Molecular and morphological characterisation of Longidoruspolyae sp. n. and L. pisi Edward, Misra & Singh, 1964 (Dorylaimida, Longidoridae) from Bulgaria Author Lazarova, Stela S. Author Elshishka, Milka Author Radoslavov, Georgi Author Lozanova, Lydmila Author Hristov, Peter Author Mladenov, Alexander Author Zheng, Jingwu Author Fanelli, Elena Author Francesca De Luca, Author Peneva, Vlada K. text ZooKeys 2019 830 75 98 http://dx.doi.org/10.3897/zookeys.830.32188 journal article http://dx.doi.org/10.3897/zookeys.830.32188 1313-2970-830-75 802300DF12834852B60A0F883605CF42 Longidorus pisi Edward, Misra & Singh, 1964 = Longidorus latocephalus Lamberti, Choleva & Agostinelli, 1983 Notes. Morphological and morphometric data for females and juvenile stages are presented in Table 4 and in Figure 8. The morphometric data obtained in this study agreed with those of L. latocephalus from its type locality ( Lamberti et al. 1983 ) and several additional populations studied by Lamberti et al. (1997) . Furthermore, when compared to the type population of L. pisi ( Edward et al. 1964 ), specimens from our populations revealed longer odontostyle (average 75 (72-79) and average 76 (74-78) vs 58 (56-61) μm ) and odontophore (average 50 (45-53) and average 49 (46-53) vs average 42.7 (35-43) μm ); longer distances anterior end to guide (average 44 (42-46) vs average 32 (31-35) μm ) and nerve ring (average 147 (138-151) and 141 (132-150) vs average 133 μm ); wider (average 10.6 vs 7.5 μm ) and higher (5-6 vs 3.5 μm ) lip region and lower c' ratio (average 1.9 (1.5-2.0) and average 1.7 (1.5-1.8) vs average 2.5 (2.4-2.6). The morphometrics of our populations were more similar to the Iranian population of L. pisi (Saveh, Markazi province) for which a D2-D3 expansion domain of 28S rRNA gene sequence identical to ours is available ( Pedram et al. 2012 ). Differences in a few characters were observed, e.g. smaller a (average 125.2 (117.2-132.1) and 127.3 (119.8-132.9) vs 139.4 (134.8-144.6) and c' (average 1.9 (1.5-2.0) and average 1.7 (1.5-1.8) vs average 2.5 (2.3-2.9) values, larger diameter at anus level (average 22.9 (21-24.5) and average 20.5 (20-22) vs average 18.1 (16-19) μm ) and slightly shorter tail (average 38.9 (36-43) and average 39.1 (37-42) vs average 45 (43-47) μm ) compared to the latter population. Table 4. Measurements of adults and juveniles of Longidorus pisi (mean +/- standard deviation, with range) from different crops in Bulgaria. All measurements in micrometers except body length (mm).
Host Character Petrich Sandanski Kromidovo Petrich
Nicotiana tabacum Vitis vinifera Nicotiana tabacum
female female female J1 J2 J3
n = 9 n = 6 n = 2 n = 2 n = 9 n = 2
Figure 8. Bulgarian populations of Longidorus pisi Edward, Misra & Singh, 1964, anterior end of female (A) and male (B) female C, D vagina and part of reproductive system (different populations) E-G pre-rectum inclusions H labial region I vagina J-M variations in tail shape, female (J, L, M) and male (K). Populations: A, L Petrich C, E-G , M Sandanski D, H, I, J Kromidovo B, K male specimens from a tobacco field, Petrich region. Scale bars: 20 μm ( A-G , J-M ); 12 μm (H, I). Sequence and phylogenetic analyses. Three ribosomal DNA regions (D2-D3 expansion segments of 28S rRNA gene, 18S rRNA gene, and ITS1-5.8S-ITS2 regions) of L. pisi were amplified and sequenced. The D2-D3 expansion segments of 28S rRNA gene sequences from all populations were identical to that of the Iranian population (JQ240274, Pedram et al. 2012 ) and differed slightly (1 and 3 bp) from those of other populations (Greece (AY601569) and South Africa (AY601568), respectively) identified as L. latocephalus ( He et al. 2005 ). In the phylogenetic analysis, all aforementioned sequences formed a clade with maximal Bayesian posterior probability (1.0) and showed a close relationship with L. mindanaoensis . The BLASTn search using 18S rRNA gene sequence revealed highest identity (99%) with five accessions (two Longidorus (HQ735099 L. mindanaoensis Coomans, Tandingan De Ley, Angsinco Jimenez & De Ley, 2012 and AY283163 L. ferrisi Robbins, Ye & Pedram, 2009) and three Paralongidorus species (JN032586 P. bikanerensis (Lal & Mathur, 1987) Siddiqi, Baujard & Mounport, 1993; AJ875152 P. maximus ( Buetschli , 1874) Siddiqi, 1964 and KJ427794 P. rex Andrassy , 1986). A pairwise comparison of L. pisi sequence with the closest sequences (AY283163, HQ735099, JN032586, AJ875152 and KJ427794) revealed 14-22 different nucleotides. The 18S rDNA phylogenetic tree is presented in Figure 10. Figure 9. Phylogenetic tree using D2-D3 expansion segments of the 28S rRNA gene inferred from a Bayesian analysis with GTR+I+G model. Numbers represent the Bayesian posterior probabilities. Figure 10. Phylogenetic tree using 18S rRNA gene inferred from a Bayesian analysis with GTR+I+G model. Numbers represent the Bayesian posterior probabilities.