Xiphinema browni, Lazarova, Stela, Peneva, Vlada & Kumari, Shesh, 2016

Lazarova, Stela, Peneva, Vlada & Kumari, Shesh, 2016, Morphological and molecular characterisation, and phylogenetic position of X. browni sp. n., X. penevi sp. n. and two known species of Xiphinemaamericanum-group (Nematoda, Longidoridae), ZooKeys 574, pp. 1-42 : 4-11

Taxon classification Animalia Dorylaimida Longidoridae

Xiphinema browni sp. n. Figures 1, 2, 3, 4, 5, 6, 7, 8, 15, 16, 17, 18

Xiphinema pachtaicum (Tulaganov, 1938) Kirjanova, 1951 apud Kumari et al. 2005, syn. n.

Measurements.

See Tables 3-5.

Description.

Females. Body slender C to open spiral shaped. Cuticle with fine transverse striae. Thickness of the cuticle at postlabial region 1-1.5 μm, 1.5 rarely 2 μm at mid-body and 2 μm at post-anal region. Labial region set-off from the rest of the body by a constriction, expanded, rounded laterally, 5.0 ± 1.1 (4-7) μm high. Amphideal fovea hardly visible, funnel-shaped, its opening c. 5 μm (50%) wide visible posterior the constriction level. Distance between first and second guide ring in specimens with retracted odonostyle 5-10 μm long. Odontophore with moderately developed basal flanges 6.1 ± 0.6 (5.5-7) μm wide. A small vestigium observed occasionally in slender part of pharynx. Pharyngeal characters presented at Table 4. Dorsal pharyngeal gland nucleus 2 μm diam. Ventrosublateral nuclei barely visible. Rectum 20.8 ± 1.5 (18-23) μm, n=7, or c 1.3 times anal body diameter. Reproductive system amphidelphic, symbiont bacteria present in the ovaries. Separate uteri and ovejector present (Table 5), oviduct 90.5 ± 13.0 (68-101) μm; vagina bell-shaped 39.5% of the corresponding body width (33-50%, n=14), vulva post-equatorial. Numerous sperm observed in one female from Kurdějov (Figs 2B, 4B). Tail conical, dorsally convex, ventrally straight or slightly concave with narrowly rounded to pointed terminus. Two pairs of caudal pores.

Male. Very rare. One specimen found in Sokolnice population. Male similar to the female with posterior region more strongly curved. Lip region and tail shape as in females, differences were observed within body width and tail length, which reflected a and c’ values. Spicules robust, slightly curved, lateral guiding piece 7 μm long. Adanal pair preceeded by a row of 5 irregularly spaced supplements, the two anteriormost weakly developed. Tail conoid, ventrally straight, dorsally convex with pointed terminus, caudal pores not visible. The slide of the only male specimen, described by Kumari et al. (2005), was subsequently damaged.

Juveniles. The scatter diagram based on functional and replacement odontostyle, and body length revealed the presence of four juvenile stages (Fig. 8). Tail shape and length similar in all stages and females with c’ slightly decreasing in successive stages (Kumari 2005, Fig. 3, Table 3).

Type locality and plant association.

Kurdějov, Břeclav County, South Moravia, Czech Republic, associated with grapevine. Other localities: Mohyla míru, Brno-Venkov County, South Moravia, the Czech Repbulic, in the rhizosphere of apple trees; Sokolnice, Brno-Venkov County, South Moravia, the Czech Repbulic, in the rhizosphere of grapevine; Moča, Komárno County, Nitra, Slovak Republic, in the rhizosphere of grapevine.

Type material.

The holotype, 9 paratype females and juveniles from all stages are deposited in the nematode collection of the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Other paratypes deposited as follows: 15 females in the Crop Research Institute, Prague, the Czech Republic; 5 females in the USDA Nematode Collection, Beltsville, Maryland, USA; 5 females in the Nematode Collection of the Institute of Plant Protection, Bari, Italy; 5 females in the Wageningen Nematode Collection (WANECO), Wageningen, the Netherlands. The ribosomal and mtDNA sequences (18S rDNA, ITS1, ITS2, D2-D3, cox1, nad4) of Xiphinema browni sp. n. are deposited in GenBank (for accession numbers see Table 2).

Sequence and phylogenetic analyses.

There was no sequence variation between populations for 18S and D2-D3, ITS1 and ITS2 rDNA regions of Xiphinema browni sp. n. Of all four populations studied cox1 region of three population from the Czech Republic ( Kurdějov, Mohyla Míru, Sokolnice) were sequenced by Kumari et al. (2010b) and all populations were identical therefore only one population was submitted to GenBank (accession number GU222424). The Slovakian population was sequenced in this study and it was identical to previously published sequence of Kurdějov the population identified as Xiphinema pachtaicum (GU222424, Kumari et al. 2010b). All four sequenced populations were also identical for nad4 part.

BLAST at NCBI using 18S and D2-D3 region sequences as queries revealed highest similarity (99 and 87%) to the corresponding sequences of Xiphinema simile Lamberti, Choleva & Agostinelli, 1983 from Serbia (AM086681) and two Spanish populations of Xiphinema opisthohysterum Siddiqi, 1961 (JQ990040 and KP268967), respectively. The es timated divergences (p-distance) between the 18S rDNA sequences of the new species and the closest species, Xiphinema parasimile from Bulgaria (this study) and Xiphinema simile from Serbia (AM086681) were 0.3 (6 nt) and 1.2% (21 nt), respectively. Again, the new D2-D3 sequence of Xiphinema parasimile from Bulgaria was most similar (p-distance = 4.6%), followed by the Serbian populations of Xiphinema parasimile (p-distance = 7.6-7.9%, calculated for D2 region only) and various populations of Xiphinema simile (14.1-14.7%). The partial cox1 sequences of Xiphinema browni sp. n revealed highest similarity to Xiphinema simile from Slovakia (AM086708). Surprisingly, these two species showed very high similarity 99% (2 nts difference) in cox1 sequences and higher dissimilarity in 18S rDNA (p-distance = 1.2%, 21 nts). Other authors ( Gutiérrez-Gutiérrez et al. 2012) have also reported similar observation namely, 100% identity in cox1 part of two different species Xiphinema duriense Lamberti, Lemos, Agostinelli & D’Addabo, 1993 (JQ990053) and Xiphinema opistohysterum (JQ990054) and clear separation in D2-D3 28S sequences (or 96 % identity). Further, the cox1 sequences of Xiphinema browni sp. n. and the closest species Xiphinema parasimile , Xiphinema simile (GU222425, Czech Republic) and Xiphinema pachtaicum (HM921369, Spain) were translated to amino acids and aligned (Fig. 9). The estimated p-distances between Xiphinema browni sp. n. and the three species were 10.1%, 21.7% and 23.3%, respectively.

In all three phylogeny reconstructions (18S, D2-D3 and cox1) Xiphinema parasimile from Bulgaria was a sister species of Xiphinema browni sp. n. and both species were part of a well supported clade with other European populations of Xiphinema simile (Figs 10-12). The recently described species Xiphinema vallense Archidona-Yuste, Navas-Cortes, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016 presented only with D2-D3 and ITS1 rDNA sequences seems also to be evolutionary very closely related (Figs 11 and 13), however amplifying additional sequences for other molecular markers (e.g. 18S and cox1) could help to better clarify its relationships. The position of the new species in the phylogeny trees based on ITS1 and ITS2 sequences was unstable (Figs 13 and 14). The analyses resulted in various tree topologies when using different alignment algorithms and reconstruction methods (ML and BI) and because of the absence of homologous sequences from closely related species. In most cases Xiphinema browni sp. n. was part of a clade of European Xiphinema americanum -group species considered as group II in a previous publication ( Archidona-Yuste et al. 2016). Due to insufficient number of nad4 sequences of species belonging to the Xiphinema americanum -group at NCBI no phylogenetic reconstructions are presented.

Diagnosis and relationships. Xiphinema browni sp. n. is characterised by a unique combination of traits: slender and medium sized body (1.6-2.41 mm) and odontostyle (73-85 μm), lip region expanded, laterally rounded, separated from the rest of body by a constriction, post-equatorial vulva position (V=52-58 %), symbiotic bacteria present, female tail conical dorsally convex, with narrow rounded to pointed tip, 24-35 μm long, (c=53.4-86.8; c’=1.5– 2.1), and specific ribosomal and mtDNA sequences (Table 2). The alpha-numeric codes based on average values (ranges given in parentheses) using the polytomous key by Lamberti et al. (2004) are: A3 (2), B3 (2), C3 (4), D2 (1/3), E2 (3), F2 (1/3), G2, H1, I2 (1/3).

Species having similar morphometrics to Xiphinema browni sp. n. based on type populations are presented in Table 6. Recently described species Xiphinema parasimile , Xiphinema parabrevicolle Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, Decraemer, Vovlas, Prior, Palomares-Rius & Castillo, 2012, Xiphinema parapachydermum Gutiérrez-Gutiérrez, Cantalapie dra-Navarrete, Decraemer, Vovlas, Prior, Palomares-Rius & Castillo, 2012, Xiphinema paratenuicutis Gutiérrez-Gutiérrez, Cantalapiedra-Navarrete, Decraemer, Vovlas, Prior, Palomares-Rius & Castillo, 2012, Xiphinema plesiopachtaicum Archidona-Yuste, Navas-Cortes, Cantalapiedra-Navarrete, Palomares-Rius & Castillo, 2016 and Xiphinema vallense ( Barsi and Lamberti 2004, Gutiérrez-Gutiérrez et al. 2012, Archidona-Yuste et al. 2016) have also been compared. Six of these species have non-European distribution (Table 6) whereas the others were described from and/or found mainly in Europe. Xiphinema simile was also included in the table comparing morphometrical data because of the close relationships based on sequence and phylogenetic analyses and its wide distribution in many European countries.

Based both on morphology and molecular data Xiphinema browni sp. n. is most similar with Xiphinema parasimile , Xiphinema simile and Xiphinema vallense . Morphologically, it can be distinguished from:

Xiphinema parasimile by its different lip region shape (expanded vs not expanded), somewhat longer odontostyle av. 79-83 (73-85) μm vs av. 70 (64-74) μm in the type population, avs. 69-70 (63-74) in Bulgarian populations and avs. 68-70 (67-72) μm in females from Romania ( Barsi and Lamberti 2004, Lazarova et al. 2008, Bontă et al. 2012);

Xiphinema simile by its longer odontostyle av. 79-83 (73-85) vs av. 66 (62-69) in type population, avs. 68.5-70 (66-72.5) in other Bulgarian populations, 67.5 (65-70) μm in a population from Bosna and Herzegovina, and avs. 67-68 (61-73) μm in females from the Czech Republic ( Lamberti et al. 1983, Barsi and Lamberti 2004, Kumari 2006, Lazarova et al. 2008). However, it should be noted that females from Serbia and Crete (odontostyle 71.5 (66-74) μm and 75-77 μm, respectively) have slightly overlapping values between Xiphinema browni sp. n. and Xiphinema simile for this character ( Barsi and Lamberti 2004, Tzortzakakis et al. 2014). Further Xiphinema browni sp. n. differs from Xiphinema simile in the length and structure of uteri (in the new species separate uteri and ovejector present vs separate uteri not present), different tail shape (conoid vs bluntly conoid), and in the shorter bulbus (53-69 vs 76-92 μm) ( Lazarova et al. 2008) (Table 4). Finally, Xiphinema browni sp. n. develops though 4 vs 3 juvenile stages in Xiphinema simile ;

Xiphinema vallense by the position of amphideal fovea aperture (posterior constriction level vs on the lips); higher lip region (4-7 μm vs 2-3.5 μm); presence of symbiont bacteria in ovaries vs ovaries without symbionts; somewhat higher c’ values ( c’ =1.8 (1.53-2.07) vs c’ =1.6 (1.4-1.7); the different tail shape (dorsoventral depression at hyaline region level not present vs present); shorter spicules in males (29 μm vs 38 μm).

Additionally, Xiphinema browni sp. n. can be differentiated from:

Xiphinema pachtaicum by the different vagina shape (bell-shaped vs funnel shaped, (Figs 16, 18) and shorter pars distalis vaginae, shorter pharyngeal bulb (53-69 vs 75-80 μm), more posterior location of the dorsal nucleus (DN=13-21% vs 9-10 %) (Table 4), different tail shape in both sexes (conical vs subdigitate). Illustrations of selected features of the closest species Xiphinema pachtaicum , Xiphinema parasimile and Xiphinema penevi sp. n. are presented in Figs 15-18 for comparison.

Xiphinema paratenuicutis in having symbionts in its ovaries vs absent, males rare vs abundant, higher values for c’ (1.8 (1.5-2.1) vs 1.4 (1.2-1.6), different location of dorsal nucleus (DN after beginning of the stronger cuticular lining of the bulbus vs before, see Fig. 1 D1-F and Fig. 2E in Gutiérrez- Gutiérrez et al. 2012);

Xiphinema plesiopachtaicum by the position of the amphideal aperture (posterior vs at the constriction level); somewhat shorter bulbus (avs. 59-60 (53-69) vs av. 73 (60-86); shorter uteri (av. 81 vs av. 138 μm); higher c’ values ( c’ =1.8 (1.53-2.07) vs c’ =1.4 (1.3-1.7); and differently shaped vagina (bell-shaped vs funnel shaped).

For comparison between Xiphinema browni sp. n. and Xiphinema penevi sp. n. see below.

Etymology.

The species is named after Prof Derek JF Brown, an outstanding nematologist, for his significant contributions to the knowledge of plant parasitic nematodes and the development of nematology in Bulgaria.