Molecular characterisation of some plant-parasitic nematodes (Nematoda: Tylenchida) from Belgium

Catherine Malike Etongwe; Phougeishangbam Rolish Singh; Wim Bert; Sergei A. Subbotin

Russian Journal of Nematology, 2020, 28 (1), 1 - 28

Molecular characterisation of some plant-parasitic nematodes (Nematoda: Tylenchida) from Belgium

Catherine Malike Etongwe1, Phougeishangbam Rolish Singh1, Wim Bert1 and

Sergei A. Subbotin2, 3

hematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium 2Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA 3A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 117071, Moscow, Russia e-mail: sergei.a. [email protected]

Accepted for publication 20 February 2020

Summary. Using morphological, morphometric and molecular analysis, eleven valid nematode species from nine genera: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, P. nanus, Rotylenchus montanus and R. robustus together with twelve unidentified species, were identified in samples collected from eighteen locations in Belgium. The unidentified species include six Paratylenchus species, one Helicotylenchus species, three criconematid species and two Rotylenchus species. A total of new partial 21 18S rRNA, 69 28S rRNA, 10 ITS rRNA and 51 COI mtDNA gene sequences were obtained and used for phylogenetic and sequence analysis. Short descriptions, morphometrics and light and scanning microscopic photos are presented for selected species. Based on the results of molecular analysis, Hemicriconemoides promissus syn. n. was proposed as a junior synonym of H. pseudobrachyurus.

Key words: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, Paratylenchus nanus, Rotylenchus montanus, Rotylenchus robustus, phylogeny, 18S rRNA, ITS rRNA, 28S rRNA, COI mtDNA.

The rapid identification of potentially harmful plant-parasitic nematodes is of critical importance in the design of effective management strategies and control measures (Gon?alves de Oliveira et al., 2011). Despite the immense risk potential, the morphological diagnosis of plant-parasitic nematodes is often rendered a difficult task due to their high phenotypic plasticity and absence of clear diagnostic characters (Vovlas et al., 2008; Palomares-Rius et al., 2017).

Molecular taxonomy and DNA barcoding provide powerful tools for the identification of organisms, especially those organisms for which morphological diagnostic characteristics are scarce (Hebert et al., 2003). Their use is particularly advantageous because of the rapidity, accuracy and transferability of the methods, allied with their ability to be used in identification irrespective of life stage of the organism. Furthermore, they enable the discrimination of cryptic, very closely-related and

even distorted species (Eyualem & Blaxter, 2003; Bhadury et al., 2006; Miller, 2007; Ferri et al., 2009; Yao et al., 2010; Ahmed et al., 2016; Subbotin et al., 2018). At the same time, classification, species delineation, revision and understanding of phylogeny currently depends greatly on the usage of a suitable DNA domain within a nuclear or mitochondrial genome (Bae et al., 2009; Van den Berg et al., 2013). Due to the decreasing price and increased availability of sequencing instruments, the number of nematode species that have been sequenced has grown exponentially the last decades. However, despite this growth in work rate, the vast majority of morphospecies remain unlinked to DNA sequences. Moreover, a substantial part of the existing sequence data appears to be incorrect, with faults ranging from sequencing errors over misassembles to misidentified, mislabelled, or chimera sequences. Therefore, to design a reliable diagnostic strategy, it

is of utmost importance to safeguard the link between morphospecies and species-specific sequences, i.e. molecular barcodes (Janssen et al., 2017a; Qing et al., 2020).

Belgium has a long-standing tradition of conducting diverse nematode-based studies, resulting in a relatively well-studied nematofauna. Coomans (1989) provided a thorough list of Belgian nematofauna, albeit excluding animal-parasitic nematodes and Bert et al. (2003) listed the Tylenchomorpha (Tylenchida and Aphelenchida) of Belgium. More recently, Steel et al. (2014) have provided an updated list of Belgian nematofauna comprising 418 species, of which 170 are Tylenchida. At the time of writing this paper, the authors are aware of 13 tylenchid species that should be added to the current Belgian nematofauna list: Heterodera ustinovi (Subbotin et al., 2000), H. aucklandica (Subbotin et al., 2003); H. betae

(Amiri et al., 2003), Meloidogyne artiellia (Damme et al., 2013), Paratrophurus bursifer (Consoli et al., 2017), Pratylenchus brzeskii (Janssen et al., 2017a), Pratylenchus convallariae (Janssen et al., 2017b), Abursanema quadrilineatum (Qing et al., 2017), Malenchus cylindricus (Qing et al., 2018), Pratylenchus horti (Nguyen et al., 2019a), Rotylenchus buxophilus (Nguyen et al., 2019c), Scutellonema brachyurus and Meloidogyne incognita (Nguyen et al., 2019b).

The goals of the present study were: i) to carry out morphological and morphometric characterisation of species from the Criconematidae, Hoplolaimidae, Hemicycliophoridae, Dolicho-doridae and Paratylenchidae families collected in Belgium; and ii) to provide molecular characterisation of selected species using the D2-D3 of the 28S rRNA, ITS rRNA, 18S rRNA and COI mtDNA gene sequences.

Table 1. Nematode species and populations used in this study.

Sample code Locality GPS location Species

BE1 Ghent University Botanical Garden 51°2'7.53" N; 3°43'20.07" E Helicotylenchus sp. A, Paratylenchus nanus

BE2 Ghent University Botanical Garden 51°2'7.53" N; 3°43'20.07" E Paratylenchus sp. C, Rotylenchus robustus

BE4 Ghent University Botanical Garden 51°2'7.10" N; 3°43'19.28" E Paratylenchus sp. B, R. robustus

BE5 Ghent University Botanical Garden 51°2'7.55" N; 3°43'20.47" E Helicotylenchus sp. A

BE7 Blaarmeersen (Ghent) 51°02'39"N; 3°41'08"E Helicotylenchus varicaudatus

BE9 Ghent, Citadel Park 51°02'05"N; 3°43'10"E Paratylenchus sp. E, H. varicaudatus, Mesocriconema xenoplax

BE11 Zwijnaarde 51°00'19"N; 3°42'11"E Hemicriconemoides pseudobrachyurus, P. nanus, R. robustus, H. varicaudatus

BE13 Zwijnaarde 51°00'17"N; 3°42'10"E Mesocriconema xenoplax, R. robustus, Criconema annuliferum

BE14 De Panne 51°07'14"N; 2°39'29"E Criconemoides informis, Helicotylenchus sp. A, H. varicaudatus

BE15 Kortrijk 50°47'58" N; 3°11'37" E C. annuliferum, criconematid sp. B, Rotylenchus sp. B, Amplimerlinius icarus

BE16 Kortrijk 50°48'29.81" N; 3°12'30.79" E C. annuliferum

BE18 Blaarmeersen 51°07'14"N; 2°39'29"E C. informis, Paratylenchus sp. F

BE19 BE20 Blaarmeersen Blaarmeersen 51°02'18.9" N; 3°41'17.2" E 51°02'14"N; 3°41'23"E Hemicycliophora thienemanni, criconematid sp. B, Paratylenchus bukowinensis, Paratylenchus sp. 8, Paratylenchus sp. B, Paratylenchus sp. F Mesocriconema xenoplax, Paratylenchus sp. D, Paratylenchus sp. B, criconematid sp. A, criconematid sp. C, C. annuliferum, Rotylenchus sp. A, H. varicaudatus

BE21 Merendree 51°4'30.49" N; 3°34'39.65" E M. xenoplax, C. annuliferum

BE22 Merendree 51°04'12" N; 3°34'37" E M. xenoplax, C. informis, Paratylenchus sp. F

BE23 Heusden, Destelbergen 51°01'24.01" N; 3°80'69.86" E Rotylenchus montanus

BE24 Eine, Oudenaarde 50°87'73.07" N; 3°58' 17.62" E A. icarus

Fig. 1. Hemicriconemoides pseudobrachyurus female from Belgium. A & B: Scanning electron microscopic (SEM) photos of anterior region; C & D: SEM photos of posterior region; E: Light microscopic (LM) photos of posterior region; F & G: LM photos of anterior region. F and G have same scale.

MATERIAL AND METHODS

Nematode populations sampling and extraction. Soil samples were collected using an auger at a depth of 15-30 cm from several locations in Belgium (Table 1). Samples were randomly collected from the rhizosphere of several unidentified plants and trees with different soil characteristics, vegetation and topology. Approximately 500 g of soil was put in plastic containers, labelled and kept at 4°C. Nematodes were extracted from soil at the Nematology Research Unit of Ghent University, using the modified Baermann tray technique (Whitehead & Hemming, 1965), Cobb's sieving and decanting method using two sets of sieves (1000 ^m mesh to remove debris, then 38 jum mesh for the collection of the nematodes) followed by sugar centrifugation method (Jenkins, 1964).

Light microscopy. Nematodes used for light microscopy were killed by gentle heat, fixed in Trump's fixative [2% paraformaldehyde + 2.5% glutaraldehyde in a 0.1 M Sorenson buffer (sodium phosphate buffer at pH 7.3)], transferred to anhydrous glycerin (De Grisse, 1969) and mounted on permanent slides following the method of Singh et al. (2018). Photographs and measurements were made from both temporal and permanent slides by using an Olympus BX5 DIC Microscope equipped with an Olympus C5060Wz camera. The measurements were made using Image J 1.51 under 10x, 20x, 40x and 100x magnifications. Holotype slide R 766 and paratypes slides (R 324-665-715) of Hemicriconemoides pseudobrachyurus obtained from the Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University were used in this study for comparison with Hemicriconemoides species collected during this study.

I— Paratylenchus goldeni (KJ934186) jo rl— Paratylenchus microdorus (AY234632) ¡4 L Paratyenchus slmilis (KJ636432)

Paratylenchus microdorus (AY284633) Г Paratylenchus sp.E (MN783662, Belgium, ВЕЭ N5( П- Paratylenchus sp F (MN783663, Belgium. BE9 N6) Paratylenchus spB (MN783659, Belgium, BE2 N6) Paratylenchus dianthus (AJ 966496) Paratylenchus sp.B {P.1N 783661, Belgium, BE4 N5) Paratylenchus зр.В (MN783680, Belgium, ВЕЛ N1) г- Paratylenchus projectus (KJ636433) '— Paratylenchus conicephalus (KP966493) Paratylenchus projectus [USA, Nevada, MF094390) Paratylenchus sp.C (MN783658, Belgium, BE2 NS) ■ Paralyienchus projectus (MF094897) Paratylenchus tenufcsudalus (KU291241) r Paratylenchus sp, F (KJ63M35)' ,yf Paratylenchus sp.F (MN783669, Belgium, BE22 N1) L Paratylenchus sp.F (MN783668, Belgium, BE22 N2) 1 Paratylenchus sp.F (MN783670, Belgium, BE18 N2) Paratylenchus sp. (MF094926) i£2r Paratylenchus sp. (MF09S023)

Paratylenchus Straelen: (AY284630) — Paratylenchus sp. (JX094442)

Paratylenchus nanus type A (MN783664, Belgium, 8E1 N7) Paratylenchus nanus type A (MN783667, Belgium, BE11 N5) Paratylenchus nanus type A (MN783666, Belgium, BE1 N6) Paratylenchus nanus type A |MN783665 Belgium, BE1 N8)

Paratylenchus nanus (KY119503)

so г Ogma seymouri (KX344498) —'1 Ogma cobbi (EU669918)

Crassonema fimbriatum (MFQ95026) Cnconema longulum (KX344495)

Xenocriconemella macrodora (MF094906) 811— Ogma decaineatus (MF094952)

60

Hemicriconemoides wessoni (KJ934166 : - Hemicriconemoides peeudobrachyurus (AY284622)

Hemicriconemoides pseudobrechyurus (MN783672, Belgium, BE11 N13) ■ Hemicriconemoides pseudobrachyurus {MN783871, Belgium, BE11 N121

1— Hemicriconemoides pseudobrachyurus (AY284624)

— Hemicriconemoides hanayaensis (MG029559)

— Cnconema mutsMfe (MF094914)

г Hemicriconemoides parataiwanensis (MG029556) ■ Hemicriconemoides criiiwoodi (MF094939)

- Criconemoides parvus (MF795586)

Criconemoides sp, (JX216045)"

Criconemoides Informis (USA, MF094902) Criconemoides informis (China, KF900157) ЮР г Ogma sp. (KJ934175)

78 100

; г

J 1 Neotobocrfconema senatum (MH668971)

1— Discocriconemella sinensis (MK253543)

- 94,—

J4E,

Щ 1

— Criconemoides informis (MN783673, Belgium, BE22 N9)

Hemicycliophora conida (KJ934172) Hemicycliophora sp. (KJ934170) Hemicycliophora thienemanni (AY284&29) Bakernema inaequale (MF09490S)

Rotylenchus robustus (MN 783675, Belgium, BE2 N7)

Rotylenchus robustus (AJ966503) Rotylenchus robustus (AY593882)"* +■ Rot^enchus robustus (KJ636397) Rotylenchus robustus (KJ 636429) Rolytencrius robusius (KJ63639S) Rotylenchus robustus (MN783677, Belgium. BE2 N9) Rotylenchus robustus (MN7B3676, Belgium, BE2 N11)

85Г-

гП"

^r Rotylenchuscretensis(KT2eeo68) Rotylenc/ius urmiaensis (KP718970) Hoplolaimus galeatus (KJ934131)

r Amplimerlinius icarus (EU 306351) J1- Amplimerlinius icarus (MN783678, Belgium, BE24 N2)

|L Amplimerlinius macrurus MK9S2146) L Amplimerlinius macrurus (KX789711) Amplimerlinius globigerus (KX789713) /Imp/imeriinius macrurus (KX789710) Г Pratylenchoides alkani (MK301104) 1— Pratylenchoides nevadensis (KU854995) г Merhntus brevidens (KX7897C8) 1— Parameriinius hexagrammus (KX789720) - Nagetus leptus (KX789718) — Aglenchus ague о fa (KJ869321) - Coslenchus costatus (AY284581)

0.1

Rotylenchus cypriensis (KT288069) »[r Rotylenchus montanus (KJ636415)"" —p Rotylenchus montanus (MN783674. Belgium, BE23 N4)

30 L Rotylenchus montanus (KJ636365)""

Fig. 2. The 50% majority rule consensus tree obtained using Bayesian analysis of the 18S rRNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * - identified as Paratylenchus nanus by van Megen et al. (unpublished); ** - identified as Hemicriconemoides sp. by Pham & Zheng (unpublished); *** - identified as Rotylenchus uniformis by Helder et al. in the GenBank; **** - identified as Rotylenchus robustus by van Megen et al. (2009).

Ммоспсеьпета xenoptax (MNТаэбВг, Belgium, BE22 Mezacncanema xenapiax (USA, California. FN433B67) Mcsocrtconcma xcnoplax (MN7e3G90, Belgium, BE20 N2) Mcsocrtconcma xertopfax (MN7a3Ga9, Belgium, BE21 N4; Mesocrsconema xenapfax MN783G88, Belgium, BE21 IM2| Mesocrsconema xenaptax IMN783G87, Belgium, BE13 N9i Mesocriconema xenoplax MN783G9G, Belgium. BE13 N101 MesocricQnema xenoptax MN7S3695, Belgium. BE13 N11) Mesotrrronoma xenoptax (MN7a3G84, Belgium, BE13 N12) Mesocrrconema xenoptax (MN7a3<jfi3, Belgium, BE13 N4) Mesocriconema xenoptax (ttaly, AY76Q964) Chconemoides brevistytus

Chconerrjoides informs (Greece, AV7S0970) Chconemoides informs (1*ап KU7223BB) Crtconemotdes informts (MN783G8Q, Belgium, BE14 N1} Crtconetnotdes informs (MN783G78, Belgium, В El® N6. N8) Crrconemwdes informis (M«793591. Belgium, BE22 N9) Discocriconemetfe sinensis (MK253537)

Heruicycfiophofa fhienemanni (Italy, AY7B0&7B) Hemicycttophora tftrenemann/|M N783992, Belgium, B19 N1) Hemtcycfiaphara thienemanm (Spain, KF430467) Hemtcyctiophota thiefie/nannt (Russia, KF43M7D) HemicycHophora thienemanni (Russia, KF43fM71 j Hemicyctiophora spia(MG4>19&20) HemicysiiQpbQni gracilis (MG01&B23) Chconemoides obtusicaudatus (JG231 106)

-criconematid nematode &p, A (MN783991, Belgium, BE2Q N3}

criconema зп/ш^нда(М1Ч7В399Э> Belgium, be 15 N9L Cnconema annuhterum (MN7E3702. Belgium, BE15 N51 Criconema annutiterum (MN783701, Belgium. BE15 N4f

_____________________________________ Jflil r

Criconema annutiferum]MN783700, Belgium. BE15 N5 Criconema annuliterum (MN793997. Befgium, BE19 Ni) №cowma annutiterum (M N783698, Belgium, BE16 N3) - Criconema demam (MHB2Bl2e)

- Ogma decaHneatus (MF633230)

_r criconematid nematode sp. В (MN793695. Belgium, BE19 N7)

1Ю1 criconematid nematode sp. В (MN783998, Belgium, BE 15 N7)

- Criconema sitvum (MF5B3234)

- Hemicnconemoides califomianus (KFB5053B)

Hemicriconemoides pseudobrachyurus (МН7азбЭ4, Belgium, ВЕН N12, N19) Hemicncortemofd&i pseudobrachyurus {Spain, KF856S27)* Hemicriconemoides psoudobrachyurus (MN7S3C93, Belgium, BE11 N9) ^mj'irtcorisiraai'des pseurtobi^byurt/s (Spain, KF856529)4

- Hemicnocnemoides macrodorus (KF856523)

imt Pamtytenchus sP.2 (KF342230) "J1- Paratytencbushamatus (KF242212)

Paratylenchus tenuicaudatus (KU291239) Paratylenchus sp С |MN783709, Belgium BE? N4)

Paratylenchus bukowinensis (Italy, A.Y 730943 j 1 Paratylenchus bukowinensis (MN793793, Belgiun

—• Paratylenchus sp. (AY7oQ9J^>

BE19 N13)

Paratyfenchus sp D (MN7S37Ü4. Belgium, BE20 N10) L—— Pamiylenchus sp.B (HN783705, Belgium, BE4N1) ■ ■ 100 Г Paratyfenchus sp.F (HH733707. Belgium, be 18 N2, N5. №) iCCiL Paratyfenchus (MN7B37Ú6, BeTgium, BE22 N3, N1, N5, N2, N4)

-\ 1- Fers(yíení!hy5sp.3rKF242232)

1- Parafytensbvs ¿W/ryg (KF242229J

Paraiyienchus nanus type a (MN7B3709, Belgium, BE11 ne) Parafytenchus nanus type A (MN793710, Belgium, BE1, NB. N6. N7)

■ ParñMenchus nanus lype A (USA, California, KF2J21&6) РагэГуЫеЬиг sp.7 {KF2422J2)

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— Paratyfenchus sp.E (MN793712, Belgium, BE9 N6} _t Paratyfcnchus sp.S (HN783711, Belgium, BE19 N2)

P0r$fyienchv$ sp.BjUSA, California, KF242233)

r r P0r$ fy!$rtChu$ Sp_6 ,. .. lf ^^

Paratyfenchus sp.5 ^

100

H$liCQtyt$nehv$ Sp-A (MN783729, Belgium, BE1 N3) H&icotyfenthus sp-A ¡мN783732, B&tgium, bes Nia, N4, MS) Heticolylenchus sp.A (MN793731, Betgium, BE5 N2)

■ Helicotyienchiis sp.A MN793730 Beigium, BE14 N4) HeticvlytenQhus sp.A MN793729, Betgium, BE14 N3)

■ Heficotyfenctous spl-11 (AB6O20O2)

■ HeHcotyierKbus dyentem (КМ50Ш7) Heticotyfanchus psevdQfQbustvs (KU7223B7) HetKijtvientbuS dihyslera fKM5Q(3a341

Heticotyfenchus varicaudatus (МЙ783723, Belgium, BET N5) Helicojytettcfws varicawiiaiw (mn7a3724, Belgium, BE20 N1) -teticotyfonchus vanceudatus (Russia

HefiGotylenctous vancautiatus {mn7в3725. Belgium, BE7 N7. N1, N5, N2)

Hebcotyfenchus varicaudatus {00328754)"" Helicofyfenchus varicaudatus (Poland, MG653535) Rotyienchus cypriansis (КРв43116) 99 Rotytenchus robuslus (MN79J719, Belgium, BE13 N2) 1 Rotyfenchus robusius (Belgium, DQ328735)" Rotyfenchus robusius (1У№7&371&. Belgium, BE2 N1, N2) Roiytwcfws jnoj^stiis МЫ7&3722. eetgium, BE11 N4) RofytenQhvs /oiwsfys (USA. JJiOl &42C) floiy/encftus fObUStUS (HN7B3721, Belgium, BE 13 N7) Roiyienc^i/s fotoustus {HN79372&, Belgium, BE 13 N6) ■ Rotyfenchus robusfus (Belgium, DQ329740)" Rvtytenchvs robusius {HR793719, Belgium, BE4 N4) Rciyfenchus robusius HN793717, Belgium, BE11 N19)

L Rofytejrettus magmas (Еи2а07Й91 --- Roiyí&jtchuí eajflf/aertííS (EUÍÓ07ft3)

r Rotyfenchus morttartus (Russia, DQ328742px 1 Roiy/enchus montanus (MN7&3726. Belgium, BE23 N4)

1 Rotyfenchus montenvs (DQ32B74J) ■ Rotyfenchus rfteuTibcu£tes{MKl 14123)

1 Rotylenchus sp.B (MN7B3727, Belgium. BE15 NB)

г Ampfirneriinius icarus (Belgium, DQ328714J

. Cosfenchns costatus (DQ33B719) -Aglenchus agricofa (AY78G979)

j Ampfimerfinius icarus (Belgium^ DQ323714) tnn Г Ampfrmerlinius icarus ¡MN793715, Belgium, BE15 N1) JZLh Amplimerftnius icarus (MN783714, Belgium. BE15 N3) L Amplifnerlinius icarus |MN793713, Belgium. BE24 N4)

— Pratyienchoides magnicauda {KFD262fl9) ■ Amptiniitrtinius giobigerus (KP31384D) „ - Snjtyfeftcbtjs fugaiui (KP31i&51) B?- Medina brevifens (KJ5B54ie)

0.1

Fig. 3. The 50% majority rule consensus tree obtained using Bayesian analysis of the 28S rRNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * - identified as Helicotylenchus sp. IX-4 by Subbotin et al. (2015); ** -identified as Rotylenchus uniformis by Subbotin et al. (2007); *** - identified as Rotylenchus sp. by Subbotin et al. (2007); **** - identified as Helicotylenchus sp. IX-3 by Subbotin et al. (2015).

Scanning microscopy. For scanning electron microscopy (SEM), specimens fixed in Trump's fixative were washed in 0.1 M phosphate buffer (pH = 7.5) and dehydrated in a graded series of ethanol solutions, critical-point-dried with liquid CO2, mounted on stubs with carbon tabs (double conductive tapes), coated with gold of 25 nm, and photographed with a JSM-840 EM (JEOL) at 12 kV (Singh et al., 2018).

DNA extraction. Live nematode specimens were handpicked using a picking needle under a binocular microscope, put into a drop of water on a glass slide, killed by gentle heat and temporary slides sealed with paraffin wax. Pictures and measurements were made using the instruments mentioned above. This was done to maintain a link between morphology and molecular data of species obtained. Later the nematodes were removed from the temporary slides, picked and put in 20 ^l double distilled water on a glass slide, cut into two or more pieces using a stainless-steel dissecting blade under a dissecting microscope. The cut nematodes were transferred into a 0.2 ml Eppendorf tube, containing 20 ^l worm lysis buffer (50 mM KCl; 10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween 20) for DNA extraction. The tubes were kept at -20°C for 10 min after which 1 ^l proteinase K (1.2 mg ml-1) was added into the Eppendorf tubes and tubes were incubated in a PCR machine at 65°C (1 h) and 95°C (10 min) consecutively. After incubation, the tubes were centrifuged and kept at -20°C until use in the DNA database of the Nematology Research Unit of Ghent University, Belgium.

PCR and DNA sequencing. Two ^l DNA template was used from the tubes for PCR. Four gene fragments were amplified and sequenced for this study: 18S rRNA, the ITS rRNA, the D2-D3 expansion segments of 28S rRNA and the partial COI genes. The forward primer SSU18A (5'-AAA GAT TAA GCC ATG CATG-3') and the reverse primer SSU 26R (5'-CAT TCT TGG CAA ATG CTT TCG-3') as described by Mayer et al. (2007) were used for amplification of 18S rRNA gene, For the ITS rRNA the forward primer TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3'), and the reverse primer AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') as described by Curran et al. (1994) were used. The forward primer D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3'), and reverse primer D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') as described by Subbotin et al. (2006) were used for amplification of the D2-D3 expansion segments of 28S rRNA gene. For amplification of rRNA genes, the PCR reaction included Mastermix (17 ^l ddH2O; 2.5 ^l 10x buffer; 2 ^l MgCl2; 2.5 ^l Coralload; 0.5

^l dNTP (10 mM); 0.5 ^l of each primer; 0.05 ^l Toptaq) and 2 ^l of DNA template. The thermocycling profile for 18S rRNA gene consisted of 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min 30 s at 52°C, and 2 min at 68°C, followed by a final step of10 min at 68°C. The PCR amplification profile for ITS and 28S rRNA genes consisted of 4 min at 94°C, 35 cycles of 1 min at 94°C, 1 min 30 s at 55°C, and 2 min at 72°C, followed by a final step of 10 min at 72°C.

The forward primer JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and the reverse primer JB4 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') as described by Derycke et al. (2010) were used for amplification of the COI gene for the majority of nematodes, whereas the forward primer COIF5 (5'-AAT WTW GGT GTT GGA ACT TCT TG AAC-3') and the reverse primer COIR9 (5'-CTT AAA ACA TAA TGR AAA TGW GC W ACW ACA TAA TAA GTA TC-3') as described by Powers et al. (2014) were used for amplification of the COI gene from criconematids. For the partial COI gene study, 3 ^l of extracted DNA was added to a 0.2 ml Eppendorf tube containing 23 ^l of Mastermix as described above. For specimens from the Criconematidae family, 10 ^l DreamTaq Green PCR Master Mix (2x) (Thermo Fisher Scientific), 10 ^l water and 0.15 ^l of each primer (1.0 ^g ^l-1) was used. The PCR amplification profile for the JB3/JB4 primers consisted of 3 min at 94°C, 34 cycles of 30 s at 94°C, 30 s at 45°C, and 1 min at 72°C, followed by a final step of 10 min at 72°C. The PCR amplification profile for the COIF5/COIR9 primers consisted of 4 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 45°C, and 1 min 30 s at 72°C, with a final extension at 72°C for 10 min. Five ^l of the PCR product was run on a 1% TAE-buffered agarose gel (130 V, 20 min). PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and directly sequenced with the primers mentioned above by Macrogen Inc. The new sequences were submitted to the GenBank database under the accession numbers: MN783679-MN783742, MN783658-MN783678 as indicated in phylogenetic trees (Figs 2-4).

Phylogenetic and sequence analysis. Alignments with the 18S rRNA, ITS rRNA, 28S rRNA and COI gene sequences were created using Clustal X 1.83 (Thompson et al., 1997) with default parameters. New sequences were aligned with corresponding gene sequences available in the GenBank (Zeng & Ye, unpublished; van Megen et al., unpublished; Holterman et al., 2006, 2009, unpublished; Ghaderi et al., unpublished; Powers et

al., 2010, 2014, 2016a, b, 2017; Munawar et al., unpublished; Subbotin et al., 2005, 2006, 2007, 2014, 2015; Bert et al., 2008; Vovlas et al., 2008; Van den Berg et al., 2012, 2013, 2014a, b, 2015, 2017, 2018; Cantalapiedra-Navarrete et al., 2013; Ghaderi et al., 2014; Panahandeh et al., 2014; Esmaeili et al., 2016; Olson et al., 2017; Maria et al., 2018, 2019; Rybarczyk-Mydlowska et al., 2019, unpublished; Tandingan De Ley et al., unpublished; Wang et al., unpublished; Holroyd et al., unpublished; Cordero et al., 2012; Kanzaki & Futai, 2002; Pang et al., unpublished; Golhasan et al., 2016; Alvani et al., unpublished; Helder et al., unpublished; Meldal et al., 2007; Pham & Zheng, unpublished; Ortiz et al., 2016; Jin, unpublished; Afshar et al., 2019; Tzortzakakis et al., 2016; Noruzi et al., 2015; Skwiercz et al., unpublished; Schratzberger et al., unpublished; Azizi et al., 2016, unpublished; Karegar et al., unpublished; Yang, unpublished; Alvani et al., unpublished; Tabolin & Markina, unpublished; Pang et al., unpublished; Nguyen et al., 2019c).

The sequence alignments were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003). The best-fit model of DNA evolution was obtained using the program jModelTest 0.1.1 (Posada, 2008) with the Akaike Information Criterion. The BI analysis for each gene was initiated with a random starting tree and was run with four chains for 1.0 x 106 generations. The Markov chains were sampled at intervals of 100 generations. Two runs were performed for each analysis. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analysis. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) were given on appropriate clades. Trees were visualised with the TreeView 1.6 program and drawn with Adobe Illustrator v. 10. Pairwise divergence between taxa was calculated as the absolute distance value and the percent of mean distance, with adjustment for missing data, using PAUP* 4b10 (Swofford, 2003).

The alignment for ITS rRNA sequences was used to construct phylogenetic network estimation using statistical parsimony (SP) as implemented in POPART software (http://popart.otago.ac.nz) (Bandelt et al., 1999).

RESULTS AND DISCUSSION

Species identification and delimitation. During this study, 173 nematodes were taken for morphological and molecular study from 18 locations in Belgium. A total of 121 new sequences was

obtained: 21 - partial 18S rRNA gene, 69 - D2-D3 of 28S rRNA gene, 10 - ITS rRNA gene and 51 - COI mtDNA gene. Species delimitation was performed using an integrated approach that considered morphological and morphometric evaluation combined with molecular criteria based on phylogenetic inference and sequence analyses. Eleven valid species from nine genera were identified during this study namely: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, P. nanus, Rotylenchus montanus and R. robustus. Twelve putative species of several genera were also identified: six - Paratylenchus (Paratylenchus sp. B, Paratylenchus sp. C, Paratylenchus sp. E, Paratylenchus sp. 8, Paratylenchus sp. D and Paratylenchus sp. F), one Helicotylenchus (Helicotylenchus sp. A), three criconematids (criconematid sp. A, criconematid sp. B, criconematid sp. C), and two Rotylenchus (Rotylenchus sp. A, Rotylenchus sp. B). Identifications of these species at species level have not been done because of the limited number of specimens.

Samples contained from one up to eight nematode species of the different studied genera. The most diverse sample was Blaarmeersen (BE20), which had representative species from six genera. Most samples consisted of a single species per genus (monospecific), except for some (BE19 and BE20) that contained more than one species of Paratylenchus.

Morphological and molecular

CHARACTERISATION Hemicriconemoides pseudobrachyurus De Grisse, 1964 (Fig. 1)

Hemicriconemoides pseudobrachyurus was first described from sandy soil around roots of grass plants and potatoes in Huise (Kruise), Belgium (De Grisse, 1964). During our study, the attempt to collect H. pseudobrachyurus from the type locality failed; however, several female specimens of this species were collected from a location in the proximity from the type locality. Morphological and morphometric characters of this

H. pseudobrachyurus population collected from a geographically close (15 km away) and similar habitat (Eedstraat, Zwijnaarde) agreed with the original description. This population is compared with the populations of H. promissus collected from different localities by other authors (Table 2).

Measurements are given in Table 2.

Table 2. Morphometric comparison of Hemicriconemoidespseudobrachyurus. All measurements are in ¡im and in the form: mean ± s.d. (range).

Species Characters* (abbreviations are as defined by Subbotin et.aL (2014) and Geraert (2010)) H. pseudobrachyurus H. pseudobrachyurus ( = H. promissus syn. n.)

Present study Zwijnaarde, Belgium De Grisse (1964) Huise, Belgium Vovlas (1980) Torre Canne, Brindisi, Italy Vovlas et al (2006) Bolonia (Cadiz), Spain Van den Berg et al (2014b) Monteagudo Island, Spain Van den Berg et aL (2014b) Bolonia, Spain

N 10 females 20 females 20 females 10 females 10 females 16 females

L 536 ± 50 (452-591) 531 (440-580) 580 (460-750) 491 ±51 (438-700) 447 ± 43 (400-527) 491 ±51 (438-560)

a 12.9±0.8 (11.3-13.3) 14(12-17) 16(15-20) 12.2 ±0.8 (11.2-13) 13.9 ± 1.2 (11.8-15.8) 12.2 ±0.8 (11.2-13)

b 4.8 ±0.5 (4.3-5.9) 4.9 (4.2-5.7) 5.8(5.5-6.4) 4.6 ±0.5 (4.1-5.3) 4.6 ±0.5 (4.0-5.4) 4.6 ±0.5 (4.1-5.3)

c 24.7 ±5.3 (18.4-36.0) 24(17-31) 30 (27-35) 22.8 ±2.1 (20.8-25.5) 25.7 ±3.0 (20.0-28.6) 22.8 ±2.1 (20.8-25.4)

c' 0.9 ±0.1 (0.9-1.0) - - - 0.8 ±0.1 (0.7-1.0) 0.99 ±0.04 (0.95-1.05)

0 4.9 ±0.9 (4.8-5.2) - - - 8.3 ±0.8 (7.3-9.2) 9.1 ±0.9(8.3-10.4)

DGO 2.8 ±0.5 (2-3) - - - 4.4 ± 0.4 (4-5) 4.4 ±0.5 (4-5)

V 95 ±2.4 (93-95) 95 (93-96) 94-95 95 ± 1.0 (94-96) 93.2 ±0.8 (92-94) 95.0 ± 1.0 (94-96)

G1 49.1 ±5.2 (45.1-55.1) - - - 50.8 ±5.9 (40.5-58.2) 55.8 ± 1.4 (54.5-57.9)

Ovary length 258 ± 7.3 (250-268) - - - 225 ± 35.5 (162-260) 274 ±35.5 (242-324)

Stylet length 57 ± 1.6 (55-60) 55 (52-59) 49(48-51) 48 ± 0.8 (47-49) 52.9 ± 1.4 (51-55) 48.0 ± 0.8 (47-49)

Metenchium length 47.6 ± 2.0 (46-48) - - - 44.7 ± 1.8(43-47) 37.3 ± 1.0(36-38)

M 83.6 ± 1.6 (81.7-86.3) - - - 83.9 ± 1.6(81.7-85.5) 77.6 ±2.4 (75-80.8)

Stylet knob height 4.2 ±0.4 (3.9-4.7) - - - 2.9 ±0.3 (2.5-3.0) -

Stylet knob width 9.6 ±0.8 (8.7-11.5) - 8.5-9.0 - 9.3 ±0.3 (9.0-9.5) 8.5 ±0.6 (8-9)

Anterior end to excretory pore 148 ± 14.2(126-166) - 159 (155-165) - 98 ±6.1 (90-104) 130 ± 10.0(124-145)

Diam at mid body 41.4 ±2.6 (38-45) - - - 32.2 ±2.4 (28-35) 40.3 ± 1.9(39-43)

Annulus width 5.9 ±0.4 (5.1-6.3) - - 5-6 4.3 ±0.3 (4.0-4.5) 5.5 ±0.6 (5-6)

Tail length 22.2 ±3.1 (15-25) - - - 17.6 ±2.2 (14-21) 21.5 ±0.6 (21-22)

Pharynx length 111 ±6.8(100-122) 110(97-128) - 89-101 97 ±6.7 (89-102) 106 ±2.6 (104-110)

R 101 ±4.2 (95-108) 98 (91-103) 97 (89-102) 96 ±4.1 (92-100) 105 ±5.2 (97-117) 95.5 ±4.1 (92-100)

RSt 14.0 ± 1.4(12-16) 11 (9-13) 8 to 9 9± 1.0 (8-10) 12.2 ±0.9 (11-14) 8.8 ±0.9 (8-10)

Roes 21.6 ± 1.6(18-23) 15(14-16) 16 ± 1.0(15-17) 20.9 ± 1.8(19-25) 15.8 ±1.0 (15-17)

Rex 28.9 ± 1.0 (27-30) 25 (23-29) 24 (23-26) 25 ± 1.3 (23-26) 22.3 ± 1.9 (20-26) 24.5 ± 1.3 (23-26)

Rvan 1 1 1 1± 0.5 (1-2) 1 1

Ran 5.8 ±0.5 (5-6) - 6 to 7 6.3 ± 1.1 (5-9) 1.3 ±0.5 (1-2)

VL/VB 1.1 ±0.2 (0.9-1.4) - 0.9(0.8-1.0) 0.9 ±0.1 (0.9-1.0) 1.1 ±0.1 (0.9-1.2) 0.9 ±0.05 (0.9-1.0)

St%L 10.7 ± 1.2 (9.7-12.8) 10.3(9.2-11.8) 8.4 (6.8-10.4) 8.8 ±0.9 (7.4-10.2) 11.9 ± 1.1 (10.4-13.6) 9.8 ±0.8 (8.8-10.7)

VL/ST 0.5 ±0.1 (0.5-0.6) 0.5 (0.4-0.6) 1.7(1.5-1.7) - - -

St% pharynx 52 ±3.5 (48-57) 50 ±5.3 (46-53) 47 (45-49) 50 (43-54) - 51 ± 3.2 (45-54)

Description. Female. Body cylindrical and slightly curved ventrally when heat-killed. Closely fitted sheath with smooth annuli, attached at the anterior end and the vulva. No lateral line present. Anastomosis of annuli not seen. Cephalic region truncated with two sublateral legs and body annulus. Cephalic region collapsed after fixation. Stylet long and robust with strong anchor-shaped knobs anteriorly indented and rounded posteriorly. Dorsal pharyngeal gland opening situated very close to the stylet base. Excretory pore situated 7-9 annuli posterior to base of the pharyngo-intestine junction. Hemizonid not seen. Vulva located usually on the sixth (6-7) annulus from the tail terminus, open and without vulva flap. Vagina straight, oblique anteriorly directed. Spermatheca relatively large, round, filled with rounded spermatozoids. Anus one annulus away from the vulva. Tail conical ending with a small finely rounded lobe (Voucher slides UGnem 240-241; Nematology Research Unit, Ghent University).

Male and juvenile. Not found.

Molecular characterisation. Intraspecific sequence variation for H. pseudobrachyurus was 00.8% (0-7 bp), 0-0.7% (0-4 bp) and 2.1-4.6% (8-17 bp) for 18S rRNA, D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 2, 3 and 4. The ITS alignment for H. pseudobrachyurus included five sequences and was 803 bp in a length. Three new ITS sequences were obtained during this study. Maximal sequence diversity was 0.7% (5 bp). The ITS haplotype phylogenetic network is given in Figure 5A.

Synonymisation of H. promissus Vovlas, 1980 syn. n. with H. pseudobrachyurus. Morphology and morphometrics of Hemicriconemoides specimens collected in this study agreed with the original description and type material of H. pseudobrachyurus. This species is very similar with H. promissus described by Vovlas (1980) from the rhizosphere of halophilic plants from a sandy dune of Torre Canne, Brindisi, Italy. Hemicriconemoides promissus was considered to be morphologically very similar to

H. pseudobrachyurus, but was considered different based on the length of the stylet (48-51 vs 52-59 ^m), RSt (8-9 vs 9-13) and the higher value of the ratio of vulva terminus distance divided by the stylet length (VL/ST = 1.5-1.7 vs 0.39-0.62). Germani & Anderson (1991) synonymised H. promissus with H. brachyurus but Vovlas et al. (2006) reestablished H. promissus as valid species and differentiated it from H. brachyurus and H. pseudobrachyurus. Vovlas et al. (2006) indicated

that H. promissus can be further separated from H. pseudobrachyurus by its slightly shorter pharynx (89-101 vs 97-128 ^m) extending for 15-17 vs 16-23 body annuli; presence of spermatheca filled with spermatozoids in all females in both, Italian and Spanish populations vs presence of spermatheca but never clearly visible spermatozoids in H. pseudobrachyurus. The male was not known for this species. Geraert (2010) did also not support this synonymisation and listed H. promissus as a valid species. Our study also showed similar differences between H. pseudobrachyurus and H. promissus; however, because there are high similarities in the 18S rRNA (0.4%), D2-D3 of 28S rRNA (0.7%), ITS rRNA (0.5%) and COI (4.6%) gene sequences of both species, we consider these species to be co-specific and their morphological differences is a result of geographical variation. We suggest that H. promissus syn. n. should be considered as a junior synonym of H. pseudobrachyurus.

Mesocriconema xenoplax

(Raski, 1952) Loof, 1989

This nematode has a worldwide distribution and wide host range (Karanastasi et al., 2008). It is known for damaging the roots of plants. In Belgium, it was found parasitising turf grass (Vandenbossche et al., 2011). In this study, two populations of this species from different locations (Zwijnaarde and Merendree) have been morphologically and molecularly characterised.

Measurements are given in Table 3.

Description. Female. Body ventrally curved to C-shaped when fixed. Cuticular annuli retrorse, with faint longitudinal lines seen. Anastomoses seen in all specimens observed at the cephalic region, mid-body and vulva. Cephalic region continuous with body. Labial disc elevated with two sub-median lobes seen projecting anteriorly. Stylet robust with strong knobs. In one specimen the reproductive system was reflexed. Spermatheca rounded, vagina sigmoid. Tail bilobed.

Male. Not found.

Molecular characterisation. Intraspecific sequence variation for M. xenoplax was 0-0.4% (0-5 bp), and 0-0.5% (0-2 bp) for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 3 and 4. The ITS rRNA gene alignment for M. xenoplax included 30 sequences, including four new sequences obtained during this study. The alignment was 315 bp in the length. Maximum intraspecific ITS sequence variation was 1.7% (0-5 bp). The ITS haplotype phylogenetic network is given in Figure 5B.

Table 3. Morphometries of Mesocriconema xenoplax, Criconema annuliferum and Criconemoides informis from Begium.

All measurements are in [j,m and in the form: mean ± s.d. (range).

Species Mesocriconema xenoplax Criconema annuliferum Criconemoides informis

Characters Zwijnaarde Merendree Kortrijk Zwijnaarde Merendree Blaarmeersen Merendree

N 10 females 10 females 10 females 7 females 5 females 1 female 1 female

L 574 ±32.0 (526-611) 505 ± 115.4 (392-693) 542 ±40.5 (478-608) 515 ±42.6 (482-578) 564 ± 24.4 (527-586) 538 422

a 11.3 ±0.7 (10.4-12.1) 11.7 ± 1.4(10.7-13.9) 8.7 ±0.6 (8.2-9.6) 9.6 ± 1.0(8.1-10.5) 10.0 ±0.7 (9.0-11.0) 10.9 7.4

b 4.1 ±0.2 (3.9-4.3) 4.0 ±0.5 (3.5-4.6) 4.0 ±0.2 (3.7-4.4) 3.9 ±0.3 (3.6-4.3) 4.1 ±0.1 (4.0-4.3) 4.3 3.7

c 24.3 ±15.8 (17.0-44.9) 22.0 ±5.5 (17.5-30.0) 21.9 ±2.9 (18.2-26.4) 30.7 ±6.2 (23.8-39.1) 26.1 ±4.7(19.4-30.9) 23.3 32.8

c' 0.8 ±0.2 (0.5-0.9) 0.8 ±0.1 (0.7-0.9) 0.9 ±0.1 (0.7-1.1) 0.8 ±0.1 (0.7-1.0) 0.8 ±0.1 (0.7-1.0) 0.6 0.5

0 9.9 ± 1.1 (8.4-11.5) 12.1 ± 1.8(10.2-14.5) 4.9 ± 1.0 (3.6-6.1) 6.8 ± 1.1 (5.7-8.6) 9.5 ±1.5 (7.1-10.9) 8.9 11.6

DGO 7.6 ±0.8 (6.4-8.6) 6.9 ±0.7 (6.1-7.7) 4.7 ± 1.1 (3.4-6.0) 7.0 ±1.3 (6.1-9.3) 9.4 ± 1.5 (7.2-10.9) 6 7.1

V 95 ±2.0 (94-96) 93 ±0.6 (93-95) 89 ±0.5 (89-90) 90 ± 0.6 (89-90) 90 ±0.8 (89-91) 92 92

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G1 56.7 ± 11.1 (45.0-66.6) 43.7 ± 11.8 (27.6-55.2) 52.4 ±4.1 (48.0-56.4) 56.0 ± 9.4 (46.8-70.7) 63.3 ± 3.5(60.0-67.7) - 51.4

Stylet length 76.3 ±3.8 (73.1-82.6) 57.4 ±3.9 (52.7-61.3) 95.0 ±3.5 (90.3-99.5) 103.8 ±5.9 (94.0-108.3) 98.8±2.7 (95.8-101.3) 67.2 61.5

Metenchium length 58.9 ±4.3 (55.2-65.9) 44.5 ± 14.1 (29.5-44.8) 76.8 ±4.6 (68.0-80.8) 86.0 ± 5.0 (77.3-90.0) 80.5 ±7.9 (66.5-85.4) 53.0 46.5

Telenchium legth 12.5 ± 1.1 (11.1-13.9) 14.1 ±3.1 (10.5-18.1) 12.9 ± 1.9(11.3-16.6) 12.6 ± 1.1 (11.7-14.4) 15.7 ±6.2 (11.0-26.5) 10.7 10.7

M 77.2 ± 1.9 (75.5-79.8) 80.6 ±3.0 (75.3-82.7) 80.9 ±2.5 (75.3-82.7) 85.5 ±32.0 (55.9-131.0) 81.4 ±6.8 (69.3-85.5) 78.7 75.6

Stylet knob height 4.8 ±0.8 (3.8-5.7) 3.5 ± 1.2 (2.5-5.2) 5.3 ±0.7 (4.1-5.9) 5.2 ± 1.0 (4.1-6.3) 2.6 ±0.3 (2.2-2.9) 3.6 4.3

Stylet knob width 12.0 ±0.8 (10.7-12.8) 10.7 ±1.4 (9.3-12.0) 11.7 ±0.7 (11.0-12.9) 12.2 ±0.4 (11.7-12.6) 12.0 ± 1.0(11.0-13.5) 10.7 11.3

Excretory pore from anterior end 170 ± 18.4(150-191) 127 ± 13.4(118-142) 133 ±46.6 (85.0-187) 168 ± 10.6(155-181) 179 ± 13.0(170-193.0) 153 -

Width at midbody 50.9 ±2.1 (48.8-54.5) 43.7 ±6.0 (35.6-50.0) 61.6 (56.4-66.2) 53.9 ±4.4 (47.3-59.5) 56.4 ± 5.2 (52.5-65.3) 49.3 57.2

Width at excretory pore 49.7 ±0.8 (48.5-50.3) 41.0 ±4.6 (35.4-43.9) 54.0 ±7.2 (45.5-62.3) 49.5 ±5.6 (44.5-55.6) 54.8 ±5.8 (49.6-64.4) 46.5 -

Table 3 (continued). Morphometries of Mesocriconema xenoplax, Criconema annuliferum and Criconemoides informis from Begium.

All measurements are in [j,m and in the form: mean ± s.d. (range).

Width at anus

Annulus width

Tail length

Pharynx length

1st lip annulus

diameter

2nd lip annulus

diameter

1st body annulus

diameter

2nd body annulus

diameter

Spermatheca

length

Spermatheca

width

R

RSt

Rpharynx

Rex

RV

Rvan

Ran

VL/VB

St%L

33.7 ±5.8 (25.1-37.5)

6.0 ±0.3 (5.6-6.3) 28.0 ±10.3 (13.1-35.5) 140 ±8.4 (135-155)

15.3 ±0.5 (14.5-15.8) 21.0 ± 1.1 (19.5-22.6) 25.5 ± 1.2 (24.8-27.6)

28.4 ± 1.6(27.0-31.1)

14.5-23.0

14.9-16.7

98 ±2.7 (95-102) 16 ±0.8 (15-17) 25± 0.8 (24-26) 31 ± 3.0 (27-34) 6 ±1.3 (4-7) 2 ±1.3 (1-4) 8 ± 0.4 (7-8) 1.0 ±0.1 (0.7-1.0) 13.3 ±0.7(13.1-14.3)

30.5 ±3.5 (26.0-34.1) 27.6 ±2.0 (25.1-30.1) 20.8 ± 1.7(18.3-23.0) 27.0 ±4.1 (23.1-33.6) 23.1 23.4

4.7 ±1.3 (3.6-6.6) 9.3 ±0.6 (8.6-10.4) 8.5 ± 1.7 (6.3-10.7) 9.3 ±0.7 (8.5-10.2) 7.0 9.4

23.6 ±2.2 (22.1-26.8) 25.1 ±3.7(18.1-29.7) 17.2 ±2.5 (13.9-20.6) 22.2 ±4.4 (17.6-28.7) 13.6 12.8

125 ±20.4 (101-154) 133 ±5.0 (127-139) 134 ±5.8 (125-141) 139 ±7.6 (130-147) 126 113

10.9 ±2.1 (8.8-13.5) 22.3 ±0.9 (21.2-23.8) 21.1 ±0.9(20.4-22.4) 22.4 ± 1.1 (21.5-24.4) 11.0 16.5

15.4 ±3.2 (12.4-19.8) 20.6 ± 1.8(17.9-22.9) 19.1 ±0.4(18.6-19.6) 21.7 ±2.4 (19.5-25.2) 19.1 22.5

20.1 ±4.5 (15.5-24.4) 30.7 ± 1.9(28.5-33.2) 28.8 ±01.3 (26.9-30.7) 30.8 ± 1.8(28.6-33.4) 23.0 28.3

22.7 ±4.7(17.6-26.9) 35.4 ± 1.8(33.2-38.2) 32.9 ± 1.6 (30.8-35.1) 35.6 ±2.5 (32.9-39.1) 28.2 34.1

11.4-18.9 18.5-21.3 - - 15.4 -

13.3-15.0 19.3-18.9 - - 18.8 -

101 ±2.5 (97-103) 63 ± 0.9 (62-64) 61 ± 1.6 (60-63) 62 ± 3.6 (59-68) 67 48

17± 1.5 (16-19) 12 ± 1.0(11-13) 10 ± 1.1 (8-11) 10 ±2.2 (8-13) 11 9

27 ±2.3 (23-29) 16 ± 1.9(14-19) 17± 1.5 (15-19) 15 ±0.8 (14-16) 18 14

32 ± 1.2 (31-33) 16 ±4.3 (10-20) 21 ± 1.0(20-22) 20 ±2.2 (17-20) 21 -

8 ± 0.6 (8-9) 8 ±0.9 (8-10) 8 ±0.5 (8-9) 9± 1.1 (7-10) 6 6

2 ± 1.0(1-3) 4 ± 0.4 (3-4) 4 ± 0.4 (4-5) 4 ±0.8 (3-5) 2 3

7± 1.0 (5-7) 5 ±0.8 (4-6) 4 ±0.8 (3-5) 5 ± 0.8 (4-6) 4 3

1.1 ±0.1 (1.0-1.1) 1.4 ±0.2 (1.2-1.6) 1.4 ±0.1 (1.2-1.5) 1.3 ±0.2 (1.1-1.5) 1.2 0.9

11.7 ±3.1 (7.6-15.3) 17.6 ± 1.7(14.9-20.2) 20.3 ±2.2 (18.6-22.4) 17.6 ±0.8 (16.4-18.2) 12.5 14.6

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LoDocncontma sp {KU236S70)

ctKonematid wmitodt »p A («N782396. Belgium. BE20 N1)

HimicrKowiSii«^«^ 7M3w! BEii Mil)

Mojocnconomi icwpiJuUSA. CsMorna KJ7879II) ' 'v I McioctKonem* zeneplti (UN7«2*00. Belgium, BED N4) Wijocncoiwru itftoptax (USA. Temeiieo KYS74648I Ueioenoentma teooptax (Canada. Brtiih CoSumbo. KJ7S7S07)

1- Cneorwmexdoi parvus (MF770468)

'CO ■— Cnconemokiei Informls (MN7B2184. Belgium. 8E18 N9) ^J I- CrfcwwmoM» tntormn IMN78218S, Belgium. BE14 N1)

'— cfKorwmjttd nematode ip. B (MtJ 782386, Belgium BE 19 NT) crkonematld nematode ip C (MN7823S». Belgium. BE» HI) Wj f/wnwnarvn (Ruivj M G0148«)

1 HemtcycUophof* thKnemtnm (MN7B2J97. Belgium. EE19 N1)

100

too

HemKyMrptnra cahfomKa (UG019S54) L Mwucvciopftora cetJoxnct (WG0194531 Cncanemt mnuliterum (MNW1M7, Belgium, BE1S N1) Cruoncmt tnnuMcrum [UN 78239 5. Belgium, BE13 Nt) Crrcortcmt tnnuliftrum (MN782394. Belgium. BE21 N2) CrKonemt tnnuliterum (MN782393, Belgium, BE20 N10| Cnconema tnnuMfmm (MN782392, Belgium. BE1S N5) CrKonemt tnnuMcrum (MN782H1, Belgium. BE1S Nt) Criconem«*nn1/(ifc™m|MN782»0, Belgium. BE21 N4) Crtcontms tnnuliterum (MN 782389. Belgium, BE1S N4) CrKontmt tnnuliterum IMN762388. Belgium, BE 15 N9) Ctxxmrmi <MF770»10)

- 0»ma mi*™« (MF 770907) WncnconenxMM atitomienus (KMSI6I93) MMMMMMMMMM iWcloffwcafui (KW5l«1»71 U r P*fifylenebus ip. F (MN782409. Belgium, BE18 NS) |L Ptrirylenchut ip. F IMN782410, Belgium, BE19 Nla) PtrtlyiHKhus IP F <M N782407, Belgium, BE22 N4) • -,- { Pintyienchui ip F <M N782413 Belgium, BE22 N1) —"i Pt/ityttnchut »p F (MN782412. Belgium. 6E22 N1) Ptrttylenchus ip. F (MN78241V Belgium. BE22 NS] 1 PirtlytotKhui ip F <MN782409 Belgium. BE22 N2) 52| PirttyHMKhut »p. B IMN782402. Belgium. BE4 N1) J 1 Ptnrylenehus ip B (MN782403. Belgium. BE4 Nil

Mi- Ptrtt/ttnchut *P 8 (MN 782404. Belgium. BE19 N41)

H Ptrttylenchus »p, B (MN782405. Belgium. BE20 N11) |_i— Parat)foncl>u3 prcjtctia (MF770889I

77"- ParaMtnchu* prcMctus (M F 7708*3)

Ptrttylenchus tp E (MN 782406. Belgium, BE» N8) Parttybnctius tp (MF77090S)

1 Ptrttyhnthut ntnus type A («N782414. Belgium. BE11 WSJ MJ Ptrttylenchus ntnus type A (M N782417. Belgium. BEi N7) Ptrttylenchus ntnus type A IMN782416. Belgium. BEI N8) Ptrttylenchus ntnus type A (MN78241S, Belgium, BE1 N6)

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Ptrttylenchus tp s (MN782401, Belgium, BE 19 N2)

Pttttvttncfius sp. (MF770960)

u I HetKQtylcnchui vtrKtudatus (MN782373, Belgium, BE7 N1) Hcflcotyfenchui vtrlctuditus (MN782376. Belgium. BE7 N7) HclKotylcnchut vtrlcaudalus (MN782175. Belgium. BE7 N5) 100-1 ' HetKOtytenchut vahctudttus (MN782374, Belgium, BE 14 N2) Hcltcoiylencbut vtrKtudttui (MN78IS77. Belgium. BE9 N2|

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"»j Hclicoiyfenenui J1 Hehcotytenchut vtricaudttus (MN782178. Belgium. 8E9 N4) HelKotytenchui vtnetudttut («N78217», Belgium. 8E11 N1 L— HtfKotyHmchui vthctudttut (MN782180. Belgium. BE7 N6)

I- Htticotylencnus x> IMG66310J)"

Hei^otyiorKhus ranctutUTLK (Pt4and. MC«311 i)

83,

Mi

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i

ScuttKotwD tc A (JX47J101) ffofyfencfw ip B (MN782381. Belgium, BEtS N!|

Rotytonchus rctrustus (USA. Ca№mu JX015412) Rotytenchut robustus IMN782172, Belgium. C02974. BE11) DoiyicrKtws robustus IMN782171, Belgium. BE11NS)

Rofylenchus robvstus (USA. CaMcnn JX015411) Rotytencbus rotHjsiui (Poland, MG663I21)* Rotyiencbus rotmstus (Poland. MG663122)" fodutfua (Spain. JX015414) FKtyanchvs mtxistus (Spa«. JX015413)

100

Rotyttnclms »p. A (MN 782382. Belgium 8E20 N8) Roijflenthui monttnus IMN782381. Belgium. BE23 N4)

Ampitnettinlut Ktrus (MN782418. Belgium. BE24 N8) Amptmertmtus Ktrus (MN7SZ419. Belgium, BE24 N2) Ampimeritnius Ktrui (MN782420, Belgium. BE1S N2) Amplmcttmiui Ktrui (MN7S2421. Belgium, BE1i N1)

PratylencfKXJcs. ntttn (KX34M29)

Panagretus r«*wvuj (AP017484) — Surjapfwipnc/iuj ¡¡bruptus (ASOS7762!

0.1

Fig. 4. The 50% majority rule consensus tree obtained using Bayesian analysis of the COI mtDNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * - identified as Rotylenchus uniformis by Rybarczyk-Mydlowska et al. (2019); ** - identified as Helicotylenchus digonicus by Rybarczyk-Mydlowska et al. in the GenBank.

Remark. Morphology and morphometrics of the specimen observed were very similar with that of the original description (Loof & De Grisse, 1989) and other populations from Russia and Greece (Peneva et al., 2000; Karanastasi et al., 2008).

Criconema annuliferum (de Man, 1921) Micoletzky, 1925 (Fig. 6)

This species is known as a common species found in many localities in Europe (Geraert, 2010). Females from three different populations were collected during this study from Zwijnaarde, Kortrijk and Merendree. The morphological and molecular data are presented in this study. Measurements are given in Table 3.

Description. Female. Body annuli retrorse, thick, rounded and with smooth edges. Anastomoses seen in some specimens. First annulus collar-like, anteriorly directed, or straight, with second annuli narrower forming a collar-like appearance. Lips distinct and domed-shaped projecting anteriorly from the first annuli. Stylet strong with rounded knobs. Vulva with overlapping lips, vagina sigmoid. Anus located 3-6 annuli from the tail terminus. Tail conical ending with a rounded or truncate lobe (Voucher slides UGnem 242-243).

Male. Not found.

Juveniles with collar like first annuli, body with spines irregularly arranged in eight rows.

Molecular characterisation. Intraspecific sequence variation for Criconema annuliferum was 0-1.1% (0-8 bp) and 0-1.9% (0-7 bp) for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 3 and 4. In the D2-D3 28S rRNA gene tree, C. annuliferum formed a clade with Ogma decalineatus and Criconema demani and in the COI tree, this species clustered with C. longulum and O. murrayi.

Remark. The morphological and morphometrical data of the three populations collected during this study are within the ranges of the original description as well as that of Peneva et al. (2000) from an oak forest in Russia.

Criconemoides informis (Micoletzky, 1922) Taylor, 1936 (Fig. 7)

De Grisse (1963) first described this species as C. flandriensis from sandy soil collected around roots of unknown grass plants around a pear tree at Eine (Marollen, Oudenaarde) in Belgium. Loof (1965) later synonymised it with C. informis. The morphometric and molecular data of the populations from Blaarmeersen (Ghent) and Merendree are presented here.

Measurements are given in Table 3.

Description. Female. Body cylindrical with, thick, retrorse annulus with rounded edges. Labial region oval shape, slightly elevated, submedian lobes present and projecting anteriorly. Stylet robust, knobs anchor-shaped projecting anteriorly. Hemizonid not seen. Anastomoses seen. Spermatheca round and empty, vulva slit-like with non-projected bulging lips. Tail plump with rounded tip, terminal annulus bi-lobed.

Male and juvenile. Not found.

Molecular characterisation. Intraspecific sequence variation of C. informis was 0.5-3.1% (318 bp) and 4.7% (17 bp) for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 2, 3 and 4. In the D2-D3 28S rRNA gene tree, this species clustered with Discocriconemella sinensis (MK253537).

Remarks. The morphometrics and morphology of specimens collected during this study agree very well with those of Geraert (2010) and Cordero et al. (2012).

Hemicycliophora thienemanni (W. Schneider, 1925) Loos, 1948

This species was initially found inhabiting moist soil in East Holstein, Germany by Schneider in 1925 and is known to inhabit a wide range of terrestrial as well as aquatic habitats. It is extensively distributed in Europe including Belgium (Subbotin et al., 2014). Males of this species are extremely rare and Chitambar & Subbotin (2014) stated males have been reported only from Ens, The Netherlands since its original description. A single female specimen of this species was found in a sample from Blaarmeersen (Ghent).

Description. Female. Slender body, 921 ^m long, straight after fixation, cuticular sheath closely attached to inner one all along the body and loose around the vulva. Cephalic region continuous, lateral field with three longitudinal lines and few irregular breaks seen but no anastomoses observed. Labial region conoid with distinct non-protruding labial disc slightly elevated. Sclerotisation not observed on lip region. Stylet slender and straight, 88.5 ^m long with rounded indistinct knobs that are sloping posteriorly. Hemizonid not seen. Distance from excretory pore to anterior end 192.3 ^m. Vulval lips unmodified with non-elongated vulval sleeve. V = 86%. Spermatheca large, 22.1 ^m long and empty. Tail 88 ^m long, elongated and conoid tapering abruptly in posterior one-third to a distinctly offset narrower and elongated attenuated distal portion with a narrowly rounded terminus. Tail annulation distinct.

Table 4. Morphometries of Rotylenchus montanus and R. robustus from Belgium. All measurements are in ¡iin and in the form: mean ± s.d. (range).

Species

Characters

R. montanus

Destelbergen

It. robustus

Ghent University Botanical Garden

Zwijnaarde

N L a b c c' V

Lip height Lip width Stylet length Conus length Shaft length Knob height Knobs width DGO

Anterior end to secretory excretory pore Anterior end to nerve ring

Anterior end to pharyngeal gland end Pharynx overlapping Maximum body diameter (MBD)

Anal body diameter

(ABD)

Tail length

Number of tail annuli Phasmid to tail terminus Spicule length Gubernaculum length

7 females 1096 ±97.3 (1012-1200) 24.6 ±0.8 (23.6-25.4)

7.2 ±0.3 (6.8-7.4) 40.3 ±5.0 (33.9-45.7) 0.9 ±0.1 (0.9-1.0) 58 ±3.1 (53-60) 6.7 ±0.5 (6.5-7.4) 12.6 ±0.3 (12.4-12.9) 36.6 ± 1.2 (35.5-37.9) 16.6 ± 1.1 (15.5-17.9) 16.1 ±0.4(15.6-16.5) 3.9 ±0.4 (3.4-4.4)

7.4 ±0.5 (7.0-8.1)

2.5 ±0.1 (2.4-2.6)

152 ±8.0 (144-163)

153 ±8.4 (143-161) 22.2 ± 10.9(7.1-31.0) 44.4 ±3.0 (41.3-48.1)

29.9 ±2.4 (28.1-33.3)

27.6 ±4.5 (24.0-34.2)

20 ±3.2 (16-22) 28.9 ±3.6 (22.8-34.7)

1 female 1647

36.6 7.6 71.3 0.8 53

8.3 17.0

43.7 19.0 20.3

4.4

5.5

217 62.9

45.0

30.5

23.1 12

2 females 1475;1607 33.9; 37.4 8.0; 8.2 55.9 0.8 56

7.7 13.9 47.8 25.4 18.7 3.7

8.4

14.6 49.8

25.7 19.6

4.5

5.4; 5.9 179; 187

183; 196 32.9; 43.5 42.9; 43.5

33.7

26.4 16; 18

1 male 1309 31.7

7.6 41.2 1.2

6.8

13.4 45.1

23.5 17.4 4.1

8.7 153

172 32.9 41.3

26.1 31.8

42.2 19.2

8 females 1435 ±134.7 (1265-1570)

33.4 ±2.4 (29.5-36.5) 7.4 ±0.6 (6.5-8.3)

70.0 ± 18.9(50.0-109.5) 0.7 ±0.1 (0.5-0.8) 55 ± 2.4 (52-60) 8.2 ±0.4 (7.6-8.9) 13.1 ±0.6(12.1-13.7) 45.3 ±2.1 (42.2-48.6)

18.0 ±0.7 (17.2-19.1) 24.3 ± 1.0 (22.7-25.9)

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4.2 ±1.2 (3.4-6.6)

4.8 ±0.6 (3.5-5.4) 157 ± 15.7(128-175)

122 ± 11.7(109-136)

194 ± 14.1 (172-219)

39.1 ±5.8(31.1-46.9) 42.9 ±1.5 (41.2-45.6)

31.5 ±3.4 (26.1-36.3)

21.3 ±4.1 (17.9-25.5) 12 ± 1.4(10-14)

10 females 1565 ±156.8 (1344-1748)

34.1 ± 1.9(32.5-35.9)

7.7 ±0.7 (6.9-8.6) 73 ±9.1 (70.8-81.2)

0.7 ±0.1 (0.6-0.7) 56 ±0.7 (55-57) 8.4 ±0.2 (8.2-8.6)

14.2 ±0.9 (12.9-14.9) 47.7 ±2.6 (44.6-50.6) 19.9 ±2.4 (18.1-24.4) 21.4 ±2.3 (18.7-24.6)

3.8 ±0.3 (3.3-4.1)

6.1-1.1 (5.1-7.4) 170 ± 21.6 (139-191)

204 ±20.2 (176-225) 52.3 ±13.1 (43.7-71.6) 44.9 ±3.4 (41.4-49.0)

32.5 ±2.8 (29.4-34.8)

21.1 ± 1.1 (20.1-22.4) 11 ± 1.7(9-13)

3 males 1150-1335 34.7 5.9 38.0 1.3

7.4-8.1

11.6-12.5 40.1-43.6 16.1-17.9

20.7-23.2 3.2-4.1

3.6-6.1 145-150

115-136

168-199 33.1-47.9 34.1-40.5

23.3 28.1-31.2

43.2-44.3 16.7-19.9

Fig. 5. Statistical parsimony network showing the phylogenetic relationships between ITS rRNA gene haplotypes. A: Hemicriconemoides pseudobrachyurus; B: Mesocriconema xenoplax and other Mesocriconema species; C: Rotylenchus robustus. Pies (circles) represent sequences of each species with the same haplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black circles represent missing haplotypes. New sequences are indicated in bold.

Male. Not found.

Molecular characterisation. Intraspecific sequence variation for this species was 0-0.7% (0-5 bp) and 0.27% (0-1 bp) for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic position of this species within some tylenchids is given in Figures 3 and 4. The sequence of H. thienemanni from Belgium clusters with sequences of this species from other countries in the D2-D3 28S rRNA gene tree.

Remarks. Morphology and morphometrics of the specimen observed were very similar to that of other populations from different countries (Chitambar & Subbotin, 2014).

Rotylenchus montanus Vovlas, Subbotin,

Troccoli, Liebanas & Castillo, 2008 (Fig. 8)

This nematode was originally described from apple orchards in Val di Non, Trento province, Italy (Vovlas et al., 2008). It is distinct from other Rotylenchus spp. based on the morphology of the lip region, tail shape, stylet length and vulva position. In Belgium, a population was collected around grasses at the border of a ditch at Heusden (Destelbergen). This is the first record of R. montanus in Belgium. The morphological and molecular data of this population collected are presented in this study.

Measurements are given in Table 4.

Description. Body habitus spiral after fixation. Labial framework well developed and hemispherical with six to seven annuli. Stylet robust with shaft equal length as the conus, basal knobs rounded. In some specimens, the excretory pore is located at the level, or posterior to the end of the pharynx. Pharyngeal glands, overlapping intestine dorsally, relatively long with one large nucleus seen. Hemizonid not seen. Lateral field with four lateral lines, regularly areolated at the pharyngeal region and smooth along the rest of the body with four lateral lines present. Reproductive system didelphic with well-developed genital branches. Spermatheca spherical and empty. Vulva of some specimens with protruding epiptygma. Phasmid pore-like, and anterior to anus. Tail rounded, regularly annulated with depression seen in some specimens.

Male. Not found.

Molecular characterisation. Intraspecific sequence variation for this species was 0-0.1% (0-1 bp) for D2-D3 of 28S rRNA gene. Phylogenetic positions of this species within some tylenchids are given in Figures 2, 3 and 4. In the 18S rRNA gene tree, the sequence of R. montanus clustered with two sequences identified as R. robustus. We assumed

that these sequences might not be correctly identified and belong to R. montanus.

Remarks. The Belgian population of this nematode agrees very well with those of the original description, except for some traits. Variations were observed in the position of the excretory pore whereby for some specimens it was located at the base of the pharynx as in the original description, or posteriorly to the base of the pharynx. In addition, the Belgian population is characterised by slightly longer body length (1012-1200 vs 977-1135 ^m), slightly longer stylet (35.5-37.9 vs 32.5-36.5 ^m), wider stylet knobs (7.0-8.1 vs 6-7 ^m), broader maximum diameter (41.3-48.1 vs 35.5-42.5 ^m) and shorter anal body diameter (28.1-33.3 vs 24.5-30.5 ^m).

Rotylenchus robustus (de Man, 1876) Filipjev, 1936

De Man (1876) described this species from the Netherlands as Tylenchus robustus. It is characterised by a high, distinctly offset, hemispherical cephalic region with 6-7 annuli, regularly areolated lateral fields at the pharyngeal region and regularly areolated lateral fields along the rest of the body, body without longitudinal striations, hemispherical tail. Later, Thorne (1949) described R. uniformis, a morphologically similar species. Loof & Oostenbrink (1958) synonymised R. uniformis with R. robustus and Krall (1978) and Siddiqi (2000) accepted this synonymisation. However, Seinhorst (1991) stated that these species are morphologically different and listed some differential characters. Several other authors also differentiated these species from each other. Brzeski (1998) distinguished R. robustus from R. uniformis based on stylet length (less than 40 ^m vs 40 ^m or more) and lateral field areolation near vulva (absent vs present). Castillo & Vovlas (2005) recognised both species and considered that R. robustus could be differentiated from R. uniformis by a higher number of lip annuli (6-8 vs 5); lateral fields areolated in pharyngeal region and irregularly areolated at mid-body vs areolated only in pharyngeal region; and female tail hemispherical vs rounded. In the present paper, we consider these species as co-specific as has been proposed by Loof & Oostenbrink (1958).

Measurements are given in Table 4.

Description. Female. Habitus spiral or C-shaped, lateral fields areolated at the pharyngeal region and irregularly along the whole body, labial region hemispherical, distinctly offset with 6-7 distinct annuli and a terminal disc. Stylet well-developed with rounded knobs, long pharyngeal glands

Table 5. Morphometrics of Helicotylenchus varicaudatus from Belgium. All measurements are in ^m and in the form:

mean ± s.d. (range).

Character Blaarmeersen Citadel park, Ghent

N 4 females 1 female 1 female 1 female

L 785 ± 26 (758-810) 683 709 662

a 25.4 ± 0.7 (24.6-28.8) 27.4 23.4 22.9

b' 4.9 ± 0.3 (4.5-5.2) 4.6 5.0 4.9

c 52.2 ± 2.6 (50.4-54.0) 53.0 66.3 49

c' 0.8 ± 0.1 (0.7-0.9) 0.8 0.6 0.8

o 25.9 ± 3.8 (25.1-27.6) 25.6 26.0 27.8

V 61 ± 1.0 (61-62) 62 62 58

Lip height 4.2 ± 0.1 (4.1-4.2) 4.3 5.4 3.2

Lip diameter 7.6 ± 0.6 (6.9-8.0) 8.8 8.0 7.9

Stylet length 31.7 ± 0.9 (30.7-32.3) 31.7 30.5 24.0

Conus length 14.5 ± 1.4 (12.8-15.8) 15.4 15.0 9.8

DGO 8.2 ± 0.5 (7.7-8.9) 8.1 7.9 6.7

m 45.7 ± 3.1 (41.7-48.9) 48.6 49.0 40.7

Pharynx length 157 ± 7.7 (148-167) 148 143 141

Anterior end to pharyngeal gland end 27.8 ± 0.7 (27.0-28.5) - 22.1 34.3

Anterior end to excretory 121 ± 10.2 (110-134) 110 121 104

pore

Maximum body diameter 29.4 ± 3.4 (24.9-32.9) 24.9 30.4 28.9

Lateral field width 2.1 ± 0.3 (1.8-2.3) 1.8 1.7 1.6

Tail length 14.2 ± 1.4 (12.9-15.6) 12.9 10.7 13.5

Anal body diameter 17.7 ± 2.2 (15.6-20.0) 15.6 16.5 16.5

Tail annuli number 9 ± 1.4 (8-10) - 10 12

Fig. 6. LM photos of Criconema annuliferum. A-D: Anterior region variations of females; E: Anastomoses; F-G: Posterior region of females; H: Anterior region of juvenile.

Fig. 7. LM photos of Criconemoides informis. A: Anterior region; B: Posterior region.

overlapping intestine dorsally. Spermatheca rounded and packed with sperm cells. Tail hemispherical, distinctly and regularly annulated, phasmid distinct above the anus and pore-like.

Male. Habitus open C shape with a labial region more distinctly offset and elevated.

Molecular characterisation. Intraspecific sequence variation for R. robustus was 0-0.5% (0-4 bp), 0.2-0.8% (1-4 bp), 0.7-17.8% (1-70 bp) for 18S rRNA, D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids is given in Figures 2, 3 and 4. The ITS gene alignment of R. robustus included seven sequences with three new sequences obtained from this study and it was 1019 bp long. Belgian sequences of this species clustered with other R. robustus/uniformis populations from GenBank with a maximum intraspecific variation of 0.2-3.1% (1-17 bp) (Fig. 5C).

Helicotylenchus varicaudatus Yuen, 1964 (Fig. 9)

This species was described from Broadbalk Wilderness, Harpenden, England (Yuen, 1964). It has a very variable tail morphology and is distinct from the others based on its stylet length, number of head annuli and the position of phasmids. The morphological and molecular data are given for samples collected from Blaarmeersen and the Citadel Park (Ghent).

Measurements are given in Table 5.

Description. Female. Body habitus spiral or C-shaped. Lip region hemispherical and continuous, with indistinct lip annuli. Stylet slender with flat knobs. Hemizonid not seen. Excretory pore located anterior to level of pharyngo-intestinal valve. Pharyngeal gland overlapping intestine ventrally. Lateral field with four lines, areolated

A 1 \ B c i - ■ ^tí f D

E fíltk 1 F 6 Q

Fig. 8. LM photos of Rotylenchus montanus. A, E: Anterior region; B: Areolation at pharynx region; C-D: Posterior region; F: Vulva region; G: Total body.

at the pharyngeal region. Spermatheca offset, rounded, and empty. Epiptygma folded into vagina. Phasmids pore-like, located posterior to anus. Tail shape is very variable.

Molecular characterisation. Intraspecific sequence variation was 0.7-1.9% (4-22 bp) and 07.4% (0-29 bp) for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 3 and 4.

Remarks. The morphology and morphometrics of the Belgian populations of this nematode agree very well with those of the original description and other literature (Yuen, 1964; Loof, 1984; Yeates & Wouts, 1992; Brzeski, 1998; Bert & Geraert, 2000; Reis et al, 2010).

Paratylenchus nanus Cobb, 1923 (Figs 10A & B)

This species was described by Cobb (1923) from a single female collected near Devil's Lake in North Dakota, USA. Geraert (1965) proposed the synonymisation of this species with

P. bukowinensis. Raski (1975) re-described the species and validated it as a valid species. In this study, populations from Zwijnaarde and Ghent University Botanical garden have been molecularly and morphologically analysed.

Measurements are given in Table 6.

Description. Body slightly curved ventrally. Lip region conically rounded and continuous with a weak framework. Stylet long and slender. Excretory pore located between the position of the basal pharyngeal bulb or isthmus. Hemizonid seen, lateral field with four lines, lateral vulva flaps distinct. Spermatheca large, and round, and filled with sperm cells. Tail with finely rounded tip.

Molecular characterisation. The D2-D3 of 28S rRNA gene sequences of Belgian populations were identical with those of P. nanus type A from California, USA (Van den Berg et al., 2014a). Phylogenetic positions of this species within some tylenchids are given in Figures 2, 3 and 4.

Remark. The morphology and morphometrics of specimens from this study agree with that of P. nanus type A reported by Van den Berg et al. (2014a).

Table 6. Morphometrics of Paratylenchus nanus and Paratylenchus sp. F from Belgium. All measurements are in ^m

and in the form: mean ± s.d. (range).

Species Characters Paratylenchus nanus Paratylenchus sp. F

Ghent University Botanical garden Merendree Zwijnaarde

N 3 females 5 females 3 females

L 340-364 356 ± 28.4 (313-389) 330-350

a 21.2-22.0 19.4 ± 2.2 (17.1-22.0) 19.4-20.8

b 3.5-3.8 4.1 ± 0.3 (3.7-4.4) 3.7-4.0

c 14.9-17.4 12.7 ± 0.9 (11.4-13.4) 13.8-15.4

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c' 1.9-2.3 2.8 ± 0.3 (2.4-3.0) 2.1-2.5

o 12.2-14.0 25.0 ± 1.5 (23.6-26.7) 12.2-15.7

DGO 3.6-4.1 7.1 ± 0.4 (6.5-7.7) 3.6-4.6

V 83-84 82 ± 1.1 (80-83) 81-83

Stylet length 29.5-29.9 28.3 ± 1.1 (26.7-29.6) 27.9-29.7

Metenchium length 19.2-20.6 18.1 ± 1.0 (16.7-19.2) 19.9-21.8

Telenchium length 7.5-8.4 7.7 ± 1.8 (5.8-10.0) 5.6-6.5

m 65.0-69.0 64.3 ± 4.6 (57.7-69.3) 71.6-74.7

Stylet knob height 1.8-2.1 2.5 ± 0.2 (2.2-2.8) 1.5-1.8

Stylet knob width 4.1-5.0 4.3 ± 0.2 (4.0-4.6) 3.9-4.9

Pharynx length 89.1-98.7 85.8 ± 3.2 (81.1-88.7) 86.1-91.2

Anterior end to excretory pore 75.3-76.9 75.2 ± 4.1 (70.3-79.1) 71.2-78.0

Diam at mid body 15.9-17.1 18.4 ± 1.3 (16.6-20.2) 16.7-17.0

Lip region diam 6.2-6.9 6.7 ± 0.2 (6.4-6.9) 6.1-6.8

Lip region height 3.1-3.8 3.1 ± 0.3 (2.8-3.5) 3.4-3.9

Annulus width 1.4-1.7 1.3 ± 0.2 (1.1-1.5) 1.2-1.9

Tail length 20.2-22.8 28.8 ± 1.7 (27.3-30.4) 22.3-25.3

EP%L 20.8-22.2 21.2 ± 0.8 (20.3-22.5) 21.0-22.3

V-anus distance 30.0-33.3 37.9 ± 2.5 (35.5-40.3) 30.0-38.5

Spermatheca length 12.4-18.8 12.1 ± 2.0 (10.8-15.0) 16.9-14.6

Spermatheca diameter 9.9-11.0 9.5 ± 1.6 (7.8-11.5) 10.0-11.5

PUB length - 17.4 -

St%L 8.1-8.8 8 ± 0.3 (7.3-8.5) 8.0-9.0

Paratylenchus sp. F (Figs 10C & D)

This nematode was found in samples collected in Blaarmeersen and Merendree and was not identified at the species level and provisionally named as species F here.

Measurements are given in Table 6.

Description. Female. Body slender and relatively small. Cephalic region rounded to faintly truncate. Submedian lobes absent or indistinct, excretory pore located at the level of the basal pharyngeal bulb. Stylet short, spermatheca oval and empty. Tail finely rounded, one specimen seen with digitate tail. Vulval flap rounded.

Male. Not found.

Juvenile very similar to female except for the absence of a stylet.

Molecular characterisation. Intraspecific sequence variation was 0-0.2% (0-2 bp), 0.6% (4 bp) and 0-0.5% (0-2 bp) for 18S rRNA, for D2-D3 of 28S rRNA and COI genes, respectively. Phylogenetic positions of this species within some tylenchids are given in Figures 3 and 4.

Amplimerlinius icarus Wallace & Greet, 1964 (Fig. 11)

This nematode was described from grassland in Winches Farm, St. Albans, England (Wallace & Greet, 1964). In this study, a population of this species from Eine (Oudenaarde) has been morphologically and molecularly characterised.

Measurements are given in Table 7.

Description. Female. Body spiral or C-shaped after fixation. Lip region conoid and continuous.

Fig. 9. LM photos of Helicotylenchus varicaudatus. A: Anterior region; B-G: Posterior region showing variations in tail shape. Scales = 25 ^m.

Table 7. Morphometrics of Amplimerlinius carus from Belgium. All measurements are in ^m and in the form:

mean ± s.d. (range).

Species Amplimerlinius icarus

Characters Eine Geraert (2011) Wallace & Greet (1964)

N 4 females ? 20 females

L 1387 ± 23.8 (1358-1416) 1450-2010 1450-1960

a 29.8 ± 1.4 (29.0-31.2) 29-39 29-34

b 7.3 ± 1.9 (6.2-10.1) - 5.9-6.9

c 21.3 ± 1.3 (19.4-22.3) 18-25 19-25

c' 2.0 ± 0.1 (1.9-2.2) 1.8-2.7 -

V 54 ± 2.2 (51-57) 50-58 50-57

Number of head annuli 5 ± 3.3 (5-8) 4-6 8

Lip height 6.2 ± 0.5 (5.5-6.5) - -

Lip width 12.2 ± 1.0 (10.9-13.1) - -

Stylet length 37.5 ± 2.2 (34.4-39.4) 34-42 34-42

Conus length 20.4 ± 1.8 (17.7-21.8) - -

Shaft length 17.1 ± 0.7 (16.6-18.2) - -

Knob height 4.3 ± 0.3 (3.9-4.7) - -

DGO 4.6 ± 1.9 (3.7-5.4) 4-5 -

Anterior end to secretory 160 ± 8.1 (151-170)

excretory pore

Anterior end to nerve ring 143-156 - -

Anterior end to pharyngeal gland end 199 ± 43.1 (135-225) 223-299 -

Maximum body diameter (MBD) 46.5 ± 1.9 (44.4-48.9) 45-58 -

Anal body diameter (ABD) 32.3 ± 0.7 (31.5-33.1) - -

Tail length 65.4 ± 4.4 (61.0-71.6) 65-93 -

Number of tail annuli 49 ± 4.1 (45-54) 34-57 50-59

Phasmid position (posterior to the 37.8-40.0 24-44

anus)

Fig. 10. LM photos of Paratylenchus nanus. A: Anterior region; B: Posterior region; LM photos of Paratylenchus sp. F. C: Anterior region; D: Posterior region.

Fig. 11. LM photos of Amplimerlinius icarus. A: Female and male; B: Anterior region of female; C: Posterior region of female; D: Posterior region of male. B, C, D have same scale.

Labial framework heavily sclerotised with 5-8 annuli. Stylet robust with round knobs projecting posteriorly. Excretory pore seen opening at the level of the isthmus of the pharynx. Hemizonid located slightly above or same position as the opening of the excretory pore. Lateral fields present with six lines, with irregular areolations at the pharyngeal and tail regions. Deirids not seen. Basal pharyngeal bulb ovoid, and does not overlap with the intestine. Vulva located approximately at the middle of the body. Spermatheca round with spermatozoids present. Tail cylindrical, annulated, with broadly rounded terminus, in some specimens. Phasmid prominent.

Male. Not found.

Molecular characterisation. Intraspecific sequence variation was 0.3% (3 bp) for 18S rRNA,

0.2% (1 bp) for D2-D3 of 28S rRNA and up to 12.7% (50 bp) for CO1 genes. Phylogenetic positions of this species within some tylenchids are given in Figures 2, 3 and 4.

Remark. Morphology and morphometrics of the specimen observed were very similar with that of the original description of Wallace & Greet (1964) and that of Geraert (2011). However, some variations were observed as specimens in this study had shorter total body length (1387 ± 23.8 (13581416) vs 1450-2010) and no vulva lips were seen as described by Geraert (2011).

Sequence and phylogenetic analysis. The 18S rRNA gene alignment included 85 sequences and two sequences selected as outgroups from the genera and was 896 bp in length. Phylogenetic

analysis resulted in a majority consensus BI tree with four major moderately supported clades (Fig. 2).

The D2-D3 of 28S rRNA gene alignment included 112 sequences, including two sequences selected as outgroup taxa and was 781 bp in length. Phylogenetic analysis resulted in majority consensus BI tree with three major supported clades (Fig. 3).

The COI gene alignment included 88 sequences including two sequences selected as outgroups and was 393 bp in length. Phylogenetic analysis resulted in a majority consensus BI tree with three major clades (Fig. 4).

ACKNOWLEDGEMENTS

The authors thank Marj olein Couvreur (Nematology Research Unit, Department of Biology, Ghent University) for her assistance during several aspects of this project, especially scanning electron microscopy; and Prof. André De Grisse for his help to retrieve nematode type localities. Catherine Malike Etongwe was supported by a VLIR-UOS ICP scholarship.

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C.M. Etongwe, P.R. Singh, W. Bert and S.A. Subbotin. Молекулярная характеристика некоторых фитопаразитических нематод (Nematoda: Tylenchida) из Бельгии.

Резюме. Используя морфологический, морфометрический и молекулярный анализы, одиннадцать валидных видов из девяти родов: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, P. nanus, Rotylenchus montanus and R. robustus и 12 неидентифицированных видов были обнаружены в пробах, собранных в девяти местах в Бельгии. Эти неопределенные образцы включали 6 видов Paratylenchus, один вид Helicotylenchus, три криконематиды и два вида Rotylenchus. В целом, 21 новых 18S рРНК, 69 28S рРНК, 10 ВТС рРНК and 51 COI генных последовательностей было получено для филогенетического анализа. Короткие описания, морфометрия и фотографии приводятся для некоторых видов. Основываясь на результатах молекулярного анализа, Hemicriconemoides promissus syn. n. синомизируется с H. pseudobrachyurus.

Molecular characterisation of some plant-parasitic nematodes (Nematoda: Tylenchida) from Belgium Текст научной статьи по специальности «Биологические науки»

Аннотация научной статьи по биологическим наукам, автор научной работы — Catherine Malike Etongwe, Phougeishangbam Rolish Singh, Wim Bert, Sergei A. Subbotin

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Молекулярная характеристика некоторых фитопаразитических нематод (Nematoda: Tylenchida) из Бельгии

Текст научной работы на тему «Molecular characterisation of some plant-parasitic nematodes (Nematoda: Tylenchida) from Belgium»