Paratrichodorus hispanus, ROCA & ARIAS, 1986

PARATRICHODORUS HISPANUS ROCA & ARIAS, 1986 View in CoL

Topotypes ( Figs 1–4; Tables 2–4)

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 G1 (tail/length of the anterior ovary) and G2 (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 (CP1) is non-protruding and located shortly (average 6.0µ 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 CP1 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 SP2 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, A213 [A112] (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; 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.

maximum), B23 [B22], C23 [C22], D11, E0, F3, G22, H33 (2), I33, J200, K33, L88, M270, N11, O100, P1; and (2) for females presented with topotypes between [] when different from type specimens, A213 [A212], B23 [B22], C1, D1, E30, F30, G1, H88 [H86 (3)], I11, J11, K30 [K23], L11 [L12], M1, N1, O11, P11, Q1, R22 , S1, T1 .

Other populations ( Figs 5, 6; Tables 2, 4)

Several populations of this species were collected from the rhizosphere of cultivated olive at Hinojos (H0003), Huelva province, southern Spain, the rhizosphere of wild olive (AR099, AR100), stone pine and cork oak (H0027) at El Rocio, Huelva ( Spain), grapevine (388GR) at Bollullos par del Condado (Huelva, Spain), cork oak (HATR) at Villamanrique de la Condesa (Sevilla, Spain), eucalyptus (H0169) at Almonte (Huelva, Spain), stone pine (H0179) and eucalyptus (H0169) at Lucena del Puerto (Huelva, Spain), and wild olive (AR139) at Aroche (Huelva, Spain) and sample AR0140 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 SP2), of which two are situated within the retracted spicules, with SP2 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 H18 population, with its smaller onchiostyle (44 µm on average vs. 53 µm in H0003 and 56 µm in AR100) and smaller (average 40 µm vs. 48.5 µm in H3 and 52 µm in AR100), 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 H3 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; 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.

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 = A212, B22, C1, D1, E33, F33, G2 (1), H 86 (3), I1, J1 (2), K23, L2, M1, N1, O1, P 1, Q4, R2, S1, T1; and (2) for males = A212, B22, C22, D11, E0, F3 (2), G22, H33, I3 (1), J100, K33, L88, M270, N11, O100, P1.

The code for males is closer to that of P. hispanus ; similar observations were made for females, with code Q4 (body pores) as the only difference detected.