Morphological and molecular characterization of Aporcella femina sp. n. (Dorylaimida, Aporcelaimidae) from Nigeria

Abstract A new species of the genus Aporcella collected from a watermelon field in Nigeria is described, including its morphological and molecular (small subunit (SSU) and large subunit (LSU) ribosomal DNA (rDNA)) characterization. Aporcella femina sp. n. is distinguished by its 3.21–3.64 mm-long body, inner cuticle layer with fine but distinct transverse striation, lip region offset by deep constriction, 22–25 μm broad, odontostyle 20–26 μm, neck 661–811 μm long, pharyngeal expansion occupying 52–56% of the total neck length, female genital system didelphic–amphidelphic, uterus 191–350 μm or 1.9–3.3 mid-body diameters long, V = 52–57, tail short and convex conoid (35–48 μm, c = 72–98, c′ = 0.7–0.9) and males absent. Phylogenetic analyses based on the partial sequence of SSU and LSU (D2–D3) rDNA revealed a close relationship of A. femina sp. n. with other Aporcella species, confirming the monophyly of the genus as well as its association to a clade made of several taxa characterized by the absence of pars refringens vaginae.

From a 2016 survey of nematode fauna associated with watermelon in Nigeria, specimens of an Aporcella population that belong to an undescribed species were recovered. This would represent a novel geographical record for the genus, and, by means of its molecular analysis, offers the chance to confirm previous findings about its monophyly and phylogeny.

Extraction and processing of nematodes
Nematode specimens were recovered from rhizosphere soil samples that were collected from watermelon fields in south-west Nigeria during 2016. Extraction of nematodes from soil followed a modified pie pan method (Coyne et al., 2007). Nematodes were fixed in a hot 4% formaldehyde solution for morphological observations (Nico et al., 2002) and subsequently mounted in anhydrous glycerine as permanent slides (De Grisse, 1963), while others were stored in dimethyl sulphoxide, disodium ethylenediamine tetra-acetic acid and saturated sodium chloride (abbreviated here as DESS) solution for molecular analysis. Morphological features of nematodes were observed, measured and photographed using an Eclipse 80i microscope (Nikon, Tokyo, Japan) equipped with differential interference contrast optics, a drawing tube (camera lucida) and a DS digital camera (Nikon, Tokyo, Japan).

Molecular identification
Fixed specimens in DESS were rinsed using double-distilled water, and two specimens were transferred to individual 1.5 ml tubes containing 15 μl nuclease-free water. DNA were extracted using chelex-100 by adding 20 μl of chelex (5% w/v) and 5 μl proteinase K to each tube, followed by incubation at 57°C for 120 min and at 95°C for 10 min (Rashidifard et al., 2019).

Phylogenetic analyses
The new SSU and LSU sequences obtained for the new species were compared to other available sequences in the GenBank using the BLASTN search tool. One data set was prepared for  each of the SSU and LSU regions and taxa selection was carried out according to Rashidifard et al. (2020). The available sequences as well as outgroups in the SSU data set were aligned using the MUSCLE alignment tool (Edgar, 2004) in Geneious Prime® 2020.2.3 (www.geneious.com), while the LSU data set was aligned using the E-INS-I algorithm in the MAFF alignment tool (Katoh & Standley, 2013). According to the jModeTest 2.1.10 (Darriba et al., 2012), the Hasegawa-Kishino-Yano with a gamma distribution (HKY + G) was the best substitution model for the SSU dataset. However, the General Time Reversible with proportion of invariable sites and a gamma distribution (GTR + I + G) was selected as the most appropriate model for the LSU dataset. Bayesian inference was conducted in MrBayes v3.1.2 (Ronquist & Huelsenbeck, 2003) implemented in Geneious Prime® 2020.2.3 (www.geneious.com). The chain was running 3 × 10 6 generations for both the SSU and LSU data sets, and after discarding the 25% burn-in samples, the remaining samples were used for further analyses. The Markov chain Monte Carlo (MCMC) algorithm was implemented to calculate the posterior probabilities (PB) for each of the phylogenetic trees (Larget & Simon, 1999)  using the 50% majority rule. The mononchid taxa were used as outgroups for both the SSU and LSU phylogenies.

Material examined
Ten females in variable but generally good state of preservation (figs 1-3).

Measurements
All measurements are presented in table 1.

Description
Female. Moderately to slender (a = 28-38) nematodes of large size, 3.21-3.64 mm long. Body cylindrical, tapering towards both ends but substantially towards the anterior region, while the tail is short and rounded. Upon fixation, habitus regularly curved ventrad, C-to G-shaped. Cuticle two-layered, very thick throughout the entire body, 4-7 μm in anterior region, 7.5-9.5 μm at mid-body and 7.5-11.5 μm on tail, consisting of a thin outer layer with nearly smooth surface, and a thicker inner layer that displays a fine but distinct transverse striation ( fig.  2g). Lateral chords narrow, 6-17 μm wide or 6-19% of mid-body diameter. Ventral and dorsal pores restricted to cervical region, two or three in number at each side at odontostyle-odontophore level; lateral pores small. Lip region offset by a distinct constriction, 3.3-3.8 times wider than high and less than one-third (22-29%) of body diameter at neck base; lips separate, with moderately protruding papillae. Amphidial fovea stirrup-shaped, its opening 9-10.5 μm, less than one-half (38-46%) of lip region diameter. Cheilostom broad, 10.5-14 μm long, with thick walls. Odontostyle strong, 3.8-4.3 times longer than wide, barely shorter (0.9-1.0 times) than lip region diameter and 0.58-0.73% of total body length, with aperture 13-17 μm long or c. two-thirds (58-76%) of total length. Guiding ring simple but distinct, plicate. Odontophore linear, rod-like, 1.9-2.4 times longer than odontostyle. Pharynx entirely muscular, enlarging very gradually, with its basal expansion 5.8-9.0 times as long as wide, 3.1-4.5 times the body diameter at neck base and occupying 52-56% of total neck length; pharyngeal gland nuclei located as follows (  Measurements in μm except L in mm, and in the form average ± standard deviation (range). a 1: Botha & Heyns (1990); 2: De Bruin & Heyns (1992). b Calculated from other morphometrics.
distalis well-developed, 10-14 μm long; pars refringens absent. Vulva a slightly posterior transverse slit. Prerectum 2.4-4.0, rectum 0.8-1.5 times the anal body diameter long. Tail short, convex conoid, its ventral side weakly straighter than the dorsal side; caudal pores two pairs, close together, at the posterior half of tail, one lateral, another sublateral. Male. Unknown. Females do not contain sperm cells.

Molecular characterization
After sequencing and editing, two totally (100%) identical sequences per each locus (SSU and LSU) were obtained; therefore, only one sequence was used for analysis and deposited into the NCBI GenBank: a 745 bp-long LSU sequence (accession number MW237838) and another 855 bp-long SSU sequence (accession

Type material
Female holotype and nine female paratypes, deposited in the nematode collection of the University of Jaén, Spain.

Etymology
The specific epithet is a Latin term meaning woman or female, and refers to the existence of only females in this species.
The new species described herein displays intermediate morphometrics between two South African speciesnamely, A. parapapillata and A. pseudospiralisall of them described based on relatively few specimens. Aporcella femina sp. n. is very similar to A. pseudospiralis, but, as mentioned above, they differ in their overall size and especially in tail length. Therefore, the possibility of co-specificity could be raised, and the noted differences possibly due to geographical variations. The absence of molecular data for A. parapapillata and A. pseudospiralis currently prevents any molecular comparison, which, when available, would clarify any ambiguity. However, the available morphological information clearly distinguishes the three species from each other, supporting a separate status for them.
The evolutionary relations of the new species, as derived from the molecular analyses based on the partial sequence of SSU and LSU rDNA, are represented in figs 3 and 4. According to the SSU phylogeny, the new species is in a highly supported (BPP: 97%) sister relation with Aporcella vitrinus. Furthermore, Bayesian tree using partial LSU sequence shows that the new species forms part of a fully supported (BPP: 100%) subclade with Aporcella charidemiensis and A. vitrinus, the former being the closest relative, which is recorded from Spain. A highly supported (BPP: 94%) Aporcella clade is made of this subclade along with three Aporcella sequences belonging to A. simplex. Therefore, the present results confirm previous findings (for instance, see Naghavi et al., 2019;Vazifeh et al., 2020) about both the monophyly of Aporcella, and the belonging of Aporcella sequences to a large clade morphologically characterized by the absence of pars refringens vaginae, which also includes discolaims in the tree provided herein.
Financial support. One author (R.P.S.) is grateful for the financial support received from the scientific project (ref.: Action 1-PAIUJA 2019-2020: EIRNM02-2017), University of Jaén. Infrastructure and partial funding by the Nematology Unit at North-West University of South Africa is also acknowledged.