New insights into the identity of Discolaimium dubium Das, Khan and Loof, 1969 (Dorylaimida) as derived from its morphological and molecular characterization, with the proposal of its transference to Aporcella Andrássy, 2002

Abstract Three Iranian populations of Discolaimium dubium are studied, including their morphological and morphometric characterization, molecular analysis (LSU-rDNA) and the description of the male for the first time. For comparative purposes, this species is distinguished by its 1.10 to 1.40 mm long body, lip region offset by constriction and 8 to 10 µm wide, odontostyle 7.5 to 10.5 µm long with aperture occupying 59 to 76% of total length, neck 300 to 362 µm, pharyngeal expansion 127 to 181 µm long or 44 to 46% of the total neck length, uterus simple and 38 to 53 µm or 1.2 to 1.5 times the body diameter long, V = 52 to 58, tail conical (32-38 µm, c = 32-43, c′ = 1.6-2.0) with rounded tip and a hyaline portion occupying 14 to 15% of tail length, spicules 30 to 32 µm long, and two or three widely space ventromedian supplements with hiatus. Both morphological and molecular data support its belonging to the genus Aporcella, whose monophyly is confirmed and to which the species is formally transferred as A. dubia (Das et al., 1969) comb. n.

In their revision of the genus Discolaimoides Heyns, 1963Das et al. (1969 described Discolaimium dubium based on the holotype plus 25 females collected from several European enclaves in Italy, the Netherlands and Switzerland. Later, it was recorded from the Netherlands again (Bongers, 1988), Spain (Liébanas et al., 2003;Peña-Santiago et al., 2005), Hungary (Andrássy, 2009) and India (Sharma, 2011). Thus, it seems to be a component of the nematode fauna inhabiting in northern territories, widely spread out in Europe.
When describing it, Das et al. (1969) characterized D. dubium by having, among other features, 1.06 to 1.33 mm long body, lateral chord without cons picuous lateral organs, lip region slightly expanded, odontostyle 8 to 10 µm long with aperture occupying slightly more than one-half of total length, anterior portion of pharynx muscular and enlarging very gradually, absence of pars refringens vaginae and conical tail. This was a peculiar combination of traits; therefore, the authors raised a doubt about its identity and named it dubium, that is, dubious or doubtful (cf. Andrássy, 2009) and pointed out (p. 486) that '… The systematic position of this species is uncertain. Because of general body shape, shape of lips, structure of cuticle and vagina it is included in Discolaimium, but it differs from all species of this genus in the thick anterior part of the esophagus, the anterior position of DO and the absence of conspicuous lateral organs. For the moment, however, it cannot be placed elsewhere; the unsclerotized vagina keeps it outside Eudorylaimus'. Patil and Khan (1982) were aware of the intricate taxonomy of D. dubium and solved the matter with the proposal of the new genus Neodiscolaimium, with N. dubium as its type and only species. Nevertheless, this action was not followed by other authors (Andrássy, 1990(Andrássy, , 2009Jairajpuri and Ahmad, 1992) who regarded Neodiscolaimium as a junior synonym of Discolaimium (Thorne, 1939).
A general survey conducted during last years to explore the dorylaimid fauna from Iran resulted in the recovering of, among other forms, several populations identified as belonging to D. dubium. Their morphological and molecular study should elucidate the evolutionary relationships of this species, and provide new information to understand better the phylogeny of these dorylaimid taxa.

Materials and methods
Extraction and processing of nematodes Several soil samples were collected from depths ranging from 10 to 40 cm, in the active plant root zone of East-Azarbaijan province, northwest Iran, during the period 2016 to 2017. Nematodes were extracted from soil samples using Whitehead and Heming (1965) method, transferred to anhydrous glycerin according to De Grisse (1969), and mounted on glass slides for handling.

Light microscopy
Measurements were made using a drawing tube attached to an Olympus BX-41 light microscope. The digital images were prepared using a DP50 digital camera attached to the same microscope with differential interference contrast (DIC). Morphometrics included Demanian indices and the usual measurements and ratios. Line illustrations were prepared using CorelDRAW ® software version 12. Photographs were edited using Adobe ® Photoshop ® CS software.

DNA extraction, PCR, and sequencing
For the molecular phylogenetic studies, DNA samples were extracted from one or two females selected from each population, studied individually on temporary slides, placed on a clean slide containing a drop of distilled water or worm lysis buffer (WLB), and crushed by a sterilized scalpel. Then, the suspension was transferred to an Eppendorf tube containing 25.65 μ l ddH2O, 2.85 μ l 10 × PCR buffer and 1.5 μ l proteinase K (600 μ g/ml) (Promega, Benelux, the Netherlands). The tubes were incubated at −80°C (1 hr), 65°C (1 hr) and 95°C (15 min). Each sample was regarded as an independent DNA sample and stored at −20°C until used as polymerase chain reaction (PCR) template. Primers for 28S rDNA D2-D3 amplification/sequencing were forward primer D2A (5´-ACAAGTACCGTGAGGGAAAGTTG-3´) and reverse primer D3B (5´-TCGGAAGGAACCAGCTACTA-3´) (Nunn, 1992). The 25 μ l PCR reaction mixture containing 10 μ l ddH2O, 12.5 μ l PCR master mix (Ampliqon, Denmark), 0.75 μ l of each forward and reverse primers and 1 μ l of DNA template. PCR was carried out using a BIO RAD thermocycler machine in accordance with Archidona-Yuste et al. (2016). The thermal cycling program for amplification were as follows: denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 sec, annealing of primers at 55°C for 45 sec and extension at 72°C for 3 min followed by a final elongation step at 72°C for 10 min. The PCR products were purified and sent for sequencing to Bioneer Company, South Korea. The recently obtained sequences were submitted into the GenBank database under the accession numbers MT079121, MT079122 and MT079123S as indicated on the phylogenetic tree (Fig. 4).

Phylogenetic analyses
The Basic Local Alignment Search Tool (BLAST) homology search program was used to compare the newly generated sequences with other available sequences in GenBank database. The recently generated sequences were aligned with the other segments of 28S rDNA gene sequences retrieved from the database using MEGA6 software (Tamura et al., 2013). Paravulvus hartingii (de Man, 1880;Heyns, 1968) (AY593062) was chosen as outgroup. Bayesian analysis (BI) was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of nucleotide substitution used for the phylogenetic analysis was selected using MrModeltest 2.3 (Nylander, 2004) with Akaike-supported model in conjunction with PAUP* v4.0b10 (Swofford, 2003). The BI analysis under GTR + I + G model was initiated with general time-reversible model with invariable sites and a gamma-shaped distribution for the 28S rDNA gene was done. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. Posterior probabilities (PP) are given on appropriate clades. The tree was visualized and saved using the program

Adult
Slender to very slender (a = 39-49) nematodes of medium size, 1.10 to 1.40 mm long. Body cylindrical, tapering toward both ends, but more so toward the posterior extremity as the tail is conical. Upon fixation, habitus regularly curved ventrad, to an open C shape in females and nearly J shape in males. Cuticle twolayered, 1 µm thick at anterior region, 1.5 to 2.5 µm in mid-body and 2.5 to 3.5 µm on tail, consisting of thin outer layer bearing fine but conspicuous transverse striation, and thicker inner layer, more appreciable at caudal region. Lateral chord 7 to 10 µm broad, occupying 21 to 28% of mid-body diameter, lacking any differentiation. Lateral pores obscure. Lip region somewhat truncate anteriorly, slightly expanded (but not discoid), that is, slightly wider than its adjoining body, 2.6 to 3.3 times as wide as high and ca onethird (26-37%) of body diameter at neck base; lips mostly amalgamated, with weakly protruding papillae. Amphidial fovea cup-like, its aperture occupying 4.5 to 6 µm or more than one-half (50-63%) of lip  Lip region diam.

8-10
Odontophore length region diameter. Cheilostom comparatively short and broad, with thick walls. Odontostyle relatively short and robust, 4.1 to 4.6 times as long as wide, nearly equal (0.9-1.0 times) to the lip region diameter, and 0.66 to 0.76% of the total body length; aperture large, 5.5 to 6.5 µm long, occupying more than onehalf (59-76%) of the odontostyle length. Guiding ring simple, but conspicuous, situated at 4.5 to 5.5 µm or 0.5 to 0.6 times the lip region diameter from the anterior end. Odontophore rod-like, 1.7 to 2.0 times the odontostyle, lacking any differentiation. Pharynx entirely muscular, consisting of a slender section gradually enlarging into the basal expansion that is 7.7 to 9.5 times as long as wide, 4.5 to 5.5 times the body diameter at neck base and occupies 44 to 46% of the total neck length; gland nuclei located as follows (n = 2): DO = 58, 60; DN = 63; S 1 N 1 = 72, 75; S 1 N 2 = ?82, S 2 N = 88, 90. Pharyngo-intestinal junction with a rounded conoid cardia 5.5−9.5 × 8.5-12.5 µm, surrounded by intestinal tissue. Tail conical, with rounded tip, ventrally nearly straight, dorsally regularly convex; inner core not reaching the tail tip, therefore a hyaline portion is present occupying 5 to 5.5 µm or 14 to 15% of tail length; caudal pores two pairs, at the middle of tail, one nearly dorsal, other subdorsal.

Female
Genital system didelphic-amphidelphic, with both branches equally and moderately developed, the anterior 111 to 203 µm long or 9 to 16% of body length, the posterior 108 to 202 µm or 8 to 17% of body length. Ovaries reflexed, relatively small, often not reaching the oviduct-uterus junction, the anterior 52 to 74 µm, the posterior 54 to 90 µm long. Oviduct 44 to 74 µm or 1.3 to 2.1 times the body diameter long, consisting of a slender distal portion made of prismatic cells and a moderately developed pars dilatata. A marked sphincter separates oviduct and uterus. Uterus a simple, tube-like structure, 38 to 53 µm or 1.2 to 1.6 times the body diameter long. Vagina extending inwards 11 to 15 µm or ca two-fifths (33-43%) of body diameter: pars proximalis 7-10 × 8-11 µm, with slightly convergent walls surrounded by weak circular musculature and pars distalis 3.5 to 4.5 µm long. Vulva a somewhat posterior, transverse slit. Prerectum 3.0 to 6.6, rectum 1.2 to 1.5 times the anal body diameter long.

Male
Genital system diorchic, with opposed testes. In addition to the adcloacal pair, located at 8 to 9 µm from the cloacal aperture, there are two or three, widely spaced 13.5 to 16 µm apart, ventromedian supplements, the most posterior of them situated far from the ad-cloacal pair, at 59 to 79 µm. Spicules dorylaimid, regularly curved ventrad and relatively robust, 3.9 to 4.9 times as long as wide; head small, 2.5 to 3 μ m long, occupying 8 to 10% of total spicule length, with its dorsal side slightly longer than the ventral one; ventral side bearing visible hump and hollow, median piece occupying 85 to 87% of spicule length. Lateral guiding pieces 8.5 to 9 µm long. Prerectum 2.4 to 4.5, cloaca 1.4 to 1.5 times body diameter at level of cloacal aperture.

Molecular characterization
Three D2-D3 28S rDNA sequences were obtained, ca 800 bp long. Their analysis has allowed the elucidation of evolutionary relationships of the species. The phylogenetic results are presented in

Discussion
Comparison of Iranian specimens with type material of D. dubium and its closely related species from the genus Aporcella The description of D. dubium by Das et al. (1969) was not excessively detailed, but available information is enough for comparative purposes. Table 1 shows that the most relevant morphometrics of Iranian nematodes and type material (consisting of specimens from at least three European enclaves, see above) are nearly identical, with totally coincident or widely overlapping ranges. General description, in particular the morphology of pharynx, genital system, and caudal region, are identical, as well. Thus, there is no doubt that the Iranian material belongs to the species. The above mentioned description therefor confirms the original one and provides an updated morphometric  Das et al., 1969. Bayesian 50% majority rule consensus tree as inferred from D2-D3 expansion segments of 28S rDNA sequence alignments under the GTR + G + I model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated by bold letters.
and morphological characterization of the species. Morphometrical cha racters of Iranian population of D. dubium and closely related species in the genus Aporcella are shown in Table 2.

Proposal of a new identity for D. dubium
Since its original description, D. dubium has been regarded as an atypical species within the genus Table 2. Main morphometrics of Iranian material of Discolaimium dubium Das et al., 1969 and its seven close species of the genus Aporcella. Discolaimium (Das et al., 1969;Patil and Khan, 1982). This study of Iranian specimens confirms this opinion as several traits (pharynx entirely muscular and enlarging gradually, anterior position of DO, absence of lateral gland bodies) are not usual in members of this genus. Besides, the odontostyle aperture is appreciably longer than one-half of the total length, a typical feature of aporcelaims, the members of the family Aporcelaimidae Heyns, 1965. Molecular analyses of LSU rDNA sequences of three Iranian specimens of D. dubium sheds some light on the identity of this species. Their evolutionary relationships, as observed in the tree provided in Figure  4, reveal that these sequences form part of a highly supported (98%) large clade including several taxa, all of them having in common the absence of pars refringens vaginae, so confirming previous findings (Álvarez-Ortega and Peña- Santiago, 2016;Álvarez-Ortega et al., 2013;Imran et al., 2019;Naghavi et al., 2019). The most relevant result is, however, that D. dubium sequences form a very robust (100%) subclade with members of the genus Aporcella (Andrássy, 2002). Other discolaims con stitute another robust (98%) subclade. This strongly supports the membership of D. dubium in Aporcella, which is totally compatible with the updated morphological characterization of the species above provided. Actually, in its general morphology, D. dubium resembles several conicaltailed members of Aporcella from which it can be easily distinguished in several relevant morphometrics (see below). Consequently, it is transferred to Aporcella as A. dubia (Das et al., 1969) comb. n.

Separation of A. dubia from similar Aporcella species
For comparative purposes and taken into consideration the results provided in the present study, A. dubia is characterized by its 1.10 to 1.40 mm long body, cuticle bearing fine but perceptible transverse striation, lip region offset by constriction and 8 to 10 µm wide, odontostyle 7.5 to 10.5 µm long with aperture occupying 59 to 76% of total length, neck 300 to 362 µm, pharyngeal expansion 127 to 181 µm long or 44 to 46% of the total neck length, female genital system didelphic-amphidelphic, uterus simple and 38 to 53 µm or 1.2 to 1.6 times the body diameter long, vulva a transverse slit (V = 52-58), tail conical (32-38 µm, c = 32-43, c′ = 1.6-2.0) with rounded tip and a hyaline portion occupying 14 to 15% of tail length, spicules 30 to 32 µm long, and two or three widely spaced, ventromedian supplements with hiatus.

Remarks
The transference of D. dubium to Aporcella brings up the taxonomy of the large clade morphologically characterized by the absence of pars refringens vaginae and robustly supported by molecular data. This clade consists of representatives of at least three traditional dorylaimid families, namely Aporcelaimidae, Qudsianematidae Jairajpuri, 1965, andTylencholaimidae Filipjev, 1934. Leaving aside the tylencholaims (Tylencholaimidae), in general easily distinguishable thanks to the leptonchid (= tylencholaimid) nature of their cuticle, the morphological separation of aporcelaims (Aporcelaimidae, represented by Aporcella sequences) and discolaims (Qudsianematidae, Discolaiminae Siddiqi, 1969, represented by Carcharolaimus, Discolaimus and Discolaimoides sequences) of this clade becomes controversial and problematic. The results obtained in the present contribution suggest that some species currently classified under Discolaimium (and perhaps other species under Discolaimoides too) might belong to Aporcella and that, as consequence, a reevaluation and/or re-definition of these genera are recommendable and necessary.