ZooKeys 1099: 29-40 (2022) A peer-reviewed open-access journal doi: |0.3897/zookeys.|099.75837 SHORT COMMUNICATION #ZooKkey S https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research Molecular analysis of Lepidopleurus cajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) from the Mediterranean and near Atlantic Mariastella Colomba', Julia D. Sigwart’, Walter Renda’, Armando Gregorini', Maurizio Sosso*, Bruno Dell’ Angelo” | University of Urbino, Dept. of Biomolecular Sciences, via I. Maggetti 22, 61029 Urbino (PU), Italy 2 Senckenberg Research Institute and Museum, Senckenberganlage 25, 60325 Frankfurt, Germany 3 Via Bologna 18/A, 87032 Amantea (CS), Italy 4 Via Bengasi 4, 16153 Genova (GE), Italy § Via Briscata 16, 16154 Genova (GE), Italy Corresponding author: Mariastella Colomba (mariastella.colomba@uniurb.it) Academic editor: Frank Kohler | Received 27 September 2021 | Accepted 28 March 2022 | Published 3 May 2022 http://zoobank. org/AD5 93 D6B-D21F-4576-A8A2-EF8E 12D3A840 Citation: Colomba M, Sigwart JD, Renda W, Gregorini A, Sosso M, Dell'Angelo B (2022) Molecular analysis of Lepidopleurus cajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) from the Mediterranean and near Atlantic. ZooKeys 1099: 29-40. https://doi.org/10.3897/zookeys.1099.75837 Abstract In the present paper we used a molecular data set (including mitochondrial partial 16S rRNA and COI gene sequences) to examine the genetic structure of Lepidopleurus cajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) - a distinctive shallow water chiton and member of the basal branching Lepidopleurida, which is widespread in and adjacent to the Mediterranean. The analyses of the two mt-standard marker fragments resolved two main discrete clusters reported as L. cajetanus s.s. and L. aff. cajetanus, respectively. Lepidopleurus cajetanus s.s. is widespread throughout the area under study, while the second distinct line- age apparently co-occurs on the eastern Spanish mainland coast of the Balearic Sea. This result is discussed comparing our data with those reported, in 2014, by Fernandez and colleagues who described L. cajetanus as exhibiting “a ‘chaotic patchiness’ pattern defined by a high genetic variability with locality-exclusive haplotypes, high genetic divergence, and a lack of geographic structure”. Although genetic data alone are not sufficient to draw any definitive conclusions, nevertheless we believe that present results shed new light on L. cajetanus which apparently shows more geographically patterned genetic structure than sup- posed so far. Keywords 16S rRNA, chitons, COI, phylogeny, standard mitochondrial markers Copyright Mariastella Colomba et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 30 Mariastella Colomba et al. / ZooKeys 1099: 29-40 (2022) Introduction Chitons (class Polyplacophora) are the third-largest class in the phylum Mollusca by species richness of living taxa (Ponder et al. 2020). The superficial similarity of the living species, with a distinctive eight-part shell armour covering a soft foot that adheres to the substratum, has created some long-term confusion in the ecological identification of species. Yet Lepidopleurus cajetanus (Poli, 1791) (Polyplacophora, Leptochitonidae) was recognised as a distinct form in the Mediterranean very early in the history of formal modern taxonomy. Although it is phylogenetically nested within the genus Leptochiton s.s. with other species in this clade from the Mediterranean and North Atlantic (Sigwart et al. 2011), taxonomists have maintained the genus Lepidopleurus in acknowledgement of its unique morphology. WoRMS (World Register of Marine Species) reports two living species of Lepidopleurus, L. cajetanus (Poli, 1791) and L. cullierti Roch, 1891 (https://www.marinespecies.org/aphia.php?p=taxdetails&id=138116). Actually, Castellanos (1988) cites L. cullierti showing also a figure of it (later taken up by Forcelli 2000), without taking into account that this species was already considered by Pilsbry (1893: 111) a nomen dubium (certainly not a Lepidopleurus, but belonging probably to the genus Chaetopleura). The species is not reported in recent papers dealing with Leptochitonidae from Chile (Sirenko 2006; Schwabe 2009; Schwabe and Sellanes 2010; Sirenko 2015; Sirenko and Sellanes 2016), but it is still (erroneously) reported in various lists. Recently, Aldea et al. (2020) reported L. cullierti as a dubious species. In conclusion, at least as far as concerns living species, Lepidopleurus is presently a monotypic genus. Notably, nomenclature is still confusing and a clear distinction between Lepidopleurus and Leptochiton has not been fully achieved. Lepidopleurus was the first genus name proposed for lepidopleuran chitons, including only the species L. cajetanus. In 1847, Gray established the genus name Leptochiton. Both genera were included in the family Leptochitonidae Dall, 1889 with Leptochiton asellus as the type species. A few years later, Pilsbry (1892) listed Leptochiton as a junior subjective synonym of Lepidopleurus, and changed the family name to Lepidopleuridae. Since then, Lepidopleurus and Leptochiton (and family names) have been used more or less interchangeably (Sigwart et al. 2011). To date, there is insufficient evidence to separate Lepidopleurus and Leptochiton s.s. as distinct genera, even if they are often distinguished on the basis of shell thickness and sculpture [i.e., distinctive shell morphology with pronounced concentric ridges on the lateral areas and terminal valves (Lepidopleurus), or flat and plain shells generally lacking strong raised sculpture (Leptochiton)]. Lepidopleurus cajetanus is widespread throughout the Mediterranean (where, even if quite discontinuously, it can be very common locally) and more rarely in the Atlantic, from the Iberian Peninsula (Spain and Portugal) to Morocco and to the Canary Islands (Spain) and Berlengas Archipelago (Portugal) (Kaas and Van Belle 1985; Dell’Angelo and Smriglio 2001). The species is known as fossils in the European Neogene: from the lower Miocene (Burdigalian) of the Aquitaine Basin to the middle Miocene of the Aquitaine Basin and Paratethys and the French and Italian upper Miocene, to the Molecular insight into Lepidopleurus cajetanus 31 Pliocene of Italy, Spain and Greece, to the Pleistocene of Italy and Greece (Dell’Angelo et al. 2018a, 2018b and references therein). As has been widely described (see for ex- ample, Dell’Angelo et al. 2013, 2015, 2018a, 2018b), fossils of L. cajetanus show great variability in the morphological characters of the plates (sculpture, shape, etc.), which is much less evident in the living specimens. Living chitons are broadly divided into two main clades, the orders Lepidopleurida and Chitonida. The former group retains plesiomorphic shell forms and is therefore particularly interesting for studies of molluscan phylogeny (Sigwart et al. 2011). Most members of Lepidopleurida inhabit deep sea environments, but as Lepidopleurus cajetanus can be found intertidally it has been widely used in genetic studies including molecular phylogenetic studies of molluscs (see for example, Giribet and Wheeler 2002). In addition to data on the impact of strong biogeographical barriers on gene flow (Ayre et al. 2009), other studies on chiton population genetics have recovered well-mixed populations in spite of geographic barriers (e.g., Doonan et al. 2012), including some species in the Mediterranean such as Rhyssoplax olivacea (Spengler, 1797) (Fernandez et al. 2014). One study described a cryptic species on the basis of differential haplotype structures which were attributed to potentially different dispersal capacity of two species of Leptochiton s.l. (Sigwart and Chen 2018): L. rugatus (Carpenter in Pilsbry, 1892) and L. cascadiensis Sigwart & Chen, 2018. In contrast to previous results for chitons, a focussed study on the population genetic structure of Lepidopleurus cajetanus from the Atlantic and Mediterranean coasts found ‘chaotic patchiness’ defined by unique haplotypes, high genetic divergence, and yet no apparent geographic partitioning (Fernandez et al. 2014). In particular, the authors found two major clades, one of which was divided into two subclades. ‘The possibility that some of these lineages represented multiple cryptic species in L. cajetanus was raised. but, eventually, dismissed “because of the unique morphology of L. cajetanus”. Starting from that paper, in the present study, we collected genetic samples from additional locations, expanding the geographical coverage examined for Lepidopleurus cajetanus, in order to test whether increasing the number of samples and adding new collection sites could confirm the pattern already described as suggesting old and stable populations with, however, limited distinguishable geographical structure. Alternatively, by filling in more of the geographic range of this species, new results could help resolve a broader structure of distinctive co-occurring but separate clades. Materials and methods Thirteen (13) Lepidopleurus cajetanus specimens were sampled from the Atlantic and Mediterranean coasts of Spain, Italy, Croatia and the Canary Islands by two of the authors (BDA and WR) and other collectors (Table 1). Total genomic DNA was isolated from a small piece of tissue taken from the foot of ethanol-preserved specimens. The extractions were carried out using the Wizard Genomic DNA Purification Kit (Promega). All the DNA extractions were kept at 32 Table |. GenBank accession numbers of 16S rRNA and COI partial sequences of the specimens used in Mariastella Colomba et al. / ZooKeys 1099: 29-40 (2022) the study and reported in the phylogenetic tree. Species / sample nr L. cajetanus s.s. SO AN WWM KR W} FH 42 ZORRO HA TOMB OOM ERD COI KF052983 KF052981 KF052980 KF052979 KF052978 KF052977 KF052976 KE052975 KF052974 KF052972 KF052971 KF052970 KF052969 KF052968 KF052967 KF052966 KF052965 KF052960 KF052959 KF052957 KF052956 KF052954 KF052952 KF052951 KF052950 KF052948 KF052947 KJ500166 AF120626 KF052961 KE052955 KF052949 KF052944 KEF052945 KF052946 MW751980 MW751981 MW751982 MW751983 MW751984 MW751985 MW751986 MW751987 16S rRNA KF052732 KF052735 KF052737 KF052713 KF052724 KF052715 KF052723 KF052725 KF052729 KF052728 KF052711 KF052733 KF052714 KF052731 KF052738 KF052730 KF052734 KF052727 KF052726 KF052721 KF052722 KF052736 KF052712 KF052719 KF052720 KF052718 KF052717 KJ500177 AY377585 KF052709 KF052710 KF052716 MW748076 MW748077 MW748078 MW748079 MW748080 MW748081 MW748082 MW748083 MW748084 MW748085 Collection site (CS) Cadaques (Girona, Spain) Cadaques (Girona, Spain) Cadaques (Girona, Spain) Tossa de Mar (Girona, Spain) Tossa de Mar (Girona, Spain) Tossa de Mar (Girona, Spain) Tossa de Mar (Girona, Spain) Tossa de Mar (Girona, Spain) Tossa de Mar (Girona, Spain) Calafat (Tarragona, Spain) Calafat (Tarragona, Spain) Cabo de Palos (Murcia, Spain) Cabo de Palos (Murcia, Spain) Mar Menuda, Tossa de Mar (Girona, Spain) 10 Mar Menuda, Tossa de Mar (Girona, Spain) 10 Mar Menuda, Tossa de Mar (Girona, Spain) 10 Mar Menuda, Tossa de Mar (Girona, Spain) 10 Mar Menuda, Tossa de Mar (Girona, Spain) 10 Mar Menuda, Tossa de Mar (Girona, Spain) 10 me BB ODO OD WW WD WW WO NH NY WN Cadaques (Girona, Spain) 2, Xabia (Alicante, Spain) 5 Xabia (Alicante, Spain) 5 NA NA Rhodes (Greece) 7 Rhodes (Greece) 7 Rhodes (Greece) 7 Rhodes (Greece) 7. Santa Maria Navarrese (Sardinia, Italy) 23 NA NA Mar Menuda, Tossa de Mar (Girona, Spain) 10 Xabia (Alicante, Spain) 5 Rhodes (Greece) 7 Cabrera (Balearic Islands, Spain) 8 Cabrera (Balearic Islands, Spain) 8 Cabrera (Balearic Islands, Spain) 8 Tossa de Mar (Girona, Spain) 3 Rhodes (Greece) 7 Rhodes (Greece) i, Torre Ovo (Taranto, Italy) 24 Chia, Cagliari (Sardinia, Italy) 23A Aguilas (Murcia, Spain) 1A Playa de Las Heras (Tenerife, Canary Is.) 26 Arzachena, Sassari (Sardinia, Italy) 23A Tertenia, Nuoro (Sardinia, Italy) 23A Tertenia, Nuoro (Sardinia, Italy) 23A Poetto, Cagliari (Sardinia, Italy) 23A San Lucido (Cosenza, Italy) 24 Aguilas (Murcia, Spain) 1A Umago (Croatia) 25 Lussino Is. (Croatia) 25 Vrsar, Orsera (Croatia) 25 CS nr Reference © .O OA“ AO B90 A A OO TTT TTT TOS OF TORTS T TT OT ES TT OTT TOT TS oS Molecular insight into Lepidopleurus cajetanus 33 Species / COI 16S rRNA Collection site (CS) CS nr Reference sample nr L. aff. cajetanus 2 KF052982 KF052702 Cadaques (Girona, Spain) 2 b 11 KF052973 KF052707 Tossa de Mar (Girona, Spain) 3 b 20 KF052964 KF052708 Mar Menuda, Tossa de Mar (Girona, Spain) 10 b 21 KF052963 KF052706 Mar Menuda, Tossa de Mar (Girona, Spain) 10 b 22 KF052962 KF052703 Mar Menuda, Tossa de Mar (Girona, Spain) 10 b 25 KF052958 KF052700 Cadaques (Girona, Spain) 2 b 29 KF052953 KF052705 Xabia (Alicante, Spain) 5 b 43 KF052701 Tossa de Mar (Girona, Spain) 3 b 45 KF052699 Cabo de Palos (Murcia, Spain) 1 b Rhyssoplax olivaceus I-46 KJ500158 — KJ500165, KJ500168 — KJ500174, KF052941 — KF052942, KJ500176, KF052739 — KF052875 — KF052877, KF052740, KF052778, KF052885 — KF052887, KF052800 — KF052802, KF052889 KF052791 — KF052792 Ischnochiton spp. AY377704 — AY377709 = AY377593 — AY377596 Lepidopleurus cajetanus specimens are indicated by numbers (available data) or letters (present study) along with collection sites, collec- tion site numbers and reference: a: Giribet and Wheeler (2002); b: Fernandez et al. (2014); c: present study. 4 °C for short-time use. Undiluted or different dilutions (from 1:10 to 1:50, based on the DNA concentration) of each DNA extraction were used as templates for PCR amplification of a portion of each of the two loci: the mitochondrial large subunit ribo- somal DNA (mt-16S rRNA) and the cytochrome oxidase subunit I (mt-COI) genes. For the COI gene the primers used were LCO1490 (5’°-GGTCAACAAATCATAAA- GATATTGG-3’) and HCO2198 (5’°-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994). PCR conditions involved an initial denaturation step at 95 °C for 5 min; then 35 cycles of denaturation at 95 °C for 1 min, annealing at 42 °C for 1 min and extension at 72 °C for 1 min; followed by a final extension step at 72 °C for 5 min. For the 16S rRNA gene, the primers used were 16sF (5’-CGGCCGCCTGTT- TATCAAAAACAT-3’) and 16sR (5’--GGAGCTCCGGTTTGAACTCAGATC-3’) (Palumbi et al. 1991). The PCR conditions involved an initial denaturation step at 95 °C for 5 min; then 35 cycles of denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 1 min; followed by a final extension at 72 °C for 5 min. Amplified products were purified using the Wizard SV Gel and PCR CleanUp System (Promega). Pinna muricata Linnaeus, 1758 (Bivalvia) and Haliotis discus Reeve, 1846 (Gastropoda) were selected as outgroup for molecular analysis following the prior study by Fernandez et al. (2014). Pinna muricata and H. discus 16S rRNA and COI partial sequences (AB076929, GQ166570, AM049335 and AY146392), retrieved from GenBank, were added to homologous sequences of Lepidopleurus cajetanus previously studied (Giribet and Wheeler 2002; Fernandez et al. 2014) and of L. cajetanus examined in the present study for the first time, with a total of 60 L. cajetanus ingroup terminals. Sixteen Rhyssoplax olivacea (Spengler, 1797) and four /schnochiton spp., were also added to the analysis (Table 1). All the sequences for each gene were aligned with BioEdit 34 Mariastella Colomba et al. / ZooKeys 1099: 29-40 (2022) ClustalW. The substitution model for each partition was determined via the CIPRES Science Gateway (http://www.phylo.org/) (Miller et al. 2010) by the tool jModelTest of XSEDE. MrBayes analysis of multiple sequence alignment (COI+16S rRNA genes, in nexus format) was run on CIPRES by MrBayes on XSEDE, with the parameters for the consensus tree (50% majority rule, excluding 25% of trees as burnin) specified on the MrBayes block. All sequences generated in the present study were deposited in NCBI GenBank (Table 1). Automatic Barcode Gap Discovery (ABGD) was also used on all available L. cajetanus COI sequence data (Puillandre et al. 2012) in order to tentatively delimit potential genetic lineages. Finally, Population Analysis with Reticulate Trees (PopART; Leigh and Bryant 2015) was employed to infer the L. cajetanus haplotype networks by the TCS (Templeton, Crandall and Sing) method. Results and discussion Results from the ABGD based on the COI fragments recovered two distinct groups, plus a separate group represented by only one specimen (specimen 35, KJ500166). These two main groups correspond exactly to the two major clades of Lepidopleurus cajetanus recovered in the combined phylogenetic analysis (Fig. 1). The COI haplotype network reconstruction (Fig. 2) also resulted in two groups that also correspond to those identified by the barcode gap and phylogenetic analysis. By comparison of the outputs obtained from these analyses (ABGD and TCS haplotype networks) and considering the phylogenetic tree topology, it appears that the two groups form well resolved and distinct populations. As far as concerns specimen 35 (from Fernandez et al. 2014) it nests within the primary Lepidopleurus cajetanus clade but is quite different from the others. The phylogenetic reconstruction for L. cajetanus shows a deep split, with two major clades supported by high (100%) posterior probability values. One of the clades is composed of individuals drawn from all the sampled populations, which we refer to as Lepidopleurus cajetanus s.s. The other clade, which we refer to as Lepidopleurus aff. cajetanus is formed by specimens from the eastern Iberian Peninsula (i-e., various localities of Girona, Alicante and Murcia including Cadaqués, Tossa del Mar, Xabia and Cabo de Palos), thus suggesting the presence of two genetically divergent lineages on the eastern Spanish coast. Fernandez et al. (2014) sampled three specimens from the Balearic Islands (COI marker only, GenBank accession numbers KF052944— KF052946) which are part of the broader Lepidopleurus cajetanus s.s. lineage. Since both clades co-occur on the eastern Spanish mainland coast, hypothetically, we cannot exclude the possibility that L. aff. cajetanus may be present also in the Balearic Islands. Comparing these two clades nominally comprising Lepidopleurus cajetanus, it appears that the L. aff. cajetanus clade has a much more limited genetic variability compared to the larger, more broadly distributed clade. The pairwise distances of COI fragments for the larger clade had a maximum separation of 8.3% (or up to 20% including specimen 35) and an average distance of 3.6%; the maximum distance Molecular insight into Lepidopleurus cajetanus 35 1 * Collection Region 3 © Lepidopleurus cajetanus ® Canary Is. 1%. sensu stricto e Spain a ® Balearic Is., Spain 16 © ® Italy 18 @ 19 © ® Croatia 24} ® Greece - . not recorded 1 De Be c # E ® F @ G ® 4 @ 17 © 23 @ 38 © 37 @ 13 @ 5 @ 44 © H © ‘ 6 € 99% 410m a 6 9 @ 41 27 @ 42 7 © 15 @ 26 © 9 31 . 100% ae L e M e 33 e 34. ® 33 47 e K a A e 100% 35 @ 46 @ 36 f * Lepidopleurus aff. 400% 2 Scans 29% 22 91.75% oe 430 45@ Ryssoplax olivaceus 7 100% Ryssopiax olivaceus 3 Ryssopiax olfvacets 7 Ryssoplax olivaceus 8 Ryssoplax olivaceus 9 Ryssopiax olivaceus 10 Ryssoplax olivaceus 17 Ryssoplax olivaceus 12 Ryssopiax olivaceus 13 Ryssoplax olivaceus 14 Ryssoplax olivaceus 15 Ryssoplax olivaceus 16 Ryssopilax olivaceus 2 Ryssoplax olivaceus 5 57.74% Ryssoplax olivaceus 6 400% Ryssoplax olivaceus 4 2 Ischnochiton rissoi 100% fschnochiton comptus 100% ischnochifon australis ischnochifon efongatus Haliotis discus Pinna muricata 0.06 Figure |. Bayesian phylogenetic tree obtained with MrBayes on the basis of a multiple sequence align- ment (COI+16S rRNA genes) analysis. Nodal supports are Bayesian inference posterior probability (ex- pressed in percentage). Scale bar represents units of length in expected substitutions per site. Lepidopleurus cajetanus specimens previously analysed (Giribet and Wheeler 2002; Fernandez et al. 2014) are indicated by numbers, L. cajetanus specimens added in the present study are indicated by letters in bold. Colours correspond to the geographic distribution (see also Table 1). 36 Mariastella Colomba et al. / ZooKeys 1099: 29-40 (2022) Lepidopfeurus aff. 4 cajetanus 10 samples 1 sample Collection Region Canary Is. Spain Balearic Is., Spain Italy Croatia Greece not recorded Figure 2. COI haplotype (TCS) network showing the relationships of L. cajetanus specimens. Circle size *®eegeeeEes is proportional to the observed haplotype frequencies. Colours correspond to the geographic distribution as in Fig. 1 (see also Table 1). between members of the L. aff. cajetanus clade was 0.49% with an average of 0.22%. This is reflected in the smaller distances and smaller number of haplotypes among the L. aff. cajetanus clade specimens (Fig. 2). It may be an artefact of comparative sample numbers for the COI fragment, with only seven specimens of the L. aff. cajetanus clade compared to 43 from Lepidopleurus cajetanus s.s., but the observed differences might indicate biological separation of the two lineages. The two clades are separated by a mean distance of 17.8%, which is similar to the value of 15.7% used as part of the description to separate Leptochiton cascadiensis from Leptochiton rugatus (Sigwart and Chen 2018). Our results confirm that the population genetic structure of Lepidopleurus cajetanus based on the COI barcode marker is characterized bya high number of private haplotypes, and high genetic divergence between haplotypes and between clades, extending the Molecular insight into Lepidopleurus cajetanus 37 pattern first identified by Fernandez et al. (2014). However, with the addition of a broader geographical sampling, it seems that the “chaotic patchiness” nonetheless divides into two discrete clades, and further to some larger biogeographic patterns. The combined phylogenetic reconstruction shows one clade of specimens from Greece and Croatia, and two groupings of specimens from Italy and Spain, within the Lepidopleurus cajetanus s.s. clade. We suspect that the Lepidopleurus cajetanus s.s. clade and the L. aff. cajetanus clade might represent two distinct lineages, where Lepidopleurus cajetanus s.s. contains substantially more genetic diversity, at least in the COI marker, and the L. aff. cajetanus clade is more constrained. Whether L. aff. cajetanus could be interpreted as a possible (criptic?) species is impossible to say, as genetic data alone are not sufficient to draw any definitive conclusions. In fact, further morphological examination of Spanish specimens is certainly required to re-examine potential diagnostic characters, and to obtain additional independent sources of comparative data. Unfortunately, all of the sequence data corresponding to the L. aff. cajetanus clade came from prior work; we have not examined specimens known to be from the L. aff. cajetanus clade in the present work. In fact, new materials that we sequenced from Aguilas, Murcia, Spain, are also part of the Lepidopleurus cajetanus s.s. clade. The fossil valves of Lepidopleurus cajetanus sensu lato show remarkable variations, e.g. in the sculpture of the lateral areas of the intermediate valves (with the starting point of the concentric ribs neighbouring the lateral margin and not near the apex, as in normal valves, and consequently with a different frontal view; compare Dell’ Angelo et al. 2013: pl. 1 figs B-C and D-E), in the position of the mucro in the tail valves [almost central in juvenile specimens but moves posterior (even to the end of the valve) as individuals grew older, as well described and illustrated by Laghi (1977: fig. 3a-b) and Dell’Angelo et al. (2013: pl. 1, figs F-G)], and in the sculpture of the central area of the intermediate valves and the antemucronal area of the tail valve [normally with longitudinal and parallel chains of granules, somewhat branching or anastomosing, very irregular, transversally intersected by thinner cords that give a pitted appearance (see Dell’Angelo et al. 2015: pl. 1, figs 4-11)]. Future studies of material of living Lepidopleurus cajetanus s.\. from the eastern Spanish mainland coast (and Balearic Is.) should focus on these shell characteristics, to determine whether the two lineages can be diagnosed morphologically, and also how they compare to the extensive fossil record. It is now well known that standard barcode markers such as COI show some variability within and among species (e.g., Sigwart and Garbett 2018), and it is not appropriate to use an a priori distance cut-off to distinguish species. Taking into account the limitations of the current study (reliance of mt-DNA only) and that species status is best assessed in light of an integrative, total evidence approach, caution is required in interpreting the L. aff. cajetanus clade until a morphological diagnosis is available. However, our results seem to suggest the presence of (at least) two genetic lineages within L. cajetanus that will need to be adequately investigated in future studies including also additional (nuclear) markers and/or anatomy to arrive at systematically more robust conclusions. Importantly, this is a species (or species complex) with a very good fossil record and representing greater disparity than the living lineages (Dell’Angelo et al. 2013, 2015, 2018a, 2018b). Although 38 Mariastella Colomba et al. / ZooKeys 1099: 29-40 (2022) Lepidopleurus cajetanus s.s. has apparently high variability in these mitochondrial markers, we are cautious about making any inferences about phylogeographic patterns or potential for cryptic species or incipient speciation. These issues do require integrated evidence from the morphology of living and fossil populations, nonetheless this study indicates a novel genetic pattern in a common and phylogenetically important species. Acknowledgements Special thanks are due to Diego Viola (Muggia, Italy), Michele Pisanu (Quartu S. Elena, Italy), Ivan Mulero Méndez and Brian Cunningham Aparicio (Murcia, Spain) for the valuable material collected. This work was supported by MIUR (PRIN 2009, prot. 2009LFSNAN_003) funds to A. Gregorini. Moreover, A. Gregorini wishes to thank the Ilaria Giacomini Associa- tion (Cantiano, PU, Italy) for financial support. 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