Morphological and Molecular Characterization of Oscheius saproxylicus sp. n. (Rhabditida, Rhabditidae) From Decaying Wood in Spain, With New Insights into the Phylogeny of the Genus and a Revision of its Taxonomy
Article Category: Arts & Humanities
Online veröffentlicht: 17. Sept. 2019
Seitenbereich: 1 - 21
Eingereicht: 13. Nov. 2018
DOI: https://doi.org/10.21307/jofnem-2019-053
© 2019 Joaquín Abolafia et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
Andrássy (1976) proposed the new genus
Sudhaus and Hooper (1994) provided new ideas about the taxonomy and the phylogeny of several rhabditid species: (i) accepted
Molecular data of
An
Nematodes were collected from dead wood using a modified trays technique (Whitehead and Hemming, 1965), killed by heat, fixed in 4% formalin, transferred to pure glycerine following the Siddiqi’s (1964) method, and mounted on permanent glass slides. Moist, dead wood was maintained as a culture to extract specimens every several months.
Observations were made using a Leitz Laborlux S (Leitz, Wetzlar, Germany) and Nikon Eclipse 80i (Nikon, Tokio, Japan) microscopes. Measurements were taken with the Leitz microscope, which has a drawing tube (
Specimens preserved in glycerine were selected for observation under SEM according to Abolafia (2015). They were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).
Nematode DNA was extracted from single fresh individuals using the proteinase K protocol and PCR assays as described Castillo et al. (2003) somewhat modified. Specimen was cut in small pieces using a sterilized dental needle on a clean slide with 18 ml of AE buffer (10 mM Tris-Cl + 0.5 mM EDTA; pH 9.0), transferred to a microtube and adding 2 μl proteinase K (700 μg/ml) (Roche, Basel, Switzerland), and stored to –80°C within 15 min (for several days). The microtubes were incubated at 65°C (1 hr), then at 95°C (15 min). The microtube was centrifuged to 13,000 r.p.m. (or 15,900 ×
For phylogenetic relationships, analyses were based on 18S and 28S rDNA. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with another 18S or 28S rRNA gene sequences available in GenBank using Muscle alignment tool implemented in the MEGA7 (Kumar et al., 2016). The ambiguously aligned parts and divergent regions were known using the online version of Gblocks 0.91b (Castresana, 2000) (
Figure 1:
Figure 2:
Figure 3:
Fifty one females in generally acceptable state of preservation.
See Table 1.
Morphometrics of
| Locality | Puente de la Sierra | |
|---|---|---|
| Province | Jaén | |
| Habitat | Dead wood | |
| n | Holotype female | Paratypes 50 females |
| Body length | 829 | 837 ± 78.4 (669–994) |
| a | 37.7 | 29.6 ± 3.2 (23.9–37.1) |
| b | 5.1 | 5.0 ± 0.5 (3.9–6.5) |
| c | 11.5 | 11.0 ± 1.2 (8.7–13.8) |
| c' | 5.5 | 5.5 ± 0.5 (5.0–7.0) |
| V | 57 | 54.8 ± 2.4 (48–59) |
| Lip region width | 9 | 9.6 ± 0.5 (9–10) |
| Stoma length | 18 | 18.7 ± 1.0 (17–21) |
| Stoma width | 5 | 5.8 ± 0.6 (5–7) |
| Pharyngeal corpus length | 72 | 75.9 ± 6.5 (68–96) |
| Isthmus length | 48 | 47.7 ± 3.9 (39–54) |
| Bulbus length | 26 | 26.6 ± 1.4 (24–29) |
| Pharynx length | 146 | 149 ± 9.4 (113–165) |
| Neck length | 164 | 168 ± 9.6 (131–182) |
| Body diameter at neck base | 22 | 23.9 ± 1.7 (20–28) |
| Body diameter at midbody | 22 | 28.5 ± 3.4 (21–37) |
| Vulva - anterior end | 469 | 459 ± 49.0 (366–549) |
| Rectum length | 48 | 44.8 ± 3.8 (40–54) |
| Anal body diameter | 13 | 13.9 ± 1.1 (11–16) |
| Tail length | 72 | 76.3 ± 5.0 (67–88) |
| Phasmid - anus distance | 19 | 23.9 ± 3.4 (20–30) |
Notes: Demanian indices (de Man, 1880): a = body length/body diameter; b = body length/pharynx length; c = body length/tail length; c’ = tail length/anal body diameter; V = (distance from anterior region to vulva/body length) × 100.
Description.
Moderately slender to slender (
Unknown.
During the culture of nematodes in dead wood under wet conditions, hundreds of
One 923 bp 18S rDNA sequence (GenBank accession number MK959600) and three identical 637 bp without changes or deletions each other 28S rDNA sequences (GenBank accession numbers MK959601-MK959603) were obtained from three specimens. Both trees show
The new species is characterized by its 0.67 to 0.99 mm long body, cuticle with very fine transverse striation, lateral field with three longitudinal alae, lip region 9 to 10 µm broad and consisting of six separated lips, stoma 17–21 × 5–7 µm with barrel-shaped gymnoprostegostom, neck 131 to 182 µm long, pharynx cylindrical with metacorpus not swollen and broad isthmus, excretory pore and deirids at isthmus level, female reproductive system didelphic-amphidelphic,
The absence of males in this population of the new species does not allow its classification under the
Figure 4:
Bayesian Inference tree from known and the newly sequenced
Figure 5:
Bayesian Inference tree from known and the newly sequenced
It also resembles
Spain, Jaén province, Jaén town, Puente de la Sierra (GPS coordinates: 37°42'36.5″N and 3°45'33.2″W, elevation 439 m), in association with decaying wood from dead white poplar trees present at the boundaries of an orchard.
Fourty seven females (holotype and paratypes) deposited in Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Spain; four female paratypes deposited in nematode collection of the Swedish Museum of Natural History, Stockholm (Sweden).
Evolutionary relationships of
The molecular analysis of 18S and 28S rDNA sequences of the new species herein described, whose results are presented in the trees of Figures 4 and 5, respectively, confirm the monophyly of both species groups as well as that they are sister groups. Thus,
Regarding the outer relationships of
Both morphological (Sudhaus and Hooper, 1994; Sudhaus, 2011) and molecular (among others Darby et al., 2011; Liu et al., 2012; Campos-Herrera et al., 2015; Torrini et al., 2015; Lima de Brida et al., 2017; Ye et al., 2018) evidences support the monophyly of
In the following diagnoses of the genus and its two subgenera, a list of their species and a key to their identification are presented. The diagnoses are mainly based on Sudhaus’ (2011) ideas about the concept of the genus and the differences between the two species groups. In addition, the status of several species, in particular those described in recent years, is discussed.
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Rhabditidae. Small- to medium-sized nematodes, 0.50 to 3.25 mm long. Lateral field with three to five ridges (four to six incisures). Lip region continuous. Stoma tubular, bearing glottoid apparatus with small elongate teeth. Pharynx consisting of cylindrical corpus gradually enlarging posteriorly, with not swollen metacorpus, and basal bulb bearing duplex haustrulum. Secretory–excretory duct elongated, looped and strongly sclerotized. Female genital system didelphic–amphidelphic, with equatorial vulva. Female rectum conspicuously longer than anal body diameter, proximally dilated, forming a bladder-like expansion of the hind gut (often filled with faeces). Female tail conical to conical elongate. Testis reflexed ventrally. Bursa peloderan or leptoderan, anteriorly open, with wide velum bearing nine genital papillae arranged 1+1+1/3+3, GP5 and GP8 opening dorsally (bursa formula: v1,v2,v3/v4,ad,v5–v6,pd,v7,ph). Male tail conoid with or without a short acute terminal tip out of the bursa. Phasmid posterior to the last GP, tubular. Spicules free, dagger-shaped, head and slanted shoulder, the tip thickened.
syn.
(Fig. 6).
Figure 6:
Lip region, male and female posterior ends of the species of the subgenus
Stoma tubular with metastegostom bearing warts. Bursa leptoderan. Male tail conoid with tip out of the bursa, filiform, variable in length. Spicules distally hook-shaped, like a crochet needle.
Oscheius (Oscheius) insectivorus (Körner, 1954) Andrássy, 1976
= Rhabditis
= Heterorhabditidoides (Oscheius) insectivora (Körner, 1954) Zhang et al., 2012
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syn.
(Fig. 7).
Figure 7:
Lip region, male and female posterior ends of the species of the subgenus
Stoma tubular or barrel-shaped with metastegostom bearing setose teeth. Bursa peloderan. Male tail tip not reaching beyond bursa end. Spicule tips shaped like a probe head.
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Very similar to
Nearly identical to
Much resembling
The identity of this species has raised some controversy as Andrássy (2005) considered it as a valid species whereas Sudhaus (2011) regarded it as junior synonym of
Tabassum and Shahina (2002),
Very similar morphologically to
Similar to
Morphologically, this species and
The identity of this species maintains some doubts being regarded by Andrássy (1983) as junior synonym of
Nearly identical to
Originally described as belonging to
Very similar to
Morphologically, this species and
Tahseen and Nisa (2006) mentioned this species as
Very similar to
1a – Body length very long, 6 mm
1b – Body length shorter, less than 3.5 mm 2
2a – Stoma barrel-shaped or tubular; bursa peloderan (unknown in O
2b – Stoma tubular; bursa leptoderan or pseudopeloderan 16
3a – Each spicule visibly with different size
3b – Both spicules with similar size 4
4a – Female rectum scarcely longer than the anal diameter 5
4b – Female rectum ca. 2 to 3 times longer than the anal diameter 6
5a – Body length less than 750 µm; spicules 29 µm long
5b – Body length more than 750 µm; spicules 32 to 48 µm long
6a – Female tail short conoid (
6b – Female tail longer, elongate (
7a – Lip region slightly offset by depression; female rectum longer, 3 times anal body diam.
7b – Lip region not offset; female rectum shorter, 2 times anal body diam. 8
8a – Males as frequent as females; spicules longer, 35 µm, exceeding the GP1
8b – Males very rare; spicules shorter, 22 to 30 µm, not reaching the GP1
9a – Lip region visibly narrower than adjacent part of body; spicules longer, more than 40 µm 10
9b – Lip region equal or wider than adjacent part of body; spicules shorter, less than 30 µm (unknown in
10a – Male body more than 1mm long; spicules longer, 41 to 48 µm
10b – Male body less than 1 mm long; spicules shorter, 50 to 52 µm
11a – Gymnostom anteriorly narrower, with convex walls 12
11b – Gymnostom with parallel walls 14
12a – Lip region offset by constriction; metacorpus slightly swollen
12b – Lip region not offset or slightly offset by depression; metacorpus not swollen 13
13a – Lip region slightly offset by depression; gymnostom as long as promesostegostom; pharynx with metacorpus with sclerotized walls, valves-like; spermatheca differentiated in a sac
13b – Lip region not offset; gymnostom slightly shorter than promesostegostom; pharynx with metacorpus without sclerotized walls; spermatheca not differentiated in a sac
14a – Lip region slightly offset; female rectum ca. two times longer than ABW; GP1 very anterior, outside of the range of the spicules
14b – Lip region not offset; female rectum ca. three times longer than ABW; GP1 at spicules level 15
15a – Body length slightly larger (584–801 µm long); neck slightly shorter relative to the body length (
15b – Body length slightly smaller (505–691 µm long); neck slightly longer relative to the body length (
16a – Female rectum ca. as long or slightly longer than anal body width 17
16b – Female rectum longer than body width 22
17a – GP1 very reduced 18
17b – All GPs with similar size 20
18a – Spicules distally straight
18b – Spicules distally slightly hook-like 19
19a – Female body less than 1.7 mm long
19b – Female body more than 1.8 mm long
20a – Female stoma 21 to 28 µm long
20b – Female stoma 12 to 20 µm long 21
21a – Female body 0.9 to 1.3 mm long; female tail more slender (
21b – Female body 1.3 to 1.8 mm long; female tail shorter (
22a – Stomatal tube shorter, ca. 1.0 to 2.0 times longer than wide 23
22b – Stomatal tube longer, ca. 2.5 to 3.0 times longer than wide 30
23a – Cheilostom as long as stomatal tube length
23b – Cheilostom one third of the stomatal tube length 24
24a – GP1 very separated from GP2, and GP2-3 very close
24b – GP1-2 distance similar or slightly more than GP2-3 distance 25
25a – Spicules with ventral bent tip 26
25b – Spicules with thin tip, crochet needle-like 28
26a – Spicules 43 to 52 µm, with thin tip; GP1-2 distance similar to GP2-3 distance
26b – Spicules 50 to 62 µm, with thick tip; GP1-2 distance slightly more than GP2-3 distance 27
27a – Male tail shorter (20–32 µm long)
27b – Male tail longer (38–45 µm long)
28a – Female rectum ca. 1.5 times longer than ABW; spicules shorter, 34 to 44 µm long
28b – Female rectum ca. 2.0–2.5 times longer than ABW; spicules longer, 45 to 70µm long 29
29b – Spicules shorter (45–51 µm long)
29a – Spicules longer (57–70 µm long)
30a – Spicules ventrad curved, as long as anal body width
30b – Spicules almost straight, longer than anal body width 31
31a – Spicules ca. 1.5 times longer than the gubernaculum
31b – Spicules ca. 2–3 times longer than the gubernaculum 32
32a – Spicules with manubrium longer than wide 33
32b – Spicules with manubrium as long as wide 35
33a – Lip region higher, twice wider than high
33b – Lip region lower, three times wider than high 34
34a – Stoma shorter, 9 to 10 µm; female tail shorter,
34b – Stoma longer, 13 to 18 µm; female tail longer,
35a – Female rectum ca. 3.0 to 4.5 times longer than anal body width
35b – Female rectum ca. 1.5 times longer than anal body width 36
36a – Female tail shorter,
36b – Female tail longer,
Zhou et al. (2017) recently described this species, mainly based on molecular analyses, but a reasonable doubt persists about its true identity. Three morphological features of its diagnosis better fit the pattern observed in
Originally described as