scholar] [CrossRef]
  34. Cervera, J.L.; Templado, J.; García-Gómez, J.C.; Ballesteros, M.; Ortea, J.; García, F.J.; Ros, J.; Luque, A.A. Catálogo actualizado y comentado de los Opistobranquios (Mollusca, Gastropoda) de la Península Ibérica, Baleares y Canarias, con algunas referencias a Ceuta y la isla de Alborán. Iberus 1988, 1, 1–84. [Google Scholar]
  35. Cervera, J.L.; Calado, G.; Gavaia, C.; Malaquias, M.A.; Templado, J.; Ballesteros, M.; García-Gómez, J.C.; Megina, C. An annotated and updated checklist of the ophisthobranchs (Mollusca: Gastropoda) from Spain and Portugal (including islands and archipelagos). Boletín Inst. Español Oceanogr. 2004, 20, 1–122. [Google Scholar]
  36. Moro, L.; Ocaña, O.; Bacallado, J.J.; Martín, J.; Ortea, J. Nota sobre tres babosas marinas (Nudibranchia y Sacoglossa) colectadas en las costas atlánticas de África (Cabo Espartel, Agadir y Dakhla), con un listado de especies acompañantes. Vieraea 2017, 45, 205–214. [Google Scholar] [CrossRef]
  37. Edmunds, M. Larval development, oceanic currents, and origins of the Opisthobranch fauna of Ghana. J. Molluscan Stud. 1977, 43, 301–308. [Google Scholar] [CrossRef]
  38. Gosliner, T.M. Nudibranchs of Southern Africa. A Guide to Opisthobranchs Molluscs of Southern Africa; Sea Challengers and Jeff Hamann: Monterey, CA, USA, 1987; p. 136. [Google Scholar]
  39. Ballesteros, M.; Madrenas, E.; Pontes, M. Doris verrucosa. 2021. Available online: https://opistobranquis.info/en/?p=496 (accessed on 14 October 2023).
  40. Sammut, C.; Perrone, A.S. A preliminary check-list of Opisthobranchia (Mollusca, Gastropoda) from the Maltese Islands. Basteria 1998, 62, 221–240. [Google Scholar]
  41. Prkić, J.; Petani, A.; Iglić, Ð.; Lanča, L. Stražnjoškržnjaci Jadranskoga Mora: Slikovni Atlas i Popis Hrvatskih Vrsta/Opisthobranchs of the Adriatic Sea: Photographic Atlas and List of Croatian Species; Ronilaćki Klub Sveti Roko: Bibinje, Croatia, 2018. [Google Scholar]
  42. Antit, M.; Kalthoumi, D.; Pola, M.; Urra, J.; Azzouna, A. First Record of Doris verrucosa Linnaeus, 1758 (Mollusca: Heterobranchia: Nudibranchia) in the bay of Tunis, Tunisia (Central Mediterranean). In Recent Advances in Environmental Science from the Euro-Mediterranean and Surrounding Regions, Proceedings of the EMCEI 2017. Advances in Science, Technology & Innovation (IEREK Interdisciplinary Series for Sustainable Development); Sousse, Tunisia, 20–25 November 2017, Kallel, A., Ksibi, M., Ben Dhia, H., Khélifi, N., Eds.; Springer: Cham, Switzerland, 2018. [Google Scholar] [CrossRef]
  43. Chemello, R. Studio della malacofauna costiera dell’isola di Ustica (Gastropoda). Lav. Della Soc. Ital. Malacol. 1986, 22, 51–76. [Google Scholar]
  44. Vitale, D.; Giacobbe, S.; Spinelli, A.; De Matteo, S.; Cervera, J.L. “Opisthobranch” (mollusks) inventory of the Faro Lake: A Sicilian biodiversity hot spot. Ital. J. Zool. 2016, 83, 524–530. [Google Scholar] [CrossRef]
  45. Furfaro, G.; Vitale, F.; Licchelli, C.; Mariottini, P. Two Seas for One Great Diversity: Checklist of the Marine Heterobranchia (Mollusca; Gastropoda) from the Salento Peninsula (South-East Italy). Diversity 2020, 12, 171. [Google Scholar] [CrossRef]
  46. Renau, M.F.; Salvador, X.; Moles, J. Molecular and morpho-anatomical assessment of the family Dorididae (Mollusca, Nudibranchia) in the Mediterranean and North-East Atlantic. Eur. J. Taxon. 2024, 943, 59–79. [Google Scholar] [CrossRef]
  47. Bianchi, C.N. Proposta di suddivisione dei mari italiani in settori biogeografici. Not. SIBM 2004, 46, 57–59. [Google Scholar]
  48. Bruccoleri, M.; Cannova, P.; Cruz-Pérez, N.; Rodríguez-Martín, J.; Ioras, F.; Santamarta, J.C. Leisure Boating Environmental footprint: A study of leisure marinas in Palermo, Italy. Sustainability 2022, 15, 182. [Google Scholar] [CrossRef]
  49. Furfaro, G.; D’Elia, M.; Mariano, S.; Trainito, E.; Solca, M.; Piraino, S.; Belmonte, G. SEM/EDX analysis of stomach contents of a sea slug snacking on a polluted seafloor reveal microplastics as a component of its diet. Sci. Rep. 2022, 12, 10244. [Google Scholar] [CrossRef] [PubMed]
  50. Aljanabi, S.M.; Martinez, I. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques. Nucleic Acids Res. 1997, 25, 4692–4693. [Google Scholar] [CrossRef] [PubMed]
  51. Folmer, O.; Black, M.; Hoeh, W.; Lutz, R.; Vrijenhoek, R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994, 3, 294–299. [Google Scholar]
  52. Palumbi, S.R.; Martin, A.; Romano, S.; McMillan, W.O.; Stice, L.; Grabowski, G. The Simple Fool’s Guide to PCR; Department of Zoology and Kewalo Marine Laboratory, University of Hawaii: Honolulu, HI, USA, 2002. [Google Scholar]
  53. Colgan, D.J.; McLauchlan, A.; Wilson, G.D.F.; Livingston, S.P.; Edgecombe, G.D.; Macaranas, J.; Cassis, G.; Gray, M.R. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust. J. Zool. 1998, 46, 419–437. [Google Scholar] [CrossRef]
  54. Furfaro, G.; Renda, W.; Nardi, G.; Giacobbe, S. Integrative Taxonomy of the Bubble Snails (Cephalaspidea, Heterobranchia) Inhabiting a Promising Study Area: The Coastal Sicilian Faro Lake (Southern Italy). Water 2023, 15, 2504. [Google Scholar] [CrossRef]
  55. Staden, R.; Beal, K.F.; Bonfield, J.K. The Staden package; 1998. Methods Mol. Biol. 2000, 132, 115–130. [Google Scholar] [CrossRef]
  56. Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 1990, 215, 403–410. [Google Scholar] [CrossRef]
  57. Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef]
  58. Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol. Biol. Evol. 2000, 17, 540–552. [Google Scholar] [CrossRef]
  59. Talavera, G.; Castresana, J. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 2007, 56, 564–577. [Google Scholar] [CrossRef] [PubMed]
  60. Posada, D. JModelTest: Phylogenetic Model Averaging. Mol. Biol. Evol. 2008, 25, 1253–1256. [Google Scholar] [CrossRef]
  61. Rozas, J.; Ferrer-Mata, A.; Sánchez-DelBarrio, J.C.; Guirao-Rico, S.; Librado, P.; Ramos-Onsins, S.E.; Sánchez-Gracia, A. DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol. Biol. Evol. 2017, 34, 3299–3302. [Google Scholar] [CrossRef]
  62. Clement, M.; Snell, Q.; Walker, P.; Posada, D.; Crandall, K. TCS: Estimating gene genealogies. In Proceedings of the 16th International Parallel and Distributed Processing Symposium (IPDPS 2002), Fort Lauderdale, FL, USA, 15–19 April 2002; Volume 2, p. 184. [Google Scholar]
  63. Xia, X.; Xie, Z.; Salemi, M.; Chen, L.; Wang, Y. An index of substitution saturation and its application. Mol. Phylogenetics Evol. 2003, 26, 1–7. [Google Scholar] [CrossRef] [PubMed]
  64. Xia, X. DAMBE7: New and improved tools for data analysis in molecular biology and evolution. Mol. Biol. Evol. 2018, 35, 1550–1552. [Google Scholar] [CrossRef]
  65. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef] [PubMed]
  66. Silvestro, D.; Michalak, I. RaxmlGUI: A Graphical Front-End for RAxML. Org. Divers. Evol. 2012, 12, 335–337. [Google Scholar] [CrossRef]
  67. Stamatakis, A. RAxML Version 8: A Tool for Phylogenetic Analysis and Post-Analysis of Large Phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  68. Giacobbe, S. Biodiversity loss in Sicilian transitional waters: The molluscs of Faro Lake. Biodivers. J. 2012, 3, 501–510. [Google Scholar]
  69. Somma, R.; Spoto, S.E.; Giacobbe, S. Geological and structural framework, inventory, and quantitative assessment of geodiversity: The case study of the lake Faro and lake Ganzirri global geosites (Italy). Geosciences 2024, 14, 236. [Google Scholar] [CrossRef]
  70. Pierri, C.; Longo, C.; Falace, A.; Gravina, M.F.; Gristina, M.; Kaleb, S.; Lazic, T.; Lisco, S.; Moretti, M.; Putignano, M.; et al. Invertebrate diversity associated with a shallow rhodolith bed in the Mediterranean Sea (Mar Piccolo of Taranto, south-east Italy). Aquat. Conserv. Mar. Freshw. Ecosyst. 2024, 34, e4054. [Google Scholar] [CrossRef]
  71. Furfaro, G.; Salvi, D.; Mancini, E.; Mariottini, P. A Multilocus View on Mediterranean Aeolid Nudibranchs (Mollusca): Systematics and Cryptic Diversity of Flabellinidae and Piseinotecidae. Mol. Phylogenet. Evol. 2018, 118, 13–22. [Google Scholar] [CrossRef]
  72. Furfaro, G.; Salvi, D.; Trainito, E.; Vitale, F.; Mariottini, P. When morphology does not match phylogeny: The puzzling case of two sibling nudibranchs (Gastropoda). Zool. Scr. 2021, 50, 439–454. [Google Scholar] [CrossRef]
  73. Furfaro, G.; Schreier, C.; Trainito, E.; Pontes, M.; Madrenas, E.; Girard, P.; Mariottini, P. The Sea Slug Doriopsilla areolata Bergh, 1880 (Mollusca, Gastropoda) in the Mediterranean Sea: Another Case of Cryptic Diversity. Diversity 2022, 14, 297. [Google Scholar] [CrossRef]
  74. Pani, A.; Marongiu, M.E.; Obino, P.; Gavagnin, M.; La Colla, P. Antimicrobial and antiviral activity of xylosyl-methylthio-adenosine, a naturally occurring analogue of methylthio-adenosine from Doris verrucosa. Experientia 1991, 47, 1228–1229. [Google Scholar] [CrossRef]
  75. Furfaro, G.; Trainito, E.; De Lorenzi, F.; Fantin, M.; Doneddu, M. Tritonia nilsodhneri Marcus Ev., 1983 (Gastropoda, Heterobranchia, Tritoniidae): First records for the Adriatic Sea and new data on ecology and distribution of Mediterranean populations. Acta Adriat. 2017, 58, 261–268. [Google Scholar] [CrossRef]
  76. Van Den Berg, C.P.; Endler, J.A.; Drummond, L.; Dawson, B.R.; Santiago, C.; Weber, N.; Cheney, K.L. Colour patterns are less variable in highly unpalatable nudibranchs. bioRxiv 2023. [Google Scholar] [CrossRef]
  77. Furfaro, G.; Trainito, E.; Fantin, M.; D’Elia, M.; Madrenas, E.; Mariottini, P. Mediterranean matters: Revision of the family Onchidorididae (Mollusca, Nudibranchia) with the description of a new genus and a new species. Diversity 2022, 15, 38. [Google Scholar] [CrossRef]
  78. Garzia, M.; Mariottini, P.; Salvi, D.; Furfaro, G. Variation and Diagnostic Power of the Internal Transcribed Spacer 2 in Mediterranean and Atlantic Eolid Nudibranchs (Mollusca, Gastropoda). Front. Mar. Sci. 2021, 8, 693093. [Google Scholar] [CrossRef]
  79. Ekimova, I.A.; Nikitenko, E.; Stanovova, M.V.; Schepetov, D.M.; Antokhina, T.I.; Malaquias, M.A.E.; Valdés, Á. Unity in diversity: Morphological and genetic variability, integrative systematics, and phylogeography of the widespread nudibranch mollusc Onchidoris muricata. Syst. Biodivers. 2023, 21, 2246472. [Google Scholar] [CrossRef]
  80. Toso, Y.; Martini, F.; Riccardi, A.; Furfaro, G. Unraveling the Sea Slug Fauna from an Extremely Variable Environment, The ‘Passetto’ Rocky Tide Pools (North Adriatic Sea). Water 2024, 16, 1687. [Google Scholar] [CrossRef]
  81. Mioni, E.; Furfaro, G. Alien travel companies: The case of two sea slugs and one bryozoan in the Mediterranean Sea. Diversity 2022, 14, 687. [Google Scholar] [CrossRef]
  82. Azzola, A.; Furfaro, G.; Trainito, E.; Doneddu, M.; Montefalcone, M. Seawater warming favours the northward range expansion of Lessepsian species in the Mediterranean Sea: The cephalaspidean Lamprohaminoea ovalis. J. Mar. Biol. Assoc. U K 2022, 102, 167–173. [Google Scholar] [CrossRef]
Diversity 17 00586 g001
Figure 1. Maps of known (dark grey squares) and new (orange circles) Mediterranean records of D. verrucosa reported in the present study.
Figure 1. Maps of known (dark grey squares) and new (orange circles) Mediterranean records of D. verrucosa reported in the present study.
Diversity 17 00586 g001
Diversity 17 00586 g002
Figure 2. Images in situ (a–d) and in laboratory (e,f) of Doris verrucosa specimens and its egg masses observed during this study; (a,b) frontal and upper view of D. verrucosa (voucher RM3_ 3528) from Mar Piccolo of Taranto; (c) two specimens mating and with their newly laid egg mass, visible at the bottom left; (d) the yellow morphotype (voucher RM3_2282) from Mar Piccolo of Taranto and its yellow egg mass; (e) detail of the egg mass at a higher magnification; (f) images of the veliger larvae inside the egg capsule.
Figure 2. Images in situ (a–d) and in laboratory (e,f) of Doris verrucosa specimens and its egg masses observed during this study; (a,b) frontal and upper view of D. verrucosa (voucher RM3_ 3528) from Mar Piccolo of Taranto; (c) two specimens mating and with their newly laid egg mass, visible at the bottom left; (d) the yellow morphotype (voucher RM3_2282) from Mar Piccolo of Taranto and its yellow egg mass; (e) detail of the egg mass at a higher magnification; (f) images of the veliger larvae inside the egg capsule.
Diversity 17 00586 g002
Diversity 17 00586 g003
Figure 3. Images of Doris verrucosa specimens observed and photographed in laboratory; (a) the brown morphotype (voucher RM3_3526) observed from the Mar Piccolo of Taranto; (b) the light brown morph showed by the specimen voucher RM3_2592; (c,d) two of the three creamy coloured specimens collected from the Faro Lake (vouchers RM3_1419 and RM3_1438 in (c,d), respectively); (e,f) details of the gills (e) and the rhinophore (f) photographed from the specimen with voucher RM3_3528.
Figure 3. Images of Doris verrucosa specimens observed and photographed in laboratory; (a) the brown morphotype (voucher RM3_3526) observed from the Mar Piccolo of Taranto; (b) the light brown morph showed by the specimen voucher RM3_2592; (c,d) two of the three creamy coloured specimens collected from the Faro Lake (vouchers RM3_1419 and RM3_1438 in (c,d), respectively); (e,f) details of the gills (e) and the rhinophore (f) photographed from the specimen with voucher RM3_3528.
Diversity 17 00586 g003
Diversity 17 00586 g004
Figure 4. Bayesian inference tree based on the COI dataset. Numbers at nodes indicate Bayesian Posterior Probability (BPP; left) and bootstrap support from maximum likelihood analysis (BP; right). The ‘-‘ symbol indicates BPP < 0.50 and BP < 50%. On the right side is reported the COI haplotypes analysis (based on the TCS network) of the Doris verrucosa (orange) and its closely related D. ocelligera (blue).
Figure 4. Bayesian inference tree based on the COI dataset. Numbers at nodes indicate Bayesian Posterior Probability (BPP; left) and bootstrap support from maximum likelihood analysis (BP; right). The ‘-‘ symbol indicates BPP < 0.50 and BP < 50%. On the right side is reported the COI haplotypes analysis (based on the TCS network) of the Doris verrucosa (orange) and its closely related D. ocelligera (blue).
Diversity 17 00586 g004
Diversity 17 00586 g005
Figure 5. Bayesian phylogenetic tree based on the ConcDNA dataset (COI, 16S, H3). Bayesian posterior probability (BPP; left) and bootstrap support from maximum likelihood analysis (BP; right) are indicated at each node. The ‘-‘ symbol indicates unsupported values.
Figure 5. Bayesian phylogenetic tree based on the ConcDNA dataset (COI, 16S, H3). Bayesian posterior probability (BPP; left) and bootstrap support from maximum likelihood analysis (BP; right) are indicated at each node. The ‘-‘ symbol indicates unsupported values.
Diversity 17 00586 g005
Diversity 17 00586 g006
Figure 6. Images of Doris marmorata specimens with voucher RM3_3419 collected in Girona, Spain (Mediterranean Sea), and analysed in the present study. In situ dorsal (a) and frontal (b) view of the collected specimen. In (c,d) are the dorsal and ventral pictures taken in the laboratory.
Figure 6. Images of Doris marmorata specimens with voucher RM3_3419 collected in Girona, Spain (Mediterranean Sea), and analysed in the present study. In situ dorsal (a) and frontal (b) view of the collected specimen. In (c,d) are the dorsal and ventral pictures taken in the laboratory.
Diversity 17 00586 g006
Table 1. Date, locality of collection, voucher (except for the individuals that were non-collected ‘n.c.’), and notes on the Doris verrucosa specimens of the present study. The asterisk (*) highlights specimens that were molecularly analysed.
Table 1. Date, locality of collection, voucher (except for the individuals that were non-collected ‘n.c.’), and notes on the Doris verrucosa specimens of the present study. The asterisk (*) highlights specimens that were molecularly analysed.
SpeciesLocalityDateVoucherNote
Doris verrucosaSicily, Messina, Faro Lake7 July 2018RM3_1419 *Depth: 1 m; Temperature: 26 °C
7 July 2018RM3_1432Depth: 1 m; Temperature: 26 °C
7 July 2018RM3_1438 *Depth: 1 m; Temperature: 26 °C
26 June 2023n.c.Depth: ca. 0.5 m; Temperature: 27 °C
Apulia, Taranto, Mar Piccolo13 October 2022RM3_2592 *Depth: 4 m; Temperature: 22 °C
15 September 2021RM3_2282 *Depth: 4 m; Temperature: 25 °C
27 June 2024RM3_3525Depth: 3 m; Temperature: 27 °C
27 June 2024RM3_3526Depth: 3 m; Temperature: 27 °C
27 June 2024RM3_3527Depth: 3 m; Temperature: 27 °C
27 June 2024RM3_3528 *Depth: 3 m; Temperature: 27 °C
Table 2. Species name; voucher; locality; and GenBank COI, 16S, and H3 accession numbers of the specimens included in the molecular analyses. Highlighted with an asterisk ‘*’ are the individuals included in the concatenated and partitioned dataset (ConcDNA). In bold are the specimens collected and analysed in the present study.
Table 2. Species name; voucher; locality; and GenBank COI, 16S, and H3 accession numbers of the specimens included in the molecular analyses. Highlighted with an asterisk ‘*’ are the individuals included in the concatenated and partitioned dataset (ConcDNA). In bold are the specimens collected and analysed in the present study.
SpeciesVoucherLocalityCOI16SH3
Doris berghiG04 *Playa Viveiro, Galicia, SpainOR286435OR286514OR340969
GC40 *Piscinas de Agaete, Gran Canaria, SpainOR286436OR286515OR340970
ZSM20210024 *Coves Cala Maset, Sant Feliu de Guíxols, Girona, SpainOR286438OR286516OR340971
MCZ395161Aigua Freda, Begur, Girona, SpainOR286437––
Doris berthelotiZSM20240264/B7La Herradura, Granada, SpainOR286428––
ZSM20240263/B2 *Blanes, Girona, SpainOR286430OR286510–
As Doris verrucosa –ON716048––
Doris marmorataRM3_3419 *‘La Depuradora’, Punta del Romaní, l’Escala, Girona, SpainPX123159PX128874PX148116
ZSMMol20210023 *Coves Cala Maset, Sant Feliu de Guíxols, Girona, SpainOR286431OR286511OR340966
ZSMMol20210045 *Caleta Caballo, Lanzarote, SpainOR286429OR286509OR340965
Doris montereyensisBICSIOM12334 *USA: California, La Jolla, La Jolla CanyonKC153022KC153024–
CASIZ174493 *USA: Battery Point, Crescent City, Del Norte Co., CaliforniaMF958425MF958294–
Doris nobilisGastr 8481V–MG935354––
Doris ocelligeraX396 *Coves Cala Maset, Sant Feliu de Guíxols, Girona, SpainOR286433OR286512OR340967
X447 *Coves Cala Maset, Sant Feliu de Guíxols, Girona, SpainOR286434OR286513OR340968
MCZ395160Punta del Romaní, l’Escala, Girona, SpainOR286432––
Doris odhneriCASIZ188014USA: Duxbury Reef, Marin Co., California–MF958295–
–Munamjin-ri, Gangwon-do, South KoreaOL800585OL800585–
Doris verrucosaRM3_1438 *Faro Lake, Messina, SicilyPX123160PX128875PX148117
RM3_1419 *Faro Lake, Messina, SicilyPX123161PX128876PX148118
RM3_2282 *Mar Piccolo of Taranto, ApuliaPX123162PX128877PX148119
RM3_2592 *Mar Piccolo of Taranto, ApuliaPX123163PX128878PX148120
RM3_3528 *Mar Piccolo of Taranto, ApuliaPX123164PX128879PX148121
ZSM20210044 *Étang de Thau, Sète, FranceOR286439OR286517OR340972
DVCM1Capo Miseno, Naples, Italy–HE861892–
Homoiodoris japonicaIsolate 11ChinaOQ573572
Avaldesia albomaculaCAS:IZ:181136 *Bigej-Meck, Kwajalein Atoll, Marshall Islands, Pacific OceanMN720286MN722434MN720314
Chromodoris sp.OS-Ss2IranMN548837__
Halgerda meringuecitreaCASIZ 231100 *South Africa: KwaZulu-NatalMW223058MW220923MW414987
Goniobranchus vibratusCASIZ 175564 *USA: Hawaii, MauiJQ727859JQ727741_
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Furfaro, G.; Solca, M.; Madrenas, E.; Tiralongo, F.; Trainito, E. Molecular Updates on the ‘Warty Dorid’ Doris verrucosa Linnaeus, 1758 (Mollusca, Nudibranchia) from the Mediterranean Sea. Diversity 2025, 17, 586. https://doi.org/10.3390/d17080586

AMA Style

Furfaro G, Solca M, Madrenas E, Tiralongo F, Trainito E. Molecular Updates on the ‘Warty Dorid’ Doris verrucosa Linnaeus, 1758 (Mollusca, Nudibranchia) from the Mediterranean Sea. Diversity. 2025; 17(8):586. https://doi.org/10.3390/d17080586

Chicago/Turabian Style

Furfaro, Giulia, Michele Solca, Enric Madrenas, Francesco Tiralongo, and Egidio Trainito. 2025. "Molecular Updates on the ‘Warty Dorid’ Doris verrucosa Linnaeus, 1758 (Mollusca, Nudibranchia) from the Mediterranean Sea" Diversity 17, no. 8: 586. https://doi.org/10.3390/d17080586

APA Style

Furfaro, G., Solca, M., Madrenas, E., Tiralongo, F., & Trainito, E. (2025). Molecular Updates on the ‘Warty Dorid’ Doris verrucosa Linnaeus, 1758 (Mollusca, Nudibranchia) from the Mediterranean Sea. Diversity, 17(8), 586. https://doi.org/10.3390/d17080586

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop