Cryptic diversity and species delimitation in the Xiphinema americanum-group complex (Nematoda: Longidoridae) as inferred from morphometrics and molecular markers Author Archidona-Yuste, Antonio Author Navas-Cortés, Juan A. Author Cantalapiedra-Navarrete, Carolina Author Palomares-Rius, Juan E. Author Castillo, Pablo text Zoological Journal of the Linnean Society 2016 2016-02-29 176 2 231 265 https://academic.oup.com/zoolinnean/article-lookup/doi/10.1111/zoj.12316 journal article 10.1111/zoj.12316 0024-4082 5357381 XIPHINEMA ASTAREGIENSE SP. NOV. ( FIGS 1 , 2 , 6 , TABLE 5 ) Holotype : Female extracted from soil samples collect- ed from rhizosphere of unidentified grasses (Graminaceae) in Jerez de la Frontera , Cadiz province , southern Spain , ( 36°46′31.36″N , 6°15′15.67″W ) by J. Martín Barbarroja and G. León Ropero , mounted in pure glycerine, and deposited in the nematode collection at IAS-CSIC (collection number J174-010). Paratypes : Female , male, and juvenile paratypes extracted from the rhizosphere of unidentified grasses (Graminaceae) in Jerez de la Frontera , Cadiz province , southern Spain , were deposited in the following nematode collections: IAS-CSIC (collection numbers J174-02, J174-03, J174-04); one female and one male at the Royal Belgian Institute of Natural Sciences , Brussels , Belgium ( RIT832 ); and one female at USDA Nematode Collection (T-6288p) . Figure 5. Relationship between body length and functional and replacement odontostyle (Ost and rOst, respectively) length in all developmental stages from first-stage juveniles (J1) to mature females of: A, Xiphinema vallense sp. nov. and B, Xiphinema astaregiense sp. nov. Description of juveniles: All four juvenile stages (first, second, third, and fourth stage) were found, and were similar to adults, except for their smaller size, longer tails, and absence of sexual characteristics. Tail becoming progressively shorter and stouter in each moult; different development stages distinguishable by relative lengths of body and functional and replacement odontostyle ( Fig. 5 , Tables 3 and 4 ). Diagnosis: Xiphinema vallense sp. nov. is a amphimictic species characterized by a medium to large body size (1830–2228 μm); lip region widely rounded, separat- ed from the rest of the body by a constriction; odontostyle and odontophore 79 and 48 μm long, respectively; V = 55–59%; female tail 22.5–34.0 μm long, dorsally convex-conoid, often with dorsoventral depression at hyaline region level, with accurate pointed tip; c ratio of 58.2–86.3; c′ ratio of 1.4–1.7; and spe- Etymology: The species epithet refers to the old Latin name of the type locality, Asta Regia (Jerez de la Frontera) , where the nematode was detected. Description of female: Body large-sized, habitus coiled in a more or less closed C-shaped to open spiral when killed by heat. Body tapering very gradually toward the posterior extremity and more abruptly in the anterior region. Cuticle finely striated transversally, 2.0– 2.5 μm thick along body but thicker at tail tip ( Table 5 , Fig. 6 ). Lip region anteriorly flattened, laterally rounded, separated from the rest of body by a depression, 8.5– 10.5 μm wide and 4.0–5.5 μm high. Amphidial fovea large, stirrup-shaped with slit-like aperture, occupying c . 77.0% of corresponding lip region width. Pharynx consisting of an anterior slender narrow part, 285– 364 μm long, extending to a terminal pharyngeal basal bulb well demarcated anteriorly, cylindrical, 93.7 ± 5.3 (85–101) μm long, 17.2 ± 2.5 (15.5–20.0) μm wide, occupying about one-quarter to one-third of the total pharyngeal length ( Fig. 1 ). Glandularium 76.5–85.0 μm long. DN in anterior part of the bulb, 18.5 ± 3.2 (16.2– 20.7) % of basal bulb length, and SVN located around mid-bulb, 48.8 ± 0.4 (48.5–49.1) % of basal bulb length (location of gland nuclei according to Loof & Coomans, 1972 ). Reproductive system amphidelphic, both branches *Measurements are in μm and in the form: mean ± SD (range).L, body length; a, 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; J1, first-stage juvenile; J2, second-stage juvenile; J3, third-stage juvenile; J4, fourth-stage juvenile; –, not obtained or not performed; G1, (length of anterior genital tract/body length) × 100; G2, (length of posterior genital tract/ body length) × 100. Table 3. Morphometrics of Xiphinema vallense sp. nov. from wild olive at San José del Valle (Cádiz, Spain)*

Paratypes
Characters/ratios	Holotype	Females	Male	J1	J2	J3	J4
N	1	21	1	2	5	6	7
L	1975	2019 ± 101	1861	620 ± 12.7	933 ± 62	1210 ± 60	1613 ± 111
(1830–2228)	(611–629)	(839–992)	(1117–1283)	(1517–1850)
a	73.1	68.9 ± 4.7	67.7	37.0 ± 1.5	43.7 ± 3.2	51.0 ± 3.4	60.5 ± 1.4
(61.6–79.1)	(35.9–38.1)	(38.1–46.1)	(47.7–55.9)	(57.8–61.7)
b	7.5	7.9 ± 0.9	6.6	4.2 ± 0.6	5.2 ± 0.8	5.1 ± 0.5	6.3 ± 0.8
(6.4–9.4)	(3.7–4.6)	(3.9–5.9)	(4.4–5.8)	(5.3–7.5)
c	70.5	73.4 ± 8.1	79.2	22.4 ± 1.9	34.2 ± 2.2	41.8 ± 2.2	55.4 ± 2.3
(58.2–86.3)	(21.1–23.7)	(31.7–37.4)	(38.5–44.4)	(53.2–60.2)
c′	1.6	1.6 ± 0.1	1.2	2.6 ± 0.01	2.1 ± 0.1	1.9 ± 0.1	1.8 ± 0.1
(1.4–1.7)	(2.6–2.7)	(2.0–2.1)	(1.8–2.0)	(1.6–1.9)
V or T	55.0	57.5 ± 1.2	–	–	–	–	–
(55.0–59.5)
G1	10.1	8.7 ± 1.2	–	–	–	–	–
(7.2–9.1)
G2	10.5	8.8 ± 1.0	–	–	–	–	–
(7.0–9.4)
Odontostyle length	83.0	79.0 ± 3.7	82.0	38.3 ± 1.8	45.7 ± 2.3	55.1 ± 2.9	68.1 ± 2.7
(73.0–85.5)	(37.0–39.5)	(42.0–48.0)	(51.0–59.0)	(63.0–71.5)
Replacement odontostyle	–	–	–	46.8 ± 0.4	52.9 ± 1.7	66.4 ± 2.4	78.6 ± 3.8
length	(46.5–47.0)	(51.0–55.5)	(61.0–67.5)	(72.5–84.0)
Odontophore length	45.5	47.5 ± 2.5	43.0	26.5 ± 4.9	29.6 ± 3.0	38.5 ± 1.9	42.4 ± 2.4
(42.0–53.5)	(23.0–30.0)	(25.0–32.5)	(36.5–41.0)	(39.0–45.0)
Lip region width	8.0	8.5 ± 0.4	9.0	6.3 ± 0.4	2.5 ± 0.4	7.4 ± 0.5	7.9 ± 0.4
(8.0–9.0)	(6.0–6.5)	(2.0–3.0)	(7.0–8.0)	(7.5–8.5)
Oral aperture-guiding	73.0	69.5 ± 3.7	64.0	32.5 ± 2.1	39.1 ± 2.4	48.3 ± 3.5	60.1 ± 2.0
ring distance	(62.0–75.5)	(31.0–34.0)	(35.0–41.0)	(44.0–53.0)	(56.0–62.5)
Tail length	28.0	27.8 ± 2.8	23.5	27.8 ± 1.8	27.3 ± 1.5	29.0 ± 1.5	29.1 ± 2.2
(22.5–34.0)	(26.5–29.0)	(26.5–30.0)	(27.5–31.0)	(26.5–33.5)
J	7.5	7.6 ± 0.6	8.0	5.0 ± 0.0	5.4 ± 0.9	6.5 ± 0.5	6.3 ± 0.3
(6.5–8.5)	(5.0–5.0)	(4.5–6.5)	(6.0–7.0)	(6.0–6.5)
Spicules	–	–	38.0	–	–	–	–
Lateral accessory piece	–	–	8.0	–	–	–	–

equally developed; ovaries reflexed without symbiontic bacteria; uteri often with spindle-shaped sperm cells 2.0–3.5 μm long, without any differentiation. Vulva slitlike, clearly posterior to mid-body; vagina 14.0 ± 1.5 (12.5–16.0) μm long perpendicular to body axis; ovejector well developed, 22.0–28.5 μm wide, or 58.3–69.1% of maximum body diameter in lateral view ( Fig. 6 ). Prerectum often indistinct. Rectum 19.6 ± 1.8 (18.0– 22.5) μm long, or 0.7–1.9 times the anal body diameter. Tail short, dorsally convex-conoid, with curvature essentially dorsal with conoid-rounded terminus, bearing two and three caudal pores ( Fig. 2 ). Tail hyaline region about one-third of the tail length. Description of male: Common (almost as frequent as female, c . 45%). Morphologically similar to female except for genital system, but with posterior part of the body more curved with greater curvature in posterior part of body ( Fig. 3 ). Testis well developed, containing numerous spindle-shaped sperms. Spicules well sclerotized, ventrally curved with bifid lateral guiding pieces 10.1 ± 0.5 (9.5–11.0) μm long ( Fig. 6 ). A preanal pair *Measurements are in μm and in the form: mean ± SD (range).L, body length; a, 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; G1, (length of anterior genital tract/body length) × 100; G2, (length of posterior genital tract/ body length) × 100. Table 4. Morphometrics of Xiphinema vallense sp. nov. from wild and cultivated olive at Bolonia (Cádiz province) and Hinojos (Huelva province), Spain, respectively*

Bolonia	Hinojos
Characters/ratios	Females	Females
N	4	4
L	1857 ± 60	1903 ± 136
(1795–1912)	(1713–2011)
a	57.6 ± 2.0	63.5 ± 5.2
(55.1–59.5)	(56.2–67.4)
b	7.2 ± 0.4	8.8 ± 1.0
(6.9–7.8)	(7.3–9.7)
c	67.6 ± 9.0	75.0 ± 4.7
(67.2–81.1)	(68.5–78.9)
c′	1.5 ± 0.1	1.4 ± 0.03
(1.5–1.6)	(1.4–1.5)
V or T	59.1 ± 1.5	58.1 ± 1.3
(57.0–60.5)	(57.0–59.5)
G1	8.7 ± 0.4	11.6 ± 2.0
(8.4–9.0)	(10.2–13.0)
G2	9.5 ± 0.8	11.9 ± 2.5
(8.9–10.0)	(10.2–13.7)
Odontostyle length	78.3 ± 2.9	75.1 ± 1.4
(75.5–81.0)	(73.0–76.0)
Odontophore length	48.3 ± 2.0	49.6 ± 2.8
(46.5–51.0)	(46.0–52.5)
Lip region width	8.1 ± 0.3	9.1 ± 0.3
(8.0–8.5)	(9.0–9.5)
Oral aperture-guiding	67.3 ± 3.2	63.1 ± 2.7
ring distance	(64.0–71.5)	(59.5–66.0)
Tail length	27.8 ± 3.0	25.4 ± 0.3
(23.5–30.5)	(25.0–25.5)
J	7.6 ± 0.3	7.5 ± 0.9
(7.5–8.0)	(6.5–8.5)

of supplements 9.0 to 11.0 μm anterior to cloacal opening and a row of six to seven single ventromedian supplements, located anterior to the spicule region ( Fig. 6 ). Description of juveniles: All juvenile stages, except for the first, were detected. They are generally similar to adults, except for their smaller size, longer tails, and absence of sexual characteristics. Tail becoming progressively shorter and stouter in each moult; different developmental stages distinguishable by relative lengths of body and functional and replacement odontostyle ( Fig. 5 , Table 5 ). Diagnosis: Xiphinema astaregiense sp. nov. is a bisexual species characterized by a large body size (2740– 3018 μm); lip region anteriorly flattened and laterally rounded, separated from the body by a depression; odontostyle and odontophore 85 and 54 μm long, respectively, the latter with well-developed flanges; V = 55–59%; length of female tail 22.5–25.0 μm, relatively short, convex-conoid with curvature essentially dorsal and conoid-rounded terminus; c ratio (112.2–129.9), c′ ratio (0.9–1.1); and specific D2-D3, ITS1 -rRNA, and coxI sequences deposited in GenBank with accession numbers KP268955 , KP268972 , and KP268977 , respectively . Morphologically and morphometrically, X. astaregiense sp. nov. can be distinguished from the most similar species by a number of particular characteristics from its specific alphanumeric codes (exceptions are in parentheses): A 3, B 3, C 1(2), D 3, E 3, F2, G 2, H 2, I 1 sensu Lamberti et al . (2004) . MORPHOLOGY AND MORPHOMETRICS OF SPECIES OF THE XIPHINEMA AMERICANUM -GROUP ( FIGS S1–S 4 , TABLES S1 AND S 2 ) The morphological and morphometric data as well as molecular delineation for X. duriense Lamberti et al ., 1993 , X. incertum Lamberti et al ., 1983 , X. opisthohysterum Siddiqi, 1961 , X. pachtaicum (Tulaganov, 1938) Kirjanova, 1951 , X. parapachydermum Gutiérrez-Gutiérrez et al ., 2012 , and X. rivesi Dalmasso, 1969 , were previously studied and compared with original descriptions and paratype specimens within previous studies on the identification and molecular phylogeny of the X. americanum -group in southern Spain ( Gutiérrez-Gutiérrez et al ., 2011b , 2012 ). The new records of these species from olive in Seville and Huelva provinces and also in Almeria province presented here extend the geographical distribution of these species in southern Spain ( Gutiérrez-Gutiérrez et al ., 2012 ). For these species only the D2-D3 sequences have been report- ed here for these samples. For other known species studied, representing the first molecular characterization and/or new records for olive or for Spain , a brief description and a morphometric comparison with previous records is provided below. Xiphinema brevisicum Lamberti et al., 1994 ( Fig. S1 , Table S1 ) The Spanish population of this species is characterized by a coiled body habitus forming an open C when killed by heat, lip region expanded and offset from the body by a constriction, female reproductive system amphidelphic with two equally developed genital branches, tail elongated-conoid, slightly curved ventrally, two caudal pores on each side. Male frequent, habitus more coiled than female. Tail elongated with pointed tip, ventrally curved with four to five *Measurements are in μm and in the form: mean ± SD (range).L, body length; a, 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; J2, second-stage juvenile; J3, third-stage juvenile; J4, fourth-stage juvenile; –, not obtained or not performed; G1, (length of anterior genital tract/body length) × 100; G2, (length of posterior genital tract/body length) × 100. Figure 6. Light micrographs of Xiphinema astaregiense sp. nov. A, B, entire female and male, respectively. C–F, female neck region. G, pharyngeal bulb. H, vulval region. I–K, female tail regions from different specimens showing the morphological variability. L, M, male tail region, ventromedian supplements arrowed. Abbreviations: a, anus; gr, guiding ring; V, vulva. Scale bars: A, B = 200 μm; C–M = 20 μm. Table 5. Morphometrics of Xiphinema astaregiense sp. nov. from grasses at Jerez de la Frontera (Cádiz, Spain)*

Paratypes
Characters/ratios	Holotype	Females	Males	J2	J3	J4
N	1	6	7	4	2	4
L	2740	2981 ± 108	2649 ± 95	1102 ± 15	1681 ± 161	2075 ± 161
(2740–3018)	(2577–2840)	(1083–1111)	(1567–1795)	(1878–2261)
a	76.1	72.4 ± 3.7	75.5 ± 4.1	49.5 ± 6.5	68.0 ± 7.5	69.8 ± 4.4
(66.8–76.1)	(69.6–83.5)	(43.3–55.6)	(62.7–73.3)	(64.5–75.1)
b	7.8	9.1 ± 1.0	9.0 ± 1.7	6.4 ± 0.4	7.2 ± 1.3	7.6 ± 0.8
(7.8–10.1)	(7.4–11.8)	(5.8–6.7)	(6.2–8.1)	(6.5–8.5)
c	114.2	123.2 ± 7.8	107.5 ± 6.3	38.8 ± 3.4	66.0 ± 8.2	80.1 ± 8.2
(112.2–129.9)	(100.2–117.4)	(35.1–41.1)	(60.3–71.8)	(74.7–92.3)
c′	1.0	1.0 ± 0.1	1,0 ± 0.1	2.0 ± 0.1	1.5 ± 0.01	1.4 ± 0.1
(0.9–1.1)	(1.0–1.2)	(1.9–2.2)	(1.5–1.6)	(1.3–1.4)
V or T	55.5	56.9 ± 1.4	–	–	–	–
(55.5–59.0)
G1	11.3	11.5 ± 1.5	–	–	–	–
(10.6–12.2)
G2	10.8	11.4 ± 0.4	–	–	–	–
(10.8–11.9)
Odontostyle length	86.0	85.3 ± 2.3	87.6 ± 2.7	50.6 ± 2.8	62.8 ± 1.1	74.1 ± 1.9
(82.0–89.0)	(84.0–91.0)	(49.0–54.5)	(62.0–63.5)	(71.5–75.5)
Replacement odontostyle	–	–	–	60.5 ± 4.0	75.0 ± 0.7	89.6 ± 3.5
length	(57.5–65.5)	(74.5–75.5)	(85.0–93.0)
Odontophore length	55.5	54.1 ± 1.4	51.9 ± 1.9	30.5 ± 2.1	38.5 ± 7.8	45.0 ± 2.2
(52.0–55.5)	(49.0–54.5)	(29.0–33.0)	(33.0–44.0)	(43.0–48.0)
Lip region width	9.0	9.3 ± 0.7	9.1 ± 0.2	7.3 ± 0.3	7.8 ± 0.4	8.0 ± 0.4
(8.5–10.5)	(9.0–9.5)	(7.0–7.5)	(7.5–8.0)	(7.5–8.5)
Oral aperture-guiding	76.5	73.2 ± 3.7	75.1 ± 1.3	42.3 ± 2.1	53.3 ± 1.1	64.0 ± 2.2
ring distance	(70.0–79.0)	(73.0–77.0)	(40.0–44.0)	(52.5–54.0)	(62.0–67.0)
Tail length	24.0	23.6 ± 0.9	24.7 ± 1.5	28.6 ± 2.2	24.8 ± 0.4	26.0 ± 1.9
(22.5–25.0)	(22.0–26.0)	(27.0–31.0)	(24.5–25.0)	(24.5–28.5)
J	8.0	8.2 ± 0.5	7.4 ± 0.5	7.6 ± 0.3	7.3 ± 1.8	7.8 ± 0.6
(7.5–9.0)	(7.0–8.0)	(7.5–8.0)	(6.0–8.5)	(7.0–8.5)
Spicules	–	–	47.1 ± 2.0	–	–	–
(43.0–49.0)
Lateral accessory piece	–	–	10.1 ± 0.5	–	–	–
(9.5–11.0)

ventromedian supplements preceding the adanal pair. The morphology and morphometrics of the Spanish population agree closely with those of the original description from grapevine and natural vegetation in Portugal by Lamberti et al . (1994) ( Table S1 ), except for lower a and c ratios in females (average 79.5, 47.8 vs. average 88.5, 56.8, respectively). Nevertheless, these differences further expand the intraspecific variation but do not exceed that reported by Lamberti et al . (1994). This species was reported from north-western Spain by Abelleira, Picoaga & Mansilla (2008) but no morphometric or molecular characterization was provided. These data indicate that this species may be an Iberian endemic species associated with cultivat- ed and wild plants as suggested by Peña-Santiago et al . (2006) . The alphanumeric codes for X. brevisicum to be applied to the polytomic identification key for the X. americanum- group species by Lamberti et al . (2004) are (exceptions are in parentheses): A 1, B 2, C 4, D 1, E 3(2), F 2, G 2, H 1, I 3. Xiphinema luci Lamberti & Bleve-Zacheo, 1979 ( Fig. S3 , Table S1 ) The Spanish population of this species is characterized by a body ventrally curved in an open C when killed by heat, lip region flat-rounded, and separated from the body by a depression ( Fig. S3 ). Female reproductive system amphidelphic with two equally developed genital branches and absence of uterine differentiation, ovary contains symbiontic bacteria, and vulva a transverse slit located slightly posterior to midbody. Tail short, broadly convex-conoid with bluntly rounded terminus and bearing three pairs of caudal pores ( Fig. S3 ). Males not found. The morphology and morphometrics of this population closely agree with the original description from celery in Diourbel , Senegal ( Lamberti & Bleve-Zacheo, 1979 ), and a population from common screw pine ( Pandanus utilis Bory. ) in the Botanical Garden of Dakar , Senegal ( Faye, Barsi & Decraemer, 2012 ), except for a lower c ratio [60.9– 68.6 vs. 54.0–81.0 ( Lamberti & Bleve-Zacheo, 1979 ), 63.0–87.0 ( Faye et al ., 2012 )]. This difference should be regarded as geographical intraspecific variation. The species has been also reported in Florida but no morphometrics were provided ( Robbins, 1993 ). The alphanumeric codes for X. luci to be applied to the polytomic identification key for the X. americanumgroup species by Lamberti et al . (2004) are (exceptions are in parentheses): A 3(4), B 2, C 2, D 2, E 2(3), F 1, G 1, H 2, I 2. Xiphinema madeirense Brown et al., 1992 ( Fig. S4 , Table S1 ) The Spanish population of this species is characterized by a relatively long body ( c . 2 mm ), forming an open coiled spiral when killed by heat, lip region expanded and clearly offset from the body by a depression, and a long odontostyle (92.5–100.5 μm long). Female reproductive system with two equally developed genital branches and uterine differentiation absent, vulva slit-like, posterior to mid-body, and vagina occupying about half of the body width. Tail conoidelongate, curved ventrally, with almost pointed terminus, and bearing two pairs of caudal pores. Male not found. The morphology and morphometrics of this population agree with the original description ( Brown et al ., 1992 ) and other populations from Portugal (Lamberti et al ., 1993, 1994) ( Fig. S4 , Table S1 ). This work represents the first report of this nematode species in Spain , although it has been described previously from the rhizosphere of bay laurel ( Laurus nobilis L.) in Queimadas, Santana, on the island of Madeira , where it seems to occur in natural habitats ( Brown et al ., 1992 ). It is also quite common and widespread in grapevines, fallow soil, and the rhizosphere of peach or hop in northern and central Portugal (Lamberti et al ., 1994). The alphanumeric codes for X. madeirense to be applied to the polytomic identification key for the X. americanum- group species by Lamberti et al . (2004) are (exceptions are in parentheses): A4, B 3, C 3(4), D 2, E 3, F 2, G 2, H 1, I 3. MOLECULAR CHARACTERIZATION OF XIPHINEMA PLESIOPACHTAICUM SP. NOV. , XIPHINEMA VALLENSE