On the molecular identity of Paratylenchus nanus (Nematoda: Tylenchida)

Abstract In this study, molecular characterization of Paratylenchus nanus collected from the type locality in Four Mile Run, Fall Church, Virginia using COI, D2-D3 of 28 S rRNA and ITS rRNA gene sequences was provided. We molecularly also characterized, Paratylenchus specimens collected from grasses in Devils Lake, Ramsey County, North Dakota indicated as the type locality in the original description of P. nanus by Cobb (1923). These nematodes were identified as representatives of the species P. projectus. Populations of P. nanus belonging to the molecular types A and B, and previously designated by Van den Berg et al. (2014) should be now identified as P. nanus and P. projectus, respectively.

Paratylenchus nanus was described by Cobb (1923) from female specimens collected from soil near the roots of grasses, Devils Lake, North Dakota, in April 1915 andlater from Four Mile Run, Fall Church, Virginia, in August 1922. Some of Cobb's original notes were available to Tarjan (1960) including formula measurements of a single female from North Dakota and three females from Virginia. Tarjan (1960) also found Cobb's type specimens for study including a single female from North Dakota and one female plus the anterior part of another from Virginia. He selected the specimen from Virginia as lectotype of P. nanus (Raski, 1975). Geraert (1965) in his review of the genus Paratylenchus, suggested that P. nanus should be a synonym of P. bukowinensis. Similarity and possible synonymization with P. bukowinensis have been already mentioned by Cobb (1923) in the P. nanus species description. Later, Thorne and Smolik (1971) re-described and illustrated P. nanus from specimens collected from native sod near Devils Lake, North Dakota and designated the specimens as topotypes. However, Raski (1975) believed that the species from Devils Lake collected by Thorne and Smolik (1971) belonged to P. projectus Jenkins 1956, not to P. nanus, and designated new topotypes collected in 1958 from a grass soil sample in Fall Church, Virginia. Raski (1975) provided a detailed report of various collections and descriptions of the species. He also noticed that P. nanus was very similar to P. projectus. Van den Berg et al. (2014) provided a detailed molecular and morphological characterization of several populations identified as P. nanus from South Africa and California, USA. This study showed that the populations identified as P. nanus might indeed contain two sibling species (molecular types A and B), which were well separated using molecular criteria, but had very similar morphometrics. These authors also noticed intraspecific variation in shapes of lip region and tail. Thus, the designation of the true P. nanus species remained to be unresolved.
The objective of this work was to molecularly characterize P. nanus from the type locality, Four Mile Run, Fall Church, Virginia, USA designated by Tarjan (1960) and Paratylenchus species from the roots of grasses collected in Devils Lake, North Dakota, which was also indicated as the type locality in the original description of P. nanus by Cobb (1923).

Nematode samples
Several sampling trips were conducted to Devils Lake, Ramsey County, North Dakota and Four Mile Run, Fall Church, Virginia to collect nematodes. Soil samples were arbitrarily collected from the grassland in Devils Lake, Ramsey County, North Dakota in 2015 and 2016 (coordinates: 48.10805 N, 98.94384 W). Sampling was conducted from June to October each year. In each sampling spot, the top, dry soil (1-2 cm) was removed and the remaining soil was collected up to a depth of 30 cm using a soil probe (2.5 cm in diameter and 30 cm in depth). Each soil sample consisted of a composite of 10 to 15 soil cores and soil samples were placed in plastic bags. The soil with grasses was also placed in a glasshouse for several weeks and then sampled for nematodes (nematode sample codes = CD1902, CD1902, CD1904, CD2192 and CD2403). Several soil samples were also collected along Four Mile Run, Fall Church, Virginia in September 2020. Nematodes were extracted from soil using sieving and decanting as well as the sugar centrifugal-flotation method (Jenkins, 1964). One sample (N 4, coordinates: 38.52563 N, 77,08557 W) contained Paratylenchus nematodes (nematode sample code = CD3326)

Morphological examination
Several Paratylenchus female specimens extracted from grass soil samples were morphologically examined and photographed with an automatic Infinity 2 camera attached to a compound Olympus BX51 microscope equipped with a Nomarski interference contrast, and then these specimens were used for molecular study.
The new sequences for each gene (D2-D3 of 28 S rRNA, ITS rRNA, COI) were aligned using ClustalX 1.83 (Thompson et al., 1997) with their corresponding published gene sequences (Subbotin et al., 2006;Van den Berg et al., 2014;Munawar et al., 2018;Etongwe et al., 2020;Mwamula et al., 2020;Powers et al., 2020 and others). ClustalX with default parameters (gap opening = 15 and gap extension = 6.66) was used to generate the ITS rRNA and COI gene sequence alignments, whereas the modified parameters (gap opening = 5 and gap extension = 3) were applied to generate the D2-D3 of 28 S rRNA gene alignment. Sequence datasets were analyzed with Bayesian inference (BI) using MrBayes 3.1.2 as described by Van den Berg et al. (2014).
Morphological examination of specimens showed that P. nanus is very similar to P. projectus and the Figure 3: Phylogenetic relationships of Paratylenchus nanus with other related species. Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28 S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. New sequences are indicated in bold. * -originally identified as P. nanus. characters most important in distinguishing these two species as indicated by Raski (1975) are: (i) lip region which is truncate, often set off by slight but distinct narrowing, annuli indistinct in P. projectus, head rounded with distinct annuli, not set off, in P. nanus ( Figure 1) and (ii) tail bluntly rounded, often digitate in P. projectus; subacute in P. nanus (Figure 2).

Molecular characterization and relationships
The D2-D3 of 28 S rRNA gene The alignment generated with modified parameters was 756 bp in length and contained 41 sequences.
Phylogenetic relationships of P. nanus within selected Paratylenchus are given in Figure 3. Sequences of P. nanus from Virginia were identical to that of P. nanus type A from California provided by Van den Berg et al. (2014). Intraspecific variation for this species was up Figure 4: Phylogenetic relationships of Paratylenchus nanus with other related species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the ITS rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. New sequences are indicated in bold. * -originally identified as P. nanus. to 0.5%. New sequences of P. projectus from North Dakota were identical to those previously identified as P. nanus type B from California ( Van den Berg et al., 2014) or as P. nanus from North Dakota (Upadhaya et al., 2019a, b) and South Korea (Mwamula et al., 2020). All these sequences are now considered as representatives of P. projectus.

The ITS of rRNA gene
The alignment generated with default parameters was 885 bp in length and contained 35 sequences. Phylogenetic relationships of P. nanus within selected Paratylenchus are given in Figure 4 Sequence of P. nanus from Virginia was identical to that of P. nanus type A from California. New sequence of P. projectus from North Dakota was identical to those of previously identified as P. nanus type B from South Africa (Van den Berg et al., 2014) or as P. nanus from North Dakota (Upadhaya et al., 2019a, b) and South Korea (Mwamula et al., 2020), and now all considered as P. projectus. Figure 5: Phylogenetic relationships of Paratylenchus nanus with other related species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the COI gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. New sequences are indicated in bold. * -originally identified as P. nanus, ** − originally identified as Pratylenchus sp.

COI mtDNA gene
The alignment generated with default parameters was 721 bp in length and contained 39 sequences. Phylogenetic relationships of P. nanus within selected Paratylenchus are given in Figure 5. Sequences of P nanus from Virginia were identical to those from P. nanus type A from Belgium provided by Etongwe et al. (2020). Paratylenchus projectus formed two clades: a and b in the phylogenetic trees. New sequence of P. projectus from North Dakota was identical to those of this species from this state by Powers et al. (2020) and those previously identified as P. nanus type B from South Korea (Mwamula et al., 2020) and now considered P. projectus.
In this study, we consider Four Mile Run, Fall Church, Virginia, USA as the type locality for P. nanus. Specimens of Paratylenchus collected from this location belong to P. nanus molecular type A according to Van den Berg et al. (2014) and, thus, these nematodes should be considered as true representatives of P. nanus. Paratylenchus specimens collected from the roots of grasses collected in Devils Lake, North Dakota, in the location also mentioned in Cobb's description of P. nanus are considered here as representatives of P. projectus as they have been already identified by Raski (1975).