Molecular and Morphological Characterization of Aphelenchoides fuchsi sp. n. (Nematoda: Aphelenchoididae) Isolated from Pinus eldarica in Western Iran

Aphelenchoides fuchsi sp. n. is described and illustrated from bark and wood samples of a weakened Mondell pine in Kermanshah Province, western Iran. The new species has body length of 332 to 400 mm (females) and 365 to 395 mm (males). Lip region set off from body contour. The cuticle is weakly annulated, and there are four lines in the lateral field. The stylet is 8 to 10 mm long and has small basal swellings. The excretory pore is located ca one body diam. posterior to metacorpus valve or 51 to 62 mm from the head. The postuterine sac well developed (60–90 mm). Spicules are relatively short (15–16 mm in dorsal limb) with apex and rostrum rounded, well developed, and the end of the dorsal limb clearly curved ventrad like a hook. Themale tail has usual three pairs of caudal papillae (2+2+2) and a well-developed mucro. The female tail is conical, terminating in a complicated step-like projection, usually with many tiny nodular protuberances. The new species belongs to the Group 2 sensu Shahina, category of Aphelenchoides species. Phylogenetic analysis based on small subunit (SSU) and partial large subunit (LSU) sequences of rRNA supported the morphological results.

To find and prevent the spread of pests comprising pine wood nematode Bursaphelenchus xylophilus (Steiner andBuhrer, 1934) Nickle, 1970 in Iran, bark and wood samples must be sampled and inspected. So, during the past few years, a wide survey on dead or weakening pine trees in northern Iran was conducted and several species belonging to Aphelenchoididae Skarbilovich, 1947 were recovered and described: two new species of the genus Ektaphelenchoides Baujard, 1984 (Pedram et al., 2012;Aliramaji et al., 2014); a new species of the genus Bursaphelenchus Fuchs, 1937 (Pedram et al., 2011); and a new species of the genus Laimaphelenchus Fuchs, 1937 (Asghari et al., 2012). Up to now, B. xylophilus, which has caused serious damage to the coniferous forests, has not been found in Iran.
The genus Aphelenchoides Fischer, 1894 contains at present more than 150 nominal species (Hunt, 2008) and tends to be greatly conserved in gross morphology, making species identification a very difficult task. Also, many of the description fall below modern standards. Currently, species discrimination in Aphelenchoides is mainly based on morphology and morphometrics. In recent years, several species of the genus were described related to the pine trees in different countries, e.g., China (Cheng et al., 2009;Zhuo et al., 2010) and the United States (Kaisa, 2000), and some species isolated form packaging wood in South Korea (Cui et al., 2011;Fang et al., 2014b), India (Bina Chanu et al., 2013), South Africa (Wang et al., 2013), and Japan (Fang et al., 2014a). Recently a new species of Aphelenchoides, Aphelenchoides huntensis Esmaeili, Fang, Li, and Heydari, 2016 is described in association with pine trees in Iran. It would be likely to find more Aphelenchoides species if other substrates (e.g., bark beetles) were examined with such attention.
During 2013 and 2014, we conducted some inspections on wood and bark samples from dead or weakened pine trees in western Iran. As a result, a new species of Aphelenchoides was isolated from a weakened Mondell pine tree (Pinus eldarica L.), which is described and figured in this paper as Aphelenchoides fuchsi sp. n. It is the second new species of Aphelenchoides that is described from Iran.

Sampling, extraction, mounting, and drawing
Several bark and wood samples from weakened pine trees were collected from western Iran, which yielded an aphelenchid nematode belonging to the genus Aphelenchoides. The nematodes were recovered from the wood samples by soaking a small amount of wood in water for 48 hr and handpicked under a stereomicroscope model Olympus SZH (Japan). The nematodes were heat killed by adding boiling 4% formalin solution and then transferred to anhydrous glycerin and mounted in permanent slides according to the method by De Grisse (1969). Permanent slides were prepared and studied using a light microscope (Nikon E200). Drawings were made using a drawing tube attached to the same microscope, and photographs of live nematodes were taken by a digital camera attached to the microscope.

DNA extraction, polymerase chain reaction, and sequencing
A single live nematode specimen of A. fuchsi sp. n. was picked out, examined on a temporary slide, and then transferred to a small drop of AE buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0; Qiagen Inc., Valencia, CA) on a clean slide and squashed using a clean cover slip. The suspension was collected by adding 20 ml AE buffer. The DNA samples were stored at 2208C until used as polymerase chain reaction (PCR) templates. For the first fragment of 18S, the forward primer 1096F (59-GGT AAT TCT GGA GCT AAT AC-39) was used in combination with the reverse primer 1912R (59-TTT ACG GTC AGA ACT AGG G-39), and the second fragment was amplified with forward primers 1813F (59-CTG CGT GAG AGG TGA AAT-39) and reverse primer 2646R (59-GCT ACC TTG TTA CGA CTT TT-39) (Holterman et al., 2006). The D2-D3 expansion segments of 28S rRNA gene were amplified using forward primer D2A (59-ACA AGT ACC GTG AGG GAA AGT TG-39) and reverse primer D3B (59-TCG GAA GGA ACC AGC TAC TA-39) (Nunn, 1992). Polymerase chain reaction was performed in a final volume of 25 ml PCR mixture and contained 12.5 ml 2X GoTaq DNA polymerase mix (Promega Corporation, Madison, WI), each of a 1.2 ml forward and reverse primers solution (5 pM), 8 ml distilled water, and 2 ml of a 100 times-diluted crude DNA extract. The following PCR profile was used: 948C for 5 min; 5 3 (948C, 30 sec; 458C, 30 sec; and 728C, 70 sec) followed by 35 3 (948C, 30 sec; 548C, 30 sec; 728C, 70 sec) and 728C, 5 min. The PCR products were purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer (Macrogen Corporation, Seoul, South Korea). The newly obtained sequences was submitted to GenBank database under accession numbers KT003986 (18S) and KT003987 (28S).
The obtained sequences of the partial 18S and partial 28S D2-D3 region ribosomal DNA (rDNA) gene of A. fuchsi sp. n. were compared with those of other aphelenchids species available in GenBank (see Table 1 for selected sequences of SSU and Table 2 for LSU D2-D3) using BLAST homology search program. Outgroup was chosen according to previous published data (Fang et al., 2014a(Fang et al., , 2014bEsmaeili et al., 2016). The newly obtained and published sequences were aligned using MAFFT ver. 7 (Katoh et al., 2002) with default parameters. Sequence alignment was edited using BioEdit (Hall, 1999). The best fitted model of DNA evolution was obtained using jModelTest2 (Darriba et al., 2012) with the Akaike information criterion. The Akaikesupported model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the Akaike information criterion were then used in phylogenetic analyses. The tree topology was confirmed using MrBayes 3.2.3 (Ronquist and Huelsenbeck, 2003) with four chains (three heated and one cold). The number of generations for the total analysis was set to 10 million, with the chain sampled every 1,000 generations and the burn-in value was 25%. The Markov Chain Monte Carlo method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees using 50% majority rule (Larget and Simon, 1999). The consensus trees were selected to represent the phylogenetic relationships with branch length and support level and visualized using TreeGraph 2 (St€ over and M€ uller, 2010).

Measurements
See Table 3.

Description
Females: Body is cylindrical, straight, somewhat ventrally arcuate when heat relaxed. Cuticle weakly annulated, lateral field with four incisures (i.e., three ridges). Lip region is rounded, offset, ca 3 to 3.5 mm high, and 6 to 7 mm broad. Stylet with small basal swellings, procorpus cylindrical. Median bulb is strongly developed, almost rectangular, with conspicuous valve situated more or less centrally. Nerve ring is situated at ca half metacarpus (median bulb) length posterior to it. Pharyngointestinal junction is immediately posterior to metacorpus. Pharyngeal gland lobe is slender, ca five to six body diam. long, overlapping intestine dorsally. Excretory pore is at level of nerve ring or opposite the posterior level of the nerve ring, the position varying from 1/2 to 2/3 metacorpus length behind metacorpus. Hemizonid is faint, situated ca two to three times metacorpus diam. posterior to excretory pore. Monodelphic, ovary is outstretched anteriorly, developing oocytes in single row. Oviduct connecting ovary and spermatheca. Spermatheca is elongate, containing compressed disklike or oblong sperm in single row. Vagina is directed anteriad. Vulva is transverse, with slightly raised lips, and vulval flap is absent. Postuterine sac is well developed, extending for about 71% to 90% of vulva to anus distance, often containing sperm. Rectum and anus are visible. Tail is conical, terminating in a complicated step-like projection, usually with many tiny nodular protuberances.
Males: They are much less common than females; body slender, cylindrical, and J-shaped when heat relaxed. Anterior region and cuticle are similar to female. Testis is single, anteriorly outstretched, locating left of intestine, occupying 53.4% to 66.2% of body length. Lips of cloacal opening protruding slightly. Spicules are arcuate, relatively short, and apex and rostrum are rounded and well developed, end of dorsal limb is clearly curved ventrally. Gubernaculum is absent. Tail is conical, bearing a short sharp mucro ca 1.5 to 2 mm long. Three pairs of subventral caudal papillae are present: first pair located just posterior to cloacal aperture, second pair in mid-tail region, and third pair just anterior to tail end. Bursa is absent.

Diagnosis and relationships
Aphelenchoides fuchsi sp. n. is characterized by body length of 332 to 400 mm (females) and 365 to 395 mm (males). Lip region with distinct constriction from the rest body. Cuticle with four lateral lines. Medium sized (8-10 mm) stylet with small basal swellings. The excretory pore is located ca one body diam. to posterior of metacorpus valve. The female tail is conical and terminates in a complicated step-like projection, usually with many tiny nodular protuberances. Spicules are relatively short (15-16 mm in dorsal limb) with the apex and rostrum rounded and well developed, the end of the dorsal limb is clearly curved ventrally like a hook (Fig. 2G,F). Aphelenchoides fuchsi sp. n. has a tail terminus with tiny nodular protuberances in female. According to the category of Aphelenchoides species sensu Shahina (1996), the new species belongs to Group 2, which is defined as having the female tail terminus with ''one or sometimes two mucronate structures.'' On the basis of the four lateral lines, stylet length, conical female tail, and shape of spicules, the new species appears morphologically most similar to four species from Group 2 including Aphelenchoides arcticus Sanwal, 1965, Aphelenchoides blastophthorus Franklin, 1952, Aphelenchoides saprophilus Franklin, 1957, and Aphelenchoides xui Wang, Wang, Gu, Wang, and Li, 2013. It is also similar to three species from Group 4 including Aphelenchoides franklini Singh, 1969, Aphelenchoides gynotylurus Timm and Franklin, 1969, and Aphelenchoides marinus Timm and Franklin, 1969 Aphelenchoides fuchsi sp. n. differs from A. arcticus, A. blastophthorus, A. franklini, A. gynotylurus, A. marinus, and A. saprophilus by the female tail terminus, i.e., ending of a step-like projection/or offset mucro with many tiny nodular protuberances in A. fuchsi sp. n. vs. a shallow constriction narrowed sharply with a very fine mucro at the tip (A. arcticus), tapering to a simple conspicuous mucro (A. blastophthorus), a simple pointed ventral mucro (A. franklini), ending in a digitate to blunt/or sometimes sickle-shaped mucro (A. gynotylurus), acutely conical tapering to a point that is sometimes truncate/or subacute (A. marinus), and a short ventral mucro (A. saprophilus).
The female tail of A. fuchsi sp. n. is also similar to some Laimaphelenchus species. Laimaphelenchus contains two groups of species: one with a vulval flap and one without (Hunt, 1993). Moreover, Laimaphelenchus is also characterized by the vagina either having a cuticular annulus at the point where it joins the uterus or is surrounded by a relatively thick refractile tube or strong musculature (Zhao et al., 2007). Considering these two morphological characters (absence of vulval flap and vagina structure in new species), the new species was far from Laimaphelenchus and therefore placed in Aphelenchoides. In addition, the entire Aphelenchoides and Laimaphelenchus groups are in urgent need of revision based on sequences of full-length SSU rDNA sequences or other informative loci combined with detailed morphological diagnostic characters.

Type habitat and locality
The type population was recovered from bark and wood samples of a weakened Mondell pine tree (P. eldarica) in vicinity of Cheshmeh-e-Nezamei, city of Gilan-e-Gharb, Kermanshah Province, western Iran (GPS coordinates: N 338599, E 468129; 1,248 m above sea level).

Remark
Aphelenchoides fuchsi sp. n. was successfully cultured on Botryotinia fuckeliana growing on Potato Dextrose Agar. The nematode was not culturable on Botrytis cinerea.

Type material
Holotype female, two paratype females, and two paratype males (Slides AAF002 and AAF003) were deposited in nematode collection of the Department of Plant Protection, College of Agriculture and Natural Resources, University of Tehran, Iran. Three paratype females were deposited at each of the following collections: CABI, Egham, United Kingdom; USDA 6.6 6.6 6 0.2 (6.5-6.9) 6.8 Nematode Collection, Beltsville, MD, and Department of Nematology, WANECO Collection, Wageningen, the Netherlands.

Molecular phylogenetic status
Amplification of the D2-D3 expansion segment of 28S rDNA and partial 18S sequences from A. fuchsi sp. n. specimens yielded a single fragment of 750 and 1,700 bp based on gel size, respectively. Alignment of the sequences with MAFFT resulted in datasets of 1,752 characters for 18S and 869 characters for 28S D2-D3. The partial 18S alignment contained 59 in-group and 1 out-group taxon and was 1,685 bp in length after removing ambiguously aligned regions. The 50% majority rule consensus phylogenetic tree generated from the partial 18S rRNA alignment using Bayesian inference analysis under the GTR + I + G model is presented in Figure 3. The D2-D3 expansion segment of 28S rDNA alignment contained 47 in-group and 1 out-group taxon and was 771 bp in length after removing ambiguously aligned regions. The 50% majority rule consensus phylogenetic tree generated from the D2-D3 alignment using Bayesian inference analysis under the TIM3 + I + G model is presented in Figure 4. According to our phylogenetic trees, A. fuchsi sp. n. occupied a basal position in a strongly supported clade with a posterior probabilities support of 100% (D2-D3) and 64% (18S) with some other Aphelenchoides spp. as well as several species of other Aphelenchoididae such as Schistonchus s.l. and Laimaphelenchus. It clearly differs genetically from all members of Aphelenchoididae with available LSU and SSU sequence in GeneBank.

DISCUSSION
The genus Aphelenchoides is rich in species and has a world-wide distribution. Correct identification of Aphelenchoides species is not easy due to the inaccessibility of some references, poor literatures, inadequate description of several species, and lack of reliable detail. Moreover, this genus has few morphologically diagnostic taxonomic characters (Kanzaki, 2006).
In general, the morphology of the tail tip of A. fuchsi sp. n. is similar to some described species of the genus in Group 2, according to the category of Aphelenchoides species sensu Shahina (1996), which is defined as having the female tail terminus with ''one or sometimes two mucronate structures'' but this structure, in the new species, is not simple and the female tail terminus have a step-like projection with many tiny nodular protuberances. Moreover, the male spicule shape (dorsal limb with a hook-like tip) is a diagnostic character between similar species of the genus.
Recently, several species of the genus were described and molecularly studied (Cui et al., 2011;Wang et al., 2013;Fang et al., 2014aFang et al., , 2014bEsmaeili et al., 2016). Molecular characterization and phylogenetic analyses based on rDNA region sequences including 18S, internal transcribed spacer regions, and the D2-D3 expansion segments of 28S can assist in accurate identification of the species, although with the important proviso that most nominal Aphelenchoides species lack such reliable information.
Small subunit of rDNA contains sufficient phylogenetic signal for the identification of Aphelenchoides species (De Ley et al., 2006;Zhao et al., 2008;Van Megen et al., 2009) and have been shown to be more useful for species identification compared to D2-D3 expansion segments of 28S rRNA and internal transcribed spacer rRNA, as both of these markers showed more species variability than did partial 18S rDNA (Zhao et al 2008;Esmaeili et al., 2016). This is the second new species of the genus from Iran to be described and sequenced. Aphelenchoides fuchsi sp. n. was found in bark of a weakened pine tree but not in its wood. Therefore, the new species appears to be feeding on fungi or lichens growing on the bark of the tree.