Paratrichodorus ramblensis, Decraemer & Cantalapiedra-Navarrete & Archidona-Yuste & Varela-Benavides & Gutiérrez-Gutiérrez & Castillo & Palomares-Rius, 2019

PARATRICHODORUS RAMBLENSIS SP. NOV.

( FIGS 9, 10; TABLES 2, 6)

urn:lsid:zoobank.org:act:0FB545D8-3607-4B6A-8450-72B0CA721DA8

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 View Materials ). GoogleMaps

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 IASN2017 _2_ M157-03–IAS_M157-05); and two male paratypes at the USDA Nematode Collection , Beltsville , MD, USA (collection number T-7049p). 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; 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.

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 (3.0–4.5µm) 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 (2.0–2.5µm, except 3.0µm), 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: SP1 close to the cloacal opening, SP2 level with posterior end of calomus of retracted spicule or immediately posterior to it and SP3 2.5 times maximal body diameter (on average) anterior to SP2 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 (ST083), 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 SP1 and SP2 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 = A323 (average, minimum, maximum), B22, C22, D11, E0, F3, G22, H33, I33, J100, K33, L88, M270, N11, O100, P1; and (2) for females = A323, B22, C1, D1, E300, F300, G1, H66, I11, J11, K230, L12, M1, N1, O11, P11, Q4, R22, S1, T1. Variation in non-type population females in A223 and H86; males in A223.

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 D2–D3 expansion domains of the 28S rRNA gene, ITS1 rRNA, partial 18S 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; CP1, anterior ventromedian cervical papilla; G1 and G2, (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; SP1, SP2 and SP3, 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 D2–D3 expansion domains of the 28S rRNA gene ITS1 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 D2–D3 expansion domains of the 28S rRNA gene sequences of P. almadenensis sp. nov. ( MG739529 View Materials – MG739530 View Materials ) and P. ramblensis sp. nov. ( MG739531 View Materials – MG739536 View Materials ) were 97% similar (17 nucleotides and no indels) to each other and 97% similar to P. anemones ( AJ781505 View Materials ), with 23 nucleotides and no indels, and 21 nucleotides and no indels, respectively. The D2–D3 expansion domains of the 28S rRNA gene sequences from P. hispanus ( MG739537 View Materials – MG739553 View Materials ) showed 91% similarity with P. almadenensis sp. nov. ( MG739529 View Materials – MG739530 View Materials ) and P. ramblensis sp. nov. ( MG739531 View Materials – MG739536 View Materials ) and 89% similarity with several accessions deposited in GenBank, such as P. pachydermus ( AM180727 View Materials ), with a difference of 78 nucleotides and five indels, and from P. anemomes ( AJ781505 View Materials ), with a difference of 74 nucleotides and seven indels. Intraspecific variation in the D2–D3 expansion domains of the 28S 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 ( MG739558 View Materials – MG739561 View Materials ) and M. vangundyi ( MG739554 View Materials – MG739557 View Materials ), 92% similarity (63 nucleotides and two indels difference) between Monotrichodorus sp. 1 ( MG739558 View Materials – MG739561 View Materials ) and Monotrichodorus sp. 2 ( MG739562 View Materials – MG739567 View Materials ) and, finally, 90% similarity (73 nucleotides and two indels in difference) between Monotrichodorus sp. 2 ( MG739562 View Materials – MG739567 View Materials ) and M. vangundyi ( MG739554 View Materials – MG739557 View Materials ). Intraspecific variation in the D2–D3 expansion domains of the 28S 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 ITS1 sequences from P. hispanus ( MG739660 View Materials – MG739668 View Materials ) 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 ITS1 region for Paratrichodorus spp. ; in fact, no similarity was found among the ITS1 sequence from P. almadenensis sp. nov. ( MG739659 View Materials ) and the other ITS1 sequences of Paratrichodorus spp. deposited in GenBank using a blastn approach. Only P. macrostylus ( AY430187 View Materials ) 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 ITS1 sequences from P. ramblensis sp. nov., probably owing to primer-binding failure because of the variability in the primer sites. The two ITS1 sequences from M. vangundyi ( MG739669 View Materials – MG739670 View Materials ) did not show any similarity with accessions deposited in GenBank. Intraspecific variation of six nucleotides was found between two studied individuals.

As with the D2–D3 expansion domains of the 28S rRNA gene, 18S rRNA gene sequences from P.almaden - ensis sp. nov. ( MG739674 View Materials – MG739675 View Materials ) were 99% similar to those of P. ramblensis sp. nov. ( MG739676 View Materials – MG739678 View Materials ) and 97% similar to those of P. anemones ( AF036600 View Materials ). The 18S rRNA gene sequences from P. hispanus ( MG739679 View Materials – MG739682 View Materials ) were 99% similar to one accession of this species deposited in GenBank ( DQ345527 View Materials ). Finally, one 18S rRNA gene sequence from M. vangundyi ( MG739683 View Materials ) was obtained in the present study that was related, 96% similar to several Trichodorus spp. , such as T. sparsus ( JN123373 View Materials ), T. viruliferus ( JN123374 View Materials ), T. pakistanensis ( JN123369 View Materials ) and Trichodorus sp. CA103 ( JN123375 View Materials ).

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 ( KP984658 View Materials – KP984696 View Materials ), 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 D2–D3 expansion domains of the 28S rRNA gene were sequenced; it was not possible to obtain the other studied regions (ITS 1, 18S 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 D2–D3 expansion domains of 28S rRNA and the partial 18S 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 D2–D3 expansion domains of 28S 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. ( MG739529 View Materials – MG739530 View Materials ) and P. ramblensis sp. nov. ( MG739531 View Materials – MG739536 View Materials ), in addition to P. hispanus ( MG739537 View Materials – MG739553 View Materials ), P. anemones ( AJ781505 View Materials ) and sequences of P. pachydermus and P. porosus ( Fig. 12). The newly described species were clearly separated from each other and P. anemones ( AJ781505 View Materials ). Monotrichodorus spp. combined with other unidentified Trichodorus species ( JN123429 View Materials – JN123431 View Materials ) 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 ( MG739558 View Materials – MG739561 View Materials ) and M. vangundyi ( MG739554 View Materials – MG739557 View Materials ) and the other with Monotrichodorus sp. 2 ( MG739562 View Materials – MG739567 View Materials ).

For the partial 18S rRNA gene, 129 sequences and 1603 bp were included in the analyses, and this tree ( Fig. 13) showed a similar topology to that of D2–D3 expansion domains of the 28S rRNA gene. All species from the P. hispanus group, including the accessions from P. hispanus ( DQ345527 View Materials and AJ439577 View Materials ), clustered in the same well-supported subclade (PP = 1.00; BS = 67) together with P. anemones ( KJ636332 View Materials and AJ439573 View Materials ), P. divergens ( DQ345528 View Materials ), two unidentified species ( DQ345523 View Materials and AJ439576 View Materials ) and the two new species described here ( P. ramblensis sp. nov. and P. almadenensis sp. nov.). As in the D2–D3 expansion domains of the 28S rRNA gene tree, M. vangundyi ( MG739683 View Materials ) showed a sister relationship inside the Trichodorus major clade together with Trichodorus sp. CA103 from California ( JN123375 View Materials ), 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 D2–D3 expansion domains of the 28S 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 18S 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 D2–D3 expansion domains of the 28S rRNA gene marker but not for the partial 18S rRNA gene ( Table 8).