Research Article |
Corresponding author: Vlada Peneva ( esn.2006@gmail.com ) Academic editor: Sergei Subbotin
© 2024 Stela Altash, Aneta Kostadinova, Vlada Peneva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Altash S, Kostadinova A, Peneva V (2024) 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. ZooKeys 1206: 137-180. https://doi.org/10.3897/zookeys.1206.124237
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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 Bastian, 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, morphometric data for multiple species populations, drawings and photomicrographs, and the first taxonomically verified sequences for C. parvus (n = 6), M. truncatus (sensu stricto) (n = 4) and M. pseudoaquaticus sp. nov. (n = 3). Comparative sequence and phylogenetic 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.
Distribution, Mononchidae, morphology, phylogeny, riverine, taxonomy, 18S rDNA, 28S rDNA
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 riparian soils (
Sequence-based tools such as barcoding have proven successful in accelerating identification of previously characterised species or in detecting cryptic species (
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 molecularly. 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 delineate the species and establish their relationships within the suborder Mononchina 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 habitats. Species of the genus Mononchus are aquatic nematodes, occasionally occurring in wet terrestrial habitats (
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 (
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 × 15 m or 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 funnel 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 paraffin as a support for the cover slide (
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; G1 anterior female gonad length as % of body length; G2 posterior female gonad length as % of body length (
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 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 14,000× 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’) (
PCR amplifications were performed in a total volume of 25 µl 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 2× MyFi™ DNA Polymerase mix (Bioline Inc., Taunton, USA; Cat. # BIO-25049) in a total volume of 20 μl, containing 8 pmol of each primer and ~ 50 ng of gDNA. The amplification profile for 28S rDNA comprised an initial denaturation at 94 °C for 5 min (or 3 min when using MyFi™ DNA Polymerase mix) followed 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; 30 s 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 (
The newly generated 18S rDNA and 28S rDNA sequences were aligned separately using MUSCLE implemented in MEGA7 (
Secondly, two alignments were constructed comprising sequences for species 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 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 likelihood (ML) analyses as implemented in MEGA7. Prior to analyses, the best-fitting 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+I+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. Mermis nigrescens Dujardin, 1842 was used as the outgroup in the analyses of both alignments based on the phylogeny published by
A total of 17 populations of Coomansus spp. and Mononchus spp. were collected 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., Populus sp., Ulmus laevis Pall., and Ulmus sp.) (Table
Summary data for the populations of Coomansus parvus and Mononchus spp. studied in 17 riparian habitats in Bulgaria.
Species | River | Locality | Coordinates | Elevation (m)a | Associated tree species (habitat) | Date (Collector) |
---|---|---|---|---|---|---|
Coomansus parvus (de Man, 1880) | Lopushnitsa (Balkan Mountains) | Near Kaleytsa, Lovech Province | 42°55'34"N, 24°38'38"E | ~ 440 | Acer sp. (litter) | 9.05.2021 (VP) |
Arda (Rhodope Mountains) | Dyavolski Most, Kardzhali Province | 41°37'14"N, 25°06'53"E | ~ 460 | Ulmus sp. (soil) | 29.08.2020 (VP) | |
41°37'22"N, 25°06'54"E | ~ 440 | Populus sp. (soil) | ||||
Vedena (Vitosha Mountain) | Near Zheleznitsa, Sofia Province | 42°32'05"N, 23°20'57"E | ~ 1200 | Fagus sylvatica L. (litter) | 4.04.2022 (SA, VP) | |
Devinska (Rhodope Mountains) | Near Devin, Smolyan Province | 41°45'21"N, 24°20'02"E | ~ 880 | Carpinus betulus L. (soil) | 20.05.2019 (SA) | |
Mononchus truncatus Bastian, 1865 | Shirokoleshka (Rhodope Mountains) | Shiroka Laka, Smolyan Province | 41°40'26"N, 24°35'51"E | ~ 1120 | Salix sp. (soil) | 23.05.2019 (SA) |
Maritsa (Upper Thracian Plain) | Near Plovdiv, Plovdiv Province | 42°09'N, 25°50'E | ~ 153 | Salix sp. (soil) | 18.10.1995 (VP) | |
Trigradska (Rhodope Mountains) | Teshel, Smolyan Province | 41°40'18"N, 24°21'13"E | ~ 860 | Salix sp. (litter) | 23.05.2019 (SA) | |
Dyavolska (Strandzha Mountains) | Near Primorsko, Burgas Province | 42°15'34"N, 27°44'18"E | ~ 10 | Fraxinus excelsior L. (soil) | 6.06.2019 (SA) | |
Rezovska (Strandzha Mountains) | Slivarovo, Burgas Province | 41°57'N, 27°40'E | ~ 240 | Ulmus laevis Pall. (soil) | 22.10.2008 (RS) | |
Danube (Southern Dobruja) | Vetren, Silistra Province | 44°08'24"N, 27°01'47"E | ~ 20 | Salix sp. (soil) | 5.07.2021 (VP) | |
Veleka | Brodilovo, Burgas Province | 42°04'53"N, 27°51'33"E | ~ 15 | Alnus glutinosa (L.) (soil) | 4.06.2019 (SA) | |
Mononchus pseudoaquaticus sp. nov. | Shirokoleshka (Rhodope Mountains) | Shiroka Laka, Smolyan Province | 41°40'26"N, 24°35'51"E | ~ 1120 | Salix sp. (soil) | 23.05.2019 (SA) |
Maritsa (Upper Thracian Plain) | Near Plovdiv, Plovdiv Province | 42°09'N, 25°50'E | ~ 153 | Salix sp. (soil) | 18.10.1995 (VP) | |
Veleka | Brodilovo, Burgas Province | 42°04'53"N, 27°51'33"E | ~ 15 | Alnus glutinosa (L.) (soil) | 4.06.2019 (SA) | |
Danube (Southern Dobruja) | Vetren, Silistra Province | 44°08'24"N, 27°01'47"E | ~ 20 | Salix sp. (soil)b | 5.07.2021 (VP) | |
Danube (Southern Dobruja) | Komluka Island, Silistra Province | 44°08'03"N, 27°03'40"E | ~ 20 | Populus sp. (soil) | 5.07.2021 (VP) | |
Mononchus sp. | Grafska, inflow of River Kopilovtsi (Balkan Mountains) | Waterfall “Durshin skok”, near Kopilovtsi, Montana Province | 43°19'40"N, 22°51'01"E | ~ 1048 | Fagus sylvatica L. (soil) | 27.07.2000 (VP) |
Four species were identified based on morphological data: C. parvus (4 populations), 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
Although an attempt was made to obtain representative 28S rDNA sequences 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 representative 18S rDNA sequences (1630–1682 bp; 2 for C parvus, 1 for M. truncatus, 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.
Female [Based on 10 specimens from 3 localities; see Table
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, I reproductive system (E anterior genital branch F, I 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 µm (A); 20 µm (B–D, F–I, M); 50 µm (E, J–L).
Morphometric data for females of Coomansus parvus collected in four riparian localities in Bulgaria.
Locality | Near Kaleytsa, Lovech Province | Dyavolski Most, Kardzhali Province | Near Zheleznitsa, Sofia Province | |
---|---|---|---|---|
River | Lopushnitsa (Balkan Mountains) | Arda (Rhodope Mountains) | Vedena (Vitosha Mountain) | |
Habitat | Acer sp. (litter) | Ulmus sp. (soil) | Populus sp. (soil) | Fagus sylvatica (litter) |
n | (n = 1) | (n = 1) | (n = 2) | (n = 6) |
L (mm) | 1.05 | 0.70 | 0.90, 1.07 | 0.96 ± 0.14 (0.83–1.15) |
a | 18.7 | 12.9 | 17, 17 | 19.0 ± 2.5 (16.2–21.7) |
b | 3.6 | 3.7 | 3.5, 3.5 | 3.2, 3.4, 3.5 (n = 3) |
c | 14.8 | 11.7 | 12.2, 12.9 | 12.7 ± 1.1 (11.5–14.0) |
c’ | 2.3 | 2.1 | 2.4, 2.3 | 2.3 ± 0.3 (2.0–2.7) |
V (%) | 62.0 | 59.9 | 61.3, 62.2 | 62.5 ± 1.6 (59.6–64.5) |
G1 (%) | 9.2 | 11.4 | 12.2, 10.5 | 13.8 ± 2.4 (12.0–17.3) (n = 5) |
G2 (%) | 12.6 | 11.3 | 10.2, 11.4 | 12.6 ± 1.5 (11.6–15.1) (n = 5) |
Buccal capsule length | 24 | 22 | 23, 26 | 26 ± 1 (25–27) |
Buccal capsule width | 14 | 14 | 14, 15 | 15 ± 0.4 (14–15) |
Tooth apex from anterior end of buccal capsule | 9 | 11 | 8, 8 | 9 ± 0.4 (9–10) |
Position of tooth apex (%)a | 38 | – | 36, 32 | 35 ± 2 (33–38) |
Excretory pore from anterior end | 116 | – | 110, 113 | 117 ± 15 (97–131) |
Nerve-ring from anterior end | 92 | – | 90, – | 100 ± 4 (94–103) (n = 4) |
Pharynx length | 294 | 189 | 258, 302 | 303, 324, 324 (n = 3) |
Lip region height | 8 | 7 | 8, 8 | 8 ± 1 (7–10) (n = 4) |
Lip region width | 25 | 23 | 25, 25 | 24 ± 3 (22–28) (n = 4) |
Amphid from anterior end | 11 | 14 | 14, 11 | 10 (n = 2) |
Maximum body diameter | 56 | 54 | 53, – | 50 ± 2 (48–53) |
Body diameter at pharynx base | 49 | 49 | 51, – | 46 ± 3 (43–50) (n = 5) |
Body diameter at mid-body | 49 | 53 | 51, – | 50 ± 2 (48–53) |
Body diameter at vagina | 56 | 54 | 53, – | 50 ± 2 (48–53) |
Body diameter at anus | 31 | 28 | 31, 36 | 33 ± 2 (31–36) |
Anterior genital branch length | 96 | 80 | 110, 112 | 161 ± 53 (118–253) (n = 5) |
Posterior genital branch length | 132 | 80 | 92, 122 | 122 ± 11 (106–133) (n = 5) |
Anterior ovary length | 90 | 70 | 60, 80 | 100, 105, 105 |
Posterior ovary length | 105 | 75 | 70, 100 | 86, 110, 140 |
Vagina length | 17 | 17 | 16, 18 | 14 ± 2 (12–16) (n = 5) |
Rectum length | 23 | 21 | 24; 26 | 23 ± 2 (21–25) |
Tail length | 71 | 60 | 74; 83 | 75 ± 8 (66–85) |
Ten specimens are deposited in the Nematological Collection 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
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
Almost cosmopolitan, except in Australia (
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 Mountains showing lower values for L, a, G1, G2, the length of the genital branches, ovaries, and tail, and greater values for the distance of the amphid from anterior end compared with the population from Vitosha Mountain (Table
The morphometric data for the present material fall within the range given by
However, the material described by
Mononchus aquaticus
sensu
Mononchus
sp. 1 sensu
Female [Based on 4 specimens from the type-population and 8 voucher specimens from other populations; see Table
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 Provincea | |
---|---|---|---|---|---|---|
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 |
G1 (%) | 12.9 | 9.9, 9.7, 9.4 | 12.8, 11.7 | 9.1 | 10.1, 8.0, 9.9 | 12.5, 11.3 |
G2 (%) | 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, – |
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.)
Komluka Island (River Danube), rivers Veleka, Shirokoleshka, and Maritsa (see Table
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-8065p).
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
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.
The species is named Mononchus pseudoaquaticus because of its similarity with M. aquaticus, hence the prefix pseudo- meaning false.
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.
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
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 (
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) (
Differentiation from M. pulcher is more complicated because the original description of
Female [Based on 14 specimens from 6 localities; see Table
Morphometric data for females of Mononchus truncatus collected in six riparian localities in Bulgaria.
Locality | Shiroka Laka, Smolyan Province | Teshel, Smolyan Province | Near Primorsko, Burgas Province | Slivarovo, Burgas Province | Near Plovdiv, Plovdiv Province | Vetren, Silistra Province |
---|---|---|---|---|---|---|
River | Shirokoleshka (Rhodope Mountains) | Trigradska (Rhodope Mountains) | Dyavolska (Strandzha Mountains) | Rezovska (Strandzha Mountains) | Maritsa (Upper Thracian Plain) | Danube (Southern Dobruja) |
Habitat | Salix sp. (soil) | Salix sp. (litter) | Fraxinus excelsior (soil) | Ulmus laevis (soil) | Salix sp. (soil) | Salix sp. (soil) |
n | (n = 6) | (n = 1) | (n = 1) | (n = 3) | (n = 1) | (n = 2) |
L (mm) | 1.94 ± 1.08 (1.83–2.09) | 1.89 | 2.06 | 1.77, 1.85, 1.84 | 1.83 | 1.89, 1.91 |
a | 30.6 ± 2.5 (27–34) | 26.5 | 38.9 | 32.1, 33.7, 29.2 | 33.3 | 33.7, 32.4 |
b | 4.1 ± 0.2 (3.7–4.3) | 4.0 | 3.9 | 4.0, 4.2, 4.1 | 4.2 | 4.0, 4.2 |
c | 8.3 ± 0.2 (8.1–8.7) | 8.6 | 8.2 | 7.8, 8.0, 8.2 | 8.2 | 8.9, 9.3 |
c’ | 5.5 ± 0.4 (5.0–6.0) | 5.5 | 6.1 | 6.7, 6.3, 6.2 | 6.2 | 5.0, 5.0 |
V (%) | 55.2 ± 1.3 (53.4–57.3) | 54.0 | 53.9 | 57.6, 53.1, 53.2 | 52.6 | 54.3, 53.8 |
G1 (%) | 10.1 ± 0.6 (9.5–10.9) | 9.6 | 10.0 | 11.3, 9.6, 10.3 | 10.2 | 9.7, 9.8 |
G2 (%) | 10.4 ± 0.6 (9.7–11.6) | 11.4 | 11.6 | 11.8, 10.4, 10.2 | 10.8 | 9.9, 10.1 |
Buccal capsule length | 43 ± 2 (40–44) | 44 | 44 | 42, 42, 41 | 40 | 42, 42 |
Buccal capsule width | 20 ± 1 (19–22) | 21 | 21 | 18, 19, 19 | 19 | 19, 20 |
Tooth apex from anterior end of buccal capsule | 12 ± 1 (11–12) | 11 | 11 | 11, 11, 11 | 10 | 11, 12 |
Position of tooth apex (%)a | 27 ± 1 (26–29) | 25 | 25 | 26, 27, 27 | 26 | 26, 27 |
Excretory pore from anterior end | 148 ± 16 (137–176) (n = 5) | 150 | – | 138, 141, 142 | 134 | 156, 146 |
Nerve-ring from anterior end | 126 ± 10 (116–144) (n = 5) | – | 140 | 138, 141, 142 | 122 | 129, 129 |
Pharynx length | 479 ± 34 (423–518) | 468 | 525 | 439, 443, 446 | 437 | 468, 450 |
Lip region height | 9 ± 1 (8–11) | 9 | 10 | 10, 10, 9 | 10 | 9, 11 |
Lip region width | 29 ± 1 (28–30) | 26 | 30 | 26, 26, 25 | 26 | 27, 25 |
Amphid from base of buccal capsule | 40 ± 3 (37–44) | 41 | 44 | –, 39, 39 | 37 | 41, 38 |
Amphid from anterior end | 12 ± 1 (10–13) (n = 4) | 10 | 11 | –, 12, 10 | 13 | 12, 11 |
Maximum body diameter | 64 ± 6 (55–70) | 71 | 53 | 55, 55, 63 | 55 | 56, 59 |
Body diameter at pharynx base | 58 ± 4 (51–61) | 53 | 52 | 51, 55, 58 | 53 | 53, 56 |
Body diameter at mid-body | 61 ± 4 (53–65) | 71 | 53 | 55, 55, 63 | 55 | 55, 59 |
Body diameter at vagina | 64 ± 6 (55–70) | 71 | 53 | 53, 54, 60 | 55 | 56, 59 |
Body diameter at anus | 42 ± 3 (38–47) | 40 | 41 | 34, 37, 36 | 34 | 42, 41 |
Anterior genital branch length | 196 ± 18 (175–223) | 181 | 206 | 199, 179, 189 | 207 | 182, 188 |
Posterior genital branch length | 203 ± 13 (190–220) | 215 | 240 | 208, 193, 187 | 215 | 187, 193 |
Anterior ovary length | 98 ± 15 (75–115) (n = 5) | 125 | 120 | 108, 91, – | 142 | 107, 107 |
Posterior ovary length | 106 ± 16 (95–135) (n = 5) | 125 | 146 | 100, 109, – | 141 | 101, 120 |
Vagina length | 17 ± 1 (15–17) | 17 | 14 | –, 18, 17 | 17 | 17, 16 |
Rectum length | 31 ± 1 (29–33) | 36 | 27 | 29, 32, 31 | 28 | 32, 30 |
Tail length | 234 ± 11 (225–254) | 218 | 252 | 227, 232, 224 | 211 | 212, 205 |
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. Photovouchers for the sequenced specimens are provided in Suppl. material
Soil around roots of F. excelsior, U. laevis, A. glutinosa and Salix sp. and litter around Salix sp. along banks of the rivers Shirokoleshka, Trigradska, Dyavolska, Rezovska, Veleka, Maritsa, and Danube (see Table
According to the abundant published data for materials reported as M. truncatus, this species appears to exhibit a worldwide distribution. However, we agree with
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 (
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 region: 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 µm (A); 20 µm (B–H, L–N); 50 µm (I–K).
We are also aware of two other questionable records of M. truncatus, not included in Suppl. material
We agree with
Mononchus truncatus was first reported from Bulgaria by
Female [Based on 2 females; see Table
Species | Mononchus sp. | M. oblongus |
---|---|---|
Source | Present study |
|
Locality | Waterfall “Durshin skok”, near Kopilovtsi, Montana Province | Near Ossés, South of France |
River | Grafska (Balkan Mountains) | na |
Habitat | Fagus sylvatica (soil) | Liver moss (soil) |
n | (n = 2) | (n = 6) |
L (mm) | 1.31, 1.62 | 1.60–1.88 |
a | 27.9, 30.6 | 25–29 |
b | 3.5, 4.1 | 3.5–3.7 |
c | 5.7, 6.2 | 6.0–7.1 |
c’ | 7.9, 7.7 | 7.5–8.8 |
V (%) | 51.2, 56.2 | 52–54 |
G1 (%) | 7.2, 8.3 | 7.6–9.4 |
G2 (%) | 8.0, 9.2 | 7.6–9.4 |
Buccal capsule length | 45, 47 | 48–51 |
Buccal capsule width | 19, 20 | 18–19 |
Tooth apex from anterior end of buccal capsule | 10, 11 | 10.0–11.5 |
Position of tooth apex (%)a | 22, 23 | 21–23 |
Excretory pore from anterior end | 123, 139 | – |
Nerve-ring from anterior end | 111, 123 | – |
Pharynx length | 370, 392 | 450–504 |
Lip region height | 8, 9 | 7–9 |
Lip region width | 25, 27 | 22–23 |
Amphid from anterior end | 12, 19 | –b |
Maximum body diameter | 47, 53 | – |
Body diameter at pharynx base | 47, 53 | 60–65 |
Body diameter at mid-body | 45, 52 | 60–68 |
Body diameter at vagina | 46, 51 | – |
Body diameter at anus | 29, 34 | 30–36 |
Anterior genital branch length | 94, 135 | – |
Posterior genital branch length | 105, 149 | – |
Anterior ovary length | 40, 88 | – |
Posterior ovary length | 45, 97 | – |
Vagina length | –, 16 | 18–21 |
Rectum length | 24, 27 | – |
Tail length | 230, 263 | 264–276 |
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.
Soil around roots of F. sylvatica near a waterfall (River Grafska, inflow of River Kopilovtsi; see Table
Morphologically, the specimens resemble most Mononchus oblongus Andrássy, 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
The present specimens also show similarities with M. truncatus and M. himalayensis 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) (
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 µm (A); 20 µm (B–G, I, K); 50 µm (H, J).
Since the last identification key to the species of the genus Mononchus was published by
Main morphometric data for the nine additional species of Mononchus described after 2011 and included in the key to species.
Species | M. amplus Gagarin & Naumova, 2017 | M. baikalensis (Gagarin & Naumova, 2017) nom. nov. | M. caudatus Shah & Hussain, 2016 | M. intermedius Tahseen & Rajan, 2009 | M. labiatus Shah & Hussain, 2016 | M. minutus Naumova & Gagarin, 2018 |
M. oryzae |
M. prodentatus Shah & Hussain, 2016 | M. pseudoaquaticus sp. nov. | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
L (mm) | ♀6.74–7.24 | ♂6.90 | ♀3.35 | ♂ 3.35–3.72 | ♀1.73–1.92 | ♀1.32–1.65 | ♀1.31–1.79 | ♀2.38–2.89 | ♂2.34–2.83 | ♀1.51–1.53 | ♀1.69–1.76 | ♀1.23–1.88 |
a | 52– 61 | 50 | 22 | 22–26 | 34–37 | 20.5–28.8 | 30–36 | 26–33 | 25–33 | 28.4–31.7 | 31–33 | 21–36 |
b | 4.7– 4.8 | 5.0 | 3.5 | 3.4–3.6 | 4.0–5.0 | 3.8–4.7 | 3.0–4.0 | 3.3–3.7 | 3.2–3.8 | 3.6–3.9 | 4.0–5.0 | 4.0–4.6 |
c | 11.3–11.6 | 16.2 | 9.7 | 11.2–13.0 | 9.0–10.0 | 8.7–10.6 | 7.0–8.0 | 12.8–15.1 | 14.0–16.1 | 12.3–13.0 | 8.0–9.0 | 7.2–10.2 |
cʼ | 8.9 | 4.6 | 5.1 | 2.9–3.5 | 5.0–6.0 | 3.9–5.8 | 6.0–7.0 | 3.3–4.4 | 2.5–3.0 | 3.9–4.2 | 5.0–6.0 | 4.7–5.8 |
V (%) | 59 | – | 53 | – | 48–51 | 50–55 | 56–64 | 56–61 | – | 59–61 | 53–55 | 48–54 |
Lip region width | 58–60 | 63 | 50 | 54–60 | 24–25 | 20–26 | 24–25 | 35–40 | 34–38 | 23–24 | 16–17 | 23–26 |
Buccal capsule length | 70–78 | 80 | 110 | 105–112 | 30–33 | 36–44 | 29–43 | 65–74 | 64–72 | 34–35 | 33–34 | 29–33 |
Position of tooth apex (%)a | 27–29 | 27 | 30 | 28–30 | 18–21 | 25–30 | 27–35 | 9–14 | – | 19–20 | 24–28 | 18–21 |
Tail length | 595–625 | 425 | 345 | 275–300 | 190–195 | 145–182 | 193–231 | 175–208 | 162–188 | 117–122 | 196–200 | 171–207 |
Supplements | – | 40 | – | 31–32 | – | – | – | – | 21–25 | – | – | – |
Spicule | – | 165 | – | 220–235 | – | – | – | – | 205–215 | – | – | – |
1 | Large species, body 2.4–7.0 mm long | 2 |
– | Smaller species, body 0.9–2.1 mm long | 13 |
2 | Tail very short, about 2 anal body diameters long | 3 |
– | Tail longer, (3–) 4–9 anal body diameters long | 6 |
3 | Posterior third of tail digitate, ventrally curved | M. mulveyi Andrássy, 1985 |
– | Posterior third of tail not digitate, more or less straight | 4 |
4 | Buccal capsule 100–120 μm long, nearly 3 times as long as wide | M. tajmiris Gagarin, 1991 |
– | Buccal capsule 50–90 μm long, about twice as long as wide | 5 |
5 | Buccal capsule 80–90 μm long; spicule 300 μm long | M. angarensis Gagarin, 1984 |
– | Buccal capsule about 50 μm long; spicule 120 μm long | M. maduei Schneider, 1925 |
6 | Body 5.0–7.2 mm long | 7 |
– | Body 2.4–3.7 mm long | 9 |
7 | Body 5.0–6.4 mm long; tail as long as 5–6 (♂♂ 2.4) anal body diameters | M. superbus Mulvey, 1978 |
– | Body 6.7–7.2 mm long; tail as long as 9 (♂♂ 4.6) anal body diameters | M. amplus Gagarin & Naumova, 2017 |
9 | Buccal capsule > 80 μm long; | 10 |
– | Buccal capsule 46–74 μm long; | 11 |
10 | Buccal capsule 80–84 μm long; tail as long as 3–4 anal body diameters | M. agilis Gagarin & Mataphonov, 2004 |
– | Buccal capsule 105–112 μm long; tail as long as 5 (♂♂ 2.9–3.5) anal body diameters | M. baikalensis (Gagarin & Naumova, 2017) nom. nov. |
11 | Dorsal tooth apex at up to 16% of buccal capsule length from its anterior end; tail as long as 3–6 anal body diameters | 12 |
– | Dorsal tooth apex at 28–30% of buccal capsule length from its anterior end; tail as long as 8–9 anal body diameters | M. altiplanicus Andrássy, 2011 |
12 | Body 2.8–3.5 mm long; buccal capsule 46–56 × 20–25 μm; spicules relatively short (134–140 μm) | M. niddensis Skwarra, 1921 |
– | Body 2.4–2.9 mm long; buccal capsule 65–74 × 28–31 μm; spicules longer (205–215 μm) | M. minutus Naumova & Gagarin, 2018 |
13 | Monodelphic species | M. italicus Andrássy, 1959 |
– | Didelphic species | 14 |
14 | Tail quite short (as long as 1.5–2 anal body diameters); spinneret subdorsal | M. clarki Altherr, 1972 |
– | Tail as long as 3 anal body diameters or longer (c’ = up to 15); spinneret terminal | 15 |
15 | Buccal capsule small, 18–23 μm long | 16 |
– | Buccal capsule larger, 26–50 μm long | 17 |
16 | Buccal capsule very narrow (nearly 3 times as long as wide); dorsal tooth apex quite close to the anterior end of buccal capsule | 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 end | M. loofi Winiszewska, 1998 |
17 | Tail as long as 7–15 (mostly 9–14) anal body diameters | 18 |
– | Tail as long as 3–8 (mostly 4–7) anal body diameters | 20 |
18 | Tail 340–390 μm long, as long as 13–15 anal body diameters | M. syrmatus Andrássy, 2008 |
– | Tail 220–300 μm long, as long as 8–11 anal body diameters | 19 |
19 | Buccal capsule 40–47 μm long; one prevulval papilla present | M. himalayensis Rawat & Ahmad, 2000 |
– | Buccal capsule 28–35 μm long; prevulval papilla absent | M. sandur Eisendle, 2008 |
20 | Pars refringens vaginae not sclerotised | M. sinensis Soni & Nama, 1983 |
– | Pars refringens vaginae distinctly sclerotised | 21 |
21 | Subventral transverse ribs located anteriorly to tooth apex | 22 |
– | Subventral transverse ribs located at level of or posterior to tooth apex | 23 |
22 | Lip region relatively wide (24–28 μm); cylindrical portion of tail 10–12 μm thick | M. truncatus Bastian, 1865 |
– | Lip region narrower (20 μm); cylindrical portion of tail 5–7 μm thick | M. medius Andrássy, 2011 |
23 | Amphid aperture posterior to dorsal tooth | M. laminatus Zullini, Loof & Bongers, 2002 |
– | Amphid aperture anterior to dorsal tooth | 24 |
24 | Tail as long as 3–4 anal body diameters | 25 |
– | Tail as long as 4–7 anal body diameters | 26 |
25 | Body 1.6–2.1 mm long; dorsal tooth apex at 22–24% of buccal capsule length from its anterior end; tail 176 μm | M. nudus Gagarin, 1991 |
– | Body 1.5 mm long, dorsal tooth apex at 30–35% of buccal capsule length from its anterior end; tail 117–122 μm | M. oryzae Ishaque, Iqbal, Dawar & Kazi, 2022 |
26 | Buccal capsule two labial diameters long or longer | 27 |
– | Buccal capsule conspicuously shorter than two labial diameters | 28 |
27 | Buccal capsule oblong, 47–50 μm long, labial diameter 22–23 μm | M. oblongus Andrássy, 2011 |
– | Buccal capsule barrel-shaped, 33–34 μm long, labial diameter 16–17 μm | M. prodentatus Shah & Hussain, 2016 |
28 | Dorsal tooth apex at > 25% of buccal capsule length from its anterior end | 29 |
– | Dorsal tooth apex at < 25% of buccal capsule length from its anterior end | 30 |
29 | Buccal capsule 36–44 μm long, c = 8.7–10.6; tail 145–182 μm long | M. intermedius Tahseen & Rajan, 2009 |
– | Buccal capsule 29–43 μm long, c = 7–8; tail 193–231 μm long | M. labiatus Shah & Hussain, 2016 |
30 | Buccal capsule (33)35–38 μm long, vagina spotted in its anterior part | M. pulcher Andrássy, 1993 |
– | Buccal capsule 29–33 μm long, vagina not spotted | 31 |
31 | Buccal capsule 1.8–2.0 times as long as wide, pars refringens vaginae rhomb-shaped | M. pseudoaquaticus sp. nov. |
– | Buccal capsule 2.2–2.5 times as long as wide, pars rrefringens vaginae drop-shaped | 32 |
32 | Body 1.2–1.7 mm long, rectum length 24–25 μm | M. aquaticus Coetzee, 1968 |
– | Body 1.7–1.9 mm long, rectum length 32–36 μm | M. caudatus Shah & Hussain, 2016 |
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 sequences, e.g., several sequences of
Next, we assessed the phylogenetic relationships of the novel isolates with representatives of the suborder Mononchina using two alignments. Upon trimming 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, Iotonchus Cobb, 1916, Jensenonchus Jairajpuri & Khan, 1982, Mulveyellus Siddiqi, 1984 and Parahadronchus Mulvey, 1978), Mononchidae (Coomansus, 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 (Bastian, 1865) Jairajpuri, 1970 (domains D3-D5) could not be used due to the very small overlap. Overall, the topology of the ML tree (Fig.
The 18S rDNA alignment comprised a total of 1636 nt positions after trimming the ends to match the shortest aligned sequences and contained sequences for representatives of ten genera of the families Anatonchidae (Anatonchus and Miconchus Andrássy, 1958), Mononchidae (Actus Baqri & Jairajpuri, 1974, Clarkus Jairajpuri, 1970, Coomansus, Mononchus, Parkellus, and Prionchulus) and Mylonchulidae (Granonchulus Andrássy, 1958 and Mylonchulus). The available sequences for representatives of the genera Iotonchus, 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.
At the species level, both phylogenies (18S rDNA and 28S rDNA) supported (i) the identification based on morphology of the novel isolates of M. truncatus 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 Anatonchidae 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
At the generic level, both phylogenies recovered the three genera represented 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 genera 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 Anatonchidae nested within the second clade of the Mononchidae despite the different composition of the taxa included in the analyses. However, this is the only concordant result for the two molecular markers. Thus, the Mylonchulidae (represented by Mylonchulus alone) was recovered as monophyletic in the 28S rDNA phylogeny but as polyphyletic in the 18S rDNA phylogeny (represented by Mylonchulus 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 + Iotonchus + Parahadronchus recovered in a strongly supported clade (97% supported) and Jensenonchus and Mulveyellus nested within one of the clades of the Mononchidae.
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
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.
Divergence | Taxa | 18S rRNA gene | 28S rRNA gene | ||
---|---|---|---|---|---|
Differences (nt) | p-distance (%) | Differences (nt) | p-distance (%) | ||
Intraspecific | Mononchus truncatus | 0 | 0 | 0–7a | 0–1.1a |
Mononchus aquaticus | 0–2b | 0–0.1b | 1 | 0.2 | |
Coomansus parvus | 0–1 | 0–0.1 | 0–2 | 0–0.3 | |
Interspecific | M. truncatus vs M. aquaticus | 13–14 | 0.8–0.9 | 59–70 | 8.9–10.5 |
Mononchus spp. | 13–23b | 0.8–1.4b | 59–77 | 8.9–11.8 | |
Coomansus spp. | 70–71c | 4.3–4.4c | 6–8d | 0.9–1.2d | |
Parkellus spp. | 24 | 1.5 | 34–54 | 5.1–8.2 | |
Mylonchulus spp. | 3–52 | 0.2–3.2 | 69 | 10.1 | |
Intergeneric | Mononchus spp. vs Coomansus spp. | 76–80 | 4.7–4.9 | 140–158d | 21.2–22.8d |
Coomansus spp. vs Parkellus spp. | 27–35e | 1.7–2.2e | 64–74d | 9.7–11.2d |
Comparative sequence analyses also provided support for the position in the phylogenies of the isolate identified as C. gerlachei (GenBank: KM092523 and KM092524) by
Genetic divergence estimates in 28S rDNA also indicated that C. batxatensis 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 Mylonchulus, i.e., 34–77 nt positions or 5.1–11.8%; Table
To the best of our knowledge, the present study is the first to apply an integrative 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. parvus, 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 together with published sequences for these species irrespective of the ribosomal locus (Figs
An alternative hypothesis for the phylogenetic results based on 18S rDNA is that the specimens of M. aquaticus sequenced by
We highlight that the new species described here could be clearly distinguished morphologically from M. aquaticus (sensu stricto) and that currently M. aquaticus likely represents a composite species and this may result in misidentifications of the isolates subjected to sequencing. For example, in the 28 rDNA tree of
The isolate of C. gerlachei sequenced by
At the generic and suprageneric level, the present 18S and 28S phylogenies both recovered the Mononchidae as paraphyletic (as in
The authors declare that they have no competing interests.
Not applicable.
This study was partially funded by a PhD grant to Stela Altash (Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences).
Stela Altash: Investigation, Formal analysis, Visualisation, Writing – original draft, Writing – review and editing, Funding acquisition. Aneta Kostadinova: Conceptualization, Methodology, Data curation, Formal analysis, Supervision, Writing – review and editing. Vlada Peneva: Conceptualization, Methodology, Data curation, Visualisation, Funding acquisition, Supervision, Writing – review and editing.
Aneta Kostadinova  https://orcid.org/0000-0001-7070-4968
Photomicrographs of sequenced specimens of Coomansus parvus, Mononchus pseudoaquaticus sp. nov., and M. truncatus
Data type: pdf
Comparative morphometric data for females of Coomansus parvus, Mononchus aquaticus, M. pseudoaquaticus sp. nov., and Mononchus truncatus
Data type: pdf