Molecular and morphological characterisation of Longidoruspolyae sp. n. and L.pisi Edward, Misra & Singh, 1964 (Dorylaimida, Longidoridae) from Bulgaria

Abstract Longidoruspolyaesp. n., a bisexual nematode species found in the rhizosphere of pear tree (Pyruscommunis L.), is described and characterised using an integrative approach. The new species has a female body length of 6.8–9.1 mm; a comparatively long odontostyle (114.0–127.5 μm); a narrow lip region (14.0–15.5 μm), anteriorly flattened and almost continuous with the body profile; pocket-like amphidial pouches long, deeply bilobed, and slightly asymmetrical, a guide ring at 37–42 μm from the anterior end; normal arrangement of pharyngeal glands; and a short bluntly rounded to hemispherical tail. Four juvenile stages identified: the first stage with a digitate tail, and the second and subsequent stages with a bluntly rounded tail. Males have one adcloacal pair and a row of 10 or 11 single ventromedian supplements; spicules 71.0–74.5 μm long. Based on morphometric data, the new species belongs to a group of species spread over Europe (L.arthensis, L.silvae, L.uroshis,), Iran (L.kheirii), and Syria (L.pauli), which share common characters such as amphidial fovea, lip region and tail shapes, similar odontostyle and body length, and similar first-stage juvenile tail shape. Codes for identifying the new species are A5, B2, C34, D3, E3, F45, G12, H1, I2, J1, K7. The phylogenetic analysis based on D2-D3 expansion domains of the rRNA gene revealed that the new species has the closest relationships with L.athesinus from Italy and three unidentified Longidorus spp. from USA (Longidorus sp. 1, Longidorus sp. 2, and Longidorus sp. 6). New morphometric and molecular data (18S rRNA gene, ITS1-5.8S-ITS2 regions and D2-D3 28S rRNA gene sequences) for three populations of L.pisi from Bulgaria were obtained and variations between populations are discussed.


Introduction
Longidorus Micoletzky, 1922 is the second most diverse genus within family Longidoridae (Thorne, 1935) Meyl, 1961 occurring in all continents except Antarctica. At present, it contains 170 species (Barsalote et al. 2018;Gharibzadeh et al. 2018;Xu et al. 2018). So far, 16 species have been recorded in Bulgaria (Peneva et al. 2012(Peneva et al. , 2013. During the study of longidorids in croplands with organic and conventional management, an unknown species was recovered from the rhizosphere of a pear tree in north-central Bulgaria. Further, several populations of L. pisi Edward, Misra & Singh, 1964 were found in a tobacco field and two vineyards from the southwestern part of the country. Longidorus pisi was originally described from the rhizosphere of Pisum sativum L. growing at the Allahabad Agricultural Institute (now Sam Higginbottom University of Agriculture) in Uttar Pradesh, India . It was subsequently reported from various countries in Asia and Africa. Longidorus latocephalus Lamberti, Choleva & Agostinelli, 1983, a very similar species to L. pisi, was described from southwestern part of Bulgaria where it was associated with various crops. Choleva et al. (1991) proposed that L. latocephalus is a junior synonym of L. pisi. However, Navas et al. (1993), using the same data and those of Brown et al. (1982), carried out comprehensive analyses and distinguished two separate groups of L. latocephalus (8 populations from Bulgaria) and L. pisi (3 populations from India, 2 from South Africa, and 1 from Malawi). As a result, the taxonomic status of these two species has been debated in several subsequent studies (Robbins et al. 1995;Lamberti et al. 1996Lamberti et al. , 1997Chen et al. 1997). Although there are observed differences in certain metrical data and amphid shape, Loof and Chen (1999) accepted the synonymy of L. latocephalus and L. pisi in their revised polytomous key. In three recent studies, sequences of D2-D3 28S rRNA or the coxI gene for populations from Greece, South Africa, and Iran have been provided, but, only the last population was characterised with morphological and morphometric data (He et al. 2005;Pedram et al. 2012;. The present study aims to characterise morphologically and molecularly: i) an unknown species of genus Longidorus and ii) populations of L. pisi from Bulgaria, and iii) to evaluate these species' phylogenetic relationships by using 18S rRNA and D2-D3 expansion domains of the 28S rRNA genes.

Sampling, nematode isolation and processing
The Longidorus specimens examined originated from various croplands in Bulgaria: Balgarene village (private garden, Pyrus communis L. tree), Petrich (a small field of Nicotiana tabacum L.), and two vineyards, near Sandanski (Polenitsa village, small-scale management) and Kromidovo village (organic farm). Nematodes were isolated from soil samples by a decanting and sieving technique (Cobb 1918). The recovered Longidorus specimens were heat killed at 55 °C for 2 minutes, fixed in a 4% formalin/1% glycerol mixture, processed to anhydrous glycerol (Seinhorst 1959), and mounted on glass microscope slides. Drawings were prepared using an Olympus BX51 compound microscope with differential interference contrast (DIC). Microphotographs and selected measurements were taken with an Axio Imager.M2 Carl Zeiss microscope, digital camera (ProgRes C7), and CapturePro 2.8 software (Jenoptic). Measurements were made using a system of a light microscope (Olympus BX41), digitising tablet (CalComp Drawing Board III) and Digitrak 1.0f programme (Philip Smith, the John Hutton Institute, Dundee, UK). The alpha-numeric codes of polytomous identification key of the genus Longidorus (Chen et al. 1997) and modified partial polytomous key (Peneva et al. 2013) were used for the morphological species delimitation.

DNA extraction, amplifications and sequencing
The genomic DNA extraction, amplification, and sequencing of single female specimens from three populations of L. pisi were carried out independently in two laboratories: one at the Institute for Sustainable Plant Protection, Bari, Italy and two at the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria (IBER-BAS). Protocols used in both laboratories are presented previously (Groza et al. 2017). DNA analyses of L. polyae sp. n. were done in the IBER-BAS. One female, one male, and two first-stage juveniles were used for DNA extraction, amplification, and sequencing. The amplified products were sequenced by Eurofins MWG Operon, Germany. Sequences were deposited in GenBank with the following accession numbers: MK172047 and MK172049 for 18S rRNA gene of L. polyae sp. n. and L. pisi, respectively, MK172046 for D2-D3 expansion domains of 28S rDNA of the new species, and MK172048 and LR032064-65 for L. pisi. The partial 18S-ITS1-5.8S-ITS2 ribosomal segment was also sequenced for one population of L. pisi (LR032063, Petrich).

Sequence and phylogenetic analyses
The 18S and D2-D3 expansion segments of the 28S rRNA gene sequences were compared with those of other nematode species deposited in the GenBank database using the BLASTn similarity search tool. The homologous sequences nearest to those of the new species were aligned using the GUIDANCE2 Server (http://guidance.tau.ac.il/) with default parameters (Sela et al. 2015) and manually trimmed and edited in Mega 6 (Tamura et al. 2013). Pairwise sequence identities/similarities were computed using the Sequence Manipulation Suite online (http://www.bioinformatics.org/sms2/) (Stothard 2000). The Bayesian Inference (BI) algorithm implemented in MrBayes 3.2.5 was used for phylogenetic relationships reconstructions (Huelsenbeck and Ronquist 2001;Ronquist et al. 2012). For further details, see Lazarova et al. (2016). Female. Body assuming a spiral shape. Lip region narrow, 5-6 μm high, continuous with body profile, anteriorly flattened. Labial papillae, especially second circle, prominent, changing slightly the body contour. Cuticle 4-5 μm thick at postlabial region, 4-5 μm along the body, and 9-10 μm on tail posterior to anus. Guide ring 4-6 μm wide. Body pores conspicuous, 1 lateral pore anterior to or at the level of guide ring, 2 or 3 along odontostyle, 1 or 2 along odontophore, 3-5 in narrow part of the pharynx and 2-4 in bulb region as well as none dorsal and 5-6 ventral in pharyngeal region; numerous lateral pores observed along the rest of body. Amphidial fovea prominent, deeply bilobed, lobes long, slightly asymmetrical, amphidial aperture assumed to be a minute pore, hardly visible under light microscope. Odontostyle very slender, 1.5-2 μm wide at base. Two nerve rings observed, the first at some distance behind the odontophore and at 246.3 ± 11.1 (230-254) μm from anterior end, the second, more prominent, behind the first one at 354.8 ± 48.1 (327-440) μm from anterior end. Pharyngo-intestinal valve broadly rounded. Normal arrangement of pharyngeal glands: nuclei of the dorsal and ventrosublateral glands situated at 22.7-28.5 % and 54.3-58.5 % (n = 5) of the distance from anterior end of the bulb. Dorsal gland nuclei 2-2.5 μm in diameter, subventral gland nuclei 3-4 μm in diameter. In odontophore area of one female a small rudimentary odontostyle tip (vestigium) observed pointing forward. Peculiar crystalloid bodies of various sizes and shapes (mostly rod-like) found in the intestine of all females. Prerectum 465-497.5 μm long and rectum 33-42 μm or 0.6-0.8 of body diameter at anus. Tail bluntly conoid, rounded to hemispherical. Two pairs of lateral caudal pores. Vagina extending to ca. half the corresponding body width. Pars distalis vaginae 20-26 μm long; pars proximalis vaginae 21-27 μm long, thick walled. Uterus bipartite, moderately long, anterior uterus 230-406, posterior uterus 235-394 μm long, respectively; well-developed sphincter between uterus and pars dilatata oviductus, pars dilatata, and uteri containing numerous sperm cells; ovary small.      Male. Rarer than females. Habitus as in females, posterior part more strongly coiled ventrally. Shape of lip region similar to that in females. Cuticle 4.0-4.5 μm thick at postlabial region, 4 μm along the body and 5 μm on tail posterior to cloaca. One lateral pore anterior to or at the level of guide ring, 2 along odontostyle, 1 or 2 along odontophore, 3 or 4 in narrow part of the pharynx and 2 or 3 in pharyngeal bulb region, no dorsal and 6 ventral pores; numerous lateral pores present along the rest of the body.
Juveniles. Four juvenile stages can be differentiated based on the body, odontostyle, and replacement odontostyle length (Figs 5-7, Table 1). Habitus more or less an open C-shape, tail of the first stage juvenile digitate, with 9-11 μm long ventral peg, whereas in the subsequent developmental stages, bluntly rounded, c' decreasing.
The group of large Longidorus species (code F34) with a moderately long odontostyle (code A45), pocket-shaped amphidial fovea, bilobed, symmetrical (code E2) or asymmetrical (code E3), normal arrangement of pharyngeal glands nuclei, short rounded tail (code H1) and digitate tail or tail with mucro (code K7) (according to Peneva et al. 2013) consists of a few species, namely: L. arthensis; L. pauli Lamberti, Molinari, De Luca, Agostinelli & Di Vito, 1999; L. kheirii Pedram, Niknam, Robbins, Ye & Karegar, 2008; L. silvae Roca, 1993;and L. uroshis (Table 2). The new species differs from these by the presence of peculiar inclusions in the intestine. Furthermore, it differs from:  Note: A -odontostyle length; B -lip region width; C -distance of guide ring to anterior body length; D -shape of anterior region; E -amphidial fovea shape; F -body length; G -index "a"; H -tail shape; I -presence/absence of male; J -number of juvenile stages; K -tail shape in first stage juvenile. *Changes in code D proposed.

Longidorus polyae Lazarova, Elshishka, Radoslavov & Peneva sp. n.
The new species belongs to a group of large Longidorus species having a moderately long odontostyle, a bilobed pocket-shaped amphidial fovea, a short rounded tail in adults, and a digitate tail with mucro in first-stage juveniles. The group contains just a few species occurring in Europe (L. arthensis, L. silvae, L. uroshis), Syria (L. pauli), and Iran (L. kheirii). A very specific character observed in all female specimens of L. polyae sp. n. were the crystalloid structures of various shapes and sizes present in the intestinal lumen, especially in the posterior gut region. To our knowledge, the presence of similar crystalloid structures has been rarely observed in plant-parasitic species (e.g. L. perangustus (Roshan-Bakhsh et al. 2016) and Hirschmanniella kwazuna (Van den Berg et al. 2009)), and more often in non-parasitic nematodes (Heyns and Coomans 1983;Bird et al. 1991;Tahseen 2012;Abebe et al. 2013).  DNA data were available for all species except L. pauli. However, different ribosomal fragments have been deposited in the GenBank database, which limit the reconstruction of phylogenetic relationships within the group. Moreover, the D2-D3 expansion segments of 28S rDNA sequences of the new species showed highest similarity (93%) to two other species of Longidorus, L. attenuatus and L. dunensis, with sequences revealing a very high inter-species similarity (or 1-3 nucleotides difference). One population of L. silvae from Serbia, characterised molecularly by three sequences of D3 segment of 28S rDNA (AM412367-AM412369, Barsi et al. 2007), was very similar to L. polyae sp. n. comparing the same fragment (2 nucleotides difference at the 3' end, overall multiple sequence alignment of 300 characters). Likewise, a very high level of similarity for the same ribosomal segment of 28S rDNA are known for other Longidorus species, e.g. the multiple alignment of 10 L. aetnaeus Roca, Lamberti, Agostinelli & Vinciguerra, 1986 and five L. leptocephalus Hooper, 1961 sequences revealed one indel for an alignment length of 237 characters. However, both species showed higher inter-species dissimilarity in D2-D3 expansion segments of 28S rDNA (0.7-1.6%) and the cox1 mtDNA region (3.2%) .
In comparing the morphology of L. polaye sp. n. to the most closely related species, inaccuracies in code D values (related to the shape of anterior regions) were identified. In the original description of L. silvae, the lip region was described as "subacute, flattened frontally and continuous with the rest of the body" (Roca 1993: 211), which rather corresponds to D3 code description ("body tapering distinctly, lip region flattened, continuous, or slightly offset by depression") than to D1 ("body tapering distinctly, lip region rounded, continuous") according to Chen et al. (1997: 18). Similarly, in the diagnosis of L. kheirii, a D1 code was assigned instead of D3 ("head continuous with the contour of the rest of body, a truncate and slightly concave" Pedram et al. 2008: 206).

Logidorus pisi Edward, Misra, & Singh, 1964
Morphological and molecular data of three Bulgarian populations of L. pisi have been presented within the framework of this study. Some differences in morphometrics between these populations and the original description have been observed; however, a more comprehensive integrative study on materials from the type area would help to evaluate the divergences. New sequences for two ribosomal DNA regions (18S rRNA gene and ITS1-5.8S-ITS2) were obtained for L. pisi for the first time. The D2-D3 expansion segments of 28S rRNA gene sequences of all populations from Bulgaria were identical. In the phylogenetic analysis (Fig. 9), our populations and all populations of L.pisi (= L. latocephalus) from Greece, Iran, and South Africa clustered together with L. mindanaoensis and thus supports their evolutionary relationship as revealed by a previous study (Coomans et al. 2012). However, in the phylogenetic tree based on the 18S rRNA gene (Fig. 10), the position of L. pisi was not resolved. Both phylogenetic trees showed incongruent evolutionary relationships between L. pisi and L. mindanaoensis. The ITS sequence of L. pisi showed no similarity with the corresponding region of Longidorus species present in the database and no phylogenetic analyses were carried out.
The species appears to have a wide distribution, having been reported from three continents and numerous countries: Asia (India, Pakistan, Iran, China), Africa (Botswana, Egypt, Malawi, Mozambique, Cameroon, Libya, Namibia, Senegal, South Africa, Sudan), and Europe (Bulgaria, Macedonia, Greece). It was associated mainly with cultivated plants (pea, grapevine, mint and other medical plants, maize, potato, apple, pear, sugarcane) and rarely with natural vegetation (Marais and Swart 2002;Bohra and Baqri 2005). In Bulgaria and other Balkan countries, it was found in soils from agricultural habitats, associated with annual and perennial crops (tobacco, tomato, sweet pepper, black currant, grapevine, apple, peach, kiwifruit walnut, sweet chestnut, Pinus nigra J.F. Arnold, etc.) Choleva et al. 1991Choleva et al. , 1997, which suggest its probable introduction to Europe.