Zoosyst. Evol. 100 (4) 2024, 1269-1286 | DOI 10.3897/zse.100.122874 sia a a _______age SPENSOFT. pvt a NATURKUNDE BERLIN New data on the polyphyletic Marionina genus (Annelida, Enchytraeidae): description of three new species from European shore habitats Tamas Felféldi!*, Hajnalka Nagy!, Klara Dozsa-Farkas* Department of Microbiology, ELTE Eétvés Lordand University, H-1117 Budapest, Pazmany Péter sétany 1/C, Hungary Institute of Aquatic Ecology, HUN-REN Centre for Ecological Research, H-1113 Budapest, Karolina ut 29, Hungary Hungarian Natural History Museum, H-1088 Budapest, Baross utca 13, Hungary FP WwW NM fF Department of Systematic Zoology and Ecology, ELTE Eétvos Lordnd University, H-1117 Budapest, Pazmdny Péter sétany 1/C, Hungary https://zoobank. org/8 DB2BEF F-F'7A6-45A5-B9AE-046B735792DD Corresponding author: Hajnalka Nagy (nhajni6b@gmail.com, sparrow@staff.elte.hu) Academic editor: Greg Rouse # Received 12 March 2024 # Accepted 18 June 2024 Published 10 September 2024 Abstract 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 MZ 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 XIIJ—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. Key Words Lake Balaton, Marionina, Mediterranean Basin, molecular taxonomy, sea and lake shore, species complex Introduction of relatively large Enchytraeus and small-sized Marionina species, which are well adapted to the cavity system of the We studied the enchytraeid fauna found in the decaying sand grains and the decaying plant biomass. As a result of seagrass detritus on the Adriatic and Tyrrhenian coasts be- —_our research, we described three new Enchytraeus species tween 2019 and 2021 (Nagy etal. 2023). The characteristic from the Enchytraeus albidus species complex recently enchytraeid fauna of the coastal supralittoral zone consists (Nagy et al. 2023), and we found two Marionina species Copyright Felfoldi, T. 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. 1270 new to science based on morphological and molecular in- vestigations, which are described in this paper. Among the specimens collected from the Mediterra- nean coast, we also found worms morphologically re- sembling Marionina spicula (Leuckart, 1847) (Frey and Leuckart 1847). For comparison, we re-examined spec- imens identified previously by one of us as M. spicula (Dozsa-Farkas 1995). These specimens had been collect- ed 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 dif- ferent from the Mediterranean M. spicula. This species 1s 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 MZ. spicula was heterogeneous at the DNA lev- el, 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 world- wide distributed genus within the family Enchytraeidae, including marine, limnic, and terrestrial small-sized worms. Most of the species live in the marine littoral, su- pra- 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, and Marionina 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 1s 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 con- nected to the oesophagus or not. The heterogeneity was somewhat reduced by the fact that several species were transferred to other genera; e.g., MZ cambrensis O’ Con- nor, 1963; M. tubifera Nielsen & Christensen, 1959; and M. changbaishanensis Xie, Liang & Wang, 2000 to Ocon- orella (Rota 1995; Dézsa-Farkas 2002); M. righiana Xie & Rota, 2001 to Xetadrilus (Schmelz et al. 2011); and M. riparia Bretscher, 1899 to Globulidrilus (Christensen & Dozsa-Farkas, 2012). zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) With the introduction of DNA-based studies in the taxonomy and systematics of Enchytraeidae, it was con- firmed that the genus is polyphyletic (Erséus et al. 2010; Martinsson et al. 2017); furthermore, notable cryptic di- versity 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 histo- ry 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 diag- nosis 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. Materials and methods Study sites 1. Croatia, Istria, Kale Cove seashore, Adriatic Sea, Kamenjak Peninsula, decaying seagrass (Zostera) detritus, 44°51'13.0"N, 13°58'50.5"E, Leg. Julia Tordk, 03 Apr 2019, and 05 Sep 2020. 2. Italy, Castiglione seashore, Tyrrhenian Sea, decay- ing seagrass detritus, 42°45'56.0"N, 10°52'51.0"E, Leg. Andras Dozsa-Farkas and Kinga Dozsa-Far- kas, 13 Dec 2019. 3. Italy, Punta Ala Grosseto, Castiglione della Pes- caia, decaying seagrass detritus, 42°46'00.0"N, 10°51'31.0"E, Leg. Andras Dozsa-Farkas and Kin- ga Dozsa-Farkas, 24 Sep 2020 and 26 Nov 2020. 4. Hungary, Lake Balaton, Bélatelep, Strand Batori, lake shore, wet sand between the roots of willow trees, 46°43'51.5"N, 17°31'41.7"E, Leg. Gyorgy Reichart, 14 Feb 2021. (See details of lakewater characteristics in Somogyi et al. 2020). 5. Denmark, Niva, — seashore, 55°56'29 3" N, 12°31'39.0"E, Leg. Bent Christensen and Klara Dozsa-Farkas, 23 Nov 1999. Methods of morphological examination The enchytraeids were extracted by the wet funnel meth- od (O’Connor 1962). Enchytraeids were first investigat- ed and measured alive, then preserved in 70% ethanol. Zoosyst. Evol. 100 (4) 2024, 1269-1286 Some specimens were stained with borax-carmine and then passed through an ethanol (70% to absolute) dehy- dration series, mounted temporarily in clove oil, and then permanently in Euparal between two coverslips. All 1m- portant 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, mea- sured, and photographed as well. In all micrographs pre- sented in this study, the orientation of specimens is the same: the head 1s 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 collec- tion of the Department of Systematic Zoology and Ecolo- gy, ELTE Eotvos Lorand University (Budapest, Hungary). Methods of molecular analysis Genomic DNA was extracted from the individuals with the DNeasy Blood & Tissue Kit (Qiagen) according to the in- structions given by the manufacturer. Three regions were amplified separately with the PCR method: the mitochon- drial cytochrome c oxidase subunit I (COI) gene, the nuclear histone 3 (H3) gene, and the nuclear ribosomal ITS region using the primer pairs HCO2198 (5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’) and LCO1490 (5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’) (Folmer et al. 1994), H3a-F (5’-ATG GCT CGT ACC AAG CAG ACV GC-3’) and H3a-R (5’-ATA TCC TTR GGC ATR ATR GTGAC-3’) (Colgan et al. 1998), and ETTS1 (5’-TGC TTA AGT TCA GCG GGT-3’) and ETTS2 (5’-TAA CAA GGT TTC CGT AGG TGA A-3’) (Kane and Rollinson 1994). If amplifica- tion failed in the case of the ITS region, COI, and H3 gene, additional primer sets ITS-5 (5’-GGA AGT AAA AGT CGT AAC AAG G-3’) and ITS-4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) (White et al. 1990), COI-E" (5’-TAT ACT TCT GGG TGT CCG AAG AAT CA-3’) (Bely and Wray 2004), and H3a-new-F (5’- TGG CTC GTA CCA AGC AGA CSG-3’) with H3a-new-R (5’-ATG ATG GTG ACG CKY TTG GC-3’) (AllGenetics, A Corufia) were applied. PCRs, sequencing reactions, and phylogenetic analyses were con- ducted as described in detail previously by Dozsa-Farkas et al. (2015). The PCR cycle parameters for ITS-5, ITS-4, and COI-E were based on Matamoros et al. (2012). Sanger sequencing was performed by the LGC Genomics GmbH (Berlin, Germany), and the construction of maximum like- lihood trees, including the search for the best-fit model, was carried out with the MEGA 7 software (Kumar et al. 2016). According to the results of the ModelTest, the following nucleotide substitution models were used for the construc- tion of phylogenetic trees: ITS region: GIR+G+I; COI gene: GIR+G+I; H3 gene: T92+G. In total, 15, 21, and 18 new sequences were obtained from the studied Marionina specimens in the cases of ITS, COI, and H3 (Table 1). Un- fortunately, we failed to amplify the studied DNA regions par from some specimens (e.g., ITS sequences from Marionina spicula and H3 sequences from M. orbifera sp. n. individ- uals), which was probably due to the improper hybridiza- tion of PCR primer sequences with the extracted genomic DNA. Sequences from other Marionina species (Erséus et al. 2010; Matamoros et al. 2012; Felf6ldi et al. 2020, etc.) were used for comparison. Besides the phylogenetic tree es- timations, pairwise genetic distances between the COI, H3, and ITS sequences of the new species and the other investi- gated Marionina species were calculated in MEGA 7.0 us- ing the p-distance method. Gaps and missing data were ex- cluded using pairwise deletions. These settings are based on Martinsson and Erséus (2018). Sequences obtained in this study were deposited in GenBank under the following ac- cession codes: MZ835280—-MZ835294 (ITS), MZ750838— MZ750858 (COI), and MZ816248—MZ8 16265 (H3). Results Systematics Genus Marionina (Michaelsen in Pfeffer, 1890) Marionina puntaalanensis sp. nov. https://zoobank. org//76F756B7-3F72-4DF4-A 73 1-F769D6BAC2CF Fig. 1 Type material. Holotype: Ma. 5, slide No. 3055. Type locality: (Loc. 3.) Italy, Punta Ala Grosseto, Castiglione della Pescaia, decaying seagrass detritus, 42°46'00.0"N, 10°51'31.0"E, Leg. Andras Dozsa-Farkas and Kinga Dozsa-Farkas, 24 Sep 2020. Paratypes: in total, four specimens: P.146.1 slide No. 3022, P.146.2 slide No. 3056, P.146.3 slide No. 3077, P.146.4 slide No. 3192. Same data as for holotype, 24 Sep 2020 and 26 Nov 2020. Further material examined. Four specimens for DNA analysis, five specimens only in vivo. Diagnosis. (1) Small size (body length 2—2.5 mm, 130-185 um wide at clitellum, in vivo), segment number 19-30; (2) chaetae straight with ental hook, two chae- tae in all bundles; (3) clitellum saddle-shaped; (4) first and second pharyngeal glands united dorsally, in V with ventral lobes; the third pair free dorsally with elongat- ed ventral lobes; (5) dorsal vessel from clitellar region, blood colorless, anterior blood vessel bifurcation ante- riorly behind the pharynx; (6) three pairs of preclitellar nephridia; (7) coelomocytes disc- or lemon-shaped with granules, 13—20 um; (8) sperm funnels small, cylindrical, 100-140 um long in vivo, 1.5—2.5 times longer than wide in vivo, collar high and narrower than funnel body; (9) spermatozoa 38-43 um long, heads 15-22 um in vivo; (10) male copulatory organs small and compact; (11) small subneural glands in XHI—XIV; (12) ectal duct of spermatheca surrounded along the length by glands and one larger, 15—27 um long, sessile gland at orifice. Am- pulla oval, 24-40 um wide and 40-55 um long in vivo. zse.pensoft.net 1272 Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) 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. Species Marionina puntaalanensis sp. nov. Marionina puntaalanensis sp. nov. Marionina reicharti sp. nov. Marionina reicharti sp. nov. Marionina reicharti sp. nov. Marionina reicharti sp. nov. Marionina reicharti sp. nov. Marionina orbifera sp. nov. Marionina orbifera sp. nov. Marionina orbifera sp. nov. Marionina aestuum Marionina argentea Marionina argentea Marionina cf. argentea Marionina clavata Marionina clavata Marionina clavata Marionina communis Marionina communis Marionina communis Marionina cf. levitheca Marionina cf. minutissima Marionina filiformis Marionina fusca Marionina nevisensis Marionina cf. nevisensis Marionina nothachaeta Marionina seminuda Marionina southerni Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina spicula Marionina tumulicola Marionina vesiculata Achaeta unibulba (outgroup) zse.pensoft.net Specimen ID 1450 1451 1360 1361 1457 1465 1466 1447 1448 1449 CE12477 1193 CE807 CE22027 734 1267 CE849 904 1216 CE811 CE1339 CE843 CE1040 CE12476 CE338 CE260 LM322 1334 CE674 1338 1339 1341 1366 1377 1378 1381 1441 1442 1443 1444 1445 1446 CE2561 CE571 898 851 Locality Italy, Castiglione Italy, Castiglione Hungary, Bélatelep Hungary, Bélatelep Hungary, Bélatelep Hungary, Bélatelep Hungary, Bélatelep Italy, Castiglione Italy, Castiglione Italy, Castiglione South Georgia Island Korea, Muljangorioreum Wetland Sweden, Lerum Norway, Hordaland Hungary, Készeg Mts. Korea, Mt. Gyebangsan Sweden, Lerum Hungary, Szolnok Korea, Mt. Gyebangsan Sweden, Skara Australia, Queensland Sweden, Lerum Sweden South Georgia Island Bahamas, Lee Stocking Island New Caledonia, Loyalty Islands Sweden, Swedish West Coast Korea, Mt. Hallasan Sweden, Gotland Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Croatia, Kale Cove Sweden, Vastergotland Australia, South West coast Hungary, Készeg Mts. Hungary, Készeg Mts. Habitat decaying seagrass detritus decaying seagrass detritus wet sand between the roots of willow trees wet sand between the roots of willow trees wet sand between the roots of willow trees wet sand between the roots of willow trees wet sand between the roots of willow trees decaying seagrass detritus decaying seagrass detritus decaying seagrass detritus intertidal, 4 m up shore from low tide soil near a lake n.d. larch forest oak forest road bank under poplar trees soil of Quercus mongolica forest brown soil beach near road coastal waters intertidal, 4 m up shore from low tide intertidal sand and rubble at pond outfall beach, marine fine sand with freshwater springs intertidal sand soil of Quercus serrata copse forest coastal waters decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus om ee eee ee ee eee ee ee decaying seagrass (Zostera) detritus decaying seagrass (Zostera) detritus upper part of narrow zone of marsh- like vegetation, roots and brown soil intertidal sand soil meadow Reference this study this study this study this study this study this study this study this study this study this study Klinth et al. 2022 Felfoldi et al. 2020 Erséus et al. 2010 Klinth et al. 2019 Felfdldi et al. 2020 Felfdldi et al. 2020 Erséus et al. 2010; Schmelz et al. 2019 DézsaFarkas et al. 2018; Felfoldi et al. 2020 Felfoldi et al. 2020 Erséus et al. 2010; Klinth et al. 2017 Erséus et al. 2010 Erséus et al. 2010 Erséus et al. 2010 Klinth et al. 2022 Matamoros et al. 2012 Erséus et al. 2010; Matamoros et al. 2012 Matamoros et al. 2012 Felfoldi et al. 2020 Matamoros et al. 2012 this study this study this study this study this study this study this study this study this study this study this study this study this study Martinsson et al. 2017; Klinth et al. 2022 Matamoros et al. 2012 Nagy et al. 2023 DézsaFarkas and Felfoldi 2017 ITS Col H3 MZ835280 MZ750838 MZ816248 MZ835281 MZ750839 MZ816249 MZ835282 MZ835283 MZ835284 MZ835285 MZ835286 MZ835287 MZ835288 MT428056 MT428057 MG252215 KU894286 JN799847 JN799846 MT428060 JN799849 MZ835289 MZ835290 MZ835291 MZ835292 MZ835293 MZ835294 MZ835279 KY583112 MZ750840 MZ750841 MZ750842 MZ750843 MZ750844 MZ750845 MZ393958 MT425084 GU902092 MN395702 MT425088 GU902097 MG252151 MT425086 GU902098 GU902093 GU902094 GU902099 MZ393959 JN799911 GU902095 JN799950 MT425091 JN799913 MZ750846 MZ750847 MZ750848 MZ750849 MZ750850 MZ750851 MZ750852 MZ750853 MZ750854 MZ750855 MZ750856 MZ750857 MZ750858 KX618730 JN799912 MZ750837 KY583130 MZ816250 MZ816251 MZ816252 MZ816253 MZ816254 MZ394832 MT433804 MT433805 MN248704 MT433808 MT433810 KU894216 MZ394834 MT433811 MZ816255 MZ816256 MZ816257 MZ816258 MZ816259 MZ816260 MZ816261 MZ816262 MZ816263 MZ816264 MZ816265 KX644887 MZ816247 KY583097 Zoosyst. Evol. 100 (4) 2024, 1269-1286 L273 Figure 1. Micrograph of Marionina puntaalanensis sp. nov. A. Entire specimen; B. Brain; C. Pharyngeal glands (marked with white arrows; spermathecae marked with black arrows); D. Anterior bifurcation of the dorsal vessel in III; E. Rounded coelomocytes; F. Lemon-shaped coelomocytes; G. Preclitellar nephridium; H, I. Sperm funnels (e = egg); J, K. Spermathecae (ectal glands marked with black arrows, ampullae marked with white arrows). A, C—J. in vivo, B, K. fixed, stained. Scale bars: 500 um (A); 50 pm (B-D, F, H—J); 20 um (E, G, K). Description. Small species (Fig. 1A), holotype 1.9 mm long, 80 um wide at VHI and 107 um at clitel- lum (fixed), segment number 21. Body length of para- types 2.0-2.5 mm, width 120-180 um at VHI and 130- 185 um at clitellum, in vivo, length of fixed specimens 1.5—2.7 mm, width 80-165 um at VII and 105-165 at clitellum, segment number 19-30. Chaetae straight with ental hook. Chaetal formula: 2 - 2: 2 - 2 (in one speci- men from Castiglione della Pescaia, three chaetae were in one ventral bundle). The chaetae equal in size within the bundles; in the ventral bundles a little longer than in the lateral ones. 15—20 um in preclitellar segments and 19-20 um at the posterior end of the body. Clitellum sad- dle-shaped in XII-1/2 XII, gland cells squarish, arranged in transverse rows, midventrally absent. Head pore at 0/1, no dorsal pores. Epidermal gland cells inconspicuous in vivo. Thickness of body wall about 18—20 um, and cuticle thin <1 um. Brain (Fig. 1B) ca. 50-60 um long (fixed), slightly lon- ger than wide, incised posteriorly. Pharynx and postpha- ryngeal 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 um 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, zse.pensoft.net 1274 blood colorless. The dorsal anterior blood vessel bifur- cation in HI (Fig. 1D). All coelomocytes nucleated oval, disc-shaped (Fig. 1E) or lemon-shaped (Fig. IF) with granules, 13—20 um long in vivo and 10-18 um, fixed. Three pairs of preclitellar nephridia in 6/7—8/9, preseptal part consisting of funnel and coils of canals, postseptal part elongate, about two times as long as preseptal part, efferent duct terminal (Fig. 1G); the first postclitellar pair at 13/14 (mostly seven postclitellar pairs). Seminal vesi- cle absent or small, paired. Sperm funnels small, cylindri- cal, 100-140 um long in vivo, 40-75 um when fixed, and about 1.5—2.5 times longer than wide in vivo (1.5—2 times, when fixed), collar high and narrower than funnel body (Fig. 1, 1). Spermatozoa 38-43 um long, heads 15—22 um in vivo and 20-32 um long and heads 10-13 um, when fixed. Sperm ducts short, about four times longer than the funnel, coiled into a loose spiral, diameter 7-10 um in vivo and 4—5 um, when fixed. Male copulatory organs small and compact, 25—36 um long, 23—40 um wide, and 15-30 um high in vivo (22-30 um long, 20-27 um wide, and 25-30 um high, when fixed). Small subneural glands in XIII—XIV (in one specimen absent). Ectal duct of sper- matheca 24—38 um long, surrounded along the length by glands and one larger, 15—27 um long, sessile gland at or- ifice. Ampulla oval, 24-40 um wide and 40-55 um long in vivo (20-30 um wide, 25-35 um long, fixed), in the lumen with some sperm (Fig. 1J, K). Ampulla attached with a short ental duct to the oesophagus. One or two ma- ture eggs at a time. 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 sea- grass 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. magnifi- ca Shurova, 1978; M. mica Finogenova, 1972; M. ab- errans Finogenova, 1973; M. elgonensis Cernosvitov, 1938; M. neroutsensis Coates, 1980; and M. mesopsam- ma Lasserre, 1964. The main differences are as follows: M. istriae is larger (body length 7-10 mm, segment num- ber 38-43 vs. body length 2—2.5 mm, segment number 19-30), the chaetae are larger (65 um long vs. 15—20 um long), and a larger ectal gland is absent. M. miniampul- lacea is also larger (body length 4-5 mm), the chaetae are also larger (50 um 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—S mm), sometimes 3-4 chaetae occur, the chaetae are larger (40-50 um 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 1s in zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) VIII; and the anterior blood vessel bifurcation is prosto- mial (also known as lumbricillinae-type). In M. aberrans, the cuticle is thick (2.5 um vs. <1 um), 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). MZ e/gonensis is similarly small, but the ectal gland of the spermathecal ectal duct is absent. In M. ner- outsensis, 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. Marionina orbifera sp. nov. https://zoobank.org/56E26433-75D8-431 1-9838-B3ECA58BA1BE Fig. 2 Type material. Holotype: Ma. 6, slide No. 3081. Type locality: (Loc. 3.) Italy, Punta Ala Grosseto, Castiglione della Pescaia, decaying seagrass detritus, 42°46'00.0"N, 10°51'31.0"E, Leg. Andras Dozsa-Farkas and Kinga Dozsa-Farkas, 24 Sep 2020. Paratypes: in total, 15 specimens: P.147.1 slide No. 3027, P.147.2 slide No. 3049 (two specimens), P.147.3 slide No. 3050 (two specimens), P.147.4 slide No. 3053 (two specimens), P.147.5 slide No. 3058, P.147.6 slide No. 3060, P.147.7 slide No. 3071, P.147.8 slide No. 3072, P.147.9 slide No. 3074, P.147.10 slide No. 3075, P.147.11 slide No. 3079, P.147.12 slide No. 3080. Same data as for holotype. Further material examined. 20 specimens (10 only in Vivo). Diagnosis. (1) Small size (body length 2.3-3.3 mm, 130-220 um wide at clitellum, in vivo, segment number 17-22), (2) chaetae straight with ental hook, two chae- tae per bundle, slightly longer at the posterior end of the body; (3) clitellum saddle-shaped; (4) brain truncate posteriorly; (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 disc- shaped with granules, 14-22 um long in vivo; (9) sem- inal vesicle well developed; (10) sperm funnel 1.5—3 times longer than wide in vivo, collar high and narrow- er than funnel body, spermatozoa 44—60 um long, heads 20-25 um in vivo; (11) male copulatory organ small and compact, 30-40 um long in vivo; (12) small subneural glands are in XIIJ—XIV; (13) ectal duct of spermatheca short, surrounded by glands. Ampulla spherical, diame- ter 40-55 um in vivo, the lumen characteristically full of many spherical sperm rolls. Ampulla attached to the oe- sophagus; (14) 1-3 mature eggs at a time. Description. Small species, holotype 2.1 mm long, 134 um wide at VII and 150 um at clitellum (fixed), segment number 21. Body length 2.3-3.4 mm, width Zoosyst. Evol. 100 (4) 2024, 1269-1286 275 Figure 2. Micrograph of Marionina orbifera sp. nov. A. Brain; B. Clitellar glands absent ventrally (the male copulatory glands = m); C. Clitellum (sperm funnels = sf); D. Chaetae at the body end; E. Anterior bifurcation of the dorsal vessel in HI; F. Pharyngeal glands, lateral view (primer pharyngeal gland pairs marked with white arrows, ventral lobes marked with black arrows); G. Coelo- mocytes; H. Large seminal vesicle; I. Sperm funnels (sperm funnels = sf, seminal vesicle = sv); J. Subneural gland in XIV; K—M. Spermathecae (the sperm rolls in the ampullae marked with black arrows, the glands at the ectal duct marked with white arrows). A-E, G-—I, K—M. /n vivo; F, J. Fixed, stained. Scale bars: 50 um (A, C, E, F, I, J, K); 20 um (B, D, G, H, L, M). zse.pensoft.net 1276 110-188 um at VIII and 130-220 um at clitellum, in vivo, length of fixed specimens 1.1—2.1 mm, width 118- 160 um at VIII and 125-180 um at clitellum, segment number 17—24. Chaetae straight with ental hook. Chaetal formula: 2 - 2: 2 - 2 (in one case, three chaetae were in one ventral bundle of the segment III). The chaetae are equal in size within the bundles, a little longer in the ven- tral bundles than in the lateral ones. Chaetae are 20-30 x 2.2 um in preclitellar segments and 28-35 x 2.8—3 um at the posterior end of the body (Fig. 2D). Clitellum saddle shaped in XII-1/2 XIII, gland cells squarish, arranged in about 16—17 transverse rows (Fig. 2C), midventrally ab- sent (Fig. 2B). Head pore at 0/I, no dorsal pores. Epider- mal gland cells inconspicuous in vivo. Thickness of body wall about 15—16 um, and cuticle thin (1 um). Brain (Fig. 2A) ca. 80 um long (fix.) slightly longer than wide, truncate posteriorly. Pharynx well developed. Prostomial glanglia absent. In the ventral nerve cord, perikarya continuous. First and secondary pharyngeal glands compact and united dorsally, with small ventral lobes; the third pair elongate and free dorsally (Fig. 2F). Chloragocytes from IV forming a denser layer from VI, about 17-30 um long in vivo, filled with refractive globules. Transition between oesophagus and intestine gradual; oesophageal appendage and intestinal divertic- ula absent. Midgut pars tumida not seen. Dorsal vessel origin from clitellar region; blood colorless. The dorsal anterior blood vessel bifurcation in III (Fig. 2E). All coe- lomocytes nucleated oval or disc-shaped with granules, 14—22 um long in vivo (Fig. 2G) and 8-10 um fixed. Two pairs of preclitellar nephridia in 7/8 and 8/9, preseptal part consisting of funnel and coils of canal, postsep- tal part elongate, about four times as long as preseptal part, efferent duct terminal. The first postclitellar pair of nephridia at 13/14. Seminal vesicle well developed, paired, extending anteriorly to X or IX and posteri- orly to XII—XIII (Fig. 2H). Sperm funnels cylindrical, 85-140 um long in vivo, 60-100 um, fixed, and about 1.2—3 times longer than wide, collar high, and narrower than funnel body (Fig. 2C, I). Spermatozoa 44-60 um long (in two specimens they were 80-87 tum long), heads 20-25 um in vivo (Fig. 21), (23-28 um and 10-15 um, respectively, when fixed). Sperm ducts short, coiled into a loose spiral, diameter 5—7 um, in vivo. Male copulato- ry organs small and compact (Fig. 2B), 30-40 um long, 20-35 um wide, and 15-30 um high, in vivo (25-38 um long, 18—25 um wide, and 20-30 um high, when fixed). Small subneural glands in XIII—XIV (Fig. 2J). The ectal duct of spermatheca short, 20-35 um long, surrounded along the length by glands, somewhat larger entally, no distinct rosette around the orifice (Fig. 2M). Ampulla spherical, diameter 40-55 um in vivo (33-40 um, fixed), the lumen filled with many spherical sperm rolls (Fig. 2K—M). Ampulla attached with a short ental duct to the oesophagus. 1—3 mature eggs at a time. Etymology. The species is named after the character- istic sperm rolls (,,orb”) in the spermatheca [orbifera = orb-bearing (Latin)]. zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) Distribution and habitat. Known from the type local- ity, decaying seagrass detritus. Differential diagnosis. Among the intertidal small Marionina species, nine species (M. sjaelandica Nielsen & Christensen, 1961, M. levitheca Erséus, 1990, M. coate- sae 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 (Cernosvitov, 1937) and the new species are characterized by spherical sperm rolls in the spermathecal ampulla. The main differences are as follows: The sper- mathecae of M. sjaelandica and M. coatesae are similar to the new species; more sperm rolls are in the sperma- thecae, 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. /evitheca is larger (segment number 38-41), the sperm rolls are ar- ranged in distinct globular cavities scattered in the wall, and there are no glands at the ectal duct of the spermathe- ca. In M. swedmarki, the spermathecal orifice has a con- spicuous 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 transmit- ted light; and the spermatheca has many sessile diverticula. Marionina reicharti sp. nov. https://zoobank.org/BA8 1 E0DD-F3A 6-4480-B2ED-9A 9F523D9B5D Figs 3-4 Marionina spicula (Leuckart, 1847). Dozsa-Farkas 1995, 125-126, 128, 130. Type material. Holotype: Ma. 7, slide No. 3128. Type locality: (Loc. 4) Hungary, Lake Balaton, Bélatelep, Strand Batori, lake shore, wet sand between the roots of willow trees, 46°43'51.5"N, 17°31'41.7"E, Leg. Gyorgy Reichart, 14 Feb 2021. Paratypes: In total, 22 (20 adult and 2 subadult) spec- imens: P.148.1 slide No. 3116 (three specimens), P.148.2 Slide No. 3117 (three specimens), P.148.3 slide No. 3122 (two specimens), P.148.4 slide No. 3123 (four speci- mens), P.148.5 slide No. 3124 (two specimens), P.148.6 Slide No. 3126 (two specimens), P.148.7 slide No. 3127, P.148.8 slide No. 3129 (two specimens), and P.148.9 slide No. 3130 (three specimens). Same data as for holotype. Further material examined. 19 specimens (10 inves- tigated only in vivo) + three specimens for DNA analysis. Zoosyst. Evol. 100 (4) 2024, 1269-1286 Figure 3. Micrograph of Marionina reicharti sp. nov. A. Entire specimen (e = egg); B. Brain; C. Clitellar glands, dorsal view; D. Chaetae, anterior bifurcation of the dorsal vessel in III, ventral view; E. Chaetae posteriorly; F. Pharyngeal glands (spermathecae marked with arrows); G. Chloragogen cells; H. Preclitellar nephridium; I. Coelomocytes; J. Pars tumida of midgut in XX—XXI. A-I. Jn vivo; J. Fixed, stained. Scale bars: 200 um (A); 50 um (F—H); 20 um (B-E, G, I, J). Diagnosis. (1) Small size (body length 2—3.3 mm, 137-190 um wide at clitellum, in vivo), segment num- ber 19-29; (2) maximum five chaetae per bundle, chae- tae straight with ental hook and mostly not equal in size; (3) clitellum saddle-shaped; (4) first and second pairs of pharyngeal glands compact and united dorsally without ventral lobes; the third pair free and elongated; (5) tran- sition between oesophagus and intestine gradual, midgut pars tumida in X VIJTI—XXII, extending over 3-4 segment lengths; (6) dorsal vessel origin in XII, blood colorless, the dorsal anterior blood vessel bifurcation anteriorly be- hind the pharynx; (7) three pairs of preclitellar nephridia; (8) coelomocytes nucleated oval or lemon-shaped with fine granules, 15—24 um long in vivo; (9) seminal vesicle absent; sperm morulae occur in all segments; (10) sperm funnel 1.5—3 times longer than wide in vivo, collar high and narrower than funnel body; spermatozoa 46—70 um long, heads 20-30 um in vivo; (11) male copulatory or- gan oval or bean-shaped and compact, 33-47 um long in vivo; (12) ectal duct of spermatheca surrounded by glands, wider proximally; one larger sessile gland at or- ifice; ampulla globular, diameter 29-40 um in vivo with some sperm-threads or sperm bundle in it; ampulla at- tached to oesophagus. Description. Small species (Fig. 3A), holotype 2.5 mm long, 115 um wide at VII and 138 um at clitel- lum (fixed), segment number 28. Body length 2.0— 3.3 mm, width 130-160 um at VIII and 137-190 um at clitellum in vivo, length of fixed specimens 1.5—2.5 mm, width 90-135 um at VHI and 115-160 um at clitellum, zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) me i ae Figure 4. Micrograph of Marionina reicharti sp. nov. A. Sperm bundles in the coelom; B, C. Male copulatory organs (e = egg, sd = sperm duct); D—-F. Sperm funnels (male copulatory organs marked with a white arrow); G—J. Spermathecae (ectal glands marked with black arrows, ampulla marked with white arrows, in H. Sperm thread in ampulla, in I. Sperm bundles in ampulla). A-C, E-I. /n vivo; D, J. Fixed, stained. Scale bars: 50 um (C, F, G); 20 um (A, B, D, E, H—J). segment number 19-29. Chaetae straight with ental hook (Fig. 3D, E). Chaetal formula: 2.3,(4) - 2.3: (3)4.5,(6) - 2.3,(4). The chaetae are not exactly equal in size with- in the bundles; often the middle chaetae slightly smaller than the ental ones, e.g., in the ventral preclitellar bun- dles, 20-19.5—18-20 um or 25—22—20.4—24.2 um long and 1.6—1.9 um wide. The ventral chaetae slightly longer than lateral ones. At the posterior end of the body, chaetae are 21—27 um long. Clitellum saddle-shaped in XII-1/2 XIII, gland cells squarish, arranged in about 15-18 trans- verse rows (Fig. 3C), midventrally absent. Head pore not seen, no dorsal pores. Epidermal gland cells inconspic- uous in vivo. Thickness of body wall about 10-13 um; cuticle thin (<1 um). Brain (Fig. 3B) ca. 47-51 um long (fixed), slightly lon- ger than wide, incised posteriorly. Prostomial glanglia ab- zse.pensoft.net sent. In the ventral nerve cord, perikarya continuous. First and second pair of pharyngeal glands compact and unit- ed dorsally without ventral lobes; the third pair free and elongate (Figs 3F, 4G). Chloragocytes from IV forming a denser layer from VI, about 8-12 um long in vivo, filled with refractive globules (Fig. 3G). Transition between oe- sophagus and intestine gradual; oesophageal appendage and intestinal diverticula absent. Midgut pars tumida in XVII-XXII, extending over 3-4 segment lengths (Fig. 3J). Dorsal vessel from XII, blood colorless. The dorsal anterior blood vessel bifurcation in III. All coelomocytes nucleated oval or lemon-shaped with fine granules, 15- 24 um long in vivo (Fig. 31) and 8-10 um, fixed. Three pairs of preclitellar nephridia in 6/7—8/9, preseptal part consisting of funnel and coils of canal, postseptal part elongate, about 1.7—3 times longer than the preseptal part, Zoosyst. Evol. 100 (4) 2024, 1269-1286 efferent duct terminal (Fig. 3H). Seminal vesicle absent, sperm morulae and sperm bundles may occur in all seg- ments (Fig. 4A). Sperm funnels cylindrical, 60-95 um long in vivo, 40—70 um, fixed, and about 1.5—3 times lon- ger than wide in vivo (1.2—2 times, when fixed), collar high, and narrower than funnel body (Fig. 4D—F). Spermatozoa 46-70 um long, heads 20-30 um in vivo, 30-50 um long, heads 10—22 um, when fixed. Sperm ducts short, about 2.5—3.5 times longer than the funnel, coiled into a loose spiral, diameter 7-9 um, in vivo. Male copulatory organ oval or mostly bean-shaped and compact (Fig. 4B, C, F), 33-47 um long, 18-26 um wide, and 18-26 um high, in vivo (30-40 um long, 21-28 um wide, and 18-25 um high, when fixed). Subneural glands absent. Ectal duct of spermatheca, 26—38 um long, surrounded along the length by glands, 14-18 um wide proximally and 10-14 um wide distally in vivo (20-23 um long, 14-20 um wide proximal- ly, and 12-16 um wide distally, when fixed). One larger, 15—22 um long, sessile gland at orifice in vivo (15—20 um, fixed) (Fig. 4G—J). Ampulla globular, diameter 29-40 um in vivo (25-40 um, fixed), lumen with some sperm-threads (Fig. 4H) or 1-3 sperm bundles (Fig. 41). Ampulla at- tached with a short ental duct to the oesophagus. One or two mature eggs at a time (Fig. 4C, F). Etymology. The new species is named in the honor of Gyorgy Reichart, who collected the sample with this species. Distribution and habitat. Known from the lake shore of Lake Balaton at Bélatelep, Strand Batori, Hungary, in wet sand between the roots of willow trees (Loc. 4). Ear- lier, they were identified as Marionina spicula (Leuckart, 1847) at four stations of the Lake Balaton (between Fuzf6 and Alsoors, Balatonberény, and Bélatelep) in a fauna in- vestigation in 1990-1992 (Dozsa-Farkas 1995). Unfortu- nately, these specimens were lost. Remarks on the studied specimens. Some small enchytraeid worms during the former study of Lake Ba- laton shore fauna were identified as Marionina spicula (Dozsa-Farkas 1995); therefore, the question was raised if they really belonged to this species known typical- ly from marine habitats. However, in two papers (La- font and Juget 1976; Rodriquez 1986), the species was observed along rivers without a detailed morphological description, and its euryhalinity was also reported pre- viously (Giere 1971). This initiated our study with a freshly collected sample from the shore of Lake Balaton, combining DNA sequencing with morphological inves- tigation. 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. Fur- thermore, 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 mo- lecular 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 L279 (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. Differential diagnosis. Our species comparison 1s based on Nielsen and Christensen (1959), and it includes obser- vations on living individuals collected in Denmark on the Niva coast in 1999 (site 5) and reinvestigated (as fixed ma- terial) in this study. The morphological differences are as follows: M. reicharti sp. nov. is smaller (2—3.3 mm length vs. 4-5 mm length in M. spicula in Denmark, in vivo, 1.5—2.5 mm length vs. 2.1—2.6 mm length, fixed), segment number 19-29 vs. 27-30. Sperm funnel slightly shorter (40-70 um long vs. 67-90 um, fixed), the spermatozoa are longer in the new species (30-50 um, heads 11—22 um vs. 20-38 um, heads 10-17 um in M. spicula). The ectal duct of the spermatheca is longer in Danish M. spic- ula specimens and proximally wider (20-50 um long and 16—50 um wide vs. 20—23 um long and 14—20 um wide in the new species). Origin of the dorsal vessel from segment XII vs. from XIII in Danish M. spicula specimens. Keeping in mind that the Mediterranean M. spicula Specimens studied here may belong to a different spe- cies, and since the origin of M. spicula specimen CE2561 is from Sweden (see additional details below), we per- formed a morphological comparison of Mediterranean M. spicula with the new species: M. reicharti sp. nov. is smaller and has in general fewer segments: length 3 mm, width 137-190 um at clitellum in vivo, 19-29 segments (vs. 3-7 mm, 200-430 um, 21-39 segments, respective- ly). The maximum number of ventral chaetae is lower, 5—6 (vs. 7-8). The clitellum is saddle shaped (vs. ring- shaped, glands absent only between the male openings). The spermatozoa are 46—70 um long, heads 20-30 um in vivo (vs. 60-120 um long and 30-43 um), the male cop- ulatory organ is also smaller, 33-47 um long, 18-26 um wide, 18—26 um high (vs. 45-70 x 45-60 x 35-50 um). Furthermore, the size of the spermatheca is different: ec- tal duct 26-38 long, ectal gland 15—22 um long, diameter of ampulla 29-40 um in vivo (vs. 35-51 um, 30-40 um and 40-63 um, respectively). The coelomocytes and the size of sperm funnels are not comparable, because these traits in the specimens of M. spicula collected at the Adri- atic seashore are very variable (a sign of that possibly M. spicula is a species complex). Morphological notes on the Marionina spicula (Leuck- art, 1847) specimens of the Adriatic seashore Figs 5-6 Material examined. About 50 specimens were investi- gated in vivo, slides were made from 28 specimens, and 11 specimens were used for DNA analysis. Collecting site: (Loc. 1) Croatia, Istria, Kale Cove seashore, Adriatic Sea, Kamenjak Peninsula, decaying seagrass (Zostera) detritus, 44°51'13.0"N, 13°58'50.5"E, Leg. Julia Torok, 03 Apr 2019, and 05 Sep 2020. zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) 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 um. Description of new material. Small worms, body length 3—7 mm, width 200-430 um at clitellum in vivo; length of fixed specimens 1.9-3.6 mm, width 170- 320 um at clitellum, segment number 21-39. Chaetae straight with ental hook. Chaetal formula variable: 2-5 - 4-2: 4-8 - 6-2. The chaetae unequal in size within the bundles. Mostly the chaetae towards the midlines of the body are shorter than the lateral ones, or the chaetae in the middle of the bundles are shorter (Fig. 5A—C). Clitel- lum ring-shaped in XII-1/2 XIII, gland cells arranged in irregular transverse rows (Fig. 5E), between the male openings absent (Fig. 5F). Head pore at the middle of prostomium, no dorsal pores. Epidermal gland cells in- conspicuous in vivo. zse.pensoft.net Brain (Fig. 5D) 62-87 um long (fixed), 1.5 times lon- ger than wide, incised posteriorly. Pharynx and postpha- ryngeal bulbs well developed. Prostomial ganglia absent. In the ventral nerve cord, perikarya continuous. First and secondary pharyngeal glands compact and united dorsal- ly; the third pair free (Figs 5G, 6F). Chloragocytes from IV forming a denser layer from VI, about 15—20 um long in vivo, filled with refractive globules. Transition between oesophagus and intestine gradual; oesophageal appendages and intestinal diverticula absent. Midgut pars tumida not seen. Dorsal vessel from XII, blood colorless. The dorsal anterior blood vessel bifurcation in III, pharyngeal (Fig. 5G). Coelomocytes variable (Fig. 5H—K), nucleated, disc- shaped with gray granules; in some specimens, the coelo- Zoosyst. Evol. 100 (4) 2024, 1269-1286 a mem mocytes are with few granules (Fig. 5J), so they are pale in the coelom. In other specimens, the coelomocytes are filled with granules (Fig. 5K), and the coelomocytes are in such large numbers that they fill the entire coelom and are dark gray in transmitted light (Fig. SI), 15-24 um long in vivo, and 15—17 um, fixed. Three pairs of preclitellar nephridia in 6/7—8/9, preseptal part consisting of funnel and coils of ca- nal, postseptal part elongate, about 2—2.5 times longer than the preseptal part, efferent duct terminal (Fig. 6A). Seminal vesicle absent. Sperm funnels cylindrical, very variable; they can be about 110-150 um long and 1.7—2.4 times lon- ger than wide (Fig. 6B, C); in other specimens, they are very large, 170-300 um long and 3-4.5 times longer than wide in vivo (Fig. 6D, E), collar 10—25 um high and narrow- er than funnel body. Spermatozoa 60-120 um long, heads 1281 St Ge with ar- rows) with a short sperm duct (e = egg); D, E. Large sperm funnels; F—G. Spermathecae (ampullae marked with arrows); H. Body- end with ejected sticky mucus. A—-F, H. in vivo, G. fixed, stained. Scale bars: 50 um (A—F, H); 20 um (G). 30-43 um in vivo. Sperm ducts short, about three times lon- ger than the funnel, diameter 10-13 um, in vivo (Fig. 6C). Male copulatory organs compact (Fig. 5F), 45-70 um long, 45—60 um wide, and 35-50 um high in vivo (32-58 um long, 30-53 um wide, and 28-40 um high, when fixed). Ec- tal duct of spermatheca 35—51 um long, surrounded along the length by glands and one larger, 30-40 um long, ses- sile gland at orifice. Ampulla rounded (diameter 40-63 um wide in vivo, 35-53 um, fixed), sometimes with a rather thick wall (6-14 um) in the lumen with sperm (Fig. 6F, G). Ampulla attached with a short ental duct to the oesophagus. One or occasionally two mature eggs at a time (Fig. 6C). In this species, as already pointed out by Giere (1971), it is often observed that the coelomic fluid, released through the anus, attaches itself to the grains of sand (Fig. 6H). zse.pensoft.net 1282 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 consistent- ly 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 sequenc- es 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 de- scribed in this study. The results of the distance analyses supported the phy- logenetic investigations. The p-distances between the ITS sequences of the three new species and the other Marion- ina 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 Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) 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 be- tween M. spicula CE2561 and the three Croatian M. spic- ula clades are 15.8—-17.3%, and the H3 distances between them are 4.1-5%. The ITS distances could not be com- pared 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 data- base. The above-presented results suggest that VM. 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. or- bifera sp. nov.) from the Tyrrhenian coast. The distinctive morphological characters served as the basis of the de- scription 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 com- plex. 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 Marionina reicharti sp. nov. 1457 Marionina reichartisp. nov. 1465 1001 Marionina reichartisp. nov. 1360 Marionina reicharti sp. nov. 1361 92) Marionina reichartisp. nov. 1466 Marionina spicula 1442 Marionina spicula 1445 55) Marionina spicula 1377 100 93] Marionina spicula 1444 100 Marionina spicula 1446 Marionina puntaalanensis sp. nov. 1450 100! Marionina puntaalanensis sp. nov. 1451 Marionina spicula 1366 54 Marionina orbifera sp. nov. 1448 100L \Yarionina orbifera sp. nov. 1449 62) 99 84 Marionina nevisensis CE338 Marionina cf. nevisensis CE260 Marionina seminuda 1334 Marionina southerni CE674 Marionina vesiculata 898 Marionina clavata 734 100! Nfarionina clavata 1267 Marionina communis 904 100! \arionina communis CE811 Achaeta unibulba 851 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 in Table 1. Scale bar: 0.2 substitutions per nucleotide. zse.pensoft.net Zoosyst. Evol. 100 (4) 2024, 1269-1286 99 1283 Marionina spicula 1444 Marionina spicula 1445 Marionina spicula 1381 99 || Marionina spicula 1377 Marionina spicula 1339 Marionina spicula 1378 gq| Marionina spicula 1443 Marionina spicula 1446 Marionina spicula 1441 99' Marionina spicula 1442 Marionina spicula CE2561 99) Marionina spicula 1338 \Marionina spicula 1341 89! Varionina spicula 1366 Marionina cf. minutissima CE843 95 Marionina argentea 1193 Marionina argentea CE807 Marionina aestuum CE12477 92'——— Marionina fusca CE12476 88>—— Marionina clavata 1267 Marionina clavata CE849 2) Marionina puntaalanensis sp. nov. 1450 Marionina puntaalanensis sp. nov. 1451 Marionina reicharti sp. nov. 1361 g9| Varionina reicharti sp. nov. 1465 Marionina reicharti sp. nov. 1466 ann Achaeta unibulba 851 Marionina tumulicola CE571 — -— Marionina cf. levitheca CE1339 70 ,-__— Marionina orbifera sp. nov. 1449 Marionina orbifera sp. nov. 1447 9* Marionina orbifera sp. nov. 1448 Marionina vesiculata 898 we [————— Marionina cf. nevisensis CE260 Marionina seminuda 1334 Marionina nevisensis CE338 —— Marionina nothachaeta LM322 Marionina filiformis CE1040 =" Marionina cf. argentea CE22027 Marionina southerni CE674 Marionina communis 1216 65 Marionina communis 904 99 Marionina communis CE811 Figure 8. Maximum likelihood (ML) tree of the COI gene for Marionina species, based on 517 nucleotide positions using the Gen- eral Time Reversible substitution model. Bootstrap values greater than 50 are shown at the nodes. Accession codes of sequences with collection information are given in Table 1. Scale bar: 0.1 substitutions per nucleotide. the detected Marionina spicula clades were comparable to the interspecific differences of the other Marionina spe- cies included in this study. However, we were not able to assign this variability to distinct new species. The ob- served 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 ven- tral bundle), the size of the sperm funnel (besides the type of “two times longer than wide,” there were very large sperm funnels, which were 34.5 times longer than wide), and the granularity of coelomocytes (sometimes the coel- omocytes were less granulated, but in other cases the coe- lomocytes 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 charac- ters. To circumvent this problem, several additional sam- ples 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 MZ spicula attach to sand parti- cles (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 materi- al. 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 Chris- tensen (1959). On the other hand, Danish individuals dif- fered from the specimens collected by us from the Adriat- ic 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 um in Adriatic specimens). Taken all together, further studies are required on the M. spicula species complex, which zse.pensoft.net 1284 Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) Marionina spicula 1443 -— Marionina spicula 1445 Marionina spicula 1381 Marionina spicula 1378 Marionina spicula 1377 Marionina spicula 1339 Marionina spicula 1446 — Marionina spicula 1441 1 Marionina spicula 1338 94 Marionina spicula 1341 Marionina spicula 1366 7 Marionina reicharti sp. nov. 1360 Marionina reicharti sp. nov. 1465 Marionina reicharti sp. nov. 1466 9 Marionina reicharti sp. nov. 1361 Marionina argentea 1193 —— Marionina spicula CE2561 ita] o Marionina vesiculata 898 Marionina clavata 1267 Marionina clavata CE849 Achaeta unibulba 851 Marionina orbifera sp. nov. 1447 -- Marionina puntaalanensis sp. nov. 1450 Marionina puntaalanensis sp. nov. 1451 (> Marionina aestuum CE12477 78 Marionina fusca CE12476 Marionina seminuda 1334 -— Marionina communis 1216 gg) Marionina communis 904 Marionina communis CE811 Figure 9. Maximum likelihood (ML) tree of the H3 gene for Marionina species, based on 201 nucleotide positions using the Ta- mura 3-parameter substitution model. Bootstrap values greater than 50 are shown at the nodes. Accession codes of sequences with collection information are given in Table 1. Scale bar: 0.02 substitutions per nucleotide. should include more specimens from the North and Baltic Seas and a comparison of the morphological variations re- ported in the literature. A systematic revision of Marioni- na spicula “sensu lato” is beyond the scope of this paper. However, it should be noted that enchytraeid species hav- ing 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 problemat- ic issues related to this heterogenous group, we exam- ined 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 spe- cies is increasing from 101 to 104. Summarizing the re- cent 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. fus- ca Klinth, Rota & Erséus, 2022, M. mimula Rota, 2013, M. mendax Rota, 2013, M. naso Timm, 2012, M. noth- achaeta Matamoros, Rota & Erséus, 2012, and Marion- ina 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 ma- rine 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 re- sults based on comparative morphological and molecular data will aid the revision of the genus in the future. zse.pensoft.net Acknowledgements H. N. was supported by the UNKP-20-4 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development, and Innovation Fund, Hungary (grant no. UNKP-20-4-I-ELTE-28 1). The authors are thankful to Andras Dozsa-Farkas and Kinga Dozsa-Farkas, Dr. Julia Torok, and Gyorgy Reich- art for collecting samples and for the reviewers for their valuable suggestions. References Bely AE, Wray GA (2004) Molecular phylogeny of naidid worms (Annelida: Clitellata) based on cytochrome oxidase I. Molecular Phylogenetics and Evolution 30(1): 50-63. https://doi.org/10.1016/ S$ 1055-7903(03)00180-5 Bretscher K (1899) Beitrag zur Kenntnis der Oligochaeten-Fauna der Schweiz. Revue Suisse de Zoologie 3(2): 369-426. https://doi. org/10.5962/bhI1.part.82518 Cernosvitov L (1937) System der Enchytraeiden. Bulletin de I’ Associ- ation suisse pour les Recherches scientifiques a Prague 5: 263-294. Cernosvitov L (1938) Mission scientifique de l’Omo. Oligochaeta Memoires du Muséum National d’ Histoire Naturelle 4: 255-318. Christensen B, Dozsa-Farkas K (2012) A new genus Globulidrilus and three new enchytraeid species (Oligochaeta: Enchytraeidae) from Seoraksan National Park (Korea). Journal of Natural History 46(45— 46): 2769-2785. https://doi.org/10.1080/00222933.2012.737038 Zoosyst. Evol. 100 (4) 2024, 1269-1286 Coates KA (1980) New marine species of Marionina and Enchytraeus (Oligochaeta, Enchytraeidae) from British Columbia. Canadian Jour- nal of Zoology 58(7): 1306-1317. https://doi.org/10.1139/z80-182 Coates KA (1989) Phylogeny and origins of Enchytraeidae. Hydrobio- logia 180(1): 17-33. https://doi.org/10.1007/BF00027534 Coates KA (1990) Marine Enchytraeidae (Oligochaeta, Annelida) of the Albany area, Western Australia. In: Wells FE, Walker DI, Kirkman H, Lethbridge R (Eds) Proceedings of the Third International Ma- rine Biological Workshop: The marine flora and fauna of Albany, Western Australia. Western Australia Museum, Perth, 13-41. Colgan DJ, McLauchlan AA, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J, Cassis G, Gray MR (1998) Histone H3 and U2 sn- RNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 46(5): 419-437. https://doi.org/10.1071/Z098048 Collado R, Hass-Cordes E, Schmelz RM (2012) Microtaxonomy of fragmenting Enchytraeus species using molecular markers, with a comment on species complexes in enchytraeids. Turkish Journal of Zoology 36: 85-94. https://doi.org/10.3906/zo0-1002-70 Dozsa-Farkas K (1995) Enchytraeid fauna of the shore of Lake Balaton. In: Mishra PC (Ed.) Advances in Ecology and Enviromental Scienc- es, Ashish Pub. House, New Delhi: Chapter 8: 117-131. Dozsa-Farkas K (2002) Notes on the genus Oconnorella Rota, 1995. - Natura Jutlandica 2, Proceeding of the 4" International Symposium on Enchytraeidae, Mols Laboratory, Denmark, 24 June 2000: 86-90. Dozsa-Farkas K, Felf6ldi T (2017) Comparative morphological and molecular taxonomic study of six Achaeta species (Clitellata: En- chytraeidae) with the description of a new Achaeta species from K6észeg Mountains, Hungary. Zootaxa 4273(2): 177-194. https:// doi.org/10.11646/zootaxa.4273.2.2 Dozsa-Farkas K, Felf6ldi T, Hong Y (2015) New enchytraeid species (Enchytraeidae, Oligochaeta) from Korea. Zootaxa 4006(1): 171-— 197. https://doi.org/10.11646/zootaxa.4006. 1.9 Dozsa-Farkas K, Felf6ldi T, Nagy H, Hong Y (2018) New enchytraeid species from Mount Hallasan (Jeju Island, Korea). Zootaxa 4496(1): 337-381. https://doi.org/10.11646/zootaxa.4496. 1.27 Erséus C, Daoyuan S, Yanling L, Bin S (1990) Marine Oligochaeta of Jiaozhou Bay, Yellow Sea coast of China. Hydrobiologia 202(1—2): 107-124. https://doi.org/10.1007/BF02208131 Erséus C, Rota E, Matamoros L, De Wit P (2010) Molecular phylog- eny of Enchytraeidae (Annelida, Clitellata). Molecular Phyloge- netics and Evolution 57(2): 849-858. https://doi.org/10.1016/j. ympev.2010.07.005 Erséus C, Klinth MJ, Rota E, De Wit P, Gustafsson DR, Martinsson S (2019) The popular model annelid Enchytraeus albidus is only one species in a complex of seashore white worms (Clitellata, En- chytraeidae). Organisms, Diversity & Evolution 19(2): 105-133. https://doi.org/10.1007/s13127-019-00402-6 Felfoldi T, Dozsa-Farkas K, Nagy H, Hong Y (2020) Three new en- chytraeid species (Enchytraeidae, Annelida) from mountain soils of Korea and ten species new for the country. Zootaxa 4896: 001-045. https://do1.org/10.11646/zootaxa.4896. 1.1 Finogenova NP (1972) New species of Oligochaeta from Dnjepr and Bug Firth and Black Sea and revision of some species. Trudy Zoolog- icheskogo Intstituta (Akademiya Nauk SSSR) Leningrad 52: 94-116. Finogenova NP (1973) New species of Oligochaeta from the Caspian Sea. Zoologicheskij Zhurnal Moscow 52: 121-124. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA prim- ers for amplification of mitochondrial cytochrome c oxidase subunit 1285 I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294-299. Frey H, Leuckart R (1847) Beitrage zur Kenntnis wirbelloser Thiere mit besonderer Beriicksichtigung der Fauna des norddeutschen Meeres. Vieweg, Braunschweig. https://doi.org/10.5962/bh1 title.2128 Giere O (1971) Marionina spicula - ein 6kologisch und morphologisch spezialisierter mariner Enchytraeide (Oligochaeta). Helgolander Wissenschaftliche Meeresuntersuchungen 22(3-4): 350-361. https://doi.org/10.1007/BF01611123 Giere O (1974) Marionina istriae n. sp., ein mariner Enchytraeide (Oligochaeta) aus dem mediterranen Hygropsammal. Helgolander Wissenschaftliche Meeresuntersuchungen 26(3-4): 359-369. https://doi.org/10.1007/BF01627620 Kane RA, Rollinson D (1994) Repetitive sequences in the ribosom- al DNA internal transcribed spacer of Schistosoma haematobium, Schistosoma intercalatum and Schistosoma mattheii. Molecu- lar and Biochemical Parasitology 63(1): 153-156. https://doi. org/10.1016/0166-6851(94)90018-3 Klinth MJ, Martinsson S, Erséus C (2017) Phylogeny and species delim- itation of North European Lumbricillus (Clitellata, Enchytraeidae). Zoologica Scripta 46(1): 96-110. https://do1.org/10.1111/zsc.12187 Klinth MJ, Kreiling A-K, Erséus C (2019) Investigating the Clitella- ta (Annelida) of Icelandic springs with alternative barcodes. Fauna Norvegica 39: 119-132. https://doi.org/10.5324/fn.v3910.3043 Klinth MJ, Rota E, Martinsson S, Prantoni AL, Erséus C (2022) New insights into the systematics of Lumbricillus and Marionina (Clitel- lata: Enchytraeidae) inferred from Southern Hemisphere samples, including three new species. Zoological Journal of the Linnean Soci- ety 194(4): 1103-1133. https://do1.org/10.1093/zoolinnean/zlab073 Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolution- ary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33(7): 1870-1874. https://doi.org/10.1093/ molbev/msw054 Lafont M, Juget J (1976) Les oligochetes du Rhone. I. Releves faunis- tiques generaux. Annales de Limnologie 12(3): 253-268. https://doi. org/10.1051/limn/1976015 Lasserre P (1964) Notes sur quelques oligochetes Enchytraeidae, présents dans les plages du Bassin d’ Areachon. Proces verbaux de la Société Linnéenne de Bordeaux 101: 87-91. Lasserre P, Erséus C (1976) Oligochetes marins des Bermudes, Nou- velles espgéces et remarques sur la distribution geograhique de quelques Tubificidae et Enchytraeidae. Cahiers de Biologie Marine 17: 447-462. Marcus E (1965) Naidomorpha aus _ brasilianischem Brackwass- er. Beitrage zur Neotropischen Fauna 4(2): 61-83. https://doi. org/10.1080/01650526509360380 Martin P, Schmelz RM, Dole-Olivier M-J (2015) Groundwater oligochaetes (Annelida, Clitellata) from the Mercantour National Park (France), with the descriptions of one new genus and two new stygobiont species. Zoosystema 37(4): 551-569. https://doi.org/10.5252/z2015n4a2 Martinsson S, Erséus C (2018) Cryptic diversity in supposedly spe- cies-poor genera of Enchytraeidae (Annelida: Clitellata). Zoolog- ical Journal of the Linnean Society 183(4): 749-762. https://doi. org/10.1093/zoolinnean/zlx084 Martinsson S, Dézsa-Farkas K, Rota E, Erséus C (2017) Placing the for- gotten: on the positions of Euenchytraeus and Chamaedrilus in an updated enchytraeid phylogeny (Clitellata: Enchytraeidae). Inver- tebrate Systematics 31(1): 85—90. https://doi.org/10.1071/IS 16042 zse.pensoft.net 1286 Matamoros L, Yildiz S, Erséus C (2007) A new species within the genus Marionina (Enchytraeidae: Annelida: Clitellata) from the southern Black Sea. Marine Biology Research 3(6): 397-402. https://doi. org/10.1080/17451000701694844 Matamoros L, Rota E, Erséus C (2012) Cryptic diversity among the achaetous Marionina (Annelida, Clitellata, Enchytraeidae). Sys- tematics and Biodiversity 10(4): 509-525. https://doi.org/10.1080/ 14772000.2012.723640 Michaelsen W (1888) Die Oligochaeten von Siid-Georgien nach der Ausbeute der Deutschen Station von 1882-1883. Jahrbuch der Hamburgischen Wissenschaftlichen Anstalten 5: 53-73. Michaelsen W (1889) Oligochaeten des Naturhistorischen Museums in Hamburg. I. Jahrbuch der Hamburgischen Wissenschaftlichen An- stalten 6: 1-17. Nagy H, Dozsa-Farkas K, Felf6ldi T (2023) New insights into the Enchytraeus albidus complex (Annelida, Enchytraeidae), with the description of three new species from seashores in Italy and Cro- atia. European Journal of Taxonomy 870: 107-145. https://doi. org/10.5852/ejt.2023.870.2123 Nielsen CO, Christensen B (1959) The Enchytraeidae. Critical revision and taxonomy of European species (studies on Enchytraeidae VII). Natura Jutlandica 8-9: 1-160. Nielsen CO, Christensen B (1961) The Enchytraeidae. Critical revision and taxonomy of European species. Supplement 1. Natura Jutland- ica 10: 1-23. O’Connor FB (1962) The extraction of Enchytraeidae from soil. In: Murphy PW (Ed.) Progress in Soil Zoology. Butterworths Publish- ers, London, 279-285. O’Connor FB (1963) Marionina cambrensis sp. nov.: Anew enchytraeid worm (Oligochaeta) from North Wales. The Annals and Magaine of Natural History, including Zoology. Botany and Geology London 6(13): 761-766. https://doi.org/10.1080/0022293630865 1427 Pfeffer G (1890) Die niedere Thierwelt des antarktischen Ufergebietes. Die internationale Polarforschung 1882—1883. Die Deutschen Expe- ditionen und ihre Ergebnisse (5) 2(17): 455-574. Rodriquez P (1986) Nuevos resultados acerca de la fauna de oligoquetos acuaticos del Pais Vasco y cuenca alta del Ebro. 2. Enchytraeidae. Munibe. Ciencias Naturales 38: 81-87. Rota E (1995) Italian Enchytraeidae. I. Bollettino di Zoologia 62(2): 183-231. https://doi.org/10.1080/11250009509356067 Rota E (2013) How many lookalikes has Marionina argentea (Mi- chaelsen, 1889) (Annelida: Clitellata: Enchytraeidae)? Three new species described from morphological evidence. Zoologischer An- zeiger 252(1): 123-137. https://doi.org/10.1016/j.jcz.2012.05.001 Rota E, Manconi R (2004) Taxonomy and Ecology of Sponge-Associate Marionina spp. (Clitellata: Enchytraeidae) from the Horomatangi Geo- thermal System of Lake Taupo, New Zealand. International Review of Hydrobiology 89(1): 58-67. https://doi.org/10.1002/iroh.2003 10695 Rota E, Matamoros L, Erséus C (2008) In search of Marionina (Clitel- lata, Enchytraeidae): A taxonomic history of the genus and rede- scription of the type species Pachydrilus georgianus Michaelsen, 1888. The Italian Journal of Zoology 75(4): 417-436. https://doi. org/10.1080/11250000801930433 zse.pensoft.net Felfoldi, T. et al.: Description of three new Marionina species (Annelida, Enchytraeidae) Schmelz RM, Collado R (2008) A type-based redescription of Pachy- drilus georgianus Michaelsen, 1888, the type species of Mari- onina Michaelsen, 1890, with comments on Christensenidrilus Dozsa-Farkas & Convey, 1998 (Enchytraeidae, “Oligochaeta”, Annelida). Verhandlungen des Naturwissenschaftlichen Vereins in Hamburg 44: 7-22. Schmelz RM, Collado R (2010) A guide to European terrestrial and freshwater species of Enchytraeidae (Oligochaeta). Soil Organisms 82(1): 1-176. Schmelz RM, Collado R (2012) An updated checklist of currently ac- cepted species of Enchytraeidae (Oligochaeta, Annelida). Newslet- ter on Enchytraeidae 12: 67-87. Schmelz RM, Collado R (2015) Checklist of taxa of Enchytraeidae (Oli- gochaeta): An update. Soil Organisms 87: 149-152. Schmelz RM, Collado R, Rémbke J (2011) Mata Atlantica enchy- traeids (Enchytraeidae, Oligochaeta), Description of a new genus Xetadrilus gen. nov., with three new species, and four new species of Guaranidrilus Cernosvitov. Zootaxa 2838: 1-29. https://doi. org/10.11646/zootaxa.2838.1.1 Schmelz RM, Klinth MJ, Chalkia C, Anastasiadou P, Vavoulidou E (2019) Enchytraeus demutatus sp. nov. (Enchytraeidae, Oligochae- ta) has characters hitherto unrecorded in the genus. Soil Organisms 91: 87-96. https://doi.org/10.25674/so9 | iss3pp87 Shurova NM (1978) The intertidal oligochaetes from the eastern coast of Kamchatka, In: Litoral Beringova moray iyugo-vost Kamchatki Moscow, 98-106. [in Russian with English summary] Shurova NM (1979) Enchytraeids (Oligochaeta) of the far-east seas of the USSR. In: Studies on the pelagic and groundinhabiting organ- isms of the Far East seas, Vladivostok, 75—89. Somogyi B, Felfoldi T, Toth LG, Bernat G, Voros L (2020) Photoauto- trophic picoplankton - a review on their occurrence, role and diver- sity in Lake Balaton. Biologia Futura 71(4): 371-382. https://doi. org/10.1007/s42977-020-00030-8 Timm T (1996) Oligochaeta of Lake Taimyr: A preliminary survey. Hy- drobiologia 334(1-3): 89-95. https://doi.org/10.1007/BF00017357 Timm T (2012) Estonian Enchytraeidae 4. Sublittoral Enchytraeidae (Annelida, Oligochaeta) in the eastern Baltic Sea. Estonian Journal of Ecology 61(3): 157-172. https://doi.org/10.3176/eco.2012.3.01 Timm T, Vvedenskaya TL (2006) Oligochaeta (Annelida) of Lake Ku- rilskoe, Kamchatka Peninsula. Species Diversity 11(3): 225-244. https://doi.org/10.12782/specdiv. 11.225 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (Eds) PCR Protocols: A Guide to Methods and Applications. Academy Press, San Diego, 315-322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1 Xie ZC, Rota E (2001) Four new terrestrial species of Marionina (Clitellata, Enchytraeidae) from China and re-examination of . hoffbaueri Moller. Journal of Natural History 35(10): 1417-1431. https://doi.org/10.1080/0022293013 17067610 Xie ZC, Liang YL, Wang HZ (2000) A new species of Marionina (Oli- gochaeta: Annelida: Enchytraeidae). Acta Zootaxonomica Sinica 25: 143-146. [in Chinese with English abstract]