Two new species of the genus Sectonema Thorne, 1930 (Nematoda, Dorylaimida, Aporcelaimidae) from Iran, with new insights into its evolutionary relationships

Abstract Two new species of the genus Sectonema found in northern Iran are characterized, including morphological descriptions and molecular (18S-, 28S-rDNA) analyses. Sectonema tehranense sp. nov. is distinguished by its 7.22 – 8.53 mm long body, lip region offset by constriction and 24 – 31 μm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, mural tooth 15.5 – 17 μm long at its ventral side, neck 1091 – 1478 μm long, pharyngeal expansion occupying 61 – 71% of the total neck length, female genital system diovarian, uterus simple and 3.9 – 4.2 times the corresponding body diameter long, transverse vulva (V = 49 – 59), tail short and rounded (44 – 65 μm, c = 99 – 162, c’ = 0.6 – 0.8), spicules 111 – 127 μm long, and 7 – 10 spaced ventromedian supplements with hiatus. Sectonema noshahrense sp. nov. displays a 4.07 – 4.73 mm long body, lip region offset by constriction and 23 – 25 μm wide with perioral lobes and abundant setae- or cilia-like projections covering the oral field, odontostyle 14 – 14.5 μm long, neck 722 – 822 μm long, pharyngeal expansion occupying 66 – 68% of the total neck length, female genital system diovarian, uterus simple and 2.4 – 2.7 times the corresponding body diameter long, transverse vulva (V = 54 – 55), tail convex conoid (39 – 47 μm, c = 91 – 111, c’ = 0.8 – 0.9), spicules 82 μm long, and seven spaced ventromedian supplements with hiatus. Molecular analyses confirm a maximally supported (Epacrolaimus + Metaporcelaimus + Sectonema) clade and a tentative biogeographical pattern, with sequences of Indolamayan taxa forming a clade separated from those of Palearctic ones. Parallel or convergent evolution processes might be involved in the phylogeny of the species currently classified under Sectonema. This genus is certainly more heterogeneous than previously assumed.


Introduction
The genus Sectonema Thorne, 1930 is a diverse, widely distributed, free-living dorylaimid (order Dorylaimida) taxon.Its last published census (Álvarez-Ortega & Peña-Santiago 2019; see also Peña-Santiago 2021) consisted of 26 valid species, but, more recently, this number was increased with the inclusion of S. reyesi (Pedram et al. 2012), transferred from Epacrolaimus Andrássy, 2000 by Peña-Santiago (2023).Almost a cosmopolitan genus, it has been recorded in Afrotropical (Mauritius, South Africa), Australian (Hawaii, New Zealand, Samoa), Indomalayan (India, Vietnam), Nearctic (United States), Neotropical (Colombia, Venezuela), and Palearctic (Belgium, Bulgaria, Egypt, Georgia, Germany, Hungary, Iran, Italy, Netherlands, Romania, Russia, South Korea, Spain, Switzerland, Turkey, United Kingdom) enclaves, where it dwells soils and freshwater habitats.Sectonema representatives are large to very large nematodes, ranging from 2.00 to 10.45 (often 4 -6) mm long, and active predators.The morphology, phylogeny, and taxonomy of the genus were studied by Álvarez-Ortega and Peña- Santiago (2019), who noted that its protruding stomatal structure might be either a reduced odontostyle or a mural tooth, confirmed its monophyly with molecular (28S-rDNA) analyses, identified two species groups with possible geographical projection, and maintained some discrepancies between morphological and molecular data.
Iranian nematode fauna currently consists of three Sectonema representatives, namely S. reyesi, originally described by Pedram et al. (2012), and S. heynsi Altherr 1968 and S. demani Altherr, 1965, both recorded by Abdi Gonbary et al. (2018).Nevertheless, two new populations of the genus were collected during a nematological survey conducted in northern Iran, and their study revealed that they belonged to two undescribed species.Thus, this contribution aims to characterize these unknown taxa and provide new insights into the taxonomy and evolutionary relationships of the genus.

Soil sampling, nematode extraction, and morphological characterization
Twenty-five soil samples were collected from the plane trees in Tehran, Tehran province, and eucalyptus trees in Noshahr, Mazandaran province, northern Iran.The samples were placed in plastic bags, transferred to Tarbiat Modares University's nematology laboratory, and maintained at cool temperature conditions.Large body-sized nematode specimens were directly extracted from the soil samples using 20 and 60 mesh sieves (US standard mesh numbers, their openings equal to 841 and 250 μm, respectively).Nematodes of interest were handpicked under a Nikon SMZ1000 (Nikon, Tokyo, Japan) dissecting microscope, heat-killed by adding boiling 4% formalin solution and transferred to anhydrous glycerin, according to De Grisse (1969).The permanent slides were made, and the recovered specimens were studied/drawn morphologically using a Nikon E6000 (Nikon) light microscope equipped with a drawing tube.
The PCR mixture (35 mL) contained the following: 17.5 μL Taq DNA polymerase 2× Master Mix RED, 2-mM MgCl2 (Ampliqon, Odenese, Denmark), 9.5 μL distilled water, 1.5 μL of each primer, and 5 μL of DNA template.The thermocycling program for amplification of the ribosomal markers was as follows: denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30s, annealing at 52°C for 40s, and extension at 72°C for 80s.The thermocycling program for amplifying the cytochrome c oxidase subunit I (COI) mtDNA was similar to the previous cycle, except the annealing temperature was set to 36°C.A final extension was performed at 72°C for 10 min for both cycles.The PCR products were sequenced using the same primers used during amplification.

Phylogenetic analyses
D2 -D3 domains of the 28S rDNA and 18S rDNA sequences of the recently recovered Sectonema populations were obtained in this study.These sequences and other sequences belonging to species of Dorylaimida from GenBank, were used for phylogenetic analyses.Outgroup taxa for each dataset were chosen following previously published studies (Álvarez-Ortega & Peña-Santiago 2019;Álvarez-Ortega et al. 2021;Peña-Santiago & Castillo 2022).Multiple sequence alignments of the two ribosomal genes were made using the fast Fourier transform (FFT)-NS-2 algorithm of MAFFTversion 7.450 (Katoh et al. 2019).Sequence alignments were visualised using BioEdit (Hall 1999) and manually edited.Phylogenetic analyses of the sequence datasets were based on Bayesian inference (BI) using MrBayes 3.1.2(Ronquist & Huelsenbec 2003).The best-fit model of DNA evolution was obtained using JModelTest V.2.1.7 (Darriba et al. 2012) with the Akaike information criterion (AIC).The best-fit model, the base frequency, the proportion of invariable sites, the gamma distribution shape parameters, and substitution rates in the AIC were then used in MrBayes for the phylogenetic analyses.BI analyses were performed under a general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) for the D2 -D3 expansion segments of the 28S rRNA and 18S rRNA genes.All Bayesian analyses were run separately per dataset with four chains for 4 × 10 6 generations.The Markov chains were sampled at intervals of 100 generations.After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for in-depth analyses.The topologies were used to generate a 50% majority-rule consensus tree.Posterior probabilities (PP) were given on appropriate clades.Trees from all analyses were visualised using FigTree software version 1.42 (Rambaut 2014).A combined analysis of the three ribosomal genes was not undertaken because some sequences were unavailable for all species.

Results
Sectonema tehranense sp.nov.(Figs 1-3, morphometrics in Table 1) Material examined: Seven females and three males from one location, in good state of preservation.
A: Very slender (a = 51 -75) nematodes of very large size, 7.22 -8.53 mm long.Body cylindrical, tapering toward both ends, but more so towards the anterior end as the tail is short and rounded.Upon fixation, the habitus curved ventrad, C-to G-shaped.Cuticle dorylaimid, 8 -8.5 μm thick at anterior region and mid-body, and 18 μm at tail, three-layered, especially appreciable at caudal region, consisting of a smooth, thin outer layer, much thicker intermediate layer with weak radial striation, and a thinner inner layer.Body pores are only conspicuous in the cervical region, where two or three ventral and dorsal pores are perceptible.Lateral chord 15 -27 μm wide, occupying 12 -19% of mid-body diameter.Lip region offset by deep constriction, 2.8 -3.4 times as wide as high, up to one-fourth (23 -25%) of body diameter at neck base, lips separated, with perioral lobes, oral field covered of abundant setae-or cilia-like projections.Amphid fovea stirrup-shaped, its aperture 13 -15 μm or ca one-half (43 -52%) of lip region diameter.Cheilostom is short and wide, with less appreciable walls.Stomatal protruding element is a ventral, somewhat deltoid, mural tooth-like structure, its ventral side hardly longer than one-half (53 -65%) of the lip region diameter, the dorsal one slightly longer than the ventral, 0.65 -0.73% of lip region diameter.The guiding ring is shallow, visibly plicate, located at 8 μm or 29% of the lip region diameter from the anterior end.Odontophore is linear, lacking differentiation, and is 1.9 -2.5 times longer than mural tooth.Pharynx entirely muscular, very gradually enlarging into the basal expansion that is 9.4 -13.7 times as long as wide, 5.9 -7 times the lip region diameter at neck base, and occupies ca two-thirds (61 -71%) of the total neck length, gland nuclei located as follows: DO = 41 -42, DN = 44 -46, S 1 N 1 = 50, S 1 N 2 = 62 -63, S 2 N = 79 -80.Pharyngo-intestinal junction consisting of a short, conical to conoid, 32 -34 × 27 -29 μm cardia, its junction to pharyngeal base surrounded by weak ring-like element.F: Genital system diovarian, with both branches equally developed, the anterior 747 -965 μm long or 10 -12% of body length, the posterior 729 -912 μm or 10 -12% of body length.Ovaries are variably large, often reaching and surpassing the M: Prerectum 5.2, cloaca 1 -1.2 times longer than body diameter at the level of cloacal aperture.Genital system is diorchic, with opposite testes.Sperm cells (once inside the uterus) are spindle-shaped to somewhat ovoid, 8 × 2.5 μm.In addition to the ad-cloacal pair, situated at 21 -25 μm from the cloacal aperture, there is a series of 7 -10 spaced (21 -28 μm apart) ventromedian supplements, the last of them located at 128 -138 μm from the ad-cloacal pair, with a distinct hiatus.Spicules dorylaimid, 4.4 times longer than wide, 1.7 times the body diameter at cloacal aperture: head 23 -24 μm long or 19% of total spicule length, with its dorsal side appreciably longer than the ventral one; median piece occupying almost half (54%) of spicule maximum width; posterior end 28 -29 μm wide; ventral hump and hollow conspicuous, the former situated at 46 -52 μm or 43% of spicule anterior end; curvature 130º.Lateral guiding piece 26 -32 μm long, 8 times as long as wide, tapering at its posterior end.The caudal region is similar to that of females.

Type material
Female holotype, four female paratypes and two male paratypes were deposited with WaNeCo collection, Wageningen, The Netherlands (http://www.waneco.eu/).One female paratype and one male paratype at nematode collection of the University of Jaén, Spain.

Etymology
The species name refers to Tehran, the Iranian province where it was collected.

Sectonema noshahrense sp. nov. (Figs 4 & 5, morphometrics in Table 1)
Material examined: Four females and one male from one location, in good state of preservation.
A: Very slender (a = 41 -63) nematodes of large size, 4.07 -4.73 mm long.The body is cylindrical, tapering toward both ends, but more toward the anterior end as the tail is convex conoid.Upon fixation, habitus curved ventrad, C-to G-shaped.Cuticle dorylaimid, 9 μm thick at the anterior region, 8 μm in mid-body, and 14 μm at the tail, three-layered, especially appreciable at the caudal region, consisting of a smooth, thin outer layer, much thicker intermediate layer with weak radial striation, and a thinner inner layer.Body pores are only conspicuous in the cervical region, where two or three ventral and dorsal pores are present.Lateral chord
Lip region offset by deep constriction, 2.5 -2.6 times as wide as high, ca one-fourth (25 -27%) of body diameter at neck base, lips separated, with perioral lobes, oral field covered of abundant setaeor cilia-like projections.Amphid fovea stirrup-shaped, its aperture 16 -17 μm or ca one-half (56%) of lip region diameter.Cheilostom is short and wide, with less appreciable walls.Stomatal protruding element a reduced odontostyle, 3.2 times as long as wide (4.5 μm at its base), hardly longer than one-half (58 -59%) of lip region diameter, and 0.35% of body length.Guiding ring shallow.Odontophore is linear, lacking differentiation, and is 2.1 times longer than odontostyle.Pharynx is entirely muscular, very gradually enlarging into the basal expansion that is 9.6 -9.9 times as long as wide, 5.1 times the body diameter at the neck base, and occupies ca two-thirds (66 -68%) of the total neck length, gland nuclei located as follows: DO = 43, DN = 46, S 1 N 1 = 51, S 1 N 2 = 62, S 2 N = 81.Pharyngo-intestinal junction consists of a short, rounded, 19 × 19 μm cardia, its junction to the pharyngeal base surrounded by a weak ring-like element.F: Genital system diovarian, with both branches equally developed, the anterior 425 -438 μm long or 10 -11% of body length, the posterior 362 -462 μm or 9 -11% of body length.Ovaries are comparatively small, not reaching the oviduct-uterus junction, the anterior 129 -177, the posterior 127 -188 μm long, with oocytes first arranged in two or more rows and then in only one row.Oviduct joining subterminally the ovary, 165 -222 μm long or 1.3 -1.8 body diameters, consisting of a distal slender portion made of prismatic cells and a moderately developed proximal pars dilatata with visible lumen inside.A strong narrowing, surrounded by a distinct muscle sphincter, separates oviduct and uterus.Uterus a simple tube-like structure, 212 -275 μm long or 2.4 -2.7 times the corresponding body diameter.Vagina extending inward 52 μm, reaching 51% of body diameter: pars proximalis 35 × 30 μm, with somewhat sigmoid walls surrounded by weak circular musculature, pars refringens consisting of (in lateral view) two close together, drop-shaped sclerotized pieces 9.5 × 6.5 μm, and with a combined width of 15.5 μm, and pars distalis 8 μm long.Vulva a transverse slit.Prerectum 3.5 -4, rectum 0.8 -1 times the body diameter at the level of cloacal aperture.Caudal region convex conoid; inner core reaching up to 70% of tail length, with cuticle visibly thickened at tip; caudal pores two pairs, one lateral, another subdorsal.M: Genital system diorchic, with opposite testes.In addition to the ad-cloacal pair of supplements, located at 18.5 μm from the cloacal aperture, there is a series of seven irregularly spaced supplements, the posteriormost of which lying out the range of spicules, at 230 μm from the ad-cloacal pair.Spicules are robust and massive, especially in their posterior half, the head occupying 14% of the total length, the median piece 10 -15 μm wide.Lateral guiding pieces 21 μm long.Tail similar to that of females.

Separation from its relatives
In having a large size (less than 5 mm long), a reduced odontostyle comparatively short as protruding stomatal structure, and abundant setae-or cilia-like projections covering the oral field, the new species is very similar to S. barbatum Heyns, 1965, at present only known to occur in the United States (Heyns 1965).Nonetheless, both species differ in the length of their reduced odontostyle (14 -14.5 μm or up to three-fifths of lip region diameter vs 16 μm or three-fourths of lip region diameter in S. barbatum) and their neck (722 -822 μm, b = 5.6 -5.7 vs 589 μm, b = 6.6, in females), and their caudal region (c' = 0.8 -0.9 vs c' = 1, with regular inner core vs inner core with a terminal projection almost reaching the tail tip).The record of S. barbatum in Switzerland (Loof & Coomans 1970) is questionable as the neck of the only female studied was 970 μm, significantly different from that of type specimen, ca 589 μm.

Type material
Female holotype, two female paratypes and one male paratype deposited with with WaNeCo collection, Wageningen, The Netherlands (http://www.waneco.eu/).One female paratype at nematode collection of the University of Jaén, Spain.

Etymology
The specific epithet refers to Noshahr, the city where the new species was collected.

Derived from morphological observations
The two Iranian species herein described belong to a group of largeto very large-sized (more than 4 mm long) Sectonema representatives.They perfectly fit the general morphology of the genus (Álvarez-Ortega & Peña-Santiago 2019), including the two basic types of protrusible stomatal structure, either a reduced odontostyle (RO, S. noshahrense sp.nov.) or a mural tooth (MT, S. tehranense sp.nov.).The drastic reduction of the size of the stomatal protruding structure (and probably the corresponding increase of its aperture) is a remarkable apomorphic trait of the Sectonema morphological pattern, probably an autapomorphy within Dorylaimina.It is also its most recognizable feature to separate it from its closest relatives, for instance, Aporcelaimus Thorne & Swanger, 1936, Metaporcelaimus Lordello, 1965 and Epacrolaimus Andrássy, 2000 (see below), as these genera bear a typical odontostyle with both dorsal and ventral arms well perceptible and variably large aperture.Heyns (1965) illustrated the evolutionary process of reducing the size of odontostyle in aporcelaims (Aporcelaimidae Heyns 1965), assuming that Sectonema species derived from an ancestor that bore typical odontostyle.On this matter, two issues deserve attention.On the one hand, if this process occurred only once or happened in several taxa and/or with different intensity throughout the evolutionary history of the dorylaims/aporcelaims.On the other hand, if the two types of reduced stomatal structure (reduced odontostyle and mural tooth) represent two different (alternative) results of the reducing process, or they are two successive stages of the process.

Derived from molecular analyses
The results of molecular analyses of 28S and 18S sequences are presented in the trees of Figures 6 and 7, respectively.Previous findings (Álvarez-Ortega & Peña-Santiago 2019; Peña-Santiago & Castillo 2022) are confirmed in both cases.On the one hand, 18S tree shows that Sectonema and Epacrolaimus form a maximally (100%) supported clade, but the internal relationships are less satisfactorily resolved than in those provided by Peña- Santiago and Castillo (2022).On the other hand, 28S tree shows better resolution and provides additional information of interest.Thus, a (Sectonema + Epacrolaimus + Metaporcelaimus) clade is maximally (100%) supported, but internal resolution is imperfect.Sectonema sequences are split into three highly/maximally supported subclades.One of them, constituted by sequences of three Indomalayan (Vietnamese) species, appears as the sister group of

Integrative approach and concluding remarks
The genus Sectonema is a typical case of nematode taxon in which morphological and molecular data do not match.It is easily recognizable and distinguishable from its most relatives by the peculiar structure of its protruding stomatal structure, either a reduced odontostyle or a mural tooth (see above).Available molecular analyses show that a (Epacrolaimus + Metaporcelaimus + Sectonema) clade is maximally supported and that Sectonema sequences are split into three subclades, one of them being the sister group of Epacrolaimus clade.The study of the two Iranian species herein described reveals two interesting results.First, the confirmation of a tentative biogeographical pattern, previously observed (Álvarez-Ortega & Peña-Santiago 2019), with sequences of Indolamayan taxa forming a clade separated from those of the Palearctic ones and the sister group of Epacrolaimus sequences.Second, the process resulting in the reduction of odontostyle probably occurred at least twice throughout the evolutionary history of the genus, as it is observed in both biogeographical groups because these groups include sequences/species (Fig. 7) bearing reduced odontostyle and mural tooth.This means that processes of parallel or convergent evolution might be involved in the phylogeny of the species currently classified under Sectonema, that this genus certainly is more heterogeneous than previously assumed, and that further research is required to elucidate its taxonomy and systematics.
Competing interest.The authors have no competing interests.
Ethical standard.The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.

Figure 6 .
Figure 6.Phylogenetic relationships of two new species of the genus Sectonema Thorne, 1930 with species of Dorylaimida.Bayesian 50% majority rule consensus tree inferred from D2 -D3 expansion segments of 28S rRNA sequence alignment under the GTR + I + G model.Posterior probabilities >0.70 are given for appropriate clades.Newly obtained sequences in this study are shown in bold, and the coloured box indicate the clade association of the studied species.Scale bar = expected changes per site.

Table 1 .
Main morphometrics of two new species of the genus Sectonema Thorne, 1930 from Iran Measurements in μm except L in mm, and, when possible, in the form: average ± SD (range)