The genus
Alphabetical list of
Species | Country |
---|---|
|
The Netherlands |
|
Mexico |
|
Iran |
|
Mexico |
|
Mexico |
|
New Zealand |
|
Italy |
|
Italy |
|
New Zealand |
|
Mexico |
|
China |
|
Mexico |
|
USA (California) |
|
New Zealand |
|
New Zealand |
|
New Zealand |
|
Mexico |
|
South Africa |
|
India |
|
New Zealand |
|
China |
|
China |
Starting with the work by Zhao (2009), half of the described species have been characterized molecularly by partial sequences of either one or two of the ribosomal DNA genes (18 S and/or 28 S rDNA). The rDNA-based phylogenetic analyses revealed the well-supported monophyletic status of
The present work focuses on morphological and molecular characterisation of the
In total, ten representative soil samples composed from five subsamples were collected from the five permanent research plots (10 × 10 m) established in the undisturbed natural beech forest in Opátka (48°47’N, 21°04’E, at 776 m a.s.l.), a small village in Košice district, eastern Slovakia. Samples were collected twice, five in May 2016 and five in September 2018, and examined in two laboratories, first in Slovakia and later in Poland.
Each sample (collected in 2016) was homogenized by gentle hand mixing. A total of 100 g of soil was then soaked in 1 l of tap water for 30–60 min and processed by the Cobb sieving and decanting method (Cobb, 1918) followed by the modified Baermann technique (van Benzooijen, 2006). Subsequently, nematodes were extracted from the aqueous soil suspensions using a set of two cotton-propylene filters. Suspensions containing nematodes were collected after 24 h. The nematode suspensions were subsequently examined under a stereomicroscope (40 × and 60 × magnification). After the excess water was removed, five
The soil samples collected in 2018 were extracted in Poland by the decantation and sieving method followed by the centrifugal flotation method (van Benzooijen, 2006). Extracted nematodes were heat-killed using tap water at 60°C. Some of the selected individuals (as well as a few individuals from 2016 that arrived alive in water from Slovakia) were fixed in TAF and subsequently submitted for morphological analysis. Photographs and measurements were taken from 16 females and 14 males with a Leica DFC 500 camera on a Leica DM 5000B microscope. The remaining nematodes were fixed in DESS and temporary slides were prepared. With photographic documentation collected, the examined specimens were washed with PBS and subsequently with sterile water and then submitted for molecular studies.
Ten selected individual nematodes (five collected in 2016 and five in 2018) were transferred to separate 0.2 ml polymerase chain reaction (PCR) tubes containing 25 μl sterile water. An equal volume of the lysis buffer, as described in Holterman et al. (2006), was added. The lysis occurred in a Thermal cycler (Veriti 96
Nearly full length 18 S rDNA (1,6 kb) was amplified in two overlapping fragments using the following primer combinations: 988 F combined with 1912R and 1813F combined with 2646 R (as in Holterman et al., 2006). The 28 S rDNA (1 kb) sequence was also amplified in two parts using the 61 F primer (Holterman et al., 2006) combined with MCB1R (Dobosz et al., 2013) and the D2A primer combined with D3B (Nunn, 1992). Amplification of the 18 S and 28 S rDNA fragments was performed in reactions containing 12.5 μl Color Perpetual Taq PCR Master Mix (2x) or Color Perpetual OptiTaq PCR Master Mix (2x) (EURx, Gdańsk, Poland), 1 μl of the forward and reverse primer (5 μM each), the 3 μl DNA template and sterile Milli-Q water to 25 μl of a total volume. All PCR reactions were performed in Veriti 96
Both the 18 S and 28 S rDNA fragments were successfully amplified and sequenced from all 10 processed nematode individuals. There were no sequence variations, neither in the 18 S rDNA, nor within the 28 S rDNA region. The obtained sequence fragments of 18 S rDNA and 28 S rDNA were deposited in GenBank under the following accession numbers: MK722523 and MK733311.
The newly obtained sequences of
The final multiple-sequence alignments comprised of 1,705 overlapping characters in case of 18 S rDNA and 676 characters in case of 28 S. Substitution models for both, 18 S and 28 S rDNA data sets were tested using FindModel – an online implementation of the MODELTEST program (Posada and Crandall, 1998).
The Bayesian 18 S rDNA and 28 S rDNA phylogenies were constructed with the program MrBayes v. 3.1 (Ronquist and Huelsenbeck, 2003). Four independent runs were performed with four Markov chains per run in each analysis. The program was run for 1.5 × 106 generations in case of the bigger 18 S rDNA data set and for 400,000 generations in case of the smaller 28 S rDNA data set. The first 150,000 generations were discarded as burn-in in case of the 18 S rDNA analysis and 20,000 generations in case of the 28 S rDNA one. Sample frequency in both cases was 200 generations. The sampled trees were combined in single 50% majority-rule 18 S and 28 S rDNA trees. Stabilization of the likelihood and parameters was checked with the program Tracer (v. 1.6; Rambaut et al., 2014).
(Figs. 1–3).
Measurements of the European population of the
Morphometrics of
Iran | Slovakia | |||
---|---|---|---|---|
Character | Females | Males | Females | Males |
n | 4 | 5 | 16 | 14 |
L | 1,813.0 ± 53.9 (1,754–1,860) 2.97 | 1,675.0 ± 96.0 (1,558–1,790) 5.73 | 1,694.3 ± 173.1 (1,472–2,038) 10.22 | 1,662.2 ± 230.8 (1,358–2,017) 13.89 |
a | 52.6 ± 1.5 (51.6–54.7) 2.85 | 52.5 ± 4.9 (45.7–59.7) 9.33 | 41.0 ± 5.6 (34.4–51.5) 13.55 | 42.2 ± 3.6 (35.8–48.5) 8.45 |
b | 6.1 ± 0.1 (5.9–6.2) 1.64 | 5.6 ± 0.3 (5.2–5.6) 5.36 | 5.4 ± 0.3 (4.9–5.8) 6.12 | 5.3 ± 0.4 (4.7–6.0) 7.71 |
c | 32.1 ± 3.2 (29.1–35.8) 9.97 | 33.6 ± 6.5 (23.9–39.8) 19.35 | 20.0 ± 1.8 (18.1–25.0) 8.88 | 19.0 ± 1.9 (15.0–22.4) 9.97 |
c’ | 2.3 ± 0.3 (2.0–2.6) 13.01 | 1.9 ± 0.3 (1.6–2.5) 15.79 | 2.8 ± 0.4 (2.1–3.4) 12.62 | 2.6 ± 0.2 (2.3–3.1) 7.36 |
V (%) | 79.6 ± 1.2 (78.4–81.2) 1.51 | 77.0 ± 1.1 (74.6–78.7) 1.47 | ||
Lip region width | 23.3 ± 2.6 (21–26) 11.16 | 23.8 ± 0.8 (23–25) 3.36 | 29.2 ± 1.3 (27.4–33.0) 4.51 | 29.3 ± 2.3 (25.5–33.3) 7.88 |
Maximum body width | 34.5 ± 1.0 (34–36) 2.90 | 32.0 ± 2.1 (30–35) 6.56 | 41.6 ± 3.5 (36.3–47.8) 8.48 | 39.3 ± 3.6 (34.7–45.3) 9.04 |
Anal body width | 24.5 ± 1.9 (23–27) 7.76 | 26.6 ± 1.1 (25–28) 4.14 | 30.8 ± 3.7 (25.9–35.9) 11.96 | 34.2 ± 3.1 (29.5–39.9) 9.14 |
Dorsal tooth from anterior body end | 27.7 ± 0.9 (25.9–29.0) 3.29 | 27.5 ± 1.1 (25.1–29.3) 4.00 | ||
Nerve ring from anterior end | 124.9 ± 9.4 (106–142) 7.54 | 124.6 ± 13.5 (112–149) 10.85 | ||
Pharynx length | 297.0 ± 6.8 (288–304) 2.29 | 297.0 ± 7.6 (286–307) 2.56 | 316.5 ± 29.0 (256–365) 9.18 | 315,7 ± 38.5 (256–369) 12.21 |
Pharynx (% of body length) | 18.7 ± 1.1 (17.1–20.2) 6.11 | 19.1 ± 1.5 (16.7–21.5) 7.82 | ||
Head to vulva | 1442.0 ± 58.0 (1392–1507) 4.02 | 1304.9 ± 132.7 (1136–1598) 10.17 | ||
Vulva to anus | 313.0 ± 13.1 (295–325) 4.19 | 303.3 ± 44.9 (255–407) 14.81 | ||
Tail length | 57.0 ± 6.5 (49–64) 11.40 | 51.2 ± 9.2 (45–67) 17.97 | 84.8 ± 5.7 (75.7–94.8) 6.73 | 87.4 ± 7.6 (75.7–99.5) 8.67 |
Tail (% of body length) | 5.0 ± 0.4 (4.0–5.5) 7.94 | 5.3 ± 0.6 (4.5–6.7) 10.40 | ||
Rectum/cloaca length | 31.0 ± 2.2 (26.8–34.2) 7.21 | 42.0 ± 4.9 (34.0–49.2) 11.62 | ||
Spineret length | 2.1 ± 0.2 (1.8–2.3) 7.86 | 2.3 ± 0.4 (1.7–2.9) 17.20 |
Note: All measurements are in µm and in the form: mean ± S.D. (range) CV.
Body long and relatively slender, arcuate ventrally after gentle heat treatment with posterior part of body more curved than anterior. Cuticle smooth, 1.8 (1.4–2.3) µm thick. Body pores small and numerous, distributed along entire body. Pseudocoelomocyte cells 14.7 (10.2–20.5) µm wide and 27.2 (15.1–41.6) µm long, located along body, their number varying from one to seven pairs. Max. body diam. generally recorded at ovary level, occasionally at level of vulva. Head rounded, 29.3 (27.4–33.0) µm diam., smooth, continuous with body contour. Inner labial papillae conical, 2.2 (1.7–2.4) µm long. Six outer labial setae and four short cephalic setae arranged in a single whorl. Outer labial setae strongly developed, 21.2 (17.0–24.2) µm long, more or less arcuate, bent at tip and directed anteriorly, four cephalic setae 9.1 (7.1–12.4) µm long, thinner than outer labial setae. Stoma 31.6 (29.6–34.9) µm long, funnel-shaped with weakly sclerotised cheilostom, rest of stoma with dorsal wall thickened almost for its entire length, thickening gradually expanding from beginning of mouth to dorsal tooth, where wall thickness is largest. Dorsal tooth relatively large, triangular, directed posteriad, lying 28 (26–29.0) µm from anterior end of body. Two small subventral teeth 3.1 (2.3–3.7) µm anterior to dorsal tooth. Amphids cup-shaped, located 19.0 (17.4–20.6) µm from anterior extremity. Excretory pore 148 (133–175) µm from anterior end. Cervical region with three ventromedian setae. One female with two lateral setae lying 54 and 138 µm from anterior end of body. Pharynx cylindrical and muscular, slightly enlarged anteriorly to surround stoma. Dorsal pharyngeal gland opening at level of dorsal tooth. Nerve ring located at 39.6 (35.5–47.0)% of pharyngeal length from anterior end. Excretory pore not observed. Cardia located between pharynx and intestine, relatively large and wide with gland-like bodies. In intestine of several individuals an ingested nematode was observed. Rectum arcuate, 31.0 (26.8–34.2) µm long. Tail with poorly visible caudal glands, terminated by a small, tubular spinneret 2.1 (1.8–2.3) µm long. Two subdorsal caudal setae situated a short distance anterior and posterior to anus. Reproductive system 374 (224–726) µm long, monodelphic-prodelphic with post-vulval uterine sac 145 (103–190) µm long, occasionally containing large, oval sperm.
Morphology and morphometrics similar to that of female. Genital branch 668 (312–849) µm long. Testis outstretched, filled with oval-shaped spermatozoa. Spicule narrow with arched proximal and acute distal end, 54 (48–59) µm long, evenly tapering, sickle-shaped. Gubernaculum well developed, 10.1 (9.0–11.8) µm long, bifurcate proximally, surrounding spicules dorsally and laterally. Cloaca 42.0 (34.0–49.2) µm long. Ventromedian, precloacal supplements five in number, papillate, irregularly arranged. Distance between first and last supplement 119 (77–159) µm. First supplement lying 8.3–11.9 µm from cloacal aperture, second 28–41, third 45–83, fourth 73–132, and fifth 96–167 µm.
Iran. Soil and litter, at a depth of 0–15 cm under a hawthorn tree (
Slovak Republic. Soil and litter, at a depth of 0–20 cm under a beech tree (
Voucher specimens were deposited in the National Nematode Collection in New Zealand (NNCNZ), the Crown Research Institute Landcare Research New Zealand Ltd, New Zealand (2 females, 2 males) and in the nematode collection at the U.S.D.A, Beltsville, USA (one female, one male). The remaining specimens were deposited in the Institute of Parasitology, Slovak Academy of Sciences, Slovak Republic (4 females, 4 males) and in the nematode collection at the Museum and Institute of Zoology, PAS, Warsaw, Poland (9 females, 7 males).
Specimens collected from a natural beech forest in Slovak Republic were first identified as an undescribed species, morphologically similar to
1. Subventral denticles located posterior to dorsal tooth
Subventral denticles located anterior to dorsal tooth
2
2. Vaginal sclerotisations oval, female without ventromedian setae in cervical region, sparse somatic setae present
Vaginal sclerotisations tear-shaped to semi-spherical, female with ventromedian setae in cervical region, sparse somatic setae absent
3
3. Pharynx = 256–369 µm, b = 4.7–6.2
Pharynx = 216–242 µm, b = 6.3–7.4