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Nematology, 2011, Vol. 13(1), 17-28

Molecular and morphometric characterisation of Xiphinema

globosum Sturhan, 1978 (Nematoda: Longidoridae) from Spain

Carolina CANTALAPIEDRA-NAVARRETE,CarlosGUTIÉRREZ-GUTIÉRREZ, BlancaB.LANDA,

Juan E. PALOMARES-RIUS and Pablo CASTILLO ∗

Instituto de Agricultura Sostenible (IAS), Consejo Superior de Investigaciones Científicas (CSIC),

Apdo. 4084, 14080 Córdoba, Spain

Received: 25 January 2010; revised: 11 March 2010

Accepted for publication: 11 March 2010

Summary – During a recent nematode survey in natural environments of the Los Alcornocales Regional Park narrow valleys, viz., the

renowned ‘canutos’ excavated in the mountains that maintain a humid microclimate, in southern Spain, an amphimictic population of

Xiphinema globosum was identified. Morphological and morphometric studies on this population fit the original and previous descriptions

and represent the first report from Spain and southern Europe. Molecular characterisation of X. globosum from Spain using D2-D3

expansion regions of 28S rRNA, 18S rRNA and ITS1-rRNA is provided and maximum likelihood and Bayesian inference analysis were

used to reconstruct phylogenetic relationships within X. globosum and other Xiphinema species. A supertree solution of the different

phylogenetic trees obtained in this study and in other published studies using rDNA genes are presented using the matrix representation

parsimony method (MRP) and the most similar supertree method (MSSA). The results revealed a closer phylogenetic relationship of

X. globosum with X. diversicaudatum, X. bakeri and with some sequences of unidentified Xiphinema spp. deposited in GenBank.

Keywords – Bayesian inference, dagger nematode, matrix representation parsimony (MRP), maximum likelihood, most similar

supertree method (MSSA), new geographic record, rDNA, river bank grapevine, Vitis riparia.

The genus Xiphinema Cobb, 1913 includes a number

of moderate to large ectoparasitic species with long life

cycles. They cause damage to an extensive range of

wild and cultivated plants by their direct feeding on

root cells and by transmitting nepoviruses to a wide

range of fruit and vegetable crops (Taylor & Brown,

1997). Xiphinema comprises more than 250 valid species

(Coomans et al., 2001; Decraemer & Robbins, 2007) of

which nine are vectors of nepoviruses (Taylor & Brown,

1997; Decraemer & Robbins, 2007). The occurrence and

geographical distribution of Xiphinema spp. in the Iberian

Peninsula was reviewed by Peña Santiago et al. (2003)

who reported 49 species.

During a recent nematode survey conducted in natural

environments in southern Spain, an amphimictic population

of Xiphinema species, characterised by a rounded

tail and a uterus with pseudo-Z-organ differentiation, appeared

to be morphologically related to Xiphinema globosum

Sturhan, 1978, a fact that prompted us to undertake a

detailed morphological and molecular comparative study

with previous reported data, as well as with type speci-

∗ Corresponding author, e-mail: pcastillo@ias.csic.es

mens (female paratypes). The nematode was associated

with a characteristic natural environment of the Los Alcornocales

Regional Park, Alcalá de los Gazules, Cádiz

Province, southern Spain, viz., the renowned ‘canutos’,

narrow valleys excavated in the mountains that maintain

a humid microclimate favouring the survival of botanical

species unique in Europe and dating back to the Tertiary

Age (Carrión et al., 2003). This amphimictic species was

originally described from a forest soil in a nature reserve

in Bavaria, and from the rhizosphere of several herbaceous

plants (Angelica sp., Aegopodium sp., Asarum sp.,

Daphne sp., Berberis sp.) in Germany (Sturhan, 1978).

The species has been also reported in Slovenia (Barsi,

1992) and in the rhizosphere of grapevine and wild plants

in northern Italy (Roca & Lamberti, 1994).

The accurate identification of Xiphinema nematodes

infesting cultivated or natural soils is a prerequisite for

quarantine measures or for designing effective management

strategies and also for ecological and biogeographical

studies. The availability of molecular techniques provides

additional tools to differentiate species, and these

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/138855410X500046

Also available online - www.brill.nl/nemy 17


C. Cantalapiedra-Navarrete et al.

have significantly improved and facilitated the routine

identification of these nematodes. In addition, molecular

approaches using ribosomal DNA (rDNA) sequences

from partial 18S, ITS regions and the D2 and D3 expansion

segments of the 28S have been shown to be a useful

diagnostic tool in the characterisation and phylogenetic

relationships within plant-parasitic nematodes such

as Longidoridae, especially in cases where morphological

characters may lead to ambiguous interpretation (Ye et al.,

2004; He et al., 2005; Barsi & De Luca, 2008; Pedram et

al., 2009).

The objectives of this work were: i) to characterise morphologically

and morphometrically the Spanish population

of X. globosum and compare with previous descriptions;

ii) to characterise molecularly the Spanish population

using the D2-D3 28S rRNA, ITS1 and partial 18S

rRNA gene sequences; and iii) to study their phylogenetic

relationships with other Xiphinema species.

Materials and methods

NEMATODE POPULATIONS

The Spanish population of X. globosum was collected

in sandy soil around the roots of black alder, Alnus glutinosa

(L.) Gaertn., and river bank grapevine, Vitis riparia

Michx, in the canuto of Valdeinfierno in the Los Alcornocales

Regional Park, Alcalá de los Gazules, Cádiz

Province, southern Spain. The nematodes used in this

study were extracted from soil by centrifugal flotation

(Coolen, 1979) and by a modification of Cobb’s decanting

and sieving (Flegg, 1967) methods. Specimens for light

microscopy were killed by gentle heat, fixed in a solution

of 4% formaldehyde + 1% propionic acid and processed

to pure glycerin using Seinhorst’s (1966) method. Specimens

were examined using a Zeiss III compound microscope

with Nomarski differential interference contrast at

powers up to 1000× magnification. Measurements were

done using a drawing tube. Morphometric data were

processed using Statistix 9.0 (NH Analytical Software,

Roseville, MN, USA). Nematodes were compared with

type specimens (female paratype, slide T-2088p) kindly

provided by Dr Z. Handoo, from the USDA Nematode

Collection (Beltsville, MD, USA).

DNA EXTRACTION, PCRAND SEQUENCING

Nematode DNA was extracted from single individuals

and protocols for PCR as described by Castillo et al.

(2003) and Tanha Maafi et al. (2003) were followed. The

D2-D3 expansion segments of 28S rDNA were amplified

using the forward D2A (5 ′ -ACAAGTACCGTGAGGG

AAAGTTG-3 ′ ) and reverse D3B (5 ′ -TCGGAAGGAACC

AGCTACTA-3 ′ ) primers (Castillo et al., 2003; He et al.,

2005; Palomares-Rius et al., 2008). The forward primer

S-ITS (5 ′ -TTGATTACGTCCCTGCCCTTT-3 ′ ) and the

reverse primer A-ITS1 (5 ′ -ACGAGCCGAGTGATCCA

CCGATAAG-3 ′ ) were used for amplification of ITS1rDNA

region (Wang et al., 2002). The partial 18S rDNA

was amplified using forward primer SSU_F_04 (5 ′ -GCTT

GTCTCAAAGATTAAGCC-3 ′ ) and the reverse primer

SSU_R_81 (5 ′ -TGATCCWKCYGCAGGTTCAC-3 ′ )

(Griffiths et al., 2006). PCR products were purified

with a gel extraction kit (AccuPrep ® Gel Purification

Kit, Bioneer, Munpyeong-Dong, South Korea) according

to the manufacturer’s instructions, quantified using

a Nanodrop spectrophotometer (Nanodrop Technologies,

Wilmington, DE, USA) and used for direct DNA sequencing.

For the 18S gene the internal primer SSU_R_13 (5 ′ -

GGGCATCACAGACCTGTTA-3) (sequence available

online at http://www.nematodes.org/barcoding/sourhope/

nemoprimers.html) was also used. DNA fragments from

two independent PCR amplifications from two different

nematodes were sequenced in both directions using the

same primers with a terminator cycle sequencing ready

reaction kit (BigDye; Perkin-Elmer Applied Biosystems,

Warrington, UK) according to the manufacturer’s instructions.

The resulting products were purified and run on

a DNA multicapillary sequencer (Model 3130XL genetic

analyser; Applied Biosystems, Foster City, CA,

USA) at the Stabvida sequencing facilities (Oeiras, Portugal).

Sequences were deposited in GenBank under accession

numbers GU549474 for D2-D3 expansion segments

of 28S rDNA, GU549475 for the ITS1-rDNA and

GU549476 for partial 18S rDNA regions.

PHYLOGENETIC ANALYSES

D2-D3 expansion segments of 28S, ITS1 and partial

18S rDNA sequences of different Xiphinema spp. from

GenBank were used for phylogenetic reconstruction. Outgroup

taxa for each dataset were chosen according to previous

published data (Pedram et al., 2009). The newly

obtained and published sequences for each gene were

aligned using ClustalW (Thompson et al., 1994) with

default parameters. Sequence alignments were manually

edited using BioEdit (Hall, 1999). Phylogenetic analysis

of the sequence data sets was performed with maximum

likelihood (ML) using PAUP* 4b10 (Swofford, 2003) and

Bayesian inference (BI) using MrBayes 3.1.2 (Huelsen-

18 Nematology


eck & Ronquist, 2001). The best fit model of DNA evolution

was obtained using the program ModelTest server

(Posada, 2006) with the Akaike Information Criterion in

conjunction with PAUP*. The Akaike-supported model,

the base frequency, the proportion of invariable sites, and

the gamma distribution shape parameters and substitution

rates in the Akaike information criterion (AIC) were used

in phylogenetic analyses. BI analysis under GTR + I + G

model for each gene was initiated with a random starting

tree and was run with four chains for 2.0 × 10 6 generations.

The Markov chains were sampled at intervals of

100 generations. Two runs were performed for each analysis.

After discarding burn-in samples and evaluating convergence,

the remaining samples were retained for further

analysis. The topologies were used to generate a 50% majority

rule consensus tree. Posterior probabilities (PP) are

given on appropriate clades. Trees were visualised using

TreeView program (Page, 1996). In ML analysis the estimation

of the support for each node was made using a

bootstrap analysis with 1000 fast-step replicates.

To compare the phylogenetic trees representing the different

rDNA genes phylogenies of Xiphinema species related

to X. globosum in a global analysis using supertree

reconstruction phylogenies. We used matrix representation

parsimony (MRP) (Baum, 1992; Ragan, 1992) with

the matrix generated by Rainbow (Chen et al., 2004) and

implemented in PAUP* (Swofford, 2003) and the most

similar supertree method (MSSA) or distance fit (dfit)

(Creevey et al., 2004) as implemented in CLANN ver.

3.0.0 (Creevey & McInerney, 2005). The search for supertree

using the MRP matrix was performed using the

heuristic search with stepwise-addition options with tree

bisection and reconnection. The search for supertree using

the MSSA method was performed using the heuristic

search of supertree space for the best tree using subtree

pruning and regrafting method without input trees normalisation.

No bootstrap analysis of the data was performed

under MRP and MSSA methods because of the low occurrence

of some taxa in the scarce source of trees (n = 10).

Yet Another Permutation Tail Test (YAPTP) has been conducted

in CLANN ver. 3.0.0 implemented with PAUP*

(Swofford, 2003), in order to test the null hypothesis that

the phylogenetic signal in the tree was no better than random.

Results

Xiphinema globosum Sturhan, 1978

(Figs 1-3)

Molecular and morphometric characterisation of Xiphinema globosum

MEASUREMENTS

See Table 1.

DESCRIPTION

Female

Body cylindrical, slightly narrowing at anterior region.

Habitus ventrally curved, usually in an open C-shape

when relaxed by gentle heating. Cuticle smooth, 3.5-

5.0 μm thick at mid-body, 10-14 μm on tail tip. Lateral

chords one fifth to one-fourth of body diam. Five

to six body pores present in dorsal and ventral regions

in odontostyle region. Lip region broadly rounded, 4.5-

6.0 μm high, marked by a shallow depression. Amphidial

slit large, 9-10 μm wide, located on lip region, amphidial

fovea stirrup shaped. Odontostyle, odontophore and guiding

ring typical of genus. Pharynx dorylaimoid with basal

bulb occupying ca one-seventh of total length, basal bulb

cylindrical, measuring 95 (87-114) μm long × 29 (27-

33) μm diam. Nerve ring located at 215-316 μm from

anterior end. Dorsal pharyngeal gland nucleus in anterior

part of bulb, one pair of subventral nuclei near middle of

bulb. Gland nuclei (calculated as described by Loof and

Coomans, 1972) were positioned as follows: DN, 11.7%,

DO, 9.6%, RS1N, 53.1%, RS1O, 50.1%, LS1N, 53.2%,

and LS1O, 48.8%. Pharyngo-intestinal valve rather small,

rounded, surrounded by intestinal tissue. Reproductive

system with two equally developed genital branches. Genital

branches composed of a long ovary, a large tubular

oviduct with enlarged pars dilatata oviduct separated

from uterus by a well developed sphincter. Uterus tripartite,

filled with oval sperm, with well developed glandular

pars dilatata uteri, continuing in a narrower, muscular

tube-like portion with a pseudo-Z-organ with four

or five weakly sclerotised, irregularly shaped, bodies of

various sizes, but without angular apophyses. Vulva located

slightly anterior to mid-body, vagina perpendicular

to body axis, large, well developed, clearly offset ovejector

occupying almost complete body diam. at vulval

level. Prerectum 549 (427-671) μm or 14.1 (12.9-15.9)

anal body diam. long. Tail short, convex-conoid to hemispherical,

terminus broadly rounded, with two or three

pairs of caudal papillae, two blind canals in some specimens.

Male

Common, almost as abundant as female. Similar to

female except for reproductive system. Body ventrally

arcuate, more strongly curved in posterior region due to

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C. Cantalapiedra-Navarrete et al.

Fig. 1. Light micrographs of Xiphinema globosum Sturhan, 1978 from Spain. A: Female neck region; B: Anterior region; C: Vulval

region showing ovejector. D, E: Detail of Z-organ; F: Female tail; G: Male tail; H: Detail of spicules; I: Anterior region of first-stage

juvenile showing replacement and functional odontostyle; J-M: Tails of first-, second-, third- and fourth-stage juveniles. Abbreviations:

a = anus; gr = guiding ring; odt = odontophore; ost = odontostyle; ovj = ovejector; pdu = pars dilatata uteri; rost = replacement

odontostyle; sp = spicules; V = vulva; Z = pseudo-Z-organ. (Scale bars: A, G = 50 μm; B-F, H-M = 20 μm.)

20 Nematology


Molecular and morphometric characterisation of Xiphinema globosum

Fig. 2. Light micrographs of female paratypes of Xiphinema globosum Sturhan, 1978. A: Female anterior region; B: Lip region;

C: Detail of basal bulb; D: Vulval region showing ovejector; E, F: Detail of Z-organ; G: Female tail. Abbreviations: a = anus;

gr = guiding ring; odt = odontophore; ost = odontostyle; ovj = ovejector; pdu = pars dilatata uteri; rost = replacement odontostyle;

sp = spicules; V = vulva; Z = pseudo-Z-organ. (Scale bars: A = 50 μm; B-E, G = 20 μm; F = 10 μm.)

Vol. 13(1), 2011 21


C. Cantalapiedra-Navarrete et al.

Fig. 3. Relationship of body length to length of functional and

replacement odontostyle (ost and rost, respectively) length in all

developmental stages from J1 to mature female of Xiphinema

globosum Sturhan, 1978 from Spain.

well developed copulatory muscles. Four, usually five,

ventromedian precloacal papillae anterior to adanal pair,

equally spaced. Spicules well sclerotised, massive, lateral

accessory pieces somewhat straight or slightly ventrally

curved. Copulatory muscles and spicule protractor and

retractor muscles well developed. Sperm cells oval 5.5

(4-7) μm long. Tail convex-conoid to a hemispherical

terminus, similar to that of female.

Juveniles

All four juvenile stages distinguishable by relative

lengths of body and functional and replacement odontostyles.

First-stage juveniles (J1) clearly recognisable

by position of replacement odontostyle located within

base of odontophore. Relationship of body length, functional

and replacement odontostyle (ost and rost, respectively)

in J1-J4 and mature females is shown in Figure 3.

Table 1. Morphometrics of Xiphinema globosum from Spain. All measurements are in μm and in the form: mean ± s.d. (range).

Character Females Males J1 J2 J3 J4

n 10 6 4 3 4 6

L 2978 ± 184 2876 ± 300 851 ± 51 1157 ± 123 1596 ± 43 2236 ± 166

(2710-3210) (2645-3460) (810-920) (1045-1290) (1545-1645) (2010-2430)

a 48.8 ± 2.9 54.4 ± 6.5 35.5 ± 0.2 39.8 ± 2.9 44.2 ± 2.7 48.0 ± 3.9

(43.3-53.1) (45.5-64.9) (35.3-35.7) (37.3-42.9) (41.7-48.0) (42.8-52.9)

b 7.1± 1.1 6.8± 0.7 4.1± 0.7 4.1± 0.7 4.0± 0.4 5.4± 0.7

(5.7-9.2) (5.7-7.5) (3.6-5.1) (3.6-5.0) (3.6-4.5) (4.2-6.0)

c 90.0 ± 12.6 86.4 ± 6.8 13.2 ± 0.7 21.7 ± 0.9 43.6 ± 4.7 60.8 ± 3.4

(73.7-109.6) (78.6-98.9) (12.3-14.0) (20.7-22.3) (40.1-49.0) (56.1-64.7)

c ′ 0.9 ± 0.08 0.8 ± 0.05 3.9 ± 0.6 2.6± 0.1 1.3± 0.1 1.0± 0.1

(0.7-1.0) (0.8-0.9) (3.2-4.7) (2.5-2.8) (1.2-1.4) (0.9-1.1)

V or T 43.4 ± 1.8 59.5 ± 6.3 – – – –

(41-47) (49-68)

Odontostyle length 138 ± 4.4 140 ± 3.1 50± 1.7 63± 1.0 95± 4.3 114 ± 3.8

(128-143) (137-145) (48-52) (62-64) (89-99) (111-121)

Odontophore length 81 ± 4.5 79± 2.0 37± 1.7 47± 1.1 63± 5.7 71± 1.6

(73-87) (77-83) (35-38) (46-48) (57-69) (69-73)

Replacement odontostyle length – – 66 ± 3.6 89± 2.3 115 ± 1.9 141 ± 3.7

(61-69) (88-92) (112-116) (135-146)

Lipregiondiam. 14.5± 0.8 13.9 ± 0.9 – – – –

(13.5-16.0) (13-15)

Oral aperture-guiding ring 122 ± 11.9 127 ± 6.7 45± 2.8 52± 3.5 78± 9.5 95± 10.6

(102-139) (117-136) (43-47) (49-56) (70-87) (80-104)

Tail length 35.5 ± 4.1 33.3 ± 1.3 65± 4.1 53± 5.7 37± 4.0 37± 2.4

(29-42) (31-35) (61-70) (47-58) (33-41) (33-39)

Spicules 75 ± 4.1 – – – –

(70-80)

Lateral accessory piece 20.2 ± 1.3 – – – –

(18-21)

22 Nematology


J1 tail long with rounder terminus, J2 tail short, with

more or less subdigitate terminus, J3 and J4 with similar

tails, i.e., short, convex-conoid with rounded terminus.

REMARKS

The Spanish population of X. globosum agrees well

with the original description by Sturhan (1978) and the

studied paratypes (Fig. 2), as well as that from Slovenia

(Barsi, 1992). Nevertheless, some minor differences in

ratios and measures were noticed, i.e., odontostyle and

odontophore length, slightly smaller a and c ratios; but

slightly larger V and c ′ ratios, all of which may be

considered as intraspecific variability. The morphological

identification of Xiphinema species is mainly based on

a polytomous key (Loof & Luc, 1990, 1993) and, due

to the large number of nominal species within this

genus, overlapping of code characters in some species is

unavoidable. The alpha-numeric codes for X. globosum

to be applied to the polytomic identification key for

Xiphinema species by Loof and Luc (1990) are: A 4, B

2, C 67, D 6, E 345, F 34, G 3, H 2, I 23, J 67, K 2,

L2.

The present record of X. globosum is the first from

Spain and southern Europe and the third in Europe after

those from Germany (Sturhan, 1978), Slovenia (Barsi,

1992), and Italy (Roca & Lamberti, 1994). The current

geographical distribution of X. globosum indicates that it

may be mostly associated with natural environments in

northern regions. However, this new record extends the

geographical distribution of this species and may be considered

as an example of a relict species because of its

natural association with the characteristic canutos vegetation

in Los Alcornocales Regional Park that maintains

a humid microclimate favouring the survival of botanical

species unique in Europe, and which may be explained

as a consequence of a glacial refuge for several plant

species (Carrión et al., 2003). In this regard, Coomans

et al. (2001) also suggested that recent glaciations events

(Würm III) had an effect on the distribution and the incipient

speciation of many taxa in Europe, as already recognised

by the refuge of Ibero-Maghreb in the West and the

Ponto-Caspian in the East (Nilson, 1983). These suggestions

agree with the results obtained for the phylogeny and

biogeography of Longidorus in the Euromediterranean region

(Navas et al., 1993), in which a dispersalist model is

one of the primary explanations for the large groups of

Longidorus species found in this area.

Molecular and morphometric characterisation of Xiphinema globosum

PHYLOGENETIC RELATIONSHIPS OF XIPHINEMA

GLOBOSUM STURHAN, 1978 WITH OTHER XIPHINEMA

SPECIES

The amplification of D2-D3 expansion segments of

28S rDNA, partial 18S and ITS1 region yielded a single

fragment of ca 800 bp, 1600 bp and 1030 bp, respectively,

based on gel electrophoresis. BLAST search

from the NCBI showed a close relationship with several

Xiphinema spp. which were used for further phylogenetic

studies. Pairwise comparisons of the ITS1 sequence

of X. globosum with those of Xiphinema spp. from

the GenBank database displayed a high nucleotide dissimilarity

and considerable variation in length. The closest

species were X. diversicaudatum (Micoletzky, 1927)

Thorne, 1939 (AJ437027) (274 bp differences, 77.2%

similarity), X. bakeri Williams, 1961 (AF511427) (289

bp differences, 76.0% similarity) and X. diversicaudatum

(AY430183) (309 bp differences, 74.1% similarity). Difficulties

were experienced while aligning the ITS1 region

and consequently species with a close relationship were

only included in the phylogenetic analysis. Figure 4A

represents the position of X. globosum (GU549475) with

other Xiphinema species based on ITS1 rDNA sequences

of a multiple edited alignment of 1314 total characters.

The phylogenetic tree has resolved three major clades: i)

X. globosum and related species; ii) X. aceri Chizhov, Tiev

& Turkina, 1986 group; and iii) X. hunaniense Wang &

Wu, 1992 with X. chambersi Thorne, 1939. These clades

were also resolved in other studies that included the X.

americanum-group (Ye et al., 2004; Pedram et al., 2009).

The position of X. globosum is not well defined with some

of the X. diversicaudatum sequences (AY430183), while

the clade comprising both sequences and those from X.

diversicaudatum (AJ437027) with X. bakeri are well supported

with a good posterior probabilities (97) and lower

bootstrap values (61) (Fig. 4A). The position of X. diversicaudatum

is difficult to determine due to discrepancies

in the sequences available in GenBank, possibly due to

misidentification of some specimens.

D2-D3 expansion segments of 28S of X. globosum

(GU549474) analysis showed a similar species relationship

to that obtained for ITS1 (Fig. 4B). A BLAST search

showed similarity with some Xiphinema spp., particularly

with an undetermined Xiphinema sp. (DQ364685), X. diversicaudatum

(EF538755) and X. bakeri (AY601623),

with 29 bp differences and 96% similarity in all cases.

Figure 4B represents the position of X. globosum

(GU549475) with other Xiphinema species based on D2-

D3 region of 28S of a multiple edited alignment of 752

Vol. 13(1), 2011 23


C. Cantalapiedra-Navarrete et al.

Fig. 4. Phylogenetic relationships within some Xiphinema spp. Bayesian 50% majority rule consensus trees as inferred from (A) ITS1 (B) D2 and D3 expansion

segments of 28S rRNA and (C) 18S rRNA gene sequence alignments under the GTR + I + G model. Posterior probabilities more than 65% are given for appropriate

clades (in bold letters); bootstrap values greater than 50% are given on appropriate clades in ML analysis. Newly obtained sequences of Xiphinema globosum are

indicated in bold. ∗Populations identified on the basis of general morphology (He et al., 2005).

24 Nematology


total characters. The phylogenetic tree resolved two major

clades: i) X. globosum and related species; and ii) X. radicicola

and the rest of the species. The X. globosum clade

enclosed an unidentified Xiphinema sp. (DQ364685), X.

diversicaudatum (EF538755) and X. bakeri (AY601623)

which is not well resolved at this level with BI and ML.

However, the upper clade involving another X. diversicaudatum

sequence (AY601624) is well resolved by BI.

These results confirm the close relationship with X. diversicaudatum

and X. bakeri. This tree showed a similar

topology to those obtained by He et al. (2005) and Pedram

et al. (2009).

Partial 18S region analysis resolved a similar phylogenetic

relationship as those obtained with ITS1 and D2-D3

region of 28S. A BLAST search showed high similarity

with X. diversicaudatum (EF538761) (11 bp, 99% similarity),

Xiphinema sp. (EF207250) (14 bp, 99% similarity)

and X. bakeri (15 bp, 99% similarity). Figure 4C represents

the position of X. globosum (GU549476) with other

Xiphinema species based on partial 18S gene of a multiple

edited alignment of 1672 total characters. The phylogenetic

tree resolves two major clades: i) main clade including

the majority of species and X. globosum; and ii) clade

with X. variegatum Siddiqi, 2000, X. krugi Lordello, 1955

and X. longicaudatum Luc, 1961. The clades and subclades

with partial 18S revealed a similar phylogenetic

relationship to the other rRNA genes studied, confirming

the relationship of X. globosum with X. diversicaudatum,

X. bakeri and X. index Thorne & Allen, 1950. Xiphinema

species showed a small variability in this region in regard

to other rDNA regions and this gene has been considered

inconclusive in the X. americanum-group (Oliveira

et al., 2004). Nonetheless, the partial 18S tree is mainly

congruent with Pedram et al. (2009) and Oliveira et al.,

(2004).

The phylogenetic trees obtained using the D2-D3, ITS1

and partial 18S regions are congruent with other studies

in which similar groupings are obtained for the D2-D3 region

of 28S using secondary structures (He et al., 2005),

ITS1 (Ye et al., 2004; Pedram et al., 2009) and partial 18S

(Oliveira et al., 2004; Pedram et al., 2009). These studies

showed two distinct major groups within Xiphinema,

differentiating the X. americanum-group from the rest of

the species. He et al. (2005) also determined the monophyly

of the genus Xiphinema even though it was split

into two major clades. In our study, excluding X. americanum-group,

X. globosum was related to X. diversicaudatum,

X. bakeri and several unidentified Xiphinema

species (EU375484, DQ364685-DQ364686, EF207250

Molecular and morphometric characterisation of Xiphinema globosum

and AY397824). The position of some species is difficult

to assign with the sequence data deposited in Gen-

Bank. Such is the case for X. diversicaudatum, in which

only two sequences are available from the same population

from Slovakia (EF538755 and EF538761), while the

other populations are from different European countries

(France and Portugal). These sequences generated different

positions in the phylogenetic trees obtained in this

study. This phenomenon is a good example demonstrating

the difficulties for species identification in this complex

genus due to character overlap (He et al., 2005) and may

also indicate the presence of cryptic species (Oliveira et

al., 2006). This demands special attention as some species

are virus vectors (i.e., X. diversicaudatum), a fact that may

have critical phytopathological implications. Our results

confirm that X. vulgare Tarjan, 1964 is a junior synonym

of X. setariae Luc, 1958 (Lamberti et al., 1995; Luc &

Baujard, 1996) since both showed identical sequences for

D2-D3 region of 28S, although the partial 18S sequence

deposited for the Xiphinema setariae/vulgare complex

(Xiphinema sp. RN-2003-AY297840) generated a different

position in the tree and close to the X. globosum clade.

The X. setariae/vulgare complex has shown differences in

partial 18S sequences between specimens (Oliveira et al.,

2004) and a more exhaustive sampling and individual nematode

sequencing will probably be necessary to assess

the phylogenetic position and species definition in this

complex. Morphologically and morphometrically similar

species such as X. dentatum Sturhan, 1978 grouped in the

same main clade with X. globosum (Fig. 4B), while others

species with quite different morphology (i.e., tail shape

with mucro in X. index or X. diversicaudatum, or the presence

of spines in the uterus of X. pyrenaicum Dalmasso,

1969) also clustered together.

SUPERTREE ANALYSIS OF XIPHINEMA SPECIES

RELATED TO X. GLOBOSUM

Many plant-parasitic nematode species are not well

studied molecularly and some have only partial sequence

information, a problem when reconstructing phylogeny

based on DNA sequences (Adams et al., 2009). One

solution to this problem is to generate a supertree –

an evolutionary tree that is assembled from a group of

smaller trees that share some, but not necessarily all,

taxa present in each single tree (Bininda-Emonds et al.,

2004; Adams et al., 2009). This approach has been used

for the study of Trichinella spp. (Pozio et al., 2009) and

Meloidogyne spp. (Adams et al., 2009).

Vol. 13(1), 2011 25


C. Cantalapiedra-Navarrete et al.

Fig. 5. Supertree solutions for trees obtained in this study and from other phylogenetic studies. A: MRP (matrix representation of

parsimony) 50% majority rule consensus tree; B: MSSA-Dfit (most similar supertree method, or distance fit) 50% majority rule

consensus tree. ∗ Xiphinema diversicaudatum sequences showing similar positions in trees (Fig. 4); 1 ∗ other X. diversicaudatum

sequences available in GenBank.

In our study, the combination of our ribosomal gene

analysis (partial 18S, D2-D3 expansion segments of 28S

and ITS1) by BI and ML, as well as the ribosomal gene

analysis from other studies (Ye et al., 2004; He et al.,

2005; Pedram et al., 2009) were used to construct a supertree

of the phylogenetic relationships in Xiphinema

species related to X. globosum using the matrix representation

of MRP and MSSA methods (Fig. 5). Both MRP

and MSSA methodologies yielded multiple, equally parsimonious,

trees. However, the phylogenetic signal was

significantly better than random using the YAPTP (P <

0.01). Comparing the input trees with the resulting tree,

MRP was more conservative following the major clades

observed in the rest of trees, while MSSA maintained the

majority of clades, although some species were not well

resolved and showed an ambiguous position in the tree

(Fig. 5). Additional trees will need to be combined to assess

a better confidence level in MSSA, which is mainly

based in comparing each source tree separately to the supertree

and scoring it. Also, the introduction of taxa represented

more than once in the trees will allow bootstrap

analysis in order to assess the confidence level of the generated

tree. The most important results with this analysis

are the maintenance of several main clades obtained

by single phylogenetic tree analysis: i) X. globosum and

all the genetically-related species are maintained in the

supertree by both methods, but different positions were

observed depending on the method (Fig. 5A, B); ii) X.

26 Nematology


setariae and related species form a clade, occupying different

tree position (Fig. 5A, B); iii) X. aceri and related

species, showed a similar clade using both methods, but

some species were included or excluded from that clade

depending on the method, i.e., X. brasiliense and X. ensiculiferum

(Fig. 5A, B); and iv) the remaining Xiphinema

species showed different positions in the tree (Fig. 5A, B).

Xiphinema diversicaudatum had different positions in the

ITS1 and D2-D3 phylogenetic trees (Fig. 4A, B). In order

to resolve this incongruence, X. diversicaudatum sequences

showing similar positions in trees (Fig. 4) were

considered as a single taxon (Fig. 5), whereas the remaining

sequences were considered as different taxa (Fig. 5).

Supertree solution produced a better view from the phylogeny

of X. globosum-related species. Poorly represented

species with few sequences, or species that are not well

defined, need more sequences and additional phylogenetic

analysis to clarify their position.

Acknowledgement

The authors thank F.J. Durán Gutiérrez, G. León

Ropero and J. Martín Barbarroja from IAS-CSIC for their

excellent technical assistance.

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28 Nematology

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