Research Article |
Corresponding author: Vlada Peneva ( esn.2006@gmail.com ) Academic editor: Sergei Subbotin
© 2017 Mariana Groza, Stela Lazarova, Francesca De Luca, Elena Fanelli, Milka Elshishka, Georgi Radoslavov, Peter Hristov, Mihaela Coman, Vlada Peneva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Groza M, Lazarova S, De Luca F, Fanelli E, Elshishka M, Radoslavov G, Hristov P, Coman M, Peneva V (2017) The morphological and molecular identity of Longidorus piceicola Lišková, Robbins & Brown, 1997 from Romania (Nematoda, Dorylaimida). ZooKeys 667: 1-19. https://doi.org/10.3897/zookeys.667.12011
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Longidorus piceicola, a new geographical and host record from Romania, was described and illustrated on the basis of two populations originating from a coniferous and a deciduous forest. The main morphological characters of specimens from Romania correspond very well with the type material collected from the soil around Picea abies L. (Slovakia) except for the shorter body and tail. The D2-D3 fragment of 28S rDNA from both populations was amplified and sequenced, and the sequences were identical to L. piceicola sequence from Slovakia. The partial 18S-ITS1-5.8S-ITS2 rDNA regions from one of the populations were sequenced for the first time. The evolutionary relationships between L. piceicola and the closest species L. intermedius based on D2-D3 sequence divergence and single-nucleotide polymorphisms are discussed. Although having very low sequence dissimilarity (0.3–0.9 %) both species have distinct morphology and biology. Longidorus piceicola differs from L. intermedius in having a much longer odontostyle, body, distance anterior end - guide ring, a wider lip region, more ventromedian supplements (11 vs 5–7) in the male, and develops through four rather than three juvenile stages. Furthermore, L. piceicola occurs more frequently in association with conifers, while L. intermedius is found mainly in oak forests.
D2–D3 expansion region rDNA, ITS, juvenile stages, new record, phylogeny, SNPs
Longidorus piceicola Lišková, Robbins & Brown, 1997 was originally described from Slovakia (
Specimens were collected from the rhizosphere of a Larix decidua Mill. forest near to Bran, Braşov County, Romania (45.3050N, 25.2156E), ca 760 m a.s.l. on 15.10.2013, and from the soil around roots of deciduous trees (Quercus sp., Tilia sp., and Fraxinus sp.), Cernica forest, Ilfov County (44.2637N, 26.16514E) and ca 60 m a.s.l. on 4.08.2014. Nematodes were isolated from soil samples by a decanting and sieving technique (
The genomic DNA extraction, amplification, and sequencing of single specimens of L. piceicola from both populations in Romania were carried out independently in two laboratories: one at the Institute for Sustainable Plant Protection, Bari, Italy and the other at the Institute of Biodiversity and Ecosystem Research, Sofia, Bulgaria. Both protocols are presented separately below.
Institute for Sustainable Plant Protection (Bari Unit): specimens (Cernica locality) for molecular analysis were kept in DESS solution (
Institute of Biodiversity and Ecosystem Research: Genomic DNA was extracted from two single female worms L. piceicola from Bran locality using a standard nematode digestion protocol (
The sequences of the L. piceicola have been deposited in GenBank with the following accession numbers: KY086070 and LT669801 for D2-D3 expansion domains of 28S rRNA gene; LT669802 and LT669803 for the ITS region. The D2-D3 and ITS sequences were compared with those of other nematode species available at the GenBank sequence database using BLASTN similarity search tool revealing similar results for both regions. The closest D2-D3 sequences to L. piceicola were aligned using ClustalX 2.1 (
Eleven females and 21 juveniles, two females and one juvenile from Cernica forest, Ilfov County, Romania on slide numbers NE 35–37 stored at the reference collection of the National Phytosanitary Laboratory, Voluntari, Romania, 9 females and 20 juveniles - at the personal collection of the first author; nine females and 30 juveniles from Bran, Braşov County, Romania, stored in the nematode collection of IBER, Bulgaria, slide numbers N2-29/2/1-19.
Figures
Measurements See Tables
Females (Figs
Longidorus piceicola Female and juveniles: A Neck region – female B1–B4, C Head end with amphidial fovea B1–B3 females, B4 juvenile 4th stage (B2 right and B3 left) C, D, E1, E2, F Anterior ends of first- to fourth-stage juveniles G–K Pharyngeal bulb of female (G) and first- to fourth-stage juveniles (H–K).
Habitus spiral shaped, more strongly coiled in posterior part of body. Cuticle 3–4 μm thick at guide ring region, ca 3 μm in mid-body, and 5–6 μm on tail posterior to anus. Lip region broadly rounded anteriorly, rounded laterally, almost continuous with rest of body. Amphideal fovea pocket-shaped, varying from not lobed to symmetrically bilobed at base (according to terminology proposed by
In the population from Cernica forest two females with reserve odontostyles have been observed (Table
Male. Not found.
Juveniles (Figs
General morphology similar to adult females. Body habitus similar in all stages, open C- to J-shaped. Tail of all juvenile stages conical, but becoming more rounded and c’ decreasing in subsequent stages: tail of first stage juvenile elongate conoid with slightly digitate terminus, in the second stage – elongate conoid, in third – bluntly conoid, variable, with narrow to widely rounded terminus, in fourth – resembling that of female, bluntly conoid (Fig.
Longidorus piceicola Female from Bran locality: A Neck region, black arrows indicate nerve rings B, C Head end (different magnifications) D, E Amphideal fovea (right and left) F Odontophore G Prerectum H Pharyngeal bulb I, J Variations in tail shape. Scale bars: A, G 80 μm; B, F, H, I, J 40 μm; C–E 20 μm.
Measurements of females and juveniles (J) of Longidorus piceicola from Bran, Braşov County, Romania (mean ± standard deviation, with range). All measurements in micrometers except for body length (mm).
Character | Females | J1 | J2 | J3 | J4 |
---|---|---|---|---|---|
n | 9 | 6 | 4 | 8 | 3 |
L | 4.90±0.47 4.05–5.64 | 1.32±0.11 1.15–1.47 | 1.83±0.16 1.63–2.02 | 2.62±0.13 2.38–2.81 | 3.21, 3.91, 3.22 |
a | 84.6±8.0 71.1–97.3 | 55.4±4.6 47–60.8 | 59.3±5.5 53.5–65.9 | 67.6±3.6 62.3–71.9 | 73.6, 67.9, 75.5 |
b | 9.9 ± 0.6 9.7–11.1 | 4.3±0.2 4.1–4.5 | 5.2±0.3 4.8–5.5 | 6.4±0.5 5.8–7.3 | 6.7, 8.1, 7.1 |
c | 129.7±13.2 102.4–147.3 | 29.3±2.5 26.4–32.1 | 42.3±4.9 35–45.3 | 61.7±5.7 53.5–69.5 | 77.9, 108.4, 84.7 |
c’ | 0.97±0.06 0.89–1.10 | 2.8±0.3 2.6–3.2 | 1.9±0.2 1.64–2.15 | 1.45±0.1 1.38–1.58 | 1.23, 0.90, 1.18 |
V (%) | 49.2±1.2 47.2–51.3 | – | – | – | – |
G1 (%) | 6.7±0.7 5.8–7.8 | – | – | – | – |
G2 (%) | 6.1±0.9 5.4–7.5 | – | – | – | – |
Developing gonad | – | 16.2±1.2 15–17 | 28.3±7.2 20–33 | 33.3±2.1 31.5–37 | –, 48, 45 |
d | 2.63±0.1 2.45–2.8 | 2.6±0.2 2.5–2.7 | 2.7±0.3 2.5–3.0 | 2.37±1.0 2.5–2.8 | 2.9, 2.8, 2.9 |
d’ | 2.02±0.1 1.9–2.1 | 1.8±0.1 1.65–2.3 | 1.95±0.25 1.7–2.3 | 1.9±0.1 1.7–1.9 | 2.0, 2.1, 2.1 |
Odontostyle | 155.5±5.2 147–163 | 95.8±1.2 82–90.3 | 100.7±3.0 97.5–105 | 118.4±3.7 115–125 | 130, 143, 142 |
Replacement odontostyle | – | 103.7±3.5 99.5–110 | 115.4±6.0 109–123 | 137.8±2.7 134–143 | 151, 153, 154 |
Odontophore | 77.7±3.4 71–82 | 47.5±1.4 46–50 | 55±4.2 50–60 | 62.9±2.9 60–68 | 75, 73, 73 |
Anterior end to guide ring | 38.1±1.9 35–41 | 22.0±1.3 22–24 | 26±1.1 25–27 | 29.9±1.7 27–33 | 36, 37, 35 |
Bulbus length | 118.5±7.9 105–130 | 65.9±4.5 59–69 | 71.8±3.4 75–83 | 91.1±3.6 86–97 | 104, 116, 101 |
Bulbus width | 23.4±1.8 20–25 | 13.8±1.2 13–14 | 16.6±0.5 16–17 | 19.2±0.6 18–20 | 22, 22, 21 |
Pharynx | 478.4±29.4 440.5 –528 | 307.6±12.3 290–319 | 352±12.9 338.5–364 | 409.3±22.9 374–447 | 480, 484, 455 |
Tail | 38.2±1.8 35 – 42 | 45.4±4.2 42–51.5 | 43.5±2.9 40.5–47 | 42.7±4.2 36–48 | 41, 36, 38 |
Length of hyaline part | 11.7±0.9 10–13 | 9.5±0.6 9–10 | 8.5±0.6 8–9 | 9.3±1.2 8–11 | 9.5, 12, 8 |
Body diameter at: – lip region | 14.5±0.6 14–16 | 8.6±0.6 8–10 | 9.6±0.6 9–10 | 11.1±0.3 11–12 | 12, 14, – |
– guide ring | 29.2±1.6 27–32 | 15.3±0.7 14.5–16 | 18.5±1.3 28–31 | 21.1±1.2 19–23 | 25, 29, 26 |
– base of pharynx | 48.4±3.3 44–55 | 22.8±0.6 23–24 | 29.2±1.3 28–31 | 36.2±2.3 32–40 | 39,47, 39 |
– mid–body/at vulva | 58.7±5.4 53–71 | 23.8±0.8 23–25 | 30.9±1.8 29–33 | 38.9±2.7 33–41.5 | 44, 58, 43 |
– anus | 39.7±3.5 35–46 | 16.1±0.6 15.5–17 | 23±1.6 22–25 | 29.5±2.4 25–32 | 34, 37, 30 |
– hyaline part | 24.9±3.5 18–29 | 7.4±0.6 6.7–8.4 | 10.8±0.3 10.5–11 | 16.1±1.5 14–18 | –, 25, 18 |
Measurements of females and juvenile stages (J) of Longidorus piceicola from Cernica-Ilfov County, Romania (mean ± standard deviation, with range). All measurements in micrometers except body length (mm).
Character | Females | J1 | J2 | J3 | J4 |
---|---|---|---|---|---|
n | 9 | 11 | 2 | 3 | 5 |
L | 5.88±0.19 5.17–6.54 | 1.36±0.09 1.21–1.52 | 1.79, 2.16 | 3.29, 3.04, 2.81 | 3.95±0.47 3.6–4.7 |
a | 95.2±11.5 73.8–105.5 | 58.96±4.9 53–66.8 | 64.1, 67.7 | 68.7, 67.7, 68.7 | 77±8.2 62.5–83.4 |
b | 10.2±1.2 8.4–12.7 | 4.88±0.8 4.1–6.7 | 8.4, 9, 6.6 | 9±1.5 6.9–10.6 | |
c | 171.9±28.8 134.4–218.0 | 31.0±1.9 28.3–33.4 | – | 79, 71,64.3 | 102±8.1 89.8–109.8 |
c’ | 0.85±0.10 0.72–0.99 | 2.9±0.2 2.6–3.1 | – | 1.3, 1, 1.6 | 1±0.1 0.9–1.2 |
V (%) |
48.1±0.98 47.1–50.6 | – | – | – | |
G1 (%) | 5.8±0.8 4.7–7.1 | – | – | – | – |
G2 (%) | 5.4±0.7 4.4–6.4 | – | – | – | – |
d | 2.9±0.1 2.7–3.1 | 2.9±0.2 2.6–3.3 | 2.78, 2.99 | 3, 3, 3.4 | 3±0.2 2.8–3.2 |
d’ | 1.8±0.1 1.7–1.9 | 1.9±0.2 1.5–2.2 | 1.8, 1.7 | 2, 2, 2 | 2±0.1 1.7–2 |
Anterior end to guide ring | 42.2±1.8 40–45 | 22.8±1.4 21–26 | 25, 29 | 34.8, 34, 34 | 37.2±0.9 36–39 |
Odontostyle | 155.4±5.4 150–165 | 86.9±2.7 82–90 | 97, 102 | 122, 124, 108 | 136.8±3.4 132–141.5 |
Bulbus length | 135±4.9 126–141 | 72.7±4.3 65–78.5 | 71, 86 | 104, 104, 100 | 113.7±5.9 108–120 |
Bulbus width | 24.7±2.0 22–29 | 12.5±0.9 11–14 | 15, 14.5 | 18, 21, 19 | 19.9±2.1 17–21 |
Replacement odontostyle | – | 95.7±3.7 92–102 | 109, 111 | 142, 136, 132 | 157.3±6.7 150–165 |
Odontophore | 78.1±4.9 70–83 | 52.5±4.8 48–65 | 55, 60 | 72, 60, 65 | 72.2±3.0 69–76 |
Oesophagus length | 579.3±47.6 514–661 | 284.9±42.4 219–356 | 320, 366 | 393, 321,425 | 460.3±84.3 345–545 |
Tail | 34.8±4.4 30–41.5 | 43.58±2.3 39–47 | – | 42, 43, 44 | 38.7±3.5 34–43 |
Length of hyaline part | 12.3±1.1 11–14 | 9.5±0.9 8–11 | 10, 10 | 9, 10, 10 | 10±0.6 9.4–11.1 |
Body diameter at: – lip region |
14.7±0.4 14–15 | 7.9±0.2 7.5–8 | 9, 10 | 11, 11,10 | 12.4±0.5 12–13 |
– guide ring | 26.1±1.2 24–27 | 14.7±1.5 12–18 | 16, 16 | 20.5, 21, 20 | 23.16±0.6 22.5–24 |
– base of pharynx | 53.6±6.1 45–62 | 21.9±0.9 20–23 | 25, 28.6 | 39, 38, 38 | 43.5±3.0 38.9–46.4 |
– mid–body/at vulva | 63.2±4.5 58–70 | 23.2±1.4 21–26 | 28, 32 | 48, 45, 41 | 51.8±5.8 45–58 |
– anus | 40.8±2.1 38–44 | 15.2±1.3 14–18 | – | 31, 30, 27 | 36±3.4 33.2–41.2 |
– hyaline part | 27.8±1.9 25–31.5 | 7.4±0.6 7–8 | 7, 8 | 18, 16, 15 | 22±1.4 20–23 |
Measurements of Longidorus piceicola females (f) from Cernica, and juveniles (j) from Bran, Braşov County, Romania showing different anomalies. All measurements in micrometers except body length (mm).
Character | f | f | j | j | j | j | j | j | j | j |
---|---|---|---|---|---|---|---|---|---|---|
No | 1 | 2 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
L | 5.95 | 5.86 | 4.73 | 2.34 | 2.72 | 2.71 | 2.62 | 1.14 | 3.66 | 2.71 |
a | 99.1 | 97.7 | 93.0 | 63.6 | 77.7 | 61.9 | 67.0 | 32.6 | 75.1 | 63.8 |
b | 9.5 | 10.3 | 5.6 | 6.0 | 5.9 | 7.1 | 2.9 | 7.9 | 6.2 | |
c | 220.3 | 172.4 | 98.8 | 60.5 | 65.2 | 61.9 | 61.4 | – | 70.3 | |
c’ | 0.75 | 0.94 | 1.3 | 1.3 | 1.6 | 1.4 | 1.7 | – | 1.2 | |
V | 49.2 | 48.9 | – | – | – | – | – | – | – | – |
Developing gonade | – | – | 65 | – | – | – | 22 | 27 | ||
d | 2.93 | 2.73 | 2.6 | 2.8 | 2.9 | 2.9 | 2.5 | 2.7 | 2.8 | |
d’ | 1.79 | 1.80 | 1.4 | 2.0 | 2.0 | 2.0 | 1.8 | 1.9 | 1.8 | |
Odontostyle | 165 | 158 | 117 | 127 | 122 | 105 | 81 | 106 | 120 | 125 |
Replacement odontostyle | 175 | 158 | 131 | 165 | 165 | 135 | 108 | 130 | 140 | 156 |
Odontophore | 80 | 70 | 78.5 | 61 | 65 | 65 | 60 | 60 | 73 | |
Anterior end to guide ring | 41 | 41 | 35 | 30 | 32 | 33 | 25 | 26 | 32 | 32 |
Bulbus length | 132 | 130 | 114 | 81 | 87 | 89 | 95 | 93 | 108 | 86 |
Bulbus width | 23 | 23 | 22 | 19 | 18 | 20 | 17 | |||
Pharynx | 627 | – | 461 | 420 | 457 | 464 | 369 | 387 | 463 | 441 |
Tail | 27 | 34 | 48 | 39 | 42 | 44 | 43 | – | 39 | |
Length of hyaline part | 11 | 8 | 10 | 9 | 9 | 6 | 9 | |||
Body diameter at: - lip region |
14 | 15 | 14 | 11 | 11 | 11 | 9 | 11.5 | ||
- guide ring | 25 | 27 | 19.5 | 21 | 22 | 23 | 11 | 19 | 21 | |
- base of pharynx | 51 | 50 | 42 | 35 | 32 | 38 | 32 | 29 | 40 | 32 |
- mid-body/at vulva | 60 | 51 | 37 | 35 | 44 | 39 | 35 | 49 | 43 | |
- anus | 60 | 38 | 29 | 26 | 32 | 24 | – | 32 | ||
- hyaline part | 25 | 23 | 20 | 16 | 14 | 15 | 11 | 18 |
The amplification of the ITS and the D2-D3 expansion domains of the 28S rRNA gene yielded fragments of 1646 and 756 bps, respectively, based on sequencing. The ITS sequences of L. piceicola from Romania were obtained for the first time in the present study. They showed 98 % similarity (962/984 identities, 9 gaps) when compared with the corresponding sequence of L. intermedius (KT308890) and 86 % with the ITS sequence of L. elongatus Hooper, 1961 (AJ549986, AJ549987). Intraspecific variation for the ITS sequences was low, with only two nucleotides difference and no indels.
D2-D3 rDNA sequences obtained from both Romanian populations were identical to each other and to the sequence of L. piceicola from Slovakia (AY601577,
The SNPs analysis comparing all D2-D3 sequences of L. piceicola and L. intermedius revealed three parsimony-informative sites (i.e. nucleotide positions with transitions 89T/C, 134T/C and 297A/G) when compared to the reference sequence of L. piceicola (AY601577) (Table
The variable positions in D2-D3 28S rDNA control region sequences of Longidorus piceicola and L. intermedius. The L. piceicola sequence from Slovakia (Acc. no AY601577) was used as a reference.
SNPositions | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
89 | 129 | 134 | 197 | 255 | 285+1gap | 285+2gap | 297 | 310 | 413 | 514 | 584 | |
AY601577 reference sequence | T | C | T | A | C | – | – | A | T | G | G | C |
AY601577L. piceicola Slovakia | . | . | . | . | . | – | – | . | . | . | . | . |
KY086070L. piceicola Romania 1 | . | . | . | . | . | – | – | . | . | . | . | . |
LT669801L. piceicola Romania 2 | . | . | . | . | . | – | – | . | . | . | . | . |
AF480074L. intermedius Germany | C | . | C | . | . | – | – | . | . | . | . | . |
JX445117L. intermedius Spain | C | . | C | . | T | A | T | G | G | . | S | . |
KT308868L. intermedius Spain | C | . | C | . | T | A | T | G | G | . | T | . |
KF242312L. intermedius Russia | C | T | C | . | . | – | – | G | . | T | . | T |
KF242311L. intermedius Russia | C | . | C | C | . | – | – | G | . | T | . | . |
Morphologically, the specimens of L. piceicola from Romania are similar to the type-population from Slovakia (
The observed abnormalities (presence of reserve odontostyle) in females have been reported for other longidorids (
Longidorus piceicola was previously recovered in association with P. abies, Abies alba L., Fagus sylvatica L., Carpinus betulus L. and Vitis vinifera L. in Slovakia, West Balkans and Poland (
Based on the molecular and morphological characterization L. piceicola is closely related to L. intermedius: however, it differs in having a much longer odontostyle (151–169 μm in the type population and reported range of 144–183 μm vs 105–118 μm and 97–121 μm, respectively), generally longer body (4.22–5.97 mm in the type population and reported range of 4.42–7.99 mm vs 3.6–4.5 mm and 3.11–5.4 mm, respectively) and bigger anterior end – guide ring distance (37–45 μm in the type population and a range of 34–46 μm vs 25–34 μm and 27–36 μm, respectively); a wider lip region (14–18 vs 11–12.5 μm), more ventromedian supplements (11 vs 5–7) in the males, and four vs three juvenile stages (
This work was supported by the BAS project ANIDIV 2. The authors are thankful to Dr Nathalie Yonow from Swansea University, Wales, UK for critical reading of the manuscript and helpful suggestions.