Research Article |
Corresponding author: Vlada K. Peneva ( esn.2006@gmail.com ) Academic editor: Sergei Subbotin
© 2019 Stela S. Lazarova, Milka Elshishka, Georgi Radoslavov, Lydmila Lozanova, Peter Hristov, Alexander Mladenov, Jingwu Zheng, Elena Fanelli, Francesca De Luca, Vlada K. Peneva.
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Citation:
Lazarova SS, Elshishka M, Radoslavov G, Lozanova L, Hristov P, Mladenov A, Zheng J, Fanelli E, De Luca F, Peneva VK (2019) Molecular and morphological characterisation of Longidorus polyae sp. n. and L. pisi Edward, Misra & Singh, 1964 (Dorylaimida, Longidoridae) from Bulgaria. ZooKeys 830: 75-98. https://doi.org/10.3897/zookeys.830.32188
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Longidorus polyae sp. n., a bisexual nematode species found in the rhizosphere of pear tree (Pyrus communis L.), is described and characterised using an integrative approach. The new species has a female body length of 6.8–9.1 mm; a comparatively long odontostyle (114.0–127.5 μm); a narrow lip region (14.0–15.5 μm), anteriorly flattened and almost continuous with the body profile; pocket-like amphidial pouches long, deeply bilobed, and slightly asymmetrical, a guide ring at 37–42 μm from the anterior end; normal arrangement of pharyngeal glands; and a short bluntly rounded to hemispherical tail. Four juvenile stages identified: the first stage with a digitate tail, and the second and subsequent stages with a bluntly rounded tail. Males have one adcloacal pair and a row of 10 or 11 single ventromedian supplements; spicules 71.0–74.5 μm long. Based on morphometric data, the new species belongs to a group of species spread over Europe (L. arthensis, L. silvae, L. uroshis,), Iran (L. kheirii), and Syria (L. pauli), which share common characters such as amphidial fovea, lip region and tail shapes, similar odontostyle and body length, and similar first-stage juvenile tail shape. Codes for identifying the new species are A5, B2, C34, D3, E3, F45, G12, H1, I2, J1, K7. The phylogenetic analysis based on D2-D3 expansion domains of the rRNA gene revealed that the new species has the closest relationships with L. athesinus from Italy and three unidentified Longidorus spp. from USA (Longidorus sp. 1, Longidorus sp. 2, and Longidorus sp. 6). New morphometric and molecular data (18S rRNA gene, ITS1-5.8S-ITS2 regions and D2-D3 28S rRNA gene sequences) for three populations of L. pisi from Bulgaria were obtained and variations between populations are discussed.
18S rDNA, Bayesian inference, croplands, D2-D3 28S rDNA, ITS1-5.8S-ITS2, morphology, phylogeny
Longidorus Micoletzky, 1922 is the second most diverse genus within family Longidoridae (Thorne, 1935) Meyl, 1961 occurring in all continents except Antarctica. At present, it contains 170 species (
The present study aims to characterise morphologically and molecularly: i) an unknown species of genus Longidorus and ii) populations of L. pisi from Bulgaria, and iii) to evaluate these species’ phylogenetic relationships by using 18S rRNA and D2-D3 expansion domains of the 28S rRNA genes.
The Longidorus specimens examined originated from various croplands in Bulgaria: Balgarene village (private garden, Pyrus communis L. tree), Petrich (a small field of Nicotiana tabacum L.), and two vineyards, near Sandanski (Polenitsa village, small-scale management) and Kromidovo village (organic farm). Nematodes were isolated from soil samples by a decanting and sieving technique (
The genomic DNA extraction, amplification, and sequencing of single female specimens from three populations of L. pisi were carried out independently in two laboratories: one at the Institute for Sustainable Plant Protection, Bari, Italy and two at the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria (IBER-BAS). Protocols used in both laboratories are presented previously (
The 18S and D2–D3 expansion segments of the 28S rRNA gene sequences were compared with those of other nematode species deposited in the GenBank database using the BLASTn similarity search tool. The homologous sequences nearest to those of the new species were aligned using the GUIDANCE2 Server (http://guidance.tau.ac.il/) with default parameters (
(for measurements see Table
Female. Body assuming a spiral shape. Lip region narrow, 5–6 μm high, continuous with body profile, anteriorly flattened. Labial papillae, especially second circle, prominent, changing slightly the body contour. Cuticle 4–5 μm thick at postlabial region, 4–5 μm along the body, and 9–10 μm on tail posterior to anus. Guide ring 4–6 μm wide. Body pores conspicuous, 1 lateral pore anterior to or at the level of guide ring, 2 or 3 along odontostyle, 1 or 2 along odontophore, 3–5 in narrow part of the pharynx and 2–4 in bulb region as well as none dorsal and 5–6 ventral in pharyngeal region; numerous lateral pores observed along the rest of body. Amphidial fovea prominent, deeply bilobed, lobes long, slightly asymmetrical, amphidial aperture assumed to be a minute pore, hardly visible under light microscope. Odontostyle very slender, 1.5–2 μm wide at base. Two nerve rings observed, the first at some distance behind the odontophore and at 246.3 ± 11.1 (230–254) μm from anterior end, the second, more prominent, behind the first one at 354.8 ± 48.1 (327–440) μm from anterior end. Pharyngo-intestinal valve broadly rounded. Normal arrangement of pharyngeal glands: nuclei of the dorsal and ventrosublateral glands situated at 22.7–28.5 % and 54.3–58.5 % (n = 5) of the distance from anterior end of the bulb. Dorsal gland nuclei 2–2.5 μm in diameter, subventral gland nuclei 3–4 μm in diameter. In odontophore area of one female a small rudimentary odontostyle tip (vestigium) observed pointing forward. Peculiar crystalloid bodies of various sizes and shapes (mostly rod-like) found in the intestine of all females. Prerectum 465–497.5 μm long and rectum 33–42 μm or 0.6–0.8 of body diameter at anus. Tail bluntly conoid, rounded to hemispherical. Two pairs of lateral caudal pores. Vagina extending to ca. half the corresponding body width. Pars distalis vaginae 20–26 μm long; pars proximalis vaginae 21–27 μm long, thick walled. Uterus bipartite, moderately long, anterior uterus 230–406, posterior uterus 235–394 μm long, respectively; well-developed sphincter between uterus and pars dilatata oviductus, pars dilatata, and uteri containing numerous sperm cells; ovary small.
Male. Rarer than females. Habitus as in females, posterior part more strongly coiled ventrally. Shape of lip region similar to that in females. Cuticle 4.0–4.5 μm thick at postlabial region, 4 μm along the body and 5 μm on tail posterior to cloaca. One lateral pore anterior to or at the level of guide ring, 2 along odontostyle, 1 or 2 along odontophore, 3 or 4 in narrow part of the pharynx and 2 or 3 in pharyngeal bulb region, no dorsal and 6 ventral pores; numerous lateral pores present along the rest of the body. Two nerve rings observed, the first just behind the odontophore at a distance of 240.7 ± 12.0 (229–253) μm from anterior end, the second, more prominent behind the first one at 324 ± 12.7 (307.5–333) μm from anterior end. A small rudimentary odontostyle tip (vestigium) pointing forward observed in all males; in odontophore area (in 2 specimens) and in the slender part of pharynx (in 2 specimens). Pharyngo-intestinal valve broadly rounded. Tail short, bluntly conoid, dorsally convex, ventrally first straight then slightly concave. Three pairs of lateral pores on tail. One adclocal pair preceded by a row of 10 or 11 ventromedian supplements. Spicules slender, curved ventrally, lateral guiding piece sigmoid, 20–22 μm long. Spermatozoids oval (6–8 μm long).
Longidorus polyae sp. n. A, B anterior region C posterior region D, J variations in tail shape E vagina F anterior genital branch G lateral pieces H, I female and male pharyngeal bulb K variations in female and male habitus shapes. Female: all except C, G and I. Scale bars: 25 μm (A–E, J, G); 50 μm (F); 1 mm (K).
Longidorus polyae sp. n., holotype A anterior end B pharyngeal region, arrows point to at nerve ring, pharyngeal bulb and cardia C intestine, posterior part with inclusions D anterior genital branch E vagina and part of the posterior genital branch F sphincter G intestine inclusion at higher magnification H tail I vagina J posterior ovary. Scale bars: 20 μm (A, F–I); 40 μm (E, J); 100 μm (B–D).
Longidorus polyae sp. n., male A anterior region B amphidial fovea C labial region D junction of two testes E distal part of testis F sperm cells G nerve ring H, I posterior end, different magnifications J tail and spicules K supplements. Scale bars: 20 μm (A–C, F, G, J, K); 40 μm (D, E, H); 100 μm (I).
Juveniles. Four juvenile stages can be differentiated based on the body, odontostyle, and replacement odontostyle length (Figs
Measurements of adults and juveniles of Longidorus polyae sp. n. (mean ± standard deviation, with range). All measurements in micrometers except body length (mm).
Character | Holotype | Females | Males | J4 | J3 | J2 | J1 |
n = 10 | n = 4 | n = 8 | n = 3 | n = 7 | n = 8 | ||
L | 7.87 | 7.98 ± 0.82 6.81–9.12 | 6.90 ± 0.590 6.15–7.53 | 6.28 ± 0.476 5.87–6.87 | 4.10, 4.12, 4.43 | 2.80 ± 0.21 2.51–3.13 | 2.19 ± 0.19 1.9–2.4 |
a | 119.5 | 105.5 ± 8.4 95.7–119.5 | 115.3 ± 9.8 101.9–125.1 | 102.3 ± 11.2 89.4–116.6 | 93.8, 96.9, 99.3 | 83.1 ± 3.4 77.1–87.2 | 77.1 ± 5.8 70.4–88.2 |
b | 14.0 | 14.7 ± 1.5 2.3–17.3 | 12.2 ± 0.8 11.1–13.1 | 11.5 ± 0.4 11.2–12.0 | 8.8, 8.8, 9.0 | 7.6 ± 0.5 6.9–8.6 | 6.6 ± 0.9 5.5–8.0 |
c | 206.9 | 211.3 ± 21.5 184.8–260.0 | 171.4 ± 25.4 146.3–197.4 | 155.0 ± 9.2 145.0–166.4 | 100.2, 96.7, 102.1 | 77.2 ± 4.3 70.8–83.3 | 48.0 ± 8.1 38.0–65.0 |
c’ | 0.7 | 0.7 ± 0.04 0.7–0.8 | 0.8 ± 0.1 0.8–0.9 | 0.8 ± 0.0 0.8–0.9 | 1.1, 1.1, 1.1 | 1.3 ± 0.0 1.3–1.4 | 2.3 ± 0.3 1.8–2.6 |
V (%)/ Spicules | 51 | 50.9 ± 0.9 49.2–52.6 | 72.8 ± 1.9 71–74.5 | ||||
G1 (%) | 8.1 | 8.0 ± 1.0 6.7–10.3 | |||||
G2 (%) | 8.2 | 7.8 ± 1.0 6.0–9.6 | |||||
d | 2.71 | 2.8 ± 0.1 2.7–2.9 | 2.4, 2.8 | ||||
d’ | 2.04 | 2.1 ± 0.1 2.0–2.2 | 1.8, 2.0 | ||||
Odontostyle | 124 | 121.8 ± 3.9 114–127.5 | 117.6 ± 5.0 112–123 | 108.8 ± 3.6 105–113 | 98, 95, 98 | 80.2 ± 2.1 78–83 | 75.6 ± 2.1 71–78.5 |
Replacement odontostyle | 120.8 ± 2.0 119–123.5 | 113, 106, 115 | 93.4 ± 2.5 90–98 | 81.4 ± 2.2 77–83 | |||
Odontophore | 79 | 79.8 ± 5.2 68–84 | 80.2 ± 3.8 76–84 | 71.2 ± 8.2 65–77 | 54.5, 64, 68 | 56.7 ± 3.9 51–61 | |
Anterior end to guide ring | 40 | 40.4 ± 1.4 37–42 | 39.4 ± 2.5 37–42 | 34.5 ± 2.6 32–38 | 32, 29.5, 31 | 25.4 ± 1.5 23–27 | 21.2 ± 0.9 20–23 |
Pharyngeal bulb length | 155 | 152 ± 10.2 132–167 | 145.5 ± 3.0 142–148 | 139.6 ± 1.1 138–141 | 128, 115, 113 | 99.5 ± 2.8 95–102 | 84.1 ± 6.0 78–94 |
Pharyngeal bulb width | 29 | 26 ± 1.1 25–28 | 24.8 ± 1.7 23–27 | 23.6 ± 1.3 22–25 | 23, 19, 20 | 18.25 ± 0.5 17–19 | 14.7 ± 1.2 14–17 |
Developing gonad | – | – | – | 73.7 ± 9.9 7–85 | 35, 33, 35 | 26.6 ± 1.6 22–30 | 19.6 ± 2.4 17–23 |
Pharynx | 562 | 553.5 ± 54.1 481–646 | 565.0 ± 36.5 518–601 | 554.6 ± 26.1 524.5–571 | 468, 466, 494 | 367.9 ± 16.7 342–387 | 328.9 ± 20.9 298–359.5 |
Tail | 38 | 37.9 ± 3.0 33–43 | 41.0 ± 7.3 1–47 | 40.6 ± 3.5 37.5–45.5 | 41, 43, 43 | 36.2 ± 1.5 35–38 | 46.4 ± 6.2 2–52 |
Length of hyaline part of tail | 12 | 12.3 ± 0.7 12–13 | 12.5 ± 2.5 11–14 | ||||
Body diameter at: | |||||||
– lip region | 14.5 | 14.6 ± 0.5 4–15.5 | 14.8 ± 0.6 14–15 | 12.2 ± 1.0 11–13 | 11, 11 | 8.2 ± 0.6 7–9 | 6.8 ± 0.2 6.5–7 |
– guide ring | 30 | 30.3 ± 1.0 29–32 | 29.4 ± 2.0 27–32 | ||||
– base of pharynx | 61 | 63.1 ± 4.7 59–75 | 56.0 ± 2.7 52–58 | 54.2 ± 1.2 52.5–55 | 43, 42, 44 | 33.5 ± 1.6 31–35.5 | 27.1 ± 2.0 24.5–30 |
– mid-body/vulva | 66 | 76.0 ± 8.6 66–92.5 | 60.0 ± 5.5 53–66.5 | 61.8 ± 6.1 55–68 | 44, 42.5, 45 | 33.6 ± 1.5 32–36 | 28.6 ± 3.6 24–34 |
– anus | 53 | 53.1 ± 3.6 49– 61 | 48.2 ± 5.5 41–53 | 51.3 ± 1.7 49–53 | 36.5, 39, 40 | 27.1 ± 1.5 25–29 | 20.6 ± 2.2 18–24 |
– hyaline part | 35 | 37.4 ± 3.1 35–42 | 26.5 ± 1.2 26–27 |
Balgarene village, Pre-Balkan zone of the Balkan Mountains, north-central Bulgaria (43°2'48.08"N; 24°46'24.53"E), 386 m a.s.l., private orchard, rhizosphere of Pyrus communis L.
The holotype (PNT 42), 20 paratype females, 2 males, and 92 juveniles (overall PNT 43-101) from all stages are deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, BAS, Sofia, Bulgaria; 1 female, 1 male and 6 juveniles in the USDA Nematode Collection, Beltsville, Maryland, USA; 1 female, 1 male and 6 juveniles in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands; 1 female and 8 juveniles in the Nematode Collection of the Institute of Sustainable Plant Protection, Bari, Italy.
The NCBI BLASTn search of D2-D3 expansion segments of the 28S rRNA gene sequence showed highest similarity (93–94%) to several Longidorus species (L. attenuatus Hooper, 1961; L. dunensis Brinkmam, Loof & Barbez, 1987; L. athesinus Lamberti, Coiro & Agostinelli, 1991; L. persicus Esmaeili, Heydari, Archidona-Yuste, Castillo & Palomares-Rius, 2017; L. euonymus Mali & Hooper, 1974; Longidorus sp. 1, and Longidorus sp. 3). The highest identity (93.0–93.1%) was calculated with populations identified as L. attenuatus and L. dunensis (accessions KT755457 and AY593057, respectively). However, in the 28S rDNA phylogenetic analyses L. polyae sp. n. grouped in a clade with four Longidorus spp. (L. athesinus, Italy; Longidorus sp. 1, Longidorus sp. 2, and Longidorus sp. 6 from USA) with intermediate to high PP support (0.7–1.0) depending of the applied MSA algorithm (Fig.
Longodorus polyae sp. n. is a comparatively large bisexual species average 7.98 (6.81–9.12 mm) with the odontostyle over 100 μm (114.0–127.5 μm) long; the lip region narrow (14.0–15.5 μm), almost continuous with body profile, anteriorly flat; amphidial fovea long, pocket-shaped, deeply bilobed, and lobes slightly asymmetrical; normal arrangement of pharyngeal glands; and tail short, bluntly rounded to hemispherical, four juvenile stages present, with the tail of the first-stage juvenile digitate.
The alpha-numeric codes for L. polyae sp. n. to be applied to the polytomous identification key for Longidorus species by
A partial polytomous key to the species of Longidorus species close to L. polyae sp. n., based on the key by
Longidorus species | A | B | C | D | E | F | G | H | I | J | K |
---|---|---|---|---|---|---|---|---|---|---|---|
L. polyae sp. n. | 5 | 2 | 34 | 3 | 3 | 45 | 12 | 1 | 2 | 1 | 7 |
L. arthensis | 4 | 23 | 3 | 1 | 2 | 3 | 12 | 12 | 2 | 1 | 67 |
L. pauli | 4 | 23 | 23 | 3 | 23 | 4 | 3 | 1 | 2 | 1 | 7 |
L. silvae | 45 | 23 | 34 | 13* | 3 | 34 | 2(3) | 11 | 1 | 1 | 7 |
L. kheirii | 45 | 345 | 34 | 13* | 2 | 34 | 12 | 1 | 2 | 1 | 7 |
L. uroshis | 56 | 24 | 34 | 3 | 23 | 34 | 2 | 1 | 2 | 1 | 7 |
The group of large Longidorus species (code F34) with a moderately long odontostyle (code A45), pocket-shaped amphidial fovea, bilobed, symmetrical (code E2) or asymmetrical (code E3), normal arrangement of pharyngeal glands nuclei, short rounded tail (code H1) and digitate tail or tail with mucro (code K7) (according to
L. pauli by females having differently shaped amphidial pouches (asymmetrically vs symmetrically bilobed); plumper body (a = 95.7–119.5 vs 120.3–143.5); more posterior guide ring position (37–42 vs 27–36 μm); longer spicules (71.0–74.5 vs 61–69 μm); guiding piece in male sigmoid vs straight; different shape of tail in second- and third-stage juveniles (bluntly rounded vs conical) (
L. uroshis by females having a longer body (6.81–9.12 vs 5.6–7.6 mm); narrower lip region (average 14.6 (14.0–15.5) vs average 17 (15.0–20.5) μm), higher c (184.8–260.0 vs 120.4–162.0) and lower c’ values (0.7–0.8 vs 0.9–1.0) (
L. kheirii by females having a narrower lip region (14.0–15.5 vs 19.5–23.0 μm) and pharyngeal bulb (25–29 vs 39.5–48 μm), higher c values (184.8–260.0 vs 119.0–167.8), amphidial pouches deeply bilobed vs not to slightly bilobed; differently shaped tail of the first- and second-stage juvenile and different ovarium structure (
L. arthensis by females having longer body (6.81–9.12 vs 5.14–6.74 mm) and odontostyle (114.0–127.5 vs 102.0–111.0 μm); asymmetrically vs evenly bilobed amphidial pouches, lower c’ values (0.7–0.8 vs 1.0); males with longer spicules (71.0–74.5 vs 60.0–66.0 μm) (
L. silvae by females having two vs one nerve rings, differently shaped tail of the first-stage juvenile (subcylindrical, rarely cylindrical part/mucro with ventral position vs cylindrical mucro with central position), mucro shorter (9–11 vs 20–27 μm), higher c values (average 211.3 (184.8–260.0) vs average 166.7 (132.0–189.0) and posteriorly located vulva (50.9 (49.2–52.6) vs 48.6 (44.9–50.7) (
Additionally, it can be differentiated from L. athesinus, a phylogenetically related species (Fig.
Morphometrical data of the most similar species are presented in Table
Morphometric comparisons of Longidorus polyae sp. n. and related Longidorus spp. with similar morphological characters and DNA sequences.
Species | L (mm) | a | c’ | Odontostyle length (µm) | Lip region width (µm) | Guide ring position (µm) |
---|---|---|---|---|---|---|
L. polyae | 6.81–9.12 | 95.7–119.5 | 0.7–0.8 | 114–127.5 | 14–15.5 | 37–42 |
L. arthensis | 5.14–6.74 | 74.5–110 | 0.8–1.1 | 102–111 | 14–17 | 30–38 |
L. athesinus | 3.7–5.8 | 56.2–88.1 | 0.7–1.1 | 83.5–94 | 14–18 | 32–38 |
L. kheirii | 6.7–9.0 | 60.3–82 | 0.6–0.9 | 113–130 | 19.5–23 | 36.5–45 |
L. pauli | 6.5–8.6 | 120.3–143.5 | 0.8–1.0 | 102–118 | 14–17 | 27–36 |
L. silvae | 5.9–8.0 | 87.5–123.5 | 0.72–0.84 | 113.5–133.0 | 14.0–17.0 | 37.0–44.0 |
L. uroshis | 5.6–7.6 | 96.9–108.9 | 0.9–1.0 | 125–144 | 15–20.5 | 38–47 |
Named after the first author’s sister Mrs Polya Kadiyska, a school teacher of art and iconography at the “Nikola Obretenov” Primary school in Rousse, Bulgaria.
= Longidorus latocephalus Lamberti, Choleva & Agostinelli, 1983
Morphological and morphometric data for females and juvenile stages are presented in Table
Measurements of adults and juveniles of Longidorus pisi (mean ± standard deviation, with range) from different crops in Bulgaria. All measurements in micrometers except body length (mm).
Host Character |
Petrich | Sandanski | Kromidovo | Petrich | ||
---|---|---|---|---|---|---|
Nicotiana tabacum | Vitis vinifera | Nicotiana tabacum | ||||
female | female | female | J1 | J2 | J3 | |
n = 9 | n = 6 | n = 2 | n = 2 | n = 9 | n = 2 | |
L | 4.02 ± 0.2 (3.77–4.28) | 3.73 ± 0.2 (3.37–3.91) | 3.17, 3.91 | 1.10, 1.21 | 1.8 ± 0.2 (1.55–2.07) | 2.71, 2.65 |
a | 125.2 ± 6.3 (117.2–132.1) | 127.3 ± 5.2 (119.8–132.9) | 113.7, 129.5 | 68.7, 68.6 | 82.2 ± 4.7 (75.8–88.6) | 104.0, 99.1 |
b | 12.1 ± 0.9 (10.9–13.4) | 11.8 ± 1.2 (10.3–13.2) | 8.8, 11.9 | 5.0, 5.7 | 6.8 ± 0.7 (5.9–8.2) | 9.5, 9.1 |
c | 102.8 ± 4.2 (96.7–111.3) | 95.5 ± 5.7 (87.2–102.0) | 80.9, 94.7 | 34.1, 38.2 | 46.2 ± 3.2 (41.2–50.3) | 54.6, 63.7 |
c’ | 1.7 ± 0.1 (1.5–1.8) | 1.9 ± 0.11 (1.7–2.0) | 2.0, 2.0 | 3.0, 2.6 | 2.7 ± 0.2 2.5–2.9) | 2.4, 2.2 |
V (%) | 48.4 ± 0.9 (47.0–49.7) | 50.9 ± 0.6 (50.2–51.7) | 51.4, 51.4 | |||
G1 (%) | 5.3 ± 0.6 (4.9–6.2) | |||||
G2 (%) | 5.7 ± 0.6 (5.2–6.6) | |||||
Odontostyle | 74.8 ± 2.3 (72–79) | 76.1 ± 1.7 (74–78) | 75, 78 | 47.5, 46.5 | 52.9 ± 1.3 (50–55) | 59, 59 |
Replacement odontostyle | 53, 53 | 63.2 ± 1.5 (61–65) | 76, 77 | |||
Odontophore | 49.8 ± 2.8 (45–53) | 49.4 ± 2.8 (46–53) | 50 | 39.3 ± 2.1 (37–42) | ||
Anterior end to guide ring | 44.1 ± 1.6 (42–46) | 43.6 ± 0.9 (43–45) | 44, 42 | 25, – | 31.7 ± 1.4 (30–34) | 36, 36 |
Pharyngeal bulbus length | 69.1 ± 2.9 (65–73) | 71.3 ± 1.4 (69.5–74) | 76, 74 | 45, 37 | 51.5 ± 3.3 (47–56) | 63, 62 |
Anterior to nerve ring | 147.4 ± 5.0 (138–151) | 141.4 ± 6.2 (132–150) | –, 153 | 115.4 ± 4.4 (110–121) | 130, 138 | |
Pharynx | 330.9 ± 20.8 (292–368) | 310.7 ± 23.3 (291–342) | 361, 328 | 222, 212 | 263.4 ± 14.8 (242–285) | 286, 292 |
Tail | 38.7 ± 2.0 (36–43) | 39.1 ± 1.8 (37–42) | 39, 41 | 32, 32 | 39 ± 2.5 (35–43) | 50, 42 |
Body diameter at: | ||||||
– lip region | 11.0 ± 0.5 (10–12) | 10.3 ± 0.3 (10–11) | 10, 11 | 7, 7 | 8.5 ± 0.9 (8–9) | 10.5, 9 |
– base of pharynx | 27.9 ± 0.8 (27–29) | 25.6 ± 0.5 (25–26) | 27, 26.5 | 16, 17 | 20.5 ± 0.7 (19.5–22) | 26, 24.5 |
– mid-body | 31.9 ± 1.1 (30–34) | 29.3 ± 0.8 28–30) | 28, 30 | 16, 18 | 21.7 ± 0.9 (20–23.5) | 26, 27 |
– anus | 22.9 ± 1.2 (21–24.5) | 20.5 ± 0.8 (20–22) | 20, 21 | 11, 12 | 14.5 ± 0.5 (14–15) | 21, 19 |
Bulgarian populations of Longidorus pisi Edward, Misra & Singh, 1964, anterior end of female (A) and male (B) female C, D vagina and part of reproductive system (different populations) E–G pre-rectum inclusions H labial region I vagina J–M variations in tail shape, female (J, L, M) and male (K). Populations: A, L Petrich C, E–G, M Sandanski D, H, I, J Kromidovo B, K male specimens from a tobacco field, Petrich region. Scale bars: 20 μm (A–G, J–M); 12 μm (H, I).
Three ribosomal DNA regions (D2-D3 expansion segments of 28S rRNA gene, 18S rRNA gene, and ITS1-5.8S-ITS2 regions) of L. pisi were amplified and sequenced. The D2-D3 expansion segments of 28S rRNA gene sequences from all populations were identical to that of the Iranian population (JQ240274,
The new species belongs to a group of large Longidorus species having a moderately long odontostyle, a bilobed pocket-shaped amphidial fovea, a short rounded tail in adults, and a digitate tail with mucro in first-stage juveniles. The group contains just a few species occurring in Europe (L. arthensis, L. silvae, L. uroshis), Syria (L. pauli), and Iran (L. kheirii). A very specific character observed in all female specimens of L. polyae sp. n. were the crystalloid structures of various shapes and sizes present in the intestinal lumen, especially in the posterior gut region. To our knowledge, the presence of similar crystalloid structures has been rarely observed in plant-parasitic species (e.g. L. perangustus (Roshan-Bakhsh et al. 2016) and Hirschmanniella kwazuna (
DNA data were available for all species except L. pauli. However, different ribosomal fragments have been deposited in the GenBank database, which limit the reconstruction of phylogenetic relationships within the group. Moreover, the D2-D3 expansion segments of 28S rDNA sequences of the new species showed highest similarity (93%) to two other species of Longidorus, L. attenuatus and L. dunensis, with sequences revealing a very high inter-species similarity (or 1–3 nucleotides difference). One population of L. silvae from Serbia, characterised molecularly by three sequences of D3 segment of 28S rDNA (AM412367-AM412369,
In comparing the morphology of L. polaye sp. n. to the most closely related species, inaccuracies in code D values (related to the shape of anterior regions) were identified. In the original description of L. silvae, the lip region was described as “subacute, flattened frontally and continuous with the rest of the body” (
Morphological and molecular data of three Bulgarian populations of L. pisi have been presented within the framework of this study. Some differences in morphometrics between these populations and the original description have been observed; however, a more comprehensive integrative study on materials from the type area would help to evaluate the divergences. New sequences for two ribosomal DNA regions (18S rRNA gene and ITS1-5.8S-ITS2) were obtained for L. pisi for the first time. The D2-D3 expansion segments of 28S rRNA gene sequences of all populations from Bulgaria were identical. In the phylogenetic analysis (Fig.
The species appears to have a wide distribution, having been reported from three continents and numerous countries: Asia (India, Pakistan, Iran, China), Africa (Botswana, Egypt, Malawi, Mozambique, Cameroon, Libya, Namibia, Senegal, South Africa, Sudan), and Europe (Bulgaria, Macedonia, Greece). It was associated mainly with cultivated plants (pea, grapevine, mint and other medical plants, maize, potato, apple, pear, sugarcane) and rarely with natural vegetation (
The study was supported by the Sino-Bulgaria government cooperation project (project number 01/3-2014) and the project ANIDIV3, funded by the Bulgarian Academy of Sciences. We thank Thomas Prior (Plant Pest and Disease Programme, FERA, Sand Hutton, UK) for providing images of a L. silvae paratype and both reviewers for their constructive comments and feedback on the manuscript.