The pitfalls of molecular species identification: a case study within the genus Pratylenchus (Nematoda: Pratylenchidae)

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Comprehensive morphological and molecular analyses revealed that published ITS sequences of the economically important plant-parasitic nematode Pratylenchus goodeyi 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.


International Journal of Fundamental and Applied Nematological Research



AbascalF.ZardoyaR.TelfordM.J. (2010). TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Research 38, 7-13. DOI: 10.1093/Nar/Gkq291

Al-BannaL.PloegA.T.WilliamsonV.M.KaloshianI. (2004). Discrimination of six Pratylenchus species using PCR and species-specific primers. Journal of Nematology 36, 142-146.

AndersonR.V. (1968). Variation in taxonomic characters of a species of Acrobeloides (Cobb, 1924) Steiner and Buhrer, 1933. Canadian Journal of Zoology 46, 309-320.

BoströmS.GydemoR. (1983). Intraspecific Variability in Acrobeloides nanus (de Man) Anderson (Nematoda, Cephalobidae) and a note on external morphology. Zoologica Scripta 12, 245-255. DOI: 10.1111/j.1463-6409.1983.tb00508.x

BowlesJ.BlairD.McManusD.P. (1992). Genetic variants within the genus Echinococcus identified by mitochondrial-DNA sequencing. Molecular and Biochemical Parasitology 54, 165-174. DOI: 10.1016/0166-6851(92)90109-W

CastilloP.VovlasN. (2007). Pratylenchus (Nematoda: Pratylenchidae): diagnosis, biology, pathogenicity and management. Nematology Monographs and Perspectives 6 (Series Editors: HuntD.J.PerryR.N.). Leiden, The Netherlands, Brill.

DarribaD.TaboadaG.L.DoalloR.PosadaD. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9, 772.

de la PeñaE.KarssenG.MoensM. (2007). Distribution and diversity of root-lesion nematodes (Pratylenchus spp.) associated with Ammophila arenaria in coastal dunes of western Europe. Nematology 9, 881-901. DOI: 10.1163/156854107782331289

De LeyP.FelixM.A.FrisseL.M.NadlerS.A.SternbergP.W.ThomasW.K. (1999). Molecular and morphological characterisation of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology 1, 591-612. DOI: 10.1163/156854199508559

De LeyP.De LeyI.T.MorrisK.AbebeE.Mundo-OcampoM.YoderM.HerasJ.WaumannD.Rocha-OlivaresA.Jay BurrA.H. (2005). An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding. Philosophical Transactions of the Royal Society B: Biological Sciences 360, 1945-1958. DOI: 10.1098/rstb.2005.1726

Dell’AnnoA.CarugatiL.CorinaldesiC.RiccioniG.DanovaroR. (2015). Unveiling the biodiversity of deep-sea nematodes through metabarcoding: are we ready to bypass the classical taxonomy? PLoS ONE 10, e0144928. DOI: 10.1371/journal.pone.0144928

De LucaF.ReyesA.TroccoliA.CastilloP. (2011). Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA. European Journal of Plant Pathology 130, 415-426. DOI: 10.1007/s10658-011-9763-9

De LucaF.TroccoliA.DuncanL.W.SubbotinS.A.WaeyenbergeL.CoyneD.L.BrentuF.C.InserraR.N. (2012). Pratylenchus speijeri n. sp. (Nematoda: Pratylenchidae), a new root-lesion nematode pest of plantain in west Africa. Nematology 14, 987-1004. DOI: 10.1163/156854112X638424

de ManJ.G. (1880). Die einheimischen, frei in der reinen Erde und im süssen Wasser lebenden Nematoden. Vorläufiger Bericht und descriptivsystematischer Theil. Tijdschrift der Nederlandsche Dierkundige Vereeniging 5, 1-104.

DeryckeS.VanaverbekeJ.RigauxA.BackeljauT.MoensT. (2010). Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes. PLoS ONE 5, e13716. DOI: 10.1371/journal.pone.0013716

FilipjevI.N. (1936). On the classification of the Tylenchinae. Proceedings of the Helminthological Society of Washington 3, 80-82.

GoffartH. (1929). Beobachtungen über Anguillulina pratensis de Man. Zeitschrift für Parasitenkunde, Berlin 2, 97-120.

Gokte-NarkhedkarN.TekchandaniD.TijareA. (2013). Unpublished ITS rRNA sequences of Pratylenchys goodeyi associated with cotton deposited in genbank under accession numbers: KF275665, KF700243, KF840454, KF840455, KF840456, KF856291. Nagpur, India, Division of Crop Protection, Central Institute for Cotton Research.

HebertP.D.N.CywinskaA.BallS.L.DeWaardJ.R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences 270, 313-321. DOI: 10.1098/rspb.2002.2218

HooperD.J. (1986). Extraction of free-living stages from soil. In: SoutheyJ.F. (Ed.). Laboratory methods for work with plant and soil nematodes. London, UK, Her Majesty’s Stationery Office, pp.  5-30.

HuelsenbeckJ.P.RonquistF. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754-755. DOI: 10.1093/bioinformatics/17.8.754

InserraR.N.ZeppA.VovlasN. (1979). Pratylenchus dell’italia meridionale. Nematologia Mediterranea 7, 137-162.

JanssenT.KarssenG.VerhaevenM.CoyneD.BertW. (2016). Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution. Scientific Reports 6, 22591. DOI: 10.1038/srep22591

JanssenT.KarssenG.OrlandoV.SubbotinS.A.BertW. (2017). Molecular characterization and species delimiting of plant parasitic nematode of the genus Pratylenchus from the penetrans group (Nematoda: Pratylenchidae). Molecular Phylogenetics and Evolution. DOI: 10.1016/j.ympev.2017.07.027 [Epub ahead of print]

KressW.J.EricksonD.L. (2008). DNA barcodes: genes, genomics, and bioinformatics. Proceedings of the National Academy of Sciences of the United States of America 105, 2761-2762. DOI: 10.1073/pnas.0800476105

LalD.LalR. (2011). Wrong sequences in databases: whose fault?? Indian Journal of Microbiology 51, 413. DOI: 10.1007/s12088-011-0186-2

LarkinM.A.BlackshieldsG.BrownN.P.ChennaR.McGettiganP.A.McWilliamH.ValentinF.WallaceI.M.WilmA.LopezR. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947-2948. DOI: 10.1093/bioinformatics/btm404

LoofP.A.A. (1960). Taxonomic studies on the genus Pratylenchus (Nematoda). Tijdschrift over Plantenziekten 66, 29-90.

LoofP.A.A. (1974). Pratylenchus pratensis. In: CIH descriptions of plant parasitic nematodes, Set 4, No. 52. Farnham Royal, UK, Commonwealth Agricultural Bureaux.

LoofP.A.A. (1991). The family Pratylenchidae Thorne, 1949. In: NickleW.R. (Ed.). Manual of agricultural nematology. New York, NY, USA, Marcel Dekker, pp.  363-421.

Majd TaheriZ.Tanha MaafiZ.SubbotinS.A.PourjamE.EskandariA. (2013). Molecular and phylogenetic studies on Pratylenchidae from Iran with additional data on Pratylenchus delattrei, Pratylenchoides alkani and two unknown species of Hirschmanniella and Pratylenchus. Nematology 15, 633-651. DOI: 10.1163/15685411-00002707

MinagawaN. (1982). Descriptions of Pratylenchus gibbicaudatus n. sp. and P. macrostylus Wu, 1971 (Tylenchida: Pratylenchidae) from Kyushu. Japanese Journal of Applied Entomology and Zoology 17, 418-423.

MokriniF.WaeyenbergeL.ViaeneN.AndaloussiF.A.MoensM. (2013). Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR. European Journal of Plant Pathology 137, 403-413. DOI: 10.1007/s10658-013-0252-1

MoraC.TittensorD.P.AdlS.SimpsonA.G.B.WormB. (2011). How many species are there on Earth and in the ocean? PLoS Biology 9, e1001127. DOI: 10.1371/journal.pbio.1001127

MuirP.LiS.T.LouS.K.WangD.F.SpakowiczD.J.SalichosL.ZhangJ.WeinstockG.M.IsaacsF.RozowskyJ. (2016). The real cost of sequencing: scaling computation to keep pace with data generation. Genome Biology 17, 53. DOI: 10.1186/s13059-016-0917-0

NilssonR.H.RybergM.KristianssonE.AbarenkovK.LarssonK.H.KoljalgU. (2006). Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective. PLoS ONE 1, e59. DOI: 10.1371/journal.pone.0000059

Palomares-RiusJ.E.CastilloP.LiebanasG.VovlasN.LandaB.B.Navas-CortesJ.A.SubbotinS.A. (2010). Description of Pratylenchus hispaniensis n. sp. from Spain and considerations on the phylogenetic relationship among selected genera in the family Pratylenchidae. Nematology 12, 429-451. DOI: 10.1163/138855409x12559479585043

Palomares-RiusJ.E.GuesmiI.Horrigue-RaouaniN.Cantalapiedra-NavarreteC.LiebanasG.CastilloP. (2014). Morphological and molecular characterisation of Pratylenchus oleae n. sp. (Nematoda: Pratylenchidae) parasitizing wild and cultivated olives in Spain and Tunisia. European Journal of Plant Pathology 140, 53-67. DOI: 10.1007/s10658-014-0443-4

PleijelF.JondeliusU.NorlinderE.NygrenA.OxelmanB.SchanderC.SundbergP.ThollessonM. (2008). Phylogenies without roots? A plea for the use of vouchers in molecular phylogenetic studies. Molecular Phylogenetics and Evolution 48, 369-371. DOI: 10.1016/j.ympev.2008.03.024

PowersT. (2004). Nematode molecular diagnostics: from bands to barcodes. Annual Review of Phytopathology 42, 367-383. DOI: 10.1146/annurev.phyto.42.040803.140348

PriceN.S.BridgeJ. (1995). Pratylenchus goodeyi (Nematoda: Pratylenchidae): a plant parasitic nematode of the montane highlands of Africa. African Journal of Zoology 109, 435-442.

Rybarczyk-MydlowskaK.MaborekeH.R.van MegenH.van den ElsenS.MooymanP.SmantG.BakkerJ.HelderJ. (2012). Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes. BMC Evolutionary Biology 12, 221. DOI: 10.1186/1471-2148-12-221

Rybarczyk-MydlowskaK.van MegenH.van den ElsenS.MooymanP.KarssenG.BakkerJ.HelderJ. (2014). Both SSU rDNA and RNA polymerase II data recognise that root-knot nematodes arose from migratory Pratylenchidae, but probably not from one of the economically high-impact lesion nematodes. Nematology 16, 125-136. DOI: 10.1163/15685411-00002750

RyssA.Y. (1986). [ The influence of some soil properties on the host-parasite system ‘Pratylenchus flakkensis-Poa pratensis.] Trudy Zoologicheskogo Instituta Morfologiya, sistematika i faunistika paraziticheskikh zhivotnykh 155, 25-40.

RyssA.Y. (1988). [ World fauna of the root parasitic nematodes of the family Pratylenchidae (Tylenchida).] Leningrad, USSR, Nauka.

RyssA.Y. (1992). [ Some records of plant parasitic nematodes (Nematoda, Tylenchida) from Estonia.] Eesti Teaduste Akadeemia Toimetised, Bioloogia 41, 72-76.

SavolainenV.CowanR.S.VoglerA.P.RoderickG.K.LaneR. (2005). Towards writing the encyclopaedia of life: an introduction to DNA barcoding. Philosophical Transactions of the Royal Society B: Biological Sciences 360, 1805-1811. DOI: 10.1098/rstb.2005.1730

SchneiderC.A.RasbandW.S.EliceiriK.W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nature Methods 9, 671-675. DOI: 10.1038/nmeth.2089

SeinhorstJ. (1959). Two new species of Pratylenchus. Nematologica 4, 83-86. DOI: 10.1163/187529259X00417

SeinhorstJ. (1962). On the killing, fixation and transferring to glycerin of nematodes. Nematologica 8, 29-32. DOI: 10.1163/187529262X00981

SeinhorstJ. (1968). Three new Pratylenchus species with a discussion of the structure of the cephalic framework and of the spermatheca in the genus. Nematologica 14, 497-510. DOI: 10.1163/187529268X00183

SherS.A.AllenM.W. (1953). Revision of the genus Pratylenchus (Nematoda: Tylenchidae). University of California Publications in Zoology 57, 441-447.

SmytheA.B.NadlerS.A. (2006). Molecular phylogeny of Acrobeloides and Cephalobus (Nematoda: Cephalobidae) reveals paraphyletic taxa and recurrent evolution of simple labial morphology. Nematology 8, 819-836. DOI: 10.1163/156854106779799178

SohleniusB.SandorA. (1987). Vertical distribution of nematodes in arable soil under grass (Festuca pratensis) and barley (Hordeum distichum). Biology and Fertility of Soils 3, 19-25.

SubbotinS.A.RagsdaleE.J.MullensT.RobertsP.A.Mundo-OcampoM.BaldwinJ.G. (2008). A phylogenetic framework for root lesion nematodes of the genus Pratylenchus (Nematoda): evidence from 18S and D2-D3 expansion segments of 28S ribosomal RNA genes and morphological characters. Molecular Phylogenetics and Evolution 48, 491-505. DOI: 10.1016/j.ympev.2008.04.028

TroccoliA.De LucaF.HandooZ.A.Di VitoM. (2008). Morphological and molecular characterization of Pratylenchus lentis n. sp. (Nematoda: Pratylenchidae) from Sicily. Journal of Nematology 40, 190-196.

TroccoliA.SubbotinS.A.ChitambarJ.J.JanssenT.WaeyenbergeL.StanleyJ.D.DuncanL.W.AgudeloP.UribeG.E.M.FrancoJ. (2016). Characterisation of amphimictic and parthenogenetic populations of Pratylenchus bolivianus Corbett, 1983 (Nematoda: Pratylenchidae) and their phylogenetic relationships with closely related species. Nematology 18, 651-678. DOI: 10.1163/15685411-00002981

van BezooijenJ. (2006). Methods and techniques for nematology. Available online at

VilgalysR. (2003). Taxonomic misidentification in public DNA databases. New Phytologist 160, 4-5. DOI: 10.1046/j.1469-8137.2003.00894.x

WaeyenbergeL. (2007). Pratylenchus goodeyi, a species-complex? Communications in Agricultural and Applied Biological Sciences 72, 697-701.

WaeyenbergeL.RyssA.MoensM.PinochetJ.VrainT.C. (2000). Molecular characterisation of 18 Pratylenchus species using rDNA Restriction Fragment Length Polymorphism. Nematology 2, 135-142. DOI: 10.1163/156854100509024

WaeyenbergeL.ViaeneN.MoensM. (2009). Species-specific duplex PCR for the detection of Pratylenchus penetrans. Nematology 11, 847-857. DOI: 10.1163/156854109X428016

WangN.GuJ.WangX.LiH. (2014). Identification of Pratylenchus japonicus intercepted in Acer palmatum from Japan. Journal of Nanjing Agricultural University 37, 76-82.

WheelerQ.D. (2008). Undisciplined thinking: morphology and Hennig’s unfinished revolution. Systematic Entomology 33, 2-7.

WillK.W.MishlerB.D.WheelerQ.D. (2005). The perils of DNA barcoding and the need for integrative taxonomy. Systematic Biology 54, 844-851. DOI: 10.1080/10635150500354878

Zamora-ArayaT.PadillaW.P.Archidona-YusteA.Cantalapiedra-NavarreteC.LiebanasG.Palomares-RiusJ.E.CastilloP. (2016). Root-lesion nematodes of the genus Pratylenchus (Nematoda: Pratylenchidae) from Costa Rica with molecular identification of P. gutierrezi and P. panamaensis topotypes. European Journal of Plant Pathology 145, 973-998. DOI: 10.1007/s10658-016-0884-z

ZhangF.YanS.ZhouY.GuoG.GuoS.JinZ.ZengH.PengD.RuanL.SunM. (2015). First report of Pratylenchus goodeyi on banana in Hainan Province, China. Plant Disease 99, 731-732. DOI: 10.1094/Pdis-08-14-0874-Pdn


  • 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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  • 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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  • 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.)

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  • Phylogenetic relationships within the genus Pratylenchus as inferred from Bayesian analysis of D2-D3 of 28S rRNA gene sequences using GTR + I + G model. Posterior probabilities are given for appropriate clades. Newly obtained sequences are indicated in bold. 18S rRNA gene sequence obtained from topotype specimen of P. lentis. ITS of RNA gene sequences from the same specimen matched with ITS sequences from Troccoli et al. (2008). ∗∗Previously misidentified as P. pratensis (de la Peña et al., 2007).

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  • 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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  • 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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  • 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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  • 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.)

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  • 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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  • 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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