Trichodorus hangzhouensis, Li & Maria & Cai & Barsalote & Peneva & Zheng, 2020

Li, Xuqing, Maria, Munawar, Cai, Ruihang, Barsalote, Eda Marie, Peneva, Vlada & Zheng, Jingwu, 2020, Distribution of trichodorid species in mainland China with description of Trichodorus hangzhouensis sp. nov. (Nematoda, Triplonchida), ZooKeys 945, pp. 163-189 : 163

Trichodorus hangzhouensis sp. nov.

Description

(Figs 1 - 3 , For measurements see Table 1 ). Male. Body cylindrical with posterior end slightly curved ventrally. Cuticle slightly swollen upon fixation, 2.0-2.5 µm thick at mid-body. Lip region dome-shaped with double papillae (composed of outer labial and cephalic papillae). Amphidial aperture post-labial, slit-like, amphidial fovea cup-shaped. Stoma narrow, refractive strengthening rods 4-5 µm long. Nerve ring surrounding the anterior part of isthmus. Slender mid part of pharynx gradually widening to form a pharyngeal bulb. Five pharyngeal gland nuclei visible, the first ventrosublateral pair obscure. Pharyngeal bulb offset from intestine. Cardia conoid, difficult to observe. Three ventromedian CP present anterior to the secretory-excretory pore (S-E), the latter opposite isthmus or anterior end of pharyngeal bulb. CPl situated opposite the end of pharyngostom to mid-isthmus, distance of CPl-CP2, CP2-CP3 and CP3-SE becomes gradually shorter. Lateral cervical pores not clearly seen. Reproductive system typical of the genus, i.e., with a single anterior outstretched testis, short germinal zone, seminal vesicle packed with large round sperm cells with fibrillar structure and a sausage-shaped central nucleus. Spicules paired, relatively short 33.2 (32.0-34.5) µm, in holotype 34.5 µm, slightly ventrally curved. Capitulum widened, slightly marked, lamina partially striated, tapers gradually to the distal end, no bristles at striation. Gubernaculum having a keel-like thickening and proximal end visible between spicules (Fig. 1E ). Three ventromedian precloacal supplements (SP) present. The posterior-most one (SP1) at the level of spicule capitulum, the SP2 slightly less than, or equal to one spicule length anterior to the SP1. The anterior most (SP3), 1.0-1.5 times spicule length apart from SP2. Cloacal lip rounded; slightly protruded, post-cloacal papillae not prominent. Tail short, conoid, less than one cloacal diameter long with one pair of subterminal subventral pores.

Female. Body straight or slightly curved upon heat relaxation. Anterior region similar to that of male except for secondary male characteristics. S-E pore located opposite isthmus or anterior part of pharyngeal bulb. Reproductive system didelphic amphidelphic with reflexed ovaries. Two finely granular oviduct cells at the tip of reflexed ovary, sperm round in shape distributed in the distal part of the uteri. Vagina well developed, pars proximalis vaginae barrel shaped in lateral optical view extending less than half corresponding body diameter. Sclerotized vaginal pieces (= pars refringes vaginae) rounded triangular with tips directed towards vulva, pieces 1.5-2.0 µm sized, at about 1.0 µm distance from each other, vulva pore-like in ventral view. Copulatory plug observed in uterus of two specimens. One pair of sublateral body pores almost opposite the vulva. Tail terminus conoid to rounded, anus subterminal, caudal pores subventral, immediately posterior to anus.

Type host and locality.

The new species was detected in association with three plants, i.e., Eriobotrya japonica (Thunb.) Lindl., Morus alba L. and Toona sinensis (A. Jussieu) M. Roemer from the botanical garden of Huajiachi Campus, Zhejiang University, Hangzhou, Zhejiang Province, P. R. China. The specimens from E. japonica were regarded as a type population. The geographical position of the sampling site is: 120°19'06"E, 30°25'67"N.

Type material.

Holotype male, 8 male and 11 female paratypes (slide nos. ZJU-29-01-ZJU-29-19) deposited in the Nematode Collection of Zhejiang University, Hangzhou, China, and 2 male and 13 female paratypes deposited in Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Bulgaria (slide nos. PNT 102-104).

Molecular profiles and phylogenetic analysis.

The new species was molecularly characterized and newly obtained sequences were deposited in the GenBank with the accession numbers HM106498, MF979178 for 18S, MF979185-MF979186, HM106497 for 28S and MF979181, HM106496, MF979182 for ITS2 of rRNA gene. The available sequences of trichodorid taxa (accession numbers of 18S, D2-D3 region of 28S and ITS2 rRNA gene sequences in Suppl. material 1: Table S1) were selected to reconstruct the phylogenetic trees.

The 18S rRNA gene tree (Fig. 7 ) revealed that T. hangzhouensis sp. nov. occupied a basal placement in an unsupported clade including T. primitivus (de Man, 1880) Micoletzky, 1922 (KY119675-76, AF036609), T. similis Seinhorst, 1963 (AJ439584-85, AJ439522) and T. obtusus Cobb, 1913 (KT282335). The pairwise sequence identity of new species with the aforementioned species is 97-99% with 16-24 nucleotide differences.

In the 28S phylogenetic tree (Fig. 8 ), T. hangzhouensis sp. nov. formed a clade with four species distributed in Spain: T. giennensis Decraemer, Roca, Castillo, Peñ-Santiago & Gomez-Barcina, 1993 (JQ716452), T. illiplaensis Decraemer, Palomares-Rius, Cantalapiedra-Navarrete, Landa, Duarte, Almeida, Vovlas & Castillo, 2013 (JQ716462), T. onubensis Decraemer, Palomares-Rius, Cantalapiedra-Navarrete, Landa, Duarte, Almeida, Vovlas & Castillo, 2013 (JQ716454-55) and T. paragiennensis Decraemer, Palomares-Rius, Cantalapiedra-Navarrete, Landa, Duarte, Almeida, Vovlas & Castillo, 2013 (JQ716461); three species occurring in Iran - T. orientalis De Waele & Hashim, 1983 (KY115140); T. persicus De Waele & Sturhan, 1987 (KX348138); T. zanjanensis Asghari, Eskandari, Tanha Maafi & Decraemer, 2018 (KY115138); one species from Israel T. minzi De Waele & Cohn, 1992 (KP259801) and an undescribed Trichodorus species (KM212949) from the USA. The pairwise sequence identity of new species with the aforementioned species is 88-91% with 73-86 nucleotide differences.

In ITS2 tree (Fig. 9 ), T. hangzhouensis sp. nov. shared the same clade with T. pakistanensis Siddiqi, 1962 (GU645896, GU645897, GU645899, JN123384) and the clade of these two species was in sister relation to a clade including T. gilanensis Maafi & Decraemer, 2002 (KY115164-KY115165), T. nanjingensis Liu & Cheng, 1990 (GU645800, GU645804, GU645893, GU645895), T. viruliferus Hooper, 1963 (JN123391) and T. sparsus Szczygiel, 1968 (JN123388). The pairwise sequence identity of the new species with the aforementioned species is 94-98% with 4-9 nucleotide differences.

The other known Trichodorus , Nanidorus and Paratrichodorus species sequenced during this study clustered with their respective species available through GenBank database, thus supporting their identity.

Diagnosis and relationships.

The new species is characterized by the male having a relatively short onchiostyle (46-49 µm) and 3 ventromedian cervical papillae anterior to the S-E pore, CP1 located opposite isthmus, distance of CPl-CP2, CP2-CP3 and CP3-S-E becoming gradually shorter, S-E pore located opposite isthmus or anterior end of pharyngeal bulb, pharynx offset, spicules relatively short, slightly curved, 33.2 (32.0-34.5) µm long, with wider slightly marked capitulum, lamina partially striated and tapering gradually to the distal end, bristles at striation absent, three ventromedian precloacal supplements; female with barrel shaped vagina, vaginal scletorized pieces medium-sized (1.5-2.0 µm), rounded triangular with tips directed towards vulva, slightly separated from each other (c. 1.0 µm), vulva pore-like in ventral view.

The species-specific codes sensu Decraemer and Baujard (1998) for this new species are as follows, for the male: F3-D3-P2-A1(2)-B2-C1-E0-G1-H2-I1-J2-K3-L2-M4-N1-O5; for female: D1-C1-L2- K2-A1(2)-B2-E2 -F1 G1-H3-I2(3)-J1-M1-N1-O1-P1-Q1-R2-S5. Based on male prime diagnostic characters for fam. Trichodoridae (F = number of ventromedian precloacal supplements, D = number of ventromedian cervical papillae, P = body habitus) and female (D = type of genital system, C = vulva position, K = size of vaginal sclerotized pieces, L = position of vaginal sclerotized pieces) the new species male belongs to group 12 while the female falls in the category group 1 of subgroup 1-7 as described by Decraemer and Baujard (1998). Among several Trichodorus species of group 12 (for male) and group 1, subgroup 1-7 (for female) the new species comes close to T. cedarus Yokoo, 1964, T. guangzhouensis Xie, Feng & Zhao, 2000, T. reduncus Siddiqi & Sharma, 1995, T. tricaulatus Shishida, 1979 and T. yokooi Eroshenko & Teplyakov, 1975. It can be differentiated as follows:

T. cedarus - by having a different position of SP1 (at the level of spicules capitulum vs posterior), shorter spicules length (32.0-34.5 vs 36-53 µm), shape of vulva (pore-like vs slit-like) and shape of vagina (barrel vs pear);

T. guangzhouensis - by having a longer onchiostyle in females (48-52 vs 36.4-41.6 μm), striations on spicule (present vs absent), different spicule shape (without constriction vs with constriction) and shape of vulva in ventral view (pore-like vs a longitudinal slit);

T. reduncus - by having a longer onchiostyle in males (46-49 vs 36-40 µm) and female (48-52 vs 37-40 µm), striations on spicules (present vs absent), different proximal part of gubernaculum (not hooked vs hooked) position of vaginal pieces (close vs widely separated) and shape of vulva (pore-like vs a small transverse slit);

T. tricaulatus - by having different position of SP1 (at the level of spicule capitulum vs outside it), spicule bristles (absent vs present), longer onchiostyle in both males (49.3 (46-49) vs 42.6 (39-51) µm) and females (49.3 (48-52) vs 42.2 (39-44) µm), type of pharyngo-intestinal junction (pharyngeal bulb offset vs pharynx overlapping intestine ventrally) and shorter spicules (33.2 (32.0-34.5) vs 39 (36-53) µm);

T. yokooi - by having striation on spicule (vs absent), shorter onchiostyle both in males (46-49 vs 57-82 µm) and females (48-52 vs 62-77 µm) and spicule (32.0-34.5 vs 38-46 µm).

Etymology.

The species name is derived from the name of the city where the new species was recovered.

Figures

Figure 1. Line drawings of Trichodorus hangzhouensis sp. nov., paratypes, Male A entire body B pharyngeal region C surface view D, E posterior end F spicule G germinal zone of testis. Scale bars: 50 μm.

Figure 3. Light photomicrographs of Trichodorus hangzhouensis sp. nov., paratypes A female entire body, arrow points vulva B male entire body C, D anterior region of female E pharyngeal region of male, arrows pointing position of cervical papillae (CP) and excretory pore (exp) F, G vulval region H, I female tail, arrows pointing position of anus (a) J-L male tail, arrows pointing position of male tail supplements (SP). Scale bars: 100 μm (A, B) 20 μm (C-L).

Figure 7. Phylogenetic relationships of Trichodorus hangzhouensis sp. nov. and other trichodorid species based on partial 18 S rRNA gene sequences. The Bayesian tree was inferred under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Tripyla sp. served as an outgroup species. Posterior probability values exceeding 70 % are given on appropriate clades.

Figure 8. Phylogenetic relationships of Trichodorus hangzhouensis sp. nov. and other trichodorid species based on D 2 - D 3 28 S rRNA gene sequences. The Bayesian tree was inferred under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Tripylina tamaki served as an outgroup species. Posterior probability values exceeding 70 % are given on appropriate clades.

Figure 9. Phylogenetic relationships of Trichodorus hangzhouensis sp. nov. and other trichodorid nematodes based on ITS 2 sequences. The Bayesian tree was inferred under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G). Thoracostoma trachyga served as an outgroup species. Posterior probability values exceeding 70 % are given on appropriate clades.