Dagger nematodes (
During a routine quarantine inspection, a
In order to make the final species identification, a detailed morphological and DNA sequencing analysis was conducted which resulted in a new species and was herein described as
For DNA extraction, a single nematode was transferred to worm lysis buffer (WLB: 20 mM Tris-HCl pH 8.0, 100 mM KCl, 3.0 mM Mg2Cl, 2.0 mM DTT, 0.9% Tween) and crushed with a sterilized pipette tip. The crushed nematode was pipetted into 8
The sequences were deposited into the GenBank database. DNA sequences were aligned by MEGA7 (Kumar et al., 2016.) using default settings. The DNA sequences were compared with those of the other nematode species available at the GenBank sequence database using the BLAST homology search program. The model of base substitution was evaluated using MODELTEST (Posada and Criandall, 1998; Huelsenbeck and Ronquist, 2001). The Akaike-supported model, the base frequencies, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist, 2001) running the chain for 1 × 106 generations and setting the “burnin” at 2,500. We used the Markov Chain Monte Carlo (MCMC) method within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using 50% majority rule.
SYSTEMATICS
Morphometrics data for
Holotype | Paratype | |||
---|---|---|---|---|
Character/ratios | Female | Female | J2 or J3 | J3 or J4 |
n | 1 | 15 | 5 | 11 |
L | 1,918 | 2008.9±78.7 (1,830.0-2,109.0) | 1,127.0±52.6 (1,046.0-1,210.0) | 1,419.6±38.5 (1,349.0-1,587.0) |
a | 45.6 | 47.2±2.3 (44.2-53.1) | 42.7±3.6 (36.7-46.3) | 48.3±3.5 (41.60-55.5) |
b | 5.4 | 5.5±0.2 (5.1-6.0) | 3.9±0.5 (3.0-4.4) | 4.7±0.3 (4.2-5.0) |
c | 18.1 | 18.0±1.0 (16.1-19.6) | 11.8±1.0 (10.5-12.8) | 13.2±0.6 (12.0-15.1) |
c’ | 4.4 | 4.9±0.4 (4.2-6.0) | 6.4±0.3 (5.9-6.8) | 5.9±0.4 (5.2-6.9) |
V | 27 | 26.2±0.6 (25.2-27.7) | — | — |
Lip diam. | 10 | 10.3±0.6 (8.8-11.2) | 8.6±0.3 (8.2-9.0) | 8.9±0.5 (8.2-10.1) |
Lip height | 5 | 4.1±0.7 (2.9-5.2) | 3.6±0.4 (3.0-4.2) | 3.8±0.4 (3.1-4.7) |
Odontostyle | 115 | 110.4±2.6 (105-115.6) | 71.0±3.8 (64.4-75.6) | 85.5±2.0 (79.5-90.0) |
Odontophore | 67 | 65.5±1.6 (61.0-68.1) | 46.1±1.4 (44.5-47.8) | 53.8±1.0 (52.2-54.8) |
Replacement odontostyle | — | — | 88.0±2.5 (85.0-92.0) | 107.0±7.9 (90.5-116.3) |
Total stylet | 173 | 176±2.9 (169.0-181.2) | 116.6±4.7 (109.0-120.5) | 140.0±2.2 (137.2-142.5) |
Flanges width | 10 | 10.4±0.7 (8.8-11.5) | 8.1±0.4 (7.7-8.7) | 9.0±0.6 (7.6-10.8) |
Esophagus | 358 | 366.6±10.6 (348.4-387.2) | 289.6±28.2 (272.2-345.6) | 311.4±14.7 (275.0-344.2) |
Esophageal bulb length | 78 | 81.5±2.1 (77.4-85.1) | 63.7±2.2 (60.1-66.1) | 70.4±3.0 (64.8-76.5) |
Esophageal bulb diam. | 24 | 24.5±1.1 (22.5-26.5) | 16.5±1.4 (14.1-17.9) | 18.6±0.6 (17.6-23.0) |
Body diam. | 42 | 42.7±2.0 (38.8-46.7) | 27.2±1.9 (24.3-29.8) | 29.6±1.5 (26.4-35.1) |
Anterior genital branch length | 11 | 10.6±1.3 (8.4-12.3) | — | — |
G1% | 0.5 | 0.5±0.1 (0.4-0.6) | — | — |
Posterior genital branch length | 240 | 218.3±40.9 (119.2-292.1) | — | — |
G2% | 8.9 | 10.7±1.9 (5.8-13.9) | — | — |
Distance from anterior end to vulva | 522 | 525.2±15.9 (493.0-548.3) | — | — |
Anal body width. | 24 | 23.1±1.5 (20.1-25.0) | 15.2±1.0 (14.0-16.4) | 18.7±0.7 (16.8-20.3) |
Tail | 106 | 112.2±6.3 (98.0-120.3) | 98.0±6.0 (88.0-104.0) | 109.0±1.4 (103.5-116.0) |
Hyaline tail part | 44 | 43.0±2.2 (39.4-47.2) | 19.3±1.2 (17.9-20.7) | 26.1±2.7 (20.2-32.0) |
H% | 41 | 38.5±2.4 (34.4-43.4) | 19.5±2.0 (17.5-23.1)) | 23.8±2.9 (17.4-28.2) |
Rectum | 38 | 35.2±2.7 (30.4-40.8) | 20.0±4.5 (16.4-27.6) | 28.4±2.3 (23.1-30.8) |
Body thin, almost straight in the anterior half, posterior portion bending ventrally, open “C” shaped upon fixation, narrows gradually and evenly in the tail region. Cuticle smooth and rather thick, lateral cords not visible. Lip region flattened, cephalic region rounded and, offset by weak depression. Amphids stirrup-shaped, fovea with wide slit-like aperture, slightly narrower than lip width. Five to six body pores present between anterior end and guiding ring on the dorsal and ventral side, respectively. Odontostyle (9–12) times lip region width. Odontophore with well-developed basal flanges (8.8–11.5
Not found.
Two juvenile stages, either J2/J3 orJ3/J4 were found and they were morphologically similar to adults except for their smaller size, shorter tails, and sexual characteristics. There is an immediate and quick progression of odontostyle length which makes it difficult to distinguish between juvenile stages. However, the characteristic feature of J1 as having replacement odontostyle being embedded in the base of odontophore was not observed in any of the juvenile specimens. Based on the information obtained through scatter graph (Fig. 5), it is not clear whether three or four juvenile stages are present in this species because only two juvenile stages were available to study.
The type specimens were extracted from the rhizosphere of
Holotype female and nine female paratypes (slide numbers 2186-1 to 2186-8) were deposited in the nematode collection of Ningbo Entry–Exit Inspection and Quarantine Bureau, China. Five paratype females (slide numbers T550a-e) were deposited in the Canadian National Collection of Nematodes, Ottawa, Canada.
Morphospecies group 1 represents species having single gonad (monodelphic), all the known species in this group are small and have an anteriorly located vulva, simple or undifferentiated posterior uterus, lacking Z organ (Cohn and Sher, 1972). With the most recent described
The new species can be differentiated from
To help identifying Tail short round to almost hemisphere with c′<1 ……………………………………….2 Tail conoid shaped with 1 < c′ < 4……………..….….……….……..…………3 Tail elongated with c′ > 4………………………….………………..…………….6 Tail ends with a digit………………………….………..…………………X. Tail ends round and hemispherical…….………..………………..X. Tail ends swollen…………………………………………..…………….. Tail conoid shaped tapers off evenly with c′ = 2.6…………….…… Tail with c′ < 2……………..………….………………….…..……….………….4 Cardia visible……………………………………………………………….. Cardia not visible……………….……………………..…..…….……………….5 Tail tapers off evenly and with hyaline part 20 µm long…..………..…… Tail tapers off unevenly and without hyaline part………..…………. Tail very long almost filiform c′ > 7……………………………………..….. Tail elongated with 4 < c′ < 7…………………………….………..…..…………7 Tail straight or bent dorsally……………..…..……………………………… Tail bent ventrally……………………..……….………………………………….8 Tail hyaline part > 50 µm long…..………………….……………….. Tail hyaline part < 50 µm long……………………………..……..……………..9 Tail hyaline part < 30 µm long………..………………………….………. Tail hyaline part > 30 and <50 µm long………………………………..…..…..10 Cardia weak with wide lumen………………….……………..… Cardia strong with narrow lumen…………………………………………
The species epithet is formed from the Latin word para = beside or near, and
The sequenced fragments of near-full-length 18S, 28S D2/D3 and ITS1 are of
Phylogenetic relationships among
Sequences of nematode species used for the phylogenetic analyses.
Species | 18S | 28S D2-D3 | ITS |
---|---|---|---|
|
- | AY601625 | - |
|
- | - | KX244924 |
|
EU477381 | - | EU477385 |
|
GU725084 | GU725075 | GU725063 |
|
KC567149 | KC567168 | KC567157 |
KC567148 | - | KC567156 | |
|
AY283173 | KF292277 | KF292281 |
- | KF292278 | AF511426 | |
- | KF292276 | - | |
- | AY601623 | - | |
|
- | KM199691 | KM199694 |
- | - | KM199693 | |
|
- | AY601629 | - |
|
- | KC567171 | KC567158 |
|
- | - | EU375483 |
|
AY297836 | AY601616 | - |
- | KP793050 | - | |
|
- | - | KX244932 |
|
- | KF446655 | - |
|
AY283174 | AY601617 | HM138503 |
HM138503 | KU680967 | HM191718 | |
HM191718 | DQ299512 | KJ934160 | |
KJ934157 | - | KJ934157 | |
KJ934160 | - | AF511428 | |
- | - | KU764410 | |
- | - | KU764405 | |
- | - | KU764406 | |
- | - | KU764407 | |
- | - | KU764408 | |
- | - | KU764409 | |
- | - | KU764411 | |
|
- | KC567173 | KC567159 |
|
- | KX931057 | KX931069 |
|
- | AY601631 | - |
|
KC567152 | - | KC567161 |
KC567153 | - | - | |
|
- | KJ802878 | KJ802895 |
|
- | AY601627 | - |
|
EF538761 | JQ780365 | KF292282 |
JQ780349 | - | - | |
JQ780348 | - | - | |
JQ780347 | - | - | |
JQ780346 | - | - | |
|
AY297825 | - | - |
|
AY297824 | EF140790 | AY524971 |
KP407872 | - | EF140789 | |
|
KC567154 | KC567180 | - |
|
GU549476 | GU549474 | GU549475 |
|
- | JQ240273 | - |
|
- | MF538772 | MF538770 |
|
KM586356 | - | KM586353 |
KM586357 | - | KM586354 | |
|
HM921368 | - | - |
|
GU725083 | GU725074 | GU725061 |
- | - | HM821367 | |
|
- | KP793049 | AY579205 |
- | EF188839 | - | |
|
AY297826 | - | - |
|
EF207249 | HM921349 | HM921334 |
AY687997 | - | AY430175 | |
HM921342 | - | - | |
|
- | - | KM893399 |
- | - | KJ9566387 | |
|
- | AY601619 | AY553980 |
- | - | AY563427 | |
|
EU477384 | - | - |
|
- | KJ802886 | |
|
HM921343 | HM921351 | HM921335 |
FJ713154 | - | HM921341 | |
- | - | AJ437029 | |
|
KY131241 | KY131240 | KY131244 |
- | KU052864 | - | |
|
AY297827 | KX931063 | DQ017154 |
AY297828 | - | KX931070 | |
|
AY297829 | - | - |
|
- | HM921352 | - |
- | KC567183 | - | |
|
HQ658630 | - | - |
|
EU477383 | - | - |
|
- | HF546081 | HF546078 |
|
- | DQ299515 | - |
|
GU725078 | GU725070 | GU725056 |
GU725079 | - | GU725057 | |
GU725080 | - | - | |
GU725081 | - | - | |
|
|
|
|
|
AY297831 | - | - |
|
- | - | KX244939 |
|
GU725085 | AY601626 | GU725060 |
- | GU725073 | - | |
|
- | AY601622 | - |
|
- | AY601620 | - |
|
- | AY601621 | KX931075 |
- | - | AY430179 | |
- | - | KX931077 | |
|
GU725082 | GU725076 | GU725062 |
|
AY297833 | - | - |
|
GU725086 | KC567185 | - |
|
KC567155 | KC567186 | KC567163 |
|
AY297834 | - | - |
|
- | DQ299514 | - |
|
AY552979 | - | HG329722 |
EF614267 | - | AJ437028 | |
|
JN153100 | JN153101 | - |
|
AY297840 | - | DQ364686 |
EF207250 | - | - | |
EU477382 | - | - | |
|
|||
|
AM086679 | - | - |
|
- | AY580056 | - |
|
- | HM163209 | - |
- | AY601604 | - | |
- | KP793051 | - | |
- | AY601605 | - | |
|
- | AY601594 | - |
- | AY601596 | - | |
|
- | AY601592 | - |
|
- | DQ285668 | - |
|
- | AY601600 | - |
|
- | JQ990032 | - |
|
- | DQ299508 | - |
|
- | DQ299496 | - |
|
- | JQ990031 | - |
|
- | HM163210 | - |
|
- | DQ299504 | - |
|
- | HM163211 | - |
|
- | JQ990040 | - |
|
- | HM921393 | - |
- | HM921356 | - | |
|
- | AY601590 | - |
|
- | JQ990042 | - |
|
- | JQ990036 | - |
|
- | AY210845 | - |
|
- | AY601587 | - |
|
AM086680 | KJ802889 | - |
AM086681 | - | - | |
JQ780350 | - | - | |
|
- | DQ299511 | - |
|
- | AY601602 | - |
|
- | AY601595 | - |
|
- | AY601598 | - |
|
AY297838 | - | - |
|
AY297839 | - | - |
|
AY283163 | - | - |
|
- | - | AJ549984 |
|
- | - | AJ549987 |
The 28S D2/D3 gene tree (Fig. 7), based on a multiple alignment of 85 sequences, revealed two major clades consisting of
The ITS1 tree (Fig. 8), was constructed from multiple sequence alignments of 68 sequences. In this tree,
Mono-opisthodelphic dagger nematodes in the genus
Our phylogenetic studies suggested that species in the morphospecies group 1 are not monophyletic, and morphospecies grouping was only established for the convenience of identification and do not always reflect the evolutionary history, which is consistent with other studies (Handoo et al., 2016; Cai et al., 2018). Considering the high variability in the morphological characters in
Morphospecies group 1 species are mainly distributed in Brazil (Oliveira et al., 2003; Silva et al., 2008; De Jesus et al., 2015), China (Zeng et al., 2016), USA, Canada (Ye, 2002; Yu et al., 2010), Malaysia (Rahman-Razak and Loof, 1998), France (Luc and Coomans, 1992) and India (Loof et al., 2001). This new species is described from the ornamental plants imported from Japan, which represents the first group 1 species from this country.