Research Article |
Corresponding author: Carlos Gutiérrez-Gutiérrez ( carlosg@uevora.pt ) Academic editor: Andreas Schmidt-Rhaesa
© 2020 Carlos Gutiérrez-Gutiérrez, Margarida Teixeira Santos, Maria Lurdes Inácio, Jonathan D. Eisenback, Manuel Mota.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Gutiérrez-Gutiérrez C, Teixeira Santos M, Inácio ML, Eisenback JD, Mota M (2020) Description of Longidorus bordonensis sp. nov. from Portugal, with systematics and molecular phylogeny of the genus (Nematoda, Longidoridae). Zoosystematics and Evolution 96(1): 175-193. https://doi.org/10.3897/zse.96.49022
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The genus Longidorus currently comprises 176 species of polyphagous plant ectoparasites, including eight species that vector nepoviruses. Longidorus is one of the most difficult genera to accurately identify species because of the similar morphology and overlapping measurements and ratios among species. Sequences of ribosomal RNA (rRNA)-genes are a powerful level-species diagnostic tool for the genus Longidorus. From 2015 to 2019, a nematode survey was conducted in vineyards and agro-forest environments in Portugal. The populations of Longidorus spp. were characterized through an integrative approach based on morphological data and molecular phylogenetic analysis from rRNA genes (D2-D3 expansion segments of the 28S, ITS1, and partial 18S), including the topotype of L. vinearum. Longidorus bordonensis sp. nov., a didelphic species recovered from the rhizosphere of grasses, is described and illustrated. Longidorus vineacola, with cork oak and wild olive as hosts, is also characterized. This is the first time that L. wicuolea, from cork oak, is reported for Portugal. Bayesian inference (BI) phylogenetic trees for these three molecular markers established phylogenetic relationships among the new species with other Longidorus spp. Phylogenetic trees indicated that i) L. bordonensis sp. nov. is clustered together with other Longidorus spp. and forms a sister clade with L. pini and L. carpetanensis, sharing a short body and odontostyle length, and elongate to conical female tail, and ii) all the other species described and illustrated are phylogenetically associated, including the topotype isolate of L. vinearum.
Bayesian inference, D2–D3 expansion segments of large ribosomal subunit 28S, genomic data, needle nematodes, internal transcribed spacer 1, partial small ribosomal subunit
Dorylaimida Pearse, 1942 is one of the most diverse orders in terms of number of species within the phylum Nematoda (
Most Longidorus species have a restricted geographical distribution. However, this genus is a cosmopolitan group, with South America being the less diverse region, followed in increasing order by Oceania, China, South Africa, North America, India, and Europe (
According to the morphological features and morphometric measurements of adult (mainly females) and that of juveniles [mainly first-stage juveniles (J1)], each Longidorus species is defined by 11 matrix codes (A–K) in the polytomous key published by
Members of the genus Longidorus have not been studied in detail during the past 18 years in Portugal, and updated information on the present occurrence and distribution is lacking, as well as molecular data (
Nematode surveys were conducted in spring and autumn from 2015 to 2019 in vineyards (Vitis sp.) and agro-forestry soils and included several host plants (Table
Nematodes were placed in a drop of water, killed in a hot fixative solution (4% formaldehyde + 1% glycerol + 85% distilled water), maintained for 48–72 h at room temperature (25 °C), and processed into pure glycerine by a modification of Seinhorst’s method (
After nematodes were extracted from the soil, specimens were examined by light microscopy (LM) on temporary glass slide mounts and digital images were recorded. These photomicrographs were used to match each phenotype with its associated genotype. Temporary slides were dismantled and individual nematodes were placed in a 2 µl drop of sterile water on the cover of a PCR tube, and the specimen was cut into six small pieces with a surface-sterilized scalpel. Subsequently, they were centrifuged in 18 µl of solution containing 10 µl ddH2O, 6 µl 10× PCR buffer, and 2 µl of proteinase K (20 mg/ml) (Nalgene), and frozen at −80 °C (15 min). Samples were mixed for 15 sec and PCR assays were conducted as described by
PCR cycle conditions for markers of ribosomal DNA included one cycle of 95 °C for 3 min; followed by 30 cycles of 94 °C for 30 s; an annealing temperature of 54 °C (D2A/D3B or 28LeX/ D3B), 53 °C (rDNA1/18S), and 50 °C (988F/1912R, 1813F/2646R) for 30 s, 72 °C for 15–45 s; and one cycle of 72 °C for 7 min. PCR products were purified after amplification using NZYGelpure (NZYTech Genes & Enzymes, Portugal) following the manufacturer’s instructions and used as template for direct sequencing at Eurofins Genomic (Germany) using the primers listed (Suppl. material
D2–D3 expansion segments of 28S rRNA, partial 18S rRNA, and ITS1 rRNA sequences from L. bordonensis sp. nov. and all other known Longidorus spp. found in the survey (Table
Measurements and morphological images are presented here in the description of species. Molecular data also helped to discriminate species within the genus Longidorus in this study. For the description of the new species L. bordonensis sp. nov., DNA data were added to the previously established morphological and morphometric analysis. Here, we provided metrical and non-metrical morphological data as well as molecular phylogenetic analyses of two known species previously reported from Portugal, L. vinearum and L. vineacola. Unfortunately, only one specimen was found in both L. wicuolea Archidona-Yuste, Navas Cortés, Cantalapiedra-Navarrete, Palomares-Rius, & Castillo, 2016 and one unidentified Longidorus species (Longidorus sp. 3 isolate ST), which were used for sequencing of the D2–D3 expansion domains of 28S rRNA or/and ITS1. In this study, in addition to the description of the new species, the molecular characterization of L. wicuolea comprises the first report of this species for Portugal. For both L. vineacola and L. vinearum, a brief description and comparison with the original description and other previous records are provided; however, for the description of the second species, topotype material was included too. Paratypes of L. vinearum were provided by Maria L. Inácio (INIAV, Oeiras, Lisbon, Portugal); however, they were not incorporated in this study because the specimens were in poor condition.
Slide PLB001.
6 females and 6 males (slides PLB002-PLB 013) mounted on glass slides.
Line drawings of Longidorus bordonensis sp. nov. paratypes from the rhizosphere of grass (unknown species) at São Pedro do Sul, Viseu district, northern Portugal (1–7). 1. Female anterior end. 2. Female lip region. 3. Female tail region. 4. Spicule and lateral guiding piece of gubernaculum. 5. Vulva region. 6. Male tail region. 7. Detail of basal pharyngeal bulb. Scale bars: 23 μm (1–3); 24 μm (4, 6); 29 μm (5); 15 μm (7).
The holotype (PLB001) and 8 paratypes (4 females and 4 males) (slides PLB002–PLB005 and PLB008–PLB011) are deposited in the Nematode Collection of the Nematology Lab, Institute for Mediterranean Agricultural and Environment Sciences, ICAAM, University of Évora, Évora; 2 paratypes (1 female and 1 male) (slides PLB006 and PLB012) in the Royal Belgian Institute of Natural Sciences, Brussels, Belgium; 2 paratypes (1 female and 1 male) (slides PLB007 and PLB013) in the Istituto per la Protezione delle Piante (IPP) of Consiglio Nazionale delle Ricerche (C.N.R.), Sezione di Bari, Bari, Italy.
Holotype and paratype specimens were extracted from a soil sample collected from the rhizosphere of an unidentified grass species at Bordonhos, São Pedro do Sul, Viseu district, Beira Alta province, northern Portugal (40°45'53"N, 8°5'12"W) (Table
Species | Sample code | Locality | Host | Genbank accessions | ||
D2D3 | ITS1 | 18S | ||||
Longidorus sp. 3 | ST | Carregueira, Santarém | cork oak tree | MN082424 | – | – |
L. bordonensis sp. nov. | 70-08-18 | Bordonhos, S.Pedro Sul | grass | MN082421 MN082422 | MN150062 | MN1297570 |
*L. vinearum | LISB-03-04 | Dois Portos, Torres Vedras | grapevine | MN082431 MN082432 | MN150065 MN150067 MN150068 | – |
L. vinearum | **LISB-22 | Picanceira, Mafra | grapevine | MN082434 | – | – |
L. vinearum | LISB-13 | Macheia, Ordasqueira, Torres Vedras | grapevine | MN082433 | – | – |
L. vinearum | M3-OLV | Valverde, Évora | wild olive | MN082430 | MN150066 | – |
L. vineacola | 119/015 | Q. da Amoreirinhas da Cima, Montemor-o-Novo | cork oak tree | MN082425 | – | – |
L. vineacola | 120/015 | Q. da Amoreirinhas da Cima, Montemor-o-Novo | cork oak tree | MN082426 MN082427 | – | – |
L. vineacola | 129/015 | Q. da Amoreirinhas da Cima, Montemor-o-Novo | cork oak tree | MN082428 | MN150064 | MN129758 |
L. vineacola | M3-OLV | Herdade da Mitra,Valverde, Évora | wild olive | MN082429 | – | – |
L. wicuolea | **Beja-16 | Linhares, Beja | cork oak tree | MN082423 | MN150063 | – |
The specific epithet of this species refers to the region of the type locality (Bordonhos) where the new species was found.
Short and slender body, slightly tapering at both ends, more pronounced in the tail. Curved in open J- or C-shaped relaxed by heat. Cuticle thin, appearing smooth under low magnifications, 1.8 ± 0.3 (1.3–2.2) μm thick at mid body, but thicker (9.1 ± 0.7 (8.1–9.8) μm) in hyaline region located at the end of tail region (Figs
Light micrographs of Longidorus bordonensis sp. nov. paratypes from the rhizosphere of grass (unknown species) at São Pedro do Sul, Viseu district, northern Portugal (1–10). 1. Anterior region. 2. Odontostyle region. 3. Lip region showing amphidial fovea. 4. Odontophore region. 5. Detail of basal bulb. 6, 7. Female tail region. 8. Male tail region. 9. vulva region. 10. Detail of spicule region. Abbreviations: a anus, af amphidial fovea, cd cardia, gr guiding ring, ost odontostyle, odph odontophore, sp spicules, spl ventromedian supplements, V vulva, vg vagina. Scale bars: 23 μm (1, 4); 15 μm (2, 3, 5); 50 μm (6, 7); 25 μm (8, 10); 30 μm (9).
Males are as common as females. Appearance of body similar to female, except for reproductive organs. Male diorchic with testes paired and opposed. Tail conoid, more convex-curved ventrally than that of the female, with rounded terminus at the end of tail (Figs
Morphometrics of females and males of Longidorus bordonensis sp. nov. from the rhizosphere of grass (unknown) at Bordonhos, São Pedro do Sul, Viseu district, Beira Alta province, northern Portugal.
Characters/ratios | Holotype | Females | Males |
---|---|---|---|
n | 1 | 7 | 6 |
L | 4115.8 | 4443.0 ± 593.1 (3671.4–5396.2) | 4560.0 ± 357.4 (4188.6–5201.0) |
a | 126.7 | 135.8 ± 13.1 (115.4–153.6) | 155.3 ± 11.5 (133.4–164.7) |
b | 12.8 | 13.8 ± 1.8 (10.7–15.9) | 13.9 ± 1.1 (12.3–15.7) |
c | 78.6 | 88.0 ± 13.7 (67.7–110.1) | 83.4 ± 9.7 (72.4–95.0) |
c’ | 2.3 | 2.3 ± 0.4(1.9–3.1) | 2.3 ± 0.2(1.9–2.5) |
V or T | 45.6 | 46.7 ± 2.0 (44.4–50.2) | 35.3 ± 8.7 (24.5–42.0) |
G1 (%) | 7.5 | 8.4 ± 0.5 (7.5–8.7) | – |
G2 (%) | 8.0 | 8.0 ± 1.2 (6.7–9.5) | – |
Odontostyle length | 67.1 | 69.8 ± 2.4 (67.1–73.5) | 69.5 ± 2.3 (67.1–73.4) |
Odontophore length | 45.6 | 47.0 ± 3.7 (42.4–52.2) | 45.5 ± 7.1 (37.0–54.6) |
Lip region width | 9.7 | 10.1 ± 0.4 (9.6–10.7) | 9.9 ± 0.7 (8.8–10.8) |
Oral aperture-guiding ring | 23.0 | 24.7 ± 1.4 (23.0–26.7) | 25.9 ± 1.0 (24.8–27.6) |
Tail length | 52.3 | 51.0 ± 6.8 (43.1–63.8) | 55.0 ± 4.6 (46.9–59.5) |
Spicules | – | – | 37.0 ± 3.4 (32.6–41.8) |
Lateral accessory piece | – | – | 8.4 ± 0.5 (8.1–8.7) (n = 2) |
J | 8.0 | 9.2 ± 0.7 (8.1–9.8) | 9.1 ± 0.6 (8.2–9.8) |
Longidorus bordonensis sp. nov. is characterized by a short body within the genus Longidorus (average = 4443 µm and 4560 µm in females and males, respectively), short odontostyle within the genus Longidorus (average = 70.0 µm and 69.5 µm in females and males, respectively), lip region anteriorly flattened, expanded (average = 10.0 µm in both females and males) and rounded laterally, set-off from body contour by a constriction, asymmetrically bilobed amphidial pouches with lobes occupying from 2/3 to 3/4 part of the distance from oral aperture to guiding ring, tail long (average = 51.0 µm and 55.0 µm in females and males, respectively), bluntly conoid, slightly ventrally curved with rounded terminus, short to medium spicules (average = 37.0 µm) with one pair of adanal and 9–11 mid-ventral supplements (Figs
Longidorus vinearum was originally described from around roots of grapevine (Vitis L.) in Dois Portos, Torres Vedras, Portugal (
Longidorus vinearum populations are characterized by a lip region, which is broadly rounded frontally, and more so laterally, and almost totally continuous with the outline of the body; a vulva near mid-body; the amphidial fovea large and clearly asymmetrically bilobed; the odontostyle long and robust; short tail characterized by having a bluntly rounded to hemispherical shape, dorsal side quite more convex than ventral side with rounded terminus; males characterized by large-sized spicules (average = 112.0 µm) and a large number of supplements, one pair of adanal and 18 or 19 mid-ventral supplements (Suppl. material
Three populations of L. vineacola from cork oak (Quercus suber L.) trees at Amoreirinhas da Cima, Montemor-o-Novo, Portugal and one population from wild olive (Olea europaea var. sylvestris) at Valverde, Évora, Portugal, are characterized morphometrically and morphologically: body medium-length to long (6.9–9.6 mm in females and 6.4–8.2 mm in males); odontostyle long (87.0–99.5 µm in females and 91.5–100.9 µm in males); lip region slightly set off from body contour by a depression; amphidial pouches asymmetrically bilobed; two equally developed female genital branches; females with broadly rounded tail usually as long as anal diameter; vulva posterior to mid-body; males with spicules well developed (69.9–79.9 µm long), and supplements consisting of an adanal pair and 14 or 15 ventromedians (Suppl. material
Polymerase chain reaction (PCR) was used to amplify the D2–D3 expansion segments of 28S rRNA, ITS1 rRNA, and partial 18S rRNA from L. bordonensis sp. nov. and four other Longidorus spp. For each of the species studied, these three genes had an approximate size of 700–800, 900–1000, and 1600 bp, respectively, based on visualization of the band on the electrophoresis gel and the subsequent direct sequencing.
D2–D3 sequences of L. bordonensis sp. nov. (MN082421–MN082422) matched well with the Longidorus spp. deposited in GenBank. Both D2–D3 sequences of L. bordonensis sp. nov. (MN082421 and MN082422) were almost identical, with a 99.31 % of sequence similarity. D2–D3 sequences of the new species were 95, 95, and 87%, similar to L. pini (MH430028, Spain), L. carpetanensis (MH430019–MH430020, Spain), and Longidorus sp. 1 FG-2018 isolate (MG765547, Iran), respectively, and differed in 27, 28–30, and 75 nucleotides, respectively. ITS1 sequence of L. bordonensis sp. nov. (MN150062) appropriately matched with other Longidorus spp. deposited in GenBank. This ITS1 sequence was 83–82, and 83% similar to L. carpetanenesis (MH429991–MH429993, Spain) and L. pini (MH430001, Spain), respectively. The variations among the ITS1 sequences of these species were from 143 to 157 nucleotides. The partial 18S rRNA gene sequences of L. bordonensis sp. nov. (MN129757) showed a high homology (more than 99% similarity) with two sequences deposited in GenBank belonging to L. carpetanensis (MH430006, Spain) and L. pini (MH430011, Spain). The variations among the 18S sequences of these species were from 8 to 15 nucleotides.
The D2–D3 expansion segments of 28S rRNA, ITS1 rRNA, and the partial 18S rRNA gene sequences obtained in this study for L. vinearum, L. vineacola, and L. wicuolea matched well with sequences from the same species previously deposited in GenBank, increasing knowledge of the genotypic diversity in Longidorus (Table
Using Bayesian inference (BI), we compared the phylogenetic position of L. bordonensis sp. nov. and other Longidorus spp. by using the D2–D3 expansion segments of 28S rRNA, the ITS1 region, and the partial 18S rRNA gene sequences (Figs
Phylogenetic relationships of Longidorus bordonensis sp. nov., L. wicuolea Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, L. lusitanicus Macara, 1986, L. vinearum Bravo & Roca and L. vineacola Sturhan & Weischer, 1964 within the genus Longidorus. Bayesian 50% majority rule consensus trees as inferred from D2–D3 expansion segments of 28S rRNA sequences alignments under the SYM model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are coloured in purple. Scale bar: expected changes per site.
Similarly, the BI tree (50% majority rule consensus tree) of a multiple-edited alignment, including 116 18S rRNA sequences and 1690 total characters (Fig.
Phylogenetic relationships of Longidorus bordonensis sp. nov. and L. vineacola Sturhan & Weischer, 1964 within the genera Longidorus and Paralongidorus. Bayesian 50% majority rule consensus trees as inferred from 18S rRNA sequences alignments under the SYM model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are coloured in light blue. Scale bar: expected changes per site.
Phylogenetic relationships of Longidorus bordonensis sp. nov., L. wicuolea Archidona-Yuste, Navas-Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, L. vinearum Bravo & Roca and L. vineacola Sturhan & Weischer, 1964 within the genus Longidorus. Bayesian 50% majority rule consensus trees as inferred from ITS1 rRNA sequences alignments under the SYM model. Posterior probabilities more than 70% are given for appropriate clades. Newly obtained sequences in this study are coloured in green. Scale bar: expected changes per site.
The main goal of our study was to identify and describe, morphologically and molecularly, Longidorus spp. parasitizing herbaceous and woody plants in vineyards and agro-forestry systems in Portugal. This was conducted in a nematological survey that included 150 sampling sites, with 43% of them in vineyards and the rest in agro-forestry areas. Eleven soil samples, each infested with only one needle nematode population, were selected for this study (Table
The comparative morphological taxonomic study of the 11 Portuguese populations of Longidorus spp. confirmed that the identification of species from phenotypic features including morphometric and morphometrical data is not easy due to inter- and intra- variability, overlapping of measurements and de Man ratios between species, and ambiguity caused by the presence of hundreds of species. As for previous biogeographic studies (
Twenty sequences belonging to three nuclear rRNA markers were used in this study: 11 D2–D3, seven ITS1, and two partial 18S sequences (Table
Phylogenetic trees reconstructed by the BI approach using new sequences and others from Genbank inferred similar patterns. For all trees generated, L. bordonensis sp. nov. was clearly grouped with L. pini and L. carpetanensis, two species sharing a similar morphology characterized by a short body and odontostyle, with an elongate to conical female tail. However, as is common in phylogenetic studies, other morphologically similar species to L. bordonensis sp. nov. were outside this sub-clade, such as L. distintus (KF242317, Italy), L. iliturgiensis Archidona-Yuste, Cantalapiedra-Navarrete, Castillo & Palomares-Rius, 2019 (MH430012, MH430013, Spain), L. indalus (KT308852–KT308854, Spain), L. juvenilis (AY601579, DQ364599, Slovenia), and L. pisi (MK172048, Bulgaria; LR032064–LR032065, Italy). The sub-clade composed of L. bordonensis sp. nov., L. pini, and L. carpetanensis shared a similar outward appearance characterized by the same code for most of five characters in the polytomous key (A2, B1, C2, H56, I2), and clustered with other members of the sub-clade composed of several other Longidorus species (i.e. for the D2–D3 tree: L. athesinus Lamberti, Coiro & Agostinelli, 1991, L. edmundsi Hunt & Siddiqi, 1977, L. polyae, L. sturhani Rubtsova, Subbotin, Brown & Moens, 2001, L. raskii Lamberti & Agostinelli, 1993), sharing a common ancestor. Our results agree with
Our work contributes greater understanding of the biodiversity within the genus Longidorus, describes Longidorus spp. by utilizing both morphological and molecular data, and establishes these species’ phylogenetic relationships within the genus. Our study also establishes the value of using rRNA molecular markers, especially from topotype specimens, for the identification of Longidorus spp., when other methods are difficult and inconclusive. In addition, we establish molecular markers for precise and unequivocal diagnosis of a new species, L. bordonensis sp. nov., and show that molecular markers are useful to differentiate this species from other species that are virus vectors. Additionally, these markers were used to characterize the topotype of L. vinearum. To our knowledge, this is also the first time that L. wicuolea is reported for Portugal.
This research was financially supported by National Funds through Foundation for Science and Technology under the project UID/AGR/00115/2019 (Portugal). We thank M.I. Ferreira and G. Albarrán from Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade of Évora for their excellent technical assistance.
Figure S1
Data type: TIF file
Explanation note: Light micrographs of female topotypes Longidorus vinearum Bravo & Roca, 1995 (1–9) infesting the grapevine (Vitis sp.) rhizosphere from Portugal. 1, 2. Female anterior ends. 3. Lip region showing amphidial fovea at different level of focus. 4. Vulva region. 5. Male tail region. 6. Female tail regions. 7–9. Juvenile tails (J2, J3, and J4, respectively). Abbreviations: a anus, af amphidial fovea, gr guiding ring, ost odontostyle, sp spicules, V vulva. Scale bars: (S1.1–3) 40 μm; (S1.4) 130 μm; (S1.5–6) 50 μm; (S1.7) 50 μm; (S1.8) 55 μm; (S1.9) 45 μm.
Figure S2
Data type: TIF file
Explanation note: Light micrographs of Longidorus vineacola Sturhan & Weischer, 1964 (1–11) infesting the rhizosphere from cork oak (Quercus suber L.) tree and wild olive (Olea europaea var. sylvestris L.) from Portugal. 1–3. Female anterior ends. 4. Lip region showing amphidial fovea at different focus. 5. Odontophore region. 6. Male tail, ventromedian supplements arrowed. 7. Vulva region. 8–10. Female tail regions. 11. Male tail with detail of spicules. Abbreviations: a anus, af amphidial fovea, gr guiding ring, ost odontostyle, odph odontophore, sp spicules, V vulva. Scale bars: (S2.1–2) 18 μm; (S2.3–4) 30 μm; (S2.5, S2.7) 75 μm; (S2.8–10) 50 μm; (S2.11, S2.6) 50 μm.
Table S1
Data type: DOCX file
Explanation note: List of primers used in this study.
Table S2
Data type: DOCX file
Explanation note: Comparison of the type population of the eight closest species to L. bordonensis sp. nov. for the most important diagnostic features.
Table S3
Data type: DOCX file
Explanation note: Morphometrics of Longidorus vinearum Sturhan & Weischer, 1964 from the rhizosphere of grapevine (Vitis sp.) in vineyards and wild olive (Olea europaea var. sylvestris) in agro-forestry systems in Portugal. All measurements in µm and in the format: mean ± s.d. (range)*.
Table S4
Data type: DOCX file
Explanation note: Morphometrics of Longidorus vineacola Sturhan & Weischer, 1964 from the rhizosphere of uncultivated plants in agro-forestry systems from Portugal. All measurements in µm and in the format: mean ± s.d. (range)*.