Nothocriconemoides hangzhouensis, Maria & Miao & Cai & Castillo & Zheng, 2020

Maria, Munawar, Miao, Wentao, Cai, Ruihang, Castillo, Pablo & Zheng, Jingwu, 2020, A new rare nematode Nothocriconemoides hangzhouensis n. sp. (Nematoda: Criconematidae) from Hangzhou, China, Journal of Nematology 52 (1), pp. 1-14 : 3-9

Nothocriconemoides hangzhouensis n. sp. ( Figs 1–4 ; Table 1 ).

Description

Females

Body is slightly curved ventrally after heat-killing. Body annuli are wide (13-17µm thick in the middle of the body) with fine longitudinal striations that look like annulus bearing rough cuticular margins. Anastomoses are 2 to 3, located at the posterior half of the body. The first cephalic annulus is rounded and expanded enclosing the lip region. The second annulus narrow, offset, collar like. En face view shows a central elevated labial disk bearing four equal-sized submedian lobes and “I”-shaped oral aperture. Stylet is robust with anchor-shaped basal knobs, and DGO indistinct. Esophageal lumen is looped in median esophageal bulb having a medium-sized valvular apparatus. Isthmus is narrow, short, encircled by nerve ring, and basal esophageal bulb distinct. Excretory pore is 3 to 4 annuli posterior to esophageal bulb. Monodelphic gonad is outstretched, and spermatheca is spherical, filled with sperm. Vagina is straight, vulva is closed, and vulval lips do not project above body contour. The ventral side of postvulval annuli is inverted, in majority of individuals. Anus is indistinct and located on the next annuli posterior to vulva. Tail is short and conoid, longitudinal striations are more prominent on the terminal annulus that gives the appearance of forked or branched terminus.

Male

Not found.

Juveniles (n =5)

Except for the cephalic region, they are similar to females; cephalic region of juveniles are devoid of collar-shaped annuli. Two rounded annuli are present and the first annulus show slight depression in the middle. Body annuli are narrower (4.5-5.5), slightly higher in number R=(48-51) and finely crenated. Stylet is (32.5-36.7)µm long and esophageal components are similar as those of females but less developed. Anus is indistinct and located 3 to 4 annuli from tail terminus. Tail is short and ends in a single lobed terminus.

Type host and locality

This population was found in the rhizosphere of Ulmus sp. from Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province, P.R. China, on February 2019. The geographical position of the sampling site is E: 120 ° 4´54˝ N: 30° 17´5.

Type material

Holotye female and 13 female paratypes (slide numbers ZJU-30-01-ZJU-30-03) were deposited in the nematode collection of Zhejiang University, Hangzhou, China. Four females and two juveniles paratypes on two slides (Slide numbers T-7353, T-7356) and ten additional females on two slides were (T-7354-55) deposited at USDA nematode collection, Beltsville, Maryland, USA. The Zoobank code is as follows: LSID urn:lsid:zoobank.org:pub:E977B880-9EF3-4E2C-BB29-4BD970D63F0A

Etymology

The species epithet refers to the City name where the species was detected.

Diagnosis and relationships

The new species can be characterized by wider body annuli that have fine longitudinal striations that look like annulus bearing rough cuticular margins, and 2 to 3 anastomoses at the posterior half of the body. The first cephalic annulus is rounded and expanded enclosing the lip region, and the

second annulus is narrow, offset, collar like. Four prominent submedian lobes are present. Excretory pore is 3 to 4 annuli posterior to esophageal bulb. Vagina is straight and vulva is closed. The ventral side of postvulval annuli is inverted, in majority of individuals. Anus is indistinct and located on the next annuli posterior to vulva. Tail is short, with forked or branched terminus.

The genus only contains two species; It can be differentiated from N. crenulatus by having shorter stylet 64.4 to 75.5 vs 87 to 96µm long, less number of body annuli R=35.0 to 39.0 vs 60 to 68, less number of annuli between vulva and tail terminus RV=2.0 to 3.0 vs 6 to 8, location of anus (next annuli to vagina vs 3 to 4 annuli posterior to vagina), and tail terminus morphology (terminal annulus forked or branched vs button shaped).

The new species differs from N. lineolatus by less number of body annuli R=35.0 to 39.0 vs 57 to 64, less number of annuli between vulva and tail terminus RV=2.0 to 3.0 vs 7 to 9, location of anus (next annuli to vagina vs 3 to 5 annuli posterior to vagina), anastomosis (2-3 vs 1), lip annuli (2 vs 4), submedian lobes (separate as four vs connected as 2 subdorsal and sublateral lobes), position of excretory pore (3 to 4 annuli posterior to esophageal bulb vs at the same level of esophageal bulb), vulval lip ornamentation (absent vs present), and tail terminus morphology (terminal annulus forked or branched vs bifid or irregular).

Molecular profiles and phylogenetIc status

The new species was molecularly characterized using partial 18 S, D2-D3 of 28 S, ITS and cox I sequences and obtained sequences were deposited in the GenBank. As this is the sole species of genus Nothocriconemoide s with molecular characterization, in order to predict the closely related species all the named criconematid species were included in the phylogenetic analysis.

In 18S tree ( Fig. 5 ), Nothocriconemoides hangzhouensis n. sp. (MN879878-MN879881) is a sister species of Criconema demani ( Micoletzky, 1925) (MH828134), Discocriconemella sinensis ( Maria et al., 2019) (MK253543) and Neolobocriconema serratum ( Khan and Siddiqi, 1963; Mehta and Raski, 1971) (MH668972). The pairwise sequence identities of the new species with its sister species are 97.53 to 98.9% (5-20 bp difference). This clade is further grouped with Bakernema inequale ( Taylor, 1936; Mehta and Raski, 1971) (MF094923) and species of Mesocriconema ( Andrássy, 1965) , Lobocriconema ( De Grisse and Loof, 1965) and D. limitanea ( Luc, 1959; De Grisse and Loof, 1965) (MF095032).

In 28S tree ( Fig. 6 ), Nothocriconemoides hangzhouensis n. sp. (MN879889-MN879890) clustered with species of Mesocriconema , Criconemoides ( Taylor 1936) , Lobocriconema and Neobakernema ( Ebsary 1981) but it is sister species of Discocriconemella sinensis (MK253537) and Criconemoides informis ( Micoletzky, 1922; Taylor, 1936) (AY780970). The pairwise sequence identities of the new species with its sister species are 84.93-89.01% (70-82bp difference).

The majority of criconematid species were not characterized for ITS sequences, and based on the available sequences, the ITS tree ( Fig. 7 ), was constructed. It indicated that Nothocriconemoides hangzhouensis n. sp. (MN876029-MN876030) is a sister species of Discocriconemella sinensis (MK253546). The sequence similarity between the new species and the sister species is 81.80% (130bp difference). This clade further grouped with species of Criconemoides , Lobocriconema , Mesocriconema , Neobakernema and D. limitanea .

In the cox I tree ( Fig. 8 ), Nothocriconemoides hangzhouensis n. sp. (MN867795-MN867799, MN-867800) clustered with species of the genera Bakernema , Criconemoides , Lobocriconema , Mesocriconema , Neobakernema , Neolobocriconema , but it is a sister species of Discocriconemella sinensis (MK249990) and Criconemoides informis (MF770692). The pairwise sequence identities of new species with its sister species are 82.7 to 89.0%.

Figures

Figure 1: Line drawings of Nothocriconemoides hangzhouensis n. sp. Female A: esophageal region; B: Cepahlic region; C: En face view: D-F: Cuticle markings; G: Tail region under SEM; H: Tail region under LM; I: Cepahlic region of juvenile: J: Crenation on cuticle of juvenile; K: Tail region of juvenile. (Scale bars= A=50µ m, B-I=10 µ m).

Figure 2: Light photomicrographs of Nothocriconemoides hangzhouensis n. sp. Female A: Entire body; B-E: Cepahlic regions; F-H: Esophageal regions, arrow pointing on the excretory pore (exp): I-K: Cuticle markings; L-O: Tail regions, arrows pointing on vulva (v) and anus (a). (Scale bars=A=50µm, B-I=10µ m).

Figure 3: Light photomicrographs of Nothocriconemoides hangzhouensis n. sp. Juvenile A: Entire body; B: Cepahlic region; C,D: Esophageal regions: E: Crenation on cuticle; F, G: Tail regions, arrows pointing on anus (a). (Scale bars=A=50µm, B-I=10µ m).

Figure 4: Scanning electron microscopy of Nothocriconemoides hangzhouensis n. sp. Female. A: Entire body; B-D: En face view; E: Cuticle markings; F-H: tail regions arrows pointing on vulva (v) and anus (a) (Scale bars, A= 100µm; B, C=10µ m; D, H=20µ m; G-F=30µ m).

Figure 5: Phylogenetic relationships of NOtHOCriCONemOideS HaNGZHOUeNSiS n. sp. with other criconematids species as inferred from Bayesian analysis using the 18S rRNA gene sequence dataset with the GTR + I + G model (−lnL=7,315.8130; AIC = 14,859.6260; freqA=0.2371; freqC=0.2413; freqG=0.2833; freqT=0.2384; R(a)=1.5166; R(b)=2.2509; R(c)=09364; R(d)=0.7246; R(e)=6.0997; R(f)=1.0000; Pinva=0.6630; and Shape=0.6070). Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.

Figure 6: Phylogenetic relationships of NOtHOCriCONemOideS HaNGZHOUeNSiS n. sp. with other criconematids species as inferred from Bayesian analysis using the D2-D3 of 28S rRNA gene sequence dataset with the GTR + I + G model (−lnL=8,382.1334; AIC=16,972.2669; freqA=0.1451; freqC=0.2354; freqG=0.3515; freqT=0.2681; R(a)=0.8404; R(b)=2.5613; R(c)=1.6924; R(d)=0.4616; R(e)=4.7092; R(f)=1.0000; Pinva=0.2730; and Shape=0.8370). Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.

Figure 7: Phylogenetic relationships of NOtHOCriCONemOideS HaNGZHOUeNSiS n. sp. with other criconematids species as inferred from Bayesian analysis using the ITS rRNA gene sequence dataset with the GTR + I + G model (−lnL=7727.9982; AIC = 15603.9963; freqA=0.2067; freqC=0.2560; freqG=0.2814; freqT=0.2559; R(a)=1.5781; R(b)=2.9918; R(c)=1.7856; R(d)=0.6423; R(e)=2.8799; R(f)=1.0000; Pinva=0.0460; and Shape=0.6180). Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.

Figure 8: Phylogenetic relationships of NOtHOCriCONemOideS HaNGZHOUeNSiS n. sp. with other criconematids species as inferred from Bayesian analysis using the COxI gene sequence dataset with the GTR + I + G model (−lnL=13,473.0592; AIC=27,198.1184; freqA=0.3715; freqC=0.0509; freqG=0.0477; freqT=0.5299; R(a)=0.7544; R(b)=36.5547; R(c)=1.6680; R(d)=51.5187; R(e)=20.3538; R(f)=1.0000; Pinva=0.2510; and Shape=0.3470). Posterior probability more than 70% is given for appropriate clades. Newly obtained sequences are indicated in bold.

Tables

Table 1. Morphometric data for Nothocriconemoides hangZhouensis n. sp.

	Holotype	Paratype
n		17
Body Length	494.0	487.1±43.8 (419.6-572.3)
R	36.0	37.2±1.2 (35.0-39.0)
Rst	7.0	6.5±0.5 (6.0-7.0)
Rex	15.0	14.7±0.6 (13.0-15.0)
RV	3.0	2.9±0.2 (2.0-3.0)
Rvan	0.0	0.0±0.0 (0.0-0.0)
Ran	3.0	2.9±0.2 (2.0-3.0)
a	8.2	7.9±0.7 (6.3-9.4)
b	4.2	4.1±0.3 (3.5-4.5)
c	18.6	17.3±1.7 (14.2-20.0)
c'	0.8	0.9±0.1 (0.7-1.1)
V	93.2	92.6±0.9 (90.5-94.0)
VL/VB	0.9	1.0±0.1 (0.9-1.2)
Lip height	8.8	9.5±0.7 (7.8-10.6)
Lip diam.	18.4	20.2±1.4 (17.2-22.1)
Stylet length	71.0	71.1±3.0 (64.4-75.5)
Stylet percentage	14.4	14.7±1.2 (13.1-17.4)
Pharynx length	117.5	118.9±5.0 (111.8-129.6)
Body width	60.3	61.8±5.3 (52.0-69. 4)
Vulval body diam.	37.0	35.7±2.5 (31.8-38.5)
Anal body diam.	31.9	32.1±3.1 (26.4-37.4)
Vulva to tail terminus	33.4	36.0±4.0 (29.8-41.7)
Tail length	26.5	28.3±1.9 (23.3-30.5)
Annuli width	13.1	14.5±1.2 (13.1-16.9)
Notes: All measurements are in µm and in the form of mean ± SD (range).