#ZooKeys ZooKeys 1206: 137-180 (2024) DOI: 10.3897/zookeys.1206.124237 Research Article Integrative taxonomic study of mononchid nematodes from riparian habitats in Bulgaria. |. Genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 with the description of Mononchus pseudoaquaticus sp. nov. and a key to the species of Mononchus Stela Altash’, Aneta Kostadinova’®, Vlada Peneva’ 1 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria Corresponding author: Vlada Peneva (esn.2006@gmail.com) OPEN Qaccess Academic editor: Sergei Subbotin Received: 30 March 2024 Accepted: 12 May 2024 Published: 5 July 2024 ZooBank: https://zoobank.org/ C1AC9890-1735-4929-B1D5- 2056B7E8AFBE Citation: Altash S, Kostadinova A, Peneva V (2024) Integrative taxonomic study of mononchid nematodes from riparian habitats in Bulgaria. |. Genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 with the description of Mononchus pseudoaquaticus sp. nov. and a key to the species of Mononchus. Zookeys 1206: 137-180. https://doi. org/10.3897/zookeys.1206.124237 Copyright: © Stela Altash et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract The species diversity of the genera Mononchus Bastian, 1865 and Coomansus Jairajpuri & Khan, 1977 was assessed in a study of the mononchid nematodes from a wide range of riparian habitats in Bulgaria. Four species were identified based on morphological and morphometric data: Coomansus parvus (de Man, 1880), Mononchus truncatus Bas- tian, 1865, Mononchus pseudoaquaticus sp. nov., and Mononchus sp. The first three species were characterised both morphologically and molecularly (18S and 28S rRNA gene sequences) and the integration of these data and phylogenetic analyses provided support for their distinct species status. This paper provides detailed descriptions, mor- phometric data for multiple species populations, drawings and photomicrographs, and the first taxonomically verified sequences for C. parvus (n = 6), M. truncatus (sensu stric- to) (n = 4) and M. pseudoaquaticus sp. nov. (n = 3). Comparative sequence and phyloge- netic analyses suggested that the utility of the 18S rRNA gene for species delimitation is rather limited at least for some species complexes within the genus Mononchus. At the generic and suprageneric level, the 18S and 28S rDNA phylogenies both recovered the three genera represented by two or more species (Mononchus, Mylonchulus, and Parkellus) as monophyletic with strong support, the Mononchidae as paraphyletic, the Anatonchidae as monophyletic, and there was no support for a sister-group relationship between Mylonchulus and Mononchus. A key to the species of Mononchus is provided to facilitate the identification of the currently recognised 31 species. Key words: Distribution, Mononchidae, morphology, phylogeny, riverine, taxonomy, 18S rDNA, 28S rDNA Introduction Riparian zones, i.e., the ecotones between aquatic and terrestrial ecosystems, represent areas of high biodiversity caused by the diversity of habitats and heterogeneous environmental conditions they provide. Both plant and animal diversity are high in these areas with impressive levels of faunal diversity in 137 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria riparian soils (Décamps et al. 2009). Soils in these functionally unique ecosys- tems are also important for sustaining diverse nematode communities, @.g., Décamps et al. (2009) estimated the number of species of nematodes in ripar- ian soils to be greater than 5000. Because soil nematodes are abundant and functionally diverse, they can serve as useful indicators of food-web structure and complexity (Bongers and Ferris 1999; Ferris et al. 2001; Neher 2001; Fer- ris 2005); nematode communities are also important for ecosystem functions. These features justify the increased interest in studying free-living nematode communities and the ecosystem functions they perform in both undisturbed and disturbed riparian zones and riparian corridors (e.g., Young-Mathews et al. 2010; Briar et al. 2012; Hodson et al. 2014). Notably, nematodes are usually identified to the genus/family level in these ecological studies due to difficul- ties in the identification based on morphological characters and/or the lack of taxonomic expertise, so there is a lack of species-level assessments on larg- er-scale processes in riparian zones (Bardgett et al. 2001). Sequence-based tools such as barcoding have proven successful in accel- erating identification of previously characterised species or in detecting cryp- tic species (Palomares-Rius et al. 2014, 2022; Archidona-Yuste et al. 2023). Currently, two nuclear loci are considered to be most relevant to barcoding of nematodes, the small subunit ribosomal RNA gene (SSU or 18S) and the large subunit ribosomal RNA gene (LSU or 28S), the first being the best sampled gene in nematodes, and the second being the subject of increased interest. However, key for the successful application of barcoding for nematodes is the availability of a database of taxonomically verified sequences, i.e., associated with species identification based on detailed morphological characterisation and morphological vouchering (physical and “virtual” vouchers sensu De Ley et al. 2005). Although the number of species of the order Mononchida Jairajpuri, 1969 with sequences available for both nuclear loci indicated above is limited, a recent trend towards building a combined evidence database is promising (Kim et al. 2018; Tabolin and Kolganova 2020; van Rensburg et al. 2021; Vu 2021; Vu et al. 2021a, 2021b; Shokoohi and Moyo 2022). In a study of the free-living nematodes from a wide range of riparian habitats in Bulgaria, we have collected several species of three families of the order Mononchida. These have been characterised both morphologically and molec- ularly. This paper presents the results of the integrative taxonomic study of the species of Coomansus Jairajpuri & Khan, 1977 and Mononchus Bastian, 1865 (family Mononchidae Chitwood, 1937), and phylogenetic analyses that delin- eate the species and establish their relationships within the suborder Monon- china Kirjanova & Krall, 1969 based on partial sequences of the 28S and 18S rRNA genes. Species of the order Mononchida occur in both aquatic and terrestrial habi- tats. Species of the genus Mononchus are aquatic nematodes, occasionally oc- curring in wet terrestrial habitats (Zullini and Peneva 2006; Andrassy 2009) unlike the species of the second genus considered here, Coomansus, which predomi- nantly dwell in terrestrial habitats. Currently, the genus Mononchus contains 30 species (Andrassy 2011a; Shah and Hussain 2016; Gagarin and Naumova 2017; Ishaque et al. 2022). According to Andrassy (2009) the number of valid species of Coomansus is 28. Subsequently, five new species have been described (An- drassy 2011b; Shah and Hussain 2015; Vu 2021). Ahmad and Jairajpuri (2010) ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 138 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria transferred the species of the Coomansus “zschokkei-group” to the genus Par- kellus Jairajpuri, Tahseen & Choi, 2001; however, the validity of Parkellus is not widely accepted (e.g., Zullini and Peneva 2006; Andrassy 2009, 2011b). In Bulgaria, two species of the genus Coomansus, C. parvus (de Man, 1880) Jairajpuri & Khan, 1977 and Coomansus zschokkei (Menzel, 1913) Jairajpuri & Khan, 1977 have been reported (Iliev and Ilieva 2016). Two further species of the genus Mononchus, M. truncatus Bastian, 1865 and M. aquaticus Coetzee, 1968 have also been recorded; however, morphological data have not been pro- vided (Andrassy 1958; Katalan-Gateva 1962, 1965; Stoichev 1996; Lazarova et al. 2004; Stoichev and Chernev 2011; Stoichev and Varadinova 2011). Only the latter two species have been reported in aquatic habitats. Materials and methods Sampling, nematode isolation, and processing More than 150 soil and litter samples were collected at 76 localities in different riparian zones in Bulgaria. Multiple core soil samples (3 per site) were collected at a depth of 40-60 cm from each habitat (sampling site of 15 x 15 mor along the riverbank) around the roots of the dominant tree species; litter samples were collected simultaneously. Nematodes were extracted from soil (at least 400 g) and litter (at least 20 g) samples using a decanting and sieving technique and a modified Baerman fun- nel method with 48 h of exposition and counted alive. Thereafter, the nematodes were gently heated at 63 °C for 2 min and fixed in 4% formaldehyde, 1% glycerine, dehydrated, and mounted on permanent slides in anhydrous glycerine with par- affin as a support for the cover slide (Seinhorst 1959). Morphological examina- tion was carried out and measurement taken under a light microscope (Olympus BX41, Tokyo, Japan) equipped with a digitising tablet (CalComp Drawing Board III) and using the DIGITRAK 1.0 f program (Philip Smith, the John Hutton Institute, Dundee, UK). Drawings were prepared using an Olympus BX51 compound micro- scope with differential interference contrast (DIC). Photomicrographs were taken with Axio Imager.M2 microscope (Carl Zeiss, Oberkochen, Germany) equipped with a digital camera (ProgRes C7) and CapturePro 2.8 software (Jenoptic). All measurements in the descriptions and tables are in micrometres unless stated otherwise and are given as the mean + standard deviation followed by the range in parentheses. A standard set of De Man indices was calculated for each specimen as follows: L, body length; V, distance from vulva to anterior end of body as % of body length; a, body length/greatest body diameter; b, body length/distance from anterior end to pharyngo-intestinal valve; c, body length/ tail length; c’ tail length/tail diameter at anus; G7 anterior female gonad length as % of body length; G2 posterior female gonad length as % of body length (De Man 1876, 1880). DNA isolation, amplification, and sequencing Specimens intended for the molecular study were identified on temporary mounts; a standard set of photomicrographs was taken for each specimen. Genomic DNA (gDNA) was isolated using 5% suspension of deionised water ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 139 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria and Chelex®, containing 0.1 mg/ml proteinase K; samples were incubated at 56 °C for 3 h or overnight, boiled at 90 °C for 8 min, and centrifuged at 14,000x g for 10 min. Two genetic markers were sequenced, the small (18S) and the large (28S) ribosomal subunit RNA coding regions. Partial fragments of the 28S rRNA gene (domains D1-D3; ~ 1000 bp) were amplified using the forward primer LSU5 (5’-TAG GTC GAC CCG CTG AAY TTA AGC A-3’) (Littlewood et al. 2000) and the reverse primer 1500R (5’-GCT ATC CTG AGG GAA ACT TCG-3’) (Tkach et al. 1999). Nearly complete (~ 1600-1700 bp) fragments of the 18S rRNA gene were amplified in two partially overlap- ping fragments using the primer sets 988F (forward: 5’-CTC AAA GAT TAA GCC ATG C-3’) and 1912R (reverse: 5-TTT ACG GTC AGA ACT AGG G-3’) for the first fragment, and 1813F (forward: 5’-CTG CGT GAG AGG TGA AAT-3’) and 2646R (reverse: 5’-GCT ACC TTG TTA CGA CTT TT-3’) for the second fragment (Holter- man et al. 2006). PCR amplifications were performed in a total volume of 25 ul using Illustra ™ PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare, Chicago, USA; Cat. # 27- 9559-01). In the case of poor amplification, the PCR reactions were performed with 2x MyFi™ DNA Polymerase mix (Bioline Inc., Taunton, USA; Cat. # BIO- 25049) in a total volume of 20 ul, containing 8 pmol of each primer and ~ 50 ng of gDNA. The amplification profile for 28S rDNA comprised an initial denatur- ation at 94 °C for 5 min (or 3 min when using MyFi™ DNA Polymerase mix) fol- lowed by 40 cycles (30 s at 94 °C; 30 s at 55 °C; and 2 min at 72 °C), and a final extension step at 72 °C for 7 min. The following amplification profile was used for 18S rDNA: initial denaturation at 94 °C for 5 min, followed by 5 cycles (30 s at 94 °C; 30 s at 45 °C; 70 s at 72 °C) and 35 cycles (30 s at 94 °C; 30s at 54 °C; 70 s at 72 °C), and a final extension step at 72 °C for 5 min. PCR amplicons were purified and sequenced directly for both strands using the PCR primers (and in some cases the internal primers 300F, ECD2 and LSU1200R (Littlewood et al. 2000) for 28S rDNA) at Macrogen Europe (Amsterdam, the Netherlands). Contiguous sequences were assembled, quality checked, and edited manually using MEGA7 (Kumar et al. 2016) and subjected to a BLASTn search on the NCBI GenBank database. Phylogenetic analyses The newly generated 18S rDNA and 28S rDNA sequences were aligned separately using MUSCLE implemented in MEGA7 (Kumar et al. 2016) with representative sequences available in the GenBank database. First, an ex- ploratory neighbour-joining (NJ) analysis was carried out on an untrimmed 28S rDNA alignment (domains D1-D3), including representative sequences for Mononchus spp. and Coomansus spp. to assess the associations of the newly generated sequences from riparian nematode populations sampled in Bulgaria. Secondly, two alignments were constructed comprising sequences for spe- cies of three families of the suborder Mononchina: Anatonchidae Jairajpuri, 1969, Mononchidae, and Mylonchulidae Jairajpuri, 1969. These alignments were trimmed to the length of the shortest sequence. The 28S rDNA alignment (domains D2-D3) contained 33 sequences for representatives of ten genera ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 140 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria of the three families and the 18S rDNA alignment contained 32 sequences for representatives of ten genera of the three families. Phylogenetic relationships were estimated by conducting maximum likeli- hood (ML) analyses as implemented in MEGA7. Prior to analyses, the best-fit- ting models of nucleotide substitution were estimated based on the Akaike information criterion (AIC); these were the Tamura 3-parameter model (T92) including estimates of invariant sites and among-site rate heterogeneity (T92+1+G) for the 18S rDNA alignment and the Kimura 2-parameter model (K2) with among-site rate heterogeneity (K2+G) for the 28S rDNA alignment. Nodal support was estimated by performing 1000 bootstrap pseudoreplicates. Mer- mis nigrescens Dujardin, 1842 was used as the outgroup in the analyses of both alignments based on the phylogeny published by Holterman et al. (2006). Ge- netic distances (number of nucleotide positions and uncorrected p-distance) were calculated in MEGA7. Results Overview of the morphological identification and the novel molecular and distributional data A total of 17 populations of Coomansus spp. and Mononchus spp. were collect- ed in soil and litter samples from habitats with various vegetation types along 12 rivers (Arda, Danube, Devinska, Dyavolska, Grafska, Lopushnitsa, Maritsa, Rezovska, Shirokoleshka, Trigradska, Vedena, and Veleka) in eight provinces in Bulgaria (Burgas, Kardzhali, Lovech, Montana, Plovdiv, Silistra, Smolyan, and Sofia). In each locality, the nematode populations were recovered around the roots of the dominant tree species (predominantly Salix spp., but also Alnus glutinosa (L.), Carpinus betulus L., Fagus sylvatica L., Fraxinus excelsior L., Pop- ulus sp., Ulmus laevis Pall., and Ulmus sp.) (Table 1). Four species were identified based on morphological data: C. parvus (4 pop- ulations), M. truncatus (7 populations), Mononchus pseudoaquaticus sp. nov. (5 populations), and Mononchus sp. (1 population). The geographical distribution of Mononchus spp. (9 localities) did not overlap that of the single species of Coomansus recovered during the study (4 localities) (Table 1). Of note, pop- ulations of the two widespread species of Mononchus, M. truncatus and M. pseudoaquaticus sp. nov., co-occurred in four localities (along riverbanks of the rivers Danube, Maritsa, Shirokoleshka, and Veleka). Although an attempt was made to obtain representative 28S rDNA sequenc- es for all species populations, the success rate was generally low. A total of nine sequences were generated, four for C. parvus (1017-1037 bp), three for M. truncatus (987-1041 bp), and two for M. pseudoaquaticus sp. nov. (910- 1042 bp). Four of the sequenced populations were selected for generating rep- resentative 18S rDNA sequences (1630-1682 bp; 2 for C parvus, 1 for M. trun- catus, and 1 for M. pseudoaquaticus sp. nov.). No sequences were generated for Mononchus sp. The newly generated 28S rDNA sequences showed very low intraspecific genetic divergence (0-2 nt positions, i.e., 0.2% for sequences for C. parvus and M. truncatus, and identical sequences for M. pseudoaquaticus sp. nov.); the two 18S rDNA sequences for C. parvus were also identical. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 141 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 1. Summary data for the populations of Coomansus parvus and Mononchus spp. studied in 17 riparian habitats in Bulgaria. Species River Locality Coordinates Tee, pa A Date (Collector) Coomansus parvus Lopushnitsa Near Kaleytsa, 42°55'34"N, | ~ 440 Acer sp. (litter) 9.05.2021 (VP) (de Man, 1880) (Balkan Mountains) | Lovech Province 24°38'38"E Arda (Rhodope Dyavolski Most, 41°37'14'N, | ~ 460 Ulmus sp. (soil) 29.08.2020 (VP) Mountains) Kardzhali Province | 25°06'53"E 41°37'22"N, | ~440 | Populus sp. (soil) 25°06'54"E Vedena (Vitosha Near Zheleznitsa, | 42°32'05"N, | ~ 1200 | Fagus sylvatica L. | 4.04.2022 (SA, VP) Mountain) Sofia Province 23-20 5/2E (litter) Devinska (Rhodope Near Devin, 41°45'21"N, | ~ 880 Carpinus betulus | 20.05.2019 (SA) Mountains) Smolyan Province | 24°20'02"E L. (soil) Mononchus truncatus Shirokoleshka Shiroka Laka, 41°40'26"N, | ~ 1120 Salix sp. (soil) 23.05.2019 (SA) Bastian, 1865 (Rhodope Smolyan Province | 24°35'51"E Mountains) Maritsa (Upper Near Plovdiv, 42°09'N, ~ 153 Salix sp. (soil) 18.10.1995 (VP) Thracian Plain) Plovdiv Province 25°50'E Trigradska Teshel, Smolyan | 41°40'18"N, | ~ 860 Salix sp. (litter) 23.05.2019 (SA) (Rhodope Province 24°21'13'"E Mountains) Dyavolska Near Primorsko, 42°15'34'"N, ~ 10 Fraxinus excelsior 6.06.2019 (SA) (Strandzha Burgas Province 27°44'18"E L. (soil) Mountains) Rezovska Slivarovo, Burgas A157 N, ~240 | Ulmus laevis Pall. | 22.10.2008 (RS) (Strandzha Province 27°40'E (soil) Mountains) Danube (Southern Vetren, Silistra 44°08'24'N, ~ 20 Salix sp. (soil) 5.07.2021 (VP) Dobruja) Province 27°01'47'E Veleka Brodilovo, Burgas | 42°04'53'N, ~15 Alnus glutinosa 4.06.2019 (SA) Province 27°51:33' E (L.) (soil) Mononchus Shirokoleshka Shiroka Laka, 41°40'26"N, | ~ 1120 Salix sp. (soil) 23.05.2019 (SA) pseudoaquaticus sp. (Rhodope Smolyan Province | 24°35'51"E nov. Mountains) Maritsa (Upper Near Plovdiv, 42°09'N, ~ 153 Salix sp. (soil) 18.10.1995 (VP) Thracian Plain) Plovdiv Province 25°50'E Veleka Brodilovo, Burgas | 42°04'53'N, ~15 Alnus glutinosa 4.06.2019 (SA) Province 272833: E (L.) (soil) Danube (Southern Vetren, Silistra 44°08'24'N, ~ 20 Salix sp. (soil)® 5.07.2021 (VP) Dobruja) Province 27°01'47'"E Danube (Southern Komluka Island, | 44°08'03"N, ~ 20 Populus sp. (soil) 5.07.2021 (VP) Dobruja) Silistra Province 27°03'40"E Mononchus sp. Grafska, inflow of | Waterfall “Durshin | 43°19'40"N, | ~ 1048 | Fagus sylvatica. | 27.07.2000 (VP) River Kopilovtsi skok”, near 2-51-01 E (soil) (Balkan Mountains) | Kopilovtsi, Montana Province Abbreviations: RS, Rabia Soufi; SD, Stela Altash; VP, Vlada Peneva. @ Metres above sea level. > Type-population. Taxonomy Genus Coomansus Jairajpuri & Khan, 1977 Coomansus parvus (de Man, 1880) Jairajpuri & Khan, 1977 Figs 1, 2 Description. Female [Based on 10 specimens from 3 localities; see Table 2 for measurements]. Body short, 0.70-1.15 mm, J- or C-shaped upon relaxation, ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 142 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria body diameter at mid-body 47-53. Cuticle smooth under light microscope (very faint striation observed in one specimen, Fig. 2H), 2-3 thick along most of body, 3-4 thick in post-anal region. Lip region offset, cephalic and labial papil- lae prominent, conical, of almost same size. Amphid apertures oval, 5 + 0.4 (4-5) (n = 6) wide, situated anterior to dorsal tooth apex, at 10-14 from anterior end. Buccal capsule oval, somewhat flattened at base, 1.6—1.9 as long as wide or 0.9-1.2 times as long as lip region width; its ventral wall 1.5—2.5 thick, dorsal wall posterior to dorsal tooth ~ 3 thick. Dorsal tooth small, its anterior margin 3.0 + 0.5 (2-4) wide, located near middle of buccal capsule, tooth apex at 9 + 1 (8-11) from anterior end of buccal capsule. Nerve-ring at 97 + 6 (90-103) (n = 6) from anterior end of body. Excretory pore posterior to nerve-ring, small, well visible. Reproductive system amphidelphic. Genital branches almost sym- metrical; anterior branch 134 + 50 (80-253) (n = 9) long; posterior branch 115 + 19 (80-133) (n = 9) long. Ovaries well developed; anterior ovary 60-105 (n = 7) long; posterior ovary 70-140 (n = 7) long. Oviduct with marked pars dilatata oviductus, ~ 30 wide. Uteri very short. Two uterine eggs present in one female measuring 81 x 40 and 90 x 38. Vagina with straight walls, its length represent- ing 25-33% of corresponding body width; pars refringens vaginae as 2 oval to drop-shaped smooth sclerotised pieces, 3-4 long and 2-3 wide; pars distalis vaginae ~ 3 long. Vulva a transverse slit; pars refringens vaginae protruding in some specimens (Fig. 21). Rectum 0.7-0.8 times as long as body diameter at anus. Tail conoid, ventrally arcuate, with finely rounded tip. Caudal glands and spinneret absent. Caudal pores two pairs. Male: Not found. Voucher material. Ten specimens are deposited in the Nematological Col- lection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Bulgaria, under the accession numbers IBER-BAS NC 49/1, IBER-BAS NC 51/2, IBER-BAS PN 68/4 litter, IBER-BAS NC 88/4, IBER-BAS NC 88/7-9. Photovouchers for the sequenced specimens are provided in Suppl. material 1: figs S1-S3. Habitats and localities. Soil around Ulmus sp., Populus sp., and C. betulus and litter around F. sylvatica and Acer sp. along riverbanks of the rivers Arda, Lopushnitsa, Vedena, and Devinska (see Table 1 for details). Representative DNA sequences. 28S rRNA gene (GenBank: PP768895- PP768898); 18S rRNA gene (GenBank: PP768899 and PP768900). Distribution. Almost cosmopolitan, except in Australia (Andrassy 2009). In Bulgaria, C. parvus has been reported with no morphological evidence support- ing identification in soil samples from an oak forest in Burgas Province (Aleks- iev et al. 1998), from beech forests in Strandzha Mountain (Strandzha Nature Park, protected zones “Bjalata prust” and “Propada’; lliev and Ilieva 2014), and from arable lands in Sofia (Katalan-Gateva 1968) and Kazanlak provinces (Kat- alan-Gateva et al. 1981). Iliev and Ilieva (2016) described and illustrated C. par- vus based on a large population of females in soil samples from one habitat in the Rhodope Mountains. The present study provides the second documented record of C. parvus in Bulgaria, the first record of this species in litter samples, and four new localities in three provinces (Tables 1, 2). Remarks. Morphologically, the present material belongs to and was identified as C. parvus. Some variation was detected in the present material with single specimens from three populations sampled in the Rhodope and Balkan Moun- tains showing lower values for L, a, G7, G2, the length of the genital branches, ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 143 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 2. Morphometric data for females of Coomansus parvus collected in four riparian localities in Bulgaria. Locality River Habitat n L (mm) a b Cc c’ V (%) G7 (%) G2 (%) Buccal capsule length Buccal capsule width Tooth apex from anterior end of buccal capsule Position of tooth apex (%)* Excretory pore from anterior end Nerve-ring from anterior end Pharynx length Lip region height Lip region width Amphid from anterior end Maximum body diameter Body diameter at pharynx base Body diameter at mid-body Body diameter at vagina Body diameter at anus Anterior genital branch length Posterior genital branch length Anterior ovary length Posterior ovary length Vagina length Rectum length Tail length Near Kaleytsa, Lovech Dyavolski Most, Kardzhali Province Near Zheleznitsa, Sofia Province Province Lopushnitsa (Balkan Arda (Rhodope Mountains) Vedena (Vitosha Mountain) Mountains) Acer sp. (litter) Ulmus sp. (soil) Populus sp. (soil) Fagus sylvatica (litter) (n = 1) (n= 1) (n = 2) (n = 6) 1.05 0.70 0.90, 1.07 0.96 + 0.14 (0.83-1.15) 18.7 12.9 a We 19.0 + 2.5 (16.2-21.7) 3.6 3.7 3.5, 3.5 3.2, 3.4, 3.5 (n = 3) 14.8 ey 12.2, 12.9 27 e115 = 1450) eo 2a 2.4, 2.3 2.3 4: 053(2.0=2,7) 62.0 59.9 61.3, 62.2 62.5 + 1.6 (59.6-64.5) 9.2 11.4 12.2, 10.5 13.8 + 2.4 (12.0-17.3) (n= 5) 12.6 11.3 10.2, 11.4 12.6 + 1.5 (11.6-15.1) (n=5) 24 pas 23,26 26-#'1 (25-27) 14 14 14,15 15+0.4 (14-15) 9 11 8,8 9+ 0.4 (9-10) 38 = 36;-32 354 2:(33-88) 116 = TOSS 117 + 15 (97-131) 92 - 90, - 100 + 4 (94-103) (n = 4) 294 189 258, 302 303, 324, 324 (n = 3) 8 7 8,8 8 +1 (7-10) (n= 4) 25 23 25,25 24 + 3 (22-28) (n = 4) 11 14 14,11 10 (n = 2) 56 54 Soi 5022 (48-53) 49 49 51, - A6 + 3 (43-50) (n = 5) 49 53 51, - 50 + 2 (48-53) 56 54 do 50 + 2 (48-53) Sl 28 31, 36 33 #2 (311=36) 96 80 110, 112 161 + 53 (118-253) (n = 5) 132 80 92, 122 122 + 11 (106-133) (n = 5) 90 70 60, 80 100, 105, 105 105 75 70, 100 86, 110, 140 17 17 16, 18 14 + 2 (12-16) (n =5) 23 21 24; 26 PSH? (Z=25) 71 60 74; 83 75 + 8 (66-85) 2 Distance from tooth apex to anterior end of buccal capsule as % of buccal capsule length from its anterior ovaries, and tail, and greater values for the distance of the amphid from anterior end compared with the population from Vitosha Mountain (Table 2). We consid- er these small metrical differences to represent intraspecific variation; this was confirmed by the very low levels of genetic divergence (see above). The morphometric data for the present material fall within the range given by Andrassy (2011b), except for the slightly greater values for the width of the buc- cal capsule (14-15 vs 10-12 um). Comparisons with published descriptions ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 144 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 1. Line drawings of Coomansus parvus (de Man, 1880) Jairajpuri & Khan, 1977. Female specimens from popula- tions collected from riverbanks of the rivers Vedena (A, B, D) and Arda (C): A anterior region B posterior genital branch C, D tail end. Scale bar: 25 um. of C. parvus revealed an overlap with the morphometric data of the present material but also a greater variation with higher upper limits of variation for the published ranges of body length (Zullini et al. 2002; Anmad and Jairajpuri 2010; Ishaque et al. 2022) and most of the indices, and lower ranges for the width of the buccal capsule in four populations falling below the range recorded in the present specimens (Suppl. material 2: table S1). It is worth noting that there was an overall good agreement with the descriptions and morphometric data for a population of C. parvus collected in Bulgaria by lliev and Ilieva (2016) and especially with a population used for generating 18S rDNA and 28S rDNA se- quences for this species described by Tabolin and Kolganova (2020). However, the material described by Ishaque et al. (2022) showed little over- lap with the published descriptions and the present material, with ranges for a number of characters falling outside the known ranges for C. parvus: outside the upper limits of variation (L, V, buccal capsule length and width, lip region width, and rectum length); and outside the lower limits of variation (G7, G2, and position of tooth apex) (Suppl. material 2: table S1). This material keys down to C. indicus Jairajpuri & Khan, 1982 in the key to species of Coomansus by Andrassy (1993, 2009) and to C. ulsani Choi, Khan & Lee, 1999 in the key by Vu (2021) but does not agree completely with the data for these species. Clearly, the material of Ishaque et al. (2022) does not belong to C. parvus but definite identification is not possible based on the available data and illustrations (also see comments in Suppl. material 2: table $1). ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 145 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 2. Photomicrographs of Coomansus parvus (de Man, 1880) Jairajpuri & Khan, 1977. Female specimens from populations collected from riverbanks of the rivers Lopushnitsa (A, B, E-G, M), Vedena (C, D, H, I, K, L), and Arda (J): A entire body B-D, G anterior region (amphid opening arrowed in G) E, F, | reproductive system (E anterior genital branch F, | vulval region showing pars refringens vaginae) H, J—M tail (cuticle striation arrowed in H; caudal pores arrowed in J and K). Scale bars: 200 um (A); 20 um (B-D, F-I, M); 50 um (E, J-L). Genus Mononchus Bastian, 1865 Mononchus pseudoaquaticus sp. nov. https://zoobank.org/DBD4723B-BBB9-4F7D-BB79-4DDE8CECEC16 Figs 3-7 Mononchus aquaticus sensu Lazarova et al. (2004) (Syn.) Mononchus sp. 1 sensu Mejia-Madrid (2018) (Syn.) ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 146 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Description. Female [Based on 4 specimens from the type-population and 8 voucher specimens from other populations; see Table 3 for measurements.] Body slender (a = 20.2-33.6), almost straight; body diameter at mid-body 44-71. Cuticle smooth under light microscope, 2-2.5 thick along body, 3-3.5 thick in post-anal region. Lip region rounded, almost continuous with adjoining body, 2.4—3.7 as wide as high; papillae small, conical; cephalic papillae some- what larger than labial. Body at posterior end of pharynx 1.8-2.5 times as wide as body width at lip region. Amphids caliciform, with oval apertures, 4 + 0.5 (3.5-5.0; n = 10), at 8-12 from anterior end; amphid position varying from little anterior to tooth apex to level of anterior end of buccal capsule. Buccal capsule elongate-oval, slightly flattened at base, about twice as long as wide (1.8-2.0; n = 10), 1.2-1.3 times as long as the labial diameter; its ventral wall 2-3 thick, dorsal wall posterior to dorsal tooth 3-4 thick. Dorsal tooth strong, its ante- rior margin 4 + 0.5 (3-5) wide, located at 6 + 0.4 (5-6.5) from anterior end of buccal capsule, its anterior margin perpendicular to vertical plane. Buccal capsule with short transverse ridge, small tooth-like projection visible in some specimens in sublateral position (n = 2). Ventro-sublateral transverse ribs of buccal capsule weak, situated just posterior to tooth apex. Nerve-ring at 108 + 8 (96-125) from anterior end of body. Excretory pore small, not well visible, at level of posterior margin of nerve-ring. Reproductive system amphidelphic. Anterior genital branch 171 + 35 (116-226) long, posterior genital branch 166 + 32 (120-205) long. Ovaries well developed, anterior ovary 105 + 39 (65-125; n= 11) long, posterior ovary 106 + 26 (70-135; n = 11) long. Oviduct with well- marked pars dilatata oviductus, 20-30 wide. Uterus a short tube with thick walls, 25-35 long. Vagina slightly swollen, with straight walls, its length repre- senting 28-38% of corresponding body width; pars refringens vaginae as two smooth rhomb-shaped sclerotised pieces 3-6 long and 2-3 wide. Two females were recovered possessing a single large, thin-shelled uterine egg measuring 86-94 x 37-46 (specimens from River Maritsa and Komluka Island). Vulva a transverse slit. Vulva-anus distance equals 2.9-4.2 tail lengths. Tail long, slen- der, initially conoid, then almost cylindrical (10-13 wide) and slightly swollen at the tip, slightly curved ventrally in the third part; tail length represents 10-14% of body length. Caudal glands moderately developed, arranged in group. Tail tip rounded, with terminal spinneret and one small papilla. One female with abnor- mal tail, very short and almost straight. Male. Not found. Type habitat and locality. Soil around Salix sp. along River Danube at Vetren, Silistra Province, North Bulgaria (44°08'24"N, 27°01'47'"E; elevation 20 m a.s.I.) Other localities. Komluka Island (River Danube), rivers Veleka, Shirokolesh- ka, and Maritsa (see Table 1 for details). Type material. The holotype female and one paratype female are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Re- search, Bulgarian Academy of Sciences, Bulgaria, under the accession numbers IBER-BAS NTC 105 and 106. One paratype female is deposited in the Wagen- ingen Nematode Collection (WANECO), Wageningen, the Netherlands (WANE- CO accession number WT 4037), and one paratype female is deposited in the Nematode Collection of the U.S. Department of Agriculture (USDA), Beltsville, Maryland, USA (USDA accession number T-8065p). Voucher material. Eight voucher specimens are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 147 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 3. Morphometric data for females of Mononchus pseudoaquaticus sp. nov. collected in five riparian localities in Bulgaria. Locality River Habitat n L (mm) V (%) G7 (%) G2 (%) Buccal capsule length Buccal capsule width Tooth apex from anterior end of buccal capsule Position of tooth apex (%)° Excretory pore from anterior end Nerve-ring from anterior end Pharynx length Lip region height Lip region width Amphid from anterior end Body diameter at pharynx base Maximum body diameter Body diameter at mid-body Body diameter at vagina Body diameter at anus Anterior genital branch length Vetren, Silistra Province Danube (Southern Dobruja) Holotype 1.45 20.2 4.0 PS 5.0 48.3 12,9 13.3 29 16 118 96 62 72 71 72 39 187 Salix sp. (soil) Paratypes (n = 3) 152, 1260, 238 28.7, 32.0, 28.0 4.5, 4.5, 4.0 -, 8.4, 7.2 =oiee Der 50.7, 49.7, 53.9 9.9, 9.7,9.4 13.3, 10.3;9.9 31,5129 16; 1'6,1:5 2,020 21419 18 2 AZ Ay 2: 102, 106, 99 342, 359, 305 10, 8, 8 24, 24, 23 3 at DP: 49, 50, 43 53, 50, 44 93, 49, 44 50, 50, 44 34, 33, 30 1515, 155;, 11:6 Komluka Island Danube Populus sp. (soil) (n= 2) 1.72, 1.88 27.7, 33.6 4.6, 4.5 3.5, 951 9.3, 5.8 49.7, 50.0 12.8, 11.7 15-99 29533 15, 16 6, 7 19,20 108.125 371, 420 8, 10 29,20 62, 56 oy: esys 62, 56 38, 36 220, 220 ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Shiroka Laka, Smolyan Province Shirokoleshka (Rhodope Mountains) Salix sp. (soil) (n= 1) 1.61 30.9 4.4 8.9 5.3 50.7 9.1 8.7 32 16 20 126 114 146 Brodilovo, Burgas Province Veleka (Strandzha Mountains) Alnus glutinosa (soil) (n = 3) 1.60, 1.71, 1.69 21,5; 33.0; 26.0 4.4, 4.6, 4.7 7.8, 8.3, 8.0 5,1, 5.8,5.4 50.8, 50.3, 48.4 10.1, 8.0, 9.9 7,5;-4.0;.10,0 30, 30, 29 15; 16, 16 3,676 18, 20, 21 129, 131, 124 109, 111, 101 364, 372, 355 8,9, 8 24, 25, 23 10, 12, 11 92, 49, 52 98, 51, 59 98, 50, 59 98, 51, 56 40, 36, 39 162, 137, 167 Near Plovdiv, Plovdiv Province? Maritsa (Upper Thracian Plain) Salix sp. (soil) (n = 2) 1.81, 1.50 28.3, 29.4 4.6, 4.5 10,2;= 4.7, - 48.8, 50.9 12:5,.1153 11.3; 71,1 31, 30 1,.= 6, 6 19, 20 153, 107 117,110 392,335 9,8 26, 24 96, 47 64, 51 63, 51 64, 50 38, 33 226, 170 148 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Locality Vetren, Silistra Province Komluka Island Shitoke Cake ‘ Brodiove, NeAe Evel Smolyan Province Burgas Province Plovdiv Province® Shirokoleshka Veleka Maritsa (Upper River Danube (Southern Dobruja) Danube (Rhodope (Strandzha ae hee . 7 Thracian Plain) Mountains) Mountains) Habitat Salix sp. (soil) Populus sp. (soil) Salix sp. (soil) one ain Salix sp. (soil) n Holotype | Paratypes (n = 3) (n= 2) (n= 1) (n = 3) (n = 2) Posterior 194 203, 165, 122 197, 186 140 120, 128, 168 205, 167 genital branch length Anterior ovary 124 94, 65, - 193, 140 85 70, 86, 79 135, 7:7 length Posterior ovary 135 109,,95;,.= 1337 135 82 70:85, 71 TSOpT17 length Vagina length 20 195-18;15 = 1.6 17 19, 18, 16 125 = Rectum length 26 28,3129 28, 30 26 28, 26, 28 29, 28 Tail length 195 Sal he rey 201) ;207 180 204, 207, 210 ee Ae @ Material reported as M. aquaticus by Lazarova et al. (2004). ’ Distance from tooth apex to anterior end of buccal capsule as % of buccal capsule length from its anterior end. Academy of Sciences, Bulgaria, under the accession numbers IBER-BAS NC 5/2, IBER-BAS NC 18/3, IBER-BAS NC 16/6, IBER-BAS NC 18/5, IBER-BAS NC 78/1, IBER-BAS NC 80/1. Photovouchers for the sequenced specimens are pro- vided in Suppl. material 1: fig. S4. Representative DNA sequences. 28S rRNA gene (GenBank: PP768893 and PP768894); 18S rRNA gene (PP768902). Etymology. The species is named Mononchus pseudoaquaticus because of its similarity with M. aquaticus, hence the prefix pseudo- meaning false. Differential diagnosis and relationships. Females of M. pseudoaquaticus sp. nov. are characterised and distinguished from the congeners by a combination of features: a medium-sized body (1.23-1.88 mm); an elongate-oval, slightly flattened at the base buccal capsule measuring 29-33 x 15-16 um, 1.8-2.0 as long as wide and distinctly shorter than 2 labial diameters (1.2-1.3 times as long as the labial diameter); amphid openings located from slightly anterior to dorsal tooth apex to level of anterior end of buccal capsule; a strong dorsal tooth situated at 18-21% of buccal capsule length from its anterior end, its anterior margin being perpendicular to the vertical plane; subventral transverse ribs located just posterior to dorsal tooth apex; didelphic (amphidelphic) repro- ductive system with pars refringens vaginae distinctly sclerotised in the form of two smooth rhomb-shaped pieces; tail (171-210 um long, c = 7.2-10.2, c’ = 4.7—5.8) slightly curved at its posterior third, spinneret terminal. Morphologically, Mononchus pseudoaquaticus sp. nov. appears most similar to M. aquaticus, M. pulcher Andrassy, 1993, and M. caudatus Shah & Hussain, 2016. However, M. aquaticus likely represents a composite species (see also Baqri and Jairajpuri 1972) based on the wide ranges of morphometric varia- tion reported in the literature (see comparative data in Suppl. material 2: table S2). However, it is not possible to revise the identification of these materials because in many cases the findings are not documented properly and import- ant characters such as vaginal characteristics (the shape of pars refringens vaginae in particular), buccal capsule shape and length/width ratio, etc., are not described, and the voucher material is inaccessible. Therefore, the species con- ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 149 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 3. Line drawings of Mononchus pseudoaquaticus sp. nov. Holotype female (A, C, D) and paratype specimens (B, E, F): A, B anterior region C posterior genital branch D tail region E, F tail tip. Scale bar: 25 um. cept for M. aquaticus (sensu stricto) used in the present comparisons is based on the original description of Coetzee (1968) and the data by Baqri and Jaira- jpuri (1972) who re-examined and provided metrical data for some paratypes of M. aquaticus. This concept was also applied by Andrassy (2011a) in the most ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 150 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria f’) oe 4 * “ Ps —_ ati — =~ Figure 4. Line drawings of Mononchus pseudoaquaticus sp. nov. Paratype females from populations collected from riverbanks of the rivers Shirokoleshka (A, H), Maritsa (B, F), Veleka (E, G) and Danube (C, D): A-E anterior region F vulval region G anterior genital branch H vulval region and posterior genital branch. Scale bar: 25 um. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 151 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 5. Line drawings of the tail region in females of Mononchus pseudoaquaticus sp. nov. from populations collected in Komluka Island (A) and riverbanks of the rivers Veleka (B), Maritsa (C) and Danube (D). Scale bar: 25 um. recent key to the species of Mononchus (see Suppl. material 2: table S3 for de- tails) and in the updated key to the species of Mononchus provided here. The present material differs from the type material of M. aquaticus (Coetzee 1968; Bagri and Jairajpuri 1972) by having: a smaller buccal capsule length/width ratio (1.8-2.0 vs 2.2-2.5); a different shape of the base of the buccal capsule (flattened vs tapering); a different direction of the anterior margin of dorsal tooth (perpendicular to the vertical plane vs oblique); a different shape of the vaginal sclerotised pieces (pars refringens vaginae) (rhomb-shaped vs drop-shaped); and a longer tail (171-210 vs 94-156 um (mean 150 um) (Suppl. material 2: table S3). The new species differs from M. caudatus by having: a different buccal capsule length/width ratio (1.8-2.0 vs 2.0-2.5); lower a value (20.2-33.6 vs 34-38); more anteriorly situated nerve-ring (96-125 vs 125-134 um); different arrangement of the caudal glands (in a group vs in tandem); and shorter rectum (26-31 vs 32-36 um) and vagina (16-20 vs 27-29 um) (Shah and Hussain 2016; Suppl. material 2: table S3). Differentiation from M. pulcher is more complicated because the original de- scription of Andrassy (1993) is based on two, geographically largely separated populations from Chile and Hungary. However, Andrassy’s (1993: fig. 2) illustra- tions indicate that he probably dealt with different species. Unfortunately, it is im- possible to separate the rather incomplete metrical data since Andrassy (1993) provided pooled data for the position of dorsal tooth apex, the length of pharynx, the width of the lip region, the body diameter at mid-body, and tail length (Sup- pl. material 2: table S3). Still, in addition to the morphological differences (e.g., different shape of the buccal capsule and direction of the dorsal tooth), the Hun- garian population is characterised by having a smaller buccal capsule length ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 152 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 6. Photomicrographs of Mononchus pseudoaquaticus sp. nov. Holotype (A-C, E, F, H, I, L) and paratype (D, G, J, K) females: A body, total view B-E anterior region (transverse ridge arrowed in C; amphid opening arrowed in E) F, G vulval region showing pars refringens vaginae and posterior genital branch (F) H—-J tail (caudal glands arrowed in J) K, L tail tip showing one small papilla (arrowed in K) and terminal spinneret (L). Scale bars: 400 um (A); 20 um (B-E, G, J, K, L); 30 um (F, I, H). (and hence length/width ratio) and an overall smaller body length/tail length ratio (c). The size of the buccal capsule is a feature that varies in rather narrow ranges for a given population/species and is one of the most important differ- entiating characters for all mononchids. These data indicate that the Hungarian population may represent another species. However, it is impossible to identify this material given the scant data provided in Andrassy (2011a). Therefore, our ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 153 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 7. Photomicrographs of Mononchus pseudoaquaticus sp. nov. Females from populations collected from river- banks of the rivers Veleka (A, G, H), Danube (B, D, E, I, J), Shirokoleshka (C, L) and Maritsa (F, K): A—D anterior region (transverse ridge arrowed in D) E-G vulval region showing an egg (E) vulval opening, subventral view (F) and pars refrin- gens vaginae (G) H, | tail J caudal glands (arrowed) K caudal pores (arrowed) L tail tip. Scale bars: 20 um (A-G, K); 30 um (J, L); 50 um (H, 1). ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria comparisons are based on the morphology and metrical data for the type-pop- ulation of M. pulcher from Chile. The new species differs from M. pulcher (sen- su stricto) by having: a shorter (29-33 vs 35-38 um) and narrower (15-16 vs 16-18 um) buccal capsule; lower values for a (20-34 vs 35-39): anterior mar- gin of the dorsal tooth perpendicular to the vertical plane vs oblique, vagina not spotted in its anterior part vs spotted, rhomb-shaped pars refringens vaginae vs drop-shaped; and smaller egg length (86-94 vs 98-100 um). Additionally, the upper ranges for body length and tail length are greater in both populations of M. pulcher (Suppl. material 2: table S3). Mononchus truncatus Bastian, 1865 Figs 8,9 Description. Female [Based on 14 specimens from 6 localities; see Table 4 for measurements.] Body of most specimens straight, with only last part of tail ven- trally curved (body C-shaped upon fixation in a few specimens), comparatively slender, body diameter at mid-body 53-71. Cuticle smooth under light micro- scope, 2-3 thick along most of body, thicker (4—5) in post-anal region. Lip region rounded, continuous with adjoining body, papillae small, cephalic papillae very small and rounded, labial papillae somewhat larger and conical. Body at poste- rior end of pharynx 1.2-1.4 times as wide as body width at lip region. Amphids with oval apertures, situated at the beginning or middle of buccal capsule, at 11 +1 (10-13) (n = 12) from anterior end and 40 + 3 (37-44) (n = 12) from poste- rior end of buccal capsule, aperture 4.5 + 0.5 (4-5) (n = 12) wide. Buccal capsule oval, tapering at base, 2.0-2.3 as long as wide or 1.3-1.7 times as long as lip re- gion width; its ventral wall 2-3 thick, dorsal wall posterior to dorsal tooth ~ 3-5 thick. Dorsal tooth strong, its anterior margin 5 + 0.6 (4-6) (n = 12) wide, located at 11 + 0.5 (10-12) from anterior end of buccal capsule. Ventral wall with short, not so well visible rib, ventro-sublateral transverse ribs located at level of tooth apex or slightly more anterior. Nerve-ring at 127 + 8 (116-144) (n = 12) from an- terior end of body. Excretory pore weakly marked, posterior to nerve-ring. Repro- ductive system amphidelphic. Anterior genital branch 193 + 14 (175-223) long, posterior branch somewhat longer, 204 + 16 (187-240) long. Ovaries well devel- oped, not reaching uterus-oviduct junction; anterior ovary 107 + 17 (75-142) (n= 12) long, posterior ovary 114 + 18 (95-146) (n = 12) long. Oviduct with marked pars dilatata oviductus, 33 + 7 (20-45) wide. Uteri thick-walled tubes, 40-60 long, length ranges for anterior and posterior uterus almost identical. Vagina with straight walls, 28 + 3 in length representing 24-33% of corresponding body width; pars refringens vaginae as two rounded drop-shaped pieces with smooth surface, 3-5 long and 2-3 wide. Vulva transverse, not protruding. Vulva-anus distance equals 2.3-3.3 tail lengths. Tail long, slender, curved ventrally in sec- ond part, length representing 11-13% of total body length, 11-13 um wide at cylindrical part, with rounded and slightly swollen tip. Caudal glands moderately developed, arranged in group, spinneret terminal. Male. Not found. Voucher material. Ten specimens are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, under the accession numbers IBER-BAS NC 5/1, IBER-BAS NC 16/1-6, IBER-BAS NC 17/1, IBER-BAS NC 18/3, IBER-BAS NC 30/13, IBER-BAS NC 311/7-9. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 155 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 4. Morphometric data for females of Mononchus truncatus collected in six riparian localities in Bulgaria. Locality River Habitat n L (mm) V (%) G7 (%) G2 (%) Buccal capsule length Buccal capsule width Tooth apex from anterior end of buccal capsule Position of tooth apex (%)? Excretory pore from anterior end Nerve-ring from anterior end Pharynx length Lip region height Lip region width Amphid from base of buccal capsule Amphid from anterior end Maximum body diameter Body diameter at pharynx base Body diameter at mid-body Body diameter at vagina Body diameter at anus Anterior genital branch length Shiroka Laka, Smolyan Province Shirokoleshka (Rhodope Mountains) Salix sp. (soil) (n = 6) 1.94 + 1.08 (1.83-2.09) 30.6 + 2.5 (27-34) 4.1 + 0.2 (3.7-4.3) 8.3 + 0.2 (8.1-8.7) 5.5 + 0.4 (5.0-6.0) 55.2 + 1.3 (53.4-57.3) 10.1 + 0.6 (9.5-10.9) 10.4 + 0.6 (9.7-11.6) 43 + 2 (40-44) 20 + 1 (19-22) 1241 (11-12) 27 +1 (26-29) 148 + 16 (137-176) (n = 5) 126 + 10 (116-144) (n = 5) 479 + 34 (423-518) 9+1 (8-11) 29 + 1 (28-30) 40 + 3 (37-44) 12 +1 (10-13) (n = 4) 64 + 6 (55-70) 58 + 4 (51-61) 61 + 4 (53-65) 64 + 6 (55-70) 42 + 3 (38-47) 196 + 18 (175-223) Teshel, Smolyan Province Trigradska (Rhodope Mountains) Salix sp. (litter) (n= 1) 1.89 26.5 4.0 8.6 5s 54.0 9.6 11.4 44 21 11 Zo 150 468 181 ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Near Primorsko, Burgas Province Dyavolska (Strandzha Mountains) Fraxinus excelsior (soil) (n= 1) 2.06 38.9 3.9 8.2 6.1 53.9 10.0 11.6 44 21 11 25 206 Slivarovo, Burgas Province Rezovska (Strandzha Mountains) Ulmus laevis (soil) (n = 3) 1.77, 1.85, 1.84 32,15:33.7; 29:2 4.0, 4.2, 4.1 7.8, 8.0, 8.2 6.7, 6.3, 6.2 97,6, 53.15.5032 11.37 9:6,.10.3 11.8, 10.4, 10.2 42,42, 41 18, 19; 19 6 Pe 26, 27,27 138, 141, 142 138, 141, 142 439, 443, 446 10, 10, 9 26, 26, 25 =, 39, 39 =12,.10 55, 55, 63 51, 55; 58 95, 55, 63 53, 54, 60 34, 37, 36 199, 179, 189 Near Plovdiv, Plovdiv Province Maritsa (Upper Thracian Plain) Salix sp. (soil) (n= 1) 1.83 33.3 4.2 8.2 6.2 52.6 10.2 10.8 40 19 10 26 134 AZ 437 10 26 37 13 ie) 53 55 55 34 207 Vetren, Silistra Province Danube (Southern Dobruja) Salix sp. (soil) (n = 2) 189, 1:91 33.7, 32.4 4.0, 4.2 8:9; 9.3 9.0, 5.0 94.3, 53.8 9.7, 9.8 9.9101 42, 42 ates) Tes: 26, 27 156, 146 129, 1:29 468, 450 Orla LeeLo 41, 38 12,44 56, 59 53, 56 55,59 56, 59 42,41 182, 188 156 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Locality River Habitat n Posterior genital branch length Anterior ovary length Posterior ovary length Vagina length Rectum length Tail length Teshel meer Near Plovdiv, Shiroka Laka, Smolyan ‘ Primorsko, | Slivarovo, Burgas crak Vetren, Silistra : Smolyan : Plovdiv 3 Province Province Burgas Province Brovinies Province Province Shirokoleshka (Rhodope i ecsoeane ee ieee Maritsa (Upper sea Mountains) (Rhodope (Strandzha (Strandzha Thracian Plain) (Southern Mountains) Mountains) Mountains) Dobruja) . . Salix sp. Fraxinus Ulmus laevis : ; : ; Salix sp. (soil) (litter) excelsion{soil) (soil) Salix sp. (soil) | Salix sp. (soil) (n = 6) (n= 1) (n= 1) (n = 3) (n= 1) (n = 2) 203 + 13 (190-220) 215 240 208, 193, 187 215 187, 193 98 + 15 (75-115) (n= 5) 125 120 108,,917= 142 107, 107 106 + 16 (95-135) (n = 5) 125 146 100, 109, - 141 101, 120 144 15517) v7 14 9 layne 17 17,16 31 +1 (29-33) 36 2a. 29, 32, 31 28 S2) 30 234 + 11 (225-254) 218 252 227,232, 224 211 ZAZ, 209 @ Distance from tooth apex to anterior end of buccal capsule as % of buccal capsule length from its anterior end. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Photovouchers for the sequenced specimens are provided in Suppl. material 1: figs S5-S7. Habitats and localities. Soil around roots of F. excelsior, U. laevis, A. glutino- sa and Salix sp. and litter around Salix sp. along banks of the rivers Shirokolesh- ka, Trigradska, Dyavolska, Rezovska, Veleka, Maritsa, and Danube (see Table 1 for details). Representative DNA sequences. 28S rRNA gene (GenBank: PP768890- PP768892); 18S rRNA gene (PP768901). Distribution. According to the abundant published data for materials reported as M. truncatus, this species appears to exhibit a worldwide distribution. Howev- er, we agree with Andrassy (2011a) who doubted that all of the records referring to M. truncatus concern in fact this species. In Bulgaria, M. truncatus has been reported from many localities but with no morphological evidence supporting identification. Andrassy (1958) recorded this species for the first time in Rila Mountains and Varna. Subsequently, M. truncatus was reported from the North Thracian Plain (Katalan-Gateva 1962) and Pazardzhik Province (Katalan-Gateva 1965) associated with cultivated plants. In aquatic habitats, the species has been reported in sediments from 24 rivers and three lakes (Stoichev 1996; Stoichev and Chernev 2011; Stoichev and Varadinova 2011). The present study is the first to provide morphological and morphometric data for M. truncatus in Bulgaria. Remarks. Morphologically, the present material belongs to and was identified as M. truncatus. However, similar to the situation with M. aquaticus (sensu lato) considered above, M. truncatus also represents a composite species (Andrassy 2011a) based on the wide ranges of morphometric variation reported in the liter- ature (see Suppl. material 2: table S4). Andrassy (2011a) summarised the data from the original description and subsequent re-descriptions of M. truncatus and provided novel data for a population from Hungary. This concept of M. truncatus (sensu stricto) (“real M. truncatus” of Andrassy 2011a) is applied here. Compar- ative morphometric data for several records deviating from this species concept are also provided in Suppl. material 2: table S4. Typically, these include studies 157 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 8. Line drawings of Mononchus truncatus Bastian, 1865. Females from populations collected from riverbanks of the rivers Shirokoleshka (A, C, D) and Maritsa (B): A, B anterior region C anterior genital branch D tail region. Scale bar: 25 um. ZooKeys 1206: 1387-180 (2024), DOI: 10.3897/zookeys.1206.124237 158 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria eaters Figure 9. Photomicrographs of Mononchus truncatus Bastian, 1865. Females from populations collected from riverbanks of the rivers Rezovska (A, D, F, G, H, K), Danube (B, E, J, L) and Shirokoleshka (C, I, M, N): A body, total view B-F anterior region (ventro-sublateral ribs arrowed in D; amphid opening arrowed in E; transverse ridge arrowed in F) G, H, L vulval re- gion: G vulval opening, ventral view H vulva and part of posterior genital branch (posterior uterus and part of pars dilatata oviductus), lateral view L vulval region showing pars refringens vaginae I-K tail M, N tail tip showing papilla (arrowed in M) and terminal spinneret (N). Scale bars: 400 um (A); 20 um (B-H, L-N); 50 um (I-K). ZooKeys 1206: 137-180 (2024), DOI: 10.3897/2 6.124237 : Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria providing data (sometimes pooled, e.g., Nakazawa 1999; Eisendle 2008) for nem- atodes from different localities (e.g., Botha and Heyns 1992; Nakazawa 1999; Eisendle 2008; Farahmand et al. 2009). Thus, the data by Botha and Heyns (1992) show upper ranges above the upper range (b, c, and V) and lower ranges below the lower range of variation in M. truncatus (sensu stricto) (buccal capsule width, position of tooth apex, anterior end to pharyngo-intestinal valve, body diameter at mid-body and tail length). Almost all of these differences were recorded in a sin- gle sample (Crocodile River) likely containing a misidentified specimen. Similarly, both samples studied by Farahmand et al. (2009) contain specimens with largely deviating morphometric data (Suppl. material 2: table S4). We are also aware of two other questionable records of M. truncatus, not in- cluded in Suppl. material 2: table S4: Koohkan et al. (2014) reported as M. trun- catus nematodes of a population from Ghale Asgar, Kerman Province, Iran, that do not correspond to this species because all important morphometric charac- ters are outside the ranges of the “true” M. truncatus sensu Andrdassy (2011a). Probably this population represents a yet undescribed species as it cannot be identified using the available keys. Similarly, Rawat and Ahmad (2000) report- ed M. truncatus as a new geographical record for India and provided a brief description based on five females. However, the measurements of some key characters such as the length of the buccal capsule (37-38 vs 42-50 um), tail length (172-212 vs 232-283 um) are outside the ranges of the “true” M. trun- catus and the data provided are insufficient to identify the species. We consider that the records listed above are based on composite material. We agree with Andrassy (2011a) who considered the description by De Bruin and Heyns (1992) to represent the “real M. truncatus” and add to his list of re- liable records the data by Coomans et al. (1995). The present material agrees well with the characteristics of M. truncatus (sensu stricto) based on the orig- inal description, the description and data for the neotype population by Clark (1960), Coomans and Khan (1981), and Bagqri and Jairajpuri (1972), and the de- scription by Andrassy (2011a) except for the shorter tail (205 vs 240-283 ym) in a single specimen from Vetren (Table 4), resulting in a greater value for c (9.3 vs 5.8-8.6) (Suppl. material 2: tables S4, S5). Mononchus truncatus was first reported from Bulgaria by Andrassy (1958) who provided limited metrical data. However, the body length reported by this author is outside the range for M. truncatus; additionally, two largely differing measurements (22.3 and 43.3 pm) were given for the length of the buccal capsule in two females of similar size, suggesting that this report is based on more than one species. Mononchus sp. Figs 10, 11 Description. Female [Based on 2 females; see Table 5 for measurements]. Body slender, straight, with strongly ventrally curved tail; body diameter 42 at poste- rior end of buccal capsule and 45-52 at mid-body. Cuticle smooth under light microscope, 3-3.5 thick along body, thicker (4-5) around vulva and posterior to anus. Lip region rounded, continuous with adjoining body; papillae small, ce- phalic papillae round and somewhat more visible than labial. Body at posterior end of pharynx twice as wide as lip region. Amphids with oval apertures (5 wide), ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 160 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria located between dorsal tooth apex and anterior end of buccal capsule. Buccal capsule oblong, with flattened base, 2.3-2.4 as long as wide or 1.7—1.8 times as long as the labial diameter, its ventral wall around 3 thick, dorsal wall posterior to dorsal tooth 4 thick. Dorsal tooth robust, its anterior margin 4 wide, located at 10-11 from anterior end of buccal capsule. Ventral wall of buccal capsule with short, not well-visible rib, ttansverse ventro-sublateral ribs located at level of dor- sal tooth apex. Excretory pore weakly marked, posterior to nerve-ring. Reproduc- tive system amphidelphic, genital branches short. Ovaries well developed, not reaching uterus-oviduct junction. Oviduct with well-marked pars dilatata oviduc- tus, 25 wide. Uteri short, anterior uterus 30 long, posterior uterus 36 long (n = 1). Oviduct-uterus junction with moderately developed muscular sphincter. Vagina with straight walls and small spots next to pars refringens vaginae, length repre- senting 31% of corresponding body width; pars refringens vaginae as two round drop-shaped sclerotised pieces with smooth surface, 4 x 2 in size; pars distalis vaginae well visible, ~ 4 long. Vulval opening round (Fig. 11E), vulva not protrud- ing; vulva-anus distance equals 2.1 tail lengths. Tail long, cylindrical, strongly curved ventrad, length representing 16-18% of body length; cylindrical part of tail ~ 6 wide. Caudal glands moderately developed, arranged in tandem. Tail tip rounded, somewhat asymmetrical, dorsal part better developed, with terminal spinneret and one large setiform papilla. Three pairs of caudal pores present. Male. Not found. Voucher material. Two specimens are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, under the accession numbers IBER-BAS NC 316/1. Habitat and locality. Soil around roots of F sylvatica near a waterfall (River Grafska, inflow of River Kopilovtsi; see Table 1 for details). Remarks. Morphologically, the specimens resemble most Mononchus oblon- gus Andrassy, 2011 regarding the shape of the buccal capsule, the actual and relative length of the tail (as percent of body length), and the position of tooth apex (Table 5; Andrassy 2011a). However, the present specimens exhibit some differences in other morphometric features and proportions such as the total body length (1.31-1.62 vs 1.60-1.88 ym), the length of the buccal capsule (45-47 vs 48-51 um) and tail (230-263 vs 264-276 um), the width of the lip region (25-27 vs 22-23 um), and the ratios buccal capsule length/width (2.3- 2.4 vs 2.6-2.8), buccal capsule length/lip region width (1.7-1.8 vs 2.1—-2.3) and body at pharynx base/lip region width (1.9-2.0 vs 2.7-2.9). The present specimens also show similarities with M. truncatus and M. hi- malayensis Rawat & Ahmad, 2000. However, Mononchus sp. differs from M. truncatus in having a shorter body (1.31-1.62 vs 1.7-2.1 mm), a more anterior position of tooth apex (22-23 vs 25-29%), longer tail in relation to body length (16-18 vs 10-13%), smaller vulva-anus length/tail length ratio (2.1 vs 2.4-3.0), a lower c value (5.7—-6.1 vs 7.5-8.4) and a different shape of the vulva (round vs transverse) (Andrassy 2011a). Differences between Mononchus sp. and M. himalayensis include a shorter body (1.31-1.62 vs 1.6-1.9 mm), a more anteri- or position of tooth apex (22-23 vs 25-31%), lower a- and c-values (28-31 vs 33-38 and 7.7-7.9 vs 8.8-10.4, respectively) and absence of pre-vulval papilla (vs presence) (Rawat and Ahmad 2000). Probably the two females represent a species not yet described; however, additional specimens are needed to con- firm the identity of the Bulgarian population. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 161 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria * ee oa = —_ = ‘a — ——_a ee Figure 10. Line drawings of Mononchus sp. female: A anterior region B vulval region and posterior genital branch C vulval region D tail. Scale bar: 25 um. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 162 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Figure 11. Photomicrographs of Mononchus sp. females: A body, total view B-D anterior region (ventro-sublateral ribs arrowed in C; amphid opening arrowed in D) E vulval opening, subventral view F, G Pars refringens vaginae (small spots next to it arrowed in F) H reproductive system I tail tip J tail K sphincter of the oviduct-uterus junction (arrow). Scale bars: 400 um (A); 20 um (B-G, I, K); 50 um (H, J). ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 163 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 5. Morphometric data for females of Mononchus sp. and Mononchus oblongus. Species Source Locality River Habitat n L (mm) a b Cc e V (%) G7 (%) G2 (%) Buccal capsule length Buccal capsule width Tooth apex from anterior end of buccal capsule Position of tooth apex (%)* Excretory pore from anterior end Nerve-ring from anterior end Pharynx length Lip region height Lip region width Amphid from anterior end Maximum body diameter Body diameter at pharynx base Body diameter at mid-body Body diameter at vagina Body diameter at anus Anterior genital branch length Posterior genital branch length Anterior ovary length Posterior ovary length Vagina length Rectum length Tail length Mononchus sp. Present study Waterfall “Durshin skok”, near Kopilovtsi, Montana Province Grafska (Balkan Mountains) Fagus sylvatica (soil) (n = 2) 1.31, 162 27.9, 30.6 3.5, 4.1 5.7, 6:2. 7.9.97 51.2, 56.2 12,83 8.0, 9.2 A5, 47 19, 20 4 8 Fea 2273 12S; 039 WIAS23 370, 392 8,9 Zan 12,19 47,53 47,53 45, 52 46, 51 29, 34 94,135 105, 149 AO, 88 45,97 = 116 24,27 230, 263 M. oblongus Andrassy (2011a) Near Ossés, South of France na Liver moss (soil) (n = 6) 1.60-1.88 25-29 S037 6.0-7.1 75-838 52-54 7.6-9.4 7.6-9.4 48-51 18-19 10;0=1T1.5 21523 450-504 7-9 22-23 18321 264-276 @ Distance from tooth apex to anterior end of buccal capsule as % of buccal capsule length from its anterior end. > “Somewhat posterior to anterior end of buccal capsule.” (Andrassy, 2011a). Key to the species of Mononchus Since the last identification key to the species of the genus Mononchus was published by Andrassy (201 1a), eight additional species have been described and one species, M. intermedius Tahseen & Rajan, 2009, was not considered by Andrassy (2011a) (see Table 6 for the main morphometric characters of these species). The key by Andrassy (2011a) was, therefore, modified in or- der to accommodate all 31 species of the genus known to date, including ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 164 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria the new species described here. Examination of recent literature revealed that Mononchus caudatus Gagarin & Naumova, 2017 was preoccupied by Mononchus caudatus Shah & Hussain, 2016. Therefore, for the species de- scribed by Gagarin and Naumova (2017) we propose the replacement name Mononchus baikalensis (Gagarin & Naumova, 2017) nom. nov. after the type locality, Lake Baikal. 10 11 15 Largespecies, body:2.4—7.0' mmm long ort .4 haan oes etc Deedee ss toraraeses 2 Smaller species; body:0-°9-2-1 MM IONG i: -ceeze., cecseeeseneesteseeaes tte ctzeces. 13 Tail very short, about 2 anal body diameters Ong ...............::cccsseeeeeereeeeeees 3 Tail longer, (3—) 4-9 anal body diameters lONG.............:cccsccseeseceseeseeseeteees 6 Posterior third of tail digitate, ventrally curved ..... M. mulveyi Andrassy, 1985 Posterior third of tail not digitate, more or less straight...............cceceees 4 Buccal capsule 100-120 um long, nearly 3 times as long as wide ............ satire foe ce tM cael cet ae gadis tnd ac ga ROR ela ak genuine M. tajmiris Gagarin, 1991 Buccal capsule 50-90 um long, about twice as long as wide.................... 5 Buccal capsule 80-90 um long; spicule 300 Um IOng.......... ee eeeeeeeteeeees olde sad canicks nate sce CoE ead eC ceo M. angarensis Gagarin, 1984 Buccal capsule about 50 um long; spicule 120 um long............. ee eeeeeeeees SE A FP nee ret eet eR Merrett Pe er M. maduei Schneider, 1925 BOYS Oe cesta On sncetes ew cere eRe ee teen acetate ee trae Me cuees grace eee eee 7 BOC ip 2FA 3-76 MING LOI ee nak tt erect MID de cee cet NT) Seemed ceca 9 Body 5.0-6.4 mm long; tail as long as 5-6 (43 2.4) anal body diameters... bbe boeebe bb cecdestedbetbe deste dacsls sdutre ta PPPe RDI ER/DTELEIOLERIIIN: LEPTIN M. superbus Mulvey, 1978 Body 6.7—7.2 mm long; tail as long as 9 (34 4.6) anal body diameters .... Ca cced gan gy Mea saa edee yap fe Oh hee EER aaly peokesa ss M. amplus Gagarin & Naumova, 2017 Buecalcapsule >80 urm4Onig, sate ee Ae ee ee 10 BeCCal-Capsule4.6s 74 [Mt lOM G:F erecta ee Severe es anaes tan 11 Buccal capsule 80-84 um long; tail as long as 3-4 anal body diameters... Pebeiroe codeochuweuusesvogioed 1144 ether Mees actes M. agilis Gagarin & Mataphonov, 2004 Buccal capsule 105-112 um long; tail as long as 5 (3\¢ 2.9-3.5) anal body diameters............... M. baikalensis (Gagarin & Naumova, 2017) nom. nov. Dorsal tooth apex at up to 16% of buccal capsule length from its anterior end; tail as long as 3-6 anal body diamete®S.................ccsscccessstecessseeeeees 12 Dorsal tooth apex at 28-30% of buccal capsule length from its anterior end; tail as long as 8-9 anal body diametePS................ccccccessseeceesteeeeeneeeees SEI, 2 RS POAT IRD, es OTS PaO M. altiplanicus Andrassy, 2011 Body 2.8-3.5 mm long; buccal capsule 46-56 x 20-25 um; spicules rela- tivelysshort'(134=140) pM); escent eres M. niddensis Skwarra, 1921 Body 2.4-2.9 mm long; buccal capsule 65-74 x 28-31 um; spicules lon- ger(Z05=2 75: Lith) cncnccres dances M. minutus Naumova & Gagarin, 2018 Monodelphic species -.::.25 5, oat eis M. italicus Andrassy, 1959 DI GSIDIMNGS DOC ICS S ve ceerw rete tne totes tie Rey pe ecemerue tea cn spa Peces nant caer ope oeceeenesetes 14 Tail quite short (as long as 1.5—2 anal body diameters); spinneret subdor- oi: | Merce hee MG Le 5 Aes ee SE ee ee en ee be M. clarki Altherr, 1972 Tail as long as 3 anal body diameters or longer (c’ = up to 15); spinneret ET INVIIE sis0 ot werent Maan xszoell aa A Pe th sdeun's tecddahadtrcadaatte rp uat be hoc seetwes seahamyunes seatie ed 15 Buccal-capsule small 1S =234iM OMG occecdnnsuessncsactanarenarcsrenepsementecenuecctty 16 Buccalcapsulelargel 26250) MP lOnGe is aii es cers Anka igs cncomedeseaeaavencels 17 Zookeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 165 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 16 28 29 Buccal capsule very narrow (nearly 3 times as long as wide); dorsal tooth apex quite close to the anterior end of buccal capsule..................cccceeeees ek cache asta a nea nana Sea TSS aac Mea ERY Gag Ala Cale eae M. tunbridgensis Bastian, 1865 Buccal capsule wider (twice as long as wide); dorsal tooth apex at 28- 33% of buccal capsule length from its anterior @Nd ............ ce ecceeseeceeteeees Dera stercerrae A ctidsctee, dba rises acne: Batluclac sete Sirdar M. loofi Winiszewska, 1998 Tail as long as 7-15 (mostly 9-14) anal body diameters................000 18 Tail as long as 3-8 (mostly 4-7) anal body diameters ................:cccccee 20 Tail 340-390 um long, as long as 13-15 anal body diameters................... reels Sesylebsa cdl nacaweee mined’ slese cree haus vomnesstes M. syrmatus Andrassy, 2008 Tail 220-300 um long, as long as 8-11 anal body diameters................. 19 Buccal capsule 40-47 um long; one prevulval papilla present.................... Secaereune stag paar ad tree a tastaaremtvis cated M. himalayensis Rawat & Ahmad, 2000 Buccal capsule 28-35 um long; prevulval papilla absent ................ eee aisles ssAchlghs salou daa sates eroeaicsaatie ts Ako wsthcuanise ggatebiteedebasic’ M. sandur Eisendle, 2008 Pars refringens vaginae not sclerotised.... M. sinensis Soni & Nama, 1983 Pars refringens vaginae distinctly SclerotiSed ................ccccccceesseeeesseeeeees 21 Subventral transverse ribs located anteriorly to tooth apex.................005 22 Subventral transverse ribs located at level of or posterior to tooth apex23 Lip region relatively wide (24-28 um); cylindrical portion of tail 10-12 um WTEC Ree ee cet ae arch oles ee 2 I Oe cae 2 Wee Be M. truncatus Bastian, 1865 Lip region narrower (20 um); cylindrical portion of tail 5-7 um thick ......... Peres Ohare reer creates, des Air ee mF M. medius Andrassy, 2011 Amphid aperture posterior to dorsal toOth................ccccccccccsssececeesstsceeeeeseaaes Per ndicss seen nietetacths tak ties geee meme M. laminatus Zullini, Loof & Bongers, 2002 Amphid aperture anterior to dorsal toOth ..............ccccccccccccesseeeeeeesseeeeeeens 24 Tail as long as 3-4 anal body diameters.....................c::ccccsessceserteeesseeeees 25 Tail-as long.as‘4—7 anal Dody: diameters’ ...5:..:004....5. 25% of buccal capsule length from its anterior end Buccal capsule 36-44 um long, c = 8.7-10.6; tail 145-182 um long......... I iiepercleth pe adeh A ranatbie ee Boa Riexchnaee as esha M. intermedius Tahseen & Rajan, 2009 Buccal capsule 29-43 um long, c = 7-8; tail 193-231 um long.................. dudirvieee iateecbeeesleen'semagcheshs ere coicg deen eet ory pashan seat M. labiatus Shah & Hussain, 2016 ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 166 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 30 Buccal capsule (33)35-38 um long, vagina spotted in its anterior part ..... eR Pedi A icc Anh IRL she Bed os, PR ea att eed M. pulcher Andrassy, 1993 - Buccal capsule 29-33 um long, vagina not spotted............ ee eeereeees 31 31 Buccal capsule 1.8-2.0 times as long as wide, pars refringens vaginae KHONMS=SINADE Cae ite Sc cane sce et cc tencaee tate a, 2s M. pseudoaquaticus sp. nov. - Buccal capsule 2.2-2.5 times as long as wide, pars rrefringens vaginae GODS NAPS Ge ol ears seen aesnss htc Sacnenest tM stem eRe at SN ecma iW Wea teen chs 32 32 Body 1.2-1-7 mnv long, rectum: length 24-25 .um........Acsa..1.002etadeck. be sctaee Hoar Wate then Neen tenses Howe enethen, Alene dN M. aquaticus Coetzee, 1968 = "Bodys1-7-1.9 mimblong; rectum length 32-36 incest ad ceseeescccenstesaens ae Sereaee ryan ty Redesansecobmeerccten an mseathanies Baccenneen M. caudatus Shah & Hussain, 2016 Molecular phylogenies To assess the associations of the newly generated sequences (4 for C. parvus and 5 for Mononchus spp.) from the nematode populations sampled in Bulgaria, we carried out an exploratory neighbour-joining (NJ) analysis on an untrimmed 28S rDNA alignment (domains D1-D3), including representative sequences for Mononchus spp. (20 sequences) and Coomansus spp. (15 sequences). Using pairwise deletion of missing data allowed us to include more taxa and sequenc- es, e.g., several sequences of Schenk et al. (2017), including sequences for M. aquaticus, albeit with a short overlap (GenBank codes MF-XXX, D3-D5 region) (Fig. 12). The novel isolates of C. parvus formed a reciprocally monophyletic clade with C. parvus, C. batxatensis Vu, 2021 and Coomansus spp. with maxi- mum support; the clade of Coomansus spp. was recovered as sister to C. ger- lachei (de Man, 1904) Jairajpuri & Khan, 1977 (GenBank: KM092524) but with poor statistical support. The isolates of M. pseudoaquaticus sp. nov. clustered with maximum support with Mononchus sp. 1 sensu Mejia-Madrid (2018) within the strongly supported clade of Mononchus spp. comprising the novel and pub- lished isolates of M. aquaticus, M. truncatus, M. maduei, M. tunbridgensis, and Mononchus sp. sensu Schenk et al. (2017) to the exclusion of one isolate identi- fied as M. aquaticus (GenBank: MF125523; Schenk et al. 2017). This molecular prospecting analysis confirmed the identification of the novel isolates based on the detailed morphological analysis (see above) and indicated that Mononchus sp. 1 sensu Mejia-Madrid (2018) belongs to the new species described here. Next, we assessed the phylogenetic relationships of the novel isolates with representatives of the suborder Mononchina using two alignments. Upon trim- ming to the length of the shortest sequence, the 28S rDNA (domains D2-D3) alignment comprised a total of 779 nt positions and contained sequences for representatives of ten genera of the families Anatonchidae (Anatonchus Cobb, 1916, lotonchus Cobb, 1916, Jensenonchus Jairajpuri & Khan, 1982, Mulveyel- lus Siddiqi, 1984 and Parahadronchus Mulvey, 1978), Mononchidae (Cooman- sus, Mononchus, Parkellus and Prionchulus Cobb, 1916) and Mylonchulidae (Mylonchulus Jairajpuri, 1969). There were no sequence data for Miconchus spp. and Actus spp., and the available sequences for Clarkus papillatus (Bas- tian, 1865) Jairajpuri, 1970 (domains D3-D5) could not be used due to the very small overlap. Overall, the topology of the ML tree (Fig. 13) was well resolved ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 167 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria ‘(ZZ0Z) ‘Je 39 anbeys| ‘(8 Loz) uUeHeES pue erouwineN ‘(600Z) Ueley pue uaasye! ‘(9LOZ) uleSsnH pue YeUs ‘(7 LOZ) eAOWWNeN pue ulebey :saouasajay ‘pua JOUaj}Ue Ss}! Wold y}bua] ajnsdeo jeoong jo % se ajnsdeod jedonq Jo pusd JOIJa}Ue 0} XAde Y}OO} WOJJ BDUe}SIC e - - - GLZ-S0Z - - - - GEZ-02Z - GOL - ginoids - - - GZ-LZ - - - - ZE-LE - Ov - sjuatuajddns L0¢-LEL 00Z-96L aA ar ib 88L-Z9L 802-SZL LEZ-E6L C8L-SVL G6L-O6L O0E-SZZ SVE SCV G79-S6S yyGua| [lel e(%) xode y}00} LZ-8L 82-V~ O¢-6L a vL-6 GE-ZE O€-SZ LZ-8L O€-82 O€ Lo 6c-LZC $O UOIISOd yu} ajnsdeo E€-62 VE-EE GE-VE cL-09 vL-G9 Ev—-672 vy-9€ E€-OE ZLL-SOL OLL 08 8Z-02 jeoong YipIM 97-E7S LL-9L VE-EE 8E-VE Ov-SE G72-V~ 97-02 GC-V~ 09-VS OS €9 09-8S uolbad diy vS-8V GG-E€S L9-6S = L9-9G v9-9S GS-—OS LS-87 = ES = 6S (%) A 8S-LY 0°9-0'S CV-65 OE-97% VV-CE 0°'L-0°9 8'S-6€ 0°9-0'°S G'E-6'7C L’S 9” 68 no) COL=CL 06-08 OSlL-ECL | LOL-OVL | FSL=-8'oL 0'8=0'2 9OL-L'8 0'0L-0°6 OSL=CEL L6 COL OLL=o UL ?) 9v-OV 0S-O'V 66-95 SE-CE ESS 0'v-0'E LYV-8E 0'S-O'V VE-VE GE 0S 87 -LV q 9E-L?¢ co-Le LLE=7 82 E€-GTS E€-97 9€-O€ 8°82-G'0Z LE-VE 92-CG GG OS L9 -cS e 88° L-E7 LS QL L-69'L5 | EG'L-LG'LS | E8'T-VETP | 68°T-8ETS | OL L-LELS GO L-ZELS ZO L-ELLS | ZLE-GSEEL | GEES | 069P | veL-7L:96 (Www) 7 ‘Aou-ds 9LOZ ulessnH | 72202 ‘Ie 8 Loz ‘uuebe9 9LOz ‘ulessnH | 6007 ‘uefey | 9L0Z ‘UIessnH ' ‘Aou “wou (2 L0Z LLoz ‘evownen Z YyeUS ya anbeus| 3 YeUS 3 uvasyel RZ YyeUS eAoUNeN 7g ulebed) saisads snojenbeopnasd ‘jy 3 PAOWUNEN SN}NulW ‘| 3 UIIeHed snjdwe ‘;:w snjejuapold ‘zw | aezAio ‘Ww snjeige] ‘WW | SNipauuajul ‘yw | snjepneo ‘jw sisuajeyleg ‘W ‘saldads 0} Ady au} Ul papnyjou! pue L LOZ Jaye paquosap snyououopy jo saldads JeuONIppe aul aU} JO} eLep DUJaWOYdJoW Ule/\ “9 a|GeL 168 ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 100] PP768893 Mononchus pseudoaquaticus sp. nov. 100 PP768894 Mononchus pseudoaquaticus sp. nov. KY750781 Mononchus sp. 1 MF125527 Mononchus sp. MF 125528 Mononchus sp. MF 125525 Mononchus aquaticus MF 125524 Mononchus aquaticus AY593063 Mononchus tunbridgensis MF 125529 Mononchus sp. 87) '\MF125526 Mononchus maduei MF125531 Mononchus truncatus MF 125530 Mononchus truncatus AY593064 Mononchus truncatus PP768891 Mononchus truncatus PP768890 Mononchus truncatus PP768892 Mononchus truncatus MZ501582 Mononchus truncatus 100}]MZ501583 Mononchus truncatus MZ501581 Mononchus truncatus MF 125523 Mononchus aquaticus BI-LSU-228 87 92 100 89 94 KM092524 Coomansus gerlachei MT799673 Coomansus parvus PP768897 Coomansus parvus PP768898 Coomansus parvus PP768895 Coomansus parvus PP768896 Coomansus parvus MW525198 Coomansus sp. MW525199 Coomansus sp. MW525200 Coomansus sp. 92'MT705327 Coomansus parvus 0.020 Figure 12. Neighbour-joining tree based on the 28S rDNA (domains D1-D3) dataset (1293 nt positions). The newly gen- erated sequences are indicated in bold. Only bootstrap values > 70% are shown. with two strongly supported main clades: (i) Mononchus spp. (98% supported); and (ii) a large clade (80% supported) comprising the remaining genera except for Mylonchulus. Within the Mononchus clade, the novel sequences for M. trun- catus clustered with four published sequences for M. truncatus with maximum support and M. pseudoaquaticus sp. nov. clustered with a sequence for Monon- chus sp. 1 sensu Mejia-Madrid (2018), again with maximum support. The sec- ond clade had fully resolved internal topology with two large sub-clades, one (100% supported) comprising Coomansus spp. (C. parvus + C. batxatensis) plus representatives of Jensenonchus, Prionchulus, Mulveyellus, and Parkellus, and one (74% supported) comprising C. gerlachei and representatives of Ana- tonchus, lotonchus and Parahadronchus. The relationships of the strongly sup- ported (100%) clade of Mylonchulus spp. remained unresolved. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 74] PP768895 Coomansus parvus PP768896 Coomansus parvus PP768898 Coomansus parvus 85! PP768897 Coomansus parvus MT799673 Coomansus parvus MT705327 Coomansus parvus MW525198 Coomansus batxatensis 400] MW525200 Coomansus batxatensis MW525199 Coomansus batxatensis 100] MW396948 Jensenonchus sp. MW 396947 Jensenonchus sp. 98} [100 MG994945 Prionchulus punctatus OP237018 Mulveyellus sp. 100[7 MT 799672 Parkellus zschokkei 80 MT799670 Parkellus hagiangensis MT799671 Parkellus tuyenquangensis KM092524 Coomansus gerlachei 1007— AY593065 Anatonchus tridentatus 97 MG994941 Anatonchus tridentatus 100 OP997552 lotonchus parabasidontus 0OQ170962 Parahadronchus divendentus 100 MW544448 Mylonchulus sp. ON927922 Mylonchulus sigmaturus AY593063 Mononchus tunbridgensis gg] 00] PP768894 Mononchus pseudoaquaticus sp. nov. KY750781 Mononchus sp. 1 MZ501583 Mononchus truncatus MZ501582 Mononchus truncatus 100) MZ501581 Mononchus truncatus PP768892 Mononchus truncatus PP768891 Mononchus truncatus PP768890 Mononchus truncatus AY593064 Mononchus truncatus KF886018 Mermis nigrescens 100 74 99 0.10 Figure 13. Maximum likelihood phylogeny based on the 28S rDNA (domains D2-D3) dataset (779 nt positions). The newly generated sequences are indicated in bold. Only bootstrap values > 70% are shown. The 18S rDNA alignment comprised a total of 1636 nt positions after trim- ming the ends to match the shortest aligned sequences and contained sequenc- es for representatives of ten genera of the families Anatonchidae (Anatonchus and Miconchus Andrassy, 1958), Mononchidae (Actus Baqri & Jairajpuri, 1974, Clarkus Jairajpuri, 1970, Coomansus, Mononchus, Parkellus, and Prionchulus) and Mylonchulidae (Granonchulus Andrassy, 1958 and Mylonchulus). The avail- able sequences for representatives of the genera Jotonchus, Jensenonchus, Mulveyellus, and Parahadronchus were excluded from the analyses because they were too short and did not exhibit sufficient overlap with the alignment. The topology of the ML tree (Fig. 14) exhibited poorly resolved basal nodes and four strongly supported clades: (i) Mononchus spp. (100% supported) (M. tunbridgensis + M. truncatus + M. aquaticus + M. pseudoaquaticus sp. nov. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 170 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 1007 AY284747 Prionchulus punctatus il AY284745 Prionchulus muscorum AB361452 Clarkus papillatus 95 MT799665 Parkellus hagiangensis 97 MT799667 Parkellus zschokkei 70] PP768899 Coomansus parvus PP768900 Coomansus parvus 99| AY284766 Coomansus parvus AY284767 Coomansus parvus 92 AJ966474 Anatonchus tridentatus KJ636436 Miconchus sp. 1 AB361035 Actus salvadoricus 1007 AY284755 Mylonchulus sigmaturus 100 AF036596 Mylonchulus arenicolus AB361443 Mylonchulus oceanicus 27) 100(- AY284756 Mylonchulus sigmaturus AB361436 Mylonchulus brachyuris 941 [— AY284751 Mylonchulus rotundicaudatus 89'— AB361440 Mylonchulus hawaiiensis AY593954 Mononchus tunbridgensis KJ636355 Mononchus truncatus 100] gg| AJ966493 Mononchus truncatus AY284762 Mononchus truncatus 88 AB361451 Mononchus truncatus PP768901 Mononchus truncatus AY284765 Mononchus aquaticus KJ636383 Mononchus aquaticus PP768902 Mononchus pseudoaquaticus sp. nov. 100} AY284764 Mononchus aquaticus AY297821 Mononchus aquaticus KJ636382 Mononchus pulcher KM092523 Coomansus gerlachei AY593953 Granonchulus sp. KF583882 Mermis nigrescens 0.020 Figure 14. Maximum likelihood phylogeny based on the 18S rDNA dataset (1636 nt positions). The newly generated sequences are indicated in bold. Only bootstrap values > 70% are shown. + M. pulcher); (ii) the remaining mononchids (Coomansus + Clarkus + Parkellus + Prionchulus) (97% supported); (iii) Mylonchulus (100% supported); and (iv) Anatonchus + Miconchus (both Anatonchidae) (92% supported). At the species level, both phylogenies (18S rDNA and 28S rDNA) supported (i) the identification based on morphology of the novel isolates of M. trunca- tus and C. parvus both forming strongly supported reciprocally monophyletic clades, and (ii) the exclusion of C. gerlachei from Coomansus; this species was recovered as a sister taxon (74% supported) to the representatives of the Ana- tonchidae in the 28S rDNA phylogeny and as a basal taxon to the remaining taxa except for Granonchulus in the 18S rDNA analysis. However, in contrast to the clear delineation of M. pseudoaquaticus sp. nov. (100% supported) in the 28S rDNA phylogeny, the 18S rDNA phylogeny did not provide support for the delimitation of M. aquaticus (as M. pulcher sensu van Megen et al. 2009; Gen- Bank: KJ636382) and M. pseudoaquaticus sp. nov. ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria At the generic level, both phylogenies recovered the three genera represent- ed by two or more species (i.e., Mononchus, Mylonchulus, and Parkellus) as monophyletic with strong support. At the suprageneric level, both phylogenies resolved fewer relationships due to the small number of taxa (10 genera, 5 gen- era in common) and perhaps the much poorly resolved basal nodes in the 18S rDNA phylogeny. Both phylogenies recovered the Mononchidae as paraphyletic with Mononchus placed in a separate basal clade and Mylonchulidae and Anaton- chidae nested within the second clade of the Mononchidae despite the different composition of the taxa included in the analyses. However, this is the only concor- dant result for the two molecular markers. Thus, the Mylonchulidae (represented by Mylonchulus alone) was recovered as monophyletic in the 28S rDNA phylog- eny but as polyphyletic in the 18S rDNA phylogeny (represented by Mylonchu- lus and Granonchulus). Similarly, the Anatonchidae was monophyletic in the 18S rDNA phylogeny (2 genera: Anatonchus and Miconchus) but paraphyletic in the 28S rDNA phylogeny containing five genera, with Anatonchus + lotonchus + Par- ahadronchus recovered in a strongly supported clade (97% supported) and Jen- senonchus and Mulveyellus nested within one of the clades of the Mononchidae. Comparative sequence analysis The trimmed alignments of 28S rDNA and 18S rDNA allowed a comparative assessment of the genetic divergence at the level of species (intraspecific) and genus (interspecific) as well as between genera (intergeneric) based on pairwise comparisons. As shown in Table 7, the divergence levels for 18S rDNA are much lower for all three categories of comparisons: up to 10-fold for intraspecific vari- ation, up to ~ 4-fold for interspecific variation, and up to ~ 5-fold for intergeneric variation. The interspecific divergence in 18S rDNA sequences for M. pulcher sensu van Megen et al. (2009; GenBank: KJ636382), M. aquaticus, and M. pseu- doaquaticus sp. nov. was particularly low (0-1 nt positions; O-0.1%). Comparative sequence analyses also provided support for the position in the phylogenies of the isolate identified as C. gerlachei (GenBank: KM092523 and KM092524) by Elshishka et al. (2015). This isolate differed from C. parvus at 70-71 nt positions (4.3-4.4%; 18S rDNA) and from the remaining Coomansus spp. at 135-137 nt positions (20.6-20.8%; 28S rDNA), values well above the genetic divergence between congeners. The isolates identified by Kagoshima et al. (2019) (GenBank: LC457639-LC457644; LC457655-LC457661) were found to be associated with high support with the isolate of Elshishka et al. (2015) that also represented the best BLAST hit for all isolates. Genetic divergence estimates in 28S rDNA also indicated that C. batxat- ensis may be conspecific with C. parvus (difference at 6-8 nt positions, i.e., 0.9-1.2%). This difference is distinctly lower than the ranges of interspecific divergence within the genera Mononchus, Coomansus, Parkellus and Mylonchu- lus, i.e., 34-77 nt positions or 5.1-11.8%; Table 7). Furthermore, the otherwise unpublished isolate identified as M. truncatus (GenBank: MZ501582) may have been misidentified; this isolate differs from the remaining isolates of M. trunca- tus by 8-15 nt (1.2—2.0%). Finally, the intergeneric divergence between Cooman- sus spp. and Parkellus spp. falls within the range of interspecific divergence for both genes (Table 7) and this is in contrast with both model-based phylogenies supporting the distinction of Parkellus spp. at the generic level (Figs 13, 14). ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 172 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Table 7. Genetic divergence estimated for the 18S rDNA and 28S rDNA sequence pairs within and between species and between species of different genera compared in this study. pivergunee Taxa 18S rRNA gene 28S rRNA gene Differences (nt) | p-distance (%) Differences (nt) | p-distance (%) Intraspecific Mononchus truncatus 0 0 0-78 oa ed Mononchus aquaticus D2? 0-0." 1 0.2 Coomansus parvus O=1 0-0.1 0-2 0-0.3 Interspecific M. truncatus vs M. aquaticus 13-14 0.8-0.9 59-/0 8.9-10.5 Mononchus spp. 13=23° 0.8-1.4° 59=77, 8.9-11.8 Coomansus spp. 70-71% 4.3-4.4¢ 6-84 0.9-1.24 Parkellus spp. 24 1.5 34-54 5:1--8:2 Mylonchulus spp. 3-52 O2e37 69 10.1 Intergeneric Mononchus spp. vs Coomansus spp. 76-80 4.7-4.9 140-158° DQS2 2-3" Coomansus spp. vs Parkellus spp. 2ES35° La 222° 64-74¢ 9.7-11.29 @ M. truncatus (GenBank: MZ501582; unpublished) excluded from the comparison (differs from the remaining isolates of M. truncatus at 8-15 nt positions, i.e., 1.2-2.0%). > M. pulcher (GenBank: KJ636382) included in the comparison (differs from M. aquaticus at 0-1 nt positions, i.e., 0-0.1%). ° Genetic divergence between C. parvus and C. gerlachei. 4 C. gerlachei (GenBank: KM092524) excluded from the comparison (differs from the remaining Coomansus spp. at 135-137 nt posi- tions, i.e., 20.6-20.8%). °C. gerlachei (GenBank: KM092523) excluded from the comparison. Discussion To the best of our knowledge, the present study is the first to apply an integra- tive taxonomic approach to the diversity of mononchid nematodes in European riparian ecosystems. Our extensive, focused sampling in a range of riverine habitats in Bulgaria revealed a wide geographical distribution and altitudinal ranges of three species of the family Mononchidae of which one represents a species new to science; these were also associated with a range of tree species of seven genera (Alnus, Carpinus, Fagus, Fraxinus, Populus, Salix, and Ulmus). The integration of molecular and morphological data for these three species provided support for their distinct species status. Thus, our study is the first to provide taxonomically verified 18S rDNA and 28S rDNA sequences for C. par- vus, M. truncatus (sensu stricto), and M. pseudoaquaticus sp. nov. At the species level, phylogenetic analyses revealed that the newly sequenced isolates of M. truncatus (sensu stricto) and C. parvus consistently clustered to- gether with published sequences for these species irrespective of the ribosom- al locus (Figs 13, 14) or region of the 28S rRNA gene (Figs 12, 13). However, the 18S rDNA phylogeny did not allow delimitation of M. pseudoaquaticus sp. nov. and M. aquaticus (Fig. 14). Whereas M. pulcher sensu van Megen et al. (2009) (GenBank: KJ636382) not used in the analysis by these authors and by Holter- man et al. (2008) is likely a misidentification, the morphological differentiation of M. aquaticus and M. pseudoaquaticus sp. nov. was strongly supported in the 28S rDNA phylogeny, suggesting that the 18S rRNA gene does not allow reliable differentiation of closely related species at least within the genus Mononchus. Lower resolution of the 18S rRNA gene was reported in a comparative barcod- ing/metabarcoding study by Schenk et al. (2020); out of 22 nematode species identified using morphology in their study, 20 species were delineated using the ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 173 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria 28S rDNA marker and only 12 species were detected using the 18S rDNA mark- er. Our comparative sequence (Table 7) and phylogenetic (Fig. 14) analyses suggest that the utility of the 18S rRNA gene for species delimitation is rather limited at least for some species complexes within the genus Mononchus. An alternative hypothesis for the phylogenetic results based on 18S rDNA is that the specimens of M. aquaticus sequenced by van Megen et al. (2009) (GenBank: KJ636382 and KJ636383), Holterman et al. (2006) (GenBank: AY284764 and AY284765) and Oliveira et al. (2004) (GenBank: AY297821) actually represent M. pseudoaquaticus sp. nov. However, because of the lack of sequence data for the 28S rRNA gene and deposited voucher material for these sequenced isolates, neither of these hypotheses can be tested. We highlight that the new species described here could be clearly distin- guished morphologically from M. aquaticus (sensu stricto) and that currently M. aquaticus likely represents a composite species and this may result in misiden- tifications of the isolates subjected to sequencing. For example, in the 28 rDNA tree of Schenk et al. (2017) the six specimens identified as M. aquaticus (n = 3), M. maduei and Mononchus sp. juv. (n = 3) formed a reciprocally monophyletic clade with high support (97%). However, in the present NJ analysis (Fig. 12) one isolate (GenBank: MF125523) was resolved as clearly distinct from the other two isolates of M. aguaticus sequenced, and in fact, from all isolates of Mononchus spp. (Fig. 12), thus questioning the identification of this isolate. The above considerations highlight the need for generating taxonomically veri- fied 18S rDNA and 28S rDNA sequences for M. aquaticus (sensu stricto). The isolate of C. gerlachei sequenced by Elshishka et al. (2015) (GenBank: KM092523 and KM092524) was not associated with the C. parvus clade (18S rDNA; Fig. 14) or with C. parvus + C. batxatensis clade (28S rDNA; Fig. 13) and exhibited genetic divergence levels well above the levels observed between species of the mononchid genera Coomansus, Mononchus, Mylonchulus, and Parkellus considered here (Table 7). Taken together, our comparative sequence and phylogenetic analyses strongly suggest that the isolates sequenced by Elshishka et al. (2015) and Kagoshima et al. (2019) should be distinguished at the generic level. At the generic and suprageneric level, the present 18S and 28S phylogenies both recovered the Mononchidae as paraphyletic (as in Holterman et al. 2008 and van Megen et al. 2009). Further, a comparison with the phylogenies of the Mononchida based on 18S rDNA by Holterman et al. (2008) and van Megen et al. (2009) revealed that all four sub-clades identified by these authors (denoted M1-M4 in Holterman et al. 2008) were supported in the present phylogeny: My- lonchulus (sub-clade M1); Mononchus (sub-clade M2); Clarkus + Prionchulus + Coomansus (sub-clade M3); and Anatonchus (sub-clade M4). The differences between the present and published phylogenetic hypotheses represent (i) the recovery of the Anatonchidae as monophyletic in the present phylogeny vs para- phyletic in Holterman et al. (2008) and van Megen et al. (2009), and (ii) the lack of support for a sister-group relationship between Mylonchulus (sub-clade M1) and Mononchus (sub-clade M2) in the present phylogeny. Finally, the poorly rep- resented Mylonchuldae (2 genera: Mylonchulus and Granonchulus) was recov- ered as polyphyletic in the present 18S rDNA phylogeny as in Holterman et al. (2008) and van Megen et al. (2009). These results are due to a single sequence for Granonchulus sp. (AY593953) by Holterman et al. (2008). Unfortunately, no ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 174 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria voucher specimen exists to support the identification of the sequenced nema- tode. Further sequencing of Granonchulus spp., and preferably, of species from the other genera of the family, will help develop a natural hypothesis for the relationships within the Mylonchulidae and the Mononchida in general. Additional information Conflict of interest The authors declare that they have no competing interests. Ethical statement Not applicable. Funding This study was partially funded by a PhD grant to Stela Altash (Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences). Author contributions Stela Altash: Investigation, Formal analysis, Visualisation, Writing — original draft, Writing — review and editing, Funding acquisition. Aneta Kostadinova: Conceptualization, Meth- odology, Data curation, Formal analysis, Supervision, Writing — review and editing. Vlada Peneva: Conceptualization, Methodology, Data curation, Visualisation, Funding acquisition, Supervision, Writing — review and editing. Author ORCIDs Aneta Kostadinova ® https://orcid.org/0000-0001-7070-4968 Data availability All data generated or analyzed during this study are included in this published article and its supplementary files. The newly generated sequences were submitted to the GenBank database under the accession numbers PP768899-PP768902 (18S rRNA gene) and PP768890-—PP768898 (28S rRNA gene). References Ahmad W, Jairajpuri MS (2010) Mononchida: The predaceous nematodes. In: Hunt DJ, Perry RN (Eds) Nematology Monographs and Perspectives; v. 7. 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Hungarian Natural History Museum, Budapest. https://books.google.bg/books?id=WZEAYAAA- CAAJ ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 175 Stela Altash et al.: Mononchid nematodes from riprian habitats in Bulgaria Andrassy | (2011a) Three new species of the genus Mononchus (Nematoda: Mononchi- da), and the “real” Mononchus truncatus Bastian. Journal of Natural History 45(5-6): 303-326. https://doi.org/10.1080/00222933.2010.524947 Andrassy | (2011b) Three new species of the genus Coomansus Jairajpuri & Khan, 1977 from the southern hemisphere (Nematoda: Mononchida). 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The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1206.124237.suppl1 Supplementary material 2 Comparative morphometric data for females of Coomansus parvus, Mononchus aquaticus, M. pseudoaquaticus sp. nov., and Mononchus truncatus Authors: Stela Altash, Aneta Kostadinova, Viada Peneva Data type: pdf Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zookeys.1206.124237.suppl2 ZooKeys 1206: 137-180 (2024), DOI: 10.3897/zookeys.1206.124237 180