Nematology, 2003, Vol.

5(3), 441-451

Nacobbus aberrans (Thorne, 1935)

Thorne & Allen, 1944 (Nematoda: Pratylenchidae);
a nascent species complex revealed by RFLP analysis
and sequencing of the ITS-rDNA region
Alex R EID 1;¤ , Rosa H. M ANZANILLA -L ÓPEZ 2 and David J. H UNT 1
1 CABI Bioscience, Bakeham Lane, Egham, Surrey, TW20 9TY, UK
2 Nematode Interactions Unit, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK
Received: 11 November 2002; revised: 18 March 2003
Accepted for publication: 20 March 2003

Summary – Twelve populations of Nacobbus aberrans, sensu lato, from Mexico, Bolivia, Peru, Ecuador and Argentina were subjected
to molecular analysis of their genetic variability. RFLP banding patterns revealed three groups: i) Mexico, Ecuador and Argentina 1
populations; ii) Bolivia and Peru populations; iii) Argentina 2 population. These differences were conŽ rmed by sequencing the ITS
rDNA region. Depth of branching was strongly supportive of the presence of three separate species, thus supporting the hypothesis that
N. aberrans s.l. is indeed a species complex. The populations from Mexico, Ecuador and Argentina 1 are attributed to N. aberrans s.s.,
although this requires conŽ rmation by molecular characterisation of N. aberrans from the type locality in the USA; those from Bolivia
and Peru are attributed to N. bolivianus Lordello, Zamith & Boock, 1961 with Argentina 2 regarded as representing another taxon.
Nacobbus serendipiticus and N. batatiformis are removed from synonymy under N. aberrans s.l. and regarded as species inquirendae.
Consistent minor banding patterns in the RFLP proŽ les may indicate that the genus reproduces predominantly by parthenogenesis.
Keywords – Argentina, Bolivia, Ecuador, Mexico, Peru, RFLP, sequencing, species inquirendae, taxonomy.

The genus Nacobbus Thorne & Allen, 1944 com- England (Franklin, 1959), The Netherlands (de Bruijn &
prises predominantly endoparasitic, gall inciting nema- Stemerding, 1968), Finland and the former USSR (Kir-
todes that are indigenous to the Americas. It has recently janova & Lovanova, 1975), India (Prasad et al., 1965) and
been the subject of a monographic review by Manzanilla- China (Yin & Feng, 1981).
López et al. (2003). Four nominal species and one sub- Nacobbus aberrans is adapted to a wide range of
species have been placed in Nacobbus, although when climatic conditions (Alarcón & Jatala, 1977), the life
Sher (1970) revised the genus he considered only two cycle being strongly in uenced by temperature (Prasad &
species as valid, viz. the type, Nacobbus dorsalis Thorne Webster, 1967; Quimí, 1979). Many aspects of its ecology
& Allen, 1944 and N. aberrans (Thorne, 1935) Thorne are still poorly understood and there is great heterogeneity
& Allen, 1944. Nacobbus dorsalis is relatively poorly in the morphology and host range of populations from
characterised and is currently known only from the USA, the majority of the geographical areas where it has been
whereas N. aberrans, an important parasite of vegetables, found. Such variability led Jatala and Golden (1977) to
including bean, chilli pepper, potato, tomato and sugar- suggest that N. aberrans could be a species complex with
beet, is geographically widespread in the Americas (Ar- a series of biotypes or physiological races.
gentina, Bolivia, Chile, Ecuador, Mexico, Peru and the Host range has been used as the main criterion to sup-
USA). Other records and quarantine interceptions of the port and designate races of N. aberrans, but discordance
genus (some of which have not been conŽ rmed) include in the data and non-reproducibility of results are of fre-

* Corresponding author, e-mail: alex.reid@sasa.gsi.gov.uk

Current address: Scottish Agricultural Science Agency, Diagnostics and Molecular Biology, 82 Craigs Road, East Craigs, Edinburgh,
EH12 8NJ, UK.

© Koninklijke Brill NV, Leiden, 2003 441

Also available online - www.brill.nl
A. Reid et al.

quent occurrence (Boluarte & Jatala, 1993; Toledo et al., Martínez et al., 1995; Canto-Sáenz et al., 1996). Although
1993; Ortuño et al., 1997; Manzanilla-López et al., 2003). differences in chromosome number were found in some
The situation is further complicated by continuing uncer- populations, these studies were not continued. Isozyme
tainty in the taxonomy of the genus, phenotypicresolution analyses have shown inter- and intra-population genetic
at the species and subspecies level being imprecise and in- variability in N. aberrans, often with greater similarity
adequate (Baldwin & Cap, 1992). among populationsfrom different geographical areas than
Of the two species currently regarded as valid, the type from the same host (Mayorga & Jatala, 1990; Doucet &
species, N. dorsalis, is apparently of minor economic im- Gardenal, 1992; Ibrahim et al., 1997).
portance because of its limited geographical distribution, Ibrahim et al. (1997), using biochemical and molecular
being found occasionally in a few sugarbeet Ž elds in Mon- methodologies, analysed four populations of N. aberrans
terey County, California (Steele, 1984; Baldwin & Cap, s.l., two coming from Peru, one from Argentina and one
1992). Nacobbus aberrans s.l., on the other hand, has a from Mexico. Non-speciŽ c esterases indicated that the
much wider geographical distribution and is regarded as a two Peruvian populations had afŽ nities with each other
major pest of many cultivated vegetable and Ž eld crops in whilst the Mexican population showed afŽ nities with that
North and South America. from Argentina. Substantial genetic variation occurred
Morphometrical data for a range of N. aberrans popu- between some of the populations. RFLP analysis of the
lations from different geographical areas have been pub- ITS1-5.8S-ITS2 rDNA with TaqI and HinfI revealed
lished by a number of authors (Sher, 1970; Johnson, two groups, the Peruvian populations being identical
1971; Quimí, 1979; Doucet, 1989; Doucet & Di Rienzo, to one another but distinct from the populations from
1991; Manzanilla-López et al., 1999) and detailed mor- Argentina and Mexico, which in turn shared a different
phological observations of populations of N. aberrans banding pattern. Alignment of the 5.8S rDNA sequences
from Argentina were reported by Doucet and Di Rienzo revealed eight substitutions, seven of which separated the
(1991) and from Argentina, Bolivia, Mexico and Peru by populations into the same two groups revealed by RFLP,
Manzanilla-López (1997). with the eighth separating the Mexican population from
Manzanilla-López et al. (1999) subjected the morpho- the other three. Ibrahim et al. (1997) commented on the
metrics of 12 populations from South America and Mex- dissimilarity between the Peruvian and Argentina/Mexico
ico to canonical variate analysis. In the scatter plots, they populations and pointed out that the depth of divergence
found that the males and females from Bolivia, Peru, and corresponded to what might be expected at species, or
those from the Urquiza population from Argentina, were even genus, level in other Tylenchida. They regarded
consistently separated from those from Mexico. The Mex- these differences as subspeciŽ c groupings which could
ican populationsin turn grouped with a second population act as an underlying framework for congruent phenotypic,
from Argentina. The datasets for Bolivia and Peru were geographic or other characters, leading ultimately to a
closer to each other than to the Argentina (Urquiza) popu- more deŽ ned taxonomic status.
lation which was more detached. It is clear that considerable heterogeneity exists within
Jatala and Golden (1977) stated that “: : : all known N. aberrans and that certain, often congruent, groupings
species found in South America at present are consid- can be demonstrated by a variety of methodologies.There
ered to be of the Nacobbus aberrans complex”. Subse- is still dispute as to whether such variation indicates the
quent workers have regarded N. aberrans as a complex presence of a species complex, although an increasing
of species or aggregates (Jatala, 1993; Canto-Sáenz et al., body of evidence indicates that such is the case (Ibrahim
1996; Doucet, 1996). In its simplest form, a species com- et al., 1997; Manzanilla-López, 1997; Manzanilla-López
plex, or aggregate, groups together a number of species et al., 1999). The ability of molecular techniques to
which are morphologically similar and difŽ cult to dis- discriminate similar phenotypesat the genetic level is well
criminate. Species within the complex often display little known, but has yet to be fully exploited in Nacobbus.
phenotypic variation and may be of restricted geographi- No doubt this is partly due to difŽ culties in assembling
cal distribution. a collection of populations that adequately represent such
Cytogenetics has been used in the recognition of a wide geographic distribution.
aggregates (cytospecies, microspecies, etc.) and such Restriction Fragment Length Polymorphism (RFLP)
studies have been applied to N. aberrans in an attempt analysis of the internal transcribed spacer (ITS) region
to discriminate populations (Jatala & Boluarte, 1993; of the ribosomal DNA repeat unit has been widely used

442 Nematology
 Nacobbus aberrans species complex

for diagnostic purposes for a variety of nematode genera. Table 1. Nacobbus aberrans s.l. population codes, origin and
This robust technique has been used to identify Steiner- GenBank accession numbers.
nema (Reid & Podrucka, 2003), Pratylenchus (Orui & Code Country Locality Accession
Mizukubo, 1999), Bursaphelenchus (Iwahori et al., 1998) of origin number
and cyst forming nematodes (Ferris et al., 1993; Zijlstra
A1 Argentina unknown AY254359
et al., 1997). Indeed, PCR-RFLP of the ITS region has
A2 ” Urquiza, La Plata, Buenos Aires AY254360
recently been used to assess intraspeciŽ c variation within B1 Bolivia Pulquina Abajo, Santa Cruz AY254361
populations of Radopholus similis (Elbadri et al., 2002). B2 ” Siberia, Santa Cruz AY254362
In the present study, we investigated the taxonomic B3 ” Pulquina Arriba, Santa Cruz AY254363
status of N. aberrans s.l. by examining genetic variation in B4 ” Cochabamba, Cochabamba AY254364
many of the original populations studied by Manzanilla- E2 Ecuador Guayabamba, Quito AY254365
López et al. (1999). The molecular data obtained were M1 Mexico Montecillo, Chapingo AY254366
derived from more populationsfrom a wider geographical M2 ” Santa María, Oaxaca State AY254367
range than those studied by Ibrahim et al. (1997) and, M3 ” Tecamachalco, Puebla State AY254368
crucially, included a number of populations from Bolivia M4 ” Zacatecas, Zacatecas State AY254369
P1 Peru Lake Titicaca, Puno
and the distinctive population from Urquiza, Argentina.

Ecuador population (E2) was collected from tomato by

Materials and methods
Carmen Triviño in 2002 and cultured at Rothamsted Re-
search on tomato. Strict measures were employed to pre-
N EMATODE POPULATIONS vent accidental cross-contamination of cultures, individ-
Twelve populations attributed to N. aberrans s.l. were ual pots being well separated from one another with verti-
used in this study. Nematode cultures were all established cal Perspex sheets acting as splash guards. Scrupulous hy-
from infested Ž eld soil in order to establish a broad ge- giene was practised during re-potting. The locality details
netic base. At no stage were single egg masses used and codes of these populationsare summarised in Table 1.
to initiate stock cultures. Mexican populations M2 and
M4, the Bolivian B1 population, Argentinian populations M OLECULAR STUDIES
A1 and A2 and the Peruvian population (P1) were col-
lected by various individuals in 1993 and sent to Rotham- Sample preparation and PCR ampliŽ cation
sted Research, Harpenden, UK where they were cultured Individual nematodes were placed on an ethanol swab-
alternately on tomato (Lycopersicon esculentum Mill.) bed glass slide in a 10 ¹l drop of worm lysis buffer
and either aubergine (Solanum melongena L.) or potato (10 mM Tris, pH 8.3, 50 mM KCl, 2.5 mM MgCl2 , 0.45%
(Solanum tuberosum L.) under strict quarantine condi- NP40, 0.45% Tween 20, 0.01% gelatin, 60 ¹g/ml Pro-
tions in a glasshouse until 2000. Infested soil was then teinase K). Juvenile stages, adult males and immature fe-
double bagged in labelled plastic bags which were sealed males were cut in half with a sterile needle and the con-
and kept in a cold room at ca 5± C until the cultures were tents transferred to a sterile 0.5 ml thin-walled PCR tube
re-established on tomato in February 2002. The Mexican and placed on ice. Mature adult females were squashed
populations M1 and M3 were established on tomato in and pieces (e.g., gonads, cuticle, etc.) transferred to four
a separate glasshouse cubicle at Rothamsted Research in separate tubes and placed on ice. All samples were frozen
1999 using Ž eld samples collected in Mexico by the sec- at ¡20± C for at least 10 min then incubated at 65±C for
ond author. All the Mexican Ž eld populations were from 1 h followed by 10 min at 95± C in a Hybaid PCR Express
tomato except for M4 which was from beans (Phaseolus thermocycler. After incubation the samples were placed
vulgaris L.). The Peruvian population was originally col- on ice for a few minutes then brie y centrifuged to col-
lected from a potato Ž eld. Bolivian populations B1, B2 lect the contents at the bottom of the tubes and 40 ¹l of
and B3 were collected in potato Ž elds by John Bridge PCR mix added. Final reaction conditions were 10 mM
in 2000/2001 and were initially cultured on potato and Tris pH 8.3, 50 mM KCl, 2.5 mM MgCl2 , 2 mM dNTPs,
tomato under quarantine glasshouse conditions at CABI 50 pmols of each primer and two units of Taq polymerase
Bioscience, Egham, UK. Additional cultures were estab- (Sigma). Oligonucleotidesused were the ITS primers de-
lished on tomato at Rothamsted Research in 2001. The scribed by Ferris et al. (1993). The ampliŽ cation condi-

Vol. 5(3), 2003 443

A. Reid et al.

tions were 95± C for 2 min followed by 40 cycles of 95± C (Gascuel, 1997) with 100 rounds of bootstrapping and by
for 30 s, 50± C for 60 s and 72± C for 90 s. A Ž nal extension an exhaustive search to Ž nd the most parsimonious tree.
step of 72± C for 5 min was included. The forward and reverse sequences of at least three
clones from each strain were assembled using Sequencher
Restriction Fragment Length Polymorphism (RFLP) v4.0.5 (Gene Codes Corporation). Alignment of the con-
analysis sensus sequences was carried out using the ClustalW
The PCR products from several individual nematodes function in MacVector v7.0r2 (Oxford Molecular Group)
from each population were digested with the following using the slow alignment speed. Phylogenetic analysis
restriction enzymes: AluI, DdeI, HaeIII, HhaI, HinfI, was carried out using PAUP* v4b.10 by Neighbor Joining,
HpaII, and RsaI at 37± C overnight. All enzymes were as before, and by a branch and bound parsimony search
obtained from Promega and were used with buffers with 100 bootstrap replicates.
supplied by the manufacturer. The digests were loaded
onto 1.5% (w/v) agarose gels made in 0.5 £ TBE buffer
and run at 100 V for 3 h and stained with ethidium Results
bromide. A molecular weight marker (Low DNA Mass
Ladder from Gibco, band sizes 2000, 1200, 800, 400, All life cycle stages yielded ampliŽ cation products
200 and 100 base pairs) was loaded between each set of with a size of approximately 800 base pairs. Individual
samples. All gel images were stored digitally. adult females did not yield products even upon dilution
1 : 1000 with distilled water, although pieces of a mature
Cloning and sequencing female, including fragments of the cuticle, did give
The ITS PCR products were puriŽ ed using a Wizard® PCR products. With most of the restriction enzymes
PCR Preps PuriŽ cation System (Promega) and cloned us- tested the samples could be separated into three distinct
ing a pGEM ®-T Easy Vector System II (Promega) accord- RFLP types. One group comprised the Argentina 1,
ing to the manufacturer’s instructions. A number of white Ecuador 2 and the four Mexican populations; a second
colonies were picked from each plate and grown overnight group comprised the four Bolivian populations and the
in 1 ml LB containing 50 ¹g/ml ampicillin at 37± C in an Peru population whilst the third group, represented solely
orbital incubator at 200 rpm. The presence of the correct by the Argentina 2 population, yielded patterns that were
insert was checked by heating 10 ¹l from each culture at unique (Fig. 1). In addition to the main RFLP pattern
95± C in a thin-walled tube for 10 min and adding 15 ¹l for each nematode there were also faint secondary bands
PCR mix and amplifying as detailed earlier. Clones con- visible in some digests (for example the Dde I digest of the
taining inserts were then grown overnight in 5 ml LB con- B4 population, marked with arrows in Fig. 1). These faint
taining 50 ¹g/ml ampicillin at 37± C in an orbital incubator digest products were consistent within each population
at 200 rpm and the plasmids puriŽ ed using a Wizard ® Plus (even after prolonged digestion) and appear to be present
Minipreps DNA PuriŽ cation System (Promega) as per at very low levels, suggesting that some of the ITS regions
manufacturer’s instructions. The puriŽ ed plasmids were are divergent in their sequences from the majority.
sequenced using a SequiTherm Excel™ II DNA Sequenc- Phylogenetic analysis of these RFLP data conŽ rms
ing Kit (Cambio) with labelled M13 primers obtained the presence of three distinct groups by both Neighbor
from MWG-Biotech. Electrophoresis was carried out on Joining (Fig. 2A) and by parsimony analysis using an
a Li-Cor DNA Gene Reader 4200 on 8% denaturing acry- exhaustive search (Fig. 2B). The Argentina 2 population
lamide gels. is more closely related to those from Bolivia and Peru
whilst the Argentina 1 and Ecuador populations cluster
Phylogenetic analyses with those from Mexico. These groupings are strongly
The computer package GelComparII (Applied Maths) supported by bootstrap analysis in the Neighbor Joining
was used for analysis of the RFLP data. This allowed the tree.
manipulation of images to correct for any uneven running Sequence data of the major RFLP type from the
of the gels. A binary table was produced showing the complete ITS region was obtained for all populations
presence/absence of the 46 different bands yielded by with the exception of the Peru isolate (see Table 1
the 12 populations. This table was then analysed using for accession numbers). These data were aligned with
PAUP* v4b.10 (Swofford, 2002) by both the distance sequences of the following sedentary parasitic nema-
based method of Neighbor Joining using the BioNJ option todes obtained from GenBank, Globodera artemisiae

444 Nematology
 Nacobbus aberrans species complex

Fig. 1. PCR-RFLP yielded by restriction enzyme digestion of the ITS region of 12 Nacobbus populations with AluI, DdeI, HinfI and
HpaII. An example of the faint secondary banding pattern is arrowed in the DdeI digest of population B4. Molecular weight markers
are base pairs. The populations are coded as in Table 1.

(AF274415), Heterodera glycines (AF216579), Meloido- Discussion

gyne hapla (AF516722), M. incognita (AF516723) and
M. javanica (AF387095). Phylogenetic analysis by both M OLECULAR CHARACTERISATION
Neighbor Joining (Fig. 3A) and Heurisitc approaches
The ease with which RFLP were discovered (all seven
(Fig. 3B) yielded trees with similar topographies to those restriction enzymes tested yielded RFLP which could
from the RFLP analysis, although the difference between be used to separate the 12 populations into the same
the Argentina 2 population and the Bolivian isolates was three groups) strongly implies that N. aberrans s.l. is a
not as pronounced as that obtained by RFLP analysis. species complex. The results are strongly supportive of

Vol. 5(3), 2003 445

A. Reid et al.

Fig. 2. A: Neighbor Joining cladogram yielded by 100 bootstrap replicates. Bootstrap values are shown at the nodes; B: Phylogram
yielded by an exhaustive search. Best tree from 654 729 075 evaluated. Tree length 50, CI 0.920, RI 0.972.

the existence of at least three distinct taxa: one comprising sperm has long been contentious in Nacobbus. Several
the Bolivian and Peru populations; a second including authors have illustrated a spermatheca-like structure con-
the Mexican, Ecuador and Argentina 1 populations; and taining sperm, whereas others note only a small expan-
a third with the single member, Argentina 2. The presence sion at the uterus/oviduct junction devoid of sperm. Cer-
of faint ITS-RFLP within an individual suggests that tainly, the cells at this point appear rather larger than the
some of the ITS regions vary in their sequence. It is other uterine cells and form a subglobular expansion with
intended to clone and sequence these minor ITS-RFLP a more or less occluded lumen. This structure almost cer-
variants in a future study. The groupings obtained by the tainly represents the spermatheca, but whether it is func-
RFLP analysis were conŽ rmed by sequence analysis of tional remains questionable. The spermatheca-like struc-
the complete ITS region. ture is small and probably incapable of storing the large
Of great interest is the fact that minor RFLP remain quantities of sperm needed to fertilise the numerous eggs.
constant between populations, both within and between Of course, it may be that obese females are continually
countries, and future work will need to examine the se- inseminated by the numerous males that cluster around
quences of these minor bands. There could be several them, or that the sperm is stored in the lumen of the uterus,
explanations for the phenomenon. Either, these species but this has never been reported. Perhaps the current ev-
have only recently spread across the continent and insuf- idence inclines towards a parthenogenetic, rather than an
Ž cient time has elapsed for them to differentiate, or Na- amphimictic, mode of reproduction and we are currently
cobbus may reproduce virtually exclusively by partheno- investigating this matter further.
genesis. If the latter proves to be true, then the produc- The results presented here are strongly supportive of N.
tion of males represents an evolutionary throwback to aberrans s.l. being a species complex comprising at least
when the genus reproduced sexually, although under ex- three distinct genotypes. It is likely that the populationsof
treme circumstances the nematode could still reproduce Nacobbus from Mexico are conspeciŽ c with N. aberrans
sexually. The presence or absence of a spermatheca with s.s. from the USA, but this remains to be conŽ rmed. It

446 Nematology
 Nacobbus aberrans species complex

Fig. 3. A: Neighbor Joining tree obtained from aligned ITS sequence data. Numbers at the nodes are bootstrap values; B: Most
parsimonious tree obtained by branch and bound method with 100 bootstrap replicates (bootstrap values shown at nodes). Tree length
937, CI 0.871, RI 0.909.

is, therefore, vitally important that N. aberrans material attempt to discriminate the species into ‘biotypes’. Un-
from the type locality in the USA is characterised in order fortunately, different cultivars of the same species of a
to conŽ rm that the Mexican populations are, in fact, the putative host may react differently to a single population
same as that from the type locality, i.e., N. aberrans s.s. of the nematode, and populations of nematode within the
It is possible that there are other cryptic species awaiting same country may attack different host plants. The ba-
resolution, something which can only be tested by more
sis of host preference in a nematode is not necessarily a
extensive sampling of the genus within the Americas
monophyletic character and may evolve independently in
allied to molecular characterisation.
Nacobbus aberrans is currently differentiated from N. response to selection pressure. As a consequence,the con-
dorsalis, the later described, but rather poorly known type cept of host races in Nacobbus needs to be assessed with
of the genus, by several morphological/morphometric caution, particularly in view of the evidence presented
characters of questionable reliability. Re-collection of N. herein for the existence of a species complex. Such cir-
dorsalis from the type locality and its characterisation by cumspection is crucial as the Nacobbus populations from
molecular methodologiesis urgently needed to determine the USA have yet to have their lineage characterised mole-
whether the two species are conspeciŽ c. cularly and we currently have insufŽ cient geographical
data to state unequivocally whether one or more species
P OSSIBLE CONGRUE NCE OF NEMATODE GENOTYPES occurs in a particular country. What is clear is that both
WITH HOST RACES
the host range and the race concept need to be re-assessed
Because of the difŽ culties involved in segregating mor- in the light of the current research. In order to establish
phological and morphometrical variation in Nacobbus, at- congruence, the nematode populations under test must be
tention has focused on host range and this, allied to an characterised by molecular methods in order to establish
inferred existence of host races, has been utilised in an their identity.

Vol. 5(3), 2003 447

A. Reid et al.

Baldwin and Cap (1992) commented that “Validity of Many taxonomists, including Luc (1987), have accepted
species and subspeciŽ c groups is not resolved, and there this synonymy, although all the species and subspecies
is frustration by those who propose to use identiŽ cation mentioned above were listed as valid by Siddiqi (1986),
to predict pathogenicity on particular hosts”. It would albeit without further comment. Subsequently, Siddiqi
be convenient if host differences could be matched to (2000) accepted N. aberrans and N. dorsalis as the only
morphology to facilitate rapid recognition but, despite two valid species in the genus.
some reported morphometrical differences, a scheme of In the light of the results from the molecular data
properly characterised races does not yet exist. presented in this paper, it is prudent to reconsider the
taxonomic placement of several taxa previously regarded
S TATUS OF THE NOMINAL SPECIES as junior synonyms of N. aberrans s.l. One complexity
here is that at present only molecular characterisation
Nacobbusaberrans s.l., the Ž rst species to be described, offers a reliable tool to distinguish the members of
is differentiated from N. dorsalis, the type species, on the complex, although more focused morphological and
the basis of several characters. These relate mainly to morphometric studies may identify congruent diagnostic
the immature and mature female and include the number characters from the current pool of variation.
of annules between vulva and anus which, according to
The description of N. serendipiticus bolivianus is quite
Sher (1970), are fewer in N. dorsalis than in N. aberrans
short and includes measurements and illustrations, but
(8-14 vs 15-24), the body shape of the swollen adults
is not detailed enough to make an accurate assessment
(round with an elongate posterior region in N. dorsalis
of the stylet knob shape, a feature that in the other
vs spindle-shaped without an elongate posterior portion
Bolivian populations we studied is characterised by the
in N. aberrans) and the retention of embryonated eggs
posteriorly sloping anterior surfaces (Manzanilla-López,
inside the body in N. dorsalis vs being laid in a gelatinous
1997; unpubl. obs.). Unfortunately, Lordello et al. (1961)
matrix in N. aberrans. The retention of embryonatedeggs,
did not refer to any designated type material of their
however, is also known to occur in N. aberrans s.l. (see
subspecies.
Manzanilla-López et al., 2003).
Although it is not possible to molecularly characterise
Two other species, viz., N. batatiformis Thorne &
the type population of N. serendipticus bolivianus, it
Schuster, 1956 collected from sugarbeet in Nebraska
(Thorne & Schuster, 1956), and N. serendipiticusFranklin, is highly probable that this nematode is conspeciŽ c
1959 collected from tomato grown under glasshouse con- with the four populations we studied from Bolivia. This
ditions in England (Franklin, 1959) have also been pro- assumption is supported by the uniform RFLP banding
posed. Nacobbus batatiformis was separated from N. dor- patterns of our four populations from Bolivia (showing
salis by the spindle-shaped mature females and from N. that only this species is deŽ nitely known to occur there)
aberrans by the anterior position of the phasmids and and also by the fact that one of our populations(B4) came
shorter distance between vulva and anus of vermiform fe- from Cochabamba, the type locality of N. s. bolivianus.
males. Nacobbus serendipiticus was distinguished from There are several taxonomic options available, but rather
N. dorsalis by the spindle-shaped mature females and than regard N. s. bolivianus as a species inquirenda and
from N. aberrans by the better developed procorpus and propose a new binomen for the Bolivian Nacobbus, we
more anterior position of the phasmids. Lordello et al. conclude that our four Bolivian populations (all of which,
(1961) proposed N. serendipiticus bolivianus Lordello, including one from the type locality, appear identical) are
Zamith & Boock, 1961 from potato (Solanum andigenum conspeciŽ c with N. s. bolivianus. We therefore recognise
Juz. & Buk.) in Cochabamba, Bolivia, the subspecies be- N. bolivianus Lordello, Zamith & Boock, 1961 as a valid
ing characterised in part by the large males. It is also combination at speciŽ c rank (following the Principle of
pertinent to note that an unusual population of Nacob- Coordination, the authority remains the same as for the
bus with characteristics somewhat intermediate between subspecies that was initially proposed). This species is
N. dorsalis and N. aberrans, was collected from spinach currently characterised by its distinctive RFLP proŽ le and
(Spinacia oleracea L.) in Texas by Johnson (1971), al- sequence data, although a morphological redescription
though a formal description was never published. is currently in preparation. Nacobbus bolivianus is a
Sher (1970) synonymised N. batatiformis, N. serendi- widespread species in Bolivia and apparently indigenous.
piticus and N. serendipiticus bolivianus with N. aberrans It also occurs in Peru and, in view of the isolation of
because of a lack of consistent morphologicaldifferences. the Titicaca population that we studied (incidentally the

448 Nematology
 Nacobbus aberrans species complex

highest location yet recorded for Nacobbus), it is probably With these caveats in mind, the genus is currently
a native there also. conceived as follows:
Nacobbus serendipiticus, described by Franklin (1959)
from glasshouse soil in the UK, has been widely regarded T YPE SPECIES
as a synonym of N. aberrans s.l. (Sher, 1970; Stone
& Burrows, 1985; Luc, 1987; Siddiqi, 2000). As our N. dorsalis Thorne & Allen, 1944.
current research on the taxonomy of the genus is based
on molecular characterisation, and we have no access OTHER SPECIES
to DNA of the taxon, we cannot unequivocally decide N. aberrans (Thorne, 1935) Thorne & Allen, 1944
upon its status. The country of origin of this introduced = Anguillulina aberrans Thorne, 1935
population is unknown. If there was any evidence to = Pratylenchus aberrans (Thorne, 1935) Filipjev, 1936
suggest that N. serendipiticus came from Bolivia, then N. bolivianus Lordello, Zamith & Boock, 1961
there would, on current evidence, be a likelihood of it = N. serendipiticusbolivianus Lordello, Zamith & Boock,
being conspeciŽ c with N. bolivianus (all four Bolivian 1961
populationsexamined herein grouped closely together). If
this was the case, serendipiticus, being the senior nomen SPECIES INQUIRENDAE
would take priority over bolivianus. Such a scenario is,
however, speculative. What is clear is that serendipiticus N. batatiformis Thorne & Schuster, 1956
cannot be regarded as a junior synonym under N. aberrans N. serendipiticus Franklin, 1959
s.s. unless and until supporting evidence is obtained.
There being no other acceptable option we therefore Conclusion
regard it as a species inquirenda, rather than as a synonym.
For the time being it is also safer to regard N. batatiformis,
Our work conŽ rms the morphometrical grouping of
also proposed as a synonym under N. aberrans s.l., as a
Nacobbus populations reported by Manzanilla-López et
species inquirenda.
al. (1999) and, by examining many more populations in
It is also important to emphasise that re-collection
greater detail, builds upon the genetic diversity studies
and molecular characterisation of N. dorsalis from the of Ibrahim et al. (1997). We conŽ rm the conclusion of
type locality is essential in order to establish whether N. Ibrahim et al. (1997) that the Peruvian populations are
aberrans s.s. is conspeciŽ c with the type of the genus. distinct from the Mexican and Argentinian populations,
In the absence of con icting data, we refer the N. aber- which in turn display remarkable homogeneity. It is
rans s.l. populationsfrom North America (i.e., those from also clear from our results, however, that there are two
the USA and Mexico) to N. aberrans s.s. This species also distinct groups from Argentina and that the Bolivian
includes the Ecuador 1 and Argentina 1 populations. It is, and Peruvian populations cluster together. The depth of
however, crucial that populations of Nacobbus from the divergence revealed by our studies is strongly supportive
USA, including Utah, the type locality of N. aberrans, are of formal recognition at the species level, although we
characterised by molecular methodologiesin order to con- accept that much work needs to be done to assess possible
Ž rm this action. congruences in phenotype and host range. Additional
The putative new Nacobbus taxon from Urquiza, Ar- studies must be undertaken to determine whether other
gentina is currently being characterised morphologically genotypes exist within the geographic range of Nacobbus.
and morphometrically and a formal description is in Distribution of the various genotypes must be established
preparation (Manzanilla-López, Hunt & Reid, unpubl.). as it is feasible for more than one species of Nacobbus to
In view of the new evidence presented in this paper occur in a particular country or geographic region.
there is a clear requirement to redeŽ ne the status of The literature contains numerous, often contradictory,
all the nominal Nacobbus species. Additional work still statements as to the existence of host races within N. aber-
needs to be done to verify that N. aberrans from the type rans s.l. This confusion has hindered the development of
locality in the USA is conspeciŽ c with other populations control strategies. Resolution of the N. aberrans s.l. com-
currently attributed to this nomen from Mexico, Ecuador plex into discrete taxa by molecular techniques should
and Argentina and to decide whether N. aberrans s.s. and lead to the development of improved, more pragmatic di-
N. dorsalis are conspeciŽ c. agnostics, thereby facilitating a tighter research focus and

Vol. 5(3), 2003 449

A. Reid et al.

enhancing our understanding of the apparently different D O U CET, M.E. (1996). Systematics of Nacobbus in Argentina.
host ranges of the various putative species. When such Nematropica 26, 202.
data have been gathered and co-ordinated we may as- D O U CET, M.E. & D I R IE NZ O , J.A. (1991). El género Nacob-
pire to the development and implementation of a realistic bus Thorne & Allen, 1944 en Argentina. 3. Caracterización
Pan-American management strategy for this remarkable morfológica y morfométrica de poblaciones de N. aberrans
(Thorne, 1935) Thorne & Allen, 1944. Nematropica 21, 19-
genus.
35.
D O U CET, M.E. & G A RDENAL , C.E. (1992). The genus Na-
cobbus in Argentina. 4. Preliminary comparisons of popula-
Acknowledgements tions of N. aberrans (Thorne, 1935) Thorne & Allen, 1944 by
means of isoenzyme phenotypes. Nematropica 22, 243-246.
We would like to thank the following individuals for E L BADRI , G.A.A., D E L EY, P., WA EY E NBER G E , L., V IE R -
their help in supplying or maintaining the nematode cul- STR A ET E , A., M O EN S , M. & VA N FL ET ER E N , J. (2002). In-

tures which made this study feasible: Ignacio Cid del traspeciŽ c variation in Radopholus similis isolates assessed
Prado, Colegio de Postgraduados, Texcoco, Mexico; Car- with restriction fragment length polymorphism and DNA se-
quencing of the internal transcribed spacer region of the ri-
men Triviño, Ecuador; Ivan Bendezú, Peru; John Bridge,
bosomal RNA cistron. International Journal for Parasitology
Paula Nash and Judith Ashurst, CABI Bioscience, Egham,
32, 199-205.
UK; Guillermo Cap, Secretaria de Agricultura, Ganade- F E RRIS , V.R., F E RRIS , J.M. & FAG HIHI , J. (1993). Variations
ria y Pesca, INTA, Argentina; Ernesto Montellano and in spacer ribosomal DNA in some cyst-forming species
Pablo Franco, CIAT Santa Cruz de la Sierra, Bolivia; of plant parasitic nematodes. Fundamental and Applied
Javier Franco and Noel Ortuño, PROINPA, Cochabamba, Nematology 16, 177-184.
Bolivia; Olivia Antezano, LADIPLANTAS CIAT, Co- F IL IP JE V, I.N. (1936). On the classiŽ cation of the Tylenchinae.
marapa, Bolivia; Ken Evans and Janet Rowe, Rothamsted Proceedings of the Helminthological Society of Washington
Research, Harpenden, UK. Rothamsted Research receives 3, 80-82.
grant-aided support from the Biotechnology and Biologi- F RA N KL IN , M.T. (1959). Nacobbus serendipiticusn. sp., a root-
cal Sciences Research Council of the United Kingdom. galling nematode from tomatoes in England. Nematologica 4,
286-293.
G A SCUE L , O. (1997). BIONJ: An improved version of the
NJ algorithm based on a simple model of sequence data.
References
Molecular Biology and Evolution 14, 685-695.
I BRAH IM , S.K., B A LDW IN , J.G., RO BE RTS , P.A. & H Y MA N ,
A LA RCO N , C. & JATA LA , P. (1977). Efecto de la temperatura B.C. (1997). Genetic variation in Nacobbus aberrans: an ap-
en la resistencia de Solanum andigena a Nacobbus aberrans. proach toward taxonomic resolution. Journal of Nematology
Nematropica 7, 2-3. 29, 241-249.
B A LDW IN , J.G. & C A P, G.B. (1992). Systematics of Nacob- I WAHORI , H., T SU DA , K., K A N ZA KI , N., I ZU I , K. & F U TA I ,
bus, the false root-knot nematode. In: Gommers, F.J. & Maas, K. (1998). PCR-RFLP and sequencing analysis of ribosomal
P.W.T. (Eds). Nematology from molecule to ecosystem. Inver- DNA of Bursaphelenchus nematodes related to pine wilt
gowrie, Dundee, Scotland. European Society of Nematolo- disease. Fundamental and Applied Nematology 21, 655-666.
gists, pp. 101-112. JATAL A , P. (1993). Nacobbus aberrans, one species or more?
B OL UART E , T. & JATAL A , P. (1993). Revision of the interna- Nematropica 23, 120.
tional race classiŽ cation scheme for identiŽ cation of physio- JATAL A , P. & B O L UA RTE , T. (1993). Cytogenetics studies of
logical races of Nacobbus aberrans. Nematropica 23, 110. 25 Nacobbus aberrans populations from North and South
D E B RUIJN , N. & S TE M ER D ING , S. (1968). Nacobbus America. Nematropica 23, 120.
serendipiticus, a plant parasitic nematode new to the Nether- JATAL A , P. & G OL D EN , M. (1977). Taxonomic status of Na-
lands. Netherlands Journal of Plant Pathology 74, 227-228. cobbus species attacking potatoes in South America. Nema-
C AN TO -S Á EN Z , M., A RCO S , M.J., JATA L A , P. & H AD DA D , tropica 7, 9-10.
R. (1996). Morphology, biology, and management of Nacob- J OH NS O N , J.D. (1971). The taxonomy and biology of a new
bus aberrans in Peru. Nematropica 26, 197. species of Nacobbus (Hoplolaimidae: Nematoda) found par-
D O UCE T, M.E. (1989). The genus Nacobbus Thorne & Allen, asitizing spinach (Spinacia oleracea L.) in Texas. Ph.D. The-
1944 in Argentina. 1. Study of a population of N. aberrans sis, Graduate College of Texas A&M University, College Sta-
(Thorne, 1935) Thorne & Allen, 1944 on Chenopodium tion, TX, USA, 142 pp.
album L. from Rio Cuarto, Province of Córdoba. Revue de K IR JANOVA , E.S. & L OVA N OVA , N.A. (1975). [A potato
Nématologie 12, 17-26. parasite.] Zashchita Rastenii, Moscow 9, 49.

450 Nematology
 Nacobbus aberrans species complex

L OR DE LL O , L.G.E., Z A MIT H , A.P.L. & B O OCK , O.J. (1961). Q U IM Í , V.H. (1979). Studies on the false root-knot nematode
Two nematodes found attacking potato in Cochabamba, Nacobbus aberrans. Ph.D. Thesis, University of London,
Bolivia. Anais da Academia Brasileira de Ciências 33, 209- Imperial College, UK, 235 pp.
215. R E ID , A. & P O D RUCKA , K. (2003). InterspeciŽ c genetic
L UC , M. (1987). A reappraisal of Tylenchina (Nemata). 7. The diversity of 50 Steinernema species as determined by RFLP
family Pratylenchidae Thorne, 1949. Revue de Nématologie analysis of the ITS region. Nematology, in press.
10, 203-208. S H E R , S.A. (1970). Revision of the genus Nacobbus Thorne and
M A NZ AN IL LA -L ÓPEZ , R.H. (1997). Studies on the characteri- Allen, 1944 (Nematoda: Tylenchoidea). Journal of Nemato-
logy 2, 228-235.
sation and bionomics of Nacobbus aberrans (Thorne, 1935)
Thorne & Allen, 1944 (Nematoda: Pratylenchidae). Ph.D. S ID D IQ I , M.R. (1986). Tylenchida parasites of plants and in-
sects. Farnham Royal, Slough, UK, Commonwealth Insti-
Thesis. University of Reading, UK, 395 pp.
tute of Parasitology/Commonwealth Agricultural Bureaux,
M A NZ AN IL LA -L ÓPEZ , R.H., H A RDING , S. & E VAN S , K.
pp. 305-307.
(1999). Morphometric study on twelve populations of Nacob-
S ID D IQ I , M.R. (2000). Tylenchida parasites of plants and
bus aberrans (Thorne, 1935) Thorne & Allen, 1944 (Nema- insects. Second edition. Wallingford, UK, CABI Publishing,
toda: Pratylenchidae) from Mexico and South America. Ne- pp. 366-369.
matology 1, 477-498. S T E EL E , A.E. (1984). Nematode parasites of sugarbeet. In:
M A NZ AN IL LA -L ÓPEZ , R.H., C O ST ILL A , M.A., D O U CET, Nickle, W.R. (Ed.). Plant and insect nematodes. New York,
M., F RAN CO , J., I N SER RA , R.N., L E HM A N , P.S., C ID D EL USA, Marcel Dekker, pp. 507-569.
P RA DO -V ER A , I., S O UZ A , R.I. & E VAN S , K. (2003). The S T ON E , A.R. & B URROWS , P.R. (1985). Nacobbus aberrans.
genus Nacobbus Thorne & Allen, 1944 (Nematoda: Praty- CIH Descriptions of plant-parasitic nematodes, Set 8, No.
lenchidae): Systematics, distribution, biology and manage- 119, Slough, UK, CAB, 3 pp.
ment. Nematropica 33, in press. S WO FFORD , D.L. (2002). PAUP*. Phylogenetic Analysis Using
M ARTÍN EZ , R., T O RRES , R., T OVAR , A., D E L A JA RA , F. Parsimony (*and other methods), Version 4. Sunderland, MA,
& Z ERÓ N , F. (1995). Reproducción y número cromosómico USA, Sinauer Associates [software].
de dos poblaciones de Nacobbus aberrans (Thorne, 1935) T H O RNE , G.D. (1935). The sugar beet nematode and other in-
Thorne & Allen, 1944. Revista Mexicana de Fitopatología 13, digenous nemic parasites of shadscale. Journal of Agricul-
154. tural Research 51, 509-514.
M AYO RGA , A. & JATA LA , P. (1990). Utilization of polyacry- T H O RNE , G.D. & A LL EN , M.W. (1944). Nacobbus dorsalis,
lamide gel electrophoresisfor detecting differences in protein nov. gen. nov. spec. (Nematoda-Tylenchidae) producing galls
on the roots of alŽ leria, Erodium cicutarium (L.) L’Hér.
patterns of twenty Nacobbus aberrans populations. Nematro-
Proceedings of the Helminthological Society of Washington
pica 20, 11-12.
11, 27-31.
O RT UÑ O , N., O ROS , R., F RAN CO , J. & M A IN , J. (1997).
T H O RNE , G.D. & S CH US T ER , M.L. (1956). Nacobbus batat-
Razas de Nacobbus aberrans que atacan al cultivo de papa iformis n. sp. (Nematoda: Tylenchidae), producing galls on
en Bolivia. Nematropica 27, 118. the roots of sugar beets and other plants. Proceedings of the
O RUI , Y. & M IZ UK U BO , T. (1999). Discrimination of seven Helminthological Society of Washington 23, 128-134.
Pratylenchus species (Nematoda: Pratylenchidae) in Japan by T OL E DO , R.J.C., S OS A -M OS S , C. & Z AVA LE TA -M E JÍA , E.
PCR-RFLP analysis. Applied Entomology and Zoology 34, (1993). Gama de hospederos de cinco poblaciones mexicanas
205-211. de Nacobbus aberrans. Nematropica 23, 105-108.
P RA SA D , S.K. & W E BST E R , J.M. (1967). Effect of tempera- Y IN , K.C. & F EN G , Z.X. (1981). The investigation of plant
ture on the rate of development of Nacobbus serendipiticus in nematodes. Acta Phytophylactica Sinica 8, 122-123.
excised tomato roots. Nematologica 13, 85-90. Z IJ LS T RA , C., U E N K , B.J. & VA N S IL FH O UT, C.H. (1997).
P RA SA D , S.K., K HA N , E. & C H AWLA , L. (1965). New records A reliable, precise method to differentiate species of root-
of nine nematode genera from the Indian Union. Indian knot nematodes in mixtures on the basis of ITS-RFLPs.
Journal of Entomology 27, 360-361. Fundamental and Applied Nematology 20, 59-63.

Vol. 5(3), 2003 451