taxonID	type	description	language	source
340A87E4FFB4FF8871F4FD3BC24BFD96.taxon	diagnosis	Amended diagnosis: Cuticle usually clearly swollen when fixed or heat-killed. Female reproductive system didelphic – amphidelphic. Lateral body pores present in ~ 50 % of the species but rarely located post-advulvar; body pores exceptionally ventral or sub-ventral in position. Vagina short (about one-third of corresponding body diameter), constrictor muscles inconspicuous, vaginal sclerotization small to inconspicuous in lateral view. Males rare or unknown in ~ 25 % of the species. Ventromedian cervical papillae in male absent or, if present, usually one papilla near the secretory – excretory (S-E) pore, exceptionally with two. Lateral cervical pores absent or, if present, usually one pair near onchiostyle base or S-E pore. Male tail region straight; caudal alae present, obscure to distinct. Sperm cells variable, large size with large sausage-shaped nucleus, medium-sized with rounded nucleus, small with small oval to rounded nucleus or thread-like (degenerated) with nucleus obscure, rarely sperm exceptionally long, fusiform with elongated nucleus. Oblique copulatory muscles poorly developed and restricted to spicule region; capsule of spicule suspensor muscles inconspicuous. Spicules usually straight and blade about equally wide and often finely striated; no bristles nor velum. One to three ventromedian precloacal supplements, exceptionally four; usually two supplements within spicule region, rarely with a single supplement. Usually one pair, rarely two, subventral postcloacal papillae. A pair of caudal pores present, rarely absent. Type species: Paratrichodorus tunisiensis (Siddiqi, 1963) Siddiqi, 1974. Remarks: Species determination of the P. hispanus group was based upon the integrative approach, applying morphological and molecular methods in order to unravel potential cryptic species diversity. The Spanish samples revealed two main species groups among the Paratrichodorus species found: (1) species possessing large sperm cells with a sausage-shaped or rounded nucleus; and (2) species with small or non-functional sperm cells. Within the first species group, all species have males with undulating spicular calomus; the group is referred to as the P. hispanus species group. The second group of species, or the P. allius species group, will be dealt in detail in a separate paper.	en	Decraemer, Wilfrida, Cantalapiedra-Navarrete, Carolina, Archidona-Yuste, Antonio, Varela-Benavides, Ingrid, Gutiérrez-Gutiérrez, Carlos, Castillo, Pablo, Palomares-Rius, Juan E. (2019): 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). Zoological Journal of the Linnean Society 185: 656-692
340A87E4FFB4FF827217FD6BC49EF8DC.taxon	description	Paratypes and topotypes: Two slides, each with a male and a female paratype, have been kindly put at our disposal by Dr Alfonso Navas, from the Nematode Collection of the Museo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Científicas, Madrid, Spain. Topotype material was collected by one of the authors (A. A. - Y.), containing four slides with in total 14 males, 18 females and five juveniles, several not suitable for measuring (bad fixation or in oblique or dorsoventral position). Topotype specimens were extracted from soil samples collected from rhizosphere of wheat at Santa Olalla, Toledo province, central Spain (40 ′. 05 ′ 83.93 ″ N, 4 ′ 42 ′ 57.44 ″ W), mounted in pure glycerine and deposited in the Nematode Collection of the Ghent University, Ghent, Belgium (slide numbers HIGUE- 01 – HIGUE- 12). Description of male: One paratype male and one paratype female in lateral position, one paratype male in ventral view and one paratype female in oblique dorsal view. The body cuticle was partly swollen, but the specimens were still in rather good condition. They agreed well with the original description. However, owing to the swelling of the body cuticle, no lateral body pores could be observed in the female. Additional measurements are provided of the onchium, pharyngostom, the pharynx measured along the lumen, the length of the ovaries and genital branches, the G 1 (tail / length of the anterior ovary) and G 2 (tail / length of the posterior ovary) ratios and the length of vaginal sclerotized pieces in optical section (2.3 µm). In the paratype male, spicules with narrower undulated calomus 1.5 µm wide, maximum width of the blade was 3.0 µm. In addition to the original description, photomicrographs and drawings are provided of the paratype specimens investigated (Figs 2, 3). Description of female: Female largely as in male; S-E pore at level of anterior pharyngeal bulb. Vagina short, rhomboid [average 12.0 µm long, i. e. ~ 30 % of cloacal body width (cbw)], usually more or less indented at mid-level owing to contraction of well-developed vaginal constrictor muscles. Vaginal sclerotized pieces, oblique, small drop-like to rounded triangular (average 1.7 µm), with distal tips very close (average 1.5 µm). One postvulval lateral body pore observed 36 – 88 µm (54.0 µm on average) from vulva, and in one specimen with uteri filled with sperm, a secretion plug was observed within the vagina. Vulva can be sunk in some specimens, and in ventral view the vulva appears as a transverse slit at the body surface, but a clear pore at the level of the sclerotized ring; slightly more inwards the lumen appears star shaped in transverse optical section (Fig. 4). Remarks: Male and female topotype specimens are clearly shorter than the paratype material, with body size outside the lower range provided for the type material. This also results in lower values for most other features. The length of the onchiostyle is also shorter, although with overlap at the lower range of the type specimens, similar for the spicule length. All specimens show a dorsal overlap of the pharynx by the intestine, and the nerve ring is adjacent to the base of the pharyngostom. In males, the single ventromedian cervical papilla (CP 1) is non-protruding and located shortly (average 6.0 µ m) anterior to the S-E pore, located at the level of the posterior isthmus region. Lateral cervical pores (LP) are present at the level of CP 1 or immediately anterior to it. Germinal zone of testis varies from short to medium-sized and sperm nucleus is large sausage-shaped, average 7.0 µm × 2.0 µm in size. Spicules were similar to the paratype, with a narrow undulated calomus 1.0 – 2.0 µm wide and maximal width of blade 2.5 – 3.0 µm, showing some slight variation in width and undulation attributable to fixation. Bursa short, rounded, extending from level SP 2 to immeditately anterior to the pair of postcloacal supplements (PSP); dorsal anal flap bifid (Fig. 3). Tail similar to paratype, about half as long as anal body width; body cuticle dorsally thickened until the terminal end, which may appear slightly knob-like, swollen, 4.0 µm thick (average); ventrally, tail cuticle thin (2.0 – 2.5 µm). Postcloacal supplements subterminal, with ventral cuticle between PSP and cloacal opening much thinner; pair of caudal pores slightly dorsal to PSP. Despite the smaller measurements, largely attributable to the impact of fixation, the topotypes fit the type description. For the first time, well-structured secretion plugs have been observed in several females in a species of Paratrichodorus. Sturhan (1985) illustrated and described the presence of a secretory plug in the vagina, albeit without a clear structure. The plug shape is different from the plugs observed in Trichodorus species, but most closely resembles the bifid plug in Trichodorus elefjohnsoni Bernard, 1992, albeit much smaller (Decraemer, 2012). Specific alphanumeric codes (in parentheses are exceptions) of the polytomous key adapted from Decraemer & Baujard (1998): (1) for males = presented with topotypes between [] when different from type specimens, A 213 [A 112] (average, minimum, Measurements are in micrometres and in the form: range (mean). (–) Not obtained or not performed. Abbreviations: a, body length in relation to the maximum body width; L, total body length; S-E, secretory-excretory; V %, percentage of the of the vulva position from the anterior end in relation to the main body length. Measurements are in micrometres and in the form: range and mean ± SD; values of re-measured paratype males and females are given in parentheses. 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; CP 1, anterior ventromedian cervical papilla; G 1 and G 2, (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; SP 1, SP 2 and SP 3, 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. maximum), B 23 [B 22], C 23 [C 22], D 11, E 0, F 3, G 22, H 33 (2), I 33, J 200, K 33, L 88, M 270, N 11, O 100, P 1; and (2) for females presented with topotypes between [] when different from type specimens, A 213 [A 212], B 23 [B 22], C 1, D 1, E 30, F 30, G 1, H 88 [H 86 (3)], I 11, J 11, K 30 [K 23], L 11 [L 12], M 1, N 1, O 11, P 11, Q 1, R 22, S 1, T 1. Other populations (Figs 5, 6; Tables 2, 4) Several populations of this species were collected from the rhizosphere of cultivated olive at Hinojos (H 0003), Huelva province, southern Spain, the rhizosphere of wild olive (AR 099, AR 100), stone pine and cork oak (H 0027) at El Rocio, Huelva (Spain), grapevine (388 GR) at Bollullos par del Condado (Huelva, Spain), cork oak (HATR) at Villamanrique de la Condesa (Sevilla, Spain), eucalyptus (H 0169) at Almonte (Huelva, Spain), stone pine (H 0179) and eucalyptus (H 0169) at Lucena del Puerto (Huelva, Spain), and wild olive (AR 139) at Aroche (Huelva, Spain) and sample AR 0140 at Rosal de la Frontera (Huelva, Spain). Description of male: Body appearance straight, medium-sized body (average 745 µm, in type specimens). Onchiostyle medium-sized (average 53 µm, in type specimens) with onchium about half as long; pharynx with gland nuclei (dorsal and posterior ventrosublateral pair) clearly separated and with developed dorsal intestinal overlap (~ 17 µm average). One ventromedian cervical papilla shortly (6.5 µm average) anterior to S-E pore opposite mid-pharyngeal bulb. Lateral body pore at level of S-E pore in holotype, but in most paratypes shortly anterior to CP. Males monorchic with testes on average 133 µm long and sausage-shaped sperm nucleus 6.0 µm × 2.0 µm in size. Medium-sized spicules (average 48.5 µm, in type specimens) with an irregular undulated (kinking) calomus (1.5 – 2.0 µm wide), and blade maximum width 2.5 µm. Three precloacal supplements (one exception with two, i. e. no SP 2), of which two are situated within the retracted spicules, with SP 2 at mid-spicule level; a pair of subterminal postcloacal supplements adjacent to a pair of caudal pores. Tail half the anal body width in length. Description of female: Females largely similar to males. Sperm stored in a spermatheca near the oviduct; vagina (~ 27 % on average of corresponding body width), with variable degree of contraction of vaginal constrictor muscles, but usually vagina clearly constricted mid-way. Vaginal sclerotized pieces small (1.5 µm on average), rounded triangular to rarely round, obliquely oriented with tips close to very close; no lateral body pores observed. Secretory plug regularly present in vagina and similar in shape to that in P. hispanus topotype females and observed in both other populations; rarely, plug in uterus. Remarks: Apart from the absence of sublateral body pores in females of these populations of P. hispanus (code Q), no other morphological difference was observed from P. hispanus, especially when topotype specimens (see code) were examined. This could be largely attribugtable to the great impact of fixation, resulting in a wide range in certain morphometrics. However, the H 18 population, with its smaller onchiostyle (44 µm on average vs. 53 µm in H 0003 and 56 µm in AR 100) and smaller (average 40 µm vs. 48.5 µm in H 3 and 52 µm in AR 100), more slender spicules (calomus width 1.5 – 2.0 µm and blade maximum 2.5 µm, whereas in AR 100 males, calomus 1.5 – 2.0 µm wide and blade 2.5 – 3.0 µm), appears slightly different morphologically but not molecularly. Intraspecific variability was also observed within the H 3 population in the degree of anterior-dorsal intestinal overlap over the pharynx, hardly present in the holotype but 8 – 22 µm long in male paratypes. 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; CP 1, anterior ventromedian cervical papilla; G 1 and G 2, (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; SP 1, SP 2 and SP 3, (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. Furthermore, within the type population the variation in measurements was rather large and overlapped with the range known for P. hispanus when including topotypes. Several female specimens had a secretion plug in the vagina, and some showed less or more contraction of the vaginal constrictor muscle, resulting in different appearances of the vagina. Specific alphanumeric codes (in parentheses are exceptions) of the polytomous key adapted from Decraemer & Baujard (1998) are as follows: (1) for females = A 212, B 22, C 1, D 1, E 33, F 33, G 2 (1), H 86 (3), I 1, J 1 (2), K 23, L 2, M 1, N 1, O 1, P 1, Q 4, R 2, S 1, T 1; and (2) for males = A 212, B 22, C 22, D 11, E 0, F 3 (2), G 22, H 33, I 3 (1), J 100, K 33, L 88, M 270, N 11, O 100, P 1. The code for males is closer to that of P. hispanus; similar observations were made for females, with code Q 4 (body pores) as the only difference detected.	en	Decraemer, Wilfrida, Cantalapiedra-Navarrete, Carolina, Archidona-Yuste, Antonio, Varela-Benavides, Ingrid, Gutiérrez-Gutiérrez, Carlos, Castillo, Pablo, Palomares-Rius, Juan E. (2019): 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). Zoological Journal of the Linnean Society 185: 656-692
340A87E4FFBEFF98727EFAA4C256FB15.taxon	description	(FIGS 7, 8; TABLES 2, 5) urn: lsid: zoobank. org: act: 45356 A 52 - CFF 1 - 4342 - 8 EA 2 - D 4 F 958 C 6 EDAB 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 AR 110 - 1 – AR 110 - 4); and AR 110 - 5 male paratypes at the USDA Nematode Collection, Beltsville, MD, USA (collection number T- 7048 p). 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 (Sevilla, Spain) 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; CP 1, anterior ventromedian cervical papilla; G 1 and G 2, (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; SP 1, SP 2 and SP 3, 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 SP 3 immediately anterior to retracted spicule, and SP 1 and SP 2 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 49 µ 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 (SP 1 and SP 2) 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 D 2 – D 3 expansion domains of the 28 S rRNA gene, ITS-rRNA, 18 S-rRNA gene and coxI sequences are deposited in GenBank with accession numbers MG 739529 – MG 739567, MG 739659 – MG 739670, MG 739674 – MG 739683 and MG 726826 – MG 726829, 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 L 4 split up in L 4 (constriction) and L 5 (blade with narrower part mid-way), L 6 (mid-way septum present), L 7 (distal tip divided by septum) and L 8 (anterior part or calomus with irregular outline): (1) for females = A 222 (average, minimum, maximum), B 22, C 1, D 1, E 300, F 33 (1), G 1, H 88 (6), I 11, J 11, K 200, L 1, M 1, N 1, O 11, P 11, Q 4, R 22, S 1, T 1; and (2) for males = A 222 (average, minimum, maximum), B 22, C 22, D 1, E 0, F 3 (4), G 22, H 33, I 33, J 120, K 33, L 88, M 270, N 11, O 100, P 1.	en	Decraemer, Wilfrida, Cantalapiedra-Navarrete, Carolina, Archidona-Yuste, Antonio, Varela-Benavides, Ingrid, Gutiérrez-Gutiérrez, Carlos, Castillo, Pablo, Palomares-Rius, Juan E. (2019): 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). Zoological Journal of the Linnean Society 185: 656-692
340A87E4FFA4FFAC7249FB35C702FE0B.taxon	description	(FIGS 9, 10; TABLES 2, 6) urn: lsid: zoobank. org: act: 0 FB 545 D 8 - 3607 - 4 B 6 A- 8450 - 72 B 0 CA 721 DA 8 Holotype: Female extracted from soil samples collected from rhizosphere of grapevine at La Rambla, Córdoba province, southern Spain (37 ° 37 ′ 17.09 ″ N, 4 ° 42 ′ 13.03 ″ W) by J. Martín Barbarroja and G. León Ropero, mounted in pure glycerine and deposited in the Nematode Collection of Ghent University, Ghent, Belgium (slide number UGMD 104343). Paratypes: Male and female paratypes extracted from soil samples collected from the rhizosphere of grapevine at La Rambla, Córdoba province, southern Spain, were deposited in the following nematode collections: IAS-CSIC (slide numbers IASN 2017 _ 2 _ M 157 - 03 – IAS _ M 157 - 05); and two male paratypes at the USDA Nematode Collection, Beltsville, MD, USA (collection number T- 7049 p). Additional populations were collected in wild olive at Antequera, Málaga province, in cultivated olive at Dos Hermanas, Sevilla province, in cultivated olive at Setenil de las Bodegas, Cádiz province, and in black alder at Andújar, Jaén province, and deposited in the Nematode Collection of IAS-CSIC. Etymology: The species epithet refers to La Rambla, the type locality where the type specimens were collected. (Córdoba, Spain) Measurements are in µm 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; CP 1, anterior ventromedian cervical papilla; G 1 and G 2, (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; SP 1, SP 2 and SP 3, 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. Description of male: Body on average ~ 1000 µm long, largely cylindrical, anteriorly gradually tapered to a rounded lip region with protruding sets of four double papillae (four cephalic and two subdorsal and two subventral outer labial papillae); body cuticle swollen (3.0 – 4.5 µm) upon fixation. Amphid with wide transverse aperture located immediately posterior to the outer crown of anterior sensilla, fovea stirrup-shaped and amphidial canal often clearly visible. Stoma narrow; strengthening rods 4.0 – 5.0 µm long. Pharyngostom with rather long (59 µm average) ventrally curved onchiostyle with half as long onchium, well marked from slender mid-pharynx (isthmus). The isthmus gradually widens to a glandular elongated bulb with three of the five gland nuclei clearly visible: the dorsal nucleus located at the anterior bulb region, the two posterior ventrosublateral gland nuclei in the posterior third of the bulb. Pharyngeal bulb overlapped (10 – 22 µm) dorsally by intestine; cardia poorly developed. A single non-protruding ventral cephalic papilla (CP) present shortly (4 – 7 µm) anterior to the S-E pore, located opposite the anterior portion of the pharyngeal bulb, i. e. near dorsal gland nucleus; on each side, a lateral body pore (LP) at about the level of the S-E pore. Nerve ring around narrowest part or isthmus, close to posterior base of pharyngostom. Reproductive system monorchic; short germinal zone, vesicular seminalis well developed (average 208 µm long) with sperm cells with sausage-shaped nucleus (6.7 – 7.5 µm × 2.3 µm in longitudinal optical section). Spicules ~ 50 µm on average, manubrium slightly widened, calomus short, narrower (1.0 – 1.5 µm) and undulating, continuing in a wider blade (2.0 – 2.5 µm, except 3.0 µm), distally tapered, undulation may be more or less pronounced; spicules with fine transverse striation except for manubrium and distal end. Gubernaculum with slightly thickened keel, usually less than one-third of the length of the spicule. Anterior anal lip bifid. Spicular capsule of suspensor muscles narrow, elongated; copulatory muscles hardly developed. Bursa medium-sized, extending anteriorly to the level of the calomus and posteriorly to the pair of postcloacal supplements, subterminally on the tail. Three precloacal supplements: SP 1 close to the cloacal opening, SP 2 level with posterior end of calomus of retracted spicule or immediately posterior to it and SP 3 2.5 times maximal body diameter (on average) anterior to SP 2 and hardly developed. One pair of caudal pores subterminally, near postcloacal supplements. Tail shorter than anal body width. Description of female: General appearance as in male apart from secondary sexual features. Reproductive system didelphic – amphidelphic, about equally developed reflexed ovaries, finely granular oviduct cells at tip of ovary; sperm stored in spermatheca adjacent to oviduct; uterus with hardly marked ovejector; vagina ~ 30 % of corresponding body width long, more or less indented mid-way; vaginal sclerotized pieces (pars refringens vaginae) in optical section mostly fine, drop-like, rounded triangular to oval with average size 1.9 µm, obliquely oriented with tips very close (average 1.2 µm); vulva pore-like in ventral view. Well-developed sclerotized plug observed in vagina (Fig. 10). No lateral body pores observed. Tail minute, anus subterminal, with a pair of caudal pores present. Remarks: In the population of Dos Hermanas, Sevilla (ST 083), one postvulvar body pore was observed in three females at 148, 164 and 198 µm posterior to the vulva; vaginal sclerotized pieces varied in shape and size from rounded triangular to rounded rectangular (1.5 – 2.3 µm, average 1.7 µm), with tips on average 0.7 µm apart (Fig. 10). Diagnosis and relationships: Paratrichodorus ramblensis sp. nov. is characterized by a rather long and slender body [body length (L) = average 1015 µm; a = 29.5 average] and onchiostyle (average 52.5 µm), a pharynx with gland nuclei (dorsal and posterior ventrosublateral pair) clearly separated and with developed dorsal intestinal overlap. Males possess 50.5 µm long and rather slender spicules with undulating calomus, one ventromedian cervical papilla shortly anterior to the S-E pore opposite the anterior one-third of the pharyngeal bulb, sperm cells with a large sausage-shaped nucleus, and three precloacal supplements, of which two are within the region of retracted spicules; a pair of subterminal postcloacal supplements adjacent to a terminal pair of caudal pores; and a tail shorter than anal body width. Females are characterized by sperm stored in a spermatheca near the oviduct, a short vagina (about one-third of corresponding body width), mid-way indented and with fine drop-like, rounded triangular to oval (1.9 µm), obliquely oriented vaginal sclerotized pieces with tips close to one another; and no lateral body pores in the type population. The new species most closely resembles P. divergens in the slender spicule shape compared with the somewhat broader spicules in P. almadenensis sp. nov., P. hispanus and P. anemones. It differs from P. divergens and from P. anemones by the location of the two posterior precloacal supplements, more spread in the region of the retracted spicules, i. e. similar to P. hispanus and P. almadenensis, instead of SP 1 and SP 2 close together. In females, the new species differs from P. divergens in the orientation of the vaginal sclerotized pieces with tips not diverged, and from P. anemones in the shape and size of the vaginal sclerotized pieces, more developed, wider triangular compared with P. anemones, with very fine triangular pieces in optical section and in general with a less indented vagina than in P. anemones. Lateral body pores appear to be absent in the new species (except in a non-type population) compared with usually two (one prevulvar, one postvulvar) in P. anemones. The new species also differs from the other P. hispanus - like species by the longer (average> 1000 µm) and more slender body (a ~ 30 µm on average in male), the S-E pore located a bit more posterior in the neck region (average at 73 % of pharynx from anterior end in male) and the slightly larger but fine vaginal sclerotized pieces. Specific alphanumeric codes (in parentheses are exceptions) of the polytomous key adapted from Decraemer & Baujard (1998) are as follows: (1) for males = A 323 (average, minimum, maximum), B 22, C 22, D 11, E 0, F 3, G 22, H 33, I 33, J 100, K 33, L 88, M 270, N 11, O 100, P 1; and (2) for females = A 323, B 22, C 1, D 1, E 300, F 300, G 1, H 66, I 11, J 11, K 230, L 12, M 1, N 1, O 11, P 11, Q 4, R 22, S 1, T 1. Variation in non-type population females in A 223 and H 86; males in A 223. Diagnosis: Cuticle usually not swollen when fixed. One pair of caudal pores. Female reproductive system monodelphic – prodelphic; spermatheca present; postvulvar uterine sac present, minute to large. Vagina well developed, length about half to more than one-half body width and anteriorly directed. Vaginal sclerotization small to medium-sized, often weakly sclerotized. One pair of lateral advulvar body pores present. Males with one ventromedian cervical papilla and one pair of lateral cervical pores. Spicules long, slender; without a spicule capitular extension, shaft striated, with or without bristles. Caudal alae absent or at most rudimentary, as in Monotrichodorus sacchari Baujard & Germani, 1985. Oblique copulatory muscles moderately developed, extending to slightly anterior to the retracted spicules. Three medioventral precloacal supplements present. One pair of large subventral postcloacal papillae. Type species: Monotrichodorus monohystera (Allen, 1957) Andrássy, 1976 (syn. Trichodorus monohystera Allen, 1957; syn. Monotrichodorus acuparvus Siddiqi, 1991; syn. Monotrichodorus parvus Siddiqi, 1991; syn. Monotrichodorus proporifer Siddiqi, 1991. MONOTRICHODORUS VANGUNDYI RODRIGUEZ- M, SHER & SIDDIQI, 1978 (FIG. 11; TABLE 7) Two populations of this species were collected from the rhizosphere of a forest at Aguas Zarcas, San Carlos, Alajuela and Toro Amarillo, Valverde Vega, Costa Rica. Description of male: Body appearance slightly curved ventrally, more abruptly curved posteriorly. Cuticle not swollen after fixation. Onchiostyle large-sized (average 65 µm) with onchium about half as long; pharynx with gland nuclei (dorsal and posterior ventrosublateral pair) clearly visible and without intestinal overlap. Secretoryexcretory pore located at about the beginning of the basal pharyngeal bulb. Paired lateral cervical pores immediately posterior to the S-E pore. Large sperm cells. Three ventromedian supplementary papillae (two supplements clearly within the region of the retracted spicule; the third supplement at a quarter of the length of the spicule head). Spicules conspicuously cephalated, slender, slightly curved ventrally; no transverse striae. Gubernaculum almost linear. Description of female: Body slightly curved ventrally. Cuticle, pharynx and S-E pore as in male. Paired lateral cervical pores immediately posterior to the amphid openings. Gonad single, with flexure at oviduct; uterus elongated, with vagina directed anteriorly. Vulva transverse slit, vaginal cuticularization conspicuous, rod-like with tips close to one another. Paired lateral body pores within one vulvar body width anterior to vulval level. Anus subterminal. Remarks: The morphology and morphometrics of the Costa Rican populations agree closely with those of the original description from Ecuador and Panama by Rodriguez-M et al. (1978) (Table 7), except for longer body length in females and males (692 – 913 and 760 – 941 vs. 650 – 810 and 670 – 830 µm, respectively), longer onchiostyle in females and males (60.0 – 68.5 and 59.0 – 72.5 vs. 48 – 57 and 49 – 56 µm, respectively), and spicules and gubernaculum length (55.0 – 60.0 and 19.5 – 26.5 vs. 50 – 57 and 13 – 16 µm, respectively), and no transverse striae in spicules vs. striae in fixed type specimens. Nevertheless, these differences should be regarded as intraspecific geographical variation. Monotrichodorus vangundyi was described from soil around the roots of oil palm (Elaeis guineensis Jacq.) near Rosa Zarate (Ecuador) and from citrus, banana and native forest soils near the type locality. It was also found in Rio Corutu riverbed soil, Puerto Armuelles, and around roots of the Kapok tree [Ceiba pentandra (L.) Gaertn.], Barro Colorado, Panamá (Rodriguez-M et al., 1978). This work represents the first report of the species in Costa Rica and, together with the report of M. monohystera in banana in Costa Rica (Rodriguez-M et al., 1978), confirms the Monotrichodorus species distribution in Central America, where it seems to occur in cultivated and natural habitats. MOLECULAR CHARACTERIZATION OF THE PARATRICHODORUS HISPANUS GROUP AND MONOTRICHODORUS SPECIES Amplification of the D 2 – D 3 expansion domains of the 28 S rRNA gene, ITS 1 rRNA, partial 18 S rRNA 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; CP 1, anterior ventromedian cervical papilla; G 1 and G 2, (anterior, posterior gonad length, respectively / body length) × 100; L, total body length; LP, labial papilla; mbd, maximal body diameter; N, number of specimens studied; S-E pore, excretory pore; SP 1, SP 2 and SP 3, posterior, second and third precloacal supplements, respectively; V, (distance from anterior end to vulva / body length) × 100. gene and coxI regions from the new and previously known Trichodoridae spp. yielded single fragments of approximately 900, 1000, 1800 and 300 bp, respectively, based on gel electrophoresis. The D 2 – D 3 expansion domains of the 28 S rRNA gene ITS 1 and coxI sequences were obtained for the first time in the present study. No molecular data were available in GenBank for the genus Monotrichodorus. The D 2 – D 3 expansion domains of the 28 S rRNA gene sequences of P. almadenensis sp. nov. (MG 739529 – MG 739530) and P. ramblensis sp. nov. (MG 739531 – MG 739536) were 97 % similar (17 nucleotides and no indels) to each other and 97 % similar to P. anemones (AJ 781505), with 23 nucleotides and no indels, and 21 nucleotides and no indels, respectively. The D 2 – D 3 expansion domains of the 28 S rRNA gene sequences from P. hispanus (MG 739537 – MG 739553) showed 91 % similarity with P. almadenensis sp. nov. (MG 739529 – MG 739530) and P. ramblensis sp. nov. (MG 739531 – MG 739536) and 89 % similarity with several accessions deposited in GenBank, such as P. pachydermus (AM 180727), with a difference of 78 nucleotides and five indels, and from P. anemomes (AJ 781505), with a difference of 74 nucleotides and seven indels. Intraspecific variation in the D 2 – D 3 expansion domains of the 28 S rRNA gene detected in these P. hispanus - group species was low; zero, and from one to four nucleotides among the five populations of P. ramblensis sp. nov., one nucleotide between the two studied populations of P. almadenensis sp. nov., zero, and from one to 14 nucleotides among the 15 studied populations of P. hispanus. Likewise, pairwise sequence comparisons among the three Monotrichodorus species included in this study showed 93 % similarity (54 nucleotides and six indels difference) between Monotrichodorus sp. 1 (MG 739558 – MG 739561) and M. vangundyi (MG 739554 – MG 739557), 92 % similarity (63 nucleotides and two indels difference) between Monotrichodorus sp. 1 (MG 739558 – MG 739561) and Monotrichodorus sp. 2 (MG 739562 – MG 739567) and, finally, 90 % similarity (73 nucleotides and two indels in difference) between Monotrichodorus sp. 2 (MG 739562 – MG 739567) and M. vangundyi (MG 739554 – MG 739557). Intraspecific variation in the D 2 – D 3 expansion domains of the 28 S rRNA gene detected in these three studied Monotrichodorus species was zero, and from one to six nucleotides among different individuals of two populations of Monotrichodorus sp. 2, and zero or four nucleotides for M. vangundyi; and zero or one nucleotide in Monotrichodorus sp. 1 populations from Ecuador. Nine ITS 1 sequences from P. hispanus (MG 739660 – MG 739668) and for P. almadenensis sp. nov. were obtained in the present study, and all showed limited similarity and coverage values with the rest of the Paratrichodorus spp. deposited in GenBank. High molecular variability was detected in the ITS 1 region for Paratrichodorus spp.; in fact, no similarity was found among the ITS 1 sequence from P. almadenensis sp. nov. (MG 739659) and the other ITS 1 sequences of Paratrichodorus spp. deposited in GenBank using a blastn approach. Only P. macrostylus (AY 430187) showed a coverage value> 50 % (85 % similarity). Molecular intra-variability was from one to 19 nucleotides for the 15 studied populations of P. hispanus. It was not possible to obtain any ITS 1 sequences from P. ramblensis sp. nov., probably owing to primer-binding failure because of the variability in the primer sites. The two ITS 1 sequences from M. vangundyi (MG 739669 – MG 739670) did not show any similarity with accessions deposited in GenBank. Intraspecific variation of six nucleotides was found between two studied individuals. As with the D 2 – D 3 expansion domains of the 28 S rRNA gene, 18 S rRNA gene sequences from P. almaden - ensis sp. nov. (MG 739674 – MG 739675) were 99 % similar to those of P. ramblensis sp. nov. (MG 739676 – MG 739678) and 97 % similar to those of P. anemones (AF 036600). The 18 S rRNA gene sequences from P. hispanus (MG 739679 – MG 739682) were 99 % similar to one accession of this species deposited in GenBank (DQ 345527). Finally, one 18 S rRNA gene sequence from M. vangundyi (MG 739683) was obtained in the present study that was related, 96 % similar to several Trichodorus spp., such as T. sparsus (JN 123373), T. viruliferus (JN 123374), T. pakistanensis (JN 123369) and Trichodorus sp. CA 103 (JN 123375). Four new coxI sequences from Trichodoridae spp. were sequenced in the present study. High interspecific variation was detected between the different studied species, including T. obtusus (KP 984658 – KP 984696), and no similarity values> 60 % were found among P. almadenensis sp. nov., P. ramblensis sp. nov. and P. hispanus. Unfortunately, owing to a shortage of Monotrichodorus sp. 1 and Monotrichodorus sp. 2 material, only the D 2 – D 3 expansion domains of the 28 S rRNA gene were sequenced; it was not possible to obtain the other studied regions (ITS 1, 18 S rRNA gene and coxI). PHYLOGENETIC RELATIONSHIPS AMONG THE PARATRICHODORUS HISPANUS GROUP AND MONOTRICHODORUS SPECIES The phylogenetic relationships among the species in the family Trichodoridae inferred from analyses of the D 2 – D 3 expansion domains of 28 S rRNA and the partial 18 S rRNA gene sequences using BI and ML are given in Figures 12 and 13, respectively. No significant differences in topology were found using the BI or ML approaches, and only a few species in some minor clades with low bootstrap support were not congruent with the general topology tree. The 50 % majority rule consensus BI and ML tree (Fig. 12) of a multiple alignment included 174 D 2 – D 3 expansion domains of 28 S rRNA gene sequences and 743 bp, and revealed two major well-supported clades (PP = 1.00; BS = 73), (I) formed by Trichodorus, Nanidorus and Monotrichodorus species and (II) formed by accessions from the genus Paratrichodorus including a subclade (IIa; PP = 0.84; BS = 97) with the two new species, P. almadenensis sp. nov. (MG 739529 – MG 739530) and P. ramblensis sp. nov. (MG 739531 – MG 739536), in addition to P. hispanus (MG 739537 – MG 739553), P. anemones (AJ 781505) and sequences of P. pachydermus and P. porosus (Fig. 12). The newly described species were clearly separated from each other and P. anemones (AJ 781505). Monotrichodorus spp. combined with other unidentified Trichodorus species (JN 123429 – JN 123431) from California formed subclade Ib, which showed a sister relationship inside the Trichodorus major clade, but its position was not well resolved (PP <0.75; BS = 73). The clade including all Monotrichodorus species (PP = 1.00; BS = 97) formed two well-supported subclades, one of them clustering Monotrichodorus sp. 1 (MG 739558 – MG 739561) and M. vangundyi (MG 739554 – MG 739557) and the other with Monotrichodorus sp. 2 (MG 739562 – MG 739567). For the partial 18 S rRNA gene, 129 sequences and 1603 bp were included in the analyses, and this tree (Fig. 13) showed a similar topology to that of D 2 – D 3 expansion domains of the 28 S rRNA gene. All species from the P. hispanus group, including the accessions from P. hispanus (DQ 345527 and AJ 439577), clustered in the same well-supported subclade (PP = 1.00; BS = 67) together with P. anemones (KJ 636332 and AJ 439573), P. divergens (DQ 345528), two unidentified species (DQ 345523 and AJ 439576) and the two new species described here (P. ramblensis sp. nov. and P. almadenensis sp. nov.). As in the D 2 – D 3 expansion domains of the 28 S rRNA gene tree, M. vangundyi (MG 739683) showed a sister relationship inside the Trichodorus major clade together with Trichodorus sp. CA 103 from California (JN 123375), but in this case, the clade was highly supported (PP = 0.99; BS = 85). The SH and AU tests of different tree topologies derived from the D 2 – D 3 expansion domains of the 28 S rRNA gene support the hypothesis (P = 0.158 and P = 0.389, respectively) that the four genera studied (Trichodorus, Nanidorus, Monotrichodorus and Paratrichodorus) are valid, because a tree topology that places each genus in a separate clade is not significantly worse than the ML tree without constraints (Table 8). However, this result was not obtained for the partial 18 S rRNA gene (P = 0.002 and P = 0.001, SH and AU test, respectively), for which the constrained tree was significantly worse than the tree without constraints. When Monotrichodorus and Paratrichodorus * Selection of sequences from every clade in their respective phylogenetic tree. † Only one sequence for available for Monotrichodorus. ‡ P <0.05 indicates the significant differences among the inferred tree topologies. § P <0.05 indicates the significant differences among the inferred tree topologies. are in separate clades and Trichodorus – Nanidorus are forced to form one clade, the tree is not significantly different from the tree without constraints according to the SH test for both markers and significantly worse according to AU test. The monophyly of Trichodorus is supported in the D 2 – D 3 expansion domains of the 28 S rRNA gene marker but not for the partial 18 S rRNA gene (Table 8).	en	Decraemer, Wilfrida, Cantalapiedra-Navarrete, Carolina, Archidona-Yuste, Antonio, Varela-Benavides, Ingrid, Gutiérrez-Gutiérrez, Carlos, Castillo, Pablo, Palomares-Rius, Juan E. (2019): 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). Zoological Journal of the Linnean Society 185: 656-692
