Fundam. appl. NemalOl., 1996,19 (4), 309-313

Forum article

MOLECUlAR SYSTEMATICS AND POPUL<\TION BIOLOGY OF PHYTONEMATODES: SOl\ΠUNIFYING PRlNCIPLES

BRADLEY C. HYMAN Department ofBiology, University of Califomia, RiveTside, CA 92521, USA. Accepted for publication 27 July 1995.

Sununary - From the perspective of nematologists conducting their research in America, our discipline is receiving diminishing attention and a renewed commirment towards demonstrating the importance and impact ofagricultural nematology to our society is urgently necessary. This forum article examines how molecular approaches to nematode sysrematics and population biology can strengrhen the perception of our science through sorne simple coherence of focus.

Réswné - Systénwtique moléculaire et biologie des populations de nématodes phytoparasites: quelques principes unificateurs - Du point de vue des nématologisres conduisant leurs recherches en Amérique, notre discipline reçoit une attention décroissante et un engagement renouvelé pour démontrer l'importance et l'impact de la nématologie agricole pour notre société est instamment nécessaire. Cet article éditorial examine comment les approches moléculaires dans la systématique des nématodes et la biologie des populations peuvent consolider la perception de notre science à travers une simple cohérence des points de vue. Key-words: Nematode systematics, population biology, diagnostics.

The assimilation of molecular data into investigation lost concept, just at a most propltlous time when the of nematode systematics and population dynamics has biotechnology necessary to address these problems has escalated dramatically in the last few years. Ir cornes as emerged. no surprise that this trend has paralleled the increased The implicit message to be discovered within " The accessibility of " user-friendly" DNA analytical tech­ Future of Nematode Systematics" (Ferris, 1994) and niques, which include molecular c1oning, polymerase echoed by the Committee on National Needs and Pri­ chain reaction (PCR) amplification methodologies, and orities in Agricultural Nematology (Barker el al., 1994) DNA sequencing. The application of these techniques is a renewed commitment towards demonstrating the ta nematological problems has propelled the publication importance and impact ofour discipline on the needs of of several useful review articles that specifically target society. Admittedly, this is a perspective from nematol­ the international nematology community (Hyman, ogists conducting their research in the current funding 1990; Curran, 1991; Williamson, 1991; Hyman & Pow­ climate of the United States, but evidence of a unified ers, 1991; Powers, 1992; Caswell-Chen el al., 1993). discipline is a worldwide responsibility, necessitating an These papers typically discuss the utility of DNA-based ever increasing dialogue among nematologists with characters for diagnostics and phylogenetic studies at ail common interests but trained in different generations. taxonomie levels. This editarial perspective examines how nematode sys­ One such thoughtful and timely cornmentary that ap­ tematics and population genetics might be employed to peared in this Forum last year (Ferris, 1994) traveled strengthen the perception of our science through sorne beyond distilling the current status of biochemical ap­ simple coherence of focus. proaches to nematode diagnostics, population biology and phylogenetics. Rather, Ferris alerted us to an alarm­ Nematode systematics : classical versus molecu­ ing concern that confronts everyone involved in study­ lar data revisited ing nematodes: the apparent decline in emphasis on nematode systematics. The problem is an enigmatic Hyman and Powers (1991) and Ferris (1994) briefly one. This decade represents an era of expanded interest review what can be considered a rwo-tiered history of in bioctiversity and ecology (Wilson, 1992), replete with nematode systematics. Early on, the understanding of the understanding that rigorous taxonomie definitions taxonomie affinities and the generation of rational, testa­ are the foundation that supports the development of ble phylogenetic hypotheses were often based on reliable diagnostics necessary to deduce relationships morphological characters available for description by among organisms. Yet, the importance of nematodes to light, and occasionally, e1ectron microscopy. These fun­ agriculture and the reliance of management regimes on damental anatomical and cytologieal studies comprised sound identification and classification appears to be a an initial phase of nematode systematics and many of

1SSN 1164-5571196/04 S 4.00/ © GaUlhier-Villars - ORSTOM 309 B. C. Hyman our own academic pedigrees can be traced to the strong molecular tools to address problems in nematode phylo­ foundation provided by these pioneering nematologists. genetics will be required to resolve the different hy­ The application of biochemical methodologies (Hus­ potheses supported by these data sets. sey, 1979) to taxonomic questions catalyzed a second era of investigation into nematode identification and Genetic variability ofnematode populations: dia­ systematics. In many cases, protein techniques includ­ gnostics or difIerentiation? ing serology, isozymes, t\vo-dimensional electrophoretic gel patterns, and more recently DNA analyses, have Microevolutionary patterning, or the genetic struc­ provided support for established taxonomic frame­ ture of nematode populations, remains the least under­ works. However, in sorne instances, morphology and stood aspect of nematode population biology (CasweU molecules do not agree, and neither approach can be & Roberts, 1987). This realization is disappointing giv­ used in isolation. The foUowing example underscores en the steady advancements reported for animal parasît• this problem. ic nematodes (Blouin el al., 1992; Dame el al., 1993; Ascaris suum (an animal pathogen), Caenorhabdùis Anderson el al., 1993; NadJer, 1996). These studies elegans (a free-Iiving nematode) and Melm"dogyne inco­ have revealed significant differences in how conspecific gnita (a phytonematode) represent three different sub­ populations of two different nematode parasites of live­ classes (Spiruria, Rhabditia and Diplogasteria, respec­ stock, Ascaris suum and Oslerlagia oSlenagi, are structur­ tively) within the Secernentia, one of the two major ed. Movement with their cattle hosts provides the op­ nematode taxonomic classes. Mitochondrial DNA se­ portunity for extensive gene flow among populations of quences were compared among these three nematodes both nematodes, which is anticipated to diminish genet­ and with Romanomeris culicivorax, a mermithid parasite ic variation between isolates. This appears to be the case of insects that represents the second major nematode for OSlertagia, where most of the genetic variability is class, the Adenophorea. Nucleotide sequence for the apportioned within, but not between geographically large mitochondrial ribosomal RNA (lg-rRNA) gene separated populations. In conrrast, Ascaris reveals signifi­ and both nucleotide and amino acid sequences for the cant, measurable differentiation among sampled iso­ genes encoding the mitochondrial proteins NADH de­ lates. These distinct patterns of genetic architecture hydrogenase subunit 3 and cytochrome b derived from structure may be a consequence of the relative effective these four nematodes were aligned (Powers el al., 1993). population sizes estimated for these two nematodes, ge­ Low similarity scores were generated among these norypic selection by varying environrnental pressures, or four nematodes with any pairwise alignment involving other factors which may influence the extent of genetic M. incognita or R. culicivomx. This result was antici­ drift within and between conspecific populations. pated, given that the Nematoda is an evolutionarily an­ It is essential that the molecular population genetics of cient phylum; subclass divergence within the Secernen­ phytonematodes becornes elevated to the level ofunder­ tia has been depicted in a hypothetical evolutionary tree standing that has been attained for their animal parasite to have occurred over 550 million years aga (mya) (Poi­ counterparts. Quantitative description of genetic vari­ nar, 1983), and a split from the Adenophorea must have ability among plant pathogenic nematode populations pre-dated that event. UnexpectedJy, mtDNA compari­ will play a vital role in understanding critical operational sons between C. elegans and A. suum indicate a more phenotypes, and the underlying genotypes, defmed by recent separation about 65 million years, assuming a plant nematologists such as sibling species, sub-specific mammalian mtDNA molecular clock (Okimoto el al., races of Meloidogyne, Heterodera and Ditylenchus spp., 1992); an updated estimation of nucleotide substitution and pathotypes of Globodem isolates. Moreover, the se­ rate within the mtDNA of these two lineages (Okimoto lection of suitable management srrategies, and the effi­ el al., 1994) suggests divergence of the subclasses Rhab­ cacy of these practices, wiU be directly impacted by the ditia and Spiruria may be even more recent than origi­ genetic variability within and between targeted nema­ nally measured. tode populations. It is necessary to understand the ge­ Here, we are presented with conflicting morphologi­ netic substructure that underlies these isolates and ad­ cal and molecular data. However, because gene se­ dress whether management regimes are capable of quences evolve at different rates (Avise, 1994) and the controUing oruy a portion of the lineages that contribute genes sampled in these studies represent but a small to the composition of inbreeding populations. portion of the nematodes' DNA, the apparent incongru­ Until recently, allozyme analysis as a measure ofaUele ence between anatomical and biochemical charaeters frequencies has been the method of choice for geneti­ may be overstated. Alternatively, the molecular results cists who studied the genetic srructure of populations suggest the foUowing questions: Is subclass divergence and diversity among the îndividuals that comprise them. within the Secernentian nematodes unequal? Have the Adoption of this approach to describe the architecture ascarids been raxonomically misplaced within the Spiru­ ofphytonematode populations has been impeded by the ria? Clearly, a productive dialogue among the rradition­ small size of these organisms. In most cases, aUozyme ally-rrained nematode systematists and those adapting analysis proves too insensitive to genorype individual

310 Fundam. appl. NemalOl. IvIolecular systemalics ofphylOnemalOdes nematodes. However, in stuclies that permit analysis of to deducing phylogenetic affinities from RAPD markers pooled individuais, this approach has proven usefLÙ in (Clark & Lanigan, 1993). studies at higher taxonomic levels such as species identi­ Unfortunately, the imporrant clistinction between fication in the genus A1eloidogyne (Fargette & Braaksma, " diagnostics" and "genetic differentiation " has be­ 1990), when conservation of morphological characters come less defined as molecLÙar techniques have been does not permit differentiation. used to develop phytonematode species identifiers. We Contemporary DNA technology, including molecu­ must continue to define our studies that employ molecu­ lar cloning, polymerase chain reaction (PCR) amplifica­ lar markers with explicitly stated objectives; cliagnostics tion, and nucleic acid sequencing have largely sur­ and population genetics are interdependent, but they are mounted the earlier methodological limitations that not " seamless ,). hindered measurement of genetic variabüity among ne­ The genetic structure of popLÙations is typically ex­ matode isolates (Hyman, 1990; Curran, 1991; Hyman pressed in quantitative terms by measuring the frequen­ & Powers, 1991; Williamson, 1991; Casweil-Chen et aL, cies of different alleles at targeted loci. Genetic differ­ 1993). Moreover, DNA analysis has the potential to entiation can be identified by electrophoretic variants of provide a broader census of genetic variabüity because allozymes, RFLP and RAPD DNA bands, or by scoring protein phenotypes survey only a portion of the genome nucleotide substitutions using DNA sequencing meth­ and because nucleotide substitutions that do not result ods. Several statistical methods, first pioneered by the in allozyme electrophoretic variants would remain un­ F-statistics of Wright (1922), partition measured allele detected. frequencies within and among populations, thereby re­ Although phytonematologists have enthusiastically vealing population substructure. Such quantitative de­ adapted new molecLÙar techniques to their studies of scriptions have been used to describe the decidely differ­ population and evolutionary genetics, an unforrunate ent genetic architectures in the animal parasitic confusion has arisen as to how we define our studies in nematode Ascaris and Ostertagia. the context of the data that molecular tools can generate. We have been studying a higWy polymorphic locus Often, molecularly-oriented work on plant pathogenic within the Meloidogyne (root-knot nematode) rnitochon­ nematodes emphasizes the identification ofDNA mark­ drial genome. The genetic variability of this mitochon­ ers that correlate with species within genera, or with drial DNA (mtDNA) segment is not due to nucleotide subspecific phenotypes such as races and biotypes. Re­ substitution, the typical " measure " of genetic variabil­ striction fragment length polymorphisms (RFLP) and ity. Rather, small portions of the mtDNA have become modified PCR procedures that generate random ampli­ duplicated to generate short, repeated regions (Okimoto fied polymorphic DNA (RAPD) markers have become et aL, 1991). Nematodes maintain mtDNA molecules increasingly popular for identifying different nematode that carry alleles with clifferent copy numbers of these isolates. However, the characterization of molecular short repeats, termed variable number tandem repeats markers that may discriminate among nematode popu­ (VNTRs). We have employed the computational logic lations (Powers, 1992) does not necessarily provide a developed by Birky et al. (1989) to characterize vari­ rigorous definition ofpopulation sub-strueture nor offer ability within and among samples of organelle genomes, astringent foundation to formLÙate phylogenetic affin­ and have applied this methodology to analyze a size­ ities. In her Forum Article, Ferris (1994) explains that polymorphic 63 base pair (bp) VNTR represented at " proper understanding of such [phylogenetic] relation­ different copy numbers within root-knot nematode ships is ... the basis for sound classification and diag­ mtDNA molecules. By employing this approach, nostic procedures". The opposite circumstance, when mtDNA variation can be apportioned among different diagnostic RFLP-or RAPD-generated molecular mark­ levels of population hierarchy, providing a quantitative ers are employed to infer taxonomic relationships, re­ signature ofgenetic structure among nematode isolates : quires a clear understanding of the avaüable method­ variation of Meloidogyne mitochondrial genomes can be ological and analytical procedures (Hadrys et aL, 1992). quantitated within individual nematodes, between incli­ One common simplification is the immediate assump­ viduals within a population and importantly, among tion that DNA bands of the same electrophoretic mobil­ conspecific popLÙations. ity are" shared " characters bet\veen taxa. Care must be Based on our stuclies to date (Whipple & Hyman, taken to demonstrate that these bands represent homo­ 1995), an emergent trend has appeared. Among the logous portions of the genomes under cornparison so popLÙations that we have analyzed, most of the genetic that false affinities are not derived. This practice will variability at this size-variable mtDNA locus resides ensure that the accumulated data is representative of the within individuals, not within or among popLÙations. organismal genealogies. In addition, it is essential to em­ Such genetic structuring is similar to what has been ploya genetic analysis most appropriate for the form of observed for Ostertagia (Blouin et aL, 1992). This obser­ data under evaluation. For example, algorithms de­ vation can be interpreted in several different contexts. In signed to interpret independent nucleotide substitution population studies, observed allele frequencies are a events (Nei & Li, 1979) may not be directly applicable consequence of balance bet\veen mutation rate (in our

Vol. 19, n' 4 - 1996 311 B. C. Hyman system the changes in VNTR copy number within a tinued extramural support, and provide attractive career mtDNA molecule) and genetic drift. Mutations that choices for the coming generations ofbiologists interest­ change the size of VNTRs will generate new alleles (dif­ ed in studying these intriguing organisms. ferent 63 bp repeat copy number in the root-knot nema­ Acknowledgements tode mitochondrial genome) and increase diversity whereas genetic drift (sorting of mtDNA molecules dur­ l thank Drs Virginia Ferris, David Lunt, Tom Powers and Lawrence Whipple for critical reading of the manuscript prior ing germ line cell divisions) will decrease variability. to submission. Our research is supported by a United States When the mutation rate greatly exceeds fixation of al­ Department of Agriculture Competitive Grant 94-37302­ leles by drift, the spectrum of mtDNA molecules will 0571. appear similar because the same collection of size-vari­ able alleles will be present in populations under compar­ References ison; this is precisely what we have observed among the ANDERSON, T. J, c., ROMERO-ABAL, M. E. & ]AENIKE, J, Meloidogyne isolates currently examined. Interestingly, (1993). Genetic structure and epidemiology ofAscaris popu­ when Hugall et al. (1994) used RFLP analysis as a mea­ lations : Patterns of host clifferentiation in Guatemala. Para­ sure of nucleotide substitution among Australian root­ sùology, 107: 319-334. knot nematode isolates, minimal nucleotide sequence AV1SE, J, C. (1994). Moleeular markers, natuml history and divergence among mitochondrial genome haplotypes evolution. New York & London, Chapman & Hall, xiv + was also observed among conspecific Meloido­ 511 p. gyne populations. Together, it appears size-variable mu­ BARKER, K. R., HUSSEY, R. S., KRUSBERG, L. R, BIRD, G. tation rate within the mitochondrial genome may be W., DUNN, R A., FERRIS, H., FERRlS, V. R, FRECK,vtAl'l, excessively rapid, while mtDNA nucleotide divergence D. W., GABRIEL, c.]., GREWAL, P. S., MACGUID\XI1N, A. may be too minimal to provide quantitative signatures E., RIDDLE, D .L., ROBERTS, P. A. & SCHl\'UTT, D. P. for reproductively isolated Meloidogyne populations. Be­ (1994). Plant and soil nematodes : Societal impact and focus yond that ofspecies diagnostics that typifies much of the for the future. J. Nematol., 26: 127-137. current nematode "molecular marker" literature, an BECKENBACH, K., SMITH, M. J, & WEBSTER, J, M. (1992). expanded quantitative treatrnent of phytonematode nu­ Taxonomic affmities and intra-and interspecific variation in clear DNA markers, such as those employed in studies Bursaphelenehus spp. as determined by polymerase chain ofBursaphelenchus (Beckenbach et al., 1992); Heterodera reaction. J. Nematol., 24: 140-147. (Caswell-Chen et al., 1992) and Xiphinema (Vrain et al., BIRKY, C. W., FUERST, P. & MARUYAMA, T. (1989). Orga­ 1992) isolates, will be an essential component of our nelle gene diversity under migration and drift: equilibrium collective efforts to address the genetic structuring of expectations, approach to equilibrium, effects of heteroplas­ nematade populations. mic cells and cornparison of nuclear genes. Geneties, 121 : 613-627. BLOUIN, M. S., DAME,J, B., TARRANT, C. A. & COURTNEY, Conclusions C. H. (1992). Unusual population genetics of a parasitic With this brief commentary, 1 have identified molecu­ nematode : MtDNA variation within and among popula­ lar approaches ta nematode systematics and population tions. Evolution, 46: 470-486. genetics as examples where focusing our priorities is CAS\XfELL, E. P. & ROBERTS, P. A. (1987). Nematode popu­ both scientifically sound and strategically essential if we lation genetics. ln: Veech, J, A. & Dixon, D. W. (Eds). are ta present ourselves as a unified, contemporary dis­ Vistas on nematology. Hyattsville, Md, USA, Society of Ne­ cipline. When viewed in isolation, molecules alone may matologists !nc : 390-397. not generate a true representation of phylogenetic affin­ CASWELL-CHEN, E. P., WILLlAMSON, V. M. & Wu, F. F. ities among nematodes at any taxonomic level. We must (1992). Random arnplified polymorphic DNA analysis of overlay the current molecular work upon a rich, descrip­ Heterodera erueiferae and H. sehaehtii populations. J. 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