CC BY
47Russian Journal of Nematology, 2017, 25 (2), 101 - 119
Morphological and molecular characterisation of some Criconematidae (Nematoda, Tylenchida): Ogma decalineatus (Chitwood, 1957) Andrâssy, 1979, Criconema silvum (van den Berg, 1984) Raski & Luc, 1985 and Neobakernema variabile (Raski & Golden, 1966) Ebsary, 1981 from South
Africa and the USA
Esther van den Berg1, Louwrens R. Tiedt2 and Sergei A. Subbotin3' 4
'National Collection of Nematodes, Biosystematics Programme, ARC-Plant Protection Research Institute, Private Bag X134,
0121, Queenswood, South Africa ^Laboratory for Electron Microscopy, North-West University, Potchefstroom Campus, 2520, Potchefstroom, South Africa 3Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento, CA
95832-1448, USA
4Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 117071, Moscow, Russia; e-mail: sergei.a.subbotin@gmail.com
Accepted for publication 13 November 2017
Summary. During recent nematological surveys Ogma decalineatus and Criconema silvum were found in South Africa and Neobakernema variabile was found in California, USA. These three species are morphologically and morphometrically described. The SEM photos are also given for the species. Molecular characterisations of these three species using the D2-D3 expansions segments of 28S rRNA, ITS rRNA and COI mtDNA gene sequences are provided. Key words: COI, D2-D3 28S rRNA, ITS rRNA, phylogeny, SEM.
During nematological surveys several criconematid species were found in South Africa and California, USA. Samples were collected along the Maretlwana River, near Mooinooi in the Magaliesberg Mountains, and from a swamp forest on the shores of Lake Mgobezeleni in the Isimangaliso Wetland Park, KwaZulu Natal, South Africa and from a willow tree growing in Napa County, California, USA. The objectives of this work were to: i) carry out a morphological and morphometric characterisation of these criconematids; ii) provide molecular characterisation of these species using sequences of the D2-D3 expansion segments of the 28S nuclear ribosomal RNA, the ITS of rRNA gene and partial COI gene; iii) analyse phylogenetic relationships within criconematid nematodes using these genes.
MATERIAL AND METHODS
Light and scanning electron microscopy.
Specimens were extracted from soil samples using the rapid centrifugal-flotation method (Jenkins, 1964), fixed in FPG (Netscher & Seinhorst, 1969), transferred
to anhydrous glycerin (De Grisse, 1969) and mounted on Cobb slides. Measurements were made with a research microscope (Nikon Labophot-2) equipped with a drawing tube. Light micrographs were taken with an automatic Infinity 2 camera attached to a compound Olympus BX51 microscope equipped with Nomarski differential interference contrast.
For scanning electron microscopy specimens were transferred to TAF (40% formalin, triethanolamine, distilled water), then dehydrated in increasing concentrations of alcohol in distilled water and finally into pure alcohol. Following conventional critical point drying and gold/palladium coating (15 nm), specimens were viewed with a FEI ESEM Quanta 200 scanning electron microscope at 10 kV.
DNA extraction, PCR and sequencing. DNA was extracted from several dead specimens of each species using the proteinase K protocol. Detailed protocols for DNA extraction, PCR, cloning and sequencing were as described by Tanha Maafi et al. (2003). Two rRNA gene fragments, ITS rRNA and D2-D3 expansion segments of 28S rRNA gene and
partial COI gene were amplified. The following primers were used for amplification in the present study: ITS rRNA gene - TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') (Tanha Maafi et al., 2003); D2-D3 of 28S rRNA gene - D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and D3B (5'-TCG GA GGA ACC AGC TAC TA-3') (Subbotin et al., 2006) and partial COI gene - COIF5 (5'-AAT WTW GGT GTT GGA ACT TCT TGA AC-3') and COIR9 (5'-CTT AAA ACA TAA T GR AAA TGW GCW ACW ACA TAA TAA GTA TC-3') (Powers et al., 2014). The obtained sequences were submitted to the GenBank database under the following accession numbers: MF683230-MF683242.
Phylogenetic analysis. The newly obtained sequences for each gene were aligned using ClustalX 1.83 with default parameters with corresponding published gene sequences of criconematids and related genera (Subbotin et al., 2005, 2006; Powers et al., 2010, 2014, 2016a, b; van den Berg et al., 2012). Outgroup taxa for each dataset were chosen according to the results of previously published data (Subbotin et al., 2005, 2006). Sequence datasets were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). BI analysis under the GTR + I + G model for each gene was initiated with a random starting tree and was run with four
chains for 1.0 * 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. The log-likelihood values of the sample points stabilised after approximately 1000 generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades.
DESCRIPTIONS
Ogma decalineatus (Chitwood, 1957) Andrassy, 1979
= Criconema decalineatum Chitwood, 1957 = Criconema (Variasquamata) decalineatum (Chitwood, 1957) Mehta & Raski, 1971
= Variasquamata decalineatum (Chitwood, 1957) Kahn, Chawla & Saha, 1976
= Criconema coffeae Edward, Misra & Rai, 1970 = Ogma coffeae (Edward, Misra & Rai, 1970) Andrassy, 1979
= Criconema (Variasquamata) gracile Mehta & Raski, 1971
= Variasquamata gracile (Mehta & Raski, 1971) Khan, Chawla & Saha, 1976
Ogma gracile (Mehta & Raski, 1971) (Figs 1A-C & 2)
Fig. 1. Ogma decalineatus. Female. A: anterior part of body; B: annuli at mid-body; C: tail region. Neobakernema variabile. Male. D: anterior part of body. Juvenile. E: juvenile cuticle with male tail inside; F: tail of another juvenile. Scale bar = 30 ^m.
Table 1. Measurements of females of Ogma decalineatus from Mooinooi (Tvl 1970), South Africa compared with measurements from some literature. All measurements are in ^m and in the form: mean ± s.d. (range).
Population O. decalineatus O. decalineatus O. decalineatus
Character Mooinooi (Tvl 1970) a b
n 15 26 40
L 357 ± 32.3 (311-408) 309-451 310-451
a 8.4 ± 0.7 (7.3-9.5) 7.1-12.1 9.2-13.9
b 3.2 ± 0.2 (2.9-3.5) 3.1-4.8 3.1-4.3
c 12.6 ± 1.4 (10.4-15.1) 11.5-21.5 10-27.7
c' 1.3 ± 0.2 (0.9-1.5) - 1.5-1.7
o 8 ± 1.5 (5.6-10.3) 6-9.5 -
DGO 6 ± 1 (4.5-7.5) 4.5-8.1 -
V 86.5 ± 1.3 (85-89) 85-90 84-91
OV1 71 ± 24.2 (57.5-95.5) 30-78.5 -
OV length 271.5 ± 54.9 (181.5-363) - -
Stylet length 75 ± 3.4 (71.5-84) 46.3-93 65-78
Metenchium 63 ± 3.5 (59.5-72) 37.5-79 54-65
Telenchium 12 ± 0.7 (10.5-13) 8.5-14 -
M 84.2 ± 1.2 (81.5-86) - 83-88
Stylet knob height 3 ± 0.4 (2.5-4) 2.2-4 3-4.7
Stylet knob width 8.5 ± 0.8 (7.5-9.5) 6-9 7.7-9.3
Excretory pore from front 123 ± 8.7 (108-134.5) 104-144 91-117
Width at mid-body 42.5 ± 2.8 (38-48) 28.7-49 -
Width at anus 21.5 ± 2.3 (19-25) - -
Annulus width 4.5 ± 0.4 (4-5.5) 3.7-7 -
Tail length 28.5 ± 3.9 (20.5-33) 19.5-33 27-33
Pharynx length 112 ± 8.5 (100-128) 93-130.5 83-100
First lip annulus width 13 ± 0.9 (11.5-14.5) 10.7-15 12-14.7
Second lip annulus width 13.5 ± 0.8 (12.5-15) 10-15 13-16
First body annulus width 17.5 ± 1.1 (16-20) 14-21 -
Second body annulus width 21 ± 1.1 (19-23) 20-26.5 -
R 83 ± 1.9 (80-87) 75-94 75-84
RSt 18.5 ± 1.5 (15-22) 13-21 15-18
ROes 26.5 ± 2.4 (23-29) 20-26 20-26
Rex 28 ± 1.3 (26-30) 23-30 23-28
Rhem 27 ± 1 (26-28) 26-28 -
RV 14 ± 1.1 (12-16) 11-16 10-14
RVan 4.5 ± 0.6 (3-5) 3-6 3-6
Ran 9 ± 1 (7-10) 5-10 5-10
VL/VB 1.5 ± 0.2 (1.2-1.8) 1.3-1.9 -
St% L 21.2 ± 1.6 (18.9-23.5) 11.9-24.9 -
a) van den Berg et al. (2004); van den Berg (1983); van den Berg & Spaull (1985); b) Minagawa (1988); Crozzoli & Lamberti (2002).
Fig. 2. Ogma decalineatus. Female. A: en face view of lip region. B: lateral view of anterior part of body. C: annuli at mid-body; D: ventral view of posterior part of body with vulva. Scale bars: A, C, D = 20 ^m, B = 10 ^m.
Fig. 3. Criconema silvum. Female. A: anterior part of body; B: vulval area with spermatheca; C, D: lateral and ventral views of tail region; E: annuli at mid-body. Juvenile. F: annuli at mid-body; G: tail area. Scale bar = 30 ^m.
This species was described from Ficus elastica Roxb. ex Hornem. 1819 roots from Florida, USA and since has been reported from various countries in the world (Geraert, 2010). It is very common in South Africa and found very often on various plants
and biomes. S. Neser collected the present specimens from sandy soil underneath various trees along the Maretlwana River, near Mooinooi (Tvl 1970, sample CD555) in the Magaliesberg Mountains, South Africa on 22.11.2008.
Measurements. See Table 1.
Female. Body slightly arcuate ventrad. Lip region with two annuli with irregularly lobed margins; first annulus directed more forward and second one more outward with rounded lobes on margin corresponding to that of the first body annulus. Two lip annuli slightly separated from the body annuli, directed posteriorly. Labial disc protruding. SEM shows a rectangular labial disc, rimmed with a narrow ridge with each corner pointing prominently outward (submedian lobes). Labial plates not seen. Amphids small, slit-like, laterally. Labial framework not very distinct. All body annuli thick and well retrorse with mostly ten longitudinal rows of rounded scales. In some specimens, twelve were counted at mid-body. Posterior margins of scales smooth. No anastomosis observed except for one or two on tail tip. Hemizonid seen in two specimens only, one annulus long and situated from directly anterior to two annuli anterior to excretory pore. Excretory pore situated from three annuli anterior to six annuli posterior to base of pharynx. Stylet long and slender with cupped basal knobs. Spermatheca small, round to oval and mostly filled with rounded sperm cells. Vagina straight to slightly sigmoid with rounded lips not projecting beyond the profile of the body.
Anterior lip bi-lobed, overlapping posterior lip slightly. Tail tapering to a small rounded knob-like terminus.
Male. Not found.
Juvenile. Not found.
Remarks. The present specimens compare very well with all the various descriptions of the species such as Chitwood (1957), van den Berg (1983), van den Berg & Spaull (1985), Vovlas (1986), Minagawa (1988), van den Berg & Cadet (1991), Crozzoli & Lamberti (2002) and van den Berg et al. (2004).
Criconema silvum (van den Berg, 1984) Raski & Luc, 1985
= Lobocriconema silvum van den Berg, 1984 (Figs 3, 4 & 5)
Originally, this species was described as Lobocriconema silvum (van den Berg, 1984b) from subtropical growth in the Dukuduku Forst Reserve, near St. Lucia Bay, KwaZulu Natal Province, South Africa. A further collection from the same site in 1985 produced 30 females and in 1989 13 females were collected from young pine trees in the Kwambonambi State Forest described by van den Berg (1992). In 2014 specimens were collected from
swamp forest on the shores of Lake Mgobezeleni in the Isimangaliso Wetland park, KwaZulu Natal (KZN4; sample CD1851), South Africa. These specimens were studied morphologically and now also molecularly for the first time. Andrassy (1979) already synonymised Lobocriconema with Criconema on various grounds. Raski and Luc (1985) discussed various Criconematidae genera and the characters separating them and finally made nine genera synonyms of Criconema, one of which was Lobocriconema. During the last decades, many of the species were moved to different genera and backed by Geraert (2010), Siddiqi (2000), Brzeski et al. (2002), Wouts (2006) and Andrassy (2007). We regard these present species as belonging to the genus Criconema as the SEM photos show that there are no real submedian lobes present.
Measurements. See Table 2.
Female. Body slightly curved ventrad. Lip region with one annulus, saucer shaped and pointing outward or slightly backward. First body annulus with a slightly smaller diameter. Second and third body annuli with larger diameters. SEM show a rectangular labial disc, but no distinct submedian lobes seen. All body annuli slightly retrorse covered with fine longitudinal lines, frequently slightly irregular or broken, margins very finely scalloped. Space between the annuli coarse with irregular nodules. No anastomosis seen except two irregular ones posterior to the vulva on one specimen. Hemizonid seen in two specimens only, two annuli posterior to excretory pore. Excretory pore situated from one annulus anterior to five annuli posterior to base of pharyngeal lobe. Stylet long, slightly curved dorsad with cupped basal knobs. Spermatheca present, varying from small to large, round to oblong and empty to be filled with rounded sperm cells. Vagina straight. Vulval lips not protruding beyond body profile. Dorsal lip straight or with two small rounded lobes and not overlapping posterior lip. Tail tapering gradually to last annulus with a small rounded lobe, sometimes this lobe is slightly irregular.
Male. Not found.
Juvenile. One female was found with a piece of the 4th stage juvenile cuticle attached to the posterior part of the body. There appeared to be 8 to 10 long pointed scales on the cuticle each ending in a sharp pointed seta.
Remarks. The present specimens fit the specimens previously described well except for having less anastomosis and fewer lobes on the tail tip and no real submedian lobes in the labial area.
Table 2. Measurements of females of Criconema silvum compared with newly collected specimens from KZN4. All measurements are in ^m and in the form: mean ± s.d. (range).
Character Population C. silvum C. silvum C. silvum
South Africa (KZN4) a b
n 33 3 43
L 560 ± 59.4 (413-657) 564 (551-577) 314-625
a 12.7 ± 1.7 (9.4-17.6) 10.5 (8.6-12.2) 6.7-14.7
b 3.8 ± 0.3 (3.2-4.6) 4(3.9-4.1) 2.8-4.4
c 49.7 ± 11.6 (30.4-76.6) 65.2 (59.1-68.2) 35.1-110.2
c' 0.7 ± 0.1 (0.5-0.9) - -
o 5.8 ± 0.8 (4.2-6.9) 6.3 (6.1-6.5) 4.2-10.6
DGO 5.5 ± 0.5 (4-6.5) 5.7 (5.5-5.9) 4-9.6
V 90 ± 2.1 (85-92) 91 (91-92) 88-92
OV1 42.4 ± 11.2 (26.9-79.9) - -
OV length 239 ± 66.8 (153-424) - -
Stylet length 95 ± 4.9 (82.5-107.5) 91.3 (90.4-92.6) 74.2-101.1
Metenchium length 79.5 ± 4.1 (69-90) 74 (73.5-75) 60.6-86.7
Telenchium length 15 ± 2.3 (9-19) 17.3 (16.9-17.6) 12.9-19.9
M 84.1 ± 2.5 (80.4-94) - -
Stylet knob height 3.5 ± 0.5 (3-5) 4.8 (4.4-5.2) 2.6-5.5
Stylet knob width 9.5 ± 1 (8-11) 11.8 (11.4-12.5) 8.1-12.5
Exc. pore from front 174 ± 16.1 (142-197) 177 (172-181) 99-204
Width at midbody 45 ± 4 (36-55) 49.6 (47.5-51.5) 38.2-51.5
Width at excretory pore - 48.3 (45.6-50) 365.8-52.9
Width at anus 18 ± 2.8 (13-25) -
Annulus width 9 ± 1.1 (6-12) 9.3 (8.8-9.6) 5.1-10.7
Tail length 12 ± 3.1 (7.5-17) 8.7 (8.1-9.6) 4-15.8
Pharynx length 148 ± 12.6 ( 123-178) - -
1st lip annulus diam. 22 ± 2.1 (18-26.5) 25 (23.9-25.7) 18-29
1st body annulus diam. 19.5 ± 2.6 (17.5-24.5) 22.9 (21.3-24.3) 16.5-27.9
2nd body annulus diam. 26 ± 2.6 (20.5-33) 31.2 (30.1-33.1) 22.1-34.6
3d body annulus diam. 29.5 ± 2.8 (23-37.5) - -
Spermatheca length 18 ± 3.5 (12-27) - -
Spermatheca width 12.5 ± 2 (10-18) - -
R 67 ± 2.8 (61-74) 65-67 57-68
RSt 12 ± 1.1 (9-14) 11-13 11-19
ROes 18 ± 1.6 (15-22) 16-17 14-25
Rex 20 ± 1(19-22) 19-22 18-24
Rhem 17; 20 (n = 2) - -
RV 9 ± 0.8 (7-10) 7-8 6-9
RVan 4 ± 1.4 (1-6) 3-5 3-5
Ran 3 ± 0.7 (2-5) 2-3 1-3
VL/VB 1.6 ± 0.2 (1-2) 1.3 (1.1-1.4) 0.9-1.7
St% L 16.6 ± 1.3 (14-21.1) 16.2 (16-16.5) 14.6-29.6
a) van den Berg (1984a); b) van den Berg (1992).
Fig. 4. Criconema silvum. Female. A, B: en face view of two lip regions; C: lateral view of anterior part of body; D: mid-body; E, F: lateral and ventral view of posterior part of body.
Fig. 5. Criconema silvum. Female. A: close up view of vulva; B: ventral view of posterior part of body with vulva; C, D: enlargement of annuli at mid-body; E, F: tail tip endings.
Neobakernema variabile (Raski & Golden, 1966) Ebsary, 1981
= Bakernema variabile Raski & Golden, 1966 = Criconemella variabilis (Raski & Golden, 1966) Raski & Luc, 1987
= Mesocriconema variabile (Raski & Golden, 1966) Loof & De Grisse, 1989
= Macroposthonia variabilis (Raski & Golden, 1966) Siddiqi, 2000 (Figs 1D-F, 6-9)
Raski and Golden (1966) originally described this species as Bakernema variabile from various hosts and localities in California, USA. The present specimens were collected from Napa County, California, USA (Capell Valley Road crossing with Wragg Canyon Road, host - Salix sp., sample CD848) and studied for the first time molecularly and with SEM.
Measurements. See Table 3. Female. Body form ranging from slightly curved ventrad to an open letter C. Lip region flattened anteriorly, first annulus margins rounded and directed more forward while second annulus is directed backward. In the SEM photos the labial area is unfortunately covered with bacteria but labial disc appears rectangular with four flattened, indistinct rounded lobes on the corners, two dorsally and two ventrally. Amphids not distinctly seen. Labial framework not very distinct. All body annuli well retrorse with wavy fringe at the posterior end. This fringe represents the fine extra cuticular layer this species possesses. The annuli appears angular when seen laterally. No anastomosis observed. Hemizonid seen in one specimen only, about one annulus long and situated one annulus anterior to excretory pore. Excretory pore situated from 13 annuli anterior to four annuli posterior to base of a pharyngeal lobe. Stylet very long, robust with cupped basal knobs. Spermatheca large, round to oblong and mostly filled with rounded sperm cells. Vagina straight to slightly sigmoid. Vulva with anterior lip slightly bilobed but not overlapping posterior lip; both lips not projecting beyond profile of body. Tail tapering slightly to a bluntly rounded tip with two to three coarsely infolded lobes. Fringe of second outer cuticle more prominent on tail with slightly longer angular projections.
Male. Five males found still within the fourth stage juvenile cuticle. Body form slightly curved ventrad. Lip region not set off but number of annuli not clear. First annulus seems to project outward. Pharynx degenerate. Stylet absent. Excretory pore seen in one specimen only, about 54 annuli from anterior end. Lateral field with four lines, outer ones
appearing slightly wavy. Annuli on body distinct. Spicules long and curved slightly irregular due to still being in the juvenile cuticle. Tail ending and number of annuli not distinct because of being cramped into the juvenile cuticle.
Juvenile. Three juveniles found, two fourth-stage juveniles with males inside. Stage of third juvenile unknown, probably third-stage juvenile. Body slightly curved ventrad. Lip region very similar to female. Annuli margins with small rounded, projections, not in longitudinal rows, difficult to count, probably about 20 present on each annulus. Tail tapering to a narrow irregular tip.
Remarks. Described as Bakernema variabile by Raski & Golden (1966). Ebsary (1981) stated that the genus Bakernema contains two species differing in several characters and created a new genus, Neobakernema for the species variabile, which has an open vulva, straight vagina and spined juveniles. Siddiqi (1986) regarded Neobakernema as a valid genus. Later, Siddiqi (2000) regarded Neobakernema as a synonym of Macroposthonia de Man, 1880. Raski and Luc (1987) gave a detailed description of the close relationship of a few genera of the Criconematidae such as Bakernema, Ogma, Blandicephalanema and Pateracephalanema. They stated that N. variabile has a closer relationship with Criconemella and, thus, transferred the species to Criconemella. Loof and De Grisse (1989) discussed the plesiomorphic and apopmorphic states of the characters of several genera and synonymised Neobakernema with Mesocriconema, a synonymy accepted by Brzeski et al. (2002). Geraert (2010) noted that the morphometrics of the seven Neobakernema species clearly separate them from Mesocriconema and regarded this genus as a valid one. The present specimens fit the descriptions of the various authors very well.
Molecular characterisation and phylogeny. The D2-D3 of 28S rRNA gene alignment included 20 sequences of criconematids and three sequences of Paratylenchus selected as outgroup taxa and was 710 bp in a length. Five new sequences were obtained in the present study. Phylogenetic analysis resulted in a majority consensus BI tree with two major highly supported clades (Fig. 10).
The ITS1 rRNA gene alignment included 21 sequences of criconematids and two sequences of Paratylenchus selected as outgroup taxa and was 422 bp in a length. Five new sequences were obtained in the present study. Phylogenetic relationships within some criconematids as inferred from BI analysis are given in Fig. 11. Sequences of Ogma decalineatus from South Africa and USA clustered together and differed in 10.1% (35 bp).
Table 3. Measurements of females, males and juveniles of Neobakernema variabile (CD848) from the USA. All measurements in |im and in the form: mean ± s.d. (range).
Population Character Neobakernema variabile N. variabile Raski & Golden (1965)
Females Juveniles (?J4) Juvenile (?J3) Males Females Males
n 13 2 1 5 67 8
L 723 ± 54.7 (647-829) 643-681 568 635 ± 45.7 (579-702) 720(530-990) 630 (550-790)
a 9.9 ± 1.9(8.1-12.7) - 14.3 22.8 ±2.6 (20.9-27.3) 12.4(10-15) 22.3 (12-27)
b 3.7 ±0.3 (3.2-4.2) - 3.9 6.9 (n= 1) 3.7(2.5-4.7) -
c 28 ±6.6 (18.4-39.3) 20.4 - 14.5 ±0.7 (13.5-15.5) 22.7 ±(18-27) 14.6(12-21)
c1 0.7 ±0.1 (0.6-0.9) 0.8 0.8 2.1 ±0.4 (1.7-2.6) - -
0 5.2 ± 1.5 (3-8.6) - 11.7 - - -
DGO 6± 1.8(4-10.5) - 8 - - -
V 93.5 ±0.9 (92-95) - - - 92(89-95) -
OV1 48 ± 9.9 (35-63) - - - - -
OV length 347.5 ±77.7 (249.5-460.5) - - - - -
Stylet length 114 ± 5 (106.5-120.5) 70-73.5 70 - 113(97-142) -
Metenchium length 88 ± 5 (80-95) - 53.5 - 89 (74-97) -
Telenchium length 26 ±3 (17.5-29.5) - 15.5 - - -
M 77.3 ±2.1 (75-83.2) - 77.7 - - -
Stylet knob height 7.5 ±0.8 (6-8) - 3 - - -
Stylet knob width 17 ± 1 (15-18.5) - 8 - 15 -
Exc. pore from front 183 ±30.4(138-236) - 141 141 ± 16.3 (119-155) 183-217 155-166
Width at midbody 78 ± 4.9 (66-84) 68.5 40 28.5 ± 1.2 (26.5-29.5) - —
Table 3. (continued)
Population Character Neobakernema variabile N. variabile Raski & Golden (1965)
Females Juveniles (?J4) Juveniles (J3?) Males Females Males
Width at anus 43.5 ±4.8 (36-50) 34.5-40 - 21 ±4 (16-27) - -
Annulus width 8.5 ±0.7 (7.5-9.5) 7.5-8 6 4 7-8 4.1-6.3
Tail length 27 ± 6.1 (18-41) 32-34.5 - 44 ± 5.2 (39-52) - -
Pharynx length 197 ± 17.7(170-224) - 146.5 - - -
1st lip annulus diam. 18 ± 1.1 (16-20) 13-14 12 - - -
2ntl lip annulus diam. 24.5 ± 1.7(22-27) 17-18.5 15 - - -
1st body annulus diam. 29.2 ±2 (26-32.5) 23-23.5 17.5 - - -
2ntl body annulus diam. 35 ± 2.1 (32-38) 27-28 20 - - -
311 body annulus diam. 40.5 ± 2.2 (37-44) - - - - -
Lip region width - - - 15 - -
Lip region height - - - 8 - -
R 94 ±4.7 (86-102) 91-94 91 - 100 (61-108) -
RSt 20 ±3.6 (17-29) - 16 - - -
ROes 31 ±4.9 (24-41) - 30 - - -
Rex 27 ± 1.8 (23-30) - 29 - 26-29 -
Rliem 25 (n= 1) - - - - -
RV 7 ±0.7 (5-7) - - - 6-10 -
Rvan 1-2 - - - - -
Ran 4 ±0.8 (3-6) 5-6 - - 3-6 -
VL/VB 0.9 ±0.1 (0.7-1.1) - - - - -
St% L 15.2 ±2.3 (13.7-18.3) - 12.2 - - -
Spermatheca length 31.5 ±2.2 (27-34.5) - - - - -
Spermatheca width 22.5 ±3.7 (15-26.5) - - - - -
Spiculum length - - 49 ±6.3 (41-55) - 56 (55-66)
Gubemaculum length - - 9; 9.5 (n = 2) - 9.5 (9-11)
J3 - third-stage juvenile; J4 - fourth-stage juvenile.
Fig. 6. Neobakernema variabile. Female. A: anterior part of body; C: another view of lip region; B, D: annuli at mid-body; E, F: tail area of two females. Juvenile. G: anterior part of body. Scale bar = 30 ^m.
Fig. 7. Neobakernema variabile. A: entire body of two females; B: entire body of male; C, D: female anterior regions; E: male anterior region; F: female body annulation; G: male lateral field; H, I: female posterior regions; J: male posterior region. Scale bars: A, B = 100 цш, C-E, H-J = 10 цш, F, G = 5 цш.
> : - -
■y ' f'y.^^Z_. irk' r .r^ leak. kjt* ___—-—-- ^Sw ^ v I , ^ __ — — i f i ! b 4 him jiiiw 11 'urn
Fig. 8. Neobakernema variabile. Female. A: lateral view of lip region; B: en face view of lip region; C: annuli at mid-body; D: tail area with vulva. Scale bars: A, B = 20 ^m, C = 50 ^m, D = 30 ^m.
Fig. 9. Neobakernema variabile. Juvenile. A: lateral view of lip region; B: en face view of lip region; C: annuli at mid-body; D: tail region. Scale bars: A, B, D = 10 цт, C = 30 цт.
Ogma decalineatus (MF683230. CD555, cl1) Ogma decalineatus (MF683231, C0555, cl2)
— Xenocnconemella macrodora (AY780960)
Crossonema aquitanense (KJ719809)
— Criconema sp. (AY780952) Criconema silvum (MF683234, CD1851)
— Hemicriconemoides alexis (AY780959.)
M
100
Criconema mutabile (AY780954) — Criconema sp. (AY780953) L Criconema sp. (FN433862) I— Ogma civellae (AY780955)
Neobakernema variabile (MF683232, CD848, cl1) Neobakernema variabile (MF683233, CD848, cl2) — Mesocriconema solivagum (AY780969) Mesocriconema xenoplax (AB933468) Mesocriconema brevistytus (KC937033) — Mesocriconema ornatum (AY780968)
Criconemoides informis (AY780970)
Mesocriconema obtusicaudatum (JQ231187) — Mesocriconema sphaerocephalum (AY780950)
Paratylenchus hamatus (KF242209) - Paratylenchus straeleni (KF242236)
- Paratylenchus dianthus (KF242228)
0.1
Fig. 10. Phylogenetic relationships within populations and species of Criconematidae as inferred from Bayesian analysis using the D2-D3 of 28S rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.
100
100
96
99
100
100
76
100
L
100
100
- Neobakernema variabile (MF683238, CD848, cl1) Neobakernema variabile (MF683239, CD848, cl3)
Lobocriconema thomei (KU236652) Lobocriconema incrassatum (KU236654) P Criconema warrenense (KU236686)
- Criconema arkaense (JQ708129) Mesocroconema vadense (JQ708102)
I— Mesocriconema xenoplax (JQ708104)
- Mesocriciconema omatum (JQ708124) Mesocriconema curvatum (HM116067) -Mesocriconema brevistylus (JQ231188)
— Mesocnconema obtusicaudatum (JQ231190) Ogma decalineatus (MF683235, South Africa, CD555, cl3)
— Ogma decalineatus (HM116075, USA) Ogma octangularis (JQ708138)
99
r~ Criconema mutabile (JQ708132)
- Criconema sp. (FN435300)
- Xenocriconemella macrodora (JQ708139) Criconema silvum (MF683236, CD1851, cl2) Criconema silvum (MF683237, CD1851, cl1)
Criconemoides informis (KF908246)
-Paratylenchus dianthus (KF242271)
-Paratylenchus hamatus (KF242247)
0 1
Fig. 11. Phylogenetic relationships within populations and species of Criconematidae as inferred from Bayesian analysis using the ITS1 rRNA gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.
Fig. 12. Phylogenetic relationships within populations and species of Criconematidae as inferred from Bayesian analysis using the partial COI gene sequence dataset with the GTR + I + G model. Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.
The COI gene alignment included 23 sequences of criconematids and two sequences of Rotylenchus used as outgroup taxa and was 721 bp in a length. Three new sequences were obtained in this study. Phylogenetic relationships within some criconematids as inferred from BI analysis are given in Fig. 12.
In the present study, we provided sequences for several representatives of Criconematina; however,
relationships within some genera remain unresolved. Greater phylogenetic resolution will require representatives of additional genera and species. Reconstruction of the phylogenetic relationships within criconematids is essential to developing a consistent classification as well as ultimately to understand genes and processes involved in plant parasitism (Subbotin et al., 2005).
REFERENCES
Abd El-Wakeil, K.F., Obuid-Allah, A.H., Mohamed, A.H. & Abd El-Aziz, F.E.A. 2013. Community structure of molluscans in River Nile and its branches in Assiut Governorate, Egypt. Egyptian Journal of Aquatic Research 39: 193-198.
Andrâssy, I. 1979. Revision of the subfamily Criconematinae Taylor, 1936 (Nematoda). Opuscula Zoologica Instituti Zoosystematici et Oecologici Universitatis Budapestinensis 16: 11-57.
Andrâssy, I. 2007. Free-Living Nematodes of Hungary (Nematoda errantia), II. Pedozoologica Hungarica, no. 4 (C. Csuzdi & S. Mahunka Eds). Hungary, Hungarian Natural History Museum. 496 pp.
Brzeski, M.W., Loof, P.A.A. & Choi, Y.E. 2002. Compendium of the genus Mesocriconema Andrâssy, 1965 (Nematoda: Criconematidae). Nematology 4: 341-360.
Chitwood, B.G. 1957. Two new species of the genus Criconema Hofmänner & Menzel, 1914. Proceedings of the Helminthological Society of Washington 24: 5761.
Crozzoli, R. & Lamberti, F. 2002. Species of Criconema Hofmänner & Menzel, 1914 and Ogma Southern, 1914 occurring in Venezuela, with description of Ogma araguaensis sp. n. Russian Journal of Nematology 10: 89-98.
De Grisse, A.T. 1969. Redescription ou modifications de quelques techniques utilisées dans l'étude des nématodes phytoparasitaires. Mededelingen van de Rijksfaculteit der Landbouwwetenschappen Gent 34: 351-369.
Ebsary, B.A. 1981. Neobakernema n. gen. (Nematoda: Criconematidae) with an emendation of Bakernema Wu, 1964. Canadian Journal of Nematology 59: 2215-2116.
Edward, J.C., Misra, S.L. & Rai, B.B. 1970. Criconema coffeae n. sp. associated with the roots of coffee in Mysore State, India. Allahabad Farmer 44: 13-15.
Geraert, E. 2010. The Criconematidae of the World: Identification of the Family Criconematidae (Nematoda). Belgium, Academia Press. 615 pp.
HUELSENBECK, J.P. & Ronquist, F. 2001. MrBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755.
Jenkins, W.R. 1964. A rapid centrifugal-flotation method for separating nematodes from soil. Plant Disease Reporter 48: 692.
Khan, E., Chawla, M.L. & Saha, M. 1976. Criconemoidea (Nematoda: Tylenchida) from India, with descriptions of nine new species, two new genera and a family. Indian Journal of Nematology 5 (1975): 70-100.
Loof, P.A.A. & De Grisse, A. 1989. Taxonomic and nomenclatorial observations on the genus
Criconemella De Grisse & Loof, 1965 sensu Luc & Raski, 1981. Mededelingen Faculteit Landbouwwetenschappen Rijksuniversiteit Gent 54: 53-74.
Mehta, U.K. & Raski, D.J. 1971. Revision of the genus Criconema Hofmänner & Menzel, 1914 and other related genera (Criconematidae: Nematoda). Indian Journal of Nematology 1: 145-198.
Minagawa, N. 1988. Taxonomic studies of Criconematidae (Nematoda: Tylenchida) of Japan. III. Genera Ogma and Pseudocriconema. Bulletin of the National Institute of Agro-Environmental Sciences 5: 123-173.
Netscher, C. & Seinhorst, J.W. 1969. Propionic acid better than acetic acid for killing nematodes. Nematologica 15: 286.
Powers, T.O., Harris, T., Higgins, R., Sutton, L. & Powers, K.S. 2010. Morphological and molecular characterization of Discocriconemella inarata, an endemic nematode from North American native tallgrass prairies. Journal of Nematology 42: 35-45.
Powers, T.O., Bernard, E.C., Harris, T., Higgins, R., Olson, M., Lodema, M., Mullin, P., Sutton, L. & Powers, K.S. 2014. COI haplotype groups in Mesocriconema (Nematoda: Criconematidae) and their morphospecies associations. Zootaxa 3827: 101146.
Powers, T.O., Mullin, P., Higgins, R., Harris, T. & Powers, K.S. 2016a. Description of Mesocriconema ericaceum n. sp. (Nematoda: Criconematidae) and notes on other nematode species discovered in an ericaceous heath bald community in Great Smoky Mountains National Park, USA. Nematology 18: 879903.
Powers, T.O., Bernard, E.C., Harris, T., Higgins, R., Olson, M., Olson, S. & Lodema, M. 2016b. Species discovery and diversity in Lobocriconema (Criconematidae: Nematoda) and related plant-parasitic nematodes from North American ecoregions. Zootaxa 4085: 301-344.
Raski, D.J & Golden, A.M. 1966. Studies on the genus Criconemoides Taylor, 1936 with descriptions of eleven new species and Bakernema variabile n. sp. (Criconematidae: Nematoda). Nematologica 11 (1965): 501-565.
Raski, D.J. & Luc, M. 1985. A reappraisal of the genus Criconema Hofmänner & Menzel, 1914 (Nematoda: Criconematidae). Revue de Nematologie 7 (1984): 323-334.
Raski, D.J. & Luc, M. 1987. A reappraisal of Tylenchina (Nemata) 10. The superfamily Criconematoidea Taylor, 1936. Revue de Nematologie 10: 409-444.
Siddiqi, M.R. 1986. Tylenchida, Parasites of Plants and Insects. UK, Commonwealth Institute of Parasitology. 645 pp.
Siddiqi, M.R. 2000. Tylenchida, Parasites of Plants and Insects (2nd edition). UK, Commonwealth Institute of Parasitology. 833 pp.
Subbotin, S.A., Vovlas, N., Crozzoli, R., Sturhan, D., Lamberti, F., Moens, M. & Baldwin, J.G. 2005. Phylogeny of Criconematina Siddiqi, 1980 (Nematoda: Tylenchida) based on morphology and D2-D3 expansion segments of the 28S-rRNA gene sequences with application of a secondary structure model. Nematology 7: 927-944.
Subbotin, S.A., Sturhan, D., Chizhov, V.N., Vovlas, N. & Baldwin, J.G. 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8: 455-474.
Tanha Maafi, Z., Subbotin, S.A. & Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on the ITS sequences of rDNA. Nematology 5: 99-111.
van den Berg, E. 1983. The genera Crossonema Mehta & Raski, 1971 and Ogma Southern, 1914 in South Africa (Nematoda: Criconematidae). Phytophylactica 15: 149-155.
van den Berg, E. 1984a. New and known species of some genera of Criconematidae (Nematoda) from South Africa. Phytophylactica 19: 399-404.
VAN DEN BERG, E. 1984b. New Lobocriconema from South Africa (Nematoda: Criconematidae). Phytophylactica 16: 93-100.
van den Berg, E. 1992. Redescriptions and new records of six known Criconema species from Natal, South
Africa (Criconematidae: Nemata). Phytophylactica 24: 29-38.
van den Berg, E. & Cadet, P. 1991. One new and some known plant parasitic species from the French Caribbean (Nemata: Tylenchida). Revue de Nematologie 14: 389-405.
van den Berg, E. & Spaull, V.W. 1985. Some Tylenchid nematodes from Aldabra Atoll with a description of a new species. Phytophylactica 17: 1925.
VAN DEN BERG, E., Mekete, T. & Tiedt, L.R. 2004. New records of Criconematidae (Nemata) from Ethiopia. Journal of Nematode Morphology and Systematics 6 (2003): 161-174.
van den Berg, E., Tiedt, L.R. & Subbotin, S.A. 2012. Morphological and molecular characterisation of Criconemoides brevistylus Singh & Khera, 1976 and C. obtusicaudatus Heyns, 1962 from South Africa (Nematoda: Criconematidae) with first description of a male C. obtusicaudatus and proposal of new synonyms. Nematology 14: 961-976.
Vovlas, N. 1986. SEM morphology of Ogma decalineatum (Nematoda: Criconematidae). Revue de Nematologie 9: 195-198.
WOUTS, W.M. 2006. Criconematina (Nematoda: Tylenchida). Fauna of New Zealand (№ 55). New Zealand, Manaaki Whenua Press, Landcare Research. 232 pp.
E. van den Berg, L. R. Tiedt and S.A. Subbotin. Морфологическая и молекулярная характеристика некоторых Criconematidae (Nematoda, Tylenchida): Ogma decalineatus (Chitwood, 1957) Andrássy, 1979, Criconema silvum (van den Berg, 1984) Raski & Luc, 1985 и Neobakernema variabile (Raski & Golden, 1966) Ebsaiy, 1981 из Южной Африки и США.
Резюме. В результате последних нематологических обследований Ogma decalineatus и Criconema silvum были обнаружены в Южной Африке и вид Neobakernema variabile был обнаружен в Калифорнии, США. Эти три вида морфологически и морфометрически описаны в статье. Фотографии с использованием электронной сканирующие микроскопии также дается в описании для этих видов. Представлены молекулярные характеристики этих трех видов, сделанные с использованием последовательностей генов сегментов D2-D3 28S рРНК, ITS и COI.
