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

Hossein Mirbabaei Karani; Ali Eskandari; Reza Ghaderi; Akbar Karegar

doi:10.21307/jofnem-2020-048

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).

Table 1.

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

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.

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).

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.

Table 2.

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

		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)	–

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.

Table 3.

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
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

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%).

Table 4.

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
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

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.

Table 5.

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 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

1 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).

Acknowledgements

The authors thank anonymous reviewers and the editor of this MS for their valuable suggestions to improve the manuscript. SEM pictures were obtained with the assistance of Dr Joaquín Abolafia and the technical staff (Amparo Martínez-Morales) and equipment of ‘Centro de Instrumentación Científico-Técnica (CICT)’ from University of Jaén, Spain, and the financial support of the research activities ‘PAIUJA 2017/2018: EI_RNM02_2017’ and ‘PAIUJA 2019/2020: EI_RNM02_2019’ of the University of Jaén.

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)