Labronema bidoupense, Vu & Elshishka & Nguyen & Le & Mladenov & Peneva, 2024

Vu, Tam T. T., Elshishka, Milka, Nguyen, Anh D., Le, Thi Mai Linh, Mladenov, Aleksandar & Peneva, Vlada, 2024, Two new species of genus Labronema Thorne, 1939 (Nematoda, Dorylaimidae) from natural parks of Vietnam with an identification key to the species with a medium-sized odontostyle, ZooKeys 1215, pp. 1-26 : 1-26

Labronema bidoupense sp. nov.

Material examined.

Six females, 6 males collected from National Park Bidoup-Nui Ba (Lac Duong District, Lam Dong Province) in good condition.

Description.

Females (for measurements see Table 1 , Figs 4 – 10 ) Nematodes of a medium size. Body cylindrical, slightly curved ventrally or adopted an open C shape after fixation. Cuticle three layered, especially obvious at caudal region, outer layer thin, intermediate layer much thicker than the outer one, particularly at the caudal region; inner layer much thinner than the intermediate layer; cuticle 7–8 μm thick at anterior region, 6–7 μm in mid-body, and 12–14 μm posterior to anus. The intermediate layer with longitudinal striations seen at a certain optical section. A narrow cervical lacuna between cuticle and epidermis observed. Ventral and lateral pores conspicuous, located all over the body, dorsal pores four or five at anterior end. After fifth or sixth ventral pore a structure resembling an excretory pore (duct with cuticularised walls) present (at 98–107 μm from anterior end). Lip region truncated, laterally somewhat angular, offset by a deep constriction, 3.2–3.6 times broader than high, less than one-third (33–38 %) of body diameter at pharynx base. Lips weakly separated, labial and cephalic papillae very low, liplets around labial opening present (Fig. 9 B ). Amphid with stirrup-shaped fovea; its aperture 11 μm wide, occupying two-fifths to one-half of lip region diameter. Cheilostom nearly cylindrical, with thick walls. Odontostyle strong and slightly sigmoid, 6–7 times as long as broad, 1.4–1.5 times longer than lip region diameter, and 1.9–2.4 % of total body length. Odontophore rod-like; 1.1 times odontostyle length. Pharynx strongly muscular, with its slender portion enlarging gradually, basal expansion 203–222 μm long, 43–46 % of pharynx length; gland nuclei and their orifices located as follows: DO = 52 %, DN = 57 %, S 2 N = 93 % (n = 1). Pharyngo-intestinal junction well developed; cardia with long conical projection into the intestinal lumen measuring 50–65 × 17–24 µm; presence of a thin and irregular disc- or belt-like structure separating the pharyngeal base from the cardia. Genital system di-ovarian, with both branches well and equally developed. Ovaries are reflexed, anterior 204–261 μm long, posterior 208–262 μm long, reaching oviduct / uterus junction, with oocytes first in two or more rows and then in a single row. Anterior oviduct 92.5–149 μm long and posterior oviduct 126–154 μm long, respectively, or 1.35–1.84 × longer than body diameter, consisting of a moderately developed proximal pars dilatata. Pars dilatata elongated and measuring 56–80 × 17–21 μm (27–33 × 16 μm in one young female), often containing round spermatozoa. Uterus complex, tripartite, anterior uterus 195–244 μm long, posterior uterus 193–241 μm long or 2.4–2.8 times longer than the body diameter; consisting of a thicker proximal region with lumen, a muscularised region (pars musculosa uteri) with Z-differentiation (Fig. 4 F – H ), ending with a tubular part. In the young female, the measurements are as follows: anterior ovary, oviduct, and uterus 106, 65, and 190 μm long, respectively, and posterior ovary, oviduct, and uterus 102, 65, and 217 μm long, respectively. Vagina extending inwards to 44 % of the body diameter: pars proximalis 22–29 × 19–26 μm in size, pars refringens consisting of (lateral view) two small triangular sclerotized pieces, with a combined width of 18–22 μm; pars distalis 9 μm long. Vulva a longitudinal slit 7–9 μm long. Prerectum 1.7–2.4 and rectum 1–1.2 times longer than anal body diameter. Tail short and rounded, three pairs of caudal pores at the posterior half of the tail. Hyaline part of tail 12–14 μm thick or 42–53 % of total tail length.

Males. General morphology similar to that of the female, except for the genital system. After the fourth or fifth ventral pore a structure resembling an excretory pore (duct with cuticularised walls) present (at 96–105 μm from anterior end). Genital system di-orchic, composed of two opposed testes, anterior 207–234 μm and posterior 191–225 μm long. Sperm oval, measuring 5 μm. Ventromedian supplements contiguous 13–15 in number, ad-cloacal pair located at 9–13 μm from cloacal aperture. Spicules 1.4–1.8 times body diameter at cloacal aperture long, 5–6 times as long as wide, spicule head 0.8–1.4 times longer than wide, occupying 9–11 % of total spicule length, and with slender walls, median piece narrow, occupying 29–36 % of spicule maximum width, and reaching the posterior end, which is 5–6 μm broad, curvature 140–150 °, ventral hump located at 20–23 μm or 23.5–30 % of spicule length from its anterior end, posterior tip 4.5–6 μm wide. Lateral guiding piece slightly curved, leaf-shaped (Fig. 6 H ), 17–19 μm long and 6 µm wide, ca 3 times longer than broad. Tail similar to that of female.

Type locality and habitat.

A pristine forest in the Bidoup mountain , Lac Duong District, Lam Dong Province, Vietnam (12 ° 04 ' 47 " N, 108 ° 39 ' 29 " E, elevation 2130 m a. s. l.).

Type material.

The holotype female, three paratype females, and two paratype males are deposited in the Nematode Collection of the Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Bulgaria, under the accession numbers IBER-BAS NTC 111 , 112, respectively. One paratype female and four paratype males are deposited in the Nematode collection of the Institute of Ecology and Biological Resources, Hanoi, Vietnam (accession number IEBR-FLN-DOR_04 and 05–08, respectively), one paratype female is deposited in the Wageningen Nematode Collection ( WANECO), Wageningen, the Netherlands (WANECO accession number WT 4040 ), and one paratype female is deposited in the Nematode Collection of the U. S. Department of Agriculture ( USDA), Beltsville, Maryland, USA (USDANC accession number T-8110 p ) .

Representative DNA sequences.

After sequencing the obtained L. bidoupense sp. nov. rDNA sequence fragments were deposited in GenBank under the following accession numbers PP 084892 (18 S rDNA) and PP 060469 (28 S rDNA), both originating from a specimen collected in NP Bidoup-NuiBa, Dak Lak, Vietnam.

Etymology.

The species is named after the Bidoup Mountain, the place from where it was recovered.

Differential diagnosis and relationships.

The new species is characterised by its medium-sizes body (1.59–2.04 mm long), lip region offset by a deep constriction and 25–28 μm wide, odontostyle 37.5–39 μm long, uterus complex (tripartite), longitudinal vulva (V = 54–57 %), short and rounded tail (26–31 μm, c = 57.8–77.2, c’ = 0.5–0.6). Males with 68–83 μm long spicules, 5–6 times as long as wide and contiguous ventromedian supplements 13–15 in number, lateral piece leaf-shaped.

In having medium body size (L = 1.5–2.5 mm) and odontostyle (31–39 μm), and lip region offset by a constriction, the new species resembles L. andrassyi , L. brevicauda , L. gerlachi , L. glandosum , L. obesum and L. porosum sp. nov. The new species differs from:

L. porosum sp. nov. by having: a thicker body cuticle, wider lip region (25–28 vs 21–24 μm), slightly sigmoid vs straight odontostyle, longer odontostyle (37.5–39 vs 32–37 μm), a peculiar cuticular fold on the dorsal site of stoma absent vs present, shorter pharyngeal expansion (203–222 vs 220–237 μm), presence of disc-shaped structure between pharynx and cardia vs absence, complex vs simple uterus, males present vs absent;

L. andrassyi by having: a shorter body length (1.59–2.04 vs 2.1–2.7 mm), thicker body cuticle, narrower lip region (25–28 vs 36–38 μm), longer prerectum (1.7–2.4 vs 3.5–5.4 times longer than anal body diameter) and tail (26–31 vs 35–45 μm), males present vs absent ( Gagarin 1992);

L. brevicauda by having: a different number of dorsal pores at anterior end (4 or 5 vs 6 or 8), wider lip region (25–28 vs 21–25 μm), longer and less robust odontostyle (37.5–39 vs 32–35.6 μm and 6–7 vs 4 times as long as broad), complex vs simple uterus, males present vs absent ( Furstenberg et al. 1993);

L. gerlachi by having: a wider lip region (25–28 vs 21–22 μm), longer odontostyle (37.5–39 vs 33–35 μm), more posterior vulva position (54–57 vs 45–48 %), rectum straight vs angular, shorter tail (26–28 vs 30–35 μm), males present vs absent ( Andrássy 2011 b);

L. glandosum by having: a differently shaped lateral chord (without vs with gland-like structures), a wider lip region (25–28 vs 20–21 μm), longer odontostyle (37.5–39 vs 32–35 μm) and tail (26–31 vs 21–24 μm), complex vs simple uterus, males present vs absent ( Rahman et al. 1986);

L. obesum by having: a shorter body length (1.59–2.04 vs 2.2 mm), longer odontostyle (37.5–39 vs 33 μm), tail without vs with distinctive central core, males present vs absent ( Thorne 1974).

Sequences and phylogenetic analyses

Molecular sequences of two specimens of L. porosum sp. nov. and one specimen of L. bidoupense sp. nov. were analysed in this study. After sequencing and editing, five sequences were obtained: a nearly full-length of 18 S rRNA gene for L. porosum sp. nov., (1641 bp; PP 084891) and L. bidoupense sp. nov., (1636 bp; GenBank: PP 084892); two nearly full-length D 2 - D 3 segment of 28 S rRNA gene for L. porosum sp. nov., (828 bp; GenBank: PP 060468, PP 060470) and for L. bidoupense sp. nov. (856 bp; GenBank: PP 060469). A BLAST search for matches to the partial 18 S rDNA sequences revealed that L. porosum sp. nov. has a difference of 34 nt with L. ferox ( AY 552972 ), 24–40 nt with L. vulvapapillatum ( AY 284807 from The Netherlands, KC 574385 from Iran), 32 nt with L. montanum ( MK 894247 – MK 894248 ) and 23 nt with the new species L. bidoupense sp. nov. The new species L. bidoupense sp. nov. has a difference of 34 nt with L. ferox ( AY 552972 ), 22–69 nt with L. vulvapapillatum ( AY 284807 from The Netherlands, KC 574385 from Iran) and 36 nt with L. montanum ( MK 894247 – MK 894248 ). A BLAST search for matches the partial 28 S rDNA sequences revealed that L. porosum sp. nov. has a difference of 76–78 nt with L. vulvapapillatum ( AY 592996 – А Y 592997 from The Netherlands, ON 685882 from Iran), 106 nt with L. montanum ( MK 894244 – MK 894246 ) and 31 nt with L. bidoupense sp. nov. Labronema bidoupense sp. nov. has 78 nt of difference with L. vulvapapillatum ( AY 592996 – AY 592997 from The Netherlands, ON 685882 from Iran), 115 nt with L. montanum ( MK 894244 – MK 894246 ) from Spain. Labronema porosum sp. nov. population from Du Gia Nature Reserve, Bac Me district, Ha Giang Province (PP 060468) was 100 % identical with Labronema population from Cuc Phuong National Park, Ninh Binh Province (PP 060470). The evolutionary relationships of the two new species as derived from the molecular analyses, are presented in the phylogenetic trees (Figs 11 , 12 ). The 18 S rDNA sequences of the two studied Labronema species from Vietnam clustered together in a group with L. ferox and were nested within the first clade (following Shokoohi et al. 2013), encompassing representatives of other species of Labronema ( L. vulvapapillatum ) and genera Paractinolaimus and Pararhyssocolpus (Fig. 11 ). Generally, this tree topology positioning was confirmed by the phylogenetic analyses based on the 28 S rDNA data (Fig. 12 ). The 28 S rDNA sequences of the two new species from Vietnam clustered together with L. vulvapapillatum from Iran ( ON 685882 ) and were positioned within a clade containing the genera belonging to the family Dorylaimidae (following the Peña-Santiago and Abolafia 2019 and Vazifeh et al. 2023) and encompassing representatives of the genera Crassolabium , Nevadanema , and Dorylaimus , as well as genera from other families Talanema ( Qudsianematidae ), Pararhyssocolpus ( Pararhyssocolpidae ), Sylphodorylaimus ( Thornenematidae ), and Paractinolaimus ( Actinolaimidae ). The close relationships of both Vietnamese species are also in agreement with their morphology. The main differences were described in detail in the diagnosis part, in the description of L. bidoupense sp. nov.

The most recent identification keys of all Labronema species known by that time is the key by Andrássy (1991). The same author also provided a key to the European species of the genus ( Andrássy 2009). However, these keys are outdated due to the numerous taxonomic changes and several new species described during the last decades. Here we elaborated a partial key for determination of a group of species with a medium-sized odontostyle (31–39 μm) to which also belong the two new Vietnamese species. Some species are excluded from the key:

Labronema virgo Monteiro 1970 : this species fits the features of the genus Labronema (the presence of offset by constriction lip region, robust odontostyle 1.5 times lip region diameter, double guiding ring), but because of the transverse vulva non-typical for the genus, this species is not included in the key.

Labronema enigmatum Baniyamuddin & Ahmad, 2007 : in the presence of a transverse vulva, spaced ventromedian supplements, and bluntly rounded tail, this species does not fit well with typical Labronema pattern. Andrássy (2009) transferred this species to the genus Labronemella Andrássy, 1985 . According to Peña-Santiago et al. (2012), the identity of this species is questionable; therefore, it should be either considered a species inquirenda or retained under Labronema . Because of the problematic position of this species, it is not included in the key.

Labronema diversum Andrássy, 2002 : this species is characterised by sexual dimorphism in the tail region, females have a tail with a dorsally curved peg, while males have a rounded conoid tail. The sexual dimorphism is atypical for the genus Labronema . According to Andrássy (2011 a) this peculiar structure of the female tail is an atavistic character. The presence of robust odontostyle, double guiding ring, longitudinal vulva and contiguous ventromedian supplements fit with genus Labronema but its position is doubtful ( Imran et al. 2021).

Labronema nemella Mushtaq & Ahmad, 2007 : according to Peña-Santiago et al. (2012) the true identity of this species is intriguing, because of the morphology of lip region and odontostyle (our remark). The authors consider that this species might be a member of Labronemella , but further data is needed to confirm this.

Two of the atypical species of genus Labronema (characterised with lip region nearly continuous with the adjacent body) – L. neopacificum Rahman, Jairajpuri, Ahmad & Ahmad, 1986 and L. pacificum (Cobb, 1906) Thorne, 1939 , which are distinguished by a medium-sized odontostyle are not included in this key because the lip region does not correspond to the typical offset lip region of members of the genus Labronema .

Figures

Figure 4. Line drawings of Labronema bidoupense sp. nov. female A, B lip region (B holotype) C – E amphids (D holotype) F, G anterior genital branch H uterine Z-differentiation (holotype) I pharyngo-intestinal junction (holotype) J, K tail ends (J holotype). Scale bars: 25 µm (A – E, H – K); 50 µm (F, G).

Figure 6. Line drawings of Labronema bidoupense sp. nov. male A, B, D lip region C amphidial fovea E odontostyle F, I tail ends G spicules H lateral guiding pieces. Scale bar: 25 µm.

Figure 9. Photomicrographs of Labronema bidoupense sp. nov. male A, B lip region C, D amphidial fovea E pharyngeal expansion F sperm cells in testis. Scale bar: 50 µm.

Figure 10. Photomicrographs of Labronema bidoupense sp. nov. male A, B tail ends. Scale bar: 50 µm.

Figure 11. Phylogenetic relationships of Labronema from the analysis of the 18 S rDNA sequences under ML (K 2 + G + I model). Numbers to the left of the branches are bootstrap values for 1000 replications.

Figure 12. Phylogenetic relationships of Labronema from the analysis of the 28 S rDNA sequences under ML (K 2 + G + I model). Numbers to the left of the branches are bootstrap values for 1000 replications.