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

Abstract Mesocriconema abolafiai n. sp. is described by morphological, morphometric, and molecular approaches. The new species is characterized by a body slightly curved with 402 to 612 μm length, 90 to 113 cuticular body annuli with smooth to irregular margins lacking of crenation with not more than one anastomoses, lip region not offset, small flattened submedian lobes, stylet robust (52.8-60.0 μm) with well-developed knobs, open vulva with simple anterior lip, straight vagina, filled spermatheca with globular sperms, presence of males, and conical-acute tail with last annulus bilobed or rounded. Discussions are made on the characterization of M. abolafiai n. sp. from the most closely related species, M. ozarkiense, and several other species having similar tail shape. Furthermore, results of phylogenetic analyses inferred from D2 to D3 expansion fragments of 28S rRNA, 18S rRNA, and ITS rRNA gene sequences revealed the phylogenetic position of the new species within representatives of Criconematidae and supported morphological justifications for considering this population from Iran as a new species in the genus Mesocriconema.

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  proposed to divide the large genus Criconemoides into several genera including Mac roposthonia 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 . Based upon SEM microscopy and discussing on plesiomorphic and apomorphic states of characters,  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; 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;Olson et al., 2017). In this paper, we describe the new species M. abolafiai n. sp., based on morphological and molecular characteristics.

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 MgCl 2 , 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).
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).

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

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 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.  (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.  For other species, see (Brzeski et al., 2002). (St measurements in µm, L measurements in mm).
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. postvulval 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. 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%).

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.