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Integrative taxonomy of Xiphinema histriae and Xiphinema lapidosum from Spain

Ruihang Cai
Ruihang Cai
, 
Antonio Archidona-Yuste
Antonio Archidona-Yuste
, 
Carolina Cantalapiedra-Navarrete
Carolina Cantalapiedra-Navarrete
, 
Juan E. Palomares-Rius
Juan E. Palomares-Rius
, 
Jingwu Zheng
Jingwu Zheng
 e 
Pablo Castillo
Pablo Castillo
   | 29 lug 2019
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Article Category: Arts & Humanities
Pubblicato online: 29 lug 2019
Pagine: 1 - 21
Ricevuto: 23 apr 2019
DOI: https://doi.org/10.21307/jofnem-2019-037
© 2019 Ruihang Cai et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

The genus Xiphinema Cobb, 1913 is a large and morphologically diverse group of plant-parasitic nematodes comprising more than 275 species (Archidona-Yuste et al., 2016a, 2016b; Peraza-Padilla et al., 2018). The economic importance of this group of nematodes is not only because of its extensive range of host plants and worldwide distribution, but for the transmission of several important plant viruses (genus Nepovirus, family Comoviridae) that cause direct damage to a wide variety of crops (Taylor and Brown, 1997; Decraemer and Robbins, 2007). Due to their economic importance, complex identification because of the sharing of a variety or morphological characters and existence of cryptic species, it is essential to identify species accurately and developing integrative taxonomy methods to control such plant pathogenic species (Archidona-Yuste et al., 2016a, 2016b). Species identification in this group is complex because of the sharing of a variety of morphological characters and the existence of cryptic species (Archidona-Yuste et al., 2016a, 2016b). According to the key for species of Xiphinema established by Loof and Luc (1990), the genus Xiphinema consists of X. americanum-group and X. non-americanum species. Later, non-americanum group was divided into eight morphospecies groups (Loof and 1990). Several authors have highlighted the great diversity of Xiphinema spp. detected in the Iberian Peninsula, in particular, around 40 species of the genus Xiphinema have been reported in Spain, mainly associated with woody, ornamental, and vegetable plant species (Gutiérrez-Gutiérrez et al., 2010, 2013, 2016; Archidona-Yuste et al., 2016a, 2016b).

Routine nematological surveys in agricultural and natural ecosystems in Spain yielded three populations of Xiphinema non-americanum group species, which were typologically different to previous reported species in Spain. Two populations of Xiphinema histriae were isolated from Quercus faginea Lam. and Pinus nigra Arnold, whereas one population of Xiphinema lapidosum was identified in association with Olea europaea subsp. europaea L. Lamberti et al. (1993a, 1993b) and Roca and Bravo (1993) described female and male stages of X. histriae and X. lapidosum, respectively, but in both species no juvenile stages were detected and described. The objectives of this study were: (i) to provide updated morphological descriptions of juvenile stages of X. histriae and X. lapidosum, (ii) to characterize the molecular data of both species using the D2 to D3 segments, ITS1 and partial CoxI gene sequences, and (iii) to determine the phylogenetic relationships of both species within the X. non-americanum group species.

Materials and methods
Nematode sampling, extraction, and morphological study

Nematodes were surveyed from 2017 to 2018 during the spring season in natural ecosystems and olive growing area in Andalucia, southern Spain (Table 1). Soil samples were collected for nematode analysis with a shovel from four to five cores randomly selected in each sampling site. Nematodes were extracted from a 500-cm3 sub-sample of soil by a modification of Cobb’s decanting and sieving method (Flegg, 1967). Specimens were killed and fixed with hot formalin (4% with 1% glycerol), and processed in glycerin (Seinhorst, 1959) as modified by De Grisse (1969). The measurements and light micrographs of nematodes were performed using a Zeiss III compound microscope.

Taxa sampled for Xiphinema species and sequences from NCBI used in this study.

Species Sampling code Locality Host-plant D2 to D3 ITS1 CoxI
1. X. histriae QUECAZ a Arroyo Frio, Cazorla, Cazorla, Jaén province, Spain Portuguese oak MK801302 MK801298 MK796911
MK801303
NAVSAP Navas de San Pedro, Cazorla, Jaén province, Spain Black pine MK801304 MK801299 MK796912
MK801305
2. X. lapidosum JAO132 Aroche, Huelva province, Spain Cultivated olive MK801306 MK801300 MK796913
MK801307 MK796914
MK796915
3. X. andalusiense 419b Andujar, Jaén province, Spain Wild olive KX244886 – –
AR108 Villaviciosa, Córdoba province, Spain Wild olive – – KY816595
4. X. abrantinum CAN223 Portugal – AY601625 – –
5. X. aceri M13 Maragheh city, Iran Wild rose – EU477385 –
6. X. adenohystherum –b Bollullos par del Condado, Huelva province, Spain Grapevine GU725075 GU725063 –
AR086 Prado del Rey, Cádiz province, Spain Wild olive – – KY816590
7. X. baetica H001 Hinojos, Huelva province, Spain Stone pine KC567169 – –
LOMAS Hinojos, Huelva province, Spain Stone pine – – KY816596
8. X. bakeri CD947 Olympic Peninsula, Washington, USA Unknown KF292276 – –
CD852 Point Reyes, Marin county, California, USA Unknown – – KF292305
9. X. barense CNR1 Brindisi province, Italy Wild olive KM199691 – –
its1IAS Brindisi province, Italy Wild olive – KM199693 –
itsLUC Brindisi province, Italy Wild olive – KM199694 –
10. X. basiri EU126 San Jose, Cuba – AY601630 – –
11. X. belmontense MOUB Merza, Coruna, Spain Pedunculate oak KC567172 – –
MOUCH Merza, Pontevedra province, Spain Chestnut – – KY816598
12. X. brasiliense SZX1305 Shenzhen, Guangdong province, China Resam KP793050 – –
13. X. cadavalense ST77 Espiel, Córdoba province, Spain Cultivated olive KX244900 – KY816599
14. X. castilloi – Gilan province, Iran Ash KF446655 – –
15. X. celtiense AR83 Penaflor, Sevilla province, Spain Wild olive KX244889 KX244926 –
AR82 Adamuz, Córdoba province, Spain Wild olive – KX244927 KY816601
16. X. chambersi 1,602 Jekyll Island, Georgia, USA Oak KU680967 – –
3,357 Jekyll Island, Georgia, USA Oak – – KU764419
17. X. cohni AR16 Sanlucar de Barrameda, Cádiz province, Spain Wild olive KX244901 KX244933 –
J126-2 El Puerto de Santa Maria, Cádiz, Spain Stone pine – KC567159 –
J0126 El Puerto de Santa Maria, Cádiz, Spain Grapevine – – KY816602
18. X. conurum ST45 Uleila del Campo, Almería province, Spain Cultivated olive KX244902 – –
ST45V Sorbas, Almería province, Spain Cultivated olive – – KY816603
19. X. costaricense ACC61 La Suiza de Turrialba, Cartago province, Costa Rica Sugarcane KX931059 – –
ACC46 Santa Rosa, Limon province, Costa Rica Cocoa – – KY816605
20. X. coxi GG10 Glynn County, Georgia, USA – AY601631 – –
21. X. coxi europaeum AR92 Alcolea, Córdoba province, Spain Wild olive KX244903 – –
AR020 Hinojos, Huelva province, Spain Wild olive – – KY816606
22. X. cretense OLI40 Hersonisos province, Greece Olive KJ802879 KJ802895 –
AR039 Hersonisos province, Greece Wild olive – – KY816608
23. X. dentatum – Silnicna, Czech Republic Hornbeam, Norway maple EU781538 – EU781537
24. X. diversicaudatum KOS Klucovec, Slovakia Unknown JQ780367 – –
AUS Marchegg, Austria Unknown – – GU222423
25. X. elongatum CD426 Brisbane, Australia Grasses MF510431 – MF510426
26. X. gersoni H0059 Almonte, Huelva province, Spain Eucalyptus KC567180 – KY816610
27. X. globosum – Alcalá de los Gazules, Cádiz Province, southern Spain Unknown GU549474 – –
28. X. granatum Xmar1 Markazi province, Iran Pomegranate JQ240273 – –
29. X. hangzhouense – Hangzhou, Zhejiang province, China Bull Bay MF538772 – MF706262
30. X. herakliense OLE18 Agiofarago, Crete Island, Greece Wild olive KM586349 – KY816613
OLE17 Agiofarago, Crete Island, Greece Wild olive – KM586355 –
OLE16 Agiofarago, Crete Island, Greece Wild olive – KM586354 –
31. X. hispanum – Andújar, Jaén province, Spain Estepa blanca GU725074 GU725061 –
00419 Andújar, Jaén province, Spain Wild olive – – KY816614
32. X. hispidum AR004 Medina Sidonia, Cádiz province, Spain Wild olive KX244906 – –
Xhi426 Bollullos par del Condado, Huelva province, Spain Grapevine – HM921367 –
H026b Rociana par del Condado, Huelva province, Spain Grapevine – – KY816616
33. X. hunaniense CD2465 Thailand – MF510432 – MF510427
34. X. ifacolum AE90 Kalutara province, Sri Lanka Grasses MH012181 – MH013396
35. X. index XinTre Trexenta, Cagliari province, Italy Grapevine HM921406 – HM921388
36. X. ingens Ps Kermanshah province, Iran Unknown KJ956388 – –
37. X. insigne CD1238 Fresno County, California, USA Grasses MF510430 – MF510425
38. X. iranicum M46 Maragheh city, Iran wild rose – EU477386 –
39. X. israeliae OLI34 Voutes province, Greece Olive KJ802886 – –
OLI13 Roufas province, Greece Olive – KJ802896 –
AR013 Roufas province, Greece Wild olive – – KY816618
40. X. italiae AR91 Puerto Real, Cádiz province, Spain Wild olive KX244912 KX244937 –
XIP12 Sbiba, Kasserine province, Tunisia Cultivated olive – KX062698 –
APUL Bari, Bari province, Italy Grapevine – – KY816623
41. X. iznajarense JAO25 Iznajar, Córdoba province, Spain Cultivated olive KX244892 KX244928 KY816624
KX244929
42. X. japonicum JH-2017 Japan Arhat pine – – KY628214
43. X. krugi CD1827 Matapalo, Puntaneras province, Costa Rica Rubber Plant KX931063 – –
ACC13 Santa Gertrudis, Alajuela province, Costa Rica Sugarcane – – KY816626
44. X. lupini H050 Hinojos, Huelva, Spain Grapevine KC567183 – –
388GD Bollullos par del Condado, Huelva, Spain Grapevine – – KY816630
45. X. macroacanthum individual 70 Adriatic Sea coast, Italy Olive HF546081 – –
ITAL Bridisi province, Italy Cultivated olive – – KY816631
46. X. macrodora JAO6 La Granjuela, Córdoba province, Spain Cultivated olive KU171041 – –
AR097 Santa Mª de Trassierra, Córdoba province, Spain Wild olive – – KY816632
47. X. meridianum XMP11 Sbitla, Kasserine province, Tunisia Cultivated olive KX062679 – –
11R16 Sbitla, Kasserine province, Tunisia Common buckthorn – – KY816635
48. X. mengibarense OC3C4 Mengibar, Jaén province, Spain Cultivated olive KX244895 – –
O3V05 Mengibar, Jaén province, Spain Cultivated olive – – KY816634
49. X. naturale N1 Fort Pierce, Florida, USA Oak DQ299515 – –
50. X. nuragicum JAO36 Casarabonela, Malaga province, Spain Wild olive KX244913 – –
RONDA Ronda, Malaga province, Spain Abete di Spagna – GU725059 –
AR113 Alcolea, Córdoba province, Spain Wild olive – – KY816640
51. X. oleae AR35 Tarifa, Cádiz province, Spain Wild olive KU171038 – –
52. X. parachambersi – Japan Cape jasmine, Spindle MG786445 – –
53. X. paradentatum 17-82 Tara Mountain, Serbia Meadow LT883658 – –
54. X. poasense AJ74 San Carlos, Alajuela province, Costa Rica Eucalyptus, Cypress and Fountain grass MF461347 – –
AP99 San Carlos, Alajuela province, Costa Rica – – MF461335
55. X. pseudocoxi AR95 Alcaracejos, Córdoba province, Spain Wild olive KX244915 – KY816643
56. X. pyrenaicum PYRDJ Cahors, Midi-Pyrenees, France Grapevine GU725073 GU725060 –
57. X. radicicola V1273 Chu’momray, Viet Nam – AY601622 – –
58. X. robbinsi Topotype TypPop Goshayesh village, East Azarbaijan, Iran Common buckthorn MH744579 – –
59. X. robbinsi Tunisia 12R28 Sbitla, Kasserine province, Tunisia Cultivated olive KX062683 – KY816647
60. X. savanicola CAN72 Dakar, Senegal – AY601620 – –
61. X. setariae AC009 Pueblo Nuevo de Duacarí, Limón, Costa Rica Banana KX931066 – KY816648
62. X. sphaerocephalum AR73 Castillo de Locubín, Jaén province, Spain Wild olive KX244917 – –
AR63 Coto Ríos, Jaén province, Spain Oak – GU725062 –
AR063 Coto Ríos, Jaén province, Spain Wild olive – – KY816649
63. X. tica ACC81 Sabanillas, San Jose province, Costa Rica Coffeae KY623491 – –
ACC32 Lagunilla, Guanacaste province, Costa Rica Soursop – – KY623501
64. X. turcicum ST149 Prado del Rey, Cádiz province, Spain Cultivated olive KX244919 – –
ST149 San José del Valle, Cádiz province, Spain Wild olive – – KY816650
65. X. turdetanense J212 Sanlucar de Barrameda, Cádiz Province, Spain Stone pine KC567186 – –
AR015 Sanlucar de Barrameda, Cádiz Province, Spain Wild olive – – KY816651
66. X. vuittenezi – Slany, Czech Republic Apple EF614266 – EF614265
– Rhein Valley, Germany – – AJ437028 –
population O Romania Grapevine – HG329722 –
67. X. zagrosense Yasooj Zagros Mountains, Iran Grass JN153101 – –

Note: aNewly sequences species in bold font; b(–) data not provided or sequence not used.

A comparative morphological and morphometrical study of type specimens of X. histriae were conducted with specimens kindly provided by Dr A. Troccoli, from the nematode collection at the Istituto per la Protezione Sostenibile delle Piante (IPSP), Consiglio Nazionale delle Ricerche (CNR), Bari, Italy; and paratypes of X. lapidosum kindly provided by Dr Z.A. Handoo from USDA Nematode Collection, Beltsville, MD, USA (T-4406p; T4407p). Spanish nematode populations of both Xiphinema species in this study are proposed as standard and reference populations for each species given until topotype material becomes available and molecularly characterized. Voucher specimens of these described species have been deposited in the nematode collection of Institute for Sustainable Agriculture, IAS-CSIC, Córdoba, Spain.
Molecular analyses

For molecular analyses, in order to avoid mistakes in the case of mixed populations, two live nematodes from each sample were temporary mounted in a drop of 1M NaCl containing glass beads (to avoid nematode crushing/damaging specimens) to ensure specimens conformed to the unidentified populations of Xiphinema. Following morphological confirmation, the specimens were removed from the slides and DNA extracted. DNA was extracted from single specimens as described by Archidona-Yuste et al. (2016a, 2016b). The D2 to D3 segments were amplified using the D2A (5’-ACAAGTACCGTGAGGGAAAGTTG-3’) and D3B (5’-TCGGAAGGAACCAGCTACTA-3’) primers (De Ley et al., 1999). The ITS1 region was amplified using forward primer 18S (5’-TTGATTACGTCCCTGCCCTTT-3’) (Vrain et al., 1992) and reverse primer rDNA1 5.8S (5’-ACGAGCCGAGTGATCCACCG-3’) (Cherry et al., 1997). And CoxI gene was amplified as described by Lazarova et al. (2006) using the primers COIF (5’-GATTTTTTGGKCATCCWGARG-3’) and COIR (5’-CWACATAATAAGTATCATG-3’). The newly obtained sequences were submitted to the GenBank database under accession numbers indicated on the phylogenetic trees and in Table 1.
Phylogenetic analysis

D2 to D3 segments, partial ITS1 rRNA, and partial CoxI sequences of different Xiphinema species belonging to the X. non-americanum group were obtained from GenBank and used for phylogenetic reconstruction. Outgroup taxa for each data set were chosen following previous published studies: Longidorus oleae (KT308871), Xiphinema americanum (KX263175); Longidorus caespiticola (KJ567469), Xiphinema duriense (KX244935), Xiphinema pachtaicum (HM921337); Scutellonema bradys (AY268114), Meloidogyne hapla (AY268113) (He et al., 2005; Holterman et al., 2006; Gutiérrez-Gutiérrez et al., 2013; Tzortzakakis et al., 2015; Archidona-Yuste et al., 2016a, 2016b; Susulovska et al. 2018: Varela-Benavides et al., 2018). Multiple sequence alignments of the different genes were made using the Q-INS-i algorithm of MAFFT V.7.205 (Katoh and Standley, 2013), which accounts for secondary RNA structure. Sequence alignments were visualized and their percentage of similarity calculated using the sequences identity matrix using BioEdit (Hall, 1999) and manually edited by Gblocks ver. 0.91b (Castresana, 2000) in Castresana Laboratory server (http://molevol.cmima.csic.es/castresana/Gblocks_server.html) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences  + 1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half).

Phylogenetic analyses of the sequence data sets were based on Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike Information Criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, the gamma distribution shape parameters, and substitution rates in the AIC were then given to MrBayes for the phylogenetic analyses. BI analyses were performed under the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR +  I  +  G) for the D2 to D3 segments of 28S, rRNA ITS1 and partial CoxI gene. These BI analyses were run separately per data set using four chains for 2 × 106 generations for all of molecular markers. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The topologies were used to generate a 50% majority rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were visualized using FigTree software V.1.42 (http://tree.bio.ed.ac.uk/software/figtree/).
Results

Systematics

Xiphinema histriae Lamberti et al. (1993a, 1993b).

(Figs. 1–3; Table 2).

Comparative morphometrics of females and males of Xiphinema histriae (Lamberti et al., 1993a, 1993b) from different localities. All measurements are in µm and in the form: mean ± sd (range)a.

Locality/host-plant Arroyo Frío, Cazorla Jaén, Spain, Portuguese oak Nava de San Pedro, Cazorla Jaén, Spain, pine Paratypes, (Lamberti et al., 1993a) Gorizia, Italy, Vitis sp. (Lamberti et al., 1993b) Trieste, Italy, Vitis sp.
Characters/ratiosb Females J2 J3 J4 Females Male Females Males Females Male
n 20 5 3 3 11 1 5 3 4 1
Lb 4,959.0 ± 253.7 (4,477.0–5,455.0) 1,715.0 ± 83.9 (1,583.0–1,795.0) 2,629.0 ± 271.4 (2,409.0–2,932.0) 4,000.0 ± 300.7 (3,659.0–4,227.0) 4,762.4 ± 322.4 (4,364.0–5,364.0) 4,773.0 4.3 ± 0.19 (4.0–4.5) 4.2 ± 0.21 (4.0–4.3) 4.2 (4.1–4.5) 4.5
a 77.6 ± 5.4 (68.2–86.4) 41.5 ± 4.0 (37.7–46.7) 57.4 ± 2.7 (54.8–60.2) 68.2 ± 1.4 (67.1–69.7) 73.0 ± 3.9 (67.1–79.2) 68.2 67.9 ± 2.2 (64.1–69.4) 65.9 ± 0.8 (65.4–66.5) 68.4 (61.0–78.1) 78.8
b 8.7 ± 0.6 (7.4–10.2) 4.3 ± 0.4 (3.7–4.7) 5.8 ± 0.5 (5.4–6.3) 7.1 ± 0.1 (7.1–7.3) 8.9 ± 0.8 (8.1–10.6) 7.6 8.3 ± 0.5 (7.7–9.0) 8.0 ± 0.4 (7.7–8.3) 8.1 (7.3–9.3) 9.1
c 111.4 ± 8.4 (98.4–130.2) 32.6 ± 1.5 (31.0–34.5) 45.3 ± 4.4 (40.4–48.9) 73.8 ± 3.4 (71.0–77.6) 114.1 ± 7.7 (99.7–128.1) 116.4 103.7 ± 8.5 (96.6–117.8) 82.5 ± 4.9 (79.0–86.0) 100.0 (86.0–108.2) 103.4
c' 1.0 ± 0.1 (0.8–1.1) 1.8 ± 0.1 (1.7–2.0) 1.9 ± 0.2 (1.8–2.1) 1.3 ± 0.1 (1.2–1.4) 1.0 ± 0.05 (0.9–1.0) 0.9 0.9 ± 0.04 (0.9–1.0) 1.0 ± 0.7 (1.0–1.1) 1.0 (0.9–1.2) 1.0
V or T (%) 53.8 ± 1.5 (51.5–57.0) – – – 53.7 ± 1.5 (52.0–57.5) 36.7 44.5 ± 0.5 (44.0–45.0) – 44.0 (42.0–46.0) –
Odontostyle 144.1 ± 4.2 (135.5–151.5) 81.6 ± 3.8 (77.5–86.5) 95.5 ± 3.5 (93.0–99.5) 116.7 ± 5.5 (111.0–122.0) 142.3 ± 6.6 (132.5–150.5) 133.5 148.5 ± 4.9 (142.9–156.5) 147.3 ± 1.2 (146.5–148.2) 151.9 (140.0–156.5) 132.3
Odontophore 89.4 ± 2.7 (85.0–95.0) 59.8 ± 2.0 (56.5–61.5) 65.5 ± 2.6 (64.0–68.5) 79.0 ± 2.6 (76.0–81.0) 85.9 ± 4.5 (80.5–93.0) 88.0 86.7 ± 2.3 (83.5–89.4) 89.4 86.4 (69.4–92.9) 74.7
Total stylet 233.5 ± 6.4 (223.5–245.0) 141.4 ± 4.9 (134.0–146.5) 161.0 ± 6.1 (157.0–168.0) 195.7 ± 7.8 (187.0–202.0) 228.2 ± 8.7 (213.5–241.0) 221.5 – – – –
Replacement Odontostyle – 100.7 ± 3.1 (95.5–104.0) 123.0 ± 0.9 (122.0–123.5) 143.0 ± 5.7 (136.5–147.0) – – – – – –
Lip width 15.4 ± 0.9 (14.0–17) 10.4 ± 0.2 (10.0–10.5) 11.8 ± 0.3 (11.5–12.0) 13.8 ± 0.4 (13.5–14.0) 15.9 ± 0.6 (14.5–16.5) 15.5 15.0 ± 0.3 (14.7–15.3) 15.0 14.0 13.5
Oa–guiding ring 128.0 ± 6.8 (116.0–139.0) 75.4 ± 2.4 (71.5–78.0) 88.0 ± 8.9 (78.0–95.0) 109.7 ± 15.5 (93.5–124.5) 117.9 ± 7.4 (108.5–129.5) 126.5 136.4 ± 6.1 (129.4–144.7) 135.0 ± 7.1 (130.0–140.0) 137.0 (117.1–145.9) 123.5
Tail length 45.3 ± 3.1 (40.5–50.0) 52.6 ± 1.8 (50.5–54.5) 58.2 ± 6.0 (51.5–63.0) 54.2 ± 2.5 (51.5–56.5) 41.8 ± 2.0 (38.5–44.5) 41.0 41.4 ± 2.3 (38.2–43.5) 50.0 42.6 (38.8–51.2) 43.5
J 16.0 ± 1.8 (13.0–19.5) 16.2 ± 2.9 (12.5–20.5) 17.3 ± 2.8 (15.5–20.5) 18.0 ± 1.3 (16.5–19.0) 17.3 ± 2.8 (13.5–23.5) 5.0 13.4 ± 0.8 (12.9–14.7) 14.4 ± 0.4 (14.1–14.7) 16.3 (14.1–18.2) 9.4
Spicules – – – – – 74.0 – 84.0 ± 2.1 (82.3–85.2) – 82.0
Lateral accessory piece – – – – – 21.0 – – – –
Suppl. 1-anus – – – – – 24.5 – – – –

Note: aMeasurements are in µm and in the form: mean  ±  standard deviation (range); ba = body length/maximum body width; b = body length/pharyngeal length; c = body length/tail length; c’ = tail length/body width at anus; V = (distance from anterior end to vulva/body length)×100; T = (distance from cloacal aperture to anterior end of testis/body length)×100; J = hyaline tail region length.

Figure 1.

Light micrographs of Xiphinema histriae (Lamberti et al., 1993a, 1993b). Females: A, Pharynx; B to D, Lip regions; E to G, I, J, Tail region; H, Gonad; K, L, Details of pseudo-Z-organ; Juveniles: M to O, Tail region of 2nd, 3rd and 4th stage juveniles; Males: P, Tail region of male. Abbreviations: a, anus; cb, crystalloid bodies; gr, guiding ring; psZ, pseudo-Z-organ; sp, spicule; spl, supplements; v, vulva. (Scale bars: A = 40 μm; B-D = 15 μm; E–G, I, J = 20 μm; H = 65 μm; K = 10 μm; L = 20 μm; M–O = 10 μm; P = 20 μm.)

Description
Female

The description of female body of Xiphinema histriae is as follows: body is cylindrical with an open C-shaped when heat relaxed; cuticle is 3.1 (2.5–3.5) μm thick at mid-body; Lip region is flatly rounded, separated from body by a slight depression, 15.4 (14.0–17.0) μm wide and 8.0 (7.5–9.0) μm high; amphids are stirrup shaped and amphidial fovea aperture extending for ca 60.7 to 70.5% of lip region diam; odontostyle long and narrow, 1.6 times longer than odontophore; odontophore with well-developed flanges 15.6 (14.0–17.0) μm wide; pharynx extending to a terminal pharyngeal bulb with three nuclei: one dorsal gland nucleus (DN) located at the beginning of basal bulb (9.0–12.7%) and two ventro-sublateral nuclei (SVN) are located near to the middle of bulb (51.9–57.5%); glandularium is 152.8 (129.5–170.5) μm long; reproductive system didelphic-amphidelphic is with equally developed branches, and vulva slit-like and situated slightly posterior to mid-body; each branch comprises a reflexed ovary and a tubular oviduct with a developed pars dilatata oviductus separated from uterus by a sphincter; uteri tripartite with a long tubular part, consisting of a developed pars dilatata uteri link with a narrower, muscular tube-like portion containing crystalloid bodies distributed over the entire length, pseudo-Z-organ with weakly muscularized wall with numerous small dense granular bodies; ovejector is well developed, 22.2 (16.0–32.5) μm wide, and vagina is 30.4 (20.0–40.0) μm long or 47.4% (34.5–57.1%) of corresponding body width; prerectum is reaching around 8.9 to 11.5% of nematode body from the anus to anterior part; rectum is extending more or less than the body width at anus; and tail is short and hemispherical with a peg 6.0 to 9.5 μm long.
Male

Very rare, only one male specimen was found in both Spanish populations. It is morphologically similar to female except for the genital system. Male genital tract is diorchic with testes with multiple rows of spermatogonia. Spicules are moderately long, curved ventrally, and lateral guiding pieces 21.0 μm long. Tail is short and hemispherical with a peg 3.5 μm long. One pair is of adanal supplements and seven of mid-ventral supplements.
Juveniles

Three juvenile stages (J2, J3, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics (Figs. 1, 2). The tails of juvenile stages become progressively wider after each moult. All of the stages are distinguishable by relative body lengths, functional, and replacement odontostyle (Robbins et al., 1996).

Figure 2:

Relationship of body length to length of functional and replacement odontostyle (▴=Odontostyle and •=Replacement odontostyle); length in three developmental stages and mature females of Xiphinema histriae.

Figure 3.

Light micrographs of Xiphinema histriae paratypes (Lamberti et al., 1993a, 1993b). Female: A, Lip region; B, C, Details of pseudo-Z-organ; D, Tail region. Abbreviations: a, anus; cb, crystalloid bodies; psZ, pseudo-Z-organ. (Scale bars: A = 15 μm; B, C = 10 μm; D = 20 μm.)

Locality and habitat

Spanish populations of Xiphinema histriae were collected in the rhizosphere of Portuguese oak (Quercus faginea Lam.) and black pine (Pinus nigra Arnold) at Arroyo Frío and Nava de San Pedro, Cazorla, Jaén Province, Spain.
Remarks

The two amphimictic populations of X. histriae agree fairly with studied paratypes (Fig. 3) and original description of X. histriae by Lamberti et al. (1993a, 1993b). According to the polytomous key (Loof and Luc, 1990), these populations belong to the X. non-americanum Group 5 and has the following specific α-numeric codes: A4, B23, C5a, D56, E6, F45, G3, H2, I3, J5, K?, L1, which fits with the original description of X. histriae, except in having bigger values of V (51.5–57.0 vs 44.0–44.5), shorter oral aperture-guiding ring length (116.0–139.0 μm vs 129.4–144.7 μm), and spicule length (74.0 μm vs 82.3–85.2 μm). No juvenile stages were described in the original description. This is the first time that J2 to J4 juvenile stages were detected and described, being similar to adults, except in body length, tail morphology, and sexual characteristics. Additionally, females of the Spanish populations of X. histriae, a pseudo-Z-organ with weakly muscularized wall, containing numerous small dense granular bodies was observed, which differ from the original description by Lamberti et al. (1993a, 1993b). This pseudo-Z-organ was also confirmed in detailed examination of paratypes (Fig. 3). Therefore, X. histriae should be placed in morphospecies Group 5. To our knowledge, this is the first report of this species in Spain.

Xiphinema lapidosum Roca and Bravo (1993).

(Figs. 4–6; Table 3).

Morphometrics of Xiphinema lapidosum (Roca and Bravo, 1993) from cultivated olive at Aroche (Huelva, Spain). All measurements are in µm and in the form: mean  ±  s.d. (range)a.

Roca and Bravo (1993) Quinta do Rogelo, Silves, Faro, Portugal/broad-beans and peas
Characters/ratios Females Males J2 J3 J4 Females Males
n 10 5 2 5 7 14 7
Lb 4,600 ± 271.5 (4,250–5,023) 4,618 ± 415.4 (4,091–4,977) (1,689–1,773) 2,292 ± 270.7 (2,068–2,704) 3,101 ± 178.9 (2,864–3,318) 4,300 ± 344 (3,700–4,600) 4,300 ± 310 (3,900–4,800)
a 71.5 ± 4.9 (64.9–75.5) 70.9 ± 6.7 (63.4–79.6) (57.3–59.1) 63.4 ± 3.5 (59.6–68.5) 67.0 ± 6.3 (59.3–77.6) 80.0 ± 5.3 (70.4–88.2) 85.0 ± 6.5 (75.5–95.5)
b 9.8 ± 0.6 (8.8–10.9) 10.9 ± 1.5 (9.1–12.3) 4.5 6.9 ± 0.9 (6.1–7.8) 7.4 ± 0.6 (6.4–8.1) 9.5 ± 0.8 (7.8–10.7) 9.0 ± 0.85 (7.5–10.0)
c 113.2 ± 8.2 (98.8–123.6) 118.4 ± 9 (109–129.8) (23.2–24.0) 40.5 ± 5.5 (35.3–49.2) 67.7 ± 4.3 (62.9–74.5) 110.0 ± 12.16 (92.0–132.0) 113.2 ± 9.73 (101.5–130.0)
c' 0.9 ± 0.07 (0.8–1.0) 0.9 ± 0.08 (0.8–1.0) (3.1–3.4) 2.2 ± 0.2 (2.0–2.5) 1.3 ± 0.1 (1.2–1.5) 1.04 ± 0.07 (0.92–1.13) 0.99 ± 0.07 (0.92–1.09)
V or T (%) 46.0 ± 2.8 (40.6–51.0) 50.3 ± 5.6 (45.2–56.3) – – – 43.4 ± 1.28 (41.0–46.0) –
Odontostyle 133.0 ± 2.5 (128.5–136.5) 133.3 ± 5.5 (127.0–139.0) (75.5–87.0) 94.6 ± 3.8 (90.5–100.0) 115.6 ± 2.8 (111.5–120.0) 134.5 ± 3.71 (124.5–139.5) 136.0 ± 3.88 (130.5–142.5)
Replacement odontostyle – – 100.0 116.1 ± 5.5 (109.5–124.0) 136.6 ± 4.7 (129.0–142.0) – –
Odontophore 82.4 ± 4.6 (72.0–88.5) 78.0 ± 3.9 (73.0–83.5) (53.0–55.5) 58.2 ± 3.5 (54.0–63.0) 70.9 ± 3.1 (66.5–76.0) 71.5 ± 1.39 (69.0–73.5) 71.5 ± 2.67 (67.5–74.5)
Lip region width 14.6 ± 0.5 (14.0–15.5) 14.7 ± 0.8 (13.5–15.5) 10.0 11.5 ± 0.6 (10.5–12.0) 12.6 ± 1.3 (10.5–14.0) 15.0 ± 0.60 (14.0–16.0) 15.0 ± 0.31 (14.5–15.5)
Oral aperture-guiding ring 122.5 ± 6.9 (111.5–135.0) 117.9 ± 3.0 (113.0–120.5) (65.0–73.0) 81.5 ± 4.2 (76.5–86.0) 98.2 ± 7.3 (84.0–106.0) 116.5 ± 7.03 (97.5–127.5) 121.5 ± 5.94 (115.5–130.5)
Tail length 40.8 ± 2.8 (36.5–44.0) 39.0 ± 2.0 (36.5–42.0) (70.5–76.5) 57.0 ± 3.5 (51.5–59.5) 46.5 ± 3.1 (43.0–52.0) 39.0 ± 2.04 (34.0–41.0) 38.5 ± 2.77 (35.4–43.0)
Hyaline tail 11.2 ± 1.5 (9.5–14.0) 9.3 ± 1.4 (7.5–10.5) 14.5 13.7 ± 3.4 (9.0–18.5) 13.3 ± 0.8 (12.0–14.0) 11.5 ± 1.8 (9.0–15.5) 11.0 ± 1.59 (8.5–13.0)
Spicules – 66.2 ± 3.1 (63.0–71.0) – – – – 64.5 ± 3.02 (60.0–68.0)
Lateral accessory piece – 18.0 ± 1.5 (16.5–19.5) – – – – 15.5 ± 1.55 (13.5–18.0)

Note: aMeasurements are in µm and in the form: mean ± standard deviation (range); ba = body length/maximum body width; b = body length/pharyngeal length; c = body length/tail length; c’ = tail length/body width at anus; V = (distance from anterior end to vulva/body length)×100; T = (distance from cloacal aperture to anterior end of testis/body length)×100; J = hyaline tail region length.

Figure 4:

Light micrographs of Xiphinema lapidosum (Roca and Bravo, 1993). Females: A, Pharynx; B to C, Lip regions; D, Gonad; E, F, Details of pseudo-Z-organ; G, H, Tail regions; Males: I, Tail region of male; Juveniles: J to L, Tail region of 2nd, 3rd and 4th stage juveniles. Abbreviations: a, anus; gr, guiding ring; psZ, pseudo-Z-organ; sb, sclerotized bodies; sp, spicule; spl, supplements; v, vulva. (Scale bars: A = 40 μm; B, C  = 15 μm; D = 65 μm; E–I = 20 μm; J–L = 10 μm.)

Figure 5:

Relationship of body length to length of functional and replacement odontostyle (▴= Odontostyle and •=  Replacement odontostyle); length in three developmental stages and mature females of Xiphinema lapidosum.

Figure 6:

Light micrographs of Xiphinema lapidosum paratypes (Roca and Bravo, 1993). Female: A, Pharynx; B, Lip region; C, D, Details of pseudo-Z-organ; E, F, Tail regions; Males: G, Tail region of male. Abbreviations: a, anus; gr, guiding ring; psZ, pseudo-Z-organ; sp, spicule; spl, supplements. (Scale bars: A = 40 μm; B = 15 μm; C-G = 20 μm.)
Description
Female

The female body of Xiphinema lapidosum is as follows: body is cylindrical, slightly tapering anteriorly and posteriorly and assuming a hook-shape upon fixation; cuticle appearing smooth, 5.0 (3.5–6.5) μm thick at the middle body; lip region is flatly rounded, separated by a weak depression; odontostyle is robust, and odontophore is with well-developed basal flanges (10.5–14 μm wide); guiding ring is double; pharynx is extending to a terminal pharyngeal bulb with three nuclei with one dorsal gland nucleus located at the beginning of pharyngeal bulb (DN = 8.5–10.5%), while two subventrolateral nuclei located at middle of bulb (SN12 = 56–60%); pharyngeal basal bulb 127 to 148 μm long and 24.5 to 35 μm diam; glandularium is 111.5 (106.5–115) μm long; female reproductive system is didelphic, with two complete genital branches equally developed, each 541 (465–580) μm long; the length of ovaries is variable, and a pars dilatata oviductus separated from the uterus by a conspicuous sphincter muscle, tripartite uterus consisting of a pars dilatata uteri followed by a tubular portion, a pseudo-Z-organ, a dilated part and an ovejector; pseudo-Z-organ well developed with a thick wall and longitudinal folding is easy to observe, comprising 15 to 20 sclerotized bodies of large size, but all of them of variable size; no spines or different structures are observed in the uterus; vulva is a transverse slit, vagina 33.0 (30.5–37.5) μm wide and perpendicular to body-axis, ovejector well developed, 50.5 (40.5–58.5) μm wide, extending inwards more than half of corresponding body diam; and tail short, convex dorsally and ending with bulge.
Male

Males are common but less frequent (50%) than female. They are morphologically similar to female except for the genital system; spicules are curved, lateral guiding pieces well sclerotized; tail is conoid with one pair of adanal supplements and five mid-ventral supplements (Table 3, Fig. 4).
Juveniles

Three juvenile stages (J2, J3, and J4) were found and they were basically similar to adults, except for their smaller size, shorter tails, and sexual characteristics (Table 3, Fig. 4). Tail becomes progressively wider and shorter after each moult.

Locality and habitat

The population was collected from the rhizosphere of cultivated olive (Olea europaea subsp. europaea L.) at Aroche, Huelva province, Spain.
Remarks

The amphimictic population of X. lapidosum from Aroche (Huelva province) corresponds fairly well with the original description (Roca and Bravo, 1993) and the studied paratypes from USDA (Fig. 6). Observations on the general morphology indicate that this Xiphinema population belongs to the X. non-americanum morphospecies Group 5 (Loof and Luc, 1990), and has the following specific α-numeric codes: A4, B2, C5b, D6, E456, F45, G3, H2, I3, J3, K?, L2. In addition, female and male morphometrics fit with those provided in the original description, except in having slightly longer values of body length (4,250–5,023 μm vs 3,700–4,600 μm), odontophore length (72.0-88.5 μm vs 69.0–73.5 μm), and slightly smaller values of c’ (0.83–1.04 vs 0.92–1.13). Since juveniles were not described in the original description, the J2 to J4 juvenile stages of Aroche population were described herein for the first time. To our knowledge, this is the first report of this species for Spain.
Phylogenetic relationships of Xiphinema histriae and Xiphinema lapidosum

Amplification of D2 to D3 expansion segments of 28S rRNA, ITS1 and the partial CoxI gene from X. histriae and X. lapidosum yielded a single fragment of ca 900, 1,100, and 500 bp, respectively. Six new D2 to D3 of 28S rRNA, three ITS1, and five partial CoxI gene sequences were obtained in the present study. Xiphinema histriae showed a high molecular similarity for D2 to D3 expansion segments of 28S rDNA, only one variable position was found between the sequences obtained from three female specimens (MK801302-MK801305). These sequences matched well with X. non-americanum group species deposited in GenBank and showed 97% similarity (differing from 21 to 24 nucleotides and from 1 to 4 indels) with X. hispidum (Roca and Bravo, 1994), X. hispanum (Lamberti et al., 1992), X. adenohystherum, and Xiphinema celtiense (Archidona-Yuste et al., 2016a, 2016b, 2016c). ITS1 region from X. histriae (MK801298-MK801299) also showed similarity with X. hispanum, X. adenohystherum, and X. hispidum displaying similarity values of 88, 86, and 84% (differing from 129 to 152 nucleotides and from 31 to 43 indels), respectively. In addition, the two new partial CoxI sequences of X. histriae (MK796911-MK796912) showed similarity values from 86% to 77% with all X. non-americanum group species in GenBank (differing from 49 to 80 nucleotides). Non intra-specific variation for this region was found among two studied individuals.

The closet species regarding D2 to D3 segments of X. lapidosum (MK801306-MK801307) were X. lupini (Roca and Pereira, 1993), 97% similar (differing from 22 to 24 nucleotides and from 3 to 4 indels), and X. turcicum, 88% similar (similarity of 83 nucleotides and 22 indels). Similarly, X. lupini was the most related species for the ITS1 rRNA region showing a similarity value of 87% with X. lapidosum (MK801300). Scarce similarity was found with the rest of Xiphinema spp. deposited in GenBank, showing coverage values below 30% with all of them. Finally, three new CoxI from X. lapidosum (MK796913-MK796915) were obtained in this study, being clearly different to the other accession from X. non-americanum group species deposited in GenBank and showing similarity values from 82 to 73% with all of them, being X. lupini the closet species (82% similar, 66 nucleotides and no indels) as in the D2 to D3 and ITS1 regions. No intra-specific variation was found between D2 and D3 and CoxI sequences from X. lapidosum obtained in this study (MK801306-MK801307, MK796913-MK796915).

Phylogenetic relationships among Xiphinema non-americanum group species inferred from analyses of D2 to D3 expansion segments of 28S, ITS1, and the partial CoxI gene sequences using BI are given in Figures 7 to 9, respectively. Poorly supported clusters were not explicitly labelled. The 50% majority rule consensus 28S rRNA gene BI tree of X. non-americanum group spp. based in a multiple edited alignment including 70 sequences and 771 total characters showed two clearly separated (PP = 1.00) major clades (Fig. 7). Clade I grouped species from all morphospecies groups, including the new accessions obtained in this study of X. histriae and X. lapidosum. Clade II was not well supported (PP = 0.84) and was mostly composed by species from the morphospecies Group 5, except for X. tica, X. bakeri, and X. index which belong to Groups 4, 7, and 8, respectively. Xiphinema histriae (MK801302-MK801305) occupies a superior position within this major clade I clustering with X. hispanum, X. celtiense, and X. cohni in a well-supported subclade (PP = 0.97). On the contrary, X. lapidosum (MK801306-MK801307) occupied a basal position and seemed to be related with X. lupini, X. turcicum, and X. oleae since all of them formed a well-supported subclade (PP = 0.99). The low similarity and small coverage between the ITS1 region from X. lapidosum and the rest of the ITS1 sequences available in GenBank made it impossible to perform a phylogenetic analysis for this region. For X. histriae, only ITS1-related sequences were used, the edited alignment generated for the 29 sequences of ITS1 was of 1,104 characters after discarding ambiguously aligned regions. This ITS phylogenetic tree (Fig. 8) showed two major clades (PP = 1.00), similar to those obtained for D2 to D3 region. Xiphinema histriae (MK801298-MK801299) appeared in the basal major clade but their phylogenetic position was not well resolved for this marker (Fig. 8). The CoxI region using a multiple alignment of 52 sequences and 390 characters after editing was used to obtain the 50% majority rule BI tree (Fig. 9). The position of X. histriae (MK796911-MK796912) was not well-defined, but clustering with X. hispanum, X. hispidum, X. cohni, and X. celtiense. By contrast, the relationship among X. lapidosum (MK796913-MK796915) and X. lupini was maintained.

Figure 7:

Phylogenetic relationships within the Xiphinema non-americanum group complex. Bayesian 50% majority rule consensus tree as inferred from D2 to D3 expansion segments of 28S rRNA sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G) (lnL = 11,543.7822; AIC = 23,383.5644; freq A = 0.2502; freq C = 0.2298; freq G = 0.2995; freq T = 0.2205; R(a) = 0.9908; R(b) = 2.7656; R(c) = 2.4778; R(d) = 0.4894; R(e) = 4.2554; R(f) = 1.0000). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

Figure 8:

Phylogenetic relationships within the Xiphinema non-americanum group complex. Bayesian 50% majority rule consensus tree as inferred from ITS1 rRNA gene sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G) (lnL = 6,024.1284; AIC = 12,068.2568; freq A = 0.2316; freq C = 0.2224; freq G = 0.3009; freq T = 0.2451; R(a) = 0.7019; R(b) = 4.6043; R(c) = 2.0272; R(d) = 0.6248; R(e) = 7.4428; R(f) = 1.0000). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.

Figure 9:

Phylogenetic relationships within the Xiphinema non-americanum group complex. Bayesian 50% majority rule consensus tree as inferred from partial cytochrome c oxidase subunit I (CoxI) gene sequence alignment under the general time-reversible model of sequence evolution with correction for invariable sites and a gamma-shaped distribution (GTR + I + G), (lnL = 8,561.5874; AIC = 17,347.1747; freq A = 0.3687; freq C = 0.1301; freq G = 0.1382; freq T = 0.3630; R(a) = 3.3231; R(b) = 22.8405; R(c) = 2.0144; R(d) = 10.5400; R(e) = 75.4228; R(f) = 1.0000). Posterior probabilities greater than 0.70 are given for appropriate clades. Newly obtained sequences in this study are shown in bold. Scale bar = expected changes per site.
Discussion

This study aimed to provide and to characterize morphometrically and molecularly two Xiphinema species belonging to Xiphinema non-americanum Group 5 from Spain, and to carry out an updated phylogenetic study of both species within the X. non-americanum group species. To date, this is the first record of the occurrence of X. histriae and X. lapidosum in Spain and the first time that describes the molecular characterization and the juvenile stages of both species.

Xiphinema histriae was originally described from Italy associated with grapevine (Lamberti et al., 1993a, 1993b, and later on, reported from the rhizosphere of wild growing grape (Vitis vinifera ssp. silvestris) in Austria (Tiefenbrunner and Tiefenbrunner, 2004). Based on the detailed study of paratypes and both Spanish populations described here, we detected that this species is characterized by having a pseudo-Z-organ with weakly muscularized wall with numerous small dense granular bodies against that initially described by Lamberti et al. (1993a, 1993b). Therefore, X. histriae must be transferred to morphospecies Group 5 (Loof and Luc, 1990). This study illustrates the importance of paratypes deposited in different official collections and reference nematology laboratories of nematodes, which are provided as a useful tool in the accurate identification and revision of nematodes species. On the other hand, X. lapidosum was, first, described from the rhizosphere of broad bean and pea in the south of Portugal (Roca and Bravo, 1993) and now it is reported from cultivated olive at Huelva, southwestern Spain. These data suggest that X. histriae may have a wider distribution than that described until now (including agricultural and natural ecosystems), and X. lapidosum may be an Iberian endemism, also associated with cultivated hosts.

The use of different ribosomal and mitochondrial markers in this study, D2 to D3, ITS1, and partial CoxI, provides a precise and unequivocal tool for the identification of X. histriae and X. lapidosum. Phylogenetic analyses based on D2 to D3, ITS1, and CoxI gene using BI resulted in a consistent position for X. histriae and X. lapidosum. Xiphinema histriae clustered with Xiphinema species from morphospecies Group 5, such as X. hispanum, X. cohni, X. celtiense, and X. hispidum, while X. lapidosum seems to be related with X. lupini because of both species clustered together in all the analyses carried out in this study. The present study on the phylogeny based on D2 to D3 segments supported a very weak correlation in the phylogenetic relationships among the different morphospecies groups within Xiphinema, a finding already reported by several authors namely, Gutiérrez-Gutiérrez et al., 2013; De Luca et al., 2014; Tzortzakakis et al., 2014, 2015; Archidona-Yuste et al., 2016a, 2016b, 2016c).

In summary, this study highlighted the diagnosis of Xiphinema non-americanum group species because a large number of species and the lack of good diagnostic characteristics among the X. non-americanum group (Loof and Luc, 1990; Loof et al., 1996). For this reason, we recommend the use of integrative taxonomy that are crucial for accurately identify species and better understanding of the present geographical distribution and host range of X. non-americanum group species. In this case, we provide new morphological and molecular data for the precise identification of these species, the first reports of these species in Spain, new hosts, and their phylogenetic position in the Xiphinema genus.
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