Nematology 19 (2017) 1179-1199 brill.com/nemy

The pitfalls of molecular species identification: a case study within the genus (Nematoda: ) ∗ Toon JANSSEN 1,2, , Gerrit KARSSEN 3, Marjolein COUVREUR 1, ∗ Lieven WAEYENBERGE 4 and Wim BERT 1, 1 Nematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium 2 Center for Medical Genetics, Reproduction and Genetics, Reproduction Genetics and Regenerative Medicine, Vrije Universiteit Brussel, UZ Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium 3 National Plant Protection Organization, Wageningen Collection, P.O. Box 9102, 6700 HC Wageningen, The Netherlands 4 ILVO, Crop Protection, Burg. Van Gansberghelaan 96 bus 2, 9820 Merelbeke, Belgium Received: 27 February 2017; revised: 13 September 2017 Accepted for publication: 13 September 2017; available online: 20 October 2017

Summary Ð Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematode are actually sequences from distantly free-living bacterivorous ‘cephalobids’. We demonstrated that this incorrect labelling resulted in a cascade of erroneous interpretations, as shown by the reports of ‘P. goodeyi’ on banana in China and on cotton in India. This clearly illustrates the risk of mislabelled sequences in public databases. Other mislabelled Pratylenchus cases are discussed to illustrate that this is not an isolated case. Herein, P. lentis n. syn. is considered a junior synonym of P. pratensis while P. flakkensis was for the first time linked to DNA sequences using topotype material. As taxonomic expertise is decreasing and sequence-based identification is growing rapidly, the highlighted problem may yet increase and a strong link between morphology and DNA sequences will be of crucial importance in order to prevent, or at least minimise, sequence-based misidentifications. Keywords Ð Acrobeloides cf. nanus, diagnostics, mislabelled sequences, molecular phylogeny, new synonym, Pratylenchus flakkensis, Pratylenchus goodeyi, Pratylenchus lentis n. syn., , topotypes.

Molecular and DNA barcoding provide a Nilsson et al. (2006) estimate that up to 20% of fungal powerful tool for the identification of organisms and for sequences are actually misidentified. For eukaryotes, er- studying biodiversity (Hebert et al., 2003; Savolainen et roneous linking of sequence and organism was thought al., 2005; Kress & Erickson, 2008). Molecular identifi- to be relatively uncommon due to the availability of mor- cation is especially important for organisms for which phological characters for their identification (Nilsson et morphological diagnostic characteristics are scarce. Due al., 2006). However, this problem appears not to be re- to the decreasing price and increased availability of se- stricted to microbiology, as demonstrated here for Ne- quence instruments, the amount of sequence data avail- matoda, using three case studies within the genus Praty- able on public DNA databases has grown exponentially lenchus (Pratylenchidae). Many species of Pratylenchus over the last 10 years (Muir et al., 2016). However, a sub- are very difficult to identify morphologically, due to a stantial part of this sequence data appears to be incor- lack of robust diagnostic characters, morphological inter- rect, with faults ranging from sequence errors over mis- specific plasticity, and incomplete taxonomic descriptions assemblies to mislabelled, unlabelled, and misidentified (Castillo & Vovlas, 2007; Subbotin et al., 2008; Troccoli sequences. In microbiology where morphological infor- et al., 2016). As a result, DNA-based identification strate- mation is almost absent and identification is often purely gies are becoming more important (Waeyenberge et al., based on 16S rDNA sequences the problem is well known 2000, 2009; Al-Banna et al., 2004; Powers, 2004; Mokrini (Vilgalys, 2003; Nilsson et al., 2006; Lal & Lal, 2011). et al., 2013).

* Corresponding authors, e-mail: [email protected]; [email protected]

© Koninklijke Brill NV, Leiden, 2017 DOI 10.1163/15685411-00003117

Downloaded from Brill.com10/06/2021 03:12:39AM via free access T. Janssen et al.

CASE STUDY 1: Pratylenchus goodeyi SHER & Castillo & Vovlas, 2007). Pratylenchus pratensis has ALLEN, 1953 been recorded in Africa (Algeria, Libya, South Africa), Asia (Azerbaijan, China, India, Pakistan, Uzbekistan) and Pratylenchus goodeyi is considered a major pest of North America (Canada, Mexico, USA) but has mainly banana and plantain (Castillo & Vovlas, 2007). It was been reported from Europe, occurring in Belgium, Bul- originally described by Sher & Allen (1953) from Kew garia, Finland, Germany, Italy, Moldavia, Poland, Rus- Gardens in London from the roots of banana trees. Since sia, Slovakia, Slovenia, Spain and The Netherlands on a then it has been reported from many banana-producing wide variety of crops (Castillo & Vovlas, 2007). Despite regions, especially from Africa and southern European the numerous morphological reports of P. pratensis, there countries: the Canary Islands (Spain), Crete (Greece) and is relatively little sequence information available for this Madeira (Portugal) (Castillo & Vovlas, 2007). In Africa, species. Nevertheless, seven 28S rDNA sequences were the parasite is often associated with higher altitudes and generated from five coastal areas in Belgium, Portugal, cooler temperatures (Price & Bridge, 1995). Surprisingly, Spain and the UK, by de la Peña et al. (2007). In addi- it has not been recorded in North or South America. tion five β-1,4-endoglucanase gene sequences, five 18S Pratylenchus goodeyi is differentiated from other root- rDNA sequences and two RNA polymerase II gene se- lesion by the presence of four lip annuli, quences were generated (Rybarczyk-Mydlowska et al., a large oblong spermathecal, and tail conoid, ventrally 2012, 2014) although morphological vouchers of these concave with a smooth tail tip (Sher & Allen, 1953; Loof, populations were not provided. In 2008, P. lentis Troccoli, 1991; Castillo & Vovlas, 2007). De Luca, Handoo & Di Vito, 2008, a taxon morphologi- In 2007, P. goodeyi was labelled as a cryptic species cally very similar to P. pratensis, was described parasitis- complex using sequence-based taxonomy (Waeyenberge, ing roots of lentil (Lens culinaris Medik.) in Sicily (Italy) 2007) and, in this study, five ITS sequences originating (Troccoli et al., 2008), the description being associated from Tenerife (Canary Islands, Spain) were made pub- with 12 ITS sequences. In later studies, P. lentis appeared licly available (FJ712922-FJ712926). In 2011, another to be embedded within the P. fallax clade (Palomares- P. goodeyi population (FR692324) was reported from Rius et al., 2010), although this relationship has recently the Canary Islands by De Luca et al. (2011). Praty- been shown to be the result of a P. fallax misidentification lenchus goodeyi was also reported by Gokte-Narkhedkar (Janssen et al., 2017). et al. (2013) from cotton in India at three different lo- cations and six P. goodeyi sequences were deposited in GenBank (KF275665, KF700243, KF840454, KF840455, CASE STUDY 3: Pratylenchus flakkensis KF840456, KF856291). In 2015, P. goodeyi was reported SEINHORST, 1968 for the first time on banana in China (Zhang et al., 2015). Both Waeyenberge (2007) and De Luca et al. (2011) re- Pratylenchus flakkensis was originally described by ported a large discrepancy between P. goodeyi sequences Seinhorst (1968) from Middelharnis (The Netherlands) from different geographical locations. This could indicate from a heavy loam soil under grass. Seinhorst (1968) either the presence of a species complex, as suggested by also reported it from Ouddorp (The Netherlands) and Waeyenberge (2007), or alternatively it could point to in- from Beckenham (UK). Later, P. flakkensis was reported correctly labelled sequences as suggested by De Luca et by Ryss from Estonia and Russia (Ryss, 1986, 1988, al. (2011). 1992). Pratylenchus flakkensis appears to be geograph- ically widespread as it has also been recorded from CASE STUDY 2: Pratylenchus pratensis (DE MAN, Guadeloupe, Pakistan, Peru, and South Africa (Castillo 1880) FILIPJEV, 1936 & Vovlas, 2007). Despite the numerous morphological records of this species, no molecular data are currently Pratylenchus pratensis was originally described as Ty- available for the taxon. lenchus pratensis by de Man (1880). The species was In order unequivocally to link DNA sequences with the subsequently transferred to Anguillulina (Goffart, 1929) above mentioned species of Pratylenchus, and to clarify and then appointed as the type species of the newly their taxonomic status, the goals of these case studies erected genus Pratylenchus by Filipjev (1936). Later, were: i) to characterise these species using a combination P. helophilus Seinhorst, 1959 and P. irregularis Loof, of morphological characteristics and molecular sequences 1960 were synonymised with P. pratensis (Loof, 1974; from geographically different locations; ii) to determine

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access Pitfalls of molecular species identification their phylogenetic position within the genus; and iii)to of 4.5% Tween-20. The mixture was heated to 95°C for enable reliable molecular diagnostics. 15 min and, after cooling to room temperature, 40 μlof double-distilled water was added. PCR amplification was performed using Toptaq DNA polymerase (Qiagen), in a Materials and methods volume of 25 μl using a Bio-Rad T100™ thermocycler. The PCR mixture was prepared according to the manu- COLLECTION OF NEMATODE POPULATIONS facturer’s protocol with 0.4 μM of each primer and 2 μl of single nematode DNA extraction. The 28S rDNA frag- Nematodes were extracted from soil and root material  using a modified Baermann funnel (Hooper, 1986) or ment D2A (5 -ACA AGT ACC GTG AGG GAA AGT TG-3) and D3B (5-TCG GAA GGA ACC AGC TAC a mistifier (van Bezooijen, 2006). An overview of the  populations collected can be found in Table 1. TA-3 ) primers were used according to the protocol of De Ley et al. (1999). The internal transcribed rDNA  MORPHOLOGICAL CHARACTERISATION spacer (ITS) was amplified using VRAIN2F (5 -CTT TGT ACA CAC CGC CCG TCG CT-3) and VRAIN2R (5- Individual specimens were studied in temporary prepa- TTT CAC TCG CCG TTA CTA AGG GAA TC-3), sub- rations sealed with nail-polish using an Olympus BX51 sequently cloned using pGEM®-T Easy Vector systems DIC microscope (Olympus Optical) and morphological (Promega) and sequenced using universal M13F (5-GTA vouchers were made using photomicrographs using a AAA CGA CGG CCA G-3) and M13R (5-CAG GAA Nikon DS-Fi1. Measurements of morphometric charac- ACAGCTATGA-3) primers. The Cytochrome c oxi- ters were directly made using NIS-Elements D mea- dase subunit 1 (COI) gene fragment was amplified using suring software or on digital specimen vouchers using JB3 (5-TTT TTT GGG CAT CCT GAG GTT TAT-3) and ImageJ (Schneider et al., 2012). Vouchered ne- JB4.5 (5-TAA AGA AAG AAC ATA ATG AAA ATG-3) matodes were subsequently picked from these tempo- according to the described protocol (Bowles et al., 1992; rary mounts and processed for DNA extraction. The Derycke et al., 2010). Sanger sequencing of purified PCR resulting digital specimen vouchers are available on- fragments was carried out in both forward and reverse line at http://nematodes.myspecies.info/taxonomy/term/ directions by Macrogen (Europe). Contigs were assem- 10645/specimens. Remaining unvouchered nematodes bled using GENEIOUS R6.1.8 (Biomatters; http://www. were fixed in TAF (2% triethanolamine, 8% formalin in Geneious.com). All contigs were subjected to BLAST distilled water) at 70°C and processed to anhydrous glyc- searches to check for possible contaminations on http:// erin, following the method of Seinhorst (1962) modified www.ncbi.nlm.nih.gov. by Sohlenius & Sandor (1987) and measured as described above with Nikon measuring software. For scanning electron microscopy (SEM) nematodes PHYLOGENETIC ANALYSIS were fixed in 600 μl fresh 4% paraformaldehyde (PFA) Ribosomal gene sequences were aligned with ClustalX fixative buffered with Phosphate Buffered Saline (PBS) (Larkin et al., 2007) using default parameters; COI se- and 1% glycerol and heated for 3 s in a 750 W microwave. Subsequently, specimen where dehydrated in a seven-step quences were translated using the TranslatorX webserver graded series of ethanol solutions and critical point-dried (http://translatorx.co.uk/) (Abascal et al., 2010) using the invertebrate genetic code, and the nucleotides aligned ac- using liquid CO2, mounted on stubs with carbon discs, and coated with gold (25 nm). Specimen were studied cording to an amino acid alignment constructed using and photographed with a JSM-840 EM (JEOL) electron ClustalW. The best fit models of DNA evolution were ob- microscope at 12 kV. tained for each dataset using the program jModeltest 2 (Darriba et al., 2012) under the Akaike information crite- rion. Bayesian phylogenetic analysis (BI) was carried out DNA EXTRACTION,PCRAMPLIFICATION AND using MrBayes 3.1.2 (Huelsenbeck & Ronquist, 2001). BI SEQUENCING analysis for each gene was initiated with a random start- Genomic DNA of individual nematodes was extracted ing tree and was run with four chains for 50 × 106 gen- using the quick alkaline lysis protocol described by erations. Two runs were performed for each analysis. The Janssen et al. (2016). Briefly, individual nematodes were Markov chains were sampled at intervals of 100 genera- transferred to a mixture of 10 μl 0.05 M NaOH and 1 μl tions. After discarding burn-in samples (25%), a 50% ma-

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access T. Janssen et al. ITS Cox1 . – – KY828318 – ––– – KY828290 – KY828320 – KY828283 – – – KY828298 KY828280 – KY828311 KY828291-KY828292 – KY828321 – KY828293-KY828294 – KY828322 – KY828305-KY828306 – KY828312 – ‘Supa’ KY828281 KY828276 – KY828308 sp. – – KY828315 – sp. KY828299 – – – sp. KY828307 – – – sp. KY828317 Lactuca sativa Malus sp. Ammophila arenaria –Musa KY828300-KY828302 – – – Dioscorea alata Plantain KY828287-KY828288 – – – Pyrus Urtica dioica Grass KY828295 – – – Grass KY828282 – KY828313 – Banana KY828285-KY828286 KY828278 – KY828309-KY828310 Grass KY828284 KY828277 KY828319 Oryza sativa Bean KY828289 – – – Maize – – – – Host 28S rDNA 18S rDNA Phoenix dactylifera Rosa Malus Prunus domestica Lens culinaris – –– Grasses KY828304 – KY828323-KY828324 – 4.186578, 1.662589, 7.19212 17.764273 3.357284 4.977920 3.723358 6.000663 4.182428 4.982603 4.182428 13.531327 36.995185 39.380026 29.887836 38.701615 51.034947, coordinates − − − Kew Gardens China Broadbalk Botanical Garden http://nematodes.myspecies.info/taxonomy/term/10645/specimens Kenya Kwale county The Netherlands Noordwijkerhout F0710 Grassland KY828296-KY828297 KY828279 KY828314 – Russia Golitsino 55.616512, Belgium Ghent University The Netherlands Slootdorp 52.823096, La Palma, Spain San Juan 28.783772, Ethiopia East Shewa 7.288950, 1 1 T307, T308 T149, T150, T155 T146T219T763, T764 The UK Netherlands Philippines Turkey Mount Ararat Rothamsted, – Grasses Grasses – KY828303 – – KY828316 – – – T216 T669, T671 ItalyT174T33, T34, T37 Solanto The Netherlands Belgium Slootdorp 38.074542, Het Zwin 52.859260, 51.366792, T222 Rwanda Nyirangarama T209 T704 The Netherlands Winssen – T151 Wageningen culture – T254, T305, T306, T142T658, The T659 Netherlands Dronten China – Baishui, Shaanxi, T104, T105 T244, T245 Nigeria Benue, Otukpo 8.13327, T683, T684 The Netherlands Doornenburg 51.894631, T210 UK London, T130, T131, T148, T616 Italy, Sicily Villalba – T73, T74 Tunisia – – T213, T214, T215, T89 The Netherlands Middelharnis 51.770211, T206, T207, T208, T93, T96, T103, T226 T165, T166, T167, T168, T169, T200 T241, T242, T243 The Netherlands Middelharnis 51.770211, T106,T107, T108, nanus cf. Species and populations of nematodes, mostly Pratylenchidae, analysed. No morphological voucher available. P. thornei P. thornei P. thornei Acrobeloides P. hexincisus P. brzeskii P. thornei P. goodeyi P. pratensis P. neglectus P. hexincisus P. hexincisus P. pratensis P. pratensis P. pratensis P. vulnus P. flakkensis P. neglectus P. goodeyi P. vulnus P. flakkensis P. goodeyi P. flakkensis Table 1. SpeciesPratylenchus goodeyi Code Country Location GPS Morphological vouchers are available1 on

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access Pitfalls of molecular species identification jority rule consensus tree was generated. Posterior proba- distant and biologically divergent populations were 99.8- bilities (PP) are given on appropriate clades. 100% similar, showing only a single heterozygous posi- tion. Morphologically, our populations displayed the typ- ical diagnostic characters of P. goodeyi, i.e., four lip an- Results nuli, large oblong spermatheca and tail conoid, ventrally concave, with a smooth tail tip (Fig. 1). In addition, mor- CASE STUDY 1: Pratylenchus goodeyi phometric characters matched well with the previously To assess further P. goodeyi as a potential cryptic reported populations of P. goodeyi (Table 2) (Castillo & species complex (Waeyenberge, 2007) newly collected Vovlas, 2007). Furthermore, the en face lip pattern, as vi- material of P. goodeyi from La Palma (Canary Islands, sualised by SEM, was identical to the originally described Spain) on banana, from plantain from the Ghent Uni- lip pattern (Sher & Allen, 1953) (Fig. 1). Intriguingly, versity botanical garden, from Rwanda on beans, and a remarkably different ITS sequence (only 54% similar) from Ethiopia on maize (Table 1) was analysed. Surpris- was observed between our populations and the popula- ingly, 28S rDNA sequences from these geographically tions described by Waeyenberge (2007) and Zhang et al.

Fig. 1. Light microscopy photomicrographs and SEM pictures of specimens of Pratylenchus goodeyi. A: Entire female body; B-D: Female anterior region; E: Female gonoduct; F, G: Spermatheca; H-I: Tail region; J: Female labial region, en face view; K: Female labial region, lateral view; L: Female lateral field at vulval region; M: Female vulval region; N: Female tail; O: Male labial region, lateral view; P: Male lateral field at mid-body; Q, R: Male tail region. (Light microscopy scale bars = 10 μm; SEM scale bars = 1 μm.)

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access T. Janssen et al. ∗ 3.8 2.0 0.5 3.6 1.8 1.3 1.6 5.1 25 9.0 0.6 0.2 3.0 8.7 15.0 6.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± s.d. (range). ± 4.5 24.7 1.7 12.1 0.5 16.2 4.4 20.2 3.2 19.4 1.1 77.6 3.1 28.1 2.6 27.6 72 541 0.6 6.4 0.2 3.9 4.1 59 7.8 85 63.9 420 14.0 140 11.5 85 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.7 28.5 0.5 12.0 0.5 15.8 0.7 20.6 1.0 19.0 1.2 78.6 1.0 28.0 2.6 28.3 31 547 0.8 6.5 0.8 3.9 1.4 54 6.2 84 23.4 420 17.5 138 4.5 92 m and in the form: mean ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± μ Russia Noordwijkerhout Doornenburg ∗ 1.7 21.6 1.1 12.6 0.6 17.5 1.1 17.3 1.7 18.9 3.1 76.6 1.5 26.8 2.5 29.4 40 508 0.5 6.7 0.4 4.2 11.9 124 5.4 54 7.0 77 28.1 389 12.8 88 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Slootdorp ∗ . All measurements are in 1.6 22.4 1.1 12.9 0.8 17.2 1.5 17.2 1.8 20.0 30 370 2.4 76.7 0.9 24.2 2.3 28.2 43 483 1.2 6.5 0.4 4.0 11.8 123 6.7 48 132 76 11.6 75 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Middelharnis P. pratensis ∗ 2 22.0 1.2 22.2 0.9 13.0 0.6 16.9 0.7 14.1 2.3 19.7 38 327 1.5 77.5 3.2 30.8 48 433 ,and 0.6 5.5 0.2 3.2 12.4 136 2.4 52 8.9 80 4.6 84 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Rwanda P. flakkensis , 1.8 15.6 1.7 11.1 0.4 14.7 2.3 15.2 3.4 20.3 3.4 73.0 2.1 24.1 1.5 32.1 68 487 6.4 78 0.2 2.9 12.0 114 2.1 54 4.9 94 47.1 357 0.43 5.2 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Botanical Garden 5.2 34.4 5.9 24.1 1.5 11.8 0.7 17.2 4.5 17.3 1.6 23.0 1.7 72.0 1.4 26.0 49 595 8.6 154 Pratylenchus goodeyi 0.8 6.5 0.4 3.8 2.1 65 6.8 91 44.4 428 9.5 100 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± (58-63)(74-89) (62-67) (84-95) (50-58) (86-108) (42-61) (58-95) (41-57) (59-86) (52-56) (70-84) (49-58) (76-93) (56-65) (73-98) (7.1-9.1) (5.9-6.8) (4.3-6.4) (4.2-8.0) (5.6-7.3) (5.8-7.8) (5.7-7.2) (5.5-7.2) (2.1-3.4) (3.6-4.0) (2.6-3.3) (2.6-3.7) (3.4-4.5) (3.3-5.6) (3.6-4.1) (3.5-4.0) (78-102) (94-106) (71-86) (68-96) (55-99) (83-93) (73-102) (76-96) 2.8 (8.6-13.0) (10.1-14.1) (9.4-12.4) (11.3-14.0) (11.5-14.8) (12.2-13.5) (9.8-14.0) (10.0-16.4) (397-507) (371-478) (304-430) (283-370) (316-415) (361-420) (331-473) (401-443) (544-675) (496-640) (424-569) (373-503) (417-534) (479-548) (432-603) (505-578) (134-154) (137-164) (99-137) (122-156) (106-138) (99-143) (120-154) (126-155) P. goodeyi P. goodeyi P. goodeyi P. flakkensis P. flakkensis P. flakkensis P. pratensis P. pratensis (23.8-39.4) (21.7-26.0) (13.3-17.1) (19.8-24.3) (19.4-24.2) (19.3-23.8) (24.6-33.8) (18.2-29.2) (16.6-18.3) (16.7-17.7) (13.9-15.5) (15.7-18.0) (16.6-18.6) (16.8-18.2) (15.2-16.5) (15.6-17.2) (15.0-27.1) (14.1-19.5) (14.0-16.2) (11.9-15.0) (15.9-19.6) (16.3-17.9) (15.1-27.2) (16.7-27.3) (19.0-23.8) (19.5-26.9) (17.4-24.9) (17.9-22.6) (17.5-24.2) (17.9-20.4) (16.2-23.4) (16.7-22.5) (72.0-76.5) (67.9-74.8) (70.7-75.7) (75.6-81.6) (72.5-82.4) (75.4-78.3) (76.7-79.6) (75.8-79.3) (26.2-30.4) (23.7-28.7) (21.4-28.2) (20.3-23.5) (22.1-26.1) (25.3-27.9) (25.0-32.3) (25.7-30.3) (24.9-36.5) (32.8-36.0) (28.2-38) (27.2-33.9) (24.2-32.0) (26.7-33.5) (24.8-31.2) (18.5-34.7) Origin: La Palma Ghent University Species: Morphometric data of females of TAF fixed specimen mounted in glycerin; other populations are measured from temporary slides.  Tail length 35.3 Anterior to cardiaAnterior to pharyngeal lobe 143 82 Post-uterine sac length 30.2 Anterior to centre of metacorpus 61 c c 20.3 Anal body diam. 11.9 Stylet length 17.7 b7.6 Max. body diam. 21.5 a 29.5 V 74.2 Anterior end to vulva 471 Table 2. n649710548 L 623 ∗ Anterior end to excretory pore 89

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(2015). A detailed comparison of the previously generated a mostly annulated tail terminus (Fig. 6) (Castillo & ‘P. goodeyi’ sequences revealed a close affinity with sev- Vovlas, 2007). Morphometric characters were measured eral Cephalobidae sequences (Fig. 2). After sequencing for populations from Doornenburg and Noordwijkerhout the ITS region for several Cephalobidae species, we found and matched well with the previously reported P. praten- that P. goodeyi sequences from Waeyenberge (2007) and sis populations (Castillo & Vovlas, 2007) (Table 2). More- Zhang et al. (2015) are 98.6% similar to Acrobeloides cf. over, our 18S rDNA sequence from Noordwijkerhout nanus (Fig. 2). The ITS sequence of this A.cf.nanus matches 99.4-100% with the P. pratensis sequences gen- is sister to the ‘P. goodeyi’ sequences and they are clus- erated by Rybarczyk-Mydlowska et al. (2014). Generated tered together within a monophyletic Cephalobidae clade 28S rDNA sequences were 99.1-99.6% similar between (Fig. 2; Fig. S1 in the Supplementary material). Addition- the two populations from Noordwijkerhout and Doornen- ally, six ‘P. goodeyi’ sequences originating from three dif- burg, confirming that both populations belong to the same ferent localities in India were also found to group in this species. Surprisingly, these 28S rDNA sequences did not monophyletic Cephalobidae clade (Gokte-Narkhedkar et match with the P. pratensis sequences deposited by de la al., 2013). Morphologically, the A.cf.nanus population Peña et al. (2007) (16.2-16.8% divergence). While both shows the same variation in lip region and tail morphology our populations and the populations from de la Peña et al. as previously reported for A. nanus (Fig. 3) (Anderson, (2007) are morphologically similar, our populations are 1968; Boström & Gydemo, 1983). Phylogenetic analysis more likely to represent the genuine P. pratensis because: of 18S rDNA and 28S rDNA sequences confirm the phy- i) morphologically, P. pratensis was described as being logenetic position of A.cf.nanus within the Cephalobidae very similar in appearance to P. pseudopratensis (Sein- and clearly visualise the genetic distance from our gen- horst, 1968) and this agrees with the position of our P. uine P. goodeyi populations (for 18S rDNA, see Fig. S2 in pratensis populations as they form a monophyletic clade the Supplementary material). According to 28S rDNA, A. based on 28S rDNA with a P. pseudopratensis popula- cf. nanus forms (as in Smythe & Nadler, 2006), a mono- tion from Majd Taheri et al. (2013); ii) the grassland phyletic clade with other Acrobeloides and Cephalobus with fruit trees in which our P. pratensis populations were sequences (data not shown). According to 28S rDNA and found is very similar to the original type locality and 18S rDNA our genuine P. goodeyi populations are posi- the habitats previously reported for P. pratensis (Sein- tioned in clade VI, forming a monophyletic group together horst, 1959; Loof, 1974; Castillo & Vovlas, 2007), while with P. zeae, P. parazeae, P. bhattii, P. delattrei, and P. bo- populations from de la Peña et al. (2007) were recovered livianus (Figs 4, 5). All these data confirm that the previ- from a very different coastal dune habitat; and iii) the 18S ously deposited sequences are cephalobid sequences that rDNA sequences from our P. pratensis population match have been misidentified as P. goodeyi. with sequences generated by Rybarczyk-Mydlowska et al. (2014). As a result, the populations from de la Peña et al. CASE STUDY 2: Pratylenchus pratensis (2007) are hypothesised to belong to a separate species of = Tylenchus pratensis DE AN M , 1880 Pratylenchus, sister to P. japonicus Ryss, 1988 according = Anguillulina pratensis (DE MAN, 1880) to our 28S rDNA phylogeny (Fig. 4) (Wang et al., 2014). GOFFART, 1929 In the summer of 2014 we sampled the type locality = Pratylenchus helophilus SEINHORST, 1959 of P. lentis (Villalba, Sicily, Italy) (Troccoli et al., 2008). = Pratylenchus irregularis LOOF, 1960 From the type locality, a species mixture of Pratylenchus = Pratylenchus lentis TROCCOLI,DE LUCA, sp. 1 (Janssen et al., 2017) and P. lentis was recovered. An HANDOO &DI VITO, 2008 N. SYN. ITS sequence of our P. lentis specimen was 96.3% simi- Pratylenchus pratensis was recovered from several lar to the ITS sequences of the original description, falling places in The Netherlands, associated with grasses (No- within the 7.1% intraspecific variability reported by Troc- ordwijkerhout, 13 km away from the type locality at coli et al. (2008). However, an 18S rDNA sequence from Leiden), Pyrus sp. (Doornenburg) and Prunus domes- the same P. lentis DNA extraction was identical to a P. tica (Winssen) (Table 1). All three populations showed pratensis sequence (KC875380) from The Netherlands the diagnostic morphological characters of P. pratensis, (Rybarczyk-Mydlowska et al., 2014) and 99.6% identi- namely: labial region with three annuli, a finely annu- cal to a P. pratensis sequences from this study. The 28S lated cuticle, four lateral lines, characteristic large elon- rDNA sequences also from the same DNA isolate matched gated spermatheca shape, tail with 20-28 annuli and with two P. pratensis populations from Noordwijkerhout

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Fig. 2. Phylogenetic position of misidentified Pratylenchus goodeyi sequences (FJ712922-FJ712926, KF275665, KF700243, KF840454, KF840455, KF840456, KF856291 and KM874803 indicated by ∗) within the Cephalobidae as inferred from Bayesian analysis of the ITS of RNA gene sequences using GTR + I + G model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold.

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Fig. 3. Light microscopy photomicrographs of specimens of Acrobeloides cf. nanus. A, B: Female anterior region, showing morphological variation of labial probolae between different specimens; C: Lateral field at mid-body; D, E: Female tail region, showing morphological variation of tail tip between different specimens. (Scale bars = 10 μm.) and Doornenburg (99.0-99.7% identical). Also note that P. lation was observed between our P. lentis population and pratensis had already been reported from Trifolium sp. in our P. pratensis populations. Based on all the above data, Italy (Puglia), and thus parasitising the same plant fam- P. lentis is regarded as being conspecific with the mor- ily (Fabaceae) as P. lentis (Inserra et al., 1979). Mor- phologically similar P. pratensis. Thus, the excellent and phologically, both P. lentis and P. pratensis were found comprehensively described P. lentis is herein proposed as to exhibit a highly variable tail tip. While Troccoli et a junior synonym of P.pratensis and the P.pratensis popu- al. (2008) reported P. pratensis to have a slightly shorter lation of de la Peña et al. (2007) is considered a different stylet and a more anterior vulva (V = 75-78) in compar- species. ison to P. lentis, these findings are contradicted by the morphometrics of our P. pratensis populations as V var- CASE STUDY 3: Pratylenchus flakkensis ied between 75.8-79.6 while stylet length varied between Pratylenchus flakkensis was recovered from its type 15.2-17.2 μm(V= 76-80, stylet length = 15.5-17.0 μm locality, a heavy loam soil under grass at Middelharnis in P. lentis) (Troccoli et al., 2008). Moreover, the sper- (The Netherlands), associated with grasses (Slootdorp, matheca is variable in shape, roundish, oval or rectangu- The Netherlands), and with Urtica dioica (Golitsina, Rus- lar for the P. pratensis populations as well as our P. lentis sia). 28S rDNA sequences from these three populations population. Additionally, no variation in cuticular annu- were 99.4-99.8% similar, confirming that these popula-

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access Pitfalls of molecular species identification G P. + I + Previously misidentified as ∗∗ . (2008). et al 18S rRNA gene sequence obtained from topotype ∗ as inferred from Bayesian analysis of D2-D3 of 28S rRNA gene sequences using GTR Pratylenchus ., 2007). et al . ITS of RNA gene sequences from the same specimen matched with ITS sequences from Troccoli P. lentis (de la Peña Phylogenetic relationships within the genus model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold. Fig. 4. specimen of pratensis

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Fig. 5. Phylogenetic relationships within the genus Pratylenchus as inferred from Bayesian analysis of 18S rRNA gene sequences using GTR + I + G model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold.

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Fig. 6. Light microscopy photomicrographs of specimens of Pratylenchus pratensis. A: Entire female; B-D: Female anterior region; E: Female gonoduct, spermatheca, uterus, vulva, and post-uterine sac; F: Lateral field at mid-body; G-J: Morphological variability in female tail region. (Scale bars: A = 100 μm; B-J = 10 μm.)

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Downloaded from Brill.com10/06/2021 03:12:39AM via free access T. Janssen et al. tions belong to the same species. Also COI sequences potentially vast impact on pest management and interna- of the P. flakkensis populations from Middelharnis and tional trade as, in this case, P. goodeyi is listed as a quar- Slootdorp were found to be 96% similar (15 variable nu- antine species in some parts of the world (for example, cleotide positions) (Fig. 7). All populations showed the in Florida). Unfortunately, the associated ‘domino effect’ characteristic morphological features of P. flakkensis, i.e., of misidentifications can result in a proliferation of further a labial region with two lip annuli, a mostly annulated wrong sequence-based identifications. Furthermore, when tail tip, a filled round to angular spermatheca, four lat- two alternative sequences are available, one tends to have eral lines and a tail with 16-27 annuli (Fig. 8) (Seinhorst, more trust in the alternative that is supported by most se- 1968; Castillo & Vovlas, 2007). Morphometric characters quences (in this case the predominant free-living nema- were found to be in agreement with those of previously re- tode sequence). However, in these ‘identification’ cases ported populations of P. flakkensis (Table 2). The morpho- the data are not independent but are, in fact, all the result logically very similar P. gibbicaudatus Minagawa, 1982 of a single mislabelling. Due to the apparently indepen- was distinguished from P. flakkensis by a higher number dent data the mislabelling error becomes more difficult to of tail annuli (24-39 vs 18-24) and this was confirmed by rectify. In this case, the misidentification was almost rec- the range of annuli number found for P. flakkensis in this tified by De Luca et al. (2011) as the authors noted a large study (17-27) (Minagawa, 1982). Phylogenetic analysis of genetic discrepancy between the mislabelled P. goodeyi 18S rDNA and 28S rDNA sequences indicate P. flakken- sequences, and a genuine P. goodeyi population. How- sis to be a member of clade V, together with P. thornei, ever, as ITS sequences failed to identify one of the popu- P. neglectus, P. hispaniensis, and P. brzeskii (Subbotin et lations as a population of Acrobeloides, the mislabelled al., 2008; Palomares-Rius et al., 2014) Within clade V, sequences were not corrected. This indicates that ITS may P. flakkensis was found to be most closely related to P. not always be such a reliable barcoding region (De Luca et brzeskii according to the 28S rDNA sequences. Interest- al., 2011) as it suffers from substitution saturation at a rel- ingly, the SEM en face lip pattern of P. flakkensis (Fig. 8) atively low taxonomic level. If a more conserved barcode was very similar to that of P. thornei, P. neglectus, and gene, such as D2-D3, had been selected the mislabelling P. hispaniensis in having an obtuse, dumbbell-shaped lip would have been obvious. pattern, indicating that this lip pattern could be an apomor- In a second mislabelling example, it was shown that phic feature of clade V (Subbotin et al., 2008; Palomares- both the genuine and a wrongly-labelled P. pratensis Rius et al., 2010). population were linked to DNA sequences. However, as both populations were connected to a different marker Discussion (18S vs 28S) the problem remained unnoticed for a long time. Furthermore, a putative new species was linked to In this study it was clearly demonstrated that 12 pub- a third marker, namely ITS. As such, the fact that P. lished ITS sequences of the economically important plant- lentis is actually conspecific with P. pratensis was over- parasitic nematode P. goodeyi are actually sequences from looked. Actually, mislabelled specimens are a symptom distant free-living bacterivorous cephalobids. Most likely, of a larger problem as 86% of the estimated existing this original mislabelling was caused by a contamination eukaryotic species on Earth have yet to be described of the DNA extraction by a free-living cephalobid. Such while the number of taxonomic experts is markedly de- contamination could happen when multiple individuals creasing (Mora et al., 2011). Nowadays, many laborato- are used for a single DNA extraction. While the use of ries are shifting towards sequence-based identification al- multiple specimens in a single DNA extraction leads to a though the majority of morphologically-described species number of problems it was, and still is, a common practice remain unlinked to DNA sequences. In this study we in order to increase DNA concentration. Problematically, have illustrated this in the case of P. flakkensis, a com- incorrect labelling of sequences can result in a cascade monly occurring species of Pratylenchus in Europe and of erroneous interpretations. It also illustrates the danger Western-Asia that was, until now, unlinked to DNA se- of mislabelled sequences in public DNA databases which quences. Within Pratylenchus diagnostics, robust mor- can lead to misidentification, as shown by the reports of phological characters are relatively scarce, greatly ham- ‘P. goodeyi’ on cotton in India (Gokte-Narkhedkar et al., pering species identification (Castillo & Vovlas, 2007). 2013) and on banana in China (Zhang et al., 2015) based Collecting topotype material is often the only option on cephalobid sequences. These misidentifications have a in order to link formerly described morphospecies to

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Fig. 7. Phylogenetic relationships within the genus Pratylenchus as inferred from Bayesian analysis of the COI gene sequences using GTR + I + G model. Posterior probabilities of over 70% are given for appropriate clades. Newly obtained sequences are indicated in bold.

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Fig. 8. Light microscopy photomicrographs and SEM pictures of specimens of Pratylenchus flakkensis. A-T: female, U-W: male. A: Entire body; B-D: Anterior region; E: Labial region, en face view; F: Spermatheca; G: Posterior gonoduct; H: Lateral field at mid-body; I: Labial region, lateral view; J: Lateral field at vulval region; K: Lateral field at mid-body; L, M: Tail region; N-T: Morphological variation of tail tip; U: Labial region, lateral view; V: Lateral field at mid-body; W: Tail region. (Scale bars: A-D, F-H, J, K, M-S = 10 μm; E, I, L, T-W = 1 μm.)

DNA sequences (De Luca et al., 2012; Troccoli et netics. A connection between morphology and DNA se- al., 2016; Zamora-Araya et al., 2016; Janssen et al., quences is therefore of crucial importance, once again em- 2017). phasising the value of depositing morphological vouch- This study clearly illustrates the importance of a strong ers associated with deposited sequences (De Ley et al., link between DNA sequences and morphological char- 2005; Pleijel et al., 2008). Mislabelling of sequences also acters. Moreover, it shows that relying solely on either poses a huge threat for pure sequence-based identifica- DNA sequences or on morphology for species identifica- tions such as environmental sequencing, as these methods tion is a flawed practice (Will et al., 2005; Wheeler, 2008). rely completely on the quality of the reference database Even a modest morphological description immediately (Dell’Anno et al., 2015). Hence, safeguarding the link be- provides possible connections to life history, ecology, be- tween DNA sequences and morphological characters is haviour and taxonomic status, while DNA sequences pro- of crucial importance in order to prevent misidentifica- vide a wealth of characters for the study of molecular tions and the presence of mislabelled sequences in public biology, phylogenetic relationships, and population ge- databases.

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Acknowledgements De Ley, P., De Ley, I.T., Morris, K., Abebe, E., Mundo-Ocampo, M., Yoder, M., Heras, J., Waumann, D., Rocha-Olivares, A., Jay Burr, A.H. et al. (2005). An integrated approach to fast We would like to thank Valeria Orlando for analysing and informative morphological vouchering of nematodes for the P. lentis population and Lisa Joos for processing applications in molecular barcoding. Philosophical Transac- the photographic data and for critical reading of this tions of the Royal Society B: Biological Sciences 360, 1945- manuscript. Alliance Nyiragatare, Adem Esimo Beriso, 1958. DOI: 10.1098/rstb.2005.1726 Qing Xue, Yao Adjiguita Kolombia and Noura Chihani Dell’Anno, A., Carugati, L., Corinaldesi, C., Riccioni, G. & are thanked for their help in collecting nematode popu- Danovaro, R. (2015). Unveiling the biodiversity of deep-sea lations. This work was supported by the special research nematodes through metabarcoding: are we ready to bypass fund UGent 01N02312. the classical taxonomy? PLoS ONE 10, e0144928. DOI: 10. 1371/journal.pone.0144928 De Luca, F., Reyes, A., Troccoli, A. & Castillo, P. (2011). References Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Abascal, F., Zardoya, R. & Telford, M.J. (2010). TranslatorX: Pratylenchidae) as inferred from the analysis of the ITS multiple alignment of nucleotide sequences guided by amino rDNA. European Journal of Plant Pathology 130, 415-426. acid translations. Nucleic Acids Research 38, 7-13. DOI: 10. DOI: 10.1007/s10658-011-9763-9 1093/Nar/Gkq291 De Luca, F., Troccoli, A., Duncan, L.W., Subbotin, S.A., Al-Banna, L., Ploeg, A.T., Williamson, V.M. & Kaloshian, I. Waeyenberge, L., Coyne, D.L., Brentu, F.C. & Inserra, (2004). Discrimination of six Pratylenchus species using PCR R.N. (2012). 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Fig. S1. Phylogenetic position of genuine (dark grey frame) and misidentified Pratylenchus goodeyi sequences (light grey frame) as inferred from Bayesian analysis of the ITS of RNA gene sequences using GTR + I + G model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold.

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Fig. S2. Phylogenetic relationships as inferred from Bayesian analysis of the 18S rRNA gene sequences using GTR + I + G model, showing the large genetic distance between genuine Pratylenchus goodeyi sequences and sequences of Acrobeloides cf. nanus and closely related misidentified ‘P. goodeyi’ sequences. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold.

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