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Description and molecular phylogeny of Mesocriconema abolafiai n. sp. (Nematoda: Criconematidae) from Iran

Hossein Mirbabaei Karani
Hossein Mirbabaei Karani
, 
Ali Eskandari
Ali Eskandari
, 
Reza Ghaderi
Reza Ghaderi
 et 
Akbar Karegar
Akbar Karegar
   | 25 mai 2020
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Publié en ligne: 25 mai 2020
Pages: 1 - 17
Reçu: 15 oct. 2019
DOI: https://doi.org/10.21307/jofnem-2020-048
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© 2020 Hossein Mirbabaei Karani et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Ring nematodes of the genus Mesocriconema (Andrássy, 1965) are damaging root ectoparasites of many economical important crops (Cordero et al., 2012). This genus was first proposed for species of the genus Criconemoides (Taylor, 1936) sensu lato with crenated margins of annuli (Andrássy, 1965). Simultaneously and independently, De Grisse and Loof (1965) proposed to divide the large genus Criconemoides into several genera including Macroposthonia with type species M. annulata (De Man, 1880) being among them (De Grisse and Loof, 1965). Luc and Raski (1981) declared Criconemoides and Macroposthonia as genera dubia and placed most of their species in the genus Criconemella (De Grisse and Loof, 1965). Based upon SEM microscopy and discussing on plesiomorphic and apomorphic states of characters, Loof and De Grisse (1989) replaced the generic name Macroposthonia by the oldest available synonym Mesocriconema and revalidated Criconemoides based on the arguments of Loof and De Grisse (1967), but Siddiqi (2000) still considered Macroposthonia as a valid name. Brzeski et al. (2002) accepted this synonymy and provided a compendium of the genus Mesocriconema with 90 species (species having open vulva and submedian lobes arising from reduced pseudolips). Moreover, they considered that Mesocriconema differs from Criconemoides (species with closed vulva and pseudolips not reduced). Geraert (2010) replaced some species in the genus Neobakernema (Ebsary, 1981b) by validation of this genus and listed 90 valid species under Mesocriconema excluding M. lamothei from Mexico (Cid del Prado Vera, 2009) that was not included in the list. After that, three other species have been identified. Mesocriconema ozarkiense (Cordero et al., 2012) was described from Ozark National Forest in Washington, USA (Cordero et al., 2012). Mesocriconema ericaceum (Powers et al., 2016) was differentiated from M. xenoplax (Raski, 1952; Loof and De Grisse, 1989) by morphological characters and mitochondrial genome (COI) analysis (Powers et al., 2016). Mesocriconema nebraskense (Olson et al., 2017) was described as a monosexual, cryptic species sympatrically distributed with its cryptic counterpart, M. curvatum (Raski, 1952; Loof and De Grisse, 1989; Olson et al., 2017). In this paper, we describe the new species M. abolafiai n. sp., based on morphological and molecular characteristics.

Material and methods
Nematode populations and morphological characterization

The specimens were recovered from two localities in Dehdasht and Basht (Kohgiluyeh and Boyer-Ahmad province, Southern Iran). The nematodes were extracted from the soil around roots of a grass (Phragmites sp.) using the combination of sieving and centrifugal-flotation method (Jenkins, 1964), killed and fixed by hot FPG (4:1:1, formaldehyde: propionic acid: glycerin), processed to anhydrous glycerin (De Grisse, 1969), and finally mounted in glycerin on permanent slides using paraffin wax. Specimens preserved in glycerin were selected for observation under SEM according to Abolafia (2015). The nematodes 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).

Morphometric and morphological characters of the nematode populations were studied by a light microscope, equipped with a Dino-eye microscope eyepiece camera in conjunction with its Dino Capture version 2.0 software. The nematode species identified by using data documented by Brzeski et al. (2002) and Geraert (2010), as well as by comparison with recently published descriptions (Cid del Prado Vera, 2009; Cordero et al., 2012; Powers et al., 2016; Olson et al., 2017).
DNA extraction

For molecular analysis, DNA was extracted from a single specimen, and three amplifications were conducted on that single specimen. A single female nematode was transferred into a drop of distilled water on a microscopic slide and examined under a light microscope. The nematode specimen was transferred into deionized water, washed three times and then put into an Eppendorf tube with 8 µl distilled water. Then, 12 µl lysis buffer (500 mM KCl, 100 mM Tris-HCL pH 8, 15 mM MgCl2, 10 mM DTT, 4.5% Tween 20) and 2 µl proteinase K were added to the Eppendorf tube. Nematode specimen was crushed with a microhomogenizer during 2 min. The tubes were incubated at 65°C for an hour and then at 95°C for 15 min (Tanha Maafi et al., 2003).
PCR amplification and sequencing

For DNA amplification the protocol described by Tanha Maafi et al. (2003) was used. The D2 to D3 expansion regions of the 28S rRNA gene was amplified with the forward D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and the reverse D3B (5´-TCGGAAGGAACCAGCTACTA-3´) primers (Nunn, 1992). The 18S rRNA was amplified as two partially overlapping fragments, using three universal and one nematode-specific primer (1912R). First 18S fragment forward primer 988F (5´-CTCAAAGATTAAGCCATGC-3´) and reverse primer 1912R (5´-TTTACGGTCAGAACTAGGG-3´) and the second fragment forward primer 1813F (5´-CTGCGTGAGAGGTGAAAT-3´) and reverse 2646R (5´-GCTACCTTGTTACGACTTTT-3´) were used in the PCR reactions for the amplification of the 18S rRNA gene (Holterman et al., 2006). The ITS1-5.8S-ITS2 regions were amplified with the forward TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) and reverse AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) primers (Joyce et al., 1994).

The PCR products were purified using the QIAquick Gel Extraction Kit (Takapozist, Iran) according to the manufacturer’s instruction and used for direct sequencing. The PCR products were sequenced in both directions (BioNeer Inc., Korea). The newly obtained sequences of the new species were submitted to GenBank database under accession numbers MN334221 for the 18S, MN334222 for the 28S D2-D3, and MN334228 for the ITS sequences.
Phylogenetic analysis

The sequences of the studied specimens were compared with sequences of other taxa in GenBank, and then, the closest sequences were selected for phylogenetic analyses. The sequences of 18S rRNA and D2 to D3 segments of 28S rRNA were aligned with ClustalX 1.83 (Thompson et al., 1997), using default parameter values and were manually edited if necessary. The best fitted model of DNA evolution was obtained using jModelTest v. 2 (Darriba et al., 2012) with the Akaike information criterion (AIC). The best-fit nucleotide substitution models were considered to be GTR + I + G for 18S and 28S, and SYM + G for ITS. The phylogenetic tree of sequences was inferred by the Bayesian method using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). Four MCMC chains for 1,000,000 generations were run. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given for appropriate clades. Pairwise divergences between taxa were computed as absolute distance values and as percentage mean distance values based on whole alignment, with adjustment for missing data with PAUP* 4.0b 10 (Swofford, 2002). Trees were visualized using TreeView (Page, 1996).
Results
Systematics
Mesocriconema abolafiai n. sp.

(Figs 1-3; Table 1).

Morphometric characters of Mesocriconema abolafiai n. sp. (measurements are in μm and in the form of average ± s.d. (range)).

Dehdasht population Basht population
Characters Holotype 10 paratype females 3 paratype males 5 females
L 540 540 ± 65 (402–612) 471 ± 33.6 (435–502) 519 ± 65 (425–605)
a 14.9 14 ± 1.5 (10.3–15.3) 24.6 ± 3.2 (20.9–27.1) 12.6 ± 1.2 (10.9–14.4)
b 4.8 4.8 ± 0.6 (3.2–5.7) 24.6 4.3 ± 0.5 (3.5–5.0)
c 14.5 15.8 ± 2.4 (12.1–19.5) 10.5 ± 0.5 (9.9–11) 19.1 ± 2.4 (16.3–21.9)
c´ 1.4 1.3 ± 0 (1.3–1.4) 3.3 ± 0.5 (2.9–3.9) 1.3 ± 0 (1.1–1.4)
V 90.5 90.3 ± 1.1 (87.8–91.9) – 90.1 ± 0.4 (89.8–90.8)
Stylet 56.9 55.2 ± 1.3 (52.8–57.2) – 56.8 ± 2.3 (54–60)
Conus 42.3 44 ± 6.3 (38.6–55.0) – 41.9 ± 1.6 (40.0–44.5)
m (conus/stylet %) 74.4 80.1 ± 11.2 (73.1–100.0) – 73.7 ± 1.7 (71.9–76.3)
Pharynx 110.7 113 ± 8 (103–124) 59.3 119 ± 8 (105–126)
Post-vulval body length (VL) 50.7 51.8 ± 4.9 (44.6–58.9) – 51.2 ± 6.7 (43–60)
Secretory-excretory pore 109 108 ± 7 (99–118) 106 ± 12 (92–113) 108 ± 8 (97–118)
Lip region-vulva 485.2 488 ± 62 (353–553) 468 ± 58.6 (382–545)
Lip region-anus 498.9 506 ± 64.1 (372–575) 426 ± 29.5 (396–455) 492 ± 63.8 (399–577)
Vulva-anus 24.0 22 ± 2.9 (17.1–26.0) – 24 ± 5.3 (17–32)
Tail length 37.0 34.3 ± 3.6 (28.0–39.3) 44.8 ± 4.5 (39.5–47.5) 27.2 ± 2.9 (23–31)
Body width 36.1 38.3 ± 2.4 (34.1–42.0) 19.2 ± 1.6 (17.5–20.7) 41.2 ± 4.3 (34–46)
Vulval body width (VB) 30.4 30.1 ± 1.6 (27.2–32.3) – 30.7 ± 1.9 (27.5–32.5)
VL/VB 1.7 1.7 ± 0.1 (1.6–1.8) – 1.6 ± 0.1 (1.5–1.8)
Annulus width 5.2 5.4 ± 0.7 (4.1–6.5) 2.8 ± 0.4 (2.3–3.2) 5.7 ± 0.6 (4.9–6.8)
R 104.0 104.2 ± 4 (97–113) 132 97 ± 4.3 (90–101)
Rst 16.0 15.5 ± 0.8 (14–17) – 14.5 ± 0.5 (14–15)
Rph 26.0 25 ± 1.4 (23–28) – 24.8 ± 1.9 (23–28)
Rexp 29 23.2 ± 4.2 (19–30) 49.6 ± 2 (48–52) 27.1 ± 4.2 (24–29)
RV 12.0 12 ± 0.8 (11–14) – 12.8 ± 0.8 (12–14)
Ran 8.0 9 ± 0.9 (8–10) – 7.4 ± 0.5 (7–8)
RVan 3.0 3.6 ± 0.4 (3–4) – 3.6 ± 0.8 (3–5)
St/L × 100 10.6 10.3 ± 1.3 (9.2–13.6) – 11 ± 1.1 (9.4–12.7)
Spicules – – 34.2 ± 0.7 (33.6–35.0) –
Gubernaculum – – 6.2 ± 0.9 (5.3–7.2) –

Figure 1:

Diagnostic drawings of Mesocriconema abolafiai n. sp. Female (A, C-E, H, I) and Male (B, F, G). A, F: entire body; B-D: anterior end and pharyngeal region; E: reproductive system; G-I: posterior end.

Figure 2:

Light micrographs of Mesocriconema abolafiai n. sp. Female (A-J) and male (K-M). A: entire body; B, C and K: anterior end and pharyngeal region; D: cuticle at mid-body; E and F: cuticle at posterior end; G: vulval region and part of reproductive system; H-J and L: posterior end; M: spicule, gubernaculum and cloaca. (Scale bars: A = 50 µm; B-M = 10 µm).

Figure 3:

SEM micrograghs of Mesocriconema abolafiai n. sp. Female (A-L). A: anterior end (arrow pointing the secretory–excretory pore); B-E: lip region in sublateral, left subventral, frontal and right subventral views, respectively (arrows pointing the amphids); F: annuli; G: cuticle at mid-body; H: entire body; I-K: posterior end in lateral, subventral and terminal views, respectively (white arrow pointing the vulva, black arrow pointing the anus); L: excretory pore (arrow).
Description
Female

Body is slightly curved ventrally when relaxed by heat, assuming an open C-shaped. Cuticular annuli are retrorse, and margins are smooth to irregular across the entire body, without any hint of crenation. Not more than one anastomosis is observed. Cephalic region is not offset, tapering and slightly conical. Oral disc has rounded edges, and slit-like amphidial apertures are located laterally on the disc. Four small flattened submedian lobes are visible at the same level with a labial plate. Submedian lobes in SEM resemble a tongue with a central, longitudinal crease, not fused with labial plates. First body annulus is slightly smaller than the second annulus, not retrorse. Stylet is robust, with well-developed knobs that possess moderate anterior projections and 9 to 11 µm in width. Secretory–excretory pore is located at 27 to 30 annuli from the anterior end, almost at 4 to 5 annuli behind the pharyngeal basal bulb, which is small, pyriform, 10 to 13.5 µm in width and 18 to 20 µm in length. Female genital gonad is outstretched and spermatheca is slightly oval, offset from gonad, filled with globular sperm cells about 1 µm in diameter. Vagina is straight, occasionally slightly curved. Vulva is open with simple anterior lip. The post-vulval region of the body tapers gradually, ending in a pointed terminus or a small bilobed end annulus. SEM reveals that the anal opening is located usually three annuli posterior to the vulva.
Male

Body is vermiform and curved ventrally when fixed by heat treatment. Lateral field has four distinct longitudinal incisures. Lip region has distinct transverse striation, 8 to 9 µm wide and 5 to 7 µm high. Stylet and pharynx are degenerated. Spicules (33.6-35.0 µm in length) are slender and tylenchoid, ending to a distinct penial tube (4-5 µm in length). Gubernaculum is simple and slightly curved. Bursa not is observed. Tail is elongated-conical, ending to a pointed terminus.
Juvenile

Similar to female in general characteristics. Body is straight or slightly curved ventrally after fixation. Annuli are retrorse, lacking any crenation and ornamentation. Total number of annuli approximately equals to that of females, but annuli are narrower than of females (average 4.4 vs 5.5 µm).
Diagnosis

Mesocriconema abolafiai n. sp. is characterized by 90 to 113 cuticular annuli with smooth to irregular margins lacking crenation, small and flattened submedian lobes, stylet 52.8 to 60.0 µm long, open vulva with a simple anterior vulval lip, straight vagina, spermatheca filled with globular sperm, presence of males, and conical-acute tail with last annulus bilobed or rounded.
Relationships

Mesocriconema abolafiai n. sp. is characterized by having flattened submedian lobes groups with M. antipolitanum (De Guiran, 1963); M. citricola (Siddiqi, 1965; Loof and De Grisse, 1989); M. juliae (Crozzoli and Lamberti, 2001); M. napoense (Talavera and Hunt, 1997); M. oostenbrinki (Loof, 1964); M. ozarkiense (Cordero et al., 2012); M. paralineolatum (Rashid et al., 1987); M. planilobatum (Ta1avera and Hunt, 1997); and M. rusticum (Khan et al., 1976) in the diagnostic compendium developed by Brzeski et al. (2002).

Mesocriconema abolafiai n. sp. can be distinguished from M. antipolitanum and M. rusticum by differences in the size of submedian lobes (small vs large), tail shape (conical vs rounded), spermatheca (filled vs empty) and occurring of males (present vs absent). It differs from M. citricola by a different shape of the anterior vulval lip (simple vs bilobed) and higher number of cuticular annuli (90-113 vs 73-78). It can be differentiated from M. juliae by stylet length (52.8-60.0 vs 79-86 µm) and shape of the anterior vulval lip (simple vs bilobed). Mesocriconema abolafiai n. sp. can be distinguished from M. napoense, M. paralineolatum, and M. planilobatum by the number of cuticular annuli (90-113 vs 73-79, 82-88, 75-84, respectively) and vagina direction (straight vs sigmoid). It differs from M. oostenbrinki by a different shape of the anterior vulval lip (simple vs bilobed), the number of cuticular annuli (90-113 vs 84-94), and vagina direction (straight vs sigmoid). Our new species can be distinguished from M. ozarkiense by differences in the posterior end of cuticular annuli on post-vulval region (smooth vs crenated), spermatheca (filled vs empty), occurring of males (presence vs absence), vagina direction (straight vs sigmoid), and VL/VB ratio (1.5-1.9 vs 1.0-1.4).

Regarding general morphometric characters and tail shape, our populations can also come similar to M. denoudeni (De Grisse, 1967; Loof and De Grisse, 1989); M. jessiense (Van den Berg, 1992, 1994); M. reedi (Diab and Jenkins, 1966; Loof and De Grisse, 1989); M. raskiense (De Grisse, 1964; Andrássy, 1965); M. vadense (Loof, 1964; Loof and De Grisse, 1989); M. kirjanovae (Andrássy, 1962; Loof and De Grisse, 1989); M. paradenoudeni (Rashid et al., 1987; Loof and De Grisse, 1989); and M. parareedi (Ebsary, 1981a; Loof and De Grisse, 1989). However, our populations can be differentiated from M. denoudeni by a different tail terminus shape (conical-acute vs conical-rounded), the number of post-vulval annuli (11-14 vs 8-11), VL/VB ratio (1.5-1.9 vs 1.0-1.3), and presence of males. The new species differs from M. jessiense and M. reedi by having more annuli at post-vulval region (11-14 vs 8-9 and 9-10) and higher value for the VL/VB ratio (1.5-1.9 vs 0.8-1.1 and 1.1-1.3), and differs from M. reedi by having a larger body size (402-612 vs 360-470 µm). In comparison with M. raskiense, it has more annuli throughout body (90-113 vs 62-72), and a different structure of cuticular annuli (smooth and without anastomoses vs crenated with anastomoses at mid-body). It also differs from M. vadense by the number of cuticular annuli (90-113 vs 70-81), the number of post-vulval annuli (11-14 vs 7-10), VL/VB ratio (1.5-1.9 vs 0.8-1.3), and tail shape (conical-acute vs conical-rounded).

M. abolafiai n. sp. can be further distinguished from M. kirjanovae, M. citricola, M. paradenoudeni, and M. parareedi by a different shape of the anterior vulval lip (simple vs bilobed), and variations in the number of cuticular annuli (90-113 vs 79-89, 73-78, 102-130, and 111-121, respectively).

The males recovered in the type population have a unique elongated tail with pointed terminus, which only could be observed in M. raskiense and M. vadense. Spicules in our population are comparable with those of M. vadense (33-35 vs 30-34 µm) but shorter than those in M. raskiense (33.6-35.0 vs 38-43 µm). Males in some other species including M. brevicauda (Van den Berg and Spaull, 1985; Loof and De Grisse, 1989); M. curvatum, M. involutum (Loof, 1987, 1989); M. irregulare (De Grisse, 1964; Loof and De Grisse, 1989); M. juliae and M. oostenbrinki have more or less similar tails but shorter in size or with a different terminus shape.
Type host and locality

The type population was found from a canebrake in Dehdasht, Kohgiloyeh and Boyer-Ahmad province (30°49.42´N, 51°28.91´E). The other population was recovered from the rhizosphere of dog-rose shrubs (Rosa canina L.) in Basht, Kohgiloyeh and Boyer-Ahmad province (30°19.29´N, 51°15.04´E) during April 2017 by the first author.
Type specimens

Holotype, 10 paratype females and three paratype males, as well as five female specimens from the other recovered population were deposited in the nematode collection of the Department of Plant Protection, College of Agriculture, University of Zanjan, Zanjan, Iran.
Etymology

The species epithet refers to the name of Dr. Joaquín Abolafia, the well-known nematologist from University of Jaén, Spain, who works on nematode systematics.
Phylogenetic relationships

The 28S alignment was 738 bp long and consisted of 58 sequences as ingroups and three sequences, including Aglenchus agricola (Andrássy, 1954; De Man 1884) (AY780979), Eutylenchus excretorius (Sher et al., 1966) (AY780980), and Merlinius brevidens (Allen, 1955; Siddiqi, 1970) (KP313844), as outgroups (Table 2). Phylogenetic relationships of M. abolafiai n. sp. with other representatives of Criconematidae (Taylor, 1936; Thorne, 1949) inferred from the analysis of D2 to D3 expansion fragments of 28S rRNA gene sequences with collapsed branches, with PP less than 50%, are given in Figure 4. In this tree, M. abolafiai n. sp. formed a cluster with an isolate of M. xenoplax (MG680454) and an unnamed population (AY780967). Partial 28S rRNA sequences of the M. abolafiai n. sp. from Iran show about 23 bp (3%) difference with the closet species according to 28S tree (M. xenoplax: MG680454), whereas two species distinguished with some characters such as tail (conical vs subcylindrical) and vagina (straight vs sigmoid) shape. There is not any record of partial 28S rRNA sequences of M. ozarkience that it is closest species to M. abolafiai n. sp. based on morphological characteristics.

List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on 28S rRNA gene.

Species name GeneBank accession no. Locality Species name GeneBank accession no. Locality
Aglenchus agricola AY780979 Belgium Hemicycliophora typica KF430515 South Africa
Caloosia longicaudata GU989627 USA H. wyei KC329574 USA
Criconema demani MH828126 Russia H. wyei KF430497 USA
C. demani MH828128 Russia Merlinius brevidens KP313844 Iran
C. mutabile MK170079 South Africa Mesocriconema abolafiai n. sp. MN334222 Iran
Criconema sp. FN433874 USA M. ornatum AY780968 Venezuela
Criconemoides brevistylus JQ231183 South Africa M. solivagum AY780969 Russia
C. brevistylus JQ231184 South Africa Mesocriconema sp. AY780967 Italy
C. brevistylus KC937033 China M. sphaerocephalum AB933464 Japan
C. informis KU722386 Iran M. sphaerocephalum AB933465 Japan
C. myungsugae MH444641 China M. sphaerocephalum AY780951 Italy
C. obtusicaudatus JQ231186 South Africa M. xenoplax AB933468 Japan
C. obtusicaudatus JQ231187 South Africa M. xenoplax AY780961 Germany
Eutylenchus excretorius AY780980 Germany M. xenoplax AY780963 USA
Hemicaloosia guangzhouensis KT381016 China M. xenoplax AY780965 Italy
H. guangzhouensis KT381017 China M. xenoplax FN433855 USA
H. vagisclera JQ246422 USA M. xenoplax FN433858 USA
Hemicriconemoides gaddi MK050500 China M. xenoplax FN433859 USA
H. rosae MK371811 India M. xenoplax KC538862 USA
H. rosae MK371813 India M. xenoplax MG680454 Portugal
H. silvaticus KF856531 Japan Ogma civellae AY780955 Venezuela
H. strictathecatus MH142613 China O. decalineatus MF683230 South Africa
H. wessoni KF856521 USA Paratylenchus tenuicaudatus KU291239 Iran
Hemicycliophora conida FN433875 Belgium Sphaeronema alni AY780978 Germany
H. epicharoides KF430512 Italy Trophonema arenarium AY780971 Italy
H. gracilis KF430482 USA Tylenchulus semipenetrans KM598334 Iran
H. halophila KF430444 New Zealand T. semipenetrans KM598335 Iran
H. lutosa GQ406240 South Africa T. semipenetrans MH156801 China
H. lutosa GQ406241 South Africa T. semipenetrans MH156802 China
H. signata MG019824 Mozambique Xenocriconemella macrodora AY780960 Italy
H. subbotini MG701275 China

Figure 4:

Bayesian 50% majority rule consensus tree as inferred from the D2 to D3 expansion segments of 28S rRNA gene dataset of Criconematoidea under the general time reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities more than 50% are given for appropriate clades. The new obtained sequence in this study is indicated in bold. Scale bar=expected changes per site.

The 18S alignment was 1538 bp long and consisted of 60 sequences as ingroups and Merlinius joctus (Thome, 1949; Sher, 1974) as outgroup (Table 3). Phylogenetic relationships of M. abolafiai n. sp. with other representatives of Criconematidae inferred from the analysis of 18S rRNA gene sequences with collapsed branches, with PP less than 50%, are given in Figure 5. The new species clustered with M. rusticum (MF094965) and Mesocriconema sp. (MF094967), all in a sister clade with M. curvatum (MF094891). The most important morphologically differences between M. abolafiai n. sp. and M. rusticum are related to lip region and post-vulval shape, respectively. The lip region in M. rusticum is set off with large submedian lobes, anteriorly flattened giving appearance of truncate anterior, but the lip region in M. abolafiai n. sp. is not offset, tapering and slightly conical. The post-vulval part is rounded in M. rusticum and tail end multi-lobed, mostly bent dorsally, whereas in M. abolafiai n. sp. post-vulval region of the body tapers gradually, ending in a pointed terminus or a small bilobed end annulus. The other interesting result inferred from the phylogenetic analysis of the present study is that the 18S rRNA gene is not a good marker for species differentiation in the Mesocriconema genus, because there is only one nucleotide difference between M. abolafiai n. sp. and M. rusticum, whereas these species are well separated based on morphological characteristics.

List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on 18S rRNA gene.

Species name GeneBank accession no. Locality Species name GeneBank accession no. Locality
Bakernema inaequale MF094908 USA H. conida AJ966471 GenBank
Criconema mutabile MF094914 USA H. conida KJ934172 USA
C. permistum MF094899 USA H. conida KJ934173 USA
C. petasum MF094927 USA H. subbotini MG701280 China
C. sphagni MF094968 USA Lobocriconema sp. MF094981 USA
Criconemoides annulatus MF095015 USA L. thornei MF094928 USA
C. annulatus MF095024 USA L. thornei MF094996 USA
C. informis MF094902 USA Merlinius joctus FJ969128 GenBank
C. informis MF095025 USA Mesocriconema abolafiai n. sp. MN334221 Iran
C. parvus MF795587 China M. curvatum MF094891 USA
Crossonema fimbriatum MF095026 USA M. discus MF094892 USA
C. fimbriatum MF094960 USA M. inaratum MF094903 USA
C. menzeli MF094937 USA M. onoense MF094909 USA
Discocriconemella limitanea MF795591 China M. ornatum MF094893 USA
D. limitanea MF095031 Costa Rica M. rusticum MF094965 USA
Gracilacus paralatescens MH200615 China Mesocriconema sp. MF094967 USA
G. wuae MF095028 Canada Mesocriconema sp. MF095012 USA
Hemicaloosia graminis JQ446376 USA M. sphaerocephalum KJ934182 USA
Hemicriconemoides. chitwoodi KJ934162 USA M. xenoplax KJ934180 USA
Hemicriconemoides. fujianensis MH444626 China M. xenoplax KJ934177 USA
H. kanayaensis MG029558 China M. xenoplax MF095021 USA
H. kanayaensis MG029559 China M. xenoplax MF094992 USA
H. parasinensis MH444635 China Ogma decalineatus MF094952 USA
H. parataiwanensis MG029556 China O. menzeli EU669919 GenBank
H. parataiwanensis MG029557 China O. seymouri MF094933 USA
H. pseudobrachyurus AY284622 GenBank Ogma sp. KJ934175 USA
Hemicriconemoides sp. MF095013 Thailand Paratylenchus straeleni AY284631 GenBank
H. wessoni KJ934163 USA Tylenchulus semipenetrans MH136626 China
H. wessoni KJ934166 USA T. semipenetrans AJ966511 UK
Hemicycliophora aquatica MF094911 USA Xenocriconemella macrodora MF095001 USA
H. conida EU669914 GenBank

Figure 5:

Bayesian 50% majority rule consensus tree as inferred from the 18S rRNA gene dataset of Criconematoidea under the general time reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Posterior probabilities more than 50% are given for appropriate clades. The new obtained sequence in this study is indicated in bold. Scale bar = expected changes per site.

The ITS alignment was 401 bp long and consisted of 70 sequences as ingroups and Coslenchus rhombus (Andrássy, 1982) as outgroup (Table 4). Phylogenetic relationships of M. abolafiai n. sp. with other representatives of Criconematidae inferred from the analysis of ITS rRNA gene sequences with collapsed branches, with PP less than 50%, are given in Figure 6. In this tree, the new species clustered with an isolate of M. curvatum (MF094891) with 37 bp difference (9.2%), and in a distant position from the morphologically related species, M. ozarkiense (JQ708122) with 129 bp difference (32%).

List of species, collection localities and GenBank accession numbers of individual specimens used in this study for phylogenetic analysis based on ITS rRNA gene.

Species name GeneBank accession no. Locality Species name GeneBank accession no. Locality
Coslenchus rhombus MK874505 South Africa Mesocriconema curvatum MF094891 USA
Criconema silvum MF683236 South Africa M. inaratum HM116070 USA
C. silvum MF683237 South Africa M. inaratum HM116069 USA
Criconemoides brevistylus KC937032 China M. inaratum HM116058 USA
C. brevistylus JQ231188 South Africa M. inaratum HM116055 USA
C. myungsugae MH444640 China M. inaratum HM116052 USA
C. myungsugae MH444639 China M. inaratum HM116051 USA
C. obtusicaudatus JQ231189 South Africa M. nebraskense MH013431 USA
C. obtusicaudatus JQ231190 South Africa M. nebraskense KY574844 USA
Crossonema sp. MK292124 USA M. nebraskense KY574860 USA
Hemicriconemoides californianus KF856558 USA M. nebraskense KY574861 USA
H. californianus KF856560 USA M. nebraskense KY574862 USA
H. chitwoodi KF856543 USA M. nebraskense KY574863 USA
H. fujianensis MH444616 China M. nebraskense KY574864 USA
H. kanayaensis EF126179 Taiwan M. nebraskense KY574865 USA
H. kanayaensis MG029566 China M. onoense JQ708120 USA
H. kanayaensis MG029568 China M. ornatum JQ708124 USA
H. ortonwilliamsi KF856552 Spain M. ozarkiense JQ708122 USA
H. paracamelliae MG029560 China Mesocriconema sp. KY574858 USA
H. promissus KF856555 Spain Mesocriconema sp. KY574857 USA
H. rosae MK371815 India Mesocriconema sp. KY574856 USA
Hemicriconemoides sp. KM516185 USA M. xenoplax JQ708112 USA
H. strictathecatus KF856565 South Africa M. xenoplax HM116073 USA
H. strictathecatus MH142617 China M. xenoplax HM116057 USA
H. strictathecatus KM516186 USA M. xenoplax MF095021 USA
H. strictathecatus KM516190 USA M. xenoplax MF094992 USA
H. strictathecatus KM516191 USA M. xenoplax MF094915 USA
Hemicycliophora californica KF430576 USA M. xenoplax MF094916 USA
H. gracilis MG019827 USA Neobakernema variabile MF683239 USA
H. raskii KF430577 USA N. variabile MF683238 USA
H. thienemanni KF430569 Russia Ogma decalineatus MF683235 USA
Mesocriconema abolafiai n. sp. MN334228 Iran Paratylenchus hamatus KF242257 USA
M. curvatum HM116062 USA Tylenchulus semipenetrans JN112274 USA
M. curvatum HM116064 USA T. semipenetrans FJ588909 China
M. curvatum HM116066 USA T. semipenetrans MH124562 China
M. curvatum HM116067 USA T. semipenetrans MH124561 China
M. curvatum HM116068 USA

Figure 6:

Bayesian 50% majority rule consensus tree as inferred from the ITS rRNA gene dataset of Criconematoidea under the symmetrical model of sequence evolution with a gamma-shaped distribution (SYM + G). Posterior probabilities more than 50% are given for appropriate clades. The new obtained sequence in this study is indicated in bold. Scale bar = expected changes per site.
Remark

The genus Mesocriconema has a large number of species, which are morphologically very close to each other. Powers et al. (2016) distinguished 24 COI haplotype groups; only five of them corresponded to morphologically characterized species. The authors further noticed that it is not unexpected that additional sampling of Mesocriconema will continue to reveal cryptic species within Linnaean morphospecies; as these species have been described in the recent works (Powers et al., 2016; Olson et al., 2017).

Mesocriconema abolafiai n. sp. comes close to M. ozarkiense and some other species bearing conical tails with narrow tails (Table 5); however, detailed morphological observations made by light microscopy and SEM, as well as molecular phylogenetic analysis using different genes allowed us to consider M. abolafiai n. sp. as a new species. The new species can be differentiated from the most closely related species, M. ozarkiense, by some morphological and morphometric characters, as well as a different phylogenetic position in the ITS rRNA tree which resulted from a 129 bp (32%) nucleotide divergence. Unfortunately, there is no molecular information on 28S rRNA and 18S rRNA genes of M. ozarkiense so the above results can be further supported by additional analyses of these gene sequences.

Summary of the diagnostic characteristics of 12 species of Mesocriconema recently described worldwide. For other species, see (Brzeski et al., 2002). (St measurements in µm, L measurements in mm).

Species St R Rex RV RVan Ran V VL/VB Anas. A. M. Vagina A. V. lip Tail shape L S. M. L. Original des.
M. abolafiai n. sp. 52–60 90–113 19–30 11–14 3–5 7–10 87–90 1.5–1.8 1 smo.–irr. straight simple con–acute 0.4–0.61 flat Present study
M. apurense 48–52 140–147 36–38 9–12 1–2 8–9 93–95 – 0 smo. straight lobulated, without projections con.–rounded 0.38–0.45 rounded Crozzoli and Lamberti (2001)
M. campbelli 58–62 102–108 25–28 11–15 – 5–7 89–93 1.2–1.8 rare smo.–ser. straight serrated con.–pointed 0.37–0.45 – Wouts (2006)
M. juliae 79–86 89–95 26 8–9 1–2 5–6 91–92 – few smo. straight with two projections conical 0.46–0.51 flat Crozzoli and Lamberti (2001)
M. lamothei 65–70 89–98 17–31 6–8 0–2 4–8 91–95 rare smo. slightly curved with two projections con.–truncate 0.40–0.49 rounded Cid del Prado Vera (2009)
M. lobellum 51–60 85–92 25 6–7 2–3 3–4 92–93 0.9–1.2 rare – straight simple rounded 0.41–0.53 rounded Pramodini et al. (2007)
M. malagutii 45–49 108–114 33–35 8–9 2 4–6 93–94 – many smo. straight lobulated, without projections con.–truncate 0.34–0.40 rounded Crozzoli and Lamberti (2001)
M. nebraskense 45–59 84–113 24–31 6–11 2–3 3–7 90–96 0.7–1.6 1–4 smo. straight with two projections rounded 0.39–0.60 rounded Olson et al. (2017)
M. ovospermatum 82 109 38 9 3 6 93 – 0 finely crenate straight ? conical 0.22 ? Mohilal and Dhanachand (1998)
M. ozarkiense 49–61 107–119 27–34 10–14 2–4 6–10 89–93 1.0–1.4 0–1 som.–irr. straight simple conical 0.38–0.51 flat Cordero et al. (2012)
M. theobromae 47–51 73–74 24–26 7 1 5 93–94 – many som. straight lobulated, without projections conical 0.27–0.32 rounded Crozzoli and Lamberti (2001)
M. waitha 66–78 117-140 26–31 9–10 4–5 4–6 92–94 0.9–1.0 many som.-irr. sigmoid simple rounded 0.43-0.53 flat? Pramodini et al. (2006)

Notes: St, stylet; Anas., Anastomoses; A. M., Annuli margin; A. V. lip, Anterior vulval lip; S. M. L, Submedian lobes; smo., smooth; ser., serrate; irr., irregular; con, conical; Original des., Original description.

Note1: Mesocriconema bakeri (Wu, 1965; Loof and De Grisse, 1989); M. calvatum (Eroshenko, 1982; Loof and De Grisse, 1989); M. hymenophorum (Wouts and Sturhan, 1999); M. longistyletum (De Grisse and Maas, 1970; Loof and De Grisse, 1989); M. paramonovi (Razzhivin, 1974; Loof and De Grisse, 1989); M. variabile (Raski and Golden, 1966; Brzeski et al., 2002) and M. yukonense (Ebsary, 1982, 1991) that have been listed in Brzeski et al. (2002), transferred to Neobakernema (Ebsary, 1981b) by Geraert (2010).

Note2: Mesocriconema incrassatum (Raski and Golden, 1966; Loof and De Grisse, 1989) that has been listed in Brzeski et al. (2002), transferred to Lobocriconema (De Grisse and Loof, 1965) by Geraert (2010).
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