Rather than by direct acquisition via lateral gene transfer, GHF5 cellulases were passed on from early Pratylenchidae to root-knot and cyst nematodes

Article (PDF Available)inBMC Evolutionary Biology 12(1):221 · November 2012with52 Reads
DOI: 10.1186/1471-2148-12-221 · Source: PubMed
Abstract
Background Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5 cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less distal family members were collected and analyzed. Results Single nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of ≈ 100 cellulase sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis further suggested a sister relationship between the lesion nematode Pratylenchus thornei and all type B cellulases from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear. Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct from type A that is characterized by two unique introns. Conclusions All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of catalytic domains are distinguished, and the presence of at least two types is relatively common among plant parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family nowadays known as the Pratylenchidae.

Figures

RES E AR C H A R T I C L E Open Access
Rather than by direct acquisition via lateral gene
transfer, GHF5 cellulases were passed on from
early Pratylenchidae to root-knot and cyst
nematodes
Katarzyna Rybarczyk-Mydłowska
1*
, Hazel Ruvimbo Maboreke
2
, Hanny van Megen
1
, Sven van den Elsen
1
,
Paul Mooyman
1
, Geert Smant
1
, Jaap Bakker
1
and Johannes Helder
1*
Abstract
Background: Plant parasitic nematodes are unusual Metazoans as they are equipped with genes that allow for
symbiont-independent degradation of plant cell walls. Among the cell wall-degrading enzymes, glycoside hydrolase
family 5 (GHF5) cellulases are relatively well characterized, especially for high impact parasites such as root-knot and
cyst nematodes. Interestingly, ancestors of extant nematodes most likely acquired these GHF5 cellulases from a
prokaryote donor by one or multiple lateral gene transfer events. To obtain insight into the origin of GHF5
cellulases among evolutionary advanced members of the order Tylenchida, cellulase biodiversity data from less
distal family members were collected and analyzed.
Results: Single nematodes were used to obtain (partial) genomic sequences of cellulases from representatives of
the genera Meloidogyne, Pratylenchus, Hirschmanniella and Globodera. Combined Bayesian analysis of 100 cellulase
sequences revealed three types of catalytic domains (A, B, and C). Represented by 84 sequences, type B is
numerically dominant, and the overall topology of the catalytic domain type shows remarkable resemblance with
trees based on neutral (= pathogenicity-unrelated) small subunit ribosomal DNA sequences. Bayesian analysis
further suggested a sister relationship between the lesion nemat ode Pratylenchus thornei and all type B cellulases
from root-knot nematodes. Yet, the relationship between the three catalytic domain types remained unclear.
Superposition of intron data onto the cellulase tree suggests that types B and C are related, and together distinct
from type A that is characterized by two unique introns.
Conclusions: All Tylenchida members investigated here harbored one or multiple GHF5 cellulases. Three types of
catalytic domains are distinguished, and the presence of at least two types is relatively common among plant
parasitic Tylenchida. Analysis of coding sequences of cellulases suggests that root-knot and cyst nematodes did not
acquire this gene directly by lateral genes transfer. More likely, these genes were passed on by ancestors of a family
nowadays known as the Pratylenchidae.
Keywords: Lateral gene transfer, Cellulase, Nematodes, Plant parasitism
* Correspondence: Kasia.Rybarczyk@wur.nl; Hans.Helder@wur.nl
1
Laboratory of Nematology, Department of Plant Sciences, Wageningen
University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands
Full list of author information is available at the end of the article
© 2012 Rybarczyk-Mydłowska et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of
the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221
http://www.biomedcentral.com/1471-2148/12/221
Background
Any movement of genetic information, ot her than by
vertical transmission from parents to their offsprin g via
conventional reproduction, is defined as horizontal or
lateral gene transfer (HGT or LGT). Although LGT
occurs frequently among members of the Archaea and
Bacteria, there are only a few probable cases of LGT be-
tween prokaryotes and multicellular eukaryotes that
have resulted in new functional genes for the recipient.
Likely cases of LGT in which the eukaryote is acting as a
donor have been described for two mosquito species,
Aedes aegypti and An opheles gambiae [1]. The transfer
of a gene related to malaria sporozoite invasion from
mosquito to its endosymbiotic bacterium Wolbachia
pipientis was demonstrated by Woolfit et al. (2008, [1]).
This gene show ed substantial divergence, and the level
of expression suggested it to be functional in the new
prokaryote host. Inter-domain gene transfers can also
happen in the reverse way. The pea aphid Acyrthisiphon
pisum probably acquired two genes from bacteria by
LGT [2]. These laterally transferred genes are expressed
in the bacteriocytes, and they con tribute to the mainten-
ance of Buchnera aphidicola, the aphids primary sym-
biont. Donors from multiple domains (bacteria , fungi
and plants) are thought to be implicated in the acquisi-
tion of at least ten protein-coding sequences by the bdel-
loid rotifer Adineta vaga [3]. A subset of these genes
were transcribed and correctly spliced. Interestingly, the
authors hypothesized that LGT could be facilitated by
mechanisms underlying the desiccation tolerance of this
rotifer.
The lateral gene transfer of prokaryotic genes has pre-
sumably also played a key role in the evolu tion of plant
parasitism in nematodes. Plant cells are protected by a
cell wall, and penetration of this wall is a prerequisite to
reach the cytosol. Potato and a soybean cyst nematode
(Globodera rostochiensis and Heterodera glycines) were
the first animals shown to harbor symbiont-independent
cellulases [4]. These cellulases are classified as members
of the glycoside hydrolase family 5 (GHF5). The nema-
tode cellulases appeared to be most similar to bacterial
cellulases. In an editorial comment Keen and Roberts [5]
suggested that lateral gene transfer may drive the mobil-
ity of pathogenicity islands (including cellulases) from
one organism to the other. Over the last decade, plant
parasitic nematodes were shown to harbor a wide
spectrum of cell wall-degrading proteins such as pectate
lyases [6], polygalacturonase [7], xylanases [8] and
expansins [ 9]. These genes are expressed during infective
life stages, and contribute to nematodes ability to exploit
plants as a food source.
Bacterivory is generally accepted as the ancestral
feeding type of nematodes. A longstanding hypthesis
suggests that bacterivores gave rise to fungivorous
nematodes, and facu ltative and obligatory plant parasites
arose from fungal feeding ancestors [10]. It is conceivable
that the evolution of plant parasitism in nematodes was
driven by the lateral transfer of genes via ingestion of the
donor (soil bacteria) by the recipient (bacterivorous
nematodes) [11]. Mechanisms underlying desiccation tol-
erance could have facilitated the uptake of prokaryotic
DNA [12]. A number of nematode species including
Aphelenchus avenae [13], Ditylenchus dipsaci [14], and
Panagrolaimus superbus [15] can develop into highly
drought resistant Dauer larva.
Among nematode genes that could have been acquired
via one or multiple HGT events, GHF5 cellulases are
best characterized. Recent genome sequencing projects
resulted in the identification of large ce llulase families in
the root-knot nematodes Meloidogyne incognita [16,17]
and Meloidogyne hapla [18]. These are highly derived
(distal) species within the family Meloidogynidae, and to
identify possible origin(s) of these genes, cellulase se-
quence information is require d from less derived repre-
sentatives of this family. Recent morphological and
molecular studies based on female gonoduct archite c-
ture [19] and small subunit ribosomal DNA sequences
[19,20] suggest that root-knot nematodes originate from -
and constitute a subclade within - the genus Pratylenchus.
By seque ncing cellulase genes from Pratylenchus spp.
(lesion n ematodes) and ba sal root-knot nematode spe-
cies - the ones that do not belong to one of the sub-
clades I, II and III a s defined in 2002 by Tandingan
De Ley et al. [21] -, we intended to generate clues
to establish the evolutionary relationship betwe en
members of the Pratylenchidae genera Pratylenchus
and Hirschmanniella, and basal root-knot nematode
species such as Meloidogyne ichinohei, M. mali and
Mulmi[20].
Several models have been proposed about HGT event
(s) underlying the acquisition of cellulases by plant para-
sitic and fungivorous nematodes. So far it is unclear
whether the distribution of cellula se-encoding genes
among Tylenchida is the result of a single HGT event,
followed by early single duplication event as suggested
by Kynd t et al. [22], or the outcome of multiple HGT
events. Comparison of the topologies of phylogenetic
trees ba sed on SSU rDNA data (e.g. [20,23]) versus
GHF5 cellulase-based tree might tell us whether the e vo-
lution within the Pratylenchidae/Meloidogynidae branch
includes one or multiple distinct cellulase lineages. Ana-
lysis of 103 paralogs and orthologs of cellulase-encoding
gene(s) (fragments) from plant parasitic Tylenchida
revealed a major clade with a topology similar to the one
revealed by SSU rDNA, a neutral gene. Moreover, a rela-
tively small, divergent subset of cellulases was found that
is probably the result of early substrate spe cificity-driven
diversification. Within the catalytic domain types A and
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221 Page 2 of 10
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B (too few type C sequences are available to make a
statement), the overall topology resembles the topologies
revealed by neutral ribosomal DNA sequences, and it is
hypothesized that root-knot, cyst and lesion nematodes
received their cellulases from more ancient Pratylenchi-
dae or even more basal members of Clade 12 [24], rather
than by direct latera l gene transfer.
Methods
Taxon sampling and microscopic identification
Pratylenchus and Hirschmanni ella species were collected
from various habitats throughout The Netherlands and
extracted from the soil using standard techniques. Individual
nematodes were identified using a light microscope (Zeiss
Axioscope) equipped with differential interference contrast
optics. Globodera pallida specimens originated from a
Dutch population named Pa3 Rookmaker. Meloidogyne
species were kindly provided by Dr. Gerrit Karssen from the
Plant Protection Service of The Netherlands: M. ichinochei
(propagated on Iris levigata; culture C2312; Japan), M.
artiellia (sampled from a field with Triticum aestivum;cul-
ture E8067; Syria), M. ardenensis (propagated on Liguster
sp.; Wageningen) and M. ulmi (isolated from Ulmus sp.;
Wageningen).
Nematode lysis
Single nematodes were transferred to a 0.2 mL poly-
merase chain reaction (PCR) tube containing 25 μL
sterile water. An equal volume of lysis buffer contain-
ing 0.2 M NaCl, 0.2 M TrisHCl (pH 8.0), 1% (v/v)
β-mercaptoethanol and 800 μg/mL proteinase K wa s
added. Lysis took place in a Thermal cycler (MyiQ,
Bio-Rad) at 65°C for 2 h foll owed by 5 min incuba-
tion at 100°C. The lysate (crude DNA extract) was
used immediately or stored at 20°C.
Amplification of cellulase-coding genes from genomic
DNA
Based on publicly available cellulase sequences (cDNA
and genomic sequences, see Additional file 1: Table S1)
from lesion, root-knot, and cyst nem atodes, six con-
served peptide motives were identified within the cata-
lytic domain, namely PPYGQLS (CD1), LKCNWN
(CD2), YVIVDW (ENG1), WCQDV (CD4), F VTEYG
(ENG2) and ISYLNWAISD (CD6) (for positioning see
Table 1). These regions were used as a starting point for
the design of eng-specific primers (Table 1). The primary
aim was to amplify the longest possible fragment, prefer-
ably from CD1 to CD6 (230 amino acids , 700bp of
the coding sequence); however, on some occasions ,
only shorter cellulase fragments could be amplified
(Table 2).
Due to differences in codon usage within the six con-
served amino acid motives mentioned above, numerous
primer variants had to be designed and subsequently
examined in various combinations. For a quick, first se-
lection of the most effective primer combinations, quan-
titative PCR was used. For this, 3 μL of template (single
nematode lysate) was mixed with relevant primers (end
concentrations for both primers 200 nM), and 12.5 μL
iQ Absolute Sybr Green Fluorescein Cat. CM-225
(Westburg). The total reaction volume was 25 μL. Ther-
mal cycling took place in the MyiQ thermal cycler (Bio-
Rad) under the following conditions: 95°C for 15 min;
followed by 60 cycles at 95°C for 30 s, 50°C for 1 min
and 72°C for 2 min. In case a possibly applicable amplifi-
cation signal was produced (criteria: C
t
value < 50 cycles,
and a melting temperature > 80°C), the amplicon was
analyzed on a 1% agarose-gel stained with GelStar
(Westburg; 2μl/100ml). For those primer combinations
that gave rise to amplification of the expected size pro-
ducts, the annealing temperature was optimized using
conventional PCR. These reactions were performed in a
final volume of 25 μl and contained 3 μl of a diluted
crude DNA extract, 0.1 μM of each PCR primer, and a
Ready-To-Go PCR bead (Amersham, Little Chalfont,
Buckinghamshire, UK). The following PCR profile was
used: 95°C for 5 min followed by 60 x (94°C, 30 sec;
specific annealing temperature, 1 min; 72°C, 2 min)
and 72°C, 10 min.
Cloning, sequencing and sequence alignment
Gel-purified amplification products (Marligen Bio-
science, Ijamsville, MD) were cloned into a TOPO TA
cloning vector (Invitrogen, Carlsbad, CA) and sequenced
using standard procedures. Newly generated sequences
were deposited at GenB ank under accession numbers
listed in Table 2.
Intron positions in the genomic sequences were iden-
tified on the basis of information about exon-intron
structure of publicly available sequences (all the ones
in the Additional file 1: Table S1 harboring at least one
intron). The newly obtained nucleotide sequences as
well as those derived from GenBank were translated
into amino acids and aligned using the ClustalW algo-
rithm as implemented in BioEdit 5.0.9 [25]. The pro-
tein alignment was improved manually and translated
back into nucleotides. The final nucleotide alignment
consisted of 103 sequences, of which 45 were generated
in this study. More than half of these sequences (66
out of 103) span almost the full catalytic domain (from
CD1 to CD6).
RNA extraction and cDNA cellulase amplification
In order to support the chosen intron extraction ap-
proach, cDNA cellulase sequence for G. pallida was
synthesized. For this purpose 100 individuals of G. pallida
were collected into a 0.2 mL PCR tube containing 25 μL
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of sterile water and lysed as specified above. The lysate
was used immediately for the RNA extraction according
to RNeasy Micro kit protocol (Qiagen). Total RNA end
concentration was approximately 7ng of RNA/μLof
water. 3 μL of the five times diluted RNA was mixed with
CDGp2F and CDGp8R primers (Table 1; end concentra-
tions for both primers 200 nM), 20 units of RNAse inhibi-
tor (Invitrogen) and the components of the SuperScript
III One-Step RT-P CR with Platinium Taq kit (12 μL
of 2X reaction Mix and 2 μLoftheSuperScriptTM
III RT/ P latinium Taq Mix). This reaction of 25 μL
in total, was used for the specific cDNA fragment
amplification u nder the following conditions : 60°C
for30min,94°Cfor2min;followedby60cyclesat
94°C for 15 s , 60°C for 30 s and 68°C for 1 min and
finished with one cycle of 68°C for 5 min. As a re-
sult of this experiment the Gp-eng-5 sequence was
acquired.
Phylogenetic analysis
The Bayesian phylogeny was constructed with the pro-
gram MrBayes 3.1.2 using a site-specific model. Data
were part itioned by codon position and gamma dis-
tribution of rate variation with a proportion of invariable
sites was used. Four independent runs were set with 4
Markov chains per run. The program was run for 5 mil-
lion generations. Stabilization of the likelihood and para-
meters were checked with the program Tracer v1.4 [26]
and the burnin was defined as 120,000 generations. For
the construction of the maximum likelihood of the cel-
lulase tree the RAxML-HPC BlackBox program [27]
available at the CIPRES S cience Gateway V. 3.1 was
used. The program FindModel (http://hcv.lanl.gov/
content/sequence/findmodel/findmodel.html from the
HC V sequence database) wa s u sed to determine the
best phylogenetic model, and the following parameters
were applied: estimated proportion of invariable sites
Table 1 Overview of PCR primers used for cellulase amplication from individual nematodes
A
CD1 CD2
TATPPPYGQLSVSGTKLVDSSGQPVQLIGNSLFWHQFQAQYWNAETVKALKCNWNANVVRAAVGVDLERGYMSDP
ENG1
TTAYNQAVAVIEAAISQGLYVIVDWHSHESHVDKAIEFFTKIAKAYGSYPHVLYETFNEPLQGVSWTDILVPYHKKVIAAI
CD4 ENG2
RALDSKNVIILGTPTWCQDVDIASQNPIKEYKNLMYTFHFYAATHFVNGLGAKLQTAINNGLPIFVTEYGTCSADGNGNI
CD6
DTNSISSWWSLMDNLKISYLNWAISDKSETCSALKPGTPAANVGVSSSWTTSGNMVADHDKKKSTGVSCS
B Primer sequence 53 * Primer sequence 53 *
Region PPYGQLS (CD1): Region WCQDV (CD4):
CD1aF ccIccItacggIcaattgtc CD4aR tccacRtcctgggacca
CD1bF ccIccItatggIcaattgtc CD4cR tccacAtcttggcacca
CD1cF ccIccItatggIcaattatc
CD1PraFa ccgccgtatgggcaa Region FVTEYG (ENG2):
CD1PraFb cctccctatggccaa ENG2 see e.g. [22] gtIccRtaYTcIgtIacRaa
CD1PraFc cg
ccgtatgggcaa
CD1MelF ctccatatgggcaattatctgt Region ISYLNWAISD (CD6):
Region LKCNWN (CD2): CD6PraFb tctcctacatcaactgggc
CD2aF ctcaaatgcaattggaacKc CD6aR gcccagttggcgtaIgaga
CD2bF ctcaaatgcaattggaatKc CD6bR gcccaIttggcRtaIgaaa
CD2cF cttaaatgcaIttggaatKc CD6cR gcccaIttgaIgtaMgaaa
CD2dF cttaaatgctIttggaatKc CD6dR gcccagttgaYgtaIgaga
Region YVIVDW (ENG1): CDGp8R gcccagttgaggtacgaa
ENG1 see e.g. [22] taYgtIatcgtIgaYtggca CD6PraRa cccagttggcgtagga
ENG1R tgccaRtcIacgatIacRta CD6MelR tgtttgagatagcccagttg
* I=inosine; K= g or t, M= a or c, R= a or g, Y= c or t. In bold: discriminative nucleotide position.
Conserved amino acid motives in GHF5 cellulases from plant parasitic nematodes residing in nematode Clade 12 (Holterman et al. 2006 [24]). Primer design was
based on these motives, and all primers used in this study are listed bellow. A. The backbone sequence given below is derived from the predicted amino acid
sequence of the potato cyst nematode ( Globodera rostochiensis) cellulase Gr-eng-1 (GenBank AF004523), amino acid positions 18 324 (mainly catalytic domain).
Underlined: part of signal peptide for secretion. B. Primer names and primer sequences.
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Table 2 Overview of GHF5 cellulase sequences generated in this study from plant parasitic nematodes belonging to
the superfamily Hoplolaimoidea
Species name Individual* Forward
primer
(1)
Reverse
primer
(1)
Fragment
length (bp)
Fragment length
after removal
putative
introns (bp)
Gene name
(2)
GenBank
Acc. No.
Meloidogyne ardenensis 1 CD1cF CD6cR 1228 727 Mard-eng-1 JN052024
Meloidogyne artiellia 1 CD1MelF CD6MelR 936 731 Mart-eng-1 JN052025
Meloidogyne ichinochei 1 CD2aF CD4aR 673 343 Mic-eng-1 JN052026
M. ichinochei 2 CD2aF CD4aR 673 343 Mic-eng-2 JN052027
Hirschmanniella gracilis 1 CD1aF CD6bR 950 728 Hgr-eng-1 JN052061
H. gracilis 1 CD1aF CD6bR 866 728 Hgr-eng-2 JN052062
H. gracilis 1 CD1aF CD6bR 950 728 Hgr-eng-3 JN052063
Hirschmanniella loofi 1 CD1bF ENG1R 282 255 Hl-eng-1 JN052057
H. loofi 1 CD1bF CD4aR 560 487 Hl-eng-2 JN052058
H. loofi 1 ENG1 CD6aR 983 461 Hl-eng-3 JN052059
H. loofi 1 ENG1 CD6aR 554 431 Hl-eng-4 JN052060
Pratylenchus crenatus 1 CD1PraFa CD6aR 820 721 Pcr-eng-1 JN052031
P. crenatus 2 CD1PraFb CD6PraRa 819 723 Pcr-eng-2 JN052030
P. crenatus 3 ENG1F CD6aR 543 449 Pcr-eng-3 JN052029
Pratylenchus neglectus 1 ENG1 CD6aR 508 452 Pn-eng-1 JN052032
P. neglectus 2 CD1PraFc CD6PraRa 791 735 Pn-eng-2 JN052033
P. neglectus 2 CD1PraFc CD6PraRa 789 733 Pn-eng-3 JN052034
Pratylenchus penetrans 1 CD2cF CD6aR 1514 588 Pp-eng-3 JN052035
P. penetrans 1 CD2cF CD6aR 695 587 Pp-eng-4 JN052036
P. penetrans 1 CD1bF CD4aR 739 484 Pp-eng-5
JN052037
P. penetrans 2 CD1PraFb CD6PraRb 841 733 Pp-eng-6 JN052038
Pratylenchus convalariae (3) 1 CD1PraFb CD6aR 874 739 Pcon-eng-1 JN052028
Pratylenchus pratensis 1 CD1bF ENG1R 256 256 Ppr-eng-1 JN052040
P. pratensis 1 CD2bF CD6dR 1124 592 Ppr-eng-2 JN052039
P. pratensis 1 CD2dF CD4aR 407 349 Ppr-eng-3 JN052041
P. pratensis 1 CD1aF CD4aR 489 489 Ppr-eng-4 JN052043
P. pratensis 1 ENG1 CD6aR 507 452 Ppr-eng-5 JN052042
P. pratensis 2 CD1PraFb CD6PraRa 801 750 Ppr-eng-6 JN052044
Pratylenchus thornei 1 CD2bF CD6dR 678 587 Pt-eng-1 JN052045
P. thornei 2 CD1PraFc CD6PraRb 824 733 Pt-eng-2 JN052046
Pratylenchus vulnus 1 CD2cF CD6aR 639 587 Pv-eng-1 JN052047
P. vulnus 1 CD2bF CD6dR 1001 614 Pv-eng-2 JN052048
P. vulnus 1 CD2bF CD6dR 639 587 Pv-eng-3 JN052049
P. vulnus 1 CD2aF CD4aR 349 349 Pv-eng-4 JN052050
P. vulnus 1 ENG1 CD6aR 958 449 Pv-eng-5 JN052051
P. vulnus 1 ENG1 CD6aR 604 312 Pv-eng-6 JN052052
P. vulnus 1 ENG1 CD6aR 505 452 Pv-eng-7 JN052053
P. vulnus 2 CD1aF CD6bR 1031 728 Pv-eng-8 JN052054
P. vulnus 3 CD1PraFb CD6aR 790 733 Pv-eng-9 JN052055
P. vulnus 3 CD1PraFb CD6PraRa 792 735 Pv-eng-10 JN052056
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(GTRGAMMA + I). To find best tree using maximum
likelihood search, bootstrapping halted automatically and
printed branch leng ths. Cellulases from Aphelenchus
avenae, a predominantly fungivorous species, were used
as outgroup as this species does not belong to the
Tylenchida (all other species used in this study belong to
this order), and - on the basis of SSU rDNA data - resides
at the very base of Cade 12.
Results and discussion
Identification of new cellulase genes in nematodes
Genomic DNA from individua ls from seven Praty-
lenchus (Pratylenchidae), two Hirschmanniella (Praty-
lenchidae), three basal Meloidogyne (Meloidogynidae)
and one Globodera (Heteroderidae) species were tested
for the presence of GHF5 cellulases. In all of the nema-
todes we identified at least one GHF5 cellulase gene
(Table 2). Recently, it was shown that GHF5 cellulases
are not the only type of cellulases present among mem-
bers of the Tylenchida (Bauters et al., pers. comm.). Ex-
cept for the very basal Meloidogyne species M.
ichinochei and Hirschmanniella loofi, we obtained at
least one complete CD1-CD6 (Table 2) sequence for
every species under investigation.
Genome sequencing of the distal tropical Meloidogyne
species M. incognita not only revealed the presence of
multiple (21) genes encoding putative cellulases, but also
showed distinct clusters of GHF5 seq uences within a
single species [15]. Here we demonstrate that this is not
unique for distal Meloidogyne species. Pratylenchus pra-
tensis harbors multiple cellulase genes, whereas both
type A or type B catalytic domains (as coined by [22];
Additional file 2: Table S2) are represented. Similar
results were obtained for Pratylenchus vulnus (Figure 1).
The inventory of all currently available catalytic domain
sequences points at a numeric al dominance of the type
B over type A and C catalytic domains. For the soybean
cyst nematode Heterodera glycines, the substrate specifi-
cities of cellulases with type A, B and C catalytic
domains were tested [28]. Hg-ENG5 (Type C) and Hg-
ENG6 (Type A) differed greatly from the most abundant
type B cellulases: their depolymerizing activity on
carboxymethylcellulose was strongly reduced (respect-
ively 40% and 20% of the activities of HG-ENG-1 and 4
(both belonging to type B cellulases)), whereas both de-
grade xylan and crystalline cellulose (Hg-ENG5 show-
ing two fold higher activity than Hg-ENG6). By contrast,
the latter two substrates were not significantly degraded
by Hg-ENG-1 and 4 [28]. As such differences have
been reported for other GHF5 cellulases as well (e.g.
[29]), we hypothesize that the different types of cata-
lytic domains could point at differences in substrate
specificities.
Phylogenetic analyses
Bayesian inference-based phylogenetic analysis of 103
coding sequences (of which 45 were generated in this
study) for - at least - a major part of the catalytic domain
of GHF5 cellulases resulted in the distinction of three major
types of catalytic domains (A, B, and C, see Figure 1). This
systematics elaborates on the evolutionary model proposed
by Kyndt et al. (2008) [22]; catalytic domain C wa s
originally presented as a well-supported group of cel-
lulases nested within the type B clade [22].
Among cellulases with a type B catalytic domain (com-
prising 84 out of the 103 sequences), we observed a top-
ology that shows a remarkable overall similarity with the
one presented by Holterman et al. (2009) [20] on the
basis of a neutral gene, viz. SSU rDNA. Also on the basis
of type B catalytic domain sequences M. artiellia and
M. ichinochei appear at the base of the family Meloi-
dogynidae, and just as observed on the basis of SSU
rDNA sequences the Meloidogynidae appear as an
elaborate subclade nested within the genus Praty-
lenchus. Unlike SSU rDNA sequences that gave no
clear answer about the nature of the link between the
Pratylenchidae and the Meloidogy nidae, a robust sister
relationship was observed between a specific Praty-
lenchus species, P. thornei, and all representatives of
the genus Meloidogyne.
Ribosomal DNA-ba sed phylogenetic analysis revealed
a sister relationship between lesion and root-knot nema-
todes on the one hand, and cyst nematodes, Hoplo-
laimidae and Rotylenchulidae on the other (e.g. [20]).
Table 2 Overview of GHF5 cellulase sequences generated in this study from plant parasitic nematodes belonging to
the superfamily Hoplolaimoidea (Continued)
Globodera pallida 1 CD1aF CD4cR 1144 481 Gp-eng-1 JN052064
G. pallida 1 CD2aF CD6cR 852 589 Gp-eng-2 JN052065
G. pallida 1 CD2aF CD6cR 853 590 Gp-eng-3 JN052066
G. pallida 2 CDGp2F CD6cR 1561 709 Gp-eng-4 JN052067
G. pallida 2 CDGp2F CDGp8R 706 706 Gp-eng-5 JN052068
Overview of GHF5 cellulase sequences generated in this study from plant parasitic nematodes belonging to the superfamily Hoplolaimoidea. Single nematodes
were used for the amplification of putative cellulase fragments. Occasionally, multiple fragments were amplified from an individual, such as in caseofPratylenchus
neglectus 2(Pn-eng-2 and 3). (1) Primers sequences are given in Table 1. (2) gene names in bold indicate fragments spanning almost the full catalytic domain
(CD1 - CD6). (3) Small subunit ribosomal DNA data suggest that Pratylenchus convalariae is identical to P. penetr ans [20].
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221 Page 6 of 10
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Remarkably, all catalytic domains from the lesion nema-
tode P. crenatus, and subsets from the P. vulnus and
P. penetrans cellulases reside in a sister position with
regard to all type B cyst nematode cellulases. Al-
though the number of sequences included is consider-
ably smaller, we observed a similar patt ern for the type
A catalytic domain. Firstly, a sister relationship was
established here between all type A catalytic domain
sequences from root-knot nematodes and a cellulase
from P. vulnus, whereas a similar relationship was
observed for a soybean cyst and a reniform nematode
cellulase on the one hand, and P. pratensis on the other
(Figure 1). Hence, at least some lesion nematode spe-
cies are equipped with both root-knot and cyst
nematode-like cellulases.
The positioning of Ditylenchus spp. is based only on
one genomic and two CDSs sequences, and should be
considered as a consequence of the virtual absence of
cellulases data from other, more basal Tylenchida. The
current positioning is not well supported, and will not
be discussed.
Exon-intron structure
The number of introns in the catalytic domains of the
nematode cellulases varied between zero and seven.
For the intron identifiers, we followed the nomencla-
ture proposed by Kyndt et al. (2008) [22]. As a conse-
quence of the increase in the number and the
diversity of catalytic domains additional predicted in-
tron positions were found. To label new intron posi-
tions without uprooting the existing systematics, we
used identifiers such as and for introns posi-
tioned between introns 1 and 2, introns 5 and 6, etc.
(see Additional file 2: Table S2) .
Most of the in silico predicted introns in the newly
generated cellulase sequences were located at positions
equivalent to the positions reported before for plant
parasitic cyst and root-knot nematodes ([30], [17], [28]
and [22]) and the outgroup Aphelenchus avenae [31].
Identifiers 1½, 12½, 15½ were added as - up to now -
unique introns in cellulase gDNAs from A. avenae,
whereas and were used to indicate new intron
positions in Hl-eng1 and Pv-eng2. Among the newly
Figure 1 Bayesian tree of GHF5 catalytic domains from members of the nematode order Tylenchida. Genomic and coding sequences
(indicated by a yellow box at the base of the relevant branch) from (partial) cellulase catalytic domains were analyzed. Sequences covering the
catalytic domain from CD1 to CD6 (as defined in Table 1) are underlined (non underlined sequences are slightly shorter). Identical colors are used
for members of the same nematode family. The tree is rooted with genomic cellulase sequences from the fungivorous nematode Aphelenchus
avenae (infraorder Tylenchomorpha). Posterior probabilities are given next to each node. Orange circles with or without a bright cross are used to
indicate the presence or absence of an intron. An orange cross behind a sequence is used to indicate that the generated piece of a sequence
was intronless. Intron numbering is essentially according to Kyndt et al. (2008) [22]. Branch length is calculated in MrBayes, and the scale bar
below represents branch length (as number of DNA substitutions/site).
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221 Page 7 of 10
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generated cellulase sequences the largest intron was
found in Gp-eng1; intron 2 with a length of 563 bp. Nu-
merous occasions of intron gain and loss were observed
in all three main types of cellulase catalytic domains tree
(A, B and C). Particular introns appear to be characteris-
tic for catalytic domain types: Type A typically contains
introns 4 and 14, whereas all type B and C catalytic
domains investigated so far have lost intron 18. Type C
representatives share the presence of intron 2. Howe ver,
this feature is not unique as it is typical for the type B
cellulases from cyst nematodes as well. Among root-
knot nematode type B cellulase the presence of intron 1
appears as a common characteristic. The two other cel-
lulase catalytic domains with an intron at position 1
were found in a type B catalytic domain Pp-eng-5
from Pratylenchus penetrans and interestingly, in a
type A catalytic domain Hg-eng-6 from soybean cyst
nematodes.
Intron phase distribution
The intron phase distribution in the catalytic domain of
nematode GHF5 cellulases was biased towards phase 0;
16 out of the 24 introns (66%) were inserted in between
two codons. Respective ly, two and four phase 1 (after
the first base of a codon) and phase 2 (after the se cond
base of a codon) introns were identified, whereas intron
positions 7 and 17 occurred in two phases (0 and 1). To
some exte nd a bias towards the phase 0 introns wa s
to be expected a s the overall f requencies of intron
phases 0, 1 and 2 in Caenorhabditis elegans are roughly
50%, 25% and 25% [32]. This p ha se bias seems to be
stronger in the case of cellulase catalytic domain from
Tylenchida.
In case of mixed phase intron 7, phase 1 was
observed only for Aphelenchus aven ae, a fungivorous
nematode that can feed on root hair or epidermal
cells of plants as well. Contrary to all other taxa
investigated here, A. avenae does not belong to the
order Tylenchida, though it is included in the infra-
order Tylenchomorpha ([33]). A phase 0 variant of in-
tron 7 is found among a subset of the type A
catalytic domains: the ones present in Meloidogyne
incognita (with one exception: Minc 19090a), and in
Pratylenchus vulnus. Among the 47 taxa harboring
mixed phase intron 17, 13 were in phase 1, and 34 in
phase 0. Phase 1 appeared to be a typical characteris-
tic for type B and type C catalytic domains of cyst
nematodes. Hence, it was also present in eng3 and
eng4 from G. rostochiensis, two otherwise highly dis-
tinct cellulases. The only other case of an intron 17
in phase 1 was obser ved for eng-1B from the banana
root nematode Radopholus similis (family Pratylenchi-
dae). This could be seen as a confirmation of a recent
SSU rDNA-based analysis suggesting a (unexpected)
close relatedness between Radopholus and cyst nema-
todes [20].
Conclusions
Addition of 45 new genomic sequences from the cata-
lytic domain of cellulases from pl ant parasitic members
of the order Tylenchida, followed by phylogenetic ana-
lyses further develops our understanding of the evolu-
tion of cellulases within a nematode order that harbors
most economically high impact plant parasites. Three
distinct types of catalytic domains were distinguished,
and we hypothesize that types of catalytic domains re-
flect distinct substrate preferences. Numerous plant
parasitic nematode species were shown to harbor two
types of GHF5 cellulases. Heterodera glycines, soybean
cyst nematode, is the only example of a plant para site
equipped with all three types of catalytic domains distin-
guished so far.
All Clade 12 members of the phylum Nematoda ana-
lyzed to date harbor one or multiple GHF5 cellulases.
This also holds for basal representatives that are not
fully dependent on plants as sole food source. Aphe-
lenchus spp., on the basis of SSU rDNA data suggested
to be sister to all Tylenchida [23], is primarily myceto-
phagous (fungal cell walls do not contain cellulose),
but can also grow and multiply on various plant spe-
cies [34]. It is noted that the necromenic nematode
species Pristionchus pacificus (Clade 9, Diplogasteridae)
is harbouring seven cellula se genes all belonging to
GHF5 ([35]). Hence, although GHF5 cellula ses are
widespread within Clade 12, they are not exclusively
present in this clade.
The family Anguinidae harbours the most ancestral
representatives of the order Tylenchida included in this
paper, and D itylenchus destructor, a memb er of this fam-
ily and the causal agent of dry rot in potato tuber, is also
known to feed on fungal hyphae. Recently, another early
branching representative of the Tylenchida, Deladenus
siridicola - a nematode with a mycetophagous and an in-
sect para sitic life cycle, was shown to harbour GHF5 cel-
lulases (dr. Bernard Slippers and co-workers, pers.
comm.). Hence, all members of Clade 12 seem to harbor
GHF5 cellulases, e ven ones that according to literature
do not feed on plants. Presuming GHF5 cellulases were
indeed acquired by lateral gene transfer, the most parsi-
monious explanation of the current cellulase tree would
be the acquisition of such a gene by an ancient represen-
tative of the Pratylenchidae. Though we realize that
current datasets are too fragmented to make a strong
statement, our results are compatible with a scenario in
which a GHF5 cellulase was acquired by the common
ancestor of Aphelenchus and all Tylenchida, followed
by one or multiple gene duplications and subsequent
diversification.
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221 Page 8 of 10
http://www.biomedcentral.com/1471-2148/12/221
Additional files
Additional file 1: Table S1. List of GHF5 endoglucanase sequences
from plant parasitic nematodes from public databases used in this paper.
Additional file 2: Table S2. Schematic overview of the (predicted)
introns in genomic sequences from the GHF5 endoglucanase genes. For
the intron identifiers, we adhered to the nomenclature proposed by
Kyndt et al. (2008) [22]. Identifiers such as and were used for
novel introns positioned between introns 1 and 2, 5 and 6, etc. The
colour scheme for the phase of the introns is explained below this Table.
Length of the introns (in bp) is given inside each box.
Competing interests
The authors have declared that there are no competing interests.
Authors contributions
KR-M and JH designed this study, HvM collected and identified the
biological material. KR-M, HRM, SvdE carried out the molecular work. KR-M
and PM did the phylogenetic analysis. KR-M, GS, JB and JH contributed to
data analyses and preparation of the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
HRM wish to thank the Erasmus Mundus Eumaine programme for financial
support. The authors wish to thank Erwin Roze for sequence information on
Meloidogyne chitwoodi cellulases. Moreover, we wish to thank dr. Yiannis
Kourmpetis and dr. Edouard Severing from Laboratory of Bioinformatics
(Wageningen University, The Netherlands).
Author details
1
Laboratory of Nematology, Department of Plant Sciences, Wageningen
University, Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands.
2
Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie,
Ökologie, Unter den Linden 6, Berlin 10099, Germany.
Received: 11 July 2012 Accepted: 4 November 2012
Published: 21 November 2012
References
1. Woolfit M, Iturbe-Ormaetxe I, McGraw EA, ONeill SL: An ancient horizontal
gene transfer between mosquito and the endosymbiotic bacterium
Wolbachia pipientis. Mol Biol Evol 2009, 26(2):367374.
2. Nikoh N, Nakabachi A: Aphids acquired symbiotic genes via lateral gene
transfer. BMC Biology 2009, 7:12.
3. Gladyshev EA, Meselson M, Arkhipova IR: Massive horizontal gene transfer
in bdelloid rotifers. Science 2008, 320(5880):12101213.
4. Smant G, Stokkermans J, Yan YT, de Boer JM, Baum TJ, Wang XH, Hussey RS,
Gommers FJ, Henrissat B, Davis EL, et al: Endogenous cellulases in animals:
Isolation of beta-1,4-endoglucanase genes from two species of
plant-parasitic cyst nematodes. Proc Natl Acad Sci USA 1998,
95(9):49064911.
5. Keen NT, Roberts PA: Plant parasitic nematodes: Digesting a page from
the microbe book. Proc Natl Acad Sci USA 1998, 95(9):47894790.
6. Popeijus H, Overmars H, Jones J, Blok V, Goverse A, Helder J, Schots A,
Bakker J, Smant G: Enzymology: Degradation of plant cell walls by a
nematode. Nature 2000, 406(6791):3637.
7. Jaubert S, Laffaire JB, Abad P, Rosso MN: A polygalacturonase of animal
origin isolated from the root- knot nematode Meloidogyne incognita.
FEBS Lett 2002, 522(13):109112.
8. Mitreva-Dautova M, Roze E, Overmars H, De Graaff L, Schots A, Helder J,
Goverse A, Bakker J, Smant G: A symbiont-independent endo-1,4-β-
xylanase from the plant-parasitic nematode Meloidogyne incognita . Mol
Plant Microbe Interact 2006, 19(5):521529.
9. Qin L, Kudla U, Roze EHA, Goverse A, Popeijust H, Nieuwland J, Overmars H,
Jones JT, Schots A, Smant G, et al: A nematode expansin acting on plants.
Nature 2004, 427(6969):30.
10. Maggenti AR: Nemic relationships and the origin of plant parasitic
nematodes.InPlant parasitic nematodes
. Edited by Zuckerman BM, Mai WF,
Rohde RA. New York: Academic Press Inc; 1971:6581.
11. Ford Doolittle W: You are what you eat: A gene transfer ratchet could
account for bacterial genes in eukaryotic nuclear genomes. Trends Genet
1998, 14(8):307311.
12. Gladyshev EA, Meselson M, Arkhipova IR: Massive horizontal gene transfer
in bdelloid rotifers. Science 2008, 320(5880):12101213.
13. Reardon W, Chakrabortee S, Pereira TC, Tyson T, Banton MC, Dolan KM,
Culleton BA, Wise MJ, Burnell AM, Tunnacliffe A: Expression profiling and
cross-species RNA interference (RNAi) of desiccation-induced transcripts
in the anhydrobiotic nematode Aphelenchus avenae. BMC Mol Biol 2010,
11:6.
14. Wharton DA, Aalders O: Desiccation stress and recovery in the
anhydrobiotic nematode Ditylenchus dipsaci (Nematoda: Anguinidae).
Eur J Entomol 1999, 96(2):199203.
15. Shannon AJ, Tyson T, Dix I, Boyd J, Burnell AM: Systemic RNAi mediated
gene silencing in the anhydrobiotic nematode Panagrolaimus superbus.
BMC Mol Biol 2008, 9:58.
16. Abad P, Gouzy J, Aury JM, Castagnone-Sereno P, Danchin EGJ, Deleury E,
Perfus-Barbeoch L, Anthouard V, Artiguenave F, Blok VC, et al: Genome
sequence of the metazoan plant-parasitic nematode Meloidogyne
incognita. Nat Biotechnol 2008, 26(8):909915.
17. Danchin EGJ, Rosso MN, Vieira P, De Almeida-Engler J, Coutinho PM,
Henrissat B, Abad P: Multiple lateral gene transfers and duplications have
promoted plant parasitism ability in nematodes. Proc Natl Acad Sci USA
2010, 107(41):1765117656.
18. Opperman CH, Bird DM, Williamson VM, Rokhsar DS, Burke M, Cohn J,
Cromer J, Diener S, Gajan J, Graham S, et al : Sequence and genetic map of
Meloidogyne hapla: A compact nematode genome for plant parasitism.
Proc Natl Acad Sci USA 2008, 105(39):1480214807.
19. Bert W, Leliaert F, Vierstraete AR, Vanfleteren JR, Borgonie G: Molecular
phylogeny of the Tylenchina and evolution of the female gonoduct
(Nematoda: Rhabditida). Mol Phylogenet Evol 2008, 48(2):728744.
20. Holterman M, Karssen G, van den Elsen S, van Megen H, Bakker J, Helder J:
Small subunit rDNA-based phylogeny of the Tylenchida sheds light on
relationships among some high-impact plant-parasitic nematodes & the
evolution of plant feeding. Phytopathology 2009, 99(3):227235.
21. Tandingan De Ley I, De Ley P, Vierstraete A, Karssen G, Moens M,
Vanfleteren J: Phylogenetic analyses of Meloidogyne small subunit rDNA.
J Nematol 2002, 34(4):319327.
22. Kyndt T, Haegeman A, Gheysen G: Evolution of GHF5 endoglucanase
gene structure in plant-parasitic nematodes: No evidence for an early
domain shuffling event. BMC Evol Biol 2008, 8:305.
23. Van Megen H, Van Den Elsen S, Holterman M, Karssen G, Mooyman P,
Bongers T, Holovachov O, Bakker J, Helder J: A phylogenetic tree of
nematodes based on about 1200 full-length small subunit ribosomal
DNA sequences. Nematology 2009, 11(6):927950.
24. Holterman M, van der Wurff A, van den Elsen S, van Megen H, Bongers T,
Holovachov O, Bakker J, Helder J: Phylum-wide analysis of SSU rDNA
reveals deep phylogenetic relationships among nematodes and
accelerated evolution toward crown clades. Mol Biol Evol 2006,
23(9):17921800.
25. Hall TA: BioEdit: a user-friendly biological sequence alignment editor and
analysis program f or Windows 95/98/ NT. Nucleic Ac ids Symp Se r 1999, 41:9 598.
26. Rambaut A, Drummond A: Tracer v1.4. 14th edition. http://beast.bio.ed.ac.uk/
Tracer 2007.
27. Stamatakis A, Hoover P, Rougemont J: A rapid bootstrap algorithm for the
RAxML web servers. Syst Biol 2008, 57(5):758771.
28. GaoB,AllenR,DavisEL,BaumTJ,HusseyRS:Developmental expression and
biochemical properties of a β-1 ,4-endo glucanase fam ily in the soybea n cyst
nematode, Heterodera glycines. Mol Plant Pathol 2004, 5(2):93104.
29. Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, Hu YL, Wei J, Tang JL, Feng
JX: Cloning and identification of novel cellulase genes from uncultured
microorganisms in rabbit cecum and characterization of the expressed
cellulases. Appl Microbiol Biotechnol 2007, 75(2):319328.
30. Yan YT, Smant G, Stokkermans J, Qin L, Helder J, Baum T, Schots A, Davis E:
Genomic organization of four beta-1,4-endoglucanase genes in plant-
parasitic cyst nematodes and its evolutionary implications. Gene 1998,
220(1
2):6170.
31. Karim N, Jones JT, Okada H, Kikuchi T: Analysis of expressed sequence
tags and identification of genes encoding cell-wall-degrading enzymes
from the fungivorous nematode Aphelenchus avenae. BMC Genomics
2009, 10:525.
Rybarczyk-Mydłowska et al. BMC Evolutionary Biology 2012, 12:221 Page 9 of 10
http://www.biomedcentral.com/1471-2148/12/221
32. Coghlan A, Wolfe KH: Origins of recently gained introns in Caenorhabditis.
Proc Natl Acad Sci USA 2004, 101(31):1136211367.
33. De Ley P, Blaxter ML: Systematic Position and Phylogeny.InThe Biology of
Nematodes. Edited by Lee DL. London, UK: Taylor & Francis; 2002:130.
34. Hunt DJ: Aphelenchida, Longidoridae and Trichodoridae: their systematics and
bionomics. Wallingford, UK: CAB International; 1993.
35. Schuster LN, Sommer RJ: Expressional and functional variation of
horizontally acquired cellulases in the nematode Pristionchus pacificus.
Gene 2012, 506(2):274282.
doi:10.1186/1471-2148-12-221
Cite this article as: Rybarczyk-Mydłowska et al.: Rather than by direct
acquisition via lateral gene transfer, GHF5 cellulases were passed on
from early Pratylenchidae to root-knot and cyst nematodes. BMC
Evolutionary Biology 2012 12:221.
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    • "In the superfamily Aphelenchoidea, GH5 were found present in Aphelenchus avenae [24], A. fragariae [8] and in A. besseyi, but not B. xylophilus [35]. Our GH5 phylogeny is consistent to previous conclusion that one or more multiple gene duplications took place since the acquisition of a GH5 gene from a common nematode ancestor (S5 Fig; [24]), and Abe-GH5-1 is placed in the catalytic domain-type B group [24]. In this revised phylogeny with more complete genomes, sub families consisting different species are observed again consistent with multiple gene duplication in the early Pratylenchidae common ancestor. "
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