Description of Deladenus brevis n. sp. (Sphaerularioidea: Neotylenchidae) from Iran: a morphological and molecular phylogenetic study

Specimens of Deladenus brevis n. sp. were obtained from the bark and rotten wood samples of a dead forest tree collected in Golestan province, northern Iran using the tray method (Whitehead and Hemming, 1965). Live specimens of interested nematodes were handpicked under a Nikon SMZ1000 stereomicroscope, heat killed by adding boiled 4% formalin solution, transferred to anhydrous glycerin, mounted on permanent slides according to De Grisse (1969), and examined using a Nikon Eclipse E600 light microscope. Photomicrographs were taken using an Olympus DP72 digital camera attached to an Olympus BX51 microscope equipped with differential interference contrast. Drawings were made using a drawing tube attached to a microscope and were redrawn using the CorelDRAW® software version 17.

Specimens preserved in glycerine were selected for observation under SEM according to Abolafia (2015). They 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).

A single live nematode specimen of D. brevis n. sp. was picked out and transferred to a small drop of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0; Qiagen) on a clean slide and squashed using a clean cover slip. The suspension was collected by adding 15 μl TE buffer. The DNA sample was stored at −20°C until used as PCR template. Three DNA samples were prepared in this manner. Primers for LSU rDNA D2-D3 expansion segments amplification were forward D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and reverse D3B (5′-TCGGAAGGAACCAGCTACTA-3) (Nunn, 1992), forward KKLSU-1 (5′-AAGGATTCCCTTAGTAACGGCGAGTG-3′) (Kiontke et al., 2004), and reverse 1006R (5′-GTTCGATTAGTCTTTCGCCCCT-3′) (Holterman et al., 2008) primers. Primers for partial amplification of SSU rDNA were forward 1813F (5′-CTGCGTGAGAGGTGAAAT-3′) and reverse 1912R (5′-TTTACGGTCAGAACTAGGG-3′) primers (Holterman et al., 2006); forward primer F22 (5′-TCCAAGGAAGGCAGCAGGC-3′) (Dorris et al., 2002) and reverse primer 18 S 1573 R (5′-TACAAAGGGCAGGGACGTAAT-3′) (Mullin et al., 2005). The COI mtDNA was amplified using HCO (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) and LCO (5′-GGTCAACAAATCATAAAGATATTGG-3′) primer pairs (Folmer et al., 1994). The PCR condition for the amplification of genomic fragments and COI mtDNA was according to Aliramaji et al. (2020), except that the annealing temperature for COI mtDNA amplification was set to 46°C. The PCR products were purified and sequenced directly for both strands using the same primers used in PCR with an ABI 3730 XL sequencer (Bioneer, South Korae). Sequences were deposited in GenBank database under the accession numbers presented in corresponding phylogenetic trees.

The molecular sequences of D. brevis n. sp. were compared with those of other nematode species available in GenBank. The previously used sequences for phylogenetic analyses (Tomar et al., 2015; Kanzaki et al., 2016; Yu et al., 2017; Morris et al., 2018) were retrieved from the database, updated by adding extra sequences and the newly generated sequences of the new species. Three independent SSU, LSU, and COI mtDNA data sets were prepared. The sequences were aligned using the Q-INS-i algorithm of the online version of MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley, 2013). The poorly aligned positions and divergent regions of SSU and LSU data sets were eliminated using the online version of Gblocks 0.91b (Castresana, 2000) using all three less stringent options. The COI alignment was edited manually using MEGA6 (Tamura et al., 2013). The model of the base substitution was selected using MrModeltest 2 (Nylander, 2004). The Akaike-supported model, a general time-reversible model including among-site rate heterogeneity and estimate of invariant sites (GTR + G + I), was selected for all phylogenetic analyses. Bayesian analysis was performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003) with a starting random tree and running the chains for four million generations for all. The burn-in phase was set at 25% of the converged runs. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to estimate the posterior probabilities of the phylogenetic tree (Larget and Simon, 1999) using the 50% majority rule. To visualize the results of each run in order to check the effective sample size of each parameter, Tracer v1.5 (Rambaut and Drummond, 2009) was used. The classic rhabditid taxa were selected as outgroups (for species names and accession numbers see trees). The output file of MrBayes was visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and redrawn in CorelDRAW version 17 (Jahanshahi Afshar et al., 2019a, 2019b).

Deladenus brevis n. sp. (Figs. 1 – 3).

Figure 1:

Figure 2:

Measurements of the new species are given in Table 1.

Females are small-sized nematodes, with slender body, gradually narrowing toward posterior end by having a conical tail. Lateral fields are with six lines. Cephalic region is low, slightly narrower than the adjacent body in permanent mounts. Stylet is small, with three small posteriorly sloping knobs (the knobs and general shape of stylet well visible in fresh material in temporary slides). Dorsal gland orifice is just posterior to stylet knobs. Hemizonid is at the level with nerve ring. Excretory pore is at the level with, or immediately behind, hemizonid. Deirid is slightly anterior to excretory pore. Pharynx is with no median bulb, lacking a chamber, slightly wider at pharyngo-intestinal junction, the latter anterior to nerve ring, and 42 to 52 µm from anterior end. Dorsal gland is long, with overlapping intestine. Intestine is simple, and the rectum and anus are functional. The reproductive system is monodelphic-prodelphic, anteriorly outstretched, composed of an ovary with maturing oocytes at single row, tubular oviduct, axial large ellipsoid spermatheca with fine spheroid sperm, crustaformeric with further than four cells in each of the four rows, uterus, vagina with moderately sclerotized wall, no PUS, and vulva with small lateral flaps. One or two eggs were observed in uterus of mature females. Tail is conical, narrowing to a rounded tip.

Not found.

Not found.

Rare: general morphology and pharynx are similar to those of female. Genital system is monorchic. They have slender spicules tylenchoid. Gubernaculum is thin and small. Bursa reaches tail terminus.

Specimens were recovered from the wood and bark samples of a dead broadleaf forest tree, collected in Golestan province, northern Iran, during October 2019. GPS coordinates were 36°44′57.5″ N, 54°19′ 12.0″ E.

Holotype female, seven paratype females, and one paratype male were deposited at USDA nematode collection (five slides with accession codes T-7475p to T-7479p) (one paratype female and one paratype male were used for SEM preparations). The LSID code of this publication is: urn: lsid:zoobank.org:pub: 34D40A8F-A13A-473C-BB24-E8AEB702EDA9.

The specific epithet refers to the short body of the new species.

Deladenus brevis n. sp. (the mycetophagous phase) is delimited in the genus by its small body size and small lateral vulval flaps. It is further characterized by six lines in the lateral fields, short 6 to 7 μm long stylet with three posteriorly sloping basal knobs well visible in fresh material in water and rare males. By having six lines in the lateral fields, the new species is comparable with five known species of the genus, namely D. apopkaetus (Chitambar, 1991), D. cocophilus (Nasira et al., 2013), D. durus, D. obtusicaudatus (Bajaj, 2015), and D. persicus having a comparable number of lines in the lateral fields. By having lateral vulval flaps, the new species was also compared with D. pakistanensis (Shahina and Maqbool, 1992). The comparisons of the new species with six aforementioned species are given as follows.

From D. apopkaetus by shorter females (415.5 (367-454) vs 743 (612-840) μm), excretory pore at the level with, or immediately behind hemizonid (vs anterior), lacking a median pharyngeal chamber (vs present), shorter stylet (6.4 (6-7) vs 10.9 (8.7-13.5) μm), anteriorly located vulva (V = 89 (87.2-90.3) vs 91 (90-93)) and shorter tail (25 (20-30) vs 37 (31-43) μm).

From D. cocophilus by shorter females (415.5 (367-454) vs 740 (550-930) µm), shorter stylet (6.4 (6-7) vs 8.9 (8-10) μm), vulval lips not protuberant and body not abruptly narrowing at posterior of vulva (vs vulval lips protuberant with sharp posterior constriction of the body after vulva).

From D. durus (the data of D. durus after Chitambar, 1991) by lacking a median pharyngeal chamber (vs present) and shorter females (415.5 (367-454) vs 796-1360 µm).

From D. obtusicaudatus by shorter females (415.5 (367-454) vs 730 μm), excretory pore at the level with, or immediately behind hemizonid (vs anterior), smaller c (16.8 (14.3-20.0) vs 29.0-29.3), greater c′ (2.8 (2.4-3.2) vs 1.5-1.7), anteriorly located vulva (V = 89 (87.2-90.3) vs 92.6-93.5), shorter stylet (6.4 (6-7) vs 9 µm) and female tail shape (conical, narrowing to a rounded tip vs cylindrical with broadly rounded to truncated terminus).

From D. persicus by shorter body (415.5 (367-454) vs 548 (474-609) μm) not remarkably narrowing posterior to vulva (vs narrowing), lacking a median pharyngeal chamber (vs present) and anteriorly located vulva (V = 89 (87.2-90.3) vs 90.3 (92.0-93.7)).

From D. pakistanensis by its body not abruptly narrowing at posterior of vulva (vs body abruptly narrowing posterior to vulva), shorter females (415.5 (367-454) vs 719 (636-761) μm), number of lines in lateral fields (6 vs 10-12) and anteriorly located vulva (V = 89 (87.2-90.3) vs 92 (92-93)).

To reconstruct the LSU rDNA tree, the newly obtained 882 nt long sequence of D2 to D3 expansion segments of LSU rDNA with accession number MT010121 was used. The BLAST search using this sequence revealed that its identity with other previously submitted sequences to the database is less than 86%. A number of 70 sphaerularid sequence and sequences of two classic rhabditids as outgroup taxa (including one sequence of the new species) were used for inferring the LSU phylogeny. The LSU alignment was composed of 447 total characters, of which 281 characters were variable, with an average nucleotide composition of 25.8% A, 20.3% C, 31.5% G, and 22.4% T. Figure 5 represents the phylogenetic tree inferred using this data set. In this tree, the new species is in sister relation with an unidentified species of the genus (KM403370) with moderate support in Bayesian inference. Deladenus appeared non-monophyletic in this tree.

Figure 5:

To reconstruct the COI tree, a newly obtained 634 nt long sequence of COI mtDNA with the accession number MT026002 was used. The BLAST search using this sequence revealed that its identity with other previously submitted sequences to the database is less than 81%. A number of 28 sphaerularid sequences (including one sequence of the new species) and one sequence of a classic rhabditid as outgroup taxon were used for inferring the COI phylogeny. The COI alignment was composed of 651 total characters, of which 262 characters were variable, with an average nucleotide composition of 21.0% A, 11.8% C, 20.7% G, and 46.5% T. Figure 6 represents the phylogenetic tree inferred using this data set. In this tree, the new species is in sister relation with a major clade including four species of Deladenus. Two other isolates of the genus (MK403374 and KY272634) are distantly related to the aforementioned clade (the new species + four other species), and based on this tree, the genus is not monophyletic.

Figure 6.