Description of Longidorus azarbaijanensis n. sp. (Dorylaimida: Longidoridae) from Iran

About 45 soil samples were collected from a depth of 5 to 30 cm, in active plant roots area in West Azarbaijan province, northwestern Iran during July 2016. Nematodes were extracted from soil using a series of 20 and 60 mesh sieves (US standard mesh numbers equal to 850 and 250 µm sized openings, respectively). The specimens of interest were hand-picked under a Nikon SMZ1000 stereomicroscope, heat-killed by adding boiling 4% formaldehyde solution and transferred to anhydrous glycerin according to De Grisse (1969). In total, four populations of the genus Longidorus were recovered. The number of females of the three populations was less than 10, and their identification at the species level needed resampling and complementary studies. Measurements of the new species were made using a drawing tube attached to a Nikon Eclipse E600 light microscope. The juvenile developmental stages were identified according to Robbins et al. (1995). The digital images were prepared using an Olympus DP72 digital camera attached to an Olympus BX51 microscope powered with differential interference contrast. Drawings were made by hand using the drawing tube and were redrawn using CorelDRAW® software version 16.

For the molecular phylogenetic studies, three female nematodes (labelled as fem.az.1-3) were picked out, studied individually on temporary slides, transferred to a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0, 100 QIAGEN Inc., Valencia CA) on separate clean slides and each was squashed using a clean slide cover glass. The suspension on each slide was collected by adding 45 μl TE buffer, each regarded as an independent DNA sample and stored at −20°C until used as polymerase chain reaction (PCR) template. Primers for the PCR amplification of the D2-D3 expansion domains of the 28S rDNA were: forward D2A (5´-ACAAGTACCGTGAGGGAAAGT-3´) (Nunn, 1992) and reverse KK28S-4 (5´-GCGGTATTTGCTACTACCAYYAMGATCTGC-3´) (Kiontke et al. 2004) (several attempts to get the expected amplified fragments using the commonly used reverse primer D3B (5´-TGCGAAGGAACCAGCTACTA-3´) were not successful). The ITS1 fragment was amplified using the forward TW81 (5´-GTTTCCGTAGGTGAACCTGC-3´) and reverse AB28 (5´-ATATGCTTAAGTTCAGCGGGT-3´) (Joyce et al. 1994) primer pairs. PCR was carried out (for both the fragments) in a total volume of 30 μl (10.6 μl distilled water, 15 μl Master, 1.2 μl of each primer (10 pMol/μl), and 2μl of DNA template). The thermal cycling program for both was as follows: denaturation at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for 40 sec and extension at 72°C for 80 sec. A final extension was performed at 72°C for 10 min (Alvani et al., 2016; Pedram, 2017). PCR products were purified and sequenced directly for both strands using the same primers with an ABI 3730XL sequencer (Bioneer Corporation, South Korea). The recently obtained sequences of three females with isolate codes fem.az.1-3 were submitted to GenBank database under accession numbers: MF677863 and MG765549 for partial 28S rDNA D2-D3 and ITS1 of female with isolate code fem.az.1, MG765547 and MF677864 for partial 28S rDNA D2-D3 and ITS1 of female with isolate code fem.az.2 and MG765548 for partial 28S rDNA D2-D3 of female with isolate code fem.az.3, respectively. The Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to inspect the relevance of the newly generated sequences with those already submitted into the database. For partial 28S tree, almost all available sequences of the genus were downloaded. The tree reconstructed from this big dataset was used to select sequences for the final 28S tree (the closely related species/sequences were selected to avoid a crowded tree). The available sequences for ITS1 rDNA of Longidorus spp. were also downloaded for reconstructing the corresponding phylogenetic tree. The sequences were aligned using the Q-INS-i algorithm of online version of MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley, 2013). The Gblocks program (version 0.91b) with all the three less stringent parameters, a server tool at the Castresana Lab (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) was used for post-editing of the alignments, i.e., to eliminate the poorly aligned regions or divergent positions. The model of base substitution was selected using MrModeltest 2 (Nylander, 2004). The Akaike-supported model, a general time-reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR+G+I), was used in the phylogenetic analyses of both 28S and ITS1 datasets. Bayesian analysis was performed with MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) running the chains for two million generations (nruns=4). After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The Markov chain Monte Carlo method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. For ML analysis, the same dataset as for the Bayesian tree was used and it was analyzed using raxmlGUI version 1.1 (Silvestro and Michalak, 2011) using the same model of nucleotide substitution (GTR+G+I) as in the previous analysis. For phylogenetic analyses of 28S dataset (both Bayesian inference (BI) and maximum likelihood (ML) methods), the species Nevadanema nevadense (Alvarez-Ortega and Peña-Santiago, 2012) (JN242245) and Prodorylaimus sp. (EF207241) were used as outgroup taxa. The species Xiphinema index (Thorne and Allen, 1950) (HG969306) and X. vuittenezi (Luc et al. 1964) (HG969309) were the out-group taxa in ITS1 tree. The output files of the used phylogenetic programs were visualized using Dendroscope V.3.2.8 (Huson and Scornavacca 2012) and redrawn in CorelDRAW software version16. The Bayesian posterior probability (BPP) and maximum likelihood bootstrap (ML BS) values exceeding 50% are given on appropriate clades in the shape BPP/ML BS.

Recovered from the soil samples collected about the rhizosphere of foxtail weed in natural grasslands and forests at 24 km distance to the city of Chaipareh, West Azarbaijan province, north-western Iran, in July 2016. GPS coordinates: 38°59.373′ N, 44°48.721′ E.

Longidorus azarbaijanensis n. sp. is a medium-length (5.4–6.8 mm) species lacking male in population. It is further characterized by having an offset lip region, anteriorly flat and separated from the rest body by constriction, funnel-shaped amphidial pouches, simple guiding ring located at 23 to 27 µm distance from the anterior end, 73 to 81 and 44 and 50 µm long odontostyle and odontophore, respectively, 95.0 to 113.5 µm long pharyngeal bulb, well-developed offset cardia, short conical tail with widely rounded terminus and four juvenile developmental stages. The matrix code of the new species according to Chen et al. (1997) is: A23-B12-C2- D34-E4-F3-G34-H23-I1. With regard to similar polytomous codes yielded from the close morphology, the new species is similar to five known species of the genus namely: L. euonymus (Mali and Hooper, 1973) L. perangustus, L. persicus, L. protae (Lamberti and Bleve-Zacheo, 1977) and L. sturhani (Rubtsova et al. 2001).

The detailed comparisons with the aforementioned species are as follows:

Compared with L. euonymus, besides distant position in both 28S and ITS1 phylogenetic trees, the new species has a lip region separated from the rest of the body by a remarkable constriction (vs somewhat concave), slightly shorter body (5.4–6.8 vs 6.0–7.6 mm), less wider lip region (10.0–12.5 vs 14 µm), shorter odontostyle (73–81 vs 81–90 µm), anteriorly located guiding ring (23–27 vs 27–33 µm distance from anterior end), and narrower female tail terminus (vs broad).

The new species differs from L. perangustus by its shorter body (5.4–6.8 vs 6.3–8.9 mm), smaller a value (133–181 vs 207–341), slightly smaller b value (12.1–17.3 vs 16–24), posteriorly located vulva (V = 52.0–56.4 vs 47.0–52.8), slightly smaller c´ value (1.2–1.7 vs 1.6–2.6), shorter tail (30–38 vs 38–51 µm) and by absence of male (vs presence). The two species also occupy distant positions in both phylogenetic trees inferred in this study.

Compared witho L. persicus, the new species has a slightly shorter body (5.4–6.8 vs 6.5–7.8 mm), shorter odontostyle and odontophore (73–81 vs 80–93 and 44–50 vs 48–72 µm, respectively), shorter tail (30–38 vs 38–50 µm), shorter pharyngeal bulb (95.0–113.5 vs 130–218 µm), absence of male (vs presence), and completely different tail characters in first three juvenile developmental stages. Both species are well distant in 28S tree, while are close relatives in ITS1 tree.

Compared with L. protae, the new species has posteriorly located vulva (V = 52.0–56.4 vs 46–51), shorter odontophore (44–50 vs 50–60 µm), shorter pharyngeal bulb (95.0–113.5 vs 125–140 µm), basic difference in tail end morphology (narrowly rounded vs broadly rounded) and shorter tail in all juvenile developmental stages.

Compared with L. sturhani, besides remarkable differences in partial sequences of partial 28S and ITS1 rDNA and distant position in both phylogeny trees, the new species has a lip region separated from the rest of the body by a constriction (vs slight depression), less wider lip region (10.0–12.5 vs 14–17 µm), shorter odontophore (44–50 vs 57–78 µm), greater a value (133.7–181.5 vs 99–138), greater c value (147.2–216.3 vs 83–155) and shorter tail (30–38 vs 35–50 µm).

Molecular characterization and phylogenetic position of Longidorus azarbaijanensis n. sp.

Sequencings of 28S rDNA D2-D3 expansion domains of three females of the new species yielded three single fragments of 634 (MF677863) and 873 (MG765547 and MG765548) nt long. The longer size of the two latter sequences was due to the longer reads of the corresponding PCR products while sequencing. There were no differences (indels or gaps) in the overlapping region of the three aforementioned sequences. Sequencing of ITS1 rDNA fragment of two females yielded two single fragments of 831 nt long (accession numbers MF677864 and MG765549). Both ITS1 sequences were identical. For molecular phylogenetic analyses of the new species, one sequence of each newly generated aforementioned genomic sequences was used to avoid crowded trees (for accession numbers see Figs. 4 and 5). A BLAST search using one of the newly obtained partial sequences of 28S rDNA D2-D3 (MF677863) showed that it has no exact identity with available sequences in the database. The highest matched sequences were an unidentified species of Longidorus (KF242335) having 100% query coverage and 96% identity (27 indels/one gap) and L. euonymus (KX062667) having 100% query coverage and 96% identity (26 indels/one gap). The identity value with all other sequences was 95% and less. The BLAST search using one of the ITS1 sequences of the new species (MF677864) revealed that the highest identity and coverage belonged to two sequences of L. euonymus (KX062692 and KX062691) with 80% identity and 60% coverage (99 different nucleotides/20 gaps) and one sequence from two species L. persicus (KU747175) and L. perangustus (KT593863) with 76% and 80% identity and 89% and 58% coverage, respectively (184 and 102 different nucleotides/55 and 21 gaps, respectively).

For partial 28S phylogeny, further than 100 sequences of almost all sequenced species/populations of Longidorus were used (the resultant tree was not shown). The related clades and species to the new species were selected to reconstruct the smaller pruned tree. A total number of 33 species/populations of Longidorus mostly belonging to the clade I in D2-D3 tree given by Subbotin et al. (2014), including one sequence of the new species, two species of Xiphinema (Cobb, 1913) and two dorylaim species (the species name and accession numbers in Fig. 4) as the outgroup taxa, were used to reconstruct the final tree. The alignment of this dataset included 749 total characters with 335 variable and 261 parsimonious informative characters. The average nucleotide composition was as follows: T, 21.5%, C, 23.1%, A, 24.5%, and G, 31.0%. Figure 4 represents the Bayesian phylogenetic tree inferred using the above-mentioned dataset. The clade A (whit 0.96% BPP and no ML BS support), inside the major clade I sensu Subbotin et al. (2014) includes the new species, five sequences of L. euonymus (KX062667, KF242332, KF242331, KF242333, AY601573), one sequence of L. perangustus (KR152295), L. persicus (KU747174) and Longidorus sp. (KF242335), three sequences of L. attenuatus (KT755457, KR911851, AY601572) and one sequence of L. sturhani (AF480071). Surprisingly, four species (L. euonymus, L. perangustus, L. persicus and L. sturhani) out of the five morphologically close species are in close phylogenetic relation with the new species in this tree. The species L. protae, the fifth morphologically close species, does currently have no available 28S sequence(s).

A total number of 18 species/populations of Longidorus including one sequence of the new species and two Xiphinema species as the outgroup taxa (the species names and accession numbers in Fig. 5) were used in the molecular phylogenetic analyses using ITS1 sequences. The alignment of this dataset included 720 total characters with 484 variable and 348 parsimonious informative characters. The average nucleotide composition was as follows: T, 26.2%, C, 20.8%, A, 24.1%, and G, 28.9%. Figure 5 represents the Bayesian phylogenetic tree inferred using the above-mentioned dataset. The clade B in this tree includes the new species, two sequences of L. euonymus (KX062692 and KX062691), one sequence of L. perangustus (KT593863) and one sequence of L. persicus (KU747175). This clade receives maximal BPP and high ML BS values (1.00 and 99%, respectively). Inside the clade B, L. azarbaijanensis n. sp. is phylogenetically more close to L. persicus both of which forming a monophyletic group with high BPP and ML BS value (0.99 and 90, respectively). From the five morphologically similar species (as already discussed), four species L. euonymus, L. perangustus, L. persicus and L. sturhani are sequence for their ITS1 fragment (KX062691 and KX062692 for two sequences of L. euonymus, KT593863 for L. perangustus, KU747175 for L. persicus and FJ009680 for L. sturhani). Similar to 28S tree, the new species has a close phylogenetic relation with three species L. euonymus, L. perangustus and L. persicus (members of clade B). In conclusion, this is one of the cases in which a group of morphologically similar species are close relatives in molecular phylogenetic analyses as well.

Figure 4

Figure 5