New record and new species of Laubierpholoe Pettibone, 1992 (Annelida, Sigalionidae) from the soft bottom of submarine caves near Marseille (Mediterranean Sea) with discussion on phylogeny and ecology of the genus

. A new species of Laubierpholoe Pettibone, 1992 (Annelida, Sigalionidae), Laubierpholoe massiliana Zhadan sp. nov., was found in two submarine caves near Marseille (France). This is the ﬁ rst record of the genus in the Mediterranean Sea. The new species di ﬀ ers from congeners by inhabiting soft sediments instead of having an interstitial lifestyle and by several morphological characters: the ventral tentacular cirri slightly shorter or of similar length to the dorsal tentacular cirri, the presence of bidentate neurochaetae, the body length, and the number of segments. Molecular phylogenetic analysis using 18S rRNA and 28S rRNA sequences con ﬁ rmed that the new species belongs to the genus Laubierpholoe , as well as the monophyly of the genus. The ecology of the new species and its adaptation to the cave-dwelling lifestyle are discussed. An identi ﬁ cation key for all known species of Laubierpholoe is provided.


Introduction
Pholoinae Kinberg, 1858 is a group of scale-worms which are small (up to 2 cm long, up to 90 segments) and common in all kinds of marine intertidal and subtidal habitats. They have been treated as a separate family or as a part of Sigalionidae Kinberg, 1856(Barnich & Fiege 2003Wiklund et al. 2005;Norlinder et al. 2012). According to recent phylogenetic analysis of Aphroditiformia Levinsen, 1883 using four molecular markers and 87 morphological characters, Pholoinae is nested within Sigalionidae and considered to be a subfamily (Gonzalez et al. 2018a). The diagnostic characters of Pholoinae are: body typically elongate and narrow with as few as 15 segments; notochaetae geniculate and fi nely tapered; simple neurochaetae absent; neuropodial stylodes possibly present; elytral brooding present in some genera (Gonzalez et al. 2018a).
Within Pholoinae, the genus Laubierpholoe Pettibone, 1992 includes fi ve meiobenthic species. The fi rst species of the genus was described as Pholoe antipoda (Hartman, 1967) from Tierra del Fuego (South Atlantic Ocean) (Fig. 1A). Its main diff erences from other species of Pholoe Johnston, 1839 were a much smaller size, a reduced number of segments and details of the elytra and neuropodial falcigers. The second species, Pholoe swedmarki (Laubier, 1975) was later described from Bermuda (Northeast Atlantic Ocean) (Fig. 1A) from 2-8 m depth. The author was unaware of the previous description of P. antipoda and compared P. swedmarki with other species of Pholoe. Laubier (1975) noticed its much smaller size (maximum length 1.6 mm, width 400 μm), smaller number of notochaetae, elongated palps but reduced ventral cirri, and smooth elytra with few papillae. He also described developing embryos inside the elytra, postulated internal fertilization and considered these characters as adaptations to interstitial life style (Laubier 1975). Pettibone (1992) performed the revision of Pholoidae Kinberg, 1858 and provided a diagnosis of this family including diagnoses of all genera and species as well as identifi cation keys to all taxa. She erected the new genus Laubierpholoe for the two above-mentioned previously described species and for two new species: L. maryae Pettibone, 1992 and L. riseri Pettibone, 1992, both from New Zealand (Fig. 1A). The new genus was distinguished by the following main characters: achaetous tentaculophores lateral to prostomium, each with a long dorsal and a very short ventral tentacular cirrus; stout, very long palps; notochaetae of a single type, slightly curved or straight; and up to 29 segments.
The last species of Laubierpholoe, L. indooceanica Westheide, 2001, was described from coral reef fl ats of Krusadai Island and the Seychelles (Indian Ocean) (Fig. 1A). It is the smallest (less than 1 mm long) of all species of the genus and diff ers by the presence of hook-like blades in some of the compound neurochaetae. Phylogenetic analysis of Aphroditiformia confi rmed the position of Laubierpholoe within Pholoinae and the clade was well supported by molecular data and by the apomorphy presence of elytral brooding (Gonzalez et al. 2018a). The other interstitial pholoin genera Imajimapholoe Pettibone, 1992 and Taylorpholoe Pettibone, 1992 also have elytral brooding and direct development. In contrast, the interstitial Metaxypsamma uebelackerae Wolf, 1986 lacks elytra. No molecular data are available for these genera at the moment.
Potential reservoirs of such easily overlooked meiobenthic taxa exist very close to the shore. In the Mediterranean, one of the best-studied areas in the world, underwater marine caves have been shown to be hotspots of unknown diversity because of their more diffi cult access and peculiar environmental conditions. Caves are numerous near Marseille (SE France) thanks to the karstic nature of the seashore (Harmelin et al. 1985). Darkness and poor water circulation create oligotrophic environmental conditions similar to the deep sea and it is remarkable that many organisms found in marine caves belong to deep water taxa (e.g., Calado et al. 2004;Janssen et al. 2013;Chevaldonné et al. 2015;Cárdenas et al. 2018;Chevaldonné & Pretus 2021). In addition, some caves with particular geomorphologies provide a cold thermal regime (mean ca 13-15°C) similar to that of the Mediterranean deep sea (Vacelet et al. 1994;Bakran-Petricioli et al. 2007). Only a few studies have focused on cave sediment fauna, often with an emphasis on targeted taxonomic groups (e.g., Janssen et al. 2013;Zeppilli et al. 2018). In the 3PP and Jarre Caves in the Calanques National Park near Marseille (Fig. 1B-С), it has been shown that sea water remained colder than the outside environment or other caves throughout the year (Bakran-Petricioli et al. 2007). For instance, though it is only 25-30 m deep, the 3PP Cave has been shown to display meiofaunal organisms usually found at abyssal sites (Janssen et al. 2013). These are also large caves with extended areas of silty bottom favourable for the study of cave sediment fauna. Therefore, they have been the focus of recent investigations of macro-and meiofauna, especially of presently poorly-studied groups (Vortsepneva et al. 2021). A diverse annelid fauna was discovered there including a new species of Laubierpholoe described here with discussion of its taxonomy, phylogeny and the ecology of the group.

Sampling and preservation
All material was sampled in the Calanques National Park near Marseille ( Fig. 1B-С) in 2018-2020. Samples were taken close to the entrance, in the middle (40 m from entrance) and the deep (60-70 m from entrance) parts of Jarre and 3PP Caves at 18-25 m depth. The coordinates of the entrance of Jarre Cave are 43.19556° N, 5.3658333° E and 43.16306° N, 5.6° E for 3PP Cave. Bottom sediment was collected by SCUBA diving with 20 cm-wide sampling boxes at about one cm sediment depth and a length of 120 cm. Samples were washed using the fl oatation method and a sieve with mesh size 130 μm. In total, about 50 specimens of Laubierpholoe were collected. They were relaxed using a magnesium chloride (MgCl 2 ) solution isotonic to seawater for 30 min and preserved in 96% ethanol or in 2.5% glutaraldehyde (Electron Microscopy Supplies, EMS, Pennsylvania, USA) buff ered with 0.1 mol phosphate buff er and transferred to 70% ethanol after washing with the same buff er.

Granulometric analysis
Subsamples of sediment were taken in 3PP and Jarre Caves in the deep, middle and near entrance parts and granulometric analysis were performed in Limited Liability Company "Laboratory" (Sankt-Petersburg, Russia).

Morphological analysis
Whole specimens were photographed via stereo microscope and compound microscope using an iPhone 6 with a Labcam (iDu Optics, Detroit, Michigan, USA) adapter for living specimens and a Leica microscope (Leica Microsystems GmbH, Germany) with a Leica adapter for preserved ones. For SEM, specimens were dehydrated in a graded ethanol series (20-25 min per step), then transferred to acetone and critical point dried. Whole specimens and their fragments were coated with platinum-palladium and examined using a Camscan S-2 (Cambridge Instruments, London, United Kingdom) scanning electron microscope at the Laboratory of Electron Microscopy at the Biological Faculty of Moscow University.

DNA amplifi cation and sequencing
The Promega Wizard SV Genomic DNA Purifi cation Kit and protocol (Promega Corporation, Madison, USA) were used for tissue lysis and DNA purifi cation. Polymerase chain reaction (PCR) amplifi cation of nuclear 18S rRNA and fragments of 28S rRNA was accomplished with the standard primers. The 18S rRNA gene was PCR amplifi ed in three overlapping fragments using primer pairs 1 F-5R, 3 F-18Sbi and 18Sa2.0-9 R (Giribet et al. 1996(Giribet et al. , 1999. For the 28S rRNA gene we used C1' and C2 primers (Le et al. 1993). The universal primers 16Sar-L and 16Sbr-H (Palumbi & Kessing 1991) did not yield products that could be sequenced, therefore for the 16S rRNA gene we tried to use the primer pair 16SAnnF-16-SAnnR (Sjölin et al. 2005), unfortunately with the same result. We were also unable to amplify the CO1 gene fragment (Folmer fragment) using the primers jgLCO1490 and jgHCO2198 (Geller et al. 2013). All loci were amplifi ed using the Encyclo PCR kit (Evrogen JointStock Company, Russia). We amplifi ed a 25 μl reaction mix containing 1x PCR buff er, 1 μl of 10 μM of primer pair mix, 1 μl of template, 0.2 mM of each dNTP and 0.5 units Taq polymerase. Reaction mixtures were heated on Veriti® Thermal Cycler to 95°C for 300 s, followed by 35 cycles of 15 s at 95°C, 20 s at a specifi c annealing temperature, and 45-60 s at 72°C, depending on the length of fragment, and then a fi nal extension of 7 min at 72°C. Annealing temperature was set to 49°C for the 18S primer pairs 1 F-5R and 18Sa2.0-9 R, 52°C for the 18S primer pair 3 F-18Sbi and for the 28S primer pair C1′-C2 (Rousset et al. 2007). We used the Promega PCR Purifi cation Kit and protocol (Promega) to purify our amplifi cation products which were sequenced in both directions. Each sequencing reaction mixture included 1 μl of BigDye (Applied Biosystems, PerkinElmer Corporation, Foster City, CA), 1 μl of 1 μM primer and 1 μl of DNA template and was processed for 40 cycles of 96°C (15 s), 50°C (30 s) and 60°C (4 min). Samples were purifi ed prior to sequencing by ethanol precipitation to remove unincorporated primers and dyes. Products were re-suspended in 12 μl formamide and electrophoresed in an ABI Prism 3500 sequencer (Applied Biosystems). All new DNA sequences have been submitted to the NCBI GenBank repository.

Data analysis
Nucleotide sequences were edited using the software CodonCode Aligner ver. 5.0.2 (CodonCode Corporation) and checked for identity against the nuclear redundant (default) database of GenBank using BLASTn (Altschul et al. 1990). Data analysis included 8 sequences from this study (four specimens of Laubierpholoe massiliana Zhadan sp. nov., 18S rRNA and 28S rRNA) and sequences of 18S rRNA and 28S rRNA obtained from GenBank for species of Sigalionidae including Pelogeniinae Chamberlin, 1919, Pholoinae, Pisioninae Ehlers, 1901, and Sigalioninae Kinberg, 1856. Harmothoe impar (Johnston, 1839), species of Polynoidae Kinberg, 1856, was included as an outgroup species. GenBank accession numbers for sequences used in the present study are provided in Table 1. The sequences were aligned with the MAFFT multiple alignment tool (Katoh & Standley 2013) with default parameters: MAFFT fl avour: auto; gap extension penalty: 0.123; gap opening penalty: 1.53; direction of nucleotide sequences: do not adjust direction; matrix selection: no matrix. Then sequences were curated with Gblocks (Castresana 2000) with default parameters: minimum number of sequences for a conserved position (b1): 50% of the number of sequences +1; minimum number of sequences for a fl ank position (b2): 85% of the number of sequences; maximum number of contiguous non-conserved positions (b3): 8; minimum length of a block (b4): 10; allowed gap positions (b5): none. The fi nal lengths for individual alignments were 1407 and 675 bp for the 18S and 28S respectively. The 18S rDNA and 28S rDNA alignments were concatenated using MEGA X software (Kumar et al. 2018). We performed phylogenetic reconstruction for concatenated 18S+28S alignment using Bayesian inference (BI) and maximum likelihood (ML) analyses. For BI the best fi t model selection was performed using MEGA X software the GTR+G+I model was selected. The BI analysis was run in MrBayes ver. 3.2.7 (Huelsenbeck & Ronquist 2001). We made one run, four chains were ran simultaneously, three heated and one cold. The number of generations was set to 1 000 000. Chains were sampled every 500 th generation and 0.25 of the samples was discarded as burnin. The ML analysis was performed in PhyML (Guindon et al. 2010) with SMS (Smart Model Selection) (Lefort et al. 2017); statistical criterion to select the model was AIC (Akaike information criterion), selected model was GTR+G+I; tree topologies were searched using SPR (Subtree pruning and regrafting); node support was assessed with 200 bootstrap replicates. All analyses were performed using NGPhylogeny.fr server (Lemoine et al. 2019). Phylogenetic trees were visualized and processed in ITOL v5 (Letunic & Bork 2021). The trees were rooted using Harmothoe impar as an outgroup. Posterior probability (PP) and bootstrap support (B) were used for nodal support in BI and ML analyses respectively. Genus Laubierpholoe Pettibone, 1992 Type species

Diagnosis (after Pettibone 1992), emended (changes in bold)
Body small, linear, with relatively few segments (up to 29). Elytra and elytrophores on segments 2, 4, 5, 7, continuing on alternate segments to 23, then on every segment to end of body. Dorsal tubercles on segments lacking elytra. Elytra delicate, with few short papillae on lateral border and on surface. Without dorsal cirri or branchiae. Prostomium and fi rst or tentacular segment fused, ventrally forming anterior lip of mouth, without facial tubercle, with or without papillae. Prostomium rounded, bilobed; median antenna with ceratophore in anterior notch of prostomium; lateral antennae absent; with or without 2 pairs of eyes. Tentaculophores lateral to prostomium, achaetous, each with long dorsal and much shorter ventral tentacular cirrus or tentacular cirri of about same length; palps stout, very long, emerging ventral and lateral to tentaculophores, rugose. Second or buccal segment with fi rst pair of large elytrophores and elytra, biramous parapodia, long ventral buccal cirri, and forming lateral and posterior lips of mouth. Muscular pharynx with 9 dorsal and 9 ventral border papillae and 2 pairs of jaws. Parapodia biramous; notopodial conical acicular lobe without subdistal bract; neuropodial conical acicular lobe without distal papillae. Notochaetae simple, slender, capillary, slightly curved and straight. Neurochaetae stouter than notochaetae, compound, falcigerous or spinigerous; shafts with or without distal spinules; blades capillary or falcate with unidentate or uni-and bidentate tips. Ventral cirri short, tapering, on all segments. Pygidium with pair of anal cirri. Development characterised by reduction of egg number and development of embryos and juveniles within elytra (elytral brooding).

Etymology
The species name refers to the type locality (Massilia -the old Roman name for Marseille).

Distribution
The Calanques, near Marseille, Jarre and 3PP marine caves.

Ecology
Inhabits the upper layer of soft sediments in the middle and deep parts of marine caves at a depth of 19-25 m. The sediment type in Jarre Cave was defi ned as silty sand in the deep part and sandy silt in the middle part, and in 3PP Cave as clayey silt in both deep and middle parts (Table 2).

Emendation of generic diagnosis of Laubierpholoe and its conseque nces
A very short ventral tentacular cirrus has been considered a diagnostic character for Laubierpholoe, in contrast to Pholoe, Taylorpholoe and Metaxypsamma Wolf, 1986 where tentacular cirri are subequal (Pettibone 1992). Since ventral tentacular cirri in L. massiliana sp. nov. are as long as or slightly shorter than the dorsal ones, this character lost its importance, leading to a modifi cation of the generic diagnosis to: "Tentaculophores … each with long dorsal and much shorter ventral tentacular cirrus or tentacular cirri of about same length". This emendation leads to a problem in distinguishing genera of Pholoinae although other characters remain useful. Laubierpholoe and Pholoe can be discerned by the number of segments (up to 90 in Pholoe, up to 29 in Laubierpholoe), the elytral brooding of embryos in Laubierpholoe and the presence of two types of notochaetae in Pholoe (shorter, strongly bent (i.e., geniculate), and longer, slightly curved or straight) whereas all notochaetae in species of Laubierpholoe are similar, slightly curved or straight. The genus Laubierpholoe has common characters with other interstitial pholoin genera such as Taylorpholoe and Imajimapholoe. They also have a small size, elytral brooding and a single type of notochaetae. Laubierpholoe is distinguished by a medial antenna inserted in the anterior notch while in Taylorpholoe and Imajimapholoe the median antenna is situated occipitally at the posterior border of the prostomium. Another interstitial pholoin genus, Metaxypsamma, is easily recognized by its uniramous parapodia and rudimental elytra, transformed to fi liform papillae. The other character of L. massiliana sp. nov. leading to an emendation of the generic diagnosis is the presence of bidentate neurochaetae. It is unique for Pholoinae and discussed below. The comparison of Pholoinae genera is summarized in Table 3. (Table 4) Laubierpholoe massiliana sp. nov. has the typical characters of the genus: small size, few segments and elytra, median antenna in anterior notch of prostomium, one type of notochaetae, and elytral embryo brooding.  Laubierpholoe massiliana sp. nov. is up to 1.2 mm long, with 17-19 segments, larger than L. indooceanica, which is shorter than 1 mm with 13-15 segments, but smaller than other species which can reach up to 1.6 mm with 26 segments (L. swedmarki) and up to 3 mm and 26-29 segments in L. antipoda, L. riseri and L. maryae.

Comparison with other species of Laubierpholoe
According to Gonzalez et al. (2018a), the genus Laubierpholoe has prostomial "cephalic" peaks. These structures, similar to those in polynoids, are illustrated by Pettibone (1992) and Westheide (2001). There the prostomium looks bilobed with the anterior lobes forming acute horns or peaks. Conversely, in L. swedmarki the prostomium is described as trapezoidal, with two lateral horns situated more ventrally. These horns, projected forward, are below the prostomium in fi g.1 (Laubier 1975). The shape of the prostomium was used to distinguish L. swedmarki from other species of Laubierpholoe: "bilobed prostomium rounded anteriorly, with small laterally projecting horns" versus "bilobed prostomium with anterior lobes projecting forward" (Pettibone 1992). SEM investigation showed that in L. massiliana sp. nov. the prostomium has an anterior depression where the medial antenna rises, but its anterior lobes are rounded, with notches above the tentaculophores. Two conical horns are situated more ventrally on the level of the ventral tentacular cirri, most probably originating from the tentacular segment. Therefore, they are not homologous with polynoid cephalic peaks. Other species of Laubierpholoe should be reinvestigated to establish the shape of their prostomium and position anterior of horns.
Unlike other species of Laubierpholoe, in L. massiliana sp. nov. ventral tentacular cirri are as long as the dorsal ones or slightly shorter. This led us to modify the generic diagnosis (discussed above) and allows to distinguish L. massiliana sp. nov. from its congeners.
One of the distinguishing characters for the species of Laubierpholoe is the number of notochaetae. It varies from 2-4 in L. swedmarki to 4-8 in L. indooceanica to numerous in L. antipoda, L. riseri and L. maryae; L. massiliana sp. nov. has a few (3-6) notochaetae, which is slightly more than in L. swedmarki but less than in other species. They can be straight or slightly curved which is typical for Laubierpholoe.
Laubierpholoe massiliana sp. nov. diff ers from all congeners by the shape of its neurochaeta. They are unusual not just for the genus Laubierpholoe but for the whole Pholoinae subfamily, as the blades of at least some of the neurochaetae are long and bidentate. Bidentate, sometimes deeply split neurochaetal blades are present in other sigalionids, e.g., Pelogeniinae subfamily, genera Sthenelais Kinberg, 1856, Fimbriosthenelais Pettibone, 1971, Willeysthenelais Pettibone, 1971, Euthalenessa Darboux, 1899(Pettibone 1970, 1971, 1997 but have not been previously described in Pholoinae. Pettibone (1992) diagnosed the former family 'Pholoidae' as having "blades short, falcate, and unidentate". Laubierpholoe indooceanica, described after this revision, has several types of neurochaetal blades, some comparatively long and hook-like (Westheide 2001: fi g. 2J-K, N). Bidentate tips in L. massiliana sp. nov. are seen only with high magnifi cation or with SEM and might have been overlooked in other species. We changed the diagnosis of the genus in that point to: "…blades unidentate or uni-and bidentate".

Ecology
All previously described species of Laubierpholoe inhabit interstitial biotopes like coarse sand, gravel, broken shells, or carpets of benthic diatoms (Table 4). They share adaptations to interstitial lifestyle like small size, very long palps, reduction of head appendages, small number of notochaetae, smooth elytra with few papillae, and intra-elytral brooding of embryos. Laubierpholoe massiliana sp. nov. is unique within the genus for living in soft sediment habitats, such as silty sand and clayey silt (Tables 2,  4). It is otherwise very similar to other species in size, morphology, and reproduction biology. Since no adaptations to digging in soft sediment were found, we suppose that L. massiliana sp. nov. moves on or just below the sediment surface. This should be confi rmed by direct observation of living worms. We have not found L. massiliana sp. nov. in samples collected outside the caves in diff erent sediments.

Adaptation to cave dwelling
Polynoidae inhabiting marine caves have long sensory parapodial cirri and no eyes or pigmentation, unlike their non-cave dwelling relatives (eyes were plausibly lost in correlation with specialization and colonization of deep-sea habitats) (Gonzalez et al. 2018b;Capa et al. 2022). Among Sigalionidae, only the genus Laubierpholoe includes cave dwelling representatives.
Laubierpholoe sp. is reported from several anchialine caves in the Canary Islands. This undescribed species was found in the Corona lava tube (Lanzarote Island) in a carpet of benthic diatoms and in the sandy bottom of the Tenerife littoral Martínez García et al. 2009). Riera et al. (2018) recorded the same species also from Los Cerebros Cave (Tenerife Island) in sand or gravel sediments. One specimen of this undescribed species from the Tenerife cave was used for phylogenetic analysis and represented as Laubierpholoe sp. C. (Gonzalez et al. 2018a). This species lacks eyes, but has short parapodial cirri; pigmentation information is absent.
Another species of Laubierpholoe lacking eyes is L. maryae, collected on coarse sediment in the intertidal and shallow subtidal zone of New Zealand (Pettibone 1992). The reason for this absence is not clear as interstitial lifestyle alone does not lead to eye loss in other species of Pholoinae.
Laubierpholoe massiliana sp. nov. has developed eyes and short parapodial cirri. Its elytra are transparent and lack pigment and its body is whitish and semitransparent. Living specimens have very long anal cirri (longer than palps, half of the body length), easily lost during sample treatment and preservation. They may play the same role as elongated parapodial cirri in cave polynoids. This hypothesis needs confi rmation through comparison with species of Laubierpholoe of other marine biotopes. Little information exists on body coloration and length of anal cirri within the genus's interstitial non-cave dwelling species. Laubierpholoe indooceanica are whitish-brown to colourless, almost transparent, some with yellowishbrown spots on the elytra and long pygidial cirri, much shorter than the palps (Westheide 2001). Laubierpholoe swedmarki is of white colour and without pygidial cirri (most probably lost) (Laubier 1975). Laubierpholoe riseri has long anal cirri, also shorter than the palps (Pettibone 1992). The body colour and pygidial cirri presence in other species of Laubierpholoe are unknown. The pigmentation loss and longer anal cirri displayed by Laubierpholoe massiliana sp. nov. have to remain unexplained for the time being. They could be regarded as adaptations to the cave dwelling lifestyle or they could be inherited from their ancestors.

Geography
Other species of Laubierpholoe were recorded in the Atlantic (off South America, Bermuda, Cuba), Indian (Seychelles) and Pacifi c (New Zealand) Oceans (Fig. 1A, Table 4). Our fi nding is the fi rst record of the genus in the Mediterranean Sea. The presence of L. massiliana sp. nov. is currently confi rmed only for the two marine caves in the Calanques, but the species is likely to be found in other caves with soft sediments in the Mediterranean Sea. However, we have not found L. massiliana sp. nov. in samples collected outside the caves.

Phylogeny
Our results inferred both from BI and ML confi rmed that Laubierpholoe massiliana sp. nov. indeed belongs to the genus Laubierpholoe, as well as the genus monophyly. It matches the previous results by Gonzalez et al. (2018a). The weak support and discordance with ML analysis of the clades within Laubierpholoe can be explained by data defi ciency for other species of Laubierpholoe (Table 1). Gonzalez et al. (2021) studied the mitochondrial genomes of Polynoidae with diff erent lifestyles and found similarity in cave and pelagic polynoids. Exciting future research could investigate the mitochondrial genome of cave dwelling Sigalionidae in comparison with other representatives of the family.