Integrative taxonomic study of mononchid nematodes from riparian habitats in Bulgaria. I. 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 Author Altash, Stela Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria Author Kostadinova, Aneta 0000-0001-7070-4968 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria Author Peneva, Vlada Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, 2 Gagarin Street, 1113 Sofia, Bulgaria text ZooKeys 2024 2024-07-05 1206 137 180 journal article 299631 10.3897/zookeys.1206.124237 e42a5b94-6c5b-4f60-b077-fe698bda6546 C1AC9890-1735-4929-B1D5-2056B7E8AFBE Mononchus pseudoaquaticus sp. nov. Figs 3 , 4 , 5 , 6 , 7 Mononchus aquaticus sensu Lazarova et al. (2004) (Syn.) Mononchus sp. 1 sensu Mejía-Madrid (2018) (Syn.) 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 somewhat 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 anterior 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 representing 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 × 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, slender, 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 abnormal tail, very short and almost straight. Male. Not found. Table 3. Morphometric data for females of Mononchus pseudoaquaticus sp. nov. collected in five riparian localities in Bulgaria.

Locality	Vetren, Silistra Province		Komluka Island	Shiroka Lakа, Smolyan Province	Brodilovo, Burgas Province	Near Plovdiv, Plovdiv Province a
River	Danube (Southern Dobruja)		Danube	Shirokoleshka (Rhodope Mountains)	Veleka (Strandzha Mountains)	Maritsa (Upper Thracian Plain)
Habitat	Salix sp. (soil)		Populus sp. (soil)	Salix sp. (soil)	Alnus glutinosa (soil)	Salix sp. (soil)
n	Holotype	Paratypes ( n = 3)	( n = 2)	( n = 1)	( n = 3)	( n = 2)
L (mm)	1.45	1.52, 1.60, 1.23	1.72, 1.88	1.61	1.60, 1.71, 1.69	1.81, 1.50
a	20.2	28.7, 32.0, 28.0	27.7, 33.6	30.9	27.5, 33.5, 28.6	28.3, 29.4
b	4.0	4.5, 4.5, 4.0	4.6, 4.5	4.4	4.4, 4.6, 4.7	4.6, 4.5
c	7.5	–, 8.4, 7.2	8.5, 9.1	8.9	7.8, 8.3, 8.0	10.2, –
c ’	5.0	–, 5.8, 5.7	5.3, 5.8	5.3	5.1, 5.8, 5.4	4.7, –
V (%)	48.3	50.7, 49.7, 53.9	49.7, 50.0	50.7	50.8, 50.3, 48.4	48.8, 50.9
G 1 (%)	12.9	9.9, 9.7, 9.4	12.8, 11.7	9.1	10.1, 8.0, 9.9	12.5, 11.3
G 2 (%)	13.3	13.3, 10.3, 9.9	11.5, 9.9	8.7	7.5, 7.5, 10.0	11.3, 11.1
Buccal capsule length	29	31, 31, 29	29, 33	32	30, 30, 29	31, 30
Buccal capsule width	16	16, 16, 15	15, 16	16	15, 16, 16	16, –
Tooth apex from anterior end of buccal capsule	6	7, 6, 5	6, 7	7	5, 6, 6	6, 6
Position of tooth apex (%) b	21	21, 19, 18	19, 20	20	18, 20, 21	19, 20
Excretory pore from anterior end	118	121, 121, 112	–	126	129, 131, 124	153, 107
Nerve-ring from anterior end	96	102, 106, 99	108. 125	114	109, 111, 101	117, 110
Pharynx length	365	342, 359, 305	371, 420	369	364, 372, 355	392, 335
Lip region height	7	10, 8, 8	8, 10	8	8, 9, 8	9, 8
Lip region width	25	24, 24, 23	25, 26	26	24, 25, 23	26, 24
Amphid from anterior end	9	11, 11, 9	8, 10	12	10, 12, 11	–
Body diameter at pharynx base	62	49, 50, 43	50, 49	48	52, 49, 52	56, 47
Maximum body diameter	72	53, 50, 44	62, 56	52	58, 51, 59	64, 51
Body diameter at mid-body	71	53, 49, 44	59, 52	50	58, 50, 59	63, 51
Body diameter at vagina	72	50, 50, 44	62, 56	52	58, 51, 56	64, 50
Body diameter at anus	39	34, 33, 30	38, 36	34	40, 36, 39	38, 33
Anterior genital branch length	187	151, 155, 116	220, 220	146	162, 137, 167	226, 170
Posterior genital branch length	194	203, 165, 122	197, 186	140	120, 128, 168	205, 167
Anterior ovary length	124	94, 65, –	193, 140	85	70, 86, 79	135, 77
Posterior ovary length	135	109, 95, –	133, 135	82	70, 85, 71	130, 117
Vagina length	20	19, 18, 15	–, 16	17	19, 18, 16	19, –
Rectum length	26	28, 31, 29	28, 30	26	28, 26, 28	29, 28
Tail length	195	–, 191, 171	201, 207	180	204, 207, 210	177, –

a Material reported as M. aquaticus by Lazarova et al. (2004) . b Distance from tooth apex to anterior end of buccal capsule as % of buccal capsule length from its anterior end. 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. l.) Other localities. Komluka Island (River Danube), rivers Veleka, Shirokoleshka, 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 Research, Bulgarian Academy of Sciences, Bulgaria, under the accession numbers IBER-BAS NTC 105 and 106 . One paratype female is deposited in the Wageningen Nematode Collection ( WANECO ), Wageningen, the Netherlands ( WANECO 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- 8065 p ) . 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 µm. Voucher material. Eight voucher specimens are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian 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 provided in Suppl. material 1 : fig. S 4. 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 µm. Representative DNA sequences. 28 S rRNA gene (GenBank: PP 768893 and PP 768894 ); 18 S rRNA gene ( PP 768902 ). 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 µm. 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 × 15–16 µm, 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) reproductive system with pars refringens vaginae distinctly sclerotised in the form of two smooth rhomb-shaped pieces; tail (171–210 µm long, c = 7.2–10.2, c ’ = 4.7–5.8) slightly curved at its posterior third, spinneret terminal. 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 Е ) 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 µm ( A ); 20 µm ( B – E, G, J, K, L ); 30 µm ( F, I, H ). Morphologically, Mononchus pseudoaquaticus sp. nov. appears most similar to M. aquaticus , M. pulcher Andrássy, 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 variation reported in the literature (see comparative data in Suppl. material 2 : table S 2). However, it is not possible to revise the identification of these materials because in many cases the findings are not documented properly and important 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 concept 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 Jairajpuri (1972) who re-examined and provided metrical data for some paratypes of M. aquaticus . This concept was also applied by Andrássy (2011 a ) in the most recent key to the species of Mononchus (see Suppl. material 2 : table S 3 for details) and in the updated key to the species of Mononchus provided here. Figure 7. Photomicrographs of Mononchus pseudoaquaticus sp. nov. Females from populations collected from riverbanks 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 refringens vaginae ( G ) H, I tail J caudal glands (arrowed) K caudal pores (arrowed) L tail tip. Scale bars: 20 µm ( A – G, K ); 30 µm ( J, L ); 50 µm ( H, I ). The present material differs from the type material of M. aquaticus ( Coetzee 1968 ; Baqri 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 µm (mean 150 µm) (Suppl. material 2 : table S 3). 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 µm); different arrangement of the caudal glands (in a group vs in tandem); and shorter rectum (26–31 vs 32–36 µm) and vagina (16–20 vs 27–29 µm) ( Shah and Hussain 2016 ; Suppl. material 2 : table S 3). Differentiation from M. pulcher is more complicated because the original description of Andrássy (1993) is based on two, geographically largely separated populations from Chile and Hungary . However, Andrássyʼs (1993: fig. 2) illustrations indicate that he probably dealt with different species. Unfortunately, it is impossible to separate the rather incomplete metrical data since Andrássy (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 (Suppl. material 2 : table S 3). Still, in addition to the morphological differences (e. g., different shape of the buccal capsule and direction of the dorsal tooth), the Hungarian population is characterised by having a smaller buccal capsule length (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 differentiating 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 Andrássy (2011 a ). Therefore, our comparisons are based on the morphology and metrical data for the type-population of M. pulcher from Chile . The new species differs from M. pulcher (sensu stricto) by having: a shorter (29–33 vs 35–38 µm) and narrower (15–16 vs 16–18 µm) buccal capsule; lower values for a (20–34 vs 35–39); anterior margin 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 µm). Additionally, the upper ranges for body length and tail length are greater in both populations of M. pulcher (Suppl. material 2 : table S 3).