New data on the polyphyletic Marionina genus (Annelida, Enchytraeidae): description of three new species from European shore habitats

Marionina (Michaelsen in Pfeffer, 1890) is a worldwide distributed genus of small enchytraeids living in mainly aquatic habitats. The genus is polyphyletic, including about 100 species with diverse morphological characters and cryptic lineages; therefore, tax - onomic revisions were performed recently, and further actions are needed in the future. In our study, Marionina individuals were investigated from decaying seagrass debris collected from seashores in Croatia and Italy using morphological characters and molec - ular markers involving the COI and H3 genes and the ITS region. Descriptions of two new Marionina species, M. puntaalanensis sp. nov. and M. orbifera sp. nov. , are presented in this paper, and in addition, the description of a third new Marionina species, M. reicharti sp. nov. , from the shore of the freshwater Lake Balaton (Hungary) is provided here. All three new species are small (2–3.5 mm in vivo with less than 30 segments), their clitellum is saddle-shaped, the dorsal anterior blood vessel bifurcation is in III, and the spermatheca is attached to the oesophagus. The main diagnostic features of M. puntaalanensis sp. nov. are: brain incised posteriorly; dorsal vessel from the clitellar region; two chaetae in all bundles; three pairs of preclitellar nephridia; small subneural glands in XIII–XIV; seminal vesicle absent or small; ectal duct of spermatheca surrounded along the length by glands and one larger. The main features of M. orbifera sp. nov. are: brain truncate posteriorly; dorsal vessel from the clitellar region; two chaetae in all bundles; two pairs of preclitellar nephridia; subneural glands in XIII–XIV; seminal vesicle well developed; the lumen of the spermathecal ampulla is characteristically full with many spherical sperm rolls. In M. reicharti sp. nov. : brain incised posteriorly, dorsal vessel origin in XII, maximum five chaetae per bundle, often the middle chaetae slightly smaller than the ental ones, three pairs of preclitellar nephridia, subneural glands absent, spermathecal ampulla globular, ectal duct surrounded along the length by glands, and one large sessile gland at the orifice.


Introduction
We studied the enchytraeid fauna found in the decaying seagrass detritus on the Adriatic and Tyrrhenian coasts between 2019 and 2021 (Nagy et al. 2023).The characteristic enchytraeid fauna of the coastal supralittoral zone consists of relatively large Enchytraeus and small-sized Marionina species, which are well adapted to the cavity system of the sand grains and the decaying plant biomass.As a result of our research, we described three new Enchytraeus species from the Enchytraeus albidus species complex recently (Nagy et al. 2023), and we found two Marionina species new to science based on morphological and molecular investigations, which are described in this paper.
Among the specimens collected from the Mediterranean coast, we also found worms morphologically resembling Marionina spicula (Leuckart, 1847) (Frey and Leuckart 1847).For comparison, we re-examined specimens identified previously by one of us as M. spicula (Dózsa-Farkas 1995).These specimens had been collected from the shore of the shallow freshwater Lake Balaton, Hungary, the largest lake in Central Europe.Applying molecular methods, it turned out that these latter worms represent a third species new to science, genetically different from the Mediterranean M. spicula.This species is also described in this paper, and the species comparison includes additional M. spicula specimens collected from a Danish seashore.On the other hand, the Mediterranean material of M. spicula was heterogeneous at the DNA level, and slight morphological differences further suggested that more than one species was involved.The genetic and morphological diversity of the Mediterranean M. spicula is presented and described here, but further evidence is needed to erect them as new species.
Marionina (Michaelsen in Pfeffer, 1890) is a worldwide distributed genus within the family Enchytraeidae, including marine, limnic, and terrestrial small-sized worms.Most of the species live in the marine littoral, supra-or sub-littoral zones, or in salt marshes.The majority of the so-called 'terrestrial' Marionina are found only in wet, moist soil, on lake shores, riverbanks, or in swamps, while some species (like Marionina clavata Nielsen &Christensen, 1961, andMarionina communis Nielsen &Christensen, 1959) are truly terrestrial.Few species occur in the profundal zone of freshwater lakes (Timm 1996;Timm and Vvedenskaya 2006), and one species (M.spongicola Rota & Manconi, 2004) lives exclusively in the interior of a sponge in a geothermal lake at a depth of more than 100 m (Rota and Manconi 2004).
Unfortunately, this genus is an artificial assemblage of several unrelated species (Coates 1989;Xie and Rota 2001;Matamoros et al. 2007;Rota et al. 2008;Schmelz and Collado 2010), so the characteristic morphological traits are highly variable between species, e.g., the shape of the chaetae (sigmoid or straight) and their distribution on the body, the shape of the brain, the location of the head pore, the number of the pharyngeal glands, the shape of nephridia, the ratio of pre-and post-septale parts, the origin of the efferent ducts, the location of the anterior bifurcation of the dorsal vessel, the structure of the male copulatory organ, and whether the spermatheca is connected to the oesophagus or not.The heterogeneity was somewhat reduced by the fact that several species were transferred to other genera; e.g., M. cambrensis O' Connor, 1963; M. tubifera Nielsen & Christensen, 1959;and M. changbaishanensis Xie, Liang & Wang, 2000 to Oconorella (Rota 1995;Dózsa-Farkas 2002); M. righiana Xie & Rota, 2001 to Xetadrilus (Schmelz et al. 2011); and M. riparia Bretscher, 1899 to Globulidrilus (Christensen & Dózsa-Farkas, 2012).
With the introduction of DNA-based studies in the taxonomy and systematics of Enchytraeidae, it was confirmed that the genus is polyphyletic (Erséus et al. 2010;Martinsson et al. 2017); furthermore, notable cryptic diversity has been detected in some species (Matamoros et al. 2012).The revision of the genus has therefore become absolutely necessary.As the initial step of the thorough revision, the type species of this genus, M. georgiana (originally Pachydrilus georgianus Michaelsen, 1888), has been re-described by Rota et al. (2008) and Schmelz and Collado (2008), providing also the taxonomic history and synonymy of the genus, and Klinth et al. (2022) supplemented the type species with additional characters recently.Molecular studies (Erséus et al. 2010;Klinth et al. 2022) revealed that a large part of the taxonomically problematic Marionina genus is not closely related to the type species.These studies also highlighted that further revisions are needed to eliminate the taxonomic problems of the genus and draw attention to the fact that even the morphological characters that could be used in the diagnosis of Marionina sensu stricto cannot be defined yet.
In this article, we present the description of three new species currently classified in the Marionina genus based on morphological and molecular data, with some remarks on the species Marionina spicula.

Methods of morphological examination
The enchytraeids were extracted by the wet funnel method (O 'Connor 1962).Enchytraeids were first investigated and measured alive, then preserved in 70% ethanol.Some specimens were stained with borax-carmine and then passed through an ethanol (70% to absolute) dehydration series, mounted temporarily in clove oil, and then permanently in Euparal between two coverslips.All important morphological characters were recorded in vivo, drawn, and photographed [Axio Imager A2 microscope with differential interference contrast illumination, Axio-Cam MRc 5 (Zeiss) digital camera, Axiovision software].The whole-mounted specimens were reinvestigated, measured, and photographed as well.In all micrographs presented in this study, the orientation of specimens is the same: the head is either on the left side or on the top of the picture.Selected material was catalogued with collection numbers, letters for the holotypes ('Ma') and paratypes ('P'), and slide numbers, and was deposited in the collection of the Department of Systematic Zoology and Ecology, ELTE Eötvös Loránd University (Budapest, Hungary).

Systematics
Brain (Fig. 1B) ca.50-60 μm long (fixed), slightly longer than wide, incised posteriorly.Pharynx and postpharyngeal bulbs well developed.Prostomial ganglia absent.In the ventral nerve cord, perikarya continuous.First and second pharyngeal glands compact and united dorsally, in V with ventral lobes; the third pair free dorsally with elongate but stout ventral lobes (Fig. 1C).Chloragocytes from IV forming a denser layer from VI, about 15-20 μm long in vivo, filled with refractive globules.Transition between oesophagus and intestine gradual; oesophageal appendage and intestinal diverticula absent.Midgut pars tumida not seen.Dorsal vessel from clitellar region,  E, G, K).
Etymology.The new species is named after the Punta Ala beach, where it was found.
Distribution and habitat.Known from Loc. 3 and Loc.2., the intertidal zone is near Punta Ala (Grosseto) and Castiglione della Pescaia, Italy, in the decaying seagrass detritus.
Differential diagnosis.Among the mostly intertidal small Marionina species with two chaetae in all chaetal bundles and without sperm rings in spermathecae, eight species are similar to the new species: M. istriae Giere, 1974;M. miniampullacea Shurova, 1978;M. magnifica Shurova, 1978;M. mica Finogenova, 1972;M. aberrans Finogenova, 1973;M. elgonensis Černosvitov, 1938; M. neroutsensis Coates, 1980;and M. mesopsamma Lasserre, 1964.The main differences are as follows: M. istriae is larger (body length 7-10 mm, segment number 38-43 vs. body length 2-2.5 mm, segment number 19-30), the chaetae are larger (65 µm long vs. 15-20 µm long), and a larger ectal gland is absent.M. miniampullacea is also larger (body length 4-5 mm), the chaetae are also larger (50 µm long), the dorsal vessel origin is in VII, and there is a rosette of glands at the orifice of the spermathecal duct.M. magnifica is larger (body length 4-5 mm), sometimes 3-4 chaetae occur, the chaetae are larger (40-50 µm long), and a larger ectal gland absent.M. mica has three ventral lobes of the pharyngeal glands in IV, V, and VI; the third pair is connected dorsally (vs.only in V; the third pair free); the dorsal vessel origin is in VIII; and the anterior blood vessel bifurcation is prostomial (also known as lumbricillinae-type).In M. aberrans, the cuticle is thick (2.5 µm vs. <1 µm), there is a large rosette of ectal glands, the preseptal part of the nephridia consists only of the funnel, and the sperm duct is long (vs.short).M. elgonensis is similarly small, but the ectal gland of the spermathecal ectal duct is absent.In M. neroutsensis, all pharyngeal glands are without ventral lobes; the ectal glands of spermathecae are absent, but a seminal vesicle is present (vs.absent).M. mesopsamma is larger (6 mm long), has a seminal vesicle, has only two pairs of preclitellar nephridia, and the spermathecae are free.(5) first and secondary pharyngeal glands united dorsally with small ventral lobes; the third pair elongate, free dorsally; (6) dorsal vessel from clitellar region, blood colorless.The dorsal anterior blood vessel bifurcation anteriorly behind the pharynx; (7) two pairs of preclitellar nephridia; (8) coelomocytes oval or discshaped with granules, 14-22 μm long in vivo; (9) seminal vesicle well developed; (10) sperm funnel 1.5-3 times longer than wide in vivo, collar high and narrower than funnel body, spermatozoa 44-60 µm long, heads 20-25 µm in vivo; (11) male copulatory organ small and compact, 30-40 µm long in vivo; (12) small subneural glands are in XIII-XIV; (13) ectal duct of spermatheca short, surrounded by glands.Ampulla spherical, diameter 40-55 µm in vivo, the lumen characteristically full of many spherical sperm rolls.Ampulla attached to the oesophagus; (14) 1-3 mature eggs at a time.
Distribution and habitat.Known from the type locality, decaying seagrass detritus.
Differential diagnosis.Among the intertidal small Marionina species, nine species (M.sjaelandica Nielsen & Christensen, 1961, M. levitheca Erséus, 1990, M. coatesae Erséus, 1990, M. swedmarki Lasserre & Erséus, 1976, M. vancouverensis Coates, 1980, M. limpida Shurova, 1979, M. cana Marcus, 1965, M. transunita Coates, 1990, M. southerni (Černosvitov, 1937) and the new species are characterized by spherical sperm rolls in the spermathecal ampulla.The main differences are as follows: The spermathecae of M. sjaelandica and M. coatesae are similar to the new species; more sperm rolls are in the spermathecae, but not in the cavity, but embedded in the walls of the ampulla.Both species have more segments (segment number 24-27 in M. sjaelandica, 27-31 in M. coatesae, vs. 18-24 segments in the new species).M. levitheca is larger (segment number 38-41), the sperm rolls are arranged in distinct globular cavities scattered in the wall, and there are no glands at the ectal duct of the spermatheca.In M. swedmarki, the spermathecal orifice has a conspicuous gland-rosette.M. vancouverensis has a maximum of six chaetae per bundle (vs.only two in the new species).M. limpida is larger (6-8 mm long, vs. 2.3-3.3 mm), the subneural glands are only in XIII (vs.XIV-XV), the sperm funnel and sperm duct are longer, and the brain is incised posteriorly.In M. cana, the sperm rolls are in the walls of the ampulla, the ectal duct is not glandular, and the dorsal vessel origin is in IX, not in the clitellar region; moreover, the brain is incised posteriorly.M. transunita is also larger (with segment numbers 26-40), and the two spermathecae are connected entally.M. southerni is 8-10 mm long with 28-36 segments; the coelomocytes are black in transmitted light; and the spermatheca has many sessile diverticula.
Etymology.The new species is named in the honor of György Reichart, who collected the sample with this species.
Distribution and habitat.Known from the lake shore of Lake Balaton at Bélatelep, Strand Bátori, Hungary, in wet sand between the roots of willow trees (Loc.4).Earlier, they were identified as Marionina spicula (Leuckart, 1847) at four stations of the Lake Balaton (between Fűzfő and Alsóörs, Balatonberény, and Bélatelep) in a fauna investigation in 1990-1992(Dózsa-Farkas 1995).Unfortunately, these specimens were lost.
Remarks on the studied specimens.Some small enchytraeid worms during the former study of Lake Balaton shore fauna were identified as Marionina spicula (Dózsa-Farkas 1995); therefore, the question was raised if they really belonged to this species known typically from marine habitats.However, in two papers (Lafont and Juget 1976; Rodriquez 1986), the species was observed along rivers without a detailed morphological description, and its euryhalinity was also reported previously (Giere 1971).This initiated our study with a freshly collected sample from the shore of Lake Balaton, combining DNA sequencing with morphological investigation.Since some specimens fitting the description of M. spicula (Nielsen and Christensen 1959) were detected in the Adriatic shore samples (collected in 2019), those specimens were also included in our comparison.Furthermore, specimens collected from a Danish seashore in 1999 (site 5) were also studied, but unfortunately, we were not able to obtain DNA sequences from them due to their fixation in Bouin's fluid.The results of the molecular analysis revealed that the DNA sequences of the Hungarian specimens (Lake Balaton) differ from those of the individuals collected by us from the Adriatic seashore (site 1, Table 1) and from the single DNA sequence of M. spicula in NCBI GenBank (see details below), so there is support at the DNA level that the Balaton specimens belong to a new species.

Results of molecular analysis
Results of the phylogenetic analyses confirmed that M. puntaalanensis sp.nov., M. orbifera sp.nov., and M. reicharti sp.nov.are genetically separated from the other (sequenced) Marionina species because their sequences formed distinct lineages on the phylogenetic trees.The Adriatic specimens, which we identified as M. spicula, fall into three separate lineages (Figs 7-9).In all trees, Mediterranean M. spicula appears as a heterogeneous group, and specimens from this species form consistently three clades (four, if CE2561 is included, a specimen from the Swedish coast) with various bootstrap support.M. reicharti sp.nov. is very similar morphologically to M. spicula as conceived by Nielsen and Christensen (1959), but in the trees based on ITS and H3, it is sister to one of the Mediterranean "M.spicula" clades.In the COI phylogenetic tree, on the other hand, it appears as the sister group of M. puntaalanensis sp.nov.It should be noted that in Fig. 8. -a tree based on the COI sequences of all identified Marionina species currently available in GenBank -reference sequences from species that are currently considered to belong to Marionina sensu stricto (namely, M. aestuum and M. fusca) (Klinth et al. 2022) form a clade separate from those species that were described in this study.
The results of the distance analyses supported the phylogenetic investigations.The p-distances between the ITS sequences of the three new species and the other Marionina species are 15.8-45.6%, the COI distances between them are 17-25.5%,and the H3 distances between them are 2.1-17.7%.The distances between the ITS sequences of M. reicharti sp.nov.and M. spicula are 15.8-26%, the COI distances between them are 17-18.7%,and the H3 distances between them are 2.9-4.8%.The ITS distances between the three Croatian M. spicula clades are 13.4-20.8%,the COI distances between them are 15.4-17.3%, and the H3 distances between them are 1.1-3.2%.The specimen M. spicula CE2561, probably from Sweden, formed a fourth clade since it separated from M. spicula individuals collected in Croatia.The COI distances between M. spicula CE2561 and the three Croatian M. spicula clades are 15.8-17.3%, and the H3 distances between them are 4.1-5%.The ITS distances could not be compared between them because the whole ITS sequence (containing ITS1, 5.8S rDNA, and ITS2) for M. spicula CE2561 is currently not available in the GenBank database.The above-presented results suggest that M. spicula is a complex of at least four species.

Discussion
As a result of our research on the supralittoral zone of the Mediterranean seashores, we described two species new to science (Marionina puntaalanensis sp.nov.and M. orbifera sp.nov.) from the Tyrrhenian coast.The distinctive morphological characters served as the basis of the description of these two new species, and their assignment was confirmed with DNA sequence analyses based on the ITS region and the COI and H3 genes.
In the case of the Marionina spicula (Leuckart, 1847) specimens found in Adriatic coastal samples, we have shown that they are probably members of a species complex.Morphological differences were detected among the specimens, and the DNA sequences also formed several clades on the phylogenetic trees based on all three studied DNA regions; furthermore, the genetic distances among the detected Marionina spicula clades were comparable to the interspecific differences of the other Marionina species included in this study.However, we were not able to assign this variability to distinct new species.The observed morphological differences included, for example, the maximum number of chaetae (which in some cases was only 4-5; in other cases, it was 7-8 chaetae per ventral bundle), the size of the sperm funnel (besides the type of "two times longer than wide," there were very large sperm funnels, which were 3-4.5 times longer than wide), and the granularity of coelomocytes (sometimes the coelomocytes were less granulated, but in other cases the coelomocytes were filled with dark grey granules).In cases where coelomocytes with dark granules filled the coelom of the worm in large numbers, it resulted in the internal organs of the animal being difficult to study in transmitted light, which hindered the comprehensive morphological characterization.Furthermore, since, after the completion of the microscopic studies, whole specimens were used for the molecular study due to the small size (few mm) of the animals, the subsequent re-investigation of the worms belonging to different clades was unfortunately not possi-ble to search for further distinctive morphological characters.To circumvent this problem, several additional samples were collected from the same location at the Adriatic coast later, but no living specimens were found in them, most probably because the seagrass detritus was removed from the beach to fulfill the requirements of tourists, and this inhibited the survival of the worms.It was reported (Giere 1971) that coelomic fluid released from the anus helps the individuals of M. spicula attach to sand particles (this was also observed by us), and this contributes to their adaptation to the sea wave-generated shore habitat consisting of a mixture of sand and decaying plant material.However, after studying the fixed specimens collected from the Danish coast in 1999, it could be concluded that they fully fit the description given by Nielsen and Christensen (1959).On the other hand, Danish individuals differed from the specimens collected by us from the Adriatic seashore since the Danish individuals have a maximum of only 4-5 chaetae in a bundle and the diameter of the spermathecal ampulla is smaller (25-32 vs. 25-55 µm in Adriatic specimens).Taken all together, further studies are required on the M. spicula species complex, which should include more specimens from the North and Baltic Seas and a comparison of the morphological variations reported in the literature.A systematic revision of Marionina spicula "sensu lato" is beyond the scope of this paper.However, it should be noted that enchytraeid species having a wide geographic distribution similarly to M. spicula (e.g., Enchytraeus albidus; Collado et al. 2012;Erséus et al. 2019;Nagy et al. 2023) represent a species complex.
Nevertheless, on the way of resolving the problematic issues related to this heterogenous group, we examined specimens previously designated as M. spicula from the littoral zone of Lake Balaton in this study, and we described them as a new species (M.reicharti sp.nov.) based on the morphological and molecular results.With the species described here, the number of Marionina species is increasing from 101 to 104.Summarizing the recent changes within the genus, 94 accepted species were reported in the checklist of Schmelz and Collado (2012), and seven new species (M.deminuta Rota, 2013, M. fusca Klinth, Rota & Erséus, 2022, M. mimula Rota, 2013, M. mendax Rota, 2013, M. naso Timm, 2012, M. nothachaeta Matamoros, Rota & Erséus, 2012, and Marionina sambugarae Schmelz, 2015) were described since then by others (Martin et al. 2015;Schmelz and Collado 2015;Klinth et al. 2022), and three new species (two marine and one freshwater) were described in this study.
All three species new to science belong to Marionina sensu lato (Klinth et al. 2022), but we hope that our results based on comparative morphological and molecular data will aid the revision of the genus in the future.

Figure 5 .
Figure 5. Micrograph of Marionina spicula. A. Chaetae maximum 5 in a ventral bundle; B. The inner chaetae shorter; C. Chaetae maximum 7-8 in a ventral bundle.A, B, and C from different specimens; D. Brain; E. Clitellar glands, dorsal view; F. Clitellar glands absent between the male copulatory organs; G. Anterior bifurcation of the dorsal vessel in III (spermathecae marked with white arrows); H. Lighter coelomocytes; I. Dark coelomocytes; J. Coelomocytes with fewer granules; K. Coelomocytes full with granules.All pictures are in vivo.Scale bars: 50 μm.

Figure 7 .
Figure 7. Maximum likelihood (ML) tree of the ITS region for Marionina species, based on 854 nucleotide positions using the General Time Reversible substitution model.Bootstrap values greater than 50 are shown at the nodes.Accession codes of sequences with collection information are given inTable 1. Scale bar: 0.2 substitutions per nucleotide.

Figure 8 .
Figure 8. Maximum likelihood (ML) tree of the COI gene for Marionina species, based on 517 nucleotide positions using the General Time Reversible substitution model.Bootstrap values greater than 50 are shown at the nodes.Accession codes of sequences with collection information are given inTable 1. Scale bar: 0.1 substitutions per nucleotide.

Table 1 .
List of specimens used for molecular taxonomic analyses with collection data and GenBank accession numbers.Sequences determined in this study appear in bold.Abbreviations: n. d. = no data.