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183Russian Journal of Nematology, 2017, 25 (2), 129 - 140
Phasmarhabditis meridionalis sp. n. (Nematoda: Rhabditidae) from a land snail Quantula striata (Gastropoda: Dyakiidae) from southern Vietnam
Elena S. Ivanova and Sergei E. Spiridonov
Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 119071, Moscow, Russia e-mail: elena_s_ivanova@rambler.ru
Accepted for publication 28 November 2017
Summary. A new nematode species, Phasmarhabditis meridionalis sp. n., was isolated from the land snail Quantula striata in Cat Tien Natural Park in southern Vietnam. The nematode is characterised by a wide stoma in adult nematodes, a cupola-shaped female tail with a filamentous spike, long, thin, slightly projecting phasmids in females and males with the longest spicules (76 (71-83) pm) within the genus featuring hook-like distal tips. Dauer larvae of P. meridionalis sp. n. are 839 (770-912) pm long; a lateral field in adults is a simple narrow band with marginal, slightly elevated ridges and, in dauer larvae, expressed as a central band flanked by 4 ridges (3 incisions) at each side. The molecular analysis based on partial sequences of LSU, SSU and ITS rDNA regions has been performed. Both morphologically and genetically, the new species is close to another Asian species, P. huizhouensis Huang, Ye, Ren & Zhao (2015).
Key words: description, ITS rDNA sequences, LSU sequence, molecular, Mollusca, morphology, morphometrics, new species, Pellioditis, phylogeny, SSU, taxonomy.
A new species of the genus Phasmarhabditis Andrassy, 1976 was found in the pallial cavity of a terrestrial bioluminescent gastropod, Quantula striata (Gray, 1834) (Gastropoda: Styllomatophora: Dyakiidae), in Cat Tien Natural Park in southern Vietnam in January 2017. Recently, a number of new species of the genus was described all but one in close association with slugs (Tandigan et al., 2014, 2016; Nermuf et al., 2016a, b, 2017; Huang et al., 2015). Except for P. huizhouensis Huang, Ye, Ren & Zhao, 2015 found in rotten leaves, all Phasmarhabditis species have similar host associations. Sudhaus (2011) has considered the relationships between these nematodes and their gastropod and earthworm hosts as necromenic. According to Huang et al. (2015), members of Phasmarhabditis either are slug-parasites or associated with slugs, snails or earthworms.
The situation with the status of Phasmarhabditis was discussed in Huang et al. (2015) who showed that the revised Pellioditis sensu Sudhaus "is a taxon basically equivalent to Phasmarhabditis sensu Andrassy containing stem species of the 'Papillosa'
group, i.e. P. papillosa, P. hermaphrodita and P. neopapillosa". Despite the priority of Pellioditis over Phasmarhabditis, all recently discovered novel species were treated under the name of Phasmarhabditis. To avoid further taxonomic confusion in the present paper, we will use this name as well. Moreover, Nermuf et al. (2016a) suggested that synonymy of Pellioditis Dougherty, 1953 and Phasmarhabditis Andrassy, 1976 'can be rejected' based on molecular studies. The opinion on the monophyly of this group of nematodes in the recent studies based on the molecular approach (Tandigan et al., 2014, 2016; Huang et al., 2015; Nermuf et al., 2016a, b, 2017) was controversial.
MATERIAL AND METHODS
Two specimens of medium-sized snail, Q. striata were collected in Cat Tien Natural Park in southern Vietnam in January 2017. Snails were dissected and nematode _ juveniles found in the mantle cavity, three in each snail. One juvenile from each snail was frozen individually for DNA extraction and the rest was used for propagation. Each pair of juveniles
was placed in a Petri dish containing small parts of the snail body on a wet filter paper and kept at room temperature. The progeny was collected and preserved in hot 4-5% formalin for morphological examination and several specimens were also frozen for DNA extraction. After depletion of the food resources, nematodes died away (after 3 weeks of growth) before the mass production of infective juveniles occurred. Live nematodes from the culture were photographed using a Leica microscope equipped with a digital camera. Nematodes preserved in formalin were processed to anhydrous glycerin for light microscopy as described by Seinhorst (1959).
Light microscopic studies and drawings were done using Nicon Eclipse 200 microscope equipped with a drawing attachment. Scale bars are given in micrometres (pm). Abbreviations: V% - distance from anterior extremity to vulva to body length in %; a, b, c - de Manian indices. Illustrations were prepared using WACOM Intuos A4 USB drawing tablet and Adobe Illustrator CS5. For the SEM studies, formalin-preserved material was dehydrated, critical point dried and coated with gold. Images were taken on a CamScan (Cambridge).
Molecular characterisation and phylogenetic analysis. For DNA extraction, juveniles recovered from the snail and mature females and males from cultures were frozen individually in 0.7 ml Eppendorf tubes. DNA was extracted according to the method described by Holterman et al. (2006).
The worm-lysis solution (950 pl of a mixture of 2 ml of 1M NaCl, 2 ml of 1M Tris-HCl, pH 8 plus 5.5 ml of deionised water plus 10 pl of mercaptoethanol and 40 pl of proteinase K, 20 mg ml-1) was prepared directly before DNA extraction. Aliquots of 25 pl of sterile water and 25 pl of worm-lysis solution were added to each tube with a nematode and incubated at 65°C for 90 min. The tubes containing the homogenate were then incubated at 99°C for 5 min to deactivate proteinase K. About 1.0 pl of homogenate was used as PCR template.
PCR reactions were performed using Encyclo Plus PCR kit (Evrogen®, Moscow, Russia) according to the manufacturer's protocol. Primer pairs Nem18S_F (5'-CGC GAA TRG CTC ATT A CA AC A GC-3') and Nem18S_R (5'-GGG CGG TAT CTG ATC GCC-3') were used to obtain partial (about 900 bp long) sequence of 5' half of the mitochondrial 18S rDNA (Floyd et al., 2005). PCR cycling parameters included primary denaturation at 94°C for 5 min followed by 34 cycles 94°C for 45 s, 54°C for 60 s and 72°C for 1 min, followed by postamplification extension at 72°C for 3 min.
A pair of primers LSU391 (5'-AGC GGA GGA AAA GAA ACT AA-3') and LSU501 (5'-TCG GAA GGA ACC AGC TAC TA-3') was used to amplify a 1100 bp long sequence of D2D3 expansion segment of 28S rDNA (Nadler et al., 2000). PCR cycling parameters included denaturation at 95°C for 4 min, followed by 35 cycles of 94°C for 30 s, 54°C for 35 s, and 72°C for 70 s.
Table 1. Morphometrics of Phasmarhabditis meridionalis sp. n. Measurements are in pm and in the form mean (range).
Female Male
Character Dauer larva
Holotype Paratypes Paratypes
n 8 9 9
L 1728 1612+294 (1057-1931) 1317+140 (1159-1526) 839+45 (770-912)
a 19.9 17.8+3.1 (14.3-24.6) 22.1+2.0 (19.2-25.8) 24.5+1.2 (23-25.8)
b 7.9 7.6+1 (5.6-8.5) 7.1+0.5 (6.5-7.6) 5.4+0.2 (5.3-5.7)
c 22.2 24.2+2.3 (19.0-32.9) 41.5+3.9 (34.2-46.8) 7.4+0.5 (7.0-8.0)
V% 56.1 52.5+1.8 (50.3-56.1) - -
Mid-body diam. 87 94+27 (43-134) 60+7 (45-67) 34+2 (32-38)
Pharynx length 220 212+16 (185-230) 185+11 (174-208) 156+5 (149-164)
Head to excretory pore 192 172+23 (142-210) 176+13 (164-190) 125+4 (120-130)
Head to nerve ring 168 154+16 (133-180) 141+7 (135-154) 114+6 (103-122)
Tail length 78 68+17 (44-97) 32+5 (25-38) 116+13 (100-135)
Spicule length (chord) - - 76+4 (71-83) -
Gubernaculum length - - 43+2 (40-46) -
Egg length 45 53+5 (45-58) - -
Egg width 35 35+3 (32-40) - -
A pair of primers TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') was used to amplify approx. 800 bp long sequence of ITS region of ribosomal DNA (Curran & Driver, 1994). PCR cycling parameters included primary denaturation at 95°C for 5 min followed by 35 cycles of 94°C for 45 s, 56°C for 60 s and 72°C for 70 s.
PCR products were visualised in agarose gel and bands were excised for DNA extraction with Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA). Samples were directly sequenced using the same primers as used for primary PCR. The sequences were combined and aligned using the Clustal_X program after the addition of sequences from the GenBank (Thompson et al., 1997). Subsequently, the sequences were edited using the Genedoc 2.7 program (Nicholas et al., 1997), to prepare a file for the analysis in MEGA5 (Tamura et al., 2011). Phylogenetic trees were obtained with different methods (MP - maximum parsimony, NJ -neighbour joining and ML - maximum likelihood) and pairwise nucleotide differences were calculated. Obtained sequences were deposited in GenBank (MG543981 for the18S rDNA sequence; MG543921 for the 28S rDNA and MG543920 for the partial 28S rDNA.
Etymology. The specific epithet reflects the southern location of Cat Tien, home to the host of the new nematode.
DESCRIPTION
Phasmarhabditis meridionalis sp. n.
(Figs 1-3)
Adults. Body robust, 1.1-2.2 mm long, straight when relaxed, slightly tapering to anterior. Cuticle about 1 pm thick, bearing rows of transversal and longitudinal striations. Longitudinal striations appearing at some distance from anterior, usually posterior to 20th annulus. Lateral fields narrow (ca 2 pm wide), simple, narrow band with 2 marginal ridges appearing posterior to 15th annulus. Deirid located at level of cardia. Head truncate, lip region short, not or very slightly offset. Six lips arranged in three groups, each lip bearing a small, short labial papilla in apical position. Each sublateral lip bearing small cephalic papilla located slightly posterior to labial one. Pore-like amphids situated close to labial papillae on lateral lips. Mouth aperture triangular. Stoma tubular, wide and short (about as long as lip region diameter). Cheilostom not cuticularised. Gymnostom walls parallel, thickened. Stegostom with glottoid apparatus, isomorphic, isotropic,
metarhabdions thickened, with three minute warts each. Pharyngeal collar narrow, covering two thirds of stoma length. Cheilostom: gymnostom: stegostom ratio 1:2.5:1. Pharynx muscular, differentiated into corpus expanded posteriorly (median bulb), stout, well defined isthmus and small, pear-shaped terminal bulb as wide as metacorpal expansion or slightly narrower. Terminal bulb with valve and haustrulum. Nerve ring surrounding middle to posterior region of isthmus. Excretory pore position variable, situated from just posterior to nerve ring to level to pharynx base, often obscure. Excretory duct short, weak. Cardia prominent, projecting into intestine. Intestine well developed, forming proventriculus at anterior. Rectal glands present.
Female. Body straight after fixation. Lateral fields running to the end of rounded part of tail or to the base of tail terminus. Stoma 7.2 ± 0.4 (7-8) pm wide and 18.8 ± 1 (18-20) pm long. Cheilostom 4 ± 0.5 (3-5) pm long, gymnostom 10.4 ± 1 (9-12) pm long, stegostom 4.3 ± 0.9 (3-6) pm long. Corpus 119 ± 7 (107-127) pm long or corresponding to 5461 (57%) of pharynx length, ca 19 pm wide at anterior, metacorpal expansion 31 ± 6 (20-40) pm; isthmus 54 ± 7 (40-60) pm long and 14 ± 2 (10-17) pm wide; terminal bulb 41 ± 5 (33-50) pm long and 35 ± 5 (26-42) pm wide. Excretory pore situated at 172 ± 23 (142-210) pm from anterior body end. Oviparous. Gonads amphidelphic, ovaries reflexed on dorsal side. Ovary branches equally long. Oocytes rounded, large, arranged in two or three rows. Oviduct short. Spermathecae filled with sperm cells. Uterus spacious, containing 20-30 eggs, 53 ± 5 (50-67) pm long and 36 ± 3 (30-40) pm wide and with thin, smooth shells. Vagina straight, muscular, less than half corresponding body diam. long or 35 ± 13 (18-55) pm. Vulva median, a wide transverse slit with flat lips. Massive copulatory plug over vulva present in fertilised specimens. Rectum inflated, about corresponding body diameter long. Anus an arcuate slit. Tail end cupola-shaped with filamentous terminus 40 ± 8 (27-50) pm long, corresponding to ca 67% of total tail length. Phasmids thin, slightly projecting, located at terminus base.
Male. Slightly shorter and much slimmer than females. Lateral field posteriorly reaching bursa level. Stoma 6 ± 1 (5-7) pm wide and 17 ± 1 (15-18) pm long. Corpus 107 ± 9 (90-118) pm long or corresponding to ca 58% of pharynx length, 15 ± 2 (12-17) pm wide anteriorly. Metacorpal expansion (bulb) prominent, 24 ± 3 (19-26) pm wide. Isthmus 41 ± 7 (35-50) pm long and 13 ± 1 (11-14) pm wide. Basal bulb 34 ± 7 (27-51) pm long and 27 ± 3 (22-30) pm
Fig. 1. Phasmarhabditis meridionalis sp. n. Female. A, B, C: head, lateral and sublateral; D: anterior end; E: gonad of a mature female; F: gonad of a young female; G: tail lateral; H: tail, ventral; I: entire worm. Scale bars = 10 pm.
Fig. 2. Phasmarhabditis meridionalis sp. n. Male (A-F) and dauer larva (G-I). A: anterior end, lateral; B, C: head, subventral and lateral; D: lateral field showing deirid; E, F: tail, lateral; G: anterior end of ensheathed larva; H: anterior end of exsheated larva; I: tail of ensheathed larva. Scale bars = 10 pm. Caudal papillae are numbered.
wide. Excretory pore poorly conspicuous, situated just posterior to isthmus. Spermatocytes rounded, arranged in two rows. Vas deferens wide, filled with large immature sperm cells ca 7 x 5 pm in size, ejaculatory duct separating from latter by constriction. Single testis reflexed at 316 + 38 (260372) pm from anterior, reflexion 271 + 33 (233338) pm long. Sperm cells ca 4-6 pm in diam. Bursa open, peloderan. Nine pairs of pedunculate genital papillae (GP) incorporated into bursa (formula 1+1+1+2/1+3+ph). GP 8 and 9 staggered. GP 4 and GP 8 longest and opening dorsally. GP 57 and GP 9 not reaching the edge of bursa. Tail tip reaching the edge of bursa. Phasmids posterior to GP 9. Three pericloacal papillae present: precloacal papilla located on the anterior cloacal lip in median or submedian position and two process-like papillae situated at lateral margins of cloacal opening. Spicules nearly straight, uniformly thick at most length, tapering to tips and at least twice longer than corresponding body diameter, with small manubria about 4 pm long and wide. Lamina mean 8 (6-10) pm wide. Velum thin, almost indiscernible. Distal tips hook-like, acute, curved towards ventral body surface. Gubernaculum boat-shaped, about twice shorter than spicules; dorsal process not present; small, distinct, ventral process bent posteriad always present.
Dauer larva (ensheathed). Body slender, tapering gently towards head end. Cuticle with transversal and longitudinal striations; longitudinal striations appearing posterior to the 20-22th annulus. Lateral field with central flat band bearing low, almost indistinct ridge in the middle. Band flanked with four closely situated ridges by each side. Head rounded, lip region flat, not offset from body contour; 6 cephalic papillae; amphidial apertures situated slightly posterior to circle of head papillae. Mouth aperture enclosed. Stoma 19 + 1 (18-22) pm long and 3.3 + 0.5 (3-4) pm wide; cheilostom ca 4 pm long, not cuticularised, gymnostom ca 15-16 pm long, strongly cuticularised, stegostom ca 2-3 pm long with cuticularised metarhabdions. Pharynx comprising straight corpus 10 + 1 (8-11) pm wide and 95 + 3 (90-98) pm long with metacorpal expansion 15 + 1 (13-17) pm wide, isthmus 38 + 3 (35-42) pm long and 8 + 1 (6-9) pm wide and pear-shaped, valvated basal bulb 23 + 3 (18-27) pm long and 19 + 4 (15-25) pm wide. Nerve ring surrounding isthmus in 114 + 6 (103-122) pm from anterior end. Cardia ca 7 pm long, elongated, protruding into intestine. Excretory pore not observed. Deirids inconspicuous. Intestine collapsed, filled with fat globules. Genital primordium at mid-body ca 32 pm long. Tail
conical, attenuated. Rectum collapsed. Phasmids pore-like, situated in 26-49 pm from anus.
Exsheathed juveniles. Similar to ensheated dauer larvae in general morphology. Stoma open. Cardia smaller, not elongated. Intestine with large proventriculus ca 45-50 pm long. Genital primordium 17-30 pm long. Rectum inflated, ca 24 pm long.
II stage juvenile or early III stage juvenile.
Body 593 pm long and 25 pm wide. Stoma 18 pm long and 3 pm wide (cheilostom 3 pm long, gymnostom 12 pm long, stegostom 3 pm long). Pharynx 125 pm long with similar to other stages structure. Genital primordium 12 pm long.
Type material. Holotype female and paratype male, accession nos 1293 and 1294 are deposited in the Museum of the Helminthological Collections of the Centre of Parasitology at the Severtsov Institute of Ecology and Evolution, Moscow.
Type host. Quantula striata (Gray, 1834) (Gastropoda, Dyakiidae).
Type locality. Cat Tien National Park, 11°30' N, 107°20' E, collected in January 2017 by I.I. Semenyuk.
Diagnosis and relationships. Phasmarhabditis
meridionalis sp. n. is recognised by its morphometrics, flat lips, a wide rhabditoid stoma, a high collar, a cupola-shaped female tail with a filamentous spike, long, thin, slightly projecting phasmids, lateral field in adults a simple narrow band with marginal, slightly elevated ridges and in dauer larvae, expressed as a central band flanked by 4 ridges (3 incisions) at each side, with nearly indistinct, low ridge running in the centre of the band; long spicules with hook-like distal tips, dauer larvae possessing a long cylindrical stoma and a filamentous tail and the distinct molecular characteristics of the new species.
The genus Phasmarhabditis Andrassy, 1976 currently comprises the following species: P. hermaphrodita (Schneider, 1859) Andrassy 1983, P. neopapillosa (Mengert in Osche, 1952) Andrassy 1983, P. papillosa (Schneider, 1866) Andrassy, 1976, P. huizhouensis Huang, Ye, Ren & Zhao, 2015, P. tawfiki (Azzam, 2003), P. californica Tandingan De Ley, Holovachov, McDonnell, Bert, Paine & De Ley, 2016, P. bonaquaense Nermuf, Puza, Mekete & Mracek, 2016, P. apuliae Nermuf, Puza & Mracek, 2016 and P. bohemica Nermuf, Puza, Mekete & Mracek, 2017.
Morphologically, Phasmarhabditis meridionalis sp. n. is most closely related to P. huizhouensis having the similar shape of a female tail albeit with the relatively longer filiform terminus (the ratio a wider part of tail/terminus being 2:3 vs 1:1). Both
species also have similarly sized spicules. However, spicules differ in the shape of the distal tip, acutely hooked in the new species vs obtuse with the shallow indentation in P. huizhouensis and female phasmids of the new species project less outside the body contour. The cuticularised cheilostom reported for P. huizhouensis was not observed in P. meridionalis sp. n. as well as in the rest of the species of Phasmarhabditis (Huang et al., 2015).
In having the similar shape of a female tail, spicules of similar length and shape and similar body length of dauer larvae (839 pm vs 902 pm), P. meridionalis sp. n. is also close to P. bonaquaense but can be distinguished by generally smaller body size of adult nematodes, less projecting and thinner phasmids in the female, lateral fields in adult nematodes representing a narrow band with elevated ridges vs more elaborated ones with about 14 incisions and much shorter tails of both sexes (32 pm vs 51 pm, male and 63 pm vs 85 pm, female). The dauer larvae of the new species, contrary to these of P. bonaquaense, possess differently structured lateral fields (formed by rows of lower ridges on the sides of a central narrow band bearing even lower central ridge vs two prominent ridges with wide band (incisure) between them) (Nermuf et al., 2016).
From another species with a cupola-shaped female tail, P. papillosa, P. meridionalis sp. n. differs by the significantly shorter female tail (mean 63 pm vs 106 pm) with different proportions, i.e. having a shorter, perfectly rounded wider part with the much longer and thinner filiform terminus while in P. papillosa, the wider part is rather conoid and the tail spike sturdier and is as long as the wider part of the tail. It is also differentiated from P. papillosa in having much longer spicules (av. 76 pm vs 27.7 pm) and a gubernaculum (av. 43 pm vs 23 pm) and flat vs inflated vulval lips (Tandingan De Ley et al., 2016).
In having the gonochoristic mode of reproduction and a dome-shaped vs a widely conoid female tail, the present species can be differentiated from hermaphroditic species of P. hermaphrodita and P. californica (Tandingan De Ley et al., 2016).
From the rest of species, i.e. P. neopapillosa, P. tawfiki, P. bohemica and P. apuliae, the new species can immediately be distinguished in having a dome-shaped with a filiform terminus female tail vs conoid one. By the body length of dauer larvae (mean 839 pm), P. meridionalis sp. n. is comparable with P. neopapillosa (mean 1010 pm), P. tawfiki (mean 965 pm), and P. apuliae (mean 812 pm and 986 pm in different strains) (Hooper et al., 1999;
Azzam, 2003, Nermuf et al., 2016a), all with a conoid female tail.
Molecular analysis and phylogenetic position of
P. meridionalis sp. n. The sequences of expected length were obtained for all three studied loci. The BLAST search for the obtained sequences supported the primary morphological identification as all the tested sequences demonstrated the closest relationships with those of Phasmarhabditis nematodes and, in some cases, nematodes of the related genus Angiostoma. All the sequences of nematodes of these two genera together with those of some other Rhabditida were downloaded as FASTA-files from NCBI site and used for alignment construction. A matrix of nucleotide data with the length of 853 bp was obtained for 18S rDNA sequences (flanking parts of unequal length were discarded). With 588 characters of this alignment being constant and 52 variable characters being parsimony-uninformative, the total number of parsimony-informative characters was 213. A matrix of nucleotide data with the length of 813 bp was obtained for D2-D3 28S rDNA. With 713 characters of this alignment being constant and 59 variable characters parsimony-uninformative, the total number of parsimony-informative characters was 41. A matrix of nucleotide data with the length 743 bp was obtained for ITS rDNA including 5.8S. With 257 characters of this alignment being constant and 235 variable characters being parsimony-uninformative, the total number of parsimony-informative characters was 251. Phylogenetic analysis of 18S rDNA data detected two closest sequences to that of Phasmarhabditis meridionalis sp. n.: KP017252 obtained for P. huizhouensis and KM510210 for Phasmarhabditis sp. 'ITD046'. Both these sequences have differed from our sequence in 26 bp while the differences with two sequences obtained from Phasmarhabditis collected in Czech Republic were equal to 27 bp. The majority of other Phasmarabditis sequences identified as the closest ones with BLAST search differed from the newly found species in 32-37 bp. Under all types of phylogenetic analysis, the 18S rDNA sequences of P. meridionalis sp. n. were found clustering together with P. huizhouensis from China under strong or medium support (Fig. 4). Surprisingly, it was a 28S rDNA sequence for Pellioditis sp. (KJ877242), which was identified with BLAST as the most similar one for the newly described species (32 bp difference). All the Phasmarhabditis species differed from the new species from Vietnam in 42-47 bp in this analysis. Still, the phylogenetic analysis demonstrated stable clustering with the low bootstrap support of P.
Fig. 3. Phasmarhabditis meridionalis sp. n. SEM images. A-D: dauer larva. A, B: head; C: cuticle; D: lateral field; E, F: female tail; G, H: male tail. Arrows indicating amphid (A) and phasmid (E). Scale bars in pm.
PelHoditis marina AF083021 100/100/100
100/100/100
G4/-/57
95/93/63
100/100/100
89/92/88
93/97/84
67/67/70
Agfaflexilis EU573704 Agfa flexiiis MF192956 Agfaflexilis HQ115063 •Phasmarhabditis sp. MF192961 Angiostoma limacis EU573705
Angiostoma margaretae MF192958 Angiostoma norvegicum KU712560
I Phasmarhabditis bohémica hapl. 1 KX017478 Phasmarhabditis bohémica haol. 2 KX017479
Phasmarhabditis bohémica hapl. Phasmarhabditis sp. EU196008 Phasmarhabditis californica KM510210 Phasmarhabditis bonaquense KX01748Q Phasmarhabditis apuliae KX017477 Phasmarhabditis papillosa KM510211 Phasmarhabditis sp, KX267673
Phasmarhabditis huizhouensis KP017252 Phasmarhabditis meridionalis sp, n. г Phasmarhabditis neopapillosa DQ639982 "IPhasmarhabditis neopapillosa FJ516754 Phasmarhabditis hermaphrodita FJ516755 Phasmarhabditis hermaphrodita KM 510209 Phasmarhabditis hermaphrodita DQ639981 Phasmarhabditis hermaphrodita DQ639980 Oscheius carolinense FJ547240 Oscheius insectívora AF083019
10
Fig. 4. Phylogenetic relationships of Phasmarhabditis meridionalis sp. n. based on 18S SSU rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), GTR+G model. For MP and NJ - 1000 bootstrap replications. Angiostoma clade with three species was absent in NJ analysis.
-Pellioditis sp. KJ877242
Phasmarhabditis sp. KX017485
99/100/95 Phasmarhabditis apuliae KX017482
Phasmarhabditis sp. MF806606
100/100/100
95/92/87
75/73/71
Phasmarhabditis sp. EU195967
55/61/73
Phasmarhabditis meridionalis sp. n.
Phasmarhabditis huizhouensis KP017253 Angiostoma norvegicum KU712561
100/100/100 I Phasmarhabditis bohémica KX017484 Phasmarhabditis sp, KX017483
Fig. 5. Phylogenetic relationships of Phasmarhabditis meridionalis sp. n. based on 28S LSU rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), K2+G model. For MP and NJ - 1000 bootstrap replications.
Ancylostoma caninum KC755029
Angiostomo margaretae MF192968
Phasmarhabditis meridionalis sp. n.
100/100/100 , Phasmarhabditis bohemica KX017490
100/100/98
100/100/100
q
100/98/100
100/100/100
100/100/100
Phasmarhabditis bohemica KX017489 Phasmarhabditis neopapillosa FJ516760 Phasmarhabditis hermaphrodita KM510201 Phasmarhabditis hermaphrodita FJ516761 Phasmarhabditis hermaphrodita KM510202
Phasmarhabditis californica KM510203 Phasmarhabditis californica KM510204 Phasmarhabditis sp. KX017491
Phasmarhabditis papillosa KM510205 Phasmarhabditis sp. KX267675
Phasmarhabditis sp. KX017486 Phasmarhabditis apuiiae KX017488 Phasmarhabditis sp. KX017487
■ rnu if
10
Fig. 6. Phylogenetic relationships of Phasmarhabditis meridionalis sp. n. based on ITS rDNA. Bootstrap support values are presented near nodes as MP/NJ/ML. ML analysis (500 bootstrap replications), T93 model. For MP and NJ - 1000 bootstrap replications.
meridionalis sp. n. with P. huizhouensis (Fig. 5). The phylogenetic tree based on the analysis of ITS rDNA demonstrated much stronger bootstrap support for the main Phasmarhabditis clades (Fig. 6) with corresponding nucleotide differences in 141152 bp.
DISCUSSION
The morphology of Phasmarhabditis species is conservative and there are few traits that proved helpful for distinguishing between the species. Pellioditis sensu Sudhaus (Sudhaus, 2011) includes P. hermaphrodita, P. incilaria (Yokoo & Shinohara in Shinohara & Yokoo, 1958), P. mairei (Maupas, 1919), P. neopapillosa, P. papillosa and P. tawfiki. Diagnostic characters given by Sudhaus (2011) for the species of Pellioditis nearly perfectly conform with the morphology of those species of Phasmarhabditis described after his publication: "Buccal tube relatively short and wide; glottoid apparatus with warts; sleeve, median bulb; amphidelphic; female tail cupola-shaped or short conical; phasmids papilliform and projecting from tail; bursa well developed, peloderan, anteriorly open, nine GPs, formula 1+1+1+2/1+3+ph, three precloacal rays evenly spaced, GP 4 and GP 5 very close and staggered, often positioned precloacally, anterior and posterior
dorsal papillae GP 5 and GP 9; phasmids posterior to GP 9, papilliform; spicules separate, dagger-shaped. Necromenic in earthworms, slugs and snails". The only departure from this is in the structure of glottoid apparatus as warts were not reported in all the species (P. huizhouensis). Such description of diagnostic characters in relation to Phasmarhabditis covers most of its morphology and leaves very little extra to add. However, the characteristics of dauer larvae are generally ignored, possibly owing to the fact that not all species descriptions included descriptions of this stage. We believe that certain morphological characteristics of dauer larvae can provide additional means for differentiation and should be taken into consideration if possible. Similar to infective juveniles of entomopathogenic nematodes of Steinernema and Heterorhabditis genera, body length, stoma size and a lateral field structure should be considered.
Three loci of ribosomal DNA were studied in the course of the study on P. meridionalis sp. n. All these three phylogenetic markers have confirmed the affiliation of the newly found species with Phasmarhabditis. In the same time, in two analyses (18S and 28S rDNA) the sequences obtained from Angiostoma nematodes formed common clades with the species of Phasmarhabditis, while such a pattern
was not observed in the ITS rDNA phylograms. However, in this analysis P. meridionalis sp. n. failed to join any of the inner Phasmarhabditis clades and formed an independent branch connected to the basal node for all other Phasmarhabditis with known ITS rDNA nucleotide data. Unfortunately, ITS rDNA data were not obtained for P. huizhouensis from China and thus this aspect could not be compared. Such pattern of phylogenetic links between Angiostoma and Phasmarhabditis as seen in 18S and 28S rDNA was reported earlier by Nermuf et al. (2016a, b, 2017). We can only speculate whether such adherence of the nematodes with prominently different morphology and life style can be explained by the low level of nucleotide differences in these DNA regions in the taxa under study. The differences in ITS rDNA are more prominent, and the majority of Phasmarhabditis species are united in well supported clades. However, the support for the basal node for three main clades of Phasmarhabditis is negligible resulting in the position of P. meridionalis sp. n. outside of these clades (Fig. 6). There is undoubtedly tight phylogenetic relationships of three genera of rhabditids with the trophic relationships with molluscs (Agfa, Angiostoma and Phasmarhabditis). More data are needed, especially for aberrant forms to explain the ambiguity of the 'molecular' gaps between nematodes with such pronounced morphological differences.
ACKNOWLEDGEMENTS
We are grateful to the staff of Cat Tien Natural Park for the permission to collect the snails used in the present study and to the Russian-Vietnamese Tropical Centre for the possibility to study these nematodes. We are indebted to Prof. A.A. Schileiko for the identification of snails. The authors also acknowledge the financial support from a grant from the Russian Fund for Basic Research 17-04-00095-a.
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