Integrative taxonomy unravels cryptic diversity in the Paratrichodorus hispanus-group complex and resolves two new species of the genus and the molecular phylogeny of the family (Nematoda: Trichodoridae) Author Decraemer, Wilfrida Author Cantalapiedra-Navarrete, Carolina Author Archidona-Yuste, Antonio Author Varela-Benavides, Ingrid Author Gutiérrez-Gutiérrez, Carlos Author Castillo, Pablo Author Palomares-Rius, Juan E. text Zoological Journal of the Linnean Society 2019 185 656 692 journal article 0024-4082 8B9F19A-F2F5-4044-86C1-11A9B78E4503 PARATRICHODORUS ALMADENENSIS SP . NOV . ( FIGS 7 , 8 ; TABLES 2 , 5 ) urn:lsid:zoobank.org:act: 45356A52-CFF1-4342-8EA2- D4F958C6EDAB Holotype : Female was extracted from soil samples collected from the rhizosphere of wild olive at Almadén de la Plata , Sevilla province , southern Spain ( 37°46′55.7″N , 6°08′05.8″W ) by J. Martín Barbarroja and G. León Ropero , mounted in pure glycerine and deposited in the Nematode Collection of the Ghent University , Ghent , Belgium (slide number UGMD 104342 ). Paratypes : Male and female paratypes extracted from soil samples collected from the rhizosphere of wild olive at Almadén de la Plata , Sevilla province , southern Spain , were deposited in the following nematode collections: IAS-CSIC (slide numbers AR110-1–AR110-4); and AR110- 5 male paratypes at the USDA Nematode Collection, Beltsville, MD , USA (collection number T-7048p) . Figure 5. Line drawings of Paratrichodorus hispanus , other localities. A–C, F, males from H18. A, neck region. B, C, posterior body region with copulatory apparatus, with C in ventral view. F, anterior testis. D, E, N, females from H18. D, anterior genital branch and vagina with copulatory plug. E, N, vaginal region. G, H–J, males from H3. G, neck region. H–J, posterior body region and copulatory apparatus. J, oblique ventral view. K–M, female from H3. K, neck region. L, posterior genital branch and vagina. M, vaginal region. Scale bar: 10 µm. Figure 6. Light micrographs of Paratrichodorus hispanus , other localities. A–C, L–P, males from AR100. A, B, neck region. C, L–P, posterior body region showing copulatory apparatus or bursa. D–K, females from AR100. D–J, vagina region. Female from H18. K, vagina with copulatory plug. Scale bars: 10 µm. Etymology: The species epithet refers to Almadén de la Plata, the type locality where the type specimens were collected. Description of male: Body appearance straight or slightly curved upon fixation, medium-sized body (average 813 µm); largely as in female. Onchiostyle medium-sized (average 47 µm) with onchium about half as long; pharynx with five gland nuclei, with the dorsal nucleus located at anterior bulb region, the two posterior ventrosublateral gland nuclei in the posterior third of the bulb, and the bulb offset or with developed dorsal intestinal overlap (34 µm; 36 µm in two males ); pharynx often retracted. One ventromedian cervical Figure 7. Line drawings of Paratrichodorus almadenensis sp. nov. A–H, male paratypes. A, neck region. B, C, posterior body region. D, anterior testis. E–H, female, holotype. E, neck region. F, vaginal region. G, tail region. H, female reproductive system. Scale bars: 20 µm in A–G; 10 µm in H. Figure 8. Light micrographs of Paratrichodorus almadenensis sp. nov. A–G, females. A, neck region, paratype. B, vaginal region, paratype. C, vaginal region, paratype. D, vaginal region, holotype. E, vaginal region with copulatory plug. F, copulatory plug at level of uterus (arrowhead). G, tail region, paratype. H–L, males, paratypes. H, neck region. J–L, posterior body region (L, bursa). Scale bars: 10 µm. Table 5. Morphometrics of Paratrichodorus almadenensis sp. nov. from wild olive at Almadén de la Plata (Sevilla, Spain )

Holotype	Paratype	Paratype
|female (AR110)	Females (AR110)	Males (AR110)
N	1	3	11
L	880	740–950	735–910
811.3 ± 98.1	813 ± 57.8
Onchium	22.5	20.0–25.5	21.0–27.5
22.75 ± 2.8	22.3 ± 2.9
Onchiostyle	48.0	44.5–54	42.0–52.0
49.3 ± 4.8	46.7 ± 3.4
Pharyngostom	63.0	59.0–67.0	47.5–72.0
63.0 ± 4.0	57.7 ± 7.0
Pharynx (along)	208.0	179–187	125–175
183 ± 4.0	153.1 ± 23.0
Anterior to guide ring	21.5	20.5, 21.5	15.0–29.0
( N = 2)	21.5 ± 3.3
Anterior to nerve ring	68.0	64.0	59.0–71.0
( N = 1)	65.3 ± 4.9
Anterior to CP1/anterior to S-E pore in female	131.0	67.5, 123.0	86.0–110.0
( N = 2)	96.0 ± 7.0
Distance CP1–S-E pore	–	–	3.0–7.0
4.7 ± 1.2
Anterior to LP	–	–	54.0–101.0
100.4 ± 7.7
mbd at cardia	49.0	37.0–42.0	29.0–45.0
38.7 ± 2.4	36.5 ± 5.62
mbd mid-body	52.0	41.0–44.5	33.0–53.0
42.8 ± 1.4	43.1 ± 5.7
abd/♀: length of anterior ovary	200.0	68.0, 96.0	17.0–25.0
( N = 2)	20.58 ± 2.3
Spicule length/ ♀: length of anterior branch	454	182, 325	42.0–52.0
( N = 2)	48.0 ± 2.9
Gubernaculum/ ♀: length of posterior ovary	200	65, 90	10–14
( N = 2)	11.7 ± 1.1
Distance SP1–cloacal opening/♀ length of posterior branch	471	248, 304	7.5–11.0
( N = 2)	8.88 ± 1.21
Distance SP1–SP2/♀ length of vagina	6.5	10.5, 11.5	11–19
( N = 2)	15.5 ± 2.6
Distance SP2–SP3/♀ vagina as a percentage of cbw	14.0	28.4	40–92
( N = 1)	64.4 ± 17.0
Tail/♀ G1	51.5	24.5, 34.2	6.5–14.5
( N = 2)	11.2 ± 2.6
a	17.0	17.3–21.4	16.7–22.9
18.9 ± 1.8	19.1 ± 1.9
b	4.2	4.0, 4.2	4.4–6.3
( N = 2)	5.5 ± 0.6
c/♀V	53.4	52.3–57.6	85.3–117.3
54.8 ± 2.2	93.7 ± 10.8
T/♀G2	53.4	32.0, 33.4	60.5–98.8
( N = 2)	72.8 ± 12.8
c′	–	–	0.4–0.7
0.5 ± 0.1
S-E pore as a percentage of L from anterior end	62.7	9.1–16.6	11.0–14.6
12.8 ± 3.7	11.3 ± 1.0
S-E pore as a percentage of pharynx from anterior end	23.6	37.7–65. 8	49.0–86.1
51.7 ± 14.0	67.8 ± 11.4

Measurements are in micrometres and in the form: range and mean ± SD. Abbreviations: a, body length/maximal body width; abw, anal body width; b, body length/pharyngeal length; c, body length/tail length; c′, tail length/ body width at anus; cbw, cloacal body width; CP, ventromedian cervical papilla; CP1, anterior ventromedian cervical papilla; G1 and G2, (anterior and posterior gonad length, respectively/body length) × 100; L, total body length; LP, labial papilla; mbw, maximal body width; N , number of specimens studied; S-E pore, excretory pore; SP1, SP2 and SP3, posterior, second and third precloacal supplements, respectively; T (distance from cloacal aperture to anterior end of testis/body length) × 100; V (distance from anterior end to vulva/body length) × 100. papilla (CP) shortly (4.5 µm average) anterior to S-E pore located opposite mid-pharyngeal bulb. Lateral cervical pores (LP) varying in position from clearly anterior to CP to shortly posterior to S-E pore. Males monorchic, with testes on average 247 µm long and sausage-shaped sperm nucleus 8.5 µm × 2.5 µm in size. Medium-sized spicules (average 48 µm) with an irregular undulated calomus (average 1.5 µm wide), and blade maximal width 2.5 µm (rarely 3 µm). Three precloacal supplements (SP); one exception with four SP with SP3 immediately anterior to retracted spicule, and SP1 and SP2 spread along distal half of spicules when retracted. A pair of subterminal postcloacal supplements adjacent to a pair of caudal pores. Tail half the anal body width in length. Description of female: General appearance as in male apart from secondary sexual features. Body appearance straight or slightly curved upon fixation; medium-sized body (average 810 µm). Body cuticle swollen (3.0–3.5 µm). Amphid with wide transverse aperture located immediately posterior to the outer crown of anterior sensilla; fovea stirrup-shaped, and amphidial canal often clearly visible. Onchiostyle medium-sized (average 49µ m) with onchium about half as long; stoma narrow; pharynx rather long (63 µm average), with gland nuclei (dorsal and posterior ventrosublateral pair) clearly separated and bulb offset or with clear dorsal intestinal overlap (36 µm, only in holotype ). Ventromedian cervical papilla; lateral cervical pores similar to those described in male specimens. Nerve ring is adjacent to the base of the pharyngostom. Reproductive system didelphic–amphidelphic, about equally developed reflexed ovaries, finely granular oviduct cells at tip ovary; sperm stored in spermatheca adjacent to oviduct; uterus without marked ovejector; vagina -30% of corresponding body width in length, trapezoid or more or less indented mid-way; vaginal sclerotized pieces (pars refringens vaginae) in optical section small (1.5 µm), rounded triangular to oval, obliquely oriented with tips very close (0.75 µm); vulva pore-like in ventral view. Well-developed sclerotized plug observed in vagina and in the uterus ( Fig. 8 ). No lateral body pores observed. Tail minute; anus subterminal, and a pair of caudal pores present. Diagnosis and relationships: Paratrichodorus almadenensis sp. nov. is characterized by a medium-sized body (~800 µm), onchiostyle (average 46.5 µm in male, 49.0 µm in female) and spicules (average 48.0 µm), narrow undulated calomus and rather slender blade (maximal width 2.5 µm); both posterior-most supplements (SP1 and SP2) spread along posterior half of spicule; in female, by size and shape of vaginal sclerotized pieces, small (1.5 µm), rounded triangular to oval and close distal tips, vagina variable but mostly trapezoid and absence of lateral body pores except for one female with one pore anterior to vulva. Furthermore, the pharyngeal bulb can be offset in a few specimens. The new species most closely resembles P. hispanus ( types ) but has a slightly shorter onchiostyle and spicule length in the male and no lateral body pores in the female; also, the size and position of the vaginal sclerotized pieces appear a bit different, i.e. being slightly smaller and closer together in P. almadenensis sp. nov. A comparison of all species within the P. hispanus group is presented in Table 2 , separately for males and females; and specific D2–D3 expansion domains of the 28S rRNA gene, ITS-rRNA, 18S-rRNA gene and coxI sequences are deposited in GenBank with accession numbers MG739529 – MG739567 , MG739659 – MG739670 , MG739674 – MG739683 and MG726826 – MG726829 , respectively. Morphologically and morphometrically, P. almadenensis sp. nov. can be distinguished from the most similar species by a number of particular characteristics resulting from its specific alphanumeric codes (in parentheses are exceptions) adapted from Decraemer & Baujard (1998) with, in males, code L4 split up in L4 (constriction) and L5 (blade with narrower part mid-way), L6 (mid-way septum present), L7 (distal tip divided by septum) and L8 (anterior part or calomus with irregular outline): (1) for females = A222 (average, minimum, maximum), B22, C1, D1, E300, F33 (1), G1, H88 (6), I11, J11, K200, L1, M1, N1, O11, P11, Q4, R22, S1, T1; and (2) for males = A222 (average, minimum, maximum), B22, C22, D1, E0, F3 (4), G22, H33, I33, J120, K33, L88, M270, N11, O100, P1.