Longidorus vineacola Sturhan & Weischer, 1964

Gutierrez-Gutierrez, Carlos, Santos, Margarida Teixeira, Inacio, Maria Lurdes, Eisenback, Jonathan D. & Mota, Manuel, 2020, Description of Longidorus bordonensis sp. nov. from Portugal, with systematics and molecular phylogeny of the genus (Nematoda, Longidoridae), Zoosystematics and Evolution 96 (1), pp. 175-193 : 175

Longidorus vineacola Sturhan & Weischer, 1964 Suppl.material 2: Fig. S2(1-11), Suppl.material 6: Table S4

Remarks.

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 L. 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 2: Fig. S2(1-11); Suppl. material 6: Table S4). The morphological and metrical traits closely agree with the original description of the species ( Sturhan and Weischer 1964) and subsequent records ( Boag and Brown 1987; Brown and Taylor 1987; Andrés et al. 1991; Roca and Bravo 1996; Bravo and Lemos 1997; Brown et al. 1997; Gutiérrez-Gutiérrez et al. 2013, 2016; Archidona-Yuste et al. 2016), except for minor intraspecific variations in a and c ratios and length of odontostyle and spicules (Suppl. material 6: Table S4). This species was originally described parasitizing grapevine roots in Germany ( Sturhan and Weischer 1964) and has since been reported in a large number of records from various Euro-Mediterranean countries from a wide range of herbaceous and woody hosts ( Bravo and Lemos 1997; Brown et al. 1997; Taylor and Brown 1997; Gutiérrez-Gutiérrez et al. 2013, 2016; Archidona-Yuste et al. 2016). The alphanumeric codes for these populations of L. vineacola were applied in the diagnostic identification key for Longidorus spp. by Chen et al. (1997) and successive supplements by Loof and Chen (1999) and Peneva et al. (2013); they are (codes in parentheses are exceptions): A3(4), B3(4), C(2)3 ,D2, E3, F45, G23, H1, I2, J?, K?. Unfortunately, we detected no juvenile stages in our surveys; however, four juvenile stages were described by Sturhan and Weischer (1964) and by Roca and Bravo (1996). Additionally, Gutiérrez-Gutiérrez et al. (2013) using an integrative strategy characterized females, males, and first-stage juveniles (J1) of several populations of southern Spain and assigned molecular markers for this species.

Molecular results and phylogenetic relationships of Longidorus bordonensis sp. nov. and other Longidorus spp.

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 1 ). For the species of Longidorus studied here, there were multiple failed attempts to sequence the ITS1 region and a partial portion of 18S rRNA gene before our study was concluded (Table 1 ). The D2-D3 expansion segments of 28S rRNA gene sequences from L. vinearum (MN082430-MN082434) matched closely (99% similarity) to sequences of Spanish populations of this species in GenBank (KT308874, KT308876-KT308877); and the variations among these D2-D3 sequences ranged from 2 to 5 nucleotides. Intra-specific variation of D2-D3 detected among the populations of L. vinearum (three from grapevine and one from wild olive) (Table 1 ) was from 0 to 2 nucleotides (99% similarity and 0-1 indels). For L. vinearum , three ITS1 sequences (MN150065, MN150067, MN150068) from Dois Portos (LISB-03-04, grapevine) and an ITS1 sequence (MN150066) from Evora (M3-OLV, wild olive) were sequenced and showed a high similarity (99%), with some minor intra-specific variations among them (2-14 nucleotides and 0-1 indels). Our ITS1 sequences (MN150065-MN150068) had 98% similarity to others deposited in GenBank for L. vinearum (KT308892-KT308893, Spain); and the variations among them ranged from 17-23 nucleotides and 1-3 indels. The D2-D3 expansion segments of 28S rRNA gene sequences from L. vineacola (MN082425-MN082429) also had 99% similarity to several sequences of Spanish populations of this species in GenBank (JX445109-JX445111; KT308872, KT308873); and the variations among them ranged from 2 to 9 nucleotides. Intra-specific variation of the D2-D3 region among the four populations of L. vineacola (three from cork oak and one from wild olive) (Table 1 ) was low, varying from 0 to 6 nucleotides (99-100% similarity and 0 indels). For L. vineacola , the ITS1 region and the partial portion of 18S gene sequenced agree with results obtained from the D2-D3 fragments. The partial 18S rRNA gene sequence of L. vineacola (MN129758) was identical (100% similarity) to several L. vineacola sequences deposited in GenBank (AY283169, UK; JX445123, Spain), and 99% similar to L. onubensis Archidona-Yuste, Navas Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016 (KT308897, Spain), L. nevesi (MH430009, Spain), L. wicuolea (KT308900, Spain), L. fasciatus (MH430008, Spain; JX445122, Spain), and L. pacensis Archidona-Yuste, Cantalapiedra-Navarrete, Castillo & Palomare-Rius, 2019 (MH430004-MH430005, Spain). The variations among the partial 18S rRNA gene sequences of these species varied from 8 to 16 nucleotides. Our ITS1 sequence of L. vineacola (MN150064) showed a variable and low sequence homology with other sequences of L. vineacola in Genbank; the homology ranged from 97% (JX445094, Spain) to 94% (JX445096, Spain). The variations among the ITS1 sequences of these three sequences ranged from 18 to 51 nucleotides. The D2-D3 expansion segments of 28S rRNA gene sequence from L. wicuolea (MN082423) were 99% similar to three sequences of Spanish populations of this same species in GenBank (KT308863-KT308865, Spain). The variations among these four D2-D3 sequences ranged from 1 to 5 nucleotides. For L. wicuolea , the ITS1 sequences agree with results from the D2-D3 region. Our ITS1 sequence of L. wicuolea (MN150063) was 100% identical to other two sequences of this species in Genbank (KT308887 and KT308889, Spain) and clearly different (90%-88% similarity) from L. silvestris Archidona-Yuste, Navas Cortés, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016 (KT308884, Spain), and L. cf. olegi Kankina & Metlitskaya, 1983 (MH429999, MH430000, Spain); and the variations among these ITS1 sequences ranged from 18 to 51 nucleotides. The D2-D3 expansion segments of 28S rRNA gene sequence from Longidorus sp. 3 isolate ST (MN082424) matched well with several Longidorus spp. deposited in GenBank, including L. lusitanicus (KT308869, Spain) as the closest with 98.55% similarity, followed by L. magnus Lamberti, Bleve-Zacheo & Arias, 1982 (JX445113 and KT308870, Spain), L. crataegi Roca & Bravo, 1996 (JX445114, Spain), L. goodeyi Hooper, 1961 (AY601581), and L. vinearum (KT308876, Spain) with 94-95% similarity; and the sequence variations among the D2-D3 sequences of these species were from 11-42 nucleotides and 1-12 indels .

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 3 - 5 ). The BI tree (50% majority rule consensus tree) of the D2-D3 domains of 28S rRNA gene (Fig. 3 ) was based on a multiple-edited alignment (135 total sequences) of 722 total characters and revealed a major clade containing the majority of these species, including L. bordonensis sp. nov. and the remaining Iberian populations of Longidorus spp. (Fig. 3 ). The generated phylogenetic tree, using sequences of these D2-D3 fragments (Fig. 3 ), showed a clearly congruent position of L. bordonensis sp. nov. (MN082421, MN082422). The clade, including L. bordonensis sp. nov., grouped morphologically related species characterized by a short body and odontostyle and elongate to conical female tail, such as in L. pini (MH430028, Spain) and L. carpetanensis (MH430019, MH430020, Spain). The D2-D3 tree showed a consistent position for L. vinearum (MN082430- MN082434), which was placed within a well-supported clade of available GenBank entries belonging to L. vinearum (KT308874, KT308876, Spain) and clearly separated from the new species and other morphologically related species, such as L. magnus (KT308870, KX445113, Spain), L. crataegi (JX445114, Spain), L. goodeyi (AY601581), L. onubensis (KT308857, KT308858, Spain), L. oakcrassus Cai, Archidona-Yuste, Cantalapiedra-Navarrete, Palomares-Rius & Pablo Castillo, 2019 (MK941187-MK941190), L. oakgracilis Cai, Archidona-Yuste, Cantalapiedra-Navarrete, Palomares-Rius & Pablo Castillo, 2019 (MK941191-MK941193), L. wicuolea (KT308863-KT308865, Spain; MN022423, Portugal), L. andalusicus (JX445101, Spain), and L. vineacola (JX445111, Spain; MN082425-MN082429 Portugal). Likewise, this D2-D3 tree also showed congruence for the phylogenetic positions of L. vineacola sequences obtained here (MN082425-MN082429), as it was positioned within a well-supported clade together an available sequence in Genbank belonging to L. vineacola (JX445111, Spain). This clade, including all L. vineacola sequences, were separated from the new species and other phenotypically similar species, such as L. cf. olegi (MH430026-MH430027, Spain), L. silvestris (KT308859, Spain), L. lusitanicus (KT308869, Spain), L. oakcrassus (MK941187-MK941190), and L. wicuolea (KT308863-KT308865, Spain; MN022423, Portugal). In addition, Longidorus sp. 3 isolate ST (MN082424) was placed in a separated position within a well-supported sub-clade, clustering together to L. lusitanicus (KT308889, Spain) and L. crataegi (JX445114, Spain).

Similarly, the BI tree (50% majority rule consensus tree) of a multiple-edited alignment, including 116 18S rRNA sequences and 1690 total characters (Fig. 4 ) and 116 ITS1 sequences and 570 total characters (Fig. 5 ), showed a topology similar to that of the D2-D3 fragments of the 28S gene. Both the partial 18S and ITS1 trees using BI (Figs 4 , 5 ) showed a close phylogenetic relationship of L. bordonensis sp. nov. (18S, MN129757; ITS1, MN150062) with L. pini (18S, MH430011, Spain; ITS1, MH430001, Spain) and L. carpetanensis (18S, MH430006 Spain; ITS1, MH429991-MH429993, Spain). Both 18S and ITS1 trees showed a congruent position for all known species found in this study. For 18S and ITS1 trees, our L. vineacola sequences (18S, MN129758; ITS1, MN150064) were grouped in a well-supported clade also containing GenBank entries belonging to L. vineacola (18S, JX445111, Spain and AY283169; ITS1, JX445094, JX445096, Spain). In the ITS1 tree, all sequences from L. vinearum , including the accessions from our sequences (MN082425-MN082429) and GenBank accessions (KT308892-KT308893, Spain) clustered in the same well-supported sub-clade; however, they also clustered together with L. magnus (HM921340, Spain). Similarly, the tree generated using the ITS1 dataset, sequences from L. wicuolea , including the accession from our population (MN150063) and other populations in GenBank (KT308886-KT308888, Spain), clustered in the same well-supported sub-clade; however, this sub-clade also included L. silvestris (KT308884, Spain).

Figures

Figure 3. Phylogenetic relationships of Longidorus bordonensis sp. nov., L. wicuolea Archidona-Yuste, Navas-Cortes, 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 D 2 - D 3 expansion segments of 28 S 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.

Figure 4. 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 18 S 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.

Figure 5. Phylogenetic relationships of Longidorus bordonensis sp. nov., L. wicuolea Archidona-Yuste, Navas-Cortes, 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 ITS 1 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.