Characterization of Nothacrobeles lanceolatus Abolafia & Peña-Santiago, 2003 (Rhabditida, Cephalobidae), a typical species of coastal dunes from Spain

Abstract The nematode Nothacrobeles lanceolatus was described in arid soils from Cabo de Gata-Níjar Natural Park, province of Almería (Spain). Since then this species has not been found again. In this study, new data on the distribution and morphological, morphometrical and molecular characteristics (based on 18S and 28S ribosomal DNA) of N. lanceolatus is provided. This species is characterized by having a tessellated cuticle, with its inner layer bearing two rows of cuticular punctations per annulus, lips quadrangular with eight pinnae, labial probolae bifurcate with divergent prongs surrounded by pinnae, pharynx with corpus 2.5–4.0 times isthmus length, post-vulval uterine sac 1.4–2.2 times the corresponding body diameter, and female and male tails conical, bearing a conical mucro with acute terminus. The phylogenetic position of this and related species of the genus Nothacrobeles is discussed. Additionally, bacteria (Pasteuria) and fungi (Catenaria, Nematocida) feeding on N. lanceolatus are showed including light microscopy and scanning electron microscopy illustrations.


Introduction
The genus Nothacrobeles was described by Allen & Noffsinger (1971) who found four new species from Israel, India and Australia, being the type species Nothacrobeles sheri Allen & Noffsinger, 1971. This genus is characterized by having lip region with labial probolae bifurcate with prongs having variable length, rarely absent, connecting basally by an expansion directed towards the secondary axil, and margins smooth or bordered by small membranous projections (pinnae). Later, Shahina & De Ley (1997) synonymized the genus Namibinema Rashid & Heyns, 1990 with it. Actually, the genus Nothacrobeles includes 21 valid species (Abolafia & Peña-Santiago, 2003;Ruiz-Cuenca & Abolafia, 2020).
One of these species, Nothacrobeles lanceolatus, was described by Abolafia & Peña-Santiago (2003) in xeric areas from Cabo de Gata-Níjar Natural Park (Spain). This species was considered as a possible endemism for this geographical area. In this study, new data about the distribution of this species, which can be considered now as Iberian littoral endemism, is provided. This new material is now characterized from the morphological, morphometric and molecular point of view.

Sampling and nematode extraction
The specimens of N. lanceolatus were extracted from the rhizosphere of xerophile plants from sand coastal dunes in the provinces of Huelva, Cádiz, Málaga, Almería, Murcia, Alicante, Valencia, Castellón, Tarragona and Barcelona (Spain) ( fig. 1). The nematodes were extracted from soil samples using a modified Baermann's (1917) funnel technique, killed by heat and fixed in a 4% formalin solution. The nematodes were processed to anhydrous glycerine according to Siddiqi's (1964) method using lactophenol-glycerine solutions and were permanently mounted on glass microscope slides with the glycerine-paraffin method (de Maeseneer & d'Herde, 1963) somewhat modified using hot liquid paraffin.

Light microscopy (LM)
Observations were made and measurements were taken using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with differential interference contrast optics. Photomicrographs were taken with a Nikon Digital Sight DS-U1 camera and processed with Adobe® Photoshop® CS (Adobe Inc., San José, California, USA) and figures mounted using Microsoft® PowerPoint® (Microsoft Corporation, Redmond, Washington, USA). Demanian indices (de Man, 1881) and other ratios were calculated. The terminology used for the morphology of probolae, stoma and spicules follows Ruiz-Cuenca & Abolafia (2021), De Ley et al. (1995) and Abolafia & Peña-Santiago (2017), respectively.

Scanning electron microscopy (SEM)
The specimens preserved in glycerine were selected for observation under SEM according to Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol-acetone series, critical-point dried, coated with gold and observed with a Zeiss Merlin microscope (5 kV) (Zeiss, Oberkochen, Germany).

Phylogenetic analyses
For phylogenetic relationships, analyses were based on 18S and 28S ribosomal DNA (rDNA) fragments. The newly obtained sequences were manually edited using BioEdit 7.2.6 (Hall, 1999) and aligned with another 18S or 28S rDNA sequence available in GenBank using the ClustalW (Thompson et al., 1994) alignment tool implemented in MEGA7 (Kumar et al., 2016). Alignment ends were trimmed using MEGA7. The best-fit model of nucleotide substitution used for the phylogenetic analysis was statistically selected using jModelTest 2.1.10 (Darriba et al., 2012). The phylogenetic trees were generated with the Bayesian inference method   (Tavaré, 1986) was initiated with a random starting tree and run with the Markov Chain Monte Carlo (MCMC) (Larget & Simon, 1999) for 1 × 10 6 generations. The trees were visualized and saved with FigTree 1.4.4 (Rambaut, 2018).

Results
Nothacrobeles lanceolatus Abolafia & Peña-Santiago, 2003 Material examined In this study, 20 females and 20 males from several Iberian coastal dunes were examined.

Measurements
For measurements, see table 1.

Description
Adult (figs 2-5). Body cylindrical, with 0.6-0.8 mm long. Cuticle with tessellated annuli; annuli 3 μm wide at midbody. Lateral fields with three longitudinal incisures (or two longitudinal alae) occupying 16-25% of the body diameter at midbody. Lip region with dentate rectangular lips, grouped in pairs. Primary axils deep with two elongate guard processes and secondary axils shallow lacking guard processes. Oral opening surrounded by three labial probolae with dentate margin bearing rounded to conoid pinnae, 10-14 μm long, bifurcated, with a prominent basal ridge protruding toward the lips; each probolae with furca elongate. Amphid openings ovoid. Stoma cephaloboid, robust, with short cheilostom with large and rounded cheilorhabdia, very reduced gymnostom with minute gymnorhabdia, and well developed stegostom with minute stegorhabdia and large dorsal tooth. Pharynx also cephaloboid, with subcylindrical pharyngeal corpus having procorpus and metacorpus well differentiated, both with similar length; pharyngeal corpus-isthmus junction well demarcated and isthmus narrower than corpus; basal bulb spheroid with valvular apparatus (grinder) well developed. Nerve ring at 70-81% of neck length, surrounding the isthmus. Excretory pore at 66-78% of neck length, at isthmus level. Deirid at 77-93% of neck length, at isthmus or basal bulb level. Intestine without distinct specializations but having narrower walls at anterior part. Female. Reproductive system monodephic-prodelphic, cephaloboid. Ovary long, without flexures with oocytes in only one row; oviduct very short; spermatheca well developed, swollen, 1.0-1.4 times the body diameter, with large spermatozoa; uterus with elongate tubular distal part having thicker walls and short proximal swollen part having thinner walls; vagina short, about one fourth of the body diameter; vulva a transversal slit; post-vulval uterine sac swollen, 1.4-2.2 times the corresponding body diameter long. Rectum shorter than the anal body diameter. Anus well developed, curved. Tail conical, spear-shaped, ending in an acute, conoid mucro, with cuticle slightly wider at posterior region. Phasmids located at 32-40% of tail length.
Male. Reproductive system monorchid, with testis ventrally reflexed anteriorly. Tail conical, ventrally curved with an acute mucro at the end. Phasmids located at 30-36% of tail length. Post-cloacal genital papillae five pairs, two at middle length of tail, one lateral and one ventrosublateral, and three near the tail tip, one subdorsal and two subventral. Spicules paired and symmetrical, ventrally curved having rounded manubrium, conoid calamus and ventrad curved lamina with reduced ventral velum and acute terminus. Gubernaculum well developed, almost straight with ventrad curved at its anterior part.

Localities and habitats
The new examined populations of N. lanceolatus have been found in coastal sand dunes from several Spanish localities and beaches (

Remarks
Eight specimens (two males, three females and three juveniles) from two localities were found with bacteria or fungi feeding on them. Thus, one specimen collected from Islantilla (province of Huelva) was found with the bacterium of the genus Pasteuria Metchnikoff, 1888 (Firmicutes, Pasteuriaceae) adhered at the cuticle of the nematode ( fig. 3e, g).
Also, two species of fungi have been found in the surface and inside the body of nematode specimens from Artola-Cabopino (province of Málaga). The fungus of the genus Catenaria Sorokin, 1876 (Blastocladiomycota, Blastocladiales) has been found in seven specimens of N. lanceolatus (figs 4a-g and 5a, b, g-k) appearing attached in groups of flagellate zoospores surrounding the oral, vulval and anal openings of the nematodes, generating long hyphae which penetrate inside the body along the digestive tract. Other fungus, found in two specimens, belonging to the genus Nematocida Troemel, Félix, Whiteman, Barrière & Ausubel, 2008 (Microsporidia, Minisporida), an obligate intracellular parasite, appears inside some intestinal cells ( fig. 4h-j). Unfortunately, it is unknown if these nematode specimens were parasitized in their natural habitat or during the extraction process, which maintains the soil samples in water for several days.

New geographical distribution of N. lanceolatus
After the original description provided by Abolafia & Peña-Santiago (2003), N. lanceolatus appears with a more extended distribution along the sand dunes of Atlantic and Mediterranean Spanish coasts. All populations examined of this species appeared in xeric areas, which have soils with very low humidity and some salinity, the Cabo de Gata zone (Southern Iberian Peninsula) being the most arid area (Capel-Molina, 1982) where the species has been found. Other additional localities have been sampled in coastal dunes from the north Iberian Peninsula, as well as numerous samples across the Iberian geography, in several habitats, and this species was not found. For this reason, it could be considered as a bioindicator of these xeric habitats. In the phylogenetic tree based on 18S rDNA ( fig. 6)  cancellatus (Thorne, 1925) Ruiz-Cuenca & Abolafia, 2020 (HM439765), Nothacrobeles hebetocaudatus Abolafia, Divsalar, Panahi & Shokoohi, 2014 (KJ08411) and Nothacrobeles spatulatus Tandingan De Ley, De Ley, Baldwin, Mundo-Ocampo & Nadler, 1999 (DQ145644), with which N. lanceolatus shares a similarity of 83.1% (113 bp differences), 83.4% (111 bp differences), 75.5% (161 bp differences) and 79.5% (137 bp differences), respectively. Also, in this phylogenetic tree, the sequences of the Spanish Nothacrobeles appear near to genera Acrobeles and Cervidellus, with similarities of 76.9% (154 bp differences) and 73.9% (175 pb differences), respectively.

Phylogenetic relationships of the Nothacrobeles species
The phylogenetic tree based on 28S rDNA fragment ( fig. 7) shows that the genus Nothacrobeles is clearly polyphyletic. Although only eight of the 21 species of the genus have been sequenced, the clades in the tree show an evolutionary development of the lip region. Thus, three species (N. abolafiai, N. cancellatus and N. hebetocaudatus), related with the genus Paracrobeles Heyns, 1968, have similar a lip region pattern appearing at the basal clades, all of which have with labial probolae smooth with long bifurcations and stipe swollen, probably a plesiomorphic condition. Nothacrobeles spatulatus, related with the genera Stegelleta Thorne, 1938 andStegellina Andrássy, 1984, has low labial probolae lacking bifurcations and pinnae. Two species (N. borregi and N. sonorensis) have bifurcated labial probolae with smooth bifurcations and low stipe with an expansion on the outer side. Another two species (N. lanceolatus and N. triniglarus), related with the genera Cervidellus and Acrobeles, have labial probolae with long or short bifurcated prongs and basal concave expansion on the outer side, both species with pinnae. This last morphological pattern is a probable apomorphic condition, appearing also in species of the genus Acrobeles, while some species of the genus Cervidellus such as Cervidellus doorsselaeri (De Clerck & De Ley, 1990) Boström & De Ley, 1996, morphologically related with Acrobeles, present labial probolae with incipient lateral pinnae.