Acrobeloides buetschli-group (de Man, 1884) Steiner & Buhrer, 1933

3.1. Species belonging to the Acrobeloides buetschli-group

3.1.1. Acrobeloides arenicola Abolafia and Pena-Santiago ˜, 2002

Fig. 1A–E View Fig and Table 1.

3.1.1.1. Material examined. Three females and two males in good state of preservation.

3.1.1.2. Description

3.1.1.2.1. Adult. Small body (0.53–0.65 mm long in females and 0.54–0.56 mm long in males). Habitus sigmoid after fixation or almost straight. Cuticle 1 μm thickness, with transverse striations; annuli 1–2 μm wide at midbody. Lateral fields 5–8 μm wide, occupying 21–30 % of midbody diameter, with four alae limited by five longitudinal incisures, extending to the phasmids, appearing less number of incisures after these. Lip region with three pairs of conoid lips, smooth. Primary axils Ushaped lacking guard processes. Secondary axils V-shaped lacking guard processes. Labial probolae three, conoid with elongate apex. Stoma cephaloboid; cheilostom with small elongated rhabdia; gymnostom very short with rhabdia poorly discernible; stegostom muscular with well sclerotized rhabdia. Pharynx cephaloboid; pharyngeal corpus subcylindrical, 3.3–4.6 times isthmus length; isthmus more slender; basal bulb ovoid, bearing well developed valves. Cardia more or less hemispherical. Nerve ring at 53–68 % of neck length, surrounding the metacorpus–isthmus junction. Excretory pore at 60–75 % of neck length, at anterior part of the isthmus level. Deirids at 71–85 % of neck length, at level of the isthmus.

3.1.1.2.2. Female. Reproductive system monodelphic-prodelphic, located to the right side with respect to the intestine; ovary posteriorly directed, with flexures posterior to vulva; oviduct alveolated; spermatheca well developed, 1.3–1.6 times body diam., with visible spermatozoa inside; uterus cylindrical, 2.3–2.6 times body diam. long, differentiated in a long distal part with a scarce lumen and thick walls, and a short proximal part with thinner walls and distinct lumen; post-vulval uterine sac small, 1.2–1.6 times the corresponding body diam. long; vagina straight, 41–60 % of body wide; vulval area sunken, vulva not protruding. Rectum 1.0–1.3 times anal body width long, with three rectal glands. Tail conoid, slightly elongate with acute tip. Phasmids at 15–25 % of tail length.

3.1.1.2.3. Male. Reproductive system monorchid with testis ventrad flexioned anteriorly. Genital papillae eight pairs, three precloacal and five postcloacal: one anterior lateral, one anterior subventral, one posterior subdorsal and two posterior subventral. Spicules with very elongate manubrium with rounded extreme, ventrad bent, calamus slightly wider than manubrium, lamina almost straight with small dorsal hump and ventral velum, and acute tip. Gubernaculum 0.5–0.7 times the spicules length, with ventrad bent manubrium and almost straight corpus. Tail conoid, slightly ventrally curved, with rounded tip. Phasmids located posteriorly, located between the lateral and subventral anterior papillae.

3.1.1.3. Localities and habitats. Species found in two populations: i) Maro-Cerro Gordo (province of M´alaga) and ii) La Manga del Mar Menor (province of Murcia), both in soil with xeric vegetation: Arthrocnemum macrostachyum (Moric.) C. Koch , Carduus tenuiflorus Curtis , Ephedra fragilis Desf. , Frankenia corymbosa Desf. , Launaea fragilis (Asso) Pau , Limbarda crithmoides (L.) Dumort., Limonium delicatulum (Girad) Kuntze. , Limonium insigne (Coss.) Kuntze , Lycium intricatum Boiss. , Lygeum spartum (L.) Kunth., Ononis natrix L., Pallenis maritima (L.) Greuter, Salsola vermiculata L., Suaeda vera Forssk. Ex J.F.Gmel. , Thymelaea hirsute (L.) Endl. After this study, the distribution area of the species is expanded, previously only found in Salinas de Cabo de Gata (province of Almería) by Abolafia and Pena-Santiago ˜(2002).

3.1.2. Acrobeloides nanus ( de Man, 1880) Anderson, 1968

Fig. 1F–I View Fig , A-E, Fig. 4A–D View Fig and Table 1.

3.1.2.1. Material examined. Ten females in good state of preservation.

3.1.2.2. Description

3.1.2.2.1. Adult. Small body (0.33–0.50 mm long in females). Habitus straight or sigmoid after fixation. Cuticle 1 μm thickness, with transverse striations; annuli 1–2 μm wide at midbody. Lateral fields 4–5 μm wide, occupying 17–28 % of midbody diameter, with four alae limited by five longitudinal incisures, extending to the phasmids, appearing with one or three incisures after these. Lip region with three pairs of conoid lips, smooth. Primary axils U-shaped lacking guard processes. Secondary axils V-shaped lacking guard processes. Labial probolae three, rounded to slightly conoid. Stoma cephaloboid; cheilostom with small rounded rhabdia; gymnostom very short with rhabdia poorly discernible; stegostom muscular with well sclerotized rhabdia. Pharynx cephaloboid; pharyngeal corpus fusiform, 2.7–3.9 times isthmus length, with subcylindrical procorpus and swollen metacorpus; isthmus more slender; basal bulb spheroid to ovoid, bearing well developed valves. Cardia more or less hemispherical. Nerve ring at 68–78 % of neck length, surrounding isthmus. Excretory pore at 75–81 % of neck length, at isthmus level. Deirids at 80–93 % of neck length, at level of the posterior part of isthmus.

3.1.2.2.2. Female. Reproductive system monodelphic-prodelphic, located to the right side with respect to the intestine; ovary posteriorly directed, with or without flexures posterior to vulva; oviduct alveolated; spermatheca poorly developed, 0.3–0.4 times body diam.; uterus cylindrical, 1-5-2.3 times body diam. long, differentiated in a long distal part with a scarce lumen and thick walls, and a short proximal part with thinner walls and distinct lumen; post-vulval uterine sac reduced, 0.1–0.3 times the corresponding body diam. long; vagina straight, 20–33 % of body wide; vulva protruding. Rectum 0.8–1.1 times anal body width long, with three rectal glands. Tail subcylindrical to conoid with wide or narrow rounded or truncated terminus. Phasmids at 26–32 % of tail length.

3.1.2.2.3. Male. Not found.

3.1.2.3. Molecular characterisation. Three sequence of Acrobeloides nanus were obtained: one 18S rDNA fragment with 946 bp (PQ002813), and two 28S rDNA fragments, one with 527 bp (PQ002811) and other with 622 bp (PQ002812). For 18S rDNA, the Spanish sequence of A. nanus show 100 % similarity with other sequences of A. nanus in a fragment in common with 927 bp (AY284672, DQ102707, KX669638) or 795 bp (MF325109). With respect to other similar species, the Spanish A. nanus show 99.9 % similarity (1 bp differences with KY119867) and 99.8 % similarity (2 bp differences with AY284673 and KY119891) with Acrobeloides apiculatus in a fragment in common with 870 bp; 100 % similarity with Acrobeloides buchneri (MF325099 and MF325101) in a fragment in common with 818 bp; 99.9 % (1 bp difference) with Acrobeloides buetschlii (EU543174) in a fragment in common with 927 bp; 99.8–99.9 % (1 or 2 pb differences with EU543175 and KY119635) and 99.1 % (8 bp differences with KY119885, probably with sequencing mistakes) with A. thornei in a fragment in common with 870 bp; and 100 % similarity with A. varius (KX889085) in a fragment in common with 927 bp.

With respect to the 28S rDNA, the two sequences of A. nanus show 99.2 % (4 bp differences with DQ903103), 98.9 % (6 bp differences with DQ903076), 98.7 % (7 bp differences with DQ903075), 98.5 % (8 bp differences with EF417139) in a fragment in common with 527 bp, and 98.6 % (7 bp differences with KX669640) in a fragment in common with 505 bp. With A. buchneri , show 98.7 % similarity (1 difference with MF325157) in a fragment in common with only 76 bp. With A. buetschlii , 99.2 % (4 bp differences with DQ903104) and 98.5 % (8 bp differences with DQ903081) in a fragment in common with 527 bp. With A. ellesmerensis , 98.5 % (8 bp differences with DQ9145624) in a fragment in common with 527 bp. With A. sexlineatus , show 98.7 % similarity (1 difference with MF325168) in a fragment in common with only 76 bp. With A. thornei , 98.5 % (8 bp differences with AF147068 and DQ903083) in a fragment in common with 523 bp. With A. tricornis , 99.8 % (10 bp differences with MW667572 and OK040504) in a fragment in common with 513 bp and 98.7 % (1 bp difference with MF125309 and MK541665) in a fragment in common with only 76 bp. With A. uberrinus , 98.5 % (8 bp differences with DQ903087) in a fragment in common with 527 bp. With A. varius , 98.6 % (7 bp differences with KX889089) in a fragment in common with 502 bp. On the other hand, with Cephalobus persegnis Bastian, 1865 (DQ903077) and Cephalobus sp. (KY750813) shows 98.9 % (6 bp differences) in a fragment in common with 527 bp.

3.1.2.4. Localities and habitats. Species found in Salinas de Cabo de Gata (province of Almería), collected in sand dunes with xeric vegetation: Arthrocnemum macrostachyum (Moric.) C. Koch , L. fragilis (Asso) Pau , Lotus creticus L., L. intricatum Boiss. , Lygeum spartum (L.) Kunth., Ononis natrix L., Onthatus maritimus Hoffmans. and Link , Salsola vermiculata L., Salsola kali L., Suaeda vera Forssk. Ex J.F.Gmel. Also found in olive tree culture ( Olea europaea var. europaea L.) in Periana (province of M´alaga).

3.1.3. Acrobeloides tricornis ( Thorne, 1925) Thorne, 1937

Fig. 1J–M View Fig , Fig. 2 View Fig F-J, Fig. 3 View Fig , Fig. 4E–L View Fig and Table 1.

3.1.3.1. Material examined. Ten females in good state of preservation.

3.1.3.2. Description

3.1.3.2.1. Adult. Small body (0.31–0.37 mm long in females). Habitus straight or sigmoid after fixation. Cuticle 1 μm thickness, with transverse striations; annuli 1–2 μm wide at midbody. Lateral fields 5–6 μm wide, occupying 25–30 % of midbody diameter, with four alae limited by five longitudinal incisures, extending to the phasmids, appearing with two incisures after these. Lip region with three pairs of conoid lips, smooth. Primary axils U-shaped lacking guard processes. Secondary axils V-shaped lacking guard processes. Labial probolae three, short conoid, rarely rounded. Stoma cephaloboid; cheilostom with small rounded rhabdia; gymnostom very short with rhabdia poorly discernible; stegostom muscular with well sclerotized rhabdia. Pharynx cephaloboid; pharyngeal corpus slightly fusiform, 2.7–3.2 times isthmus length; isthmus more slender; basal bulb rounded to ovoid, bearing well developed valves. Cardia more or less hemispherical. Nerve ring at 71–81 % of neck length, surrounding isthmus. Excretory pore at 80–87 % of neck length, at isthmus level. Deirids at 86–91 % of neck length, at level of the posterior part of isthmus.

3.1.3.2.2. Female. Reproductive system monodelphic-prodelphic, located to the right side with respect to the intestine; ovary posteriorly directed, without flexures posterior to vulva; oviduct alveolated; spermatheca poorly developed, 0.5–0.7 times body diam.; uterus cylindrical, 1.0–1.4 times body diam. long, differentiated in a long distal part with a scarce lumen and thick walls, and a short proximal part with thinner walls and distinct lumen; post-vulval uterine sac reduced, 0.2–0.4 times the corresponding body diam. long; vagina straight, 23–30 % of body wide; vulva midventral, slightly protruding. Rectum 1.0–1.1 times anal body width long, with three rectal glands. Tail conoid, slightly elongate, with finely rounded terminus or scarcely wider. Phasmids at 35–43 % of tail length.

3.1.3.2.3. Male. Not found.

3.1.3.3. Molecular characterisation. Unfortunately, sequences of the Spanish population of A. tricornis were not obtained. However, with respect to other sequences of this species obtained previously, it is very similar to those sequences identified as other species. Thus, the 18S rDNA sequence of A. tricornis (MK541681) shows 100 % similarity (AY284673 and KY119867) and 99.5 % similarity (2 bp with KY119891) with A. apiculatus in a fragment in common with 388 bp; 100 % similarity with A. buchneri (MF325099, MF325101) in a fragment in common with 342 bp; 100 % similarity with A. buetschlii (EU543174) in a fragment in commont with 532 bp (with respect to KC206039, the sequence has numerous discrepances with EU543174, probably due to sequencing mistakes); 100 % similarity with A. nanus (AY284672, DQ102707, KX669638, MK 541680 in a fragment in common with 524 bp, and MF 325109 in a fragment in common with 313 bp) and 99.7 % similarity (1 bp with PQ002813 in a fragment in common with 446 bp); 99.6 % similarity (1 bp with EU543175), 98.7 % similarity (5 bp with KY119635) and 98.2 % similarity (7 bp with KY119885) with A. thornei in a fragment in common with 396 bp; 100 % similarity with A. varius (KX889085) in a fragment in common with 524 bp.

Comparing the 28S rDNA sequences, A. tricornis from Tunisia (MW667572, MW667572) in a fragment in common with 289 bp are 100 % identical to A. tricornis from Germany (MK541665, MF125309). With respect to other species, A. tricornis from Tunisia shows 98.6 % similarity (7 bp differences with PQ002811, PQ002812) with A. nanus from Spain in a fragment in common with 502 bp while it shows 100 % similarity (with KX669640), 99.9 % similarity (1 bp difference with DQ903076, EF417139), 99.7 % similarity (2 bp differences with DQ903075), 99.3 % similarity (5 bp differences with DQ903103) with A. nanus from other countries in a fragment in common with 700 bp. Also in a fragment in common with 700 bp, it shows 99.6 % similarity (3 bp differences) with A. buetschlii (DQ903081, DQ903104), Acrobeloides ellesmerensis (DQ145624) and Acrobeloides uberrinus (DQ903087), 100 % similarity with A. thornei (AF147068, DQ903083) and A. varius (KX889089). In addition, A. tricornis shows 99.9 % similarity (1 bp difference) with Cephalobus persegnis (DQ903077) and 99.7 % similarity (2 bp differences) with Cephalobus sp. (KY750813) in a fragment in common with 700 bp. On the other hand, in a fragment in common with 289 bp, A. tricornis from Tunisia and Germany are 100 % identical to A. buchneri (MF325157) and A. sexlineatus (MF325168).

3.1.3.4. Localities and habitats. Species found in three populations: i) Maro-Cerro Gordo (province of M´alaga), ii) Algarve ( Portugal), and iii) Salobre˜na (province of Granada), all of them collected in sand dunes with xeric vegetation: Arthrocnemum macrostachyum (Moric.) C. Koch , Carduus tenuiflorus Curtis , Ephedra fragilis Desf. , Frankenia corymbosa Desf. , L. fragilis (Asso) Pau , Limbarda crithmoides (L.) Dumort., Limonium delicatulum (Girad) Kuntze. , Limonium insigne (Coss.) Kuntze , Lygeum spartum (L.) Kunth., Ononis natrix L., Pallenis maritima (L.) Greuter, Phragmites mauritianus Kunth. , Salsola vermiculata L., Suaeda vera Forssk. Ex J.F.Gmel.