Unravelling cryptic diversity in the Paratrichodorus allius- group species complex to resolve eight new species of the genus and new insights on the molecular phylogeny % Nematoda: Trichodoridae) Author Decraemer, Wilfrida Ghent University, Department of Biology, Ledeganckstraat 35, B- 9000 Ghent, Belgium palomaresje@ias.csic.es Author Archidona-Yuste, Antonio Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain palomaresje@ias.csic.es Author Clavero-Camacho, Ilenia Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain Author Vovlas, Alessio Consiglio Nazionale delle Ricerche (CNR), Istituto per la Protezione Sostenibile delle Piante (IPSP), v. G. Amendola, 122 / D, 70126 Bari, Italy Author Cantalapiedra-Navarrete, Carolina Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain Author Alba Author Ruiz-Cuenca Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain & Departament of Animal, Plant Biology and Ecology, Universidad de Jaén, Campus Las Lagunillas, Jaén, Spain palomaresje@ias.csic.es Author Castillo, Pablo Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain Author Juan Author Palomares-Rius Institute for Sustainable Agriculture (IAS), Spanish National Research Council (CSIC), Avda. Menendez Pidal s / n, 14004 Córdoba, Campus de Excelencia Internacional Agroalimentario, ceiA 3, Spain & Ghent University, Department of Biology, Ledeganckstraat 35, B- 9000 Ghent, Belgium palomaresje@ias.csic.es text Zoological Journal of the Linnean Society 2024 2024-01-24 202 3 1 44 https://doi.org/10.1093/zoolinnean/zlad194 journal article 10.1093/zoolinnean/zlad194 0024-4082 14423027 31FB687-C656-4F7F-8893-AA10FC8DC63A Paratrichodorus uliaensis sp. nov. Decraemer et al . ( Figs 1–3A, B ; Table 2 ) Zoobank registration: urn:lsid:zoobank.org:act: 7908CD13- BD88-45CE-BDEA-F24425B2590D Type material: Holotype . Male extracted from soil samples collected from the rhizosphere of grapevine ( Vitis vinifera ) at Montemayor , Córdoba province , southern Spain ( 37°38 ʹ 11.7 ʹʹ N latitude, 004°42 ʹ 21.9 ʹʹ W longitude) 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 104445 ). Paratypes : Male and female paratypes extracted from soil samples collected from the rhizosphere of grapevine at Montemayor , Córdoba province , southern Spain , were deposited in the following nematode collections: Ghent University, Ghent, Belgium (slide numbers males and females paratype UGMD 104445 - UGMD 104449 ) and one male and one female paratypes at the USDA Nematode Collection, Beltsville, MD, USA (collection number T-8015p ). Figure 1. Line drawings of Paratrichodorus uliaensis sp. nov .. Male. A, B, pharyngeal region. C, spicules. D, G, posterior body region with copulatory apparatus. E, F, anterior part of testis. Female paratypes. H, I, pharyngeal region. J, K, reproductive system: posterior branch and total system. L, N, vagina region. M, tail region. Holotype male, A, D; paratype male, F; paratype male, E. Specimens from La Rambla locality. Scale bars = 20 µm. Figure 2. Light micrographs of Paratrichodorus uliaensis sp. nov .. A–D, holotype male. A, pharyngeal region. B, total view. C, testis; D, tail region with copulatory apparatus. Female paratypes. E, reproductive system. F, G, vagina region. Scale bars A, C-G = 10 µm; B = 100 µm. Additional material examined: An additional population was collected also in grapevine at La Rambla , Córdoba province , Spain ( 37°37 ʹ 17.9 ʹʹ N latitude, 4°42 ʹ 13.3 ʹʹ W longitude). Etymology: The species epithet refers to the roman name (Ulia) of Montemayor, the type locality where the type specimens were collected. Description of male: Body more or less straight, cigar-shaped, often with swollen cuticle (3.5–7.5 µm at midbody) upon fixation and anteriorly slightly tapered to a rounded lip region appearing offset by the protruding double papillae (four cephalic and two subdorsal and two subventral outer labial papillae). Onchiostyle rather small (average 33.5 µm long). Pharynx at posterior isthmus either gradually widening to a posterior bulb or bulb more or less marked, usually with a pronounced (8–37 µm) ventral overlap of the ventrosublateral pharyngeal glands, rarely a combination of a short dorsal intestinal and a ventral pharyngeal overlap; posterior ventrosublateral gland nuclei close to pharyngo-intestinal junction. Nerve ring surrounding isthmus; secretory-excretory pore (SE-pore) at level of anterior pharyngeal bulb when pharynx relaxed. A single ventromedian CP at rather a short distance (10–22 µm) anterior to SE-pore. Reproductive system monorchic, testis with short germinal zone (25 µm in holotype ), and sperm with small oval-shaped nucleus (2.5 × 1.5 µm), closely packed with fibrillar appearance. Spicules medium-sized (average 44.5 µm), nearly straight, with marked capitulum, blade largely equally wide, but distally tapered, transversely striated, except at extremities. Copulatory muscles hardly developed; capsule of suspensor muscles very thin, around anterior half of spicule; gubernaculum with thickened distal part, parallel to spicules. Bursa narrow, rather short, extending from head of retracted spicule to subterminally on tail. Three precloacal supplements, posterior two (SP1 and SP2) opposite distal half of the retracted spicules with SP1 at about equal distance from cloacal opening and from SP2; SP3 little developed and non-protruding, located about 2.5 spicule lengths from the cloacal opening. A pair of well-developed postcloacal supplements close to the cloacal opening, the latter with a small protruding anterior lip flap (in ventral view visible as two flaps bordering the opening). Tail short, less than one anal body diameter long and rounded conical. A pair of caudal pores subterminally near tail tip; cuticle slightly thicker at tail tip (average 3.5 µm). Figure 3. Line drawings for comparison of vagina in the Paratrichodorus allius species-complex group. A, B, P. uliaensis sp. nov. ; C, D. P. benalupensis sp. nov. ; E, P. hinogerus sp. nov. ; F, G, P. tarifaensis sp. nov. ; H, P. asidonaensis sp. nov. ; I, P. rocianerus sp. nov. ; J, P. litoralis sp. nov. ; K, L, P. paraallius sp. nov. ; M–P, P. allius based on Sturhan (1989); Q, P. allius , paratype; R, S, P. teres , paratypes. Description of female: Body slightly ventrally curved; largely similar to male apart from reproductive system and male secondary features. Digestive system with obvious ventral overlap of pharyngeal glands (15–53 µm). SE-pore at posterior level of isthmus or anterior end of pharyngeal bulb. Reproductive system didelphic-amphidelphic with both branches about equally developed, or one branch may be longer in presence of developed oocytes or egg; oviduct cells finely granular; sperm cells, when present, spread along uterus, no spermathecae; vagina more or less barrel-shaped, short [average 8 µm long or 22% of corresponding body diameter (cbd)]; pars refringens vaginae in optical section with minute (1.0–1.5 µm), rounded, triangular to oval sclerotized pieces, slightly obliquely oriented, tips 2.0–2.5 µm apart; vulva at about midbody (average 55%) and a longitudinal slit in ventral view. Sublateral body pores mostly absent (observed in only three specimens from type population); could be obscured by swelling of body cuticle. Table 2. Morphometrics of Paratrichodorus uliaensis sp. nov. male and female paratype specimens and specimens from La Ramblaa .

Holotype male	Paratype males	Paratype females	La Rambla males	La Rambla females
N	9	7	11	6
L	788	660–790 708 ± 37	690–815 742 ± 43	630–895 748 ± 78	745–1000 918 ± 97
Onchiostyle	36	30–37 33.5 ± 2.3	34–37 35.4 ± 31.2	33–38 36 ± 1.8	34.5–41 36.3 ± 2.5
Onchium	16	13–17.5 15.0 ± 1.5	15–15.5 15.3 ± 0.3	14–21.5 17.1 ± 2.5	15–18 16.3 ± 1.0
Pharyngostom	44	37–45 41.2 ± 2.3	39–46 42.3 ± 2.3	38.5–48 44.6 ± 3.5	40–47 44.0 ± 2.6
Pharynx	123	88–128 111 ± 12	111–128 115 ± 9	97–151 125 ± 18	118–141 132 ± 8
Anterior to guide ring	17	13–17 14 ± 1.9	14–18 16.2 ± 1.7	12.5–19 16.5 ± 2.5	15–19 17.2 ± 1.5
Anterior to nerve ring	52	44–57 49 ± 4.9	45–53 50 ± 3.2	49–63 53.3 ± 5.6	49–55 53.2 ± 3.7
Anterior to CP1/anterior to EP in female	83	72–96 80.7 ± 7.1	87–134 100 ± 18	62–101 81.1 ± 12	74–116 96.2 ± 18
Distance CP1-EP	11	10–22 13.9 ± 4.0	9–26 15.2 ± 7.6
mbd at cardia	30	27–37 32 ± 4.7	24–38 32 ± 4.9	26–39 31.1 ± 3.3	30–35.5 32.3 ± 2.1
mbd midbody	33	29–38 34.1 ± 5.9	32–42 35.7 ± 3.9	28–41 32.6 ± 3.6	31–39 33.8 ± 2.8
abd/♀: length anterior ovary	17	16–19 18 ± 2.1	38–101 80.3 ± 24.8	15.5–19 16.9 ± 1.4	34–106 75.7 ± 27.5
Spicule length/ ♀: length anterior branch	45	42–47 44.5 ± 1.6	173–278 231 ± 40	41–49 45.55 ± 2.6	211–320 259 ± 45
Gubernaculum/ ♀: length posterior ovary	16	10–15 11.8 ± 2.3	56–113 78.7 ± 19.8	9–16 11.9 ± 2.2	34–109 67.2 ± 24.4
Distance SP1-cloacal opening/♀ length posterior branch	12.5	9.5–12 10.2 ± 0.9	153–279 211 ± 44	6–13 10.3 ± 1.8	139–276 197 ± 45
Distance SP1-SP2/ ♀ length vagina	9	10.5–14 12.1 ± 1.2	7.5–9 7.9 ± 0.7	10–16 13.0 ± 2.9	7.5–9 8.0 ± 0.6
Distance SP2-SP3/♀ vagina % cbw	104	65–120 95.3 ± 17.5	20.7–23.7 22.4 ± 1.5	76–115 87.3 ± 14.2	23.1–24.2 23.7 ± 0.5
Tail/♀G1	11	9–10 9.6 ± 0.4	24.5–37.6 31.0 ± 4.6	6.5–11 9.1 ± 1.53	23.6–33.4 28.3 ± 4.4
ab	23.88	14.5–25.0 20.8 ± 3.7	17.6–22.5 20.8 ± 1.9	16.2–26.8 22.6 ± 3.4	24.0–28.4 27.1 ± 2.0
b	6.41	5.4–7.38 6.4 ± 0.7	5.5–7.3 6.4 ± 0.7	4.8–7.7 6.3 ± 1.2	5.5–7.6 7.0 ± 0.9
c/♀V	71.64	69.6–79.6 73.4 ± 3.7	48.4–60.0 54.2 ± 4.2	66.5–110 81.3 ± 15.7	52.56–59.4 56.0 ± 2.6
T/♀G2	85.94	85.0–98.6 87.0 ± 6.2	18.8–39.0 28.6 ± 6.6	54.1–98.5 69.8 ± 18.8	15.5–27.9 21.7 ± 5.1

aMeasurements are in µm and in the form:range and mean ± SD. ba , body length/maximum body width; abw (anal body width); b, body length/pharyngeal length; c, body length/tail length;cbw (corresponding body width); CP (ventromedian cervical papilla); CP1 (anterior ventromedian cervical papilla); G1, G2 (anterior, posterior genital branch length, respectively/body length) × 100; EP (secretory-excretory pore); L (total body length); mbw (maximum body width); N (number of specimens studied); SP1, SP2 SP3 (posterior, second and third precloacal supplements, respectively);V (distance from anterior end to vulva/body length) × 100; T (distance from cloacal aperture to anterior end of testis/body length) × 100. Code oithin the tabular identification key (based on: Decraemer and Baujard 1998): For males:A 212 (average, min., max.), B11, C22, D1, E0, F3, G2, H23, I2(4), J1, K2, L2, M27, N25, O1, P1. For females: A212 (average, min., max.), B11, C11, D1, E23, F2 (4), G3, H53, I1, J13, K2, L3, M1, N1, O2, P25, Q4(1), R2, S1, T1. Remarks: In three females , lateral body pores were observed (Q1), one postvulvar sublateral pore at nearly 2.5 vaginal body diameter from vulva, and one prevulvar sublateral body pore six body diameters anterior to vulva. Diagnosis and relationships: Paratrichodorus uliaensis sp. nov. is characterized in both sexes by the rather short onchiostyle (smaller than 40 µm) of 33.5 (30–37) µm in males and 35.4 (34–37) µm in females; in males by presence of a single CP, testis with small sperm cells with small nucleus (2.5 × 1.5 µm), and often fibrillar appearance, medium-sized spicules [44.5 (42–47) µm] with marked capitulum and three precloacal supplements with the posterior two within distal half of retracted spicules, and in females by the very short, less than 25% of corresponding body diameter or 22.4% (20.7–23.7 %), barrel-shaped vagina with minor midway indentation at level of constrictor muscles, minute rounded triangular sclerotized pieces, oblique in orientation and clearly separated from one another, sublateral body pores mostly not observed and vulva in ventral view a longitudinal slit. Sperm spread along uteri. Based upon the tabular identification keys for males (Decraemer and Baujard 1998), sorting on primary features F (number of ventromedian precloacal supplements), feature D (number of CP), and P (habitus) the new species belongs to species group 6. Further sorting by feature N ( type of sperm cells: small cell with small nucleus) grouped the new species with P. catharinae Vermeulen and Heyns 1983 , P. meyeri De Waele and Kilian 1992 , P. paraporosus Khan et al . 1989 , and P. tunisiensis ( Siddiqi 1963 ) Siddiqi 1974. Paratrichodorus uliaensis sp. nov. can be distinguished from these four species by a shorter onchiostyle (feature B), average less than or equal to 40 µm. For females, sorting on primary features D and C, followed by L and K, resulted in a classification in subgroup 1-12 in Decraemer and Baujard (1998). Further sorting on type of sperm cells (feature P) grouped the new species with the known species P. acaudatus ( Siddiqi 1960 ) Siddiqi 1974, P. allius , and P. teres (subgroup 1-12) from which it can be distinguished by the shorter onchiostyle (<40 µm vs.>40µm). The new species agrees with P. acaudatus in male being common but differs in vagina shape (quadrangular in P. acaudatus ) and in male characteristics such as absence of CP and much longer spicules in P. acaudatus . Paratrichodorus uliaensis sp. nov. differs from both P. allius and P. teres by males being common, from P. allius by location of sperm, i.e. only in the uteri and not in ovarial sac, and from P. teres in shape of vagina without widened midpart.