Lamberti , 2004 ( Longidoridae ) , with a description of the male of X . parasimile

Several populations of Xiphinema simile Lamberti, Choleva et Agostinelli, 1983 and two of X. parasimile Barsi et Lamberti, 2004, originating from various habitats – natural and man-made, have been studied. Xiphinema simile was associated both with cultivated and naturally growing plants, while X. parasimile was recovered from soil around grapevine. Data on the morphological and biometrical characteristics (including juvenile stages) are presented and variations discussed. Pharyngeal bulbus and glandularium length, vaginal and uterine characteristics were shown to be good diff erentiating characters. Th is report of X. parasimile is a new record for Bulgaria as well as a new plant association for the species. Th e description of its male is provided for the fi rst time. Th e Bulgarian population of X. parasimile showed the same pattern as the Serbian population revealed by the RFLP analyses of D1-D2 region.


Introduction
Th e present study is focused on the morphology and distribution of two closely related species of the Xiphinema americanum lineage from Bulgaria -Xiphinema simile Lamberti, Choleva et Agostinelli, 1983 and X. parasimile Barsi et Lamberti, 2004. Xiphinema simile has been originally described from Bulgaria in association with poplar, grapevine, fruit-trees, black current (Lamberti et al. 1983), and subsequently has been reported from forest nurseries (Peneva and Choleva 1992). In Europe, the species has been found in various habitats: forests and grasslands in former Yugoslavia (Barsi 1994, Barsi and Lamberti 2002), hornbeam tree, maize and fi eld maple in Serbia, and dogwood shrubs in Bosnia and Herzegovina (Barsi and Lamberti 2004, Barsi and De Luca 2008); grapevine, fruit and nut trees, and raspberries in Slovakia (Lišková et al. 1993, Lišková 1995, Lišková and Brown 1996, Lamberti et al. 1999; orchards in Czech Republic (Kumari 2006); various plants in Hungary (fruit trees, grapevine, birch, wild rose and Elsbeere tree) (Repasi et al. 2008). Outside Europe X. simile has been reported from Kenya in the rhizosphere of pepper and baobab trees, and riverine forest (Coomans and Heyns 1997).
Xiphinema parasimile was described from forest habitats in Serbia, in association with woody plants (Quercus sp. and Carpinus betulus L., Barsi and Lamberti 2004). In morphology and morphometrics it showed highest similarity to populations of X. simile, with diff erences in lip region and tail shape, however the authors underlined that both species can be thoroughly diff erentiated by the number of juvenile stages (four vs three).

Materials and methods
Soil samples were collected from diff erent habitats representing various ecosystem types in Bulgaria. Nematodes were extracted from 200 cm 3 soil by a sieving and decanting technique, heat killed at 60°C for two minutes and fi xed in a 4% formaldehyde solution. For morphological studies nematodes were processed to anhydrous glycerol and mounted on permanent microscopic glass slides (Seinhorst 1959). Additionally, live specimens were fi xed in 1M NaCl for molecular studies. PCR amplifi cation of X. parasimile D1-D2 domains of the 26S rDNA was carried out by using the primers FOR and REV as described in De Luca et al. (2004). Th e RFLP analyses of the D1-D2 region were performed using the following enzymes Alu I, Ava II, Dde I, Nde II, Pst I and Rsa I according to De Luca et al. (2004).
Drawings and photographs were taken using an Olympus BX51 compound microscope powered with diff erential interference contrast (DIC). Images were taken with a ColorView IIIu camera and cell^P software (Olympus Soft Imaging Solutions Gmbh). Measurements were made using an Olympus BX 41 light microscope, a digitising tablet (CalComp Drawing Board III, GTCO CalCom Peripherals, Scottsdale, AZ, USA), and computer Digitrak 1.0f programme, (Philip Smith, Scottish Crop Research Institute, Dundee, UK).

Measurements. See
Male. One specimen was found in Vinogradets population. Male similar to the female with posterior region more strongly curved. Lip region and tail shape as in females, a diff erence was observed within anal body width, which refl ect the lower c' value. Spicules slightly curved, lateral guiding piece 4 μm long. Adanal supplement pair preceded by a row of fi ve irregularly spaced supplements. Tail conoid, ventrally straight, dorsally convex with pointed terminus.
Juveniles. Th e scatter diagram based on functional and replacement odontostyle, and body length revealed presence of four juvenile stages (Fig. 7).
PCR-RFLP analysis. Th e amplifi cation of the D1-D2 expansion domains of X. parasimile generated a unique band of about 0.8 kb. Th e restriction fragments of the amplifi ed region of X. parasimile population from Vinogradets locality using several enzymes are presented in Fig. 8.
Remarks. Metric data are within the ranges reported in the original description, with slightly lower average values for the female body length (1.78-1.82 vs 1.93-1.99 Table 1. Morphometrics of Xiphinema parasimile from two localities in Bulgaria. All measurements in micrometres, except body length and ratios, and in form: mean ± standard deviation (range).

Vitis vinifera
Th e RFLP results showed that the Bulgarian population of X. parasimile has the same restriction profi les as that from Serbia (Barsi and De Luca 2008). Th e enzyme Ava II produced in the Bulgarian population two extra bands that were absent in X. parasimile from Serbia, suggesting the existence of diff erences in restriction sites in D1-D2 sequences. Furthermore, the enzyme Alu I always showed a band of 0.8 kb, corresponding to the undigested product, along with the expected restriction fragments suggesting microheterogeneity of the D1-D2 region of Bulgarian materials of X. parasimile (Powers et al. 1997, Hugall et al. 1999, Hung et al. 1999, Subbotin et al. 2000, Morales-Hojas et al. 2001, Elbadri et al. 2002, Otranto et al. 2003, De Luca et al. 2004.  Tables 2-4 Description. Females. Body slender, slightly tapering towards both ends; C-to spiral-shaped. Th ickness of the cuticle at postlabial region 1 μm; at dorsal side of the tail cuticle thickness increases gradually from 2 to 3.6 (3-4) μm towards tail end. Lip region expanded, fl atly rounded, 4 (4-5) μm high. Amphidial opening 4-5 μm wide, occupying 44-50% of the corresponding body width (n=4), located just below the demarcation line. Odontostyle with moderately developed basal collar, guiding ring not appearing single. Pharyngeal characters presented at Table 2. Genital system with two almost equally developed branches, uteri short (Table 3); vagina 13-16 μm long or 46-56 % of the corresponding body diameter. Sperm cells observed in females from Kalimok and Orlyane populations. Ovaries contain symbiotic bacteria. Rectum 20.1 (18-22) μm long. Tail conoid, dorsally convex, terminus rounded, in some specimens pointed; presence of slight dorsal constriction at the level of hyaline part.

Xiphinema simile
Males. Similar to female apart from body more curved at the posterior end and higher lip region (5-5.5 μm). Spicules slightly curved, one adanal pair and 3 ventromedian supplements present, lateral guiding piece 6 μm long Th e spicules of the specimen from Srebarna Reserve were not well developed and the testes were not observed while the specimen from Kalimok-Brashlen locality was apperantly functional with well developed testes fi lled with sperm. Tail longer than in female, especially in the specimen from Srebarna, conoid, dorsally convex with rounded terminus.
Juveniles. Th e scatter diagram based on functional and replacement odontostyle, and body length reveal presence of three juvenile stages (Figs 11A & B). Table 3. Morphometrics of Xiphinema simile from diff erent localities in Bulgaria. All measurements in micrometres, except body length and ratios, and in form: mean ± standard deviation (range).

Locality
Host plant  Remarks. According to Barsi and Lamberti (2002) the populations of X. simile found in diff erent localities have shown a broad range of variability in body length with populations with more southern distribution being shorter. Th is study revealed one population of X. simile from Kamen brayg area with lower mean values for body and tail length, aand c'-ratios and higher c-ratio, as compared to other three populations. Th e comparisons with populations from diff erent localities, showed that this population has similar body length with other Bulgarian (Lamberti et al. 1983, Peneva andCholeva 1992) and the Kenyan populations (Coomans and Heyns 1997), but still nematodes of this population had shorter tail length, higher c-ratios, and smaller c'-ratios. Th e other populations studied were within the range of those reported from northern localities of the range (Barsi 1994, Lišková and Brown 1996, Lamberti et al. 1999, Barsi and Lamberti 2002, Barsi and Lamberti 2004, Kumari 2006, Repasi et al. 2008.

Spicules
Measurements of juvenile stages and male specimens are presented for the fi rst time for Bulgarian populations. Th e obtained values were equal or close to those reported by Barsi and Lamberti (2002) and Barsi and De Luca (2008). Th e frequency distribution graphs of functional and replacement odontostyle lengths represent four    groups, corresponding to three juvenile stages and an adult stage and confi rm the fi ndings of other authors (Coomans and Heyns 1997, Barsi and Lamberti 2002, Barsi and Lamberti 2004, Kumari 2006 for the developmental pattern of X. simile. Xiphinema simile was found to occur together with X. parasimile (Orlyane locality) and X. pachtaicum (Tulaganov, 1938) Kirjanova, 1951 (Kalimok-Brashlen protected area).

Conclusions
Apparently, X. simile has a wider geographical range and has been found in association of numerous plant species in natural habitats and arable lands compared to X. parasimile which is so far recorded only from the Balkan Peninsula. Th e males of both species regarded here, although quite rare, may play an important role in genetic variability and hence contribute to the phenotypic plasticity within and among their populations. Th e overlapping of the metric characteristics, especially when combining data from many diff erent populations of these species has been widely discussed by Barsi and De Luca (2008). Yet, some qualitative A B characters can be used to separate X. simile and X. parasimile such as the shape of lip region and tail (Barsi and Lamberti 2004). We have found that the length of pharyngeal bulbus and glandularium are good characters allowing discrimination of both species, no overlapping of the ranges reported. Comparison between the Bulgarian populations of X. simile and X. parasimile studied showed that the uteri and the pars distalis vaginae of the fi rst species are shorter. Uteri in X. simile were represented only by ovejector, while in X. parasimile ovejector and separate uteri were observed. Th us, the structure of the uteri can also be used as another diff erentiating character. Our study confi rms the results by Barsi and De Luca (2008) concerning the validity of both species adding new information about their morphology, distribution, plant associations, genetic variability and male characteristics of X. parasimile.