Collection data

For this study, we used specimens from extensive museum collections, deposited at the California State Polytechnic University Invertebrate Collection (CPIC, Pomona, USA), the Lomonosov Moscow State University (White Sea Branch, ZMMU WS, Moscow, Russia), the Museum of National Scientific Centre of Marine Biology (MIMB, Vladivostok, Russia), Museum National d’Histoire Naturelle (MNHN, Paris, France), Museo Nacional de Ciencias Naturales (MNCN, Madrid, Spain). The sampling was largely focused on the tropical Indo-West Pacific, the Eastern Pacific and the Atlantic. Samples were collected during numerous fieldwork expeditions between 2011 and 2024. Voucher numbers and sampling locations for each specimen are given in S1 Table.

DNA extraction, amplification, and sequencing

Three different protocols were used to obtain partial fragments of two mitochondrial and two nuclear genes: partial mitochondrial cytochrome c oxidase subunit I and 16S rRNA, and nuclear histone H3 and 28S rRNA. The protocols used at Lomonosov Moscow State University are described in detail in [34,36,37]; those used at California State Polytechnic University are described in [38]; and those at Instituto Universitario de Investigación Marina (INMAR), University of Cadiz, in [39]. Amplification and sequencing primers are listed in S2 Table. All novel sequences were submitted to NCBI GenBank (S1 Table).

Data processing and phylogenetic reconstruction

Raw reads for each gene were assembled and checked for ambiguities and low-quality data in Geneious R10 (Biomatters, Auckland, New Zealand). Edited sequences were verified for contamination using the BLAST-n algorithm run over the GenBank nr/nt database [40]. For phylogenetic reconstruction, previously published datasets [22,28,34,35,41] were incorporated, with Tritoniidae selected as a distant outgroup along with representatives of all major Aeolidoidea families. Original data and publicly available sequences were aligned with the MUSCLE [42] algorithm in MEGA 7 [43]. Additionally, all protein-coding sequences were translated into amino acids to verify reading frames and check for stop-codons. Saturation was checked by plotting for all specimens including outgroup the total number of pairwise differences (transitions and transversions) against uncorrected p-distances. For the COI and H3 fragments, saturation was further examined separately for the first, second and third codon positions. Indel-rich regions of the 16S and 28S alignments were identified and removed in Gblocks 0.91b [44] with the least stringent settings. The final sequence dataset contained 328 specimens, including 263 Flabellinidae s.l. specimens, and 65 outgroup taxa. Sequences were concatenated in raxmlGUI 2.0 [45]. Phylogenetic reconstructions were conducted for the concatenated multi-gene partitioned datasets. The best-fit nucleotide evolution models were tested in the MEGA 7 toolkit based on the Bayesian Information Criterion (BIC) for each partition. Multi-gene analyses were done by applying evolutionary models separately to partitions representing single markers. The Bayesian phylogenetic analysis (BI) and estimation of posterior probabilities were performed in MrBayes 3.2 [46]. Two independent analyses were carried out; they were initiated with a random starting tree with 20% of trees discarded as burn-in and ran for 2x10⁷ generations. Markov chains were sampled at intervals of 500 generations. The analysis was converged and terminated when the standard deviation of split frequencies reached <0.01. The Maximum likelihood analysis was conducted in raxmlGUI 2.0 [45] with automatically estimated pseudo-replicate number defined by the autoMRE algorithm [47] under the GTRCAT approximation, applied to each partition individually. Final phylogenetic tree images were visualized in FigTree 1.4.0 and finalized in Adobe Illustrator CS 2015. Node support was assessed with posterior probabilities (PP) for BI and bootstrap values for ML and MP analyses. Only nodes supported by BS ≥ 75 and PP ≥ 0.90 were considered significant [48].

Since our goal is to clarify the taxonomy of Flabellinidae s.l. at the genus and family level, species diversity and identity were not the focus, and no species delimitation analyses were performed. We identified the species-level clades as supported when they demonstrate reciprocal monophyly in combination with stable differences observed in nuclear markers. The p-distances between closely related species were calculated for the COI alignment in MEGA 7 [43].

Morphological studies

All collected specimens were examined for external morphology under a stereomicroscope. The internal morphology of 91 specimens was examined, including the digestive and reproductive systems. The buccal mass was removed and soaked in hypochlorite solution or in 10% NaOH to dissolve connective and muscle tissues. The radula and the jaws were rinsed in distilled water, air-dried, mounted on an aluminium stub, and sputter-coated with gold for visualization under a CamScan S2 (Cambridge), Quattro SEM (Thermo Fisher Scientific, USA), Jeol JCM7000 (Jeol, Japan), and Nova NanoSEM™ scanning electron microscopes (SEM) available at Lomonosov Moscow State University (Moscow, Russia), at the White Sea Biological Station (Primorsky, Russia), and the SC-ICYT, University of Cadiz (Puerto Real, Spain) accordingly. Features of the jaws were examined by optical stereomicroscopy and SEM. For the study of the reproductive system, specimens were dissected dorsally along the midline and examined under a stereomicroscope.

Nomenclatural acts

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub: D7D7A473-83EB-4ACF-9036–06300DDBEFB2. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Molecular phylogeny

In this section the previous classification of [22,35] is used for clarity. A total of 465 newly generated sequences were obtained from 130 specimens of Flabellinidae s.l. The final concatenated dataset included 1724 bp (650 bp for COI, 327 bp for H3, 315 bp for 28S and 396 bp for 16S, the latter two partitions were trimmed with Gblocks).

Both BI and ML analyses revealed similar topologies and compatible node support in most cases (Fig 1, S1 Fig). The analyses confirmed the monophyly of Coryphellidae, Apataidae and Paracoryphellidae (the latter only in the BI analysis, see below). At the same time, Flabellinidae s.str., Samlidae, Unidentiidae and Flabellinopsidae (all sensu [22]), the genera Edmundsella Korshunova et al., 2017 and Samla were not recovered as monophyletic. This was due to the position of Flabellina bertschi Gosliner & Kuzirian, 1990, F. engeli Marcus & Marcus, 1968, F. bulbosa Ortea & Espinosa, 1998, Samla rubropurpurata (Gosliner & Willan, 1991), S. telja (Marcus & Marcus, 1967), Piseinotecus soussi Tamsouri et al., 2014, and Bajaeolis bertschi Gosliner & Behrens, 1986. In this regard, F. bertschi from California clustered with Edmundsella sp. CPIC0643 from Florida (posterior probability from BI, PP = 1; bootstrap support from ML, BS = 100), and their clade is sister to Atlantic and Mediterranean species Edmundsella pedata and E. albomaculata (Pola et al., 2014) (PP = 1; BS = 100). Furthermore, the temperate Eastern Pacific species Samla telja was recovered as sister to Luisella babai (Schmekel, 1972) (PP = 0.92; BS = 71), and they both clustered with Flabellina engeli from Bocas del Toro, Panama (PP = 1; BS = 98) and Flabellina bulbosa from Cape Verde (PP = 1; BS = 96). This clade was sister to the main Samla group, which included the type species, Samla bicolor (Kelaart, 1858) (PP = 1; BS = 100). Samla rubropurpurata from Vietnam and New Caledonia were split into two distinct clades (PP = 1; BS = 100), which showed a close relationship with the monophyletic Coryphellidae (PP = 1; BS = 96). The Mediterranean species Piseinotecus soussi (Piseinotecidae) clustered with the genera Pacifia Korshunova et al., 2017 and Unidentia Millen & Hermosillo, 2012 (Unidentiidae, PP = 1, BS = 100) and formed a sister clade to Unidentia (PP = 1; BS = 68). Finally, Bajaeolis bertschi (Facelinidae) unexpectedly grouped in a single clade with Flabellinopsis iodinea (Cooper, 1863), Baenopsis baetica (García-Gómez, 1984) and Kynaria cynara (Marcus & Marcus, 1967) (PP = 1; BS = 86).

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Fig 1. Molecular phylogenetic hypothesis based on the Bayesian Inference (BI), concatenated dataset of four markers (COI + 16S + H3 + 28S), species-level clades and outgroups are collapsed to a single branch.

Species names on leaves are given according to [22,35], generic and family names on the right indicate revised names. Newly sequenced species are highlighted in bold. Numbers above branches indicate posterior probabilities from BI (> 0.9), numbers below branches – bootstrap support from Maximum Likelihood (ML) (> 60). Nodes supported by maximal support in BI and high support in ML (> 90) are identified with a black circle.

https://doi.org/10.1371/journal.pone.0347759.g001

Coryphellidae

The monophyly of the genus Coryphella was highly supported in BI (PP = 1) and moderate in ML (BS = 70). Within this group, the six monophyletic clades were recovered in BI, which are mostly consistent with previously recovered relationships [40], although the support was lower in most cases. The ML tree suggested a similar but unsupported topology (S1 Fig). The only addition was a single specimen from the Kuril Islands (referred here as Coryphella sp. 3), which formed a polytomy with most of Coryphella diversity. As previously mentioned, Samla rubropurpurata was recovered as sister to Coryphella (PP = 1; BS = 96).

Flabellinidae

Within Flabellinidae s.str., three large clades were recovered. The first included all described species of the genus Coryphellina, including the type species Coryphellina rubrolineata O’Donoghue, 1929, and also four putative new species (PP = 1; BS = 99). Within the genus Coryphellina, two monophyletic groups were recovered: (1) species with red lines on the dorsal and lateral sides (Coryphellina rubrolineata species complex) (PP = 1; BS = 100), and (2) species without lines, i.e., Coryphellina arveloi (Ortea & Espinosa, 1998), Coryphellina exoptata (Gosliner & Willan, 1991), and Coryphellina sp. 1 (PP = 1; BS = 100).

The second clade was sister to Coryphellina (PP = 1; BS = 99) and included the type species of the genus Edmundsella (E. pedata), its sister species Edmundsella albomaculata (PP = 1; BS = 100), and Flabellina bertschi and Edmundsella sp. clade (PP = 1; BS = 100).

The third clade (PP = 1; BS = 100) contained representatives of the monophyletic genera Paraflabellina Korshunova et al., 2017 (PP = 1; BS = 100), Calmella Eliot, 1906 (PP = 1; BS = 100), Carronella Korshunova et al., 2017 (PP = 1; BS = 100), and the type species of the genus Flabellina (F. affinis). The genera Calmella and Paraflabellina were recovered as sister taxa (PP = 0.99; BS = 98), other relationships were not supported.

Paracoryphellidae

Paracoryphellidae was recovered as a phylogenetically close group to Flabellinidae and Coryphellidae (PP = 1; BS = 84). BI analyses supported its monophyly (PP = 1), but in the ML analysis, this group received much less support (BS = 58). Overall, three major clades were identified. The first included representatives of the genera Chlamylla (PP = 1; BS = 99), Paracoryphella islandica (Odhner, 1937) (the type species of Paracoryphella) and Polaria polaris (Volodchenko, 1946) (the type species of Polaria Korshunova et al., 2017). The latter was sister to Chlamylla + Paracoryphella (PP = 1; BS = 92). The second clade was represented by members of the genus Ziminella Korshunova et al., 2017 (PP = 1; BS = 100). Within Ziminella, Z. japonica (Volodchenko, 1941) was sister to Z. vrijenhoeki Valdés et al., 2018 (PP = 1; BS = 100), but the relationships between them, Z. abyssa Korshunova et al., 2017, and Z. salmonacea (Couthouy, 1838) were not supported. The third clade was highly supported (PP = 1; BS = 100) and included four putative new species from the Kuril Islands, clustering with recently described Mgueolia almamater Korshunova, Fletcher & Martynov, 2025 [49]. This group performed a sister relationship to a clade uniting Chlamylla, Paracoryphella and Polaria in BI (PP = 0.99; BS = 31).

Flabellinopsidae

Flabellinopsidae showed a somewhat unstable position; in the final concatenated analysis, it was recovered as sister to Coryphellidae, Flabellinidae and Paracoryphellidae, but this relationship lacked support in BI (PP = 0.73) and was not recovered in ML. Flabellinopsidae sensu [22] was not recovered as monophyletic due to the position of Bajaeolis bertschi, originally described as a member of the family Facelinidae due to its uniserial radula. It formed a monophyletic group with Flabellinopsis iodinea and Baenopsis baetica (PP = 1; BS = 98). Kynaria cynara, which was initially assigned to “Flabellinidae insertae sedis” was recovered sister the two flabellinopsid genera and Bajaeolis (PP = 1; BS = 86).

Samlidae

The monotypic genus Luisella (the type species Luisella babai) formed a single clade with Samla telja, F. engeli, and F. bulbosa, as already mentioned above (PP = 1; BS = 96). With the exclusion of S. rubropurpurata and S. telja, the genus Samla was recovered as monophyletic and highly supported (PP = 1; BS = 100). Specimens initially identified as Samla bicolor, were split into five monophyletic groups and further referred as “Samla bicolor” species complex.

Unidentiidae and Piseinotecidae

The monophyly of the family Unidentiidae and both included genera Unidentia and Pacifia, including Piseinotecus soussi was well supported (PP = 1; BS = 100). Within Unidentia, one species from Vietnam, likely representing a new species for science, was sister to U. aliciae Korshunova et al., 2019 (PP = 1; BS = 95). Within Pacifia, P. goddardi (Gosliner, 2010) was not recovered as monophyletic because the specimen CAS182590 formed a polytomy with the other P. goddardi specimens and P. amica Korshunova et al., 2017 (PP = 1; BS = 100).

Apataidae

This family was recovered monophyletic with full support (PP = 1; BS = 100). In most trees it clustered with the family Cumanotidae, but the support was not high (PP = 0.94; BS = 61). Within the family, Apata pricei (MacFarland, 1966) was split into three clades, each with high support (PP = 1; BS > 95).

Notaeolidiidae

In both trees, the relationships of Notaeolidia depressa Eliot, 1905 with other Flabellinidae s.l. were unresolved. The two specimens displayed high genetic divergence from each other (the p-distance in COI is 9.1%), suggesting they may represent two distinct species.

Morphological analysis and integration of molecular and morphological data

Morphological traits of all currently accepted Flabellinidae s.l. species (including the families Apataidae, Samlidae, Unidentiidae, the Antarctic family Notaeolidiidae and Piseinotecus soussi) are summarized on Fig 2 and in S3 Table, and additionally illustrated on Figs 3–7, S2–S29 Figs. Overall, we identified several features that may vary greatly among species of a single genus, while in other genera, the same features could be identified as a single synapomorphy of each group.

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Fig 2. Summary of diagnostic characters of the flabellinid taxa and the family Notaeolididae, based on the original data and literature search for the morphology of each species represented in S3 Table.

Phylogenetic reconstruction based on Bayesian Inference is given on the left, and each chart pie on the right refers to the character states for the respective genus (center) or family (right). Character states for the family Apataidae are indicated with consideration of the diagnosis of the genus Tularia. The legend for diagnostic character states coding each color of circles is given on the bottom.

https://doi.org/10.1371/journal.pone.0347759.g002

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Fig 3. External and internal morphology of Mgueolia vladimiri sp. nov.

A – paratype MIMB52146, living photo. B – holotype MIMB52148, living photo. C – paratype MIMB52146, radula. D, G – paratype MIMB52146, fragment of radula, enlarged. E – paratype MIMB52146, left jaw plate. F – paratype MIMB52146, denticulation of the masticatory process. Scale bars: A, B = 5 mm; C = 250 µm; D = 50 µm; E, G = 100 µm; F = 10 µm.

https://doi.org/10.1371/journal.pone.0347759.g003

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Fig 4. The reproductive system morphology of representatives of Mgueolia and Launsina gen. nov.

A – Mgueolia vladimiri sp. nov., holotype MIMB52146. B – Mgueolia anastasiae sp. nov., holotype MIMB52144. C – Mgueolia ninae sp. nov. holotype MIMB52149. D – Launsina tanyae sp. nov., holotype MIMB52174, female gland mass removed. Abbreviations: amp – ampulla, bw – body wall; drs – distal seminal receptacle, dvd – distal vas deferens, fgm – female gland mass, hd – hermaphroditic duct, ov – oviduct, p – penis, pr – prostatic region, psh – penial sheath, psr – proximal seminal receptacle, pvd – prostatic vas deferens, sr – seminal receptacle, va – vagina, vd – vas deferens. Scale bars: 500 µm.

https://doi.org/10.1371/journal.pone.0347759.g004

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Fig 5. External and internal morphology of Mgueolia anastasiae sp. nov.

A, B – holotype MIMB52144, lateral (A) and dorsolateral (B) view, living photos. C – paratype MIMB52145, living photo. D – holotype MIMB52144, radula. E – holotype MIMB52144, fragment of radula, enlarged. F – holotype MIMB52144, right jaw plate. G – holotype MIMB52144, denticulation of the masticatory process. H, I – holotype MIMB52144, serrated (H) and smooth (I) lateral teeth. Scale bars: A, B = 5 mm; C = 2 mm; D = 250 µm; E = 150 µm; F = 500 µm; G = 25 µm; H, I = 50 µm.

https://doi.org/10.1371/journal.pone.0347759.g005

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Fig 6. External and internal morphology of Mgueolia ninae sp. nov.

A – holotype MIMB52149, living photo. B – MIMB52209, living photo. C – holotype MIMB52149, radula. D – holotype MIMB52149, fragment of radula, enlarged. E – holotype MIMB52149, left jaw plate. F – holotype MIMB52149, denticulation of masticatory border. G, H – holotype MIMB52149, details of lateral (G) and rachidian (H) teeth. Scale bars: A = 2,5 mm. B = 5 mm. C = 500 µm. D, G, H = 50 µm. E = 250 µm. F = 25 µm.

https://doi.org/10.1371/journal.pone.0347759.g006

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Fig 7. External and internal morphology of representatives of the genus Launsina gen. nov.

A – Launsina rupropurpurata comb. nov., MNHN-IM-2019-27166, living specimen, ca. 6 mm in length preserved. B–F – Launsina tanyae sp. nov., holotype MIMB52174, 7 mm in length. B – living specimen. C – jaws. D – Radula. E – anterior radular portion. F – posterior radular portion. Scale bars: C, D = 50 µm, E, F = 10 µm.

https://doi.org/10.1371/journal.pone.0347759.g007

1. 1] Rhinophoral morphology. This feature varies greatly within Coryphella, exhibiting mosaic diversity among most other genera or families (Fig 2). However, in the genera Coryphellina, Edmundsella and the family Unidentiidae, this character is diagnostic: the papillate rhinophores is the most important synapomorphy of Coryphellina (S12 Fig), while the smooth rhinophores of Edmundsella and Unidentiidae (in addition to other features) differentiate these species from the closely related flabellinid groups. Also, perfoliate rhinophores are characteristic of most Flabellinopsidae (S17A–C, S20A–D, S21A–C Figs), except Baenopsis baetica which has rhinophores with numerous ridges (and could be a secondary modification) (S19A Fig) and to most Samlidae, except Luisella bulbosa that has rugose rhinophores (S22A–E, S24A, F Figs).
2. 2] Body width. A wide body is apparently a plesiomorphic character, which is typical of Notaeolidiidae, representatives of Paracoryphellidae sensu [22], and several species of the genus Coryphella (e.g., Coryphella nobilis, C. trophina (Bergh, 1890)). A wide body was thought to be a diagnostic character of the family Paracoryphellidae. However, species of the genus Mgueolia (Figs 3A, B, 5A–C, 6A, B) may have a slenderer body than Chlamylla or Ziminella (S10A–C, S16A, D Figs), thus resembling wide-body representatives of the genus Coryphella (S6B, C Fig).
3. 3] Reduction of the notal edge. All Flabellinidae s.l. are characterized by different levels of reduction of the notal edge, from the plesiomorphic well-developed and continuous edge of Chlamylla, Ziminella, Notaeolidia (S10A–C, S16A, D, S27A Figs) (in Mgueolia it is continuous but less developed than in these groups (Figs 3A, B, 5A–C, 6A, B), to the reduced but continuous of the wide-bodied Coryphella (S6B, C Fig), the reduced discontinuous of most Coryphella (S6A, D–L Fig) and several members of the Flabellina s.l. clade, as well as Coryphellina, Edmundsella, Flabellinopsidae and Samla (see S2, S12, S15A–C, S17A–C, S19A, S20A–D, S21A–C, S22A–E Figs). A completely reduced notal edge could be observed in several members of the Flabellina s.l. clade (S3 Table), in Launsina gen. nov. (Fig 7A, B) and in Piseinotecidae, Apataidae and Luisella (S3 Table, S24A, F, S25A–E, S29A–C Figs). Korshunova et al. [22] reported a completely reduced notal edge in Coryphella athadona Bergh, 1875, which is not supported either by our data (S6L Fig), or by the morphological redescription of this species [50].
4. 4] Ceratal position and arrangement. This character was found to be one of the most important, not only for separating the families, but also for generic delineation. Our analysis showed that species of the Samlidae clade (Figs 1 and 2) have a similar arrangement of cerata: this could be interpreted as ‘serial’, when all cerata are placed on a single simple peduncle, but the length of cerata gradually decreases from the dorsal to the ventral side (Fig 2, S22A–E, S24A, F Figs). Furthermore, representatives of the family Apataidae are characterized by comb-shaped peduncles (Fig 2, S25A–E Fig). In Flabellinopsidae all genera have cerata placed on wide arc-shaped elevations (Fig 2, S17A–C, S19A, S20A–D, S21A–C Figs), which are not typical for members of closely related Flabellinidae s.l. Finally, across clades corresponding to Coryphellidae, Flabellinidae, and Paracoryphellidae (all sensu [22]), cerata might be arranged in rows or groups (Fig 2). In the latter case, they are either attached directly to the body (most Coryphella, all Chlamylla, Ziminella and Mgueolia (Fig 3A, B, 5A–C, 6A, B, S6, S10A–C, S16A, D Figs)), or placed on low elevations (all Coryphellina, Edmundsella, Launsina gen. nov., few Coryphella, few Flabellina s.l., see Fig 7A, B, S2B, S12, S15A–C Figs), or placed on compound peduncles (most Flabellina s.l., see S2A, C–F Fig) (Fig 2, S3 Table).
5. 5] Position of the anal opening. Most flabellinids are distinctly pleuroproctic animals, however, there are several taxa with the anus placed more dorsally, the so-called ‘pleuroproctic in a higher acleioproctic position’ (Apataidae, most Piseinotecidae) or acleioproctic (some Flabellina, Unidentia, Piseinotecus soussi). The more dorsal position of the anal opening evolved independently in different groups. Also, it is worth mentioning that in the majority of the Flabellinidae s.l. the anus is placed in a somewhat anterior position, but in the Mgueolia and Chlamylla clades as well as in the wide-bodied Coryphella it is placed closer to the middle of the body, and in Ziminella and Notaeolidia it is located laterally on the second half of the body. We reviewed the cleioproctic position of the anus in Bajaeolis bertschi as mentioned in the initial description of this species [51]. Although the anus is located right under the postcardial arc-shaped elevation with cerata, the reduced notal edge is distinct and observed above the anal opening, thus its position should be identified as pleuroproctic (S3 Table).
6. 6] Radular formula. The triserial condition of the radula is characteristic for the majority of Flabellinidae s.l. (Fig 2, S3 Table). Few exceptions are: the uniserial radula of Bajaeolis bertschi (Flabellinopsidae) (S21E–G Fig), and the uniserial radula in Piseinotecidae (including former Unidentiidae) (Figs 1, 2, S29D–F Fig). Notaeolidia possesses a polyserial radula, a unique condition among Aeolidida (Fig 2, S27B, C, F Fig). The same was also observed [18,22] in juveniles of the genus Paracoryphella sensu [22], which represents a plesiomorphic trait.
7. 7] Morphology of the central cusp of the rachidian teeth. The position of the central cusp is one of the key differences between the genera of Flabellinidae, Coryphellidae, and Paracoryphellidae as defined by [22]. The depressed central cusp is observed in Mgueolia, Edmundsella, Flabellina, and Coryphellina (Figs 2, 3D, G, 5E, 6D, H, S3A–E, G, S13A–F, S15E–G Fig), while in Coryphella it is somewhat elevated (S7 Fig), even when the cusp is slightly compressed by adjacent denticles (S7B Fig). In most Coryphella species, the cusp is longer than the lateral denticles (S7 Fig). The central cusp maybe large and broad, much larger than the lateral denticles, as in Ziminella and Chlamylla (S10D–F, S16B, E Figs). Overall, the depression/elevation of the central cusp is a more important feature for generic differentiation, while its dimensions and the number and morphology of the lateral denticles are useful features for species-level taxonomy.
8. 8] Lateral teeth shape and denticulation. This is a quite variable feature, but we identified six major patterns (Fig 2): narrow (1) or wide (2) slightly curved triangular teeth with a large central cusp and strong lateral denticles; triangular teeth with a wide base and a slender subulate tip (3); wide (4) or narrow (5) triangular teeth with reduced lateral denticles or denticles absent; and plate-like wide triangular teeth (6). The morphology of the lateral teeth in general correlates with the recovered phylogenetic relationships (Fig 2), however this feature may vary even within a single clade (e.g., in Coryphella, Ziminella two morphological types are observed) (S3 Table), and likely has less phylogenetic significance.
9. 9] Number and position of seminal receptacles. This feature was believed to have a high phylogenetic significance, however our review (S3 Table) in combination with newly obtained molecular phylogenetic hypotheses (Figs 1 and 2) shows that the number, position and morphology of the seminal receptacles vary greatly even within a single genus. For example, species of the genus Edmundsella were reported to have one bilobed seminal receptacle (E. bertschi, E. pedata, E. vansyoci (Gosliner, 1994)), while in E. albomaculata and E. dushia (Marcus & Marcus, 1963) both proximal and distal seminal receptacles are present (S14C, D Fig). The same is true for the well-defined genus Coryphellina, where usually two receptacles were present, and the proximal receptacle was bilobed (S14A Fig), but in C. albomarginata (Miller, 1971), C. arveloi and C. exoptata, only the proximal receptacle was observed (S3 Table, S14B Fig). The same pattern was observed within other flabellinid taxa, including Coryphella, the Flabellina s.l. clade, and across Samlidae (Fig 2, S5, S9, S23 Figs). High variation of this feature within these clades limits its usefulness as a distinctive trait to support the separation of monotypic genera.
10. 10] Length of the vas deferens, granulated prostate, and penial morphology. These three features were also used to support the establishment of separate genera within Coryphellidae, Flabellinidae, and Paracoryphellidae [22]. However, our analysis shows their limited phylogenetic significance. Although a long vas deferens is observed in most Paracoryphellidae (S3 Table), in Mgueolia it may be long or moderate in length (Fig 4A–C). The occurrence of short and long vas deferens within a single group is also observed within the genera Coryphella, Samla, Luisella and the families Flabellinopsidae and Apataidae (S5, S9, S18, S23, S26 Figs; S3 Table). This suggests that the occurrence of a long vas deferens across distant lineages is likely the result of retention of a plesiomorphic state rather than an apomorphic feature, and thus it cannot be used to support the distinct status of a given high-rank taxonomical group. The presence of well-developed, granulated prostatic region was used to support the distinct status of Chlamylla from the closely related Paracoryphella and Polaria [22], however, in Luisella this is just intrageneric variation (Luisella babai has a granulated prostate, while in other species the prostatic region is indistinct, S23B–D Fig). Finally, most flabellinid species have a conical penis, but in some groups, it can be very different in shape (most diverse in Coryphella) (S3 Table). The armed penis of Unidentia represents a reliable synapomorphy of this genus, differentiating it from Pacifia and Piseinotecus soussi (S28 Fig; S3 Table).

Family Flabellinopsidae Korshunova et al., 2017

Diagnosis. Head elongate; body moderately wide; anterior foot corners present; notal edge reduced discontinuous; cerata in groups on wide arc-shaped elevations; rhinophores perfoliate or with ridges, granules; anus pleuroproctic or mixed; oral glands present or absent; jaws oval-triangular to oval-rectangular with several rows of denticles on masticatory border; uniserial to triserial radula; triangular or elongated rachidian teeth with non-compressed or compressed small elevated cusp, small denticles on each side; lateral teeth present or absent; lateral teeth wide triangular or elongated, with strong denticles on inner edge or with reduced denticles, outer denticles always present; proximal seminal receptacle present. one or two; short to moderately long prostatic or non-prostatic vas deferens; penial gland absent; unarmed penis.

1. Genus Flabellinopsis MacFarland, 1966
   1. Flabellinopsis MacFarland, 1966: 308. (S17, 18A Figs)

Diagnosis. Body moderately wide; head rounded; notal edge reduced discontinuous; cerata in groups on arc-shaped elevations; rhinophores perfoliate; anus pleuroproctic; oral glands absent; jaws with several rows of denticles; radula triserial; rachidian teeth triangular with non-compressed small cusp, strong denticles on each side; lateral teeth wide, triangular, with long outer basal process, strong conical cusp, large denticles on inner edge, outer denticles present; proximal seminal receptacle serial; long vas deferens with indistinct prostate; penial gland absent; unarmed penis.

Type species: Aeolis iodinea Cooper, 1863 by original designation.

Species included: Flabellinopsis iodinea (Cooper, 1863)

1. Genus Baenopsis Korshunova et al., 2017
   1. Baenopsis Korshunova et al., 2017: 24 (S18B, S19 Figs)

Diagnosis. Body moderately wide; head rounded; notal edge reduced discontinuous; cerata in groups on broad elevations; rhinophores with numerous ridges, granules; anus pleuroproctic; oral glands absent; jaws with several rows of denticles; triserial radula; rachidian teeth triangular with non-compressed small cusp, strong denticles on each side; lateral teeth wide, triangular, with reduced tiny denticles on inner side, with massive apical cusp; small dentitions on outer edge present; proximal seminal receptacle; long vas deferens with wide prostate; penial gland absent; unarmed penis.

Type species: Flabellina baetica García-Gómez, 1984 by original designation.

Species included: Baenopsis baetica (García-Gómez, 1984)

Remarks: The reexamination of internal morphology of Baenopsis baetica revealed the presence of outer denticles on the lateral teeth (S19E, F Fig), which have never been observed before [63,64].

1. Genus Kynaria Korshunova et al., 2017
   1. Kynaria Korshunova et al., 2017: 66–67. (S18C, S20 Figs)

Diagnosis. Body moderately wide; head rounded; notal edge reduced discontinuous; cerata in groups on arc-shaped elevations; rhinophores perfoliate; anus pleuroproctic in higher acleioproctic position; oral glands absent; jaws with several rows of denticles; rachidian teeth elongated arc-shaped slightly compressed small cusp, strong denticles on each side; lateral teeth elongated, narrow, curved, with strong curved cusp, large denticles on inner edge, outer denticles present; two proximal seminal receptacles; long vas deferens with wide prostate; penial gland absent; unarmed penis.

Type species: Coryphella cynara Marcus & Marcus, 1967 by original designation.

Species included: Kynaria cynara (Marcus & Marcus, 1967)

1. Genus Bajaeolis Gosliner & Behrens, 1986
   1. Bajaeolis Gosliner & Behrens, 1986: 106. (S18D, S21 Figs)

Diagnosis. Body moderately wide; head rounded; notal edge reduced discontinuous; cerata in groups on arc-shaped elevations; rhinophores perfoliate; anus pleuroproctic, under second ceratal cluster in higher position; oral glands present; jaws rectangular, with few rows of denticles; radula uniserial; rachidian teeth triangular with non-compressed small cusp, strong denticles on each side; lateral teeth absent; proximal seminal receptacle; short vas deferens with indistinct prostate; penial gland absent; unarmed penis.

Type species: Bajaeolis bertschi Gosliner & Behrens, 1986 by original designation

Species included: Bajaeolis bertschi Gosliner & Behrens, 1986

Family Samlidae Korshunova et al., 2017

Diagnosis. Head elongate; body narrow; anterior foot corners present or reduced; notal edge reduced discontinuous or absent; cerata in groups on serial peduncles; rhinophores perfoliate or rarely rugose; anus pleuroproctic; oral glands present; jaws triangular with several rows of denticles on masticatory border; triserial radula; triangular rachidian teeth with elevated or slightly depressed small conical cusp, numerous denticles on each side; lateral teeth wide triangular with long outer basal process, strong denticles on inner edge, or denticles absent; one or two seminal receptacles; short to moderately long prostatic or non-prostatic vas deferens; penial gland absent; unarmed penis.

1. Genus Samla Bergh, 1900
   1. Samla Bergh, 1900: 237. (S22, S23A Figs)

Diagnosis. Body narrow; head elongate; notal edge reduced discontinuous; cerata in groups on serial peduncles, 2–3 cerata per group; rhinophores perfoliate; anus pleuroproctic; oral glands present; jaws with several rows of denticles; triserial radula; rachidian teeth triangular, with non-compressed elevated or slightly depressed central cusp, numerous denticles with long furrows on each side; lateral teeth wide triangular with long outer basal process, strong inner denticles; distal seminal receptacle present, proximal seminal receptacle serial, representing expanded portion of oviduct, or absent; short to moderately long vas deferens with indistinct prostate; penial gland absent; unarmed penis.

Type species: Samla annuligera Bergh, 1900 by monotypy.

Species included: Samla bicolor (Kelaart, 1858), Samla bilas (Gosliner & Willan, 1991), Samla macassarana (Bergh, 1905), Samla riwo (Gosliner & Willan, 1991), Samla takashigei Korshunova et al., 2017.

Remarks: Korshunova et al. [22] reinstated the genus Samla for species phylogenetically close to Bergh’s Samla annuligera [65] (currently considered a junior synonym of Samla bicolor). They identified several differences between Samla and other Flabellinidae: 1) absence of compound stalks, often non-compressed cusp of the rachidian teeth, short vas deferens. Korshunova et al. [22] also included Flabellina telja (considered here as Luisella telja) and F. rubropurpurata (designated here as Launsina rubropurpurata comb. nov.) in the genus Samla. Although our analysis supports the distinct status of Samla, it also shows that neither F. telja, nor F. rubropurpurata are the part of this group (Fig 1). The position of F. rubropurpurata has a crucial importance, as it questions the differences between the diagnoses of Samla and Flabellinidae suggested by [22]. Our morphological analysis instead shows that the primary difference between the Flabellinidae and Samlidae is the ceratal arrangement: in the latter family their arrangement on the common elevation or peduncle is serial: the size of the cerata gradually increases from lateral to dorsal (Fig 3, S22A–E, S24A, F Figs), this feature is not known for any species of Flabellinidae s.str, including the genus Launsina gen. nov. (Fig 7A, B). Also, our analysis shows that Samla and Luisella represent two distinct genera, which are well differentiated by the number of cerata on a common peduncle (2–3 in Samla, 4–8 in Luisella, S22A–E, S24A, F Figs). Finally, the notal edge in Samla is still present but reduced and discontinuous, whereas it is completely absent in all Luisella species studied herein.

Our analysis also reveals a considerable hidden diversity within Samla, as we identified several phylogenetically distinct groups likely representing separate species in the Samla bicolor species complex (Fig 1). Some of them were collected from Hawaii (S1 Table), which is the type locality of species Samla annuligera and Samla alisonae Gosliner, 1980. Both species are currently considered junior synonym of Samla bicolor, and therefore our results suggests that the distinct status of some of these species should be further reconsidered.

1. Genus Luisella Korshunova et al., 2017
   1. Luisella Korshunova et al., 2017: 61. (S23B–D, S24 Figs)

Diagnosis. Body narrow; head elongate; notal edge completely absent; cerata in groups on serial peduncles, 4–8 cerata per group; rhinophores perfoliate or rugose; anus pleuroproctic; oral glands absent; jaws with several rows of denticles; triserial radula; rachidian teeth triangular with small conical compressed non-depressed or slightly depressed cusp, denticles on each side; lateral teeth wide triangular with long outer basal process, denticles strong inner or absent; distal seminal receptacle present or absent, proximal seminal receptacle simple; short to moderately long vas deferens with indistinct or granulated prostate; penial gland absent; unarmed penis.

Type species: Flabellina babai Schmekel, 1972, by monotypy.

Species included: Luisella babai (Schmekel, 1972), Luisella bulbosa (Ortea & Espinosa, 1998) comb. nov., Luisella engeli (Marcus & Marcus, 1968), Luisella telja (Marcus & Marcus, 1967).

The following species are provisionally placed in Luisella based on morphological data, but need the molecular data for confirmation: Luisella llerae (Ortea, 1989) comb. nov.

Remarks: The reconstructed phylogenetic relationships (Fig 1) show that the species Flabellina bulbosa, Flabellina engeli, and Samla telja (all generic names sensu [22]) clustered with Luisella babai. The latter species was placed in the separate genus Luisella, because it has a granulated prostate [22]. None of the remaining closely-related species has this type of prostate, however all of them have a common pattern of the ceratal arrangement: serial peduncles with 4–8 cerata per group, which differentiates all these species from Samla. This, along with molecular divergence, support the distinctness of the genus Luisella. Most of Luisella species also have perfoliated rhinophores, except L. bulbosa, in which rhinophores are rugose (S3 Table).

Family Apataidae Korshunova et al., 2017

Diagnosis. Head elongate; anterior foot corners present; notal edge completely reduced; cerata arranged in groups, placed on comb-shaped peduncles; rhinophores annulate or wrinkled; anus pleuroproctic in higher acleioproctic position; oral glands present; jaws triangular with one or two rows of denticles on masticatory border; triserial radula; triangular rachidian teeth with small conical compressed non-depressed cusp, numerous denticles on each side; lateral teeth wide triangular with long outer basal process, smooth; one distal seminal receptacle, stalked; short to long vas deferens with indistinct prostate; penial gland absent; unarmed penis.

1. Genus Apata Korshunova et al., 2017
   1. Apata Korshunova et al., 2017: 64. (S25, S26A Figs)

Diagnosis. Body narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on comb-shaped peduncles; rhinophores annulate; anus pleuroproctic in higher acleioproctic position; oral glands present; jaws triangular with one row of denticles on masticatory border; triserial radula; triangular rachidian teeth with small conical compressed non-depressed cusp, numerous denticles on each side; lateral teeth wide triangular with long outer basal process, smooth; distal stalked seminal receptacle; long vas deferens with indistinct prostate; penial gland absent; unarmed penis.

Type species: Coryphella pricei MacFarland, 1966 by original designation.

Species included: Apata pricei (MacFarland, 1966).

Remarks: According to our phylogenetic analysis (Fig 1) and preliminary morphological analysis (S25 Fig), the genus Apata likely includes at least three distinct species, however more material from the NE Pacific should be studied to complete a needed comprehensive revision.

1. Genus Tularia Burn, 1966
   1. Tularia Burn, 1966: 26. (S26B Fig)

Diagnosis. Body narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on comb-shaped peduncles; rhinophores wrinkled; anus pleuroproctic in higher acleioproctic position; oral glands present; jaws triangular with two rows of denticles on masticatory border; triserial radula; triangular rachidian teeth with small conical compressed non-depressed cusp, numerous denticles on each side; lateral teeth wide triangular with long outer basal process, smooth; one stalked seminal receptacle; short vas deferens with indistinct prostate; penial gland absent; unarmed penis.

Type species: Cuthona bractea Burn, 1962 by original designation

Species included: Tularia bractea (Burn, 1962)

Remarks: The limited distribution of this species (New Zealand: [66]) restricts its availability for research. A specimen from the Victoria Museum (F113298) was loaned for molecular analyses, as morphological studies were refused. However, amplification was unsuccessful, so this data could not be included in our phylogenetic reconstruction.

Family Notaeolidiidae Eliot, 1910

Diagnosis. Head rounded; body wide; anterior foot corners present; notal edge well-developed, continuous; cerata arranged in continuous rows, attached directly to body; rhinophores annulate or with irregular ridges; anus pleuroproctic; oral glands absent; jaws oval-triangular, smooth; polyserial radula; rachidian teeth triangular or elongated, non-compressed or compressed small elevated cusp, small denticles on each side; lateral teeth present, wide triangular or elongated, with strong denticles on inner edge or with reduced denticles, with or without outer denticles; separate seminal receptacle; short to moderately long prostatic or non-prostatic vas deferens; penial gland absent; unarmed penis.

1. Genus Notaeolidia Eliot, 1905

1. Notaeolidia Eliot, 1905: 520. (S26C, S27 Figs)

Diagnosis. Same as for the family.

Type species: Notaeolidia gigas Eliot, 1905 by original designation.

Species included: Notaeolidia depressa Eliot, 1905, Notaeolidia gigas Eliot, 1905, Notaeolidia schmekelae Wägele, 1990.

Remarks: Two specimens of Notaeolidia depressa were included in the phylogenetic analysis (Fig 1), which displayed high levels of genetic divergence. These data suggest the existence of the cryptic diversity within this group.

Family Piseinotecidae Edmunds, 1970

1. = Unidentiidae Millen & Hermosillo, 2012 syn. nov.

Diagnosis. Body narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on low elevations or simple peduncles; rhinophores smooth; anus pleuroproctic or pleuroproctic in higher position, sometimes acleioproctic; oral glands commonly present; jaws elongate triangular or rectangular, with one to several rows of denticles on masticatory border or smooth; uniserial radula; triangular to almost rectangular rachidian teeth with narrow to broad non-compressed elevated, short conical cusp, numerous denticles on each side; lateral teeth absent; one or two seminal receptacles; short to moderately long vas deferens with or without distinct prostate region; penial gland absent or present; penis armed or unarmed.

Remarks. Piseinotecus soussi clustered with the genera Pacifia and Unidentia representing the sister group to the latter genus (Fig 1). The high levels of divergence between P. soussi and both Unidentia and Pacifia suggests it may belong to a separate genus within the family Unidentiidae, and this placement could indicate that the family Piseinotecidae may represent a senior synonym of the Unidentiidae. Moreover, the diagnosis of the genus Piseinotecus sensu [67] —and of the family Piseinotecidae—updated by [68] fully agrees with the diagnosis of the family Unidentiidae by [22] as follows: 1) uniserial short radula, 2) strong central cusp of the rachidian teeth, 3) a single row of denticles on masticatory process, 4) cerata in groups on low elevations, 5) smooth rhinophores, 6) acleioproctic anus position 7) proximal seminal receptacle. However, the type species of the genus Piseinotecus, P. divae is a rare species that has not been studied since its original description [67], and therefore, there is no molecular data available. The morphology of P. divae was described in detail, and it fully corresponds to the diagnosis of Unidentiidae. While the available evidence supports the synonymy of Unidentiidae and Piseinotecidae, caution is warranted due to the existence of numerous examples of rampant homoplasies and convergence in aeolid systematics, e.g., [69,70]. At the same time, retaining separate family names for Piseinotecidae and Unidentiidae due to the unavailability of molecular data for P. divae, seems to be an impractical solution for several reasons. First, it requires the introduction of a new genus for P. soussi, whose diagnosis would be identical to that of Piseinotecus s.str. Second, the genus or family affiliations of any new taxon with a piseinotecid-like morphology could not be possible without molecular data. This would create substantial confusion and uncertainty in the entire classification scheme. Therefore, we suggest tentatively regarding the family Piseinotecidae as a senior synonym of the family Unidentiidae, with the caveat that unravelling the phylogenetic position of the type species (P. divae) is crucial for the confirmation of this decision. Molecular data for the rest Piseinotecus species are also needed to confirm the monophyly of this enigmatic genus.

1. Genus Piseinotecus Marcus, 1955
   1. Piseinotecus Marcus, 1955: 176. (S28A–D Fig)

Diagnosis. Body narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on low elevations or simple peduncles; rhinophores smooth; anus acleioproctic; oral glands not described; jaws elongate triangular with a single row of denticles; uniserial radula; triangular to broad rachidian teeth with narrow to broad non-compressed conical cusp elevated cusp with denticles on each side; lateral teeth absent; proximal seminal receptacle present; distal seminal receptacle present or absent; short vas deferens with wide, distinct prostate; penial gland absent; penis unarmed.

Type species: Piseinotecus divae Er. Marcus, 1955 by original designation.

Species included: Piseinotecus divae Marcus, 1955, Piseinotecus soussi Tamsouri et al., 2014.

The following species are provisionally placed in Piseinotecus based on morphological data, but need the molecular data for confirmation: Piseinotecus ernestina Ortea & Moro, 2020, Piseinotecus gonja Edmunds, 1970, Piseinotecus kima Edmunds, 1970, Piseinotecus minipapilla Edmunds, 2015; Piseinotecus sphaeriferus (Schmekel, 1965).

Remarks: The genus Piseinotecus differ from the genus Pacifia by smooth central cusp of the rachidian tooth, and from the genus Unidentia – by unarmed penis.

1. Genus Pacifia Korshunova et al., 2017
   1. Pacifia Korshunova et al., 2017: 56 (S28E Fig)

Diagnosis. Body narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on low elevations; rhinophores smooth; anus pleuroproctic towards acleioproctic position; oral glands present; jaws elongate triangular with a single row of denticles or weak tubercles; uniserial radula; triangular to broad rachidian teeth with narrow to broad non-compressed conical cusp elevated cusp with denticles on each side; cusp of rachidian tooth bearing denticles, or smooth; lateral teeth absent; two seminal receptacles; short vas deferens with wide, distinct prostate; penial gland present; penis unarmed.

Type species: Pacifia amica Korshunova et al., 2017 by original designation.

Species included: Pacifia amica Korshunova et al., 2017; Pacifia goddardi (Gosliner, 2010)

Remarks: According to our molecular phylogenetic analysis P. goddardi did not form a monophyletic group (Fig 1), so the identity of this species should be further tested with additional molecular and morphological data.

1. Genus Unidentia Millen & Hermosillo, 2012
   1. Unidentia Millen & Hermosillo, 2012: 155. (S28F, S29 Figs)

Diagnosis. Body relatively narrow; head elongate; notal edge completely reduced; cerata arranged in groups, placed on low elevations; rhinophores smooth; anus pleuroproctic towards acleioproctic position; oral glands present; jaws elongate triangular with a single row of denticles or weak tubercles; uniserial radula; triangular to broad rachidian teeth with narrow to broad non-compressed conical cusp elevated cusp with denticles on each side; lateral teeth absent; one or two proximal seminal receptacles; short vas deferens with distinct wide prostate; penial gland absent; penis armed.

Type species: Unidentia angelvaldesi Millen & Hermosillo, 2012 by original designation.

Species included: Unidentia angelvaldesi Millen & Hermosillo, 2012, Unidentia aliciae Korshunova et al., 2019, Unidentia nihonrossija Korshunova et al., 2017, Unidentia sandramillenae Korshunova et al., 2017.

Remarks: Our analysis (Fig 1) and the results of [71] suggest that at least two additional undescribed species (from Australia and Vietnam) could be identified within Unidentia.

Supported divergence of Apataidae, Samlidae and reconsideration of Piseinotecidae

Our molecular phylogenetic analysis confirms that the traditional Flabellinidae s.l. is polyphyletic, generally corroborating the revision by [22]. Representatives of the families Apataidae, Samlidae and Piseinotecidae form three highly supported and derived clades, not related to the ‘core’ group of Flabellinidae nor to each other (Fig 1, [22]). We tentatively suggest synonymizing Unidentiidae and Piseinotecidae, but this should be reconsidered once molecular data for the type species P. divae become available.

We have identified several morphological synapomorphies for the families Apataidae, Samlidae and Piseinotecidae (Fig 2). Some of them have been already discussed (comb-shaped peduncles of Apataidae, the uniserial radula in combination with proximal seminal receptacle of Piseinotecidae, see [22]). Other synapomorphies are newly established; these are perfoliate rhinophores and serial peduncles of Samlidae, a completely reduced notal edge in Luisella in contrast to Samla, and a serial proximal seminal receptacle of Samla. We consider that these synapomorphies should facilitate the correct placement of newly described taxa, even if molecular data are not available. At the same time, the species composition of some genera needs further revision as several diverse species complexes have been identified (see above).

Furthermore, contrary to previous studies, the expected sister relationship of Cumanotidae Odhner, 1907 and Pseudovermidae Thiele, 1931 [72] was not recovered. In both analyses, the family Apataidae was sister to Cumanotidae (Fig 1, S1 Fig), but received no statistical support. The type species Cumanotus beaumonti (Eliot, 1906) has a triserial radula [72], but its morphology differs significantly from that of the family Apataidae (S25 Fig) and most taxonomic characters of Cumanotidae (position of the rhinophores, position of the anus, ceratal grouping) do not correspond the diagnostic features of Flabellindiae s.l. and, in particular, Apataidae. This relationship, however, should be considered as tentative, since our analysis lacks many aeolid taxa, including lineages of the large family Facelinidae s.l. Further resolution of the family-level phylogeny of Aeolidoidea would benefit from the use of genomic data rather than a handful of individual markers, as was already shown for cladobranch molluscs [73].

New taxa of Flabellinopsidae

One of the most surprising results of the present study is the close relationship of the genus Bajaeolis and representatives of the family Flabellinopsidae (Fig 2). Bajaeolis has a uniserial radula (S21E–F Fig) and was originally placed in the family Facelinidae [51]. In all our phylogenetic analyses, Bajaeolis is recovered in a clade including representatives of the family Flabellinopsidae (Fig 1). In addition, the monotypic genus Kynaria, established for the species Flabellina cynara and provisionally placed in “Flabellinidae incertae sedis” [22], is sister to the clade comprising Flabellinopsidae + Bajaeolis (Fig 1). The close relationship of these four monotypic genera is quite unexpected, since they differ significantly in radular morphology. In Flabellinopsis and Baenopsis, the lateral teeth are wide, triangular, and bear denticles along the inner edge (S17G, H, S19E, G Figs), while in Kynaria the lateral teeth are narrow and curved (S20G Fig), and in Bajaeolis they are reliably absent (S21E–G Fig). The lateral teeth of Flabellinopsis, Baenopsis and Kynaria bear denticles along the outer edge (S17H, S19F, S20I Figs), which may be an important synapomorphy of the group. The rachidian teeth of all four species differ in shape, and also in the number and arrangement of denticles (S17G, S19D, S20G, S21F, G Figs). At the same time, all four genera are characterized by a similar arrangement of cerata: they are arranged in wide groups, located on small horseshoe-shaped low elevations (S17A–C, S19A, S20C, S21A Figs). In addition, three of the four genera (Flabellinopsis, Kynaria, Bajaeolis) are characterized by having perfoliated rhinophores (Fig 2, S17A–C, S20A–D, S21A–C Figs). Although the rhinophores of Baenopsis differ significantly as they highly folded and granulated (Fig 2, S19A Fig), it can be hypothesized that this morphology is derived from perfoliated rhinophores, rather than being formed de novo from smooth or wrinkled ones. Taking this assumption into account, four synapomorphies can be established for this group: 1) rachidian teeth with a small protruding central cusp, 2) lateral teeth bearing outer denticles, 3) perfoliated rhinophores, 4) cerata are placed on wide arc-shaped elevations. The fundamental differences in the morphology of the radula between Flabellinopsis, Baenopsis, Kynaria, Bajaeolis, and the mosaic pattern of other internal and external traits, together with high genetic distances, suggest the need to retain four monotypic genera within the family Flabellinopsidae. More importantly, although the family Flabellinopsidae forms a sister group to Flabellinidae in the BI analysis (Fig 1, S1 Fig), this relationship was not statistically supported. This, on the one hand, supports the separate status of Flabellinopsidae as different from Flabellinidae and indicates the need for further studies using a genomic approach and an increased sample size.

Newly described taxa

In the case of the ‘core’ group of Flabellinidae s.l. (i.e., the families Paracoryphellidae, Flabellinidae, Coryphellidae sensu [22]), our analyses show significant similarities with the results obtained by [22]. However, several important discrepancies at high taxonomic levels were also identified. We have shown that the species Samla rubropurpurata sensu [22] forms a sister clade to the genus Coryphella (Fig 1) and is described herein as Launsina gen. nov. Furthermore, Launsina gen. nov. includes a species complex of at least two pseudocryptic species. These species differ from Coryphella in several features: the cerata arranged in groups on low elevations, rather than being attached directly to the body (Fig 7A, B), as in most Coryphella, and the margin of the notum is completely reduced (S6 Fig). The main difference between Launsina gen. nov. and Coryphella is the morphology of the lateral teeth of the radula (Fig 7D–F, S7 Fig). Launsina gen. nov. has a wide base with an elongated and narrow, rather subulate tip, and the inner part of teeth forms an almost right angle (Fig 7E, F), which is very similar to the lateral teeth of Edmundsella (S15E–G Fig). At the same time, lateral teeth of Coryphella are always triangular in shape, almost always slightly curved, and the tip is a well-defined strong cusp (S7 Fig). Thus, Launsina gen. nov. shows several features that do not correspond to Coryphella diagnosis, and inclusion of these two species into the Coryphella will further make its diagnosis identical to that of Edmundsella. At the same time, Edmundsella cannot be united with Coryphella, as they do not represent a monophyletic group because of the position of well-established and easily diagnosed genus Coryphellina (Fig 1, S1 Fig). As Launsina gen. nov. forms a separate clade in the phylogenetic tree (Fig 1, S1 Fig), we propose that it represents a distinct genus of Flabellinidae s.l.

Notably, the initial placement of Launsina rubropurpurata comb. nov. in the genus Samla [22] was probably due to the significant similarity of these groups in external morphology (narrow body, perforated rhinophores, a small number of cerata arranged in distinct groups). Nevertheless, in Launsina gen. nov. the cerata are located on small elevations and are disordered, while in representatives of Samlidae the arrangement of cerata on a common peduncle is serial: the size of cerata gradually increases from lateral to dorsal (S22A–E, S24A, F Figs). In addition, in Launsina gen. nov. the reproductive system is generally similar to that Coryphella as they have both distal and proximal seminal receptacles with well-defined ducts (Fig 4D, S6 Fig), whereas in Samla, the proximal receptacle is ‘serial’ [19] and represents a slight expansion of the oviduct (S23A Fig).

Based on material collected from the Kuril Islands, we discovered three new species of the recently described genus Mgueolia, which now includes four species (Figs 1, 3, 5, 6). This group is characterized by a unique combination of morphological features for Flabellinidae s.l. (S3 Table): Mgueolia and closely related Ziminella and Chlamylla, have a continuous notal margin, and their cerata are arranged in rows. But in the case of Mgueolia the notal margin is much less developed (Figs 3A, B, 5A–C, 6A, B) than in Chlamylla and Ziminella (S10A, C, S16A, D Figs) and more similar to the reduced but continuous margin of Coryphella nobilis (S6C Fig). The anus in Mgueolia is located on the posterior half of the body, which is similar to Ziminella (S16A, D Fig). At the same time, the radular morphology of Mgueolia differs significantly from the closely related Ziminella and Chlamylla: the rachidian teeth have a significantly depressed central cusp (Figs 3D, G, 5E, 6D, H) (similar to Edmundsella and Coryphellina, S13, S15E–G Figs); the lateral teeth of Mgueolia are substantially flattened and lack or have few denticles (Figs 3D, G, 5E, H, I, 6D, G), and resemble the flattened lateral teeth of the genus Eubranchus (Fionidae s.l., see [74]) instead of the typical triangular denticulated lateral plates of Flabellinidae s.l. (see S3, S7 Figs). Our phylogenetic reconstruction shows that species of Mgueolia form a monophyletic group close to Chlamylla and Ziminella. However, these relationships were not supported in the ML analysis (S1 Fig).

Redefinition of Flabellinidae

Our analyses demonstrate that adding new taxa to the phylogenetic dataset required reassessing genus-level diagnostic characters in Coryphellidae and Flabellinidae sensu [22]. This is particularly true for the placement of the genus Launsina gen. nov. Since this genus shares several diagnostic features with Coryphella and Edmundsella, its inclusion in the family Coryphellidae (in accordance with its phylogenetic relationships) renders the diagnosis of Coryphellidae identical to that of Flabellinidae sensu [22]. The same is true for Mgueolia – with the expansion of the diagnosis of Paracoryphellidae to include the diagnostic features of Mgueolia, it largely overlaps with that of Coryphellidae sensu [22]. This situation is further complicated by complex mosaic character states within the entire diversity of Flabellinidae s.l., as initially identified by [19,23,24]. In many cases, key synapomorphies or shared features exhibit significant variability. For example, the presence of papillated rhinophores is an important synapomorphy of the genus Coryphellina (Fig 2), but within the genus Coryphella the morphology of the rhinophores varies substantially, from smooth to annulated, or perfoliated (Fig 2). Notably the high variability of rhinophore morphology was used to distinguish different genera within the Coryphellidae [22], but further phylogenetic studies did not support this suggestion, as rhinophores may differ significantly even in closely related species [34,41]. For example, Coryphella fogata has annulated rhinophores, and its sister species C. cooperi has wrinkled rhinophores [41], and the same was shown for C. trophina with perfoliate rhinophores and its closely related species C. alexanderi with smooth rhinophores [34,75].

In other cases, the variability of morphological characters requires considering their combination. This is exemplified by Flabellina. In the type genus of Flabellinidae, the arrangement of cerata, along with the features of the lateral teeth play a crucial role in identifying species within this genus. Except for F. pellucida and F. enne, all species in this genus are characterized by having cerata on compound peduncles (S3 Table), a feature unique to Flabellina within Flabellinidae (Fig 2). For the sister species F. pellucida and F. enne, the smooth lateral teeth (S3C, D Fig) set them apart from the genus Edmundsella, with which both species share several common traits: smooth rhinophores and cerata placed on low elevations (S2B, S15A–C Figs). The presence of these morphological characters in these latter two species indicates a retention of plesiomorphic traits.

Thus, despite the fact that the families Coryphellidae, Paracoryphellidae, and Flabellinidae sensu [22] were recovered as monophyletic and were highly supported in our BI analyses (Fig 1) (but not in the ML analysis, S1 Fig), the morphological study indicates rampant homoplasy across all groups. Further consideration of separate families Coryphellidae, Flabellinidae, and Paracoryphellidae with significantly overlapping diagnoses, as well as the description of many monotypic genera, leads to an unstable system that introduces practical difficulties associated with incorrect interpretation of characters’ taxonomical value. Our results highlight the limitations of adopting a splitting approach in this context. For example, the species Luisella telja was placed in the genus Samla [22], and the species Luisella engeli, Edmundsella dushia and E. bertschi were considered members of the genus Flabellina, but [22] mentioned that these species may represent several separate genus-level taxa. On the one hand, our analyses confirm the affiliation of these species to the genera Luisella and Edmundsella, respectively, and also show that these groupings are supported by unique morphological synapomorphies characteristic of all species of the corresponding genera (Fig 2). In this regard, the re-definition of genus-ranking taxa to encompass larger species diversity provides significantly more practical advantages, since it eliminates the allocation of taxa based on single autapomorphies, and ultimately leads to a greater stability of the system and increases its predictability. This also eliminates the need to create a high number of monotypic genera for any new derived phylogenetic entity with unique autapomorphies. Therefore, we also propose to redefine the composition of Flabellinidae s.str. by synonymizing of the Coryphellidae and Paracoryphellidae with Flabellinidae.

We demonstrate that the traditional ‘lumping’ approach, grouping diverse taxa under the name Flabellina, obscures complex evolutionary patterns within Flabellinidae s.l. Our results generally support phylogenetic relationships recovered by [22], and we want to additionally emphasize that the referred work provided a robust baseline for phylogeny-based systematics. However, the instability of several newly added taxa highlights a weakness of their “splitting” approach, as excessive splitting produces narrow diagnoses thus decreasing predictability. Neither extreme lumping nor excessive splitting provides optimal solution. Consequently, a balanced approach provides more advantages to accommodate mosaic evolution, overlapping diagnoses, and enhance practical stability. This balance aims for a more resilient classification.

A recent phylogenetic reconstruction and taxonomic revision of Aeolidacea [35] broadly agrees with our results in terms of recovered clades, but differs substantially in translating phylogeny into taxonomy. Most notably, the clade termed Flabellinidae by [35] is not supported at a reasonable level in any analysis, whereas the clade we recognize as Flabellinidae here is consistently recovered, well supported, and assigned family rank. Similarly, the grouping of Flabellina, Paraflabellina, Calmella, and Carronella at a level of genetic divergence comparable to Coryphellina and Edmundsella is supported in both studies despite [22,35] using a smaller dataset. The main taxonomic discrepancies stem from differing approaches to defining taxa: [22,35] emphasizes splitting based on interpreted morphological differences, which risks overlooking inconsistencies and relies on inherently subjective interpretation. While integrating phylogenetic and morphological data can reveal finer evolutionary patterns, especially in well-studied groups, clades corresponding to plausible taxa can be identified in most phylogenies, and supporting arguments can usually be constructed post hoc. A more robust approach should actively test and question putative taxa, considering clade stability, support, and the limitations of morphological diagnoses. This is especially important in groups with extensive undescribed diversity, where premature high-level taxonomic revisions may destabilize classification. Given that new specimens frequently prompt reassessment of both phylogenetic relationships and morphological interpretations, current datasets and analyses remain incomplete, and early studies often underestimate true diversity. Consequently, major taxonomic changes based on [35] approach risk rapid obsolescence. In line with a Bayesian perspective, we argue that taxonomy should be revised only when successive analyses of expanded datasets consistently support proposed taxa and reveal no evidence against them.