Content uploaded by Leonid Rusin
Author content
All content in this area was uploaded by Leonid Rusin on Apr 07, 2020
Content may be subject to copyright.
Nematology, 2003, Vol. 5(4), 615-628
The 18S ribosomal RNA gene of Soboliphyme baturini Petrow,
1930 (Nematoda: Dioctophymida) and its implications for
phylogenetic relationships within Dorylaimia
Leonid Y. RUSIN 1, Vladimir V. AL ESH IN 1;¤,
Alexey V. TCHESUNOV 2and Gennadiy I. ATRASHKEV ICH 3
1A.N. Belo zersky In stitute of Physico-Ch emical Bi ology, Dep artment of Evolutio nary Bioch emistry; and
2Faculty o f Bio logy, Moscow Stat e Universit y, Mo scow, 1 19992, Ru ssia
3Institute of the Biological Problems of the North, Far-Eastern Branch of the Russian Academy of Sciences, 685000
Magadan, Russia
Received: 2 August 2002; revised: 28 April 2003
Accepted for publication:29 April 2003
Summary – Phylogenetic estimations from 18S rDNA sequence data reveal close relationships of dioctophymids with Trichinellida
(Trichocephalida), the latter represented by the families Trichinellidae and Trichocephalidae (Trichuridae). This phylogeny is congruent
with a scenario of molecular evolution deduced from conservative motifs within the V4 and V9 regions of 18S rRNA. A phylogenetic
approach to analyse data containing highly unequal rates of sequence evolution is proposed. The entire gene possesses only a few
conservative molecular synapomor phies of a clade consisting of Dorylaimida, Mononchida and Mermithida . Dioctoph ymida, togeth er
with Trichinellida, are inferred a s a sister tax on to this clade which join tly constitut e the Dor ylaimia. Molec ular data juxtaposed with
morphology were used to reconstruct some of the putative features of the common ancestor of Dorylaimia which is speculated to have
possessed a spear and, as found in extant Enoplia and Dioctophymida, pharyngeal gland outlets located in the stoma. Mononchids
are postulated to have secondarily lost the spear contrary to all previously published phylogenies. Reduction of caudal glands and
transformation of pharyngeal glands into the stichosome are not parsimonious across the tree of Dorylaimia. There are no unequivocal
adult morphological synapomorphies for Dorylaimia; the only non-molecular diagnostic feature is the unique speci cation of the
endodermal precursor in early embryogenesis.
Keywords – Bayesian inference, homoplasy, long branch attraction artefact, maximum likelihood, maximum parsimony, molecular
phyloge ny, secondary st ructure, SSU rRNA.
Phylog enetic system atics o f Ne matoda remains a d if -
cult task (see Lo renzen, 19 81; Malakh ov,1 986; De Ley &
Blaxter, 2002, for reviews). Molecular evidence has been
decisive in adducing many issues in nematode phylogeny
(Blaxter et al., 2000; Coomans, 2000; De Ley, 2000).
The 18S rRNA gene was successfully employed for in-
ferring internal phylogeny of Rhabditida, the large-scale
relationships of Chromadoria and delimitation between
Enoplia and Dorylaimia (Aleshin et al., 1998a; Blaxter
et al., 1998; Sudhaus & Fitch, 2001; De Ley & Blaxter,
2002).
The Dioctophymidais a small group of nematodes with
about 30 species in three families (Karmanova, 1968),
some of them having strong veterinarian and medical im-
¤Correspon ding author, e- mail: Aleshin @genebee.msu.s u
pact (Karmanova, 1968; Beaver & Theis, 1979; Mea-
sures, 1985; Eberhard et al., 1989; Anderson, 2000). All
dioctophymids are animal parasites with two or three
hosts in the life cycle. The larvae persist in oligochaetes
and then transfer to a vertebrate de nitive host. Diocto-
phymida is undoubtedlya monophyletic group and is cur-
rently assigned ordinal status (De Ley & Blaxter, 2002).
All representatives exhibit features unique to this taxon
(autapomorphies), amongst which are i/ muscular cau-
dal alae (bursa) in males, and ii) muscle cells (‘mesen-
teries’) stretching between the body wall and the intes-
tine. The head region in many dioctophymids is trans-
formed into a mouth sucker (Petrow, 1930) or a spinose
non-re tractile platfo rm (Schmidt-R haesa, 2000). Presence
©Koninklijke Bril l NV, Leide n, 2003 615
Also available online - www.brill.nl
L.Y. Rusin et al.
of a spear1in the JI is typical for all dioctophymids (Kar-
manova, 1968; Malakhov & Spiridonov, 1983) and links
them to Dorylaimida sensu Lorenzen (1981), while the
location of pharyngeal gland openings inside the stoma
suggests a link to Enoplia. Different systems have placed
the group inside the Enoplida as a suborder along with
Enoplina and Dorylaimina (Chitwood, 1933), as a sub-
order inside the Trichinellida (Spassky, 1956), as an in-
dependent order (Filipjev, 1934) or an order related to
Dorylaimida (Ryzhikov & Sonin, 1981; Malakhov, 1986;
De Ley & Blaxter, 2002). No molecular evidence on the
Dioctophymida has been previously examined for phylo-
genetic implication.
In this study we report analysis of the sequence of the
18S rRNA gene of Soboliphyme baturini, a representative
of the family Soboliphymidae (Petrow, 1930). We discuss
the phylogeny of Dorylaimia with respect to patterns of
evolutionarychange in the molecular structure of the gene
and discuss possible ancestral states for some important
morphologicalcharacters for this clade.
Materials and methods
Specimens of Soboliphyme baturini Petrow, 1930 were
obtain ed from the sto mach of sable ( Mustela zibellina L.),
its de nitive host, caught in Kolyma Province, Siberia.
Bodies of shot animals were stored frozen. Semi-thawed
stomachs were dissected and nematodes were rinsed with
ice cold physiological saline and xed immediately with
96% ethanol. Three out of nearly 200 sables dissected
contained specimens of S. baturini.
DNA was isolated with phenol extraction and precip-
itated with ethanol. The entire small subunit ribosomal
RNA (18S rRNA) gene was ampli ed using universal eu-
karyotic primers for nuclear 18S rRNA coding regions
(Medlin et al., 1988). PCR products were puri ed on
an agarose gel, cloned in the pGEM T-Vector System
(Promega), and sequenced using the fmol DNA sequenc-
ing system (Promega) on both strands. The sequence
was deposited with GenBank under accession number
AY277895.
1Historica lly, protractile mouth structures are referred to und er
different terms in different nematode taxa (e.g., onchiostyle in
Trichodoridae and Dioctophymidae, odontostyle in Dorylaim-
ida, (stomato) stylet in Tylenchid a). In order to avoid misleading
interpretations of the homology of this character across nema-
tode taxa discussed, the neutral term spear is used throughout
the text.
For phylogenetic analyses the 18S rRNA sequences
were manually aligned according to the eukaryotic SSU
rRNA secondary structure model of Van de Peer et al.
(2000). Taxon names with corresponding GenBank ac-
cession numbers are given in Fig. 1. All alignment posi-
tions were ut ilised in phylog enyreco nstruction ,exce pt for
some positionsin the occasionallyincomplete ankingre-
gions. In some analyses variable positions were assigned
weights inve rsely propo rtional to th e rates o f sub stitution
calculated for corresponding sites (for eight categories
with invariants) eith er with the a id of TREE-PUZZLE 5.0
(Strimmer & von Haeseler, 1996) under the HKY model
of molecula r substitu tion (Hasegawa et al., 1985), or with
RevDNArates version 1.0.3 (Korber et al., 2000) under
the REV model (Yang, 1994).
Maximum parsimony (MP) and neighbour-joining(NJ)
phylogenies were inferred with Dnapars.exe and Neigh-
bor.exe programs from the PHYLIP 3.6a2.1 package
(Felsenstein, 1993). MP search was conducted with the
‘Print out steps in each site’ option in effect. We traced
the states of predicted apomorphic characters on a large
alignment of metazoan 18S rRNA sequences compiled
from the universal 18S rRNA Database (The Univer-
sity of Antwerp, http://oberon.rug.ac.be:8080/rRNA/;Van
de Peer et al., 2000). Maximum likelihood (ML) phy-
logenies were estimated with fastDNAml version 1.2.2
(Olsen et al., 1994) and fastDNAml modi ed to incor-
porate the REV model of molecular substitution (Kor-
ber et al., 2000). MP and NJ analyses were conducted
with 1000 bootstrap replicates (Felsenstein, 1985). ML
analyses had 20 bootstrap replicates due to time limita-
tions. All suboptimal ML trees found were taken into ac-
count. Suboptimal trees obtained in ML searches were
compare d by Kishino and Hasegawa (1 989) test. Bayesian
inferenc e (BI) was condu cted with four simulta neous runs
of Markov chain Monte Carlo (MCMC) algorithm imple-
mented in MrBayes version 2.01 (Huelsenbeck & Ron-
quist, 2 001). The Marko v c hains were run for 1 000 000
generations wit h sampling every ten generations for a to-
tal of 100 000 samples per run. The states of the chain
before reaching stationarity were discarded as burn-in.
Likelihood parameters for BI corresponded to the General
Nonreversib le Model (settings nst D12, ncat D8, rates D
invgamma,shape Destimate, basefreq Destimate).
The closest living relative of Nematoda remains un-
known, and several taxa were taken as outgroups in-
cluding representatives of Ecdysozoa (Aguinaldo et al.,
1997) and of some ‘pseudocoelomate’phyla. Such a mul-
ticompound outgroup is recommended in cases when the
616 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
ancestral character states are unknown (Pavlinov, 1990;
Philippe, 2000).
Elemen ts of the 18S rRNA mo lecule’s secondary struc-
ture were modelled with mfold (Zuker et al., 1999) and
visualised with RnaVis (De Rijk et al., 2003).
Results
The sequence o f the 18 S rRNA gene of S. baturini(dis-
regarding primer sites) is 1743 bp long, which is within
the typical range for Nematoda. It does not contain exten-
sive indels that would hamper the alignment procedure.
The nal alignment inclu des 18S rRNA data for most ne-
matode orders and all non-chromadoriangenera published
so far. This dataset was processed with MP, ML, BI, and
NJ algorithms. Most of the topological elements of in-
ferred trees are identical and supported by high values of
bootstrap proportions (BP) or posterior probability (PP).
Some nodes, relating to the deeper radiations within ne-
matodes, are dependent on the inferring algorithm and/or
have BP <50%. These few ambiguous nodes are col-
lapsed in the consensus tree (Fig. 1). 18S rRNA sequence
of S. baturini is included in Dorylaimia on MP, ML,
NJ and major-consensus trees after resampling (Clade I
sensu Blaxter et al., 1998). The resulting clade repre-
sents the conventional orders Dorylaimida, Mononchida,
Mermithida, Trichinellida and Dioctophymida. The esti-
mate of the posteriorprobabilityfor the clade to be correct
equal s 1.000 when the seq uence of S. baturini is included,
and bootstrap support is about 90% with slight variations
depending on the particular inferring algorithm (Fig. 1).
When the same dataset is processed without S. baturini,
BP support for the Dorylaimia node falls drastically rang-
ing from 60 to 70% (Table 1). The latter value is similar
to BP for Clade I a s estimated in previous studies (Blaxter
et al., 1998, 2000; Rusin et al., 2001). Thus, adding S. ba-
turini to the dataset consolidates Dorylaimia. The lower
of BP for Dorylaimia without Dioctophymida is related
to the placement of Trichinellida closer to the root of the
nematode tree in many subsamples. For instance, in MP
analyses this pattern occurs in 25.1% of bootstrap repli-
cates, which is not surprising in view of the longer branch
lengths leading to trichinellids in trees (Fig. 1) and con-
sidering the computational artefacts likely to occur in such
cases (Fe lsenstein, 19 78). Despite t he branc h leading t o S.
baturini being no shorter from those of trichinellids, this
sequence is placed to the root of the tree only in 8.8% of
the replicates.
Due to the presence of the highly divergent diocto-
phymid and trichinellid lineages inside the Dorylaimia
clade, the number of molecular synapomorphies shared
by all members of this clade is very few: there is only
one such substitution – an A!C transversion within the
50branch of helix 23. In our dataset of more than 200 ne-
matode 18S rRNA sequences, only Desmodora ovigera
Ott, 1976 (accession number Y16913) also shares this
transversion.
As noted, the S. baturini branch always joins up with
Trichinellida (Fig. 1). This node is reconstructed in most
parsimonious trees (BP >90%), is present in the MCMC
consensus topology (PP D1.000) and is also robust
against modifying the dataset (data not shown). About
100 synapomorphiesare inferred for this clade with Dna-
pars, 20 of them falling on sites that are inferred with no
more than ve substitutions per the most parsimonious
tree. We traced the states of these characters on a larger
alignment of metazoan 18S rRNA sequences which were
obtai ned from the rRNA WWW Server maintained by the
University of Gent (http://oberon.rug.ac.be:8080/rRNA/).
Some apomorphiesof the node appeared to have little ho-
moplastic occurrence among nematodes and other Meta-
zoa (1766 sequences, including 172 nematode taxa avail-
able). Wh en these chara cters do oc cur outside th e (Diocto-
phymida, Trichinellida) clade, they usually represent au-
tapomorphiesof entire monophyletic groups of different
rank. Thus, a speci c A!G transition at position 3838 of
the larger al ignment is characteristi c of 15 acoelan turb el-
larians and ve myzostomids, while a G!A transition at
position 9698 occurs in 12 atworms, mostly monopisto-
cotyleanmonogeneans,36 insect taxa, mostly culicoidean
mosquitoes and reduviid bugs, and in nine representa-
tives of Acanthocephala, which constitute a monophyletic
clade according to recent ndings (Herlyn at al., 2003).
On this dataset, the molecular synapomorphies of Dioc-
tophymida and Trichinellida mentioned above have fre-
quencies of homoplastic occurrence less than 10¡2.
Three synapomorphies of the (Dioctophymida, Trichi-
nellida) node are situated within the predicted single-
stranded region of the loop of helix 18 (Fig. 2). This 17
bases long loop is hypothesised to be one of the longest
single-stranded regions of the 18S rRNA molecule sec-
ondary structure (Neefs et al., 1993; Wuyts et al., 2000).
However, some residues in this region are potentially
able to form Watson-Crick pairs, which are maintained
in Dioctophymida and Trichinellida. Comparative analy-
sis of a few substit utions known at t hese positions shows
that si ngle mutation s were xe d less freque ntly than were
Vol. 5(4), 2003 617
L.Y. Rusin et al.
Fig. 1. Phylogenetic position of Dioctophymida on the nematode tree inferred with 18S rDNA sequence data. Most nodes included in
this general consensus trees are reconstructed with all algorithms. Ambiguous nodes are collapsed. Branch lengths are estimated with
TREE PUZZLE 5.0. Values of support are given for selected nodes only. Valu es above the bar are MP, NJ and ML b ootstrap percentages
without gamma correction for rate heterogeneity, while values below the bar are bootstrap percentage for MP and ML with gamma
correction and p osterior probabil ities for BI. If a n ode was not present in the 50 %-majority rul e con sensus to pology o f a n an alysis, th e
corresponding statistics are given in brackets. Numbers of pseudoreplicates, evolutionary models employed, number of rate categories
and BI parameters are described in text.
618 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
Table 1. Effect of taxon sampling on values of statistic support for alternative clustering of dorylaimian lineages.
All tax a Dioctoph ymida exclud ed Trichinellid a excluded
MP MP ML ML BI MP MP ML ML BI MP MP ML ML
(¡0) (C0) (¡0)a(C0)b(¡0) (C0) (¡0)c(C0)d(¡0) (C0) (¡0)e(C0)f
a) Dorylaimia 89 92 92 91 1.000 66 72 75 78 1.000 89 94 95 96
b) Dioctophymida/Trichinellida 97 97 97 94 1.000 n/a n/a n/a n/a n/a n/a n/a n/a n/a
c) Mononchida/Mermithida 100 100 100 100 1.000 100 100 100 100 1.000 100 100 100 100
d) Mononchida/Mermithida/ 36 34 56 50 0.189 39 32 20 18 0.003 25 17 1 11
Dorylaimida
e) Dioctophymida/Trichinellida/ 35 29 8 18 0.627 57 62 60 69 0.984 54 45 1 8
Mononchida/Mermithida
f) Dioctophymida/Trichinellida/ 26 34 34 27 0.183 3 5 12 11 0.012 19 38 97 80
Dorylaimida
Values of MP bootstrap ping are obtained for 1 000 pseud oreplicates, values of ML b ootstrapping are o btained for 20 pseudoreplicates
(unless otherwise indicated). Values of BI support indicate posterior probabilities of a cluster to be correct. The number of suboptimal
trees (estimated with KH test) saved in ML bootstrap analyses was as follows: a6826 trees; b6313 trees; c4541 trees in ten
pseudoreplicates; d4162 trees; e3170 trees in ten pseudoreplicates; f5232 trees. Underlined values indicate clusters observed in the
best top ology, values in bo ld indica te clusters ke pt in th e major-rul e consensus tre e. For e ach dataset ana lyses were con ducted with a nd
without 0co rrection (C0and ¡0, respectively).
double compensatory mutations (data not shown). Such a
pattern of co-evolu tion is typical for residu es involve d in
molecular interaction (Woese et al., 1983). Thus, hairpin
18 actually possesses a long imperfectly paired stem and
a nine bases long single-stranded loop, rather than a 17
bases long loop (Fig. 2).
The presence of three monophyletic clades; i/ Dioc-
tophymida and Trichinellida; ii) Mononchida and Mer-
mithida, and iii) Dorylaimida, allows for three possible
basal topologies for the Dorylaimia (Fig. 3). Ironically,
all the three combinations were estimated as equally par-
simonious. Dnapars reconstructed approximately equal
amounts of putative synapomorphies for each; 43, 46 and
46 synapomorphiesbeing inferred for topologiesA, B and
C, respectively (see Fig. 3).
Two taxa can be erroneously clustered together due
to high levels of homoplasy in hypervariable regions of
the sequences compared. A conventional way to reduce
this artefact is to assign more variable positions a lower
weight in phylogenyreconstruction,e.g., by introducinga
gamma distribution approximation to evolutionary mod-
els (Yang, 1996; Whelan et al., 2001). However, in the
case of Dorylaimia, the resulting ML topology and the
consensus of suboptimal trees after bootstrap resampling
were in uenced little by the gamma correction, depend-
ing more upon taxonomic composition of the data set
(Table 1). This may be accounted for by high disparities
in nucleotide composition of 18S rRNA genes of repre-
sentatives of Dorylaimia. Of 39 taxa in our dataset, two
were rejected with 95% con dence by Â2test to t the
HKY mo del (TREE-PUZZLE 5 .0 analysis) an d both the se
taxa (MylonchulusarenicolusClark, 1961, AF036596 and
Trichuris muris Schrank, 1788, AF036637) belonged to
Dorylaimia. The corresponding Pvalues for the taxa in
the dataset are plotted in Fig. 4. Representatives of Do-
rylaimia, except for Dorylaimida, are characterised by
low Pvalues. The gure illustrates disparities in Pval-
ues among the sampled Dorylaimia. It suggests that the
18S rRNA gene in two dorylaimian lineages, (Diocto-
phymida, Trichinellida) and (Mononchida, Mermithida),
may have followed modes of molecular evolution differ-
ent from those of the majority of taxa, and perhaps from
Dorylaimi da as well. It is known that poor choices of su b-
stitution model render ML phylogenetic reconstructions
unreliable (Yang, 1996). The same applies to the BI algo-
rithm, which is susceptible to incorrect estimates of like-
lihoods of trees and actually produces similar results (Ta-
ble 1).
To resolve th e interna l structure o f Dorylaim ia, selected
molecular signatures were assayed for variability using
data from the metazoan 18S rRNA database. It appeared
that only two topologies, namely A and C in Fig. 3, are
supported by substitutions showing relatively low levels
of homoplasy. The most plausible characters that sup-
port the sister group status of (Dioctophymida,Trichinel-
lida) clade with respect to the rest of Dorylaimia are two
Vol. 5(4), 2003 619
L.Y. Rusin et al.
Fig. 2. A: Synapomorphies of Dioctophymida and Trichinellida; B: Predicted secondary structure in the V3 region of helix 18 of the
18S r RNA molecu le. Synapo morphic substit utions inferred with Dnapars are shown on the lower dotted line. Possible synapomorphies
are in black boxes, those representing possible symplesiomorphies for Nematoda are enclosed in empty boxes.
620 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
Fig. 3. Three possible internal topologies (A, B and C) for
Dorylaimia mapped wi th primary morphol ogical characters.
synapomorphiesof the Mononchida/Mermithida/Dorylai-
mida grouping:a speci c deletion of one residue in the V4
region and a relatively rare R!Y transversion within re-
gion V9 of helix 49. The altern ative topolo gy with (Di oc-
tophymida,Trichinellida)and (Mononchida,Mermithida)
as closest relatives is supported by a conservative T!C
transiti on also situ ated within h elix 49. Th e fact tha t mole-
cular characters generating con icting phylogenetic sig-
nals are located close to each other within the molecule
prompted us to model the secondary structure of the cor-
responding region. We found that the folding pattern of
helix 49 differs slightly among the groups of Dorylaimia
(Fig. 5). A plausible interpretation of this is presented in
below.
Discussion
We will focus here on four points: i/ reliability of Dioc-
tophymida as a sister taxon to Trichinellida; ii) feasibil-
ity of phylogeny reconstruction for the entire Dorylaimia
with 18S rDNA sequence data; iii) morphological im-
plications of molecular phylogenies of Dorylaimia, and
iv) possibility of molecular diagnosis of Dorylaimia with
18S rDNA sequence data.
As may be deduced from the branch lengths on inferred
trees (see Fig. 1), both the dioctophymid and trichinellid
lineages contain multiple modi cations in the 18S rRNA
gene structure with respect to other nematodes and each
other. Thus, their coalescence may be due to ‘long branch
attraction’ artefacts (Felsenstein, 1978). However, some
evidence suggests that this grouping is non-accidental.
There is no sign of clustering of the S. baturini sequence
with any of the highly divergent lineages other than
trichinellids present in the dataset (viz., trichodorids and
rhabditids, including in particular Pelodera strongyloides
(Schneider, 1860), accession number U13932). Further-
more, the clustering with trichinellids is very robust us-
ing all methods (Fig. 1) and receives high bootstrap sup-
port after correction for among-sites rate variation (Ta-
ble 1). The number of molecular synapomorphies found
in the 18S rRNA gene of S. baturini and representatives of
Trichine llida also stron gly sug gests that th ese line ages are
most closely related. The possibility of Dioctophymida
and Trichinellida representing sister groups does not con-
tradict morphological evidence. Both lineages share pe-
culiar features such as the nerve ring positioned far ante-
rior in the pharynx region, terminal (male) or subterminal
(female) position of the anus, an obtuse tail not tapering
toward the end of body, absence of lips and presence of
operculatedeggs. The most striking distinction of Diocto-
phymida, the muscular bursa in males, may be related to
circular carinae on the body wall in some males of capil-
lariids (e.g.,Capillaria anceris Madsen, 1945; C. bursata
Freitas & Almeida, 1934). In addition, some capillariids,
like dioctophymids, require an oligochaete intermediate
host in the life cycle (Spassky, 1956).
Identifying the closest relative of the (Dioctophymida,
Trichinellida) clade is complicated by the fact that the
number of reliable synapomorphies, which would sup-
port their grouping with other dorylaimian clades, is ex-
tremely scarce. To handle this situation, cladistic analy-
sis of individual informative characters may help to dis-
tinguish between competing topologies. This approach
has already been successfully applied to phylogenies of
Vol. 5(4), 2003 621
L.Y. Rusin et al.
Fig. 4. Distribu tion of Â2Pvalues for 39 taxa in the dataset. Pv alues indica te the probab ility that a sequence devi ation from the HKY
model is explained by chance.
Chromadorida, Strongylida and Trefusiidae (Aleshin et
al., 1998a, b; Rusin et al., 2001), and allows us to obtain
reliable molecular evidence for monophyly of all Dory-
laimia, as well as to surmise its possible internal topology
(Fig. 3). The inform ative substi tutions det ected in con ser-
vative regions of the gene have very rare homoplastic oc-
currenc e among nemato des and o ther Bilateria.
Within the limits of this analysis, the problem of
de ning the internal topology of Dorylaimia narrows
down to discriminating between the two conservative
sites within the 18S rRNA which contribute con icting
phylogenetic signal to the data: the deletion of one
base within the V4 region, which supports sister group
status of (Dioctophymida, Trichinellida) with the rest
of Dorylaimia, versus the T!C transition in the V9
region, which suggests Dorylaimida diverged rst within
Dorylaimia.
The V4 deletion is situated within a single-stranded
intern al loop (Van de Peer et al., 2000) or a multistemloop
(Wuyts et al., 2000) of the predicted 18S rRNA molecule
secondary structure. The apomorphic condition in all
cases can be reconstructed as shortening of this segment
and is conserved among the taxa. The T!C transition
in region V9 is located within an imperfect stem of the
native RNA. Many non-dorylaimid nematodes and non-
nematode taxa exhibit uniformity in the structure of this
predicted region. This hypothetical ancestral pattern is
marked b y a no n-convention alpyrimid ine-pyrimid ine pa ir
at the 19th position from the base of hairpin 49 (Fig. 5,
lower part). However, all Dorylaimia are characterised
by an unusual pattern of dislocation of the unpaired
pyrimidine residues and/or their unusual anking motifs
(Fig. 5, upper part). The observed structure of hairpin
49 in extant taxa cannot simply be explained by step-
wise derivations of one extant condition from another.
Thus, it may be speculated that the putative ancestral
pattern was already altered at the level of the common
ancest or of Dory laimia. The a ncestral con dition may h ave
actually be en the destabilisation of the entire stem region
between the 18th and the 20th positions of hairpin 49
caused by a T!C transition and a G!T transversion
(see Fig. 5, in empty circles). These were then presumably
followed by complete or partially compensatory changes
in different subclades of Dorylaimia, either by means
of back mutations or further substitutions within the 50-
branch of the helix (Fig. 5, in grey circles). Without such
speculation, it would be dif cult to minimise the change
in conservativesecondary structure of helix 49 in sampled
Dorylaimia without having to postulate multiple local
substitutions.According to this hypotheticalscenario, the
crucial p lesiomorphy of Dorylaimida appears to repre sent
a reversal from the condition apomorphic for all other
recent Dorylaimia.
This reversa l in rece nt Dorylai mida affects on ly the pri -
mary structure of the molecule and does not restore its
presumed ancestral secondary structure. Analogously,the
reversal at the secondary structure level observed in S.
baturini presumably occurred by means of numerous nu-
cleotid e substitu tions. This exa mple is a good illustration
of Dollo’s law of irreversible evolution in application to
622 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
Fig. 5. Hypothetical scenario of molecular evolution of helix 49 of the 18S rRNA in Dorylaimia. Folding patterns for this region
observed in outgroups are given in the lower part. Substitutions leading to destabilisation of the hypothetical ancestral state of the
loop are given in empty circles. Further substitutions leading to stabilisation of the structure are given in grey circles. YY – a non-
conventional pyrimidine – pyrimidine pair at the 19th position of the hypothetical ancestral structure of the helix.
Vol. 5(4), 2003 623
L.Y. Rusin et al.
macromo lecules. The irreversi bility in this ca se is bro ught
about by the diversity of possibilities of shortening the in-
ternal loop of helix 49. Stabilising one or two pairs out
of the three can be implemented in 18 different ways,
and ve of them are realised in the ve subtaxa of Do-
rylaimia. Only one will lead to restitution of the ancestral
state and it is not observed in any of the sampled Dory-
laimia. If the radiation of Dorylaimia occurred relatively
rapidly and gave rise to a larger number of lineages, the
record of su perimposed mut ations in t he V9 region wou ld
be dif cult to decipher (Cunninghamet al., 1998).
Thus, the only conservative character unlikely to have
experienced superimposed mutations in evolution of the
18S rRNA gene in Dorylaimia is the 1-bp deletion
within the V4 region, a synapomorphy for the clade
((Mononchida, Mermithida), Dorylaimida). Blaxter et al.
(2000) relying on statistical evidence, also conclude that
the occurrence of this clade in 18S rRNA phylogenies re-
ects true evolutionary af nities rather than the conse-
quences of long branch attraction of Trichinellida to the
basal node of the tree.
Combining sequence data and morphology has inter-
esting implications for understanding the general pat-
terns of nematode evolution. Thus, in both topologies,
Mononchida are placed as sister group to Mermithida at
a distal node of the tree. Mononchids do not possess a
spear but do have caudal glands, whilst other Dorylaimia
have a spear but lack caudal glands. The distribution of
the states of these characters over the entire clade makes
phylo genetic assignment sdi f cul t. Two sc enarios are pos-
sible; mononchids lost an ancestral spear and evolved cau-
dal glands, or all other taxa evolved the spear and lost
caudal glands. Speculating about the ancestral life strate-
gies of particular dorylaimian lineages, one could postu-
late that Mononchidaretained the ancestral attachment or-
gans due to their initial radiation in fresh water environ-
ments (and more recent ingress to the soil), whilst Dory-
laimida,one of the dominantnematodetaxa in soils, could
have ori ginated from a so il-dwelling g roup. Trichin ellida,
Dioctophymidaand Mermithidaare all highly specialised
parasite s of un known ance stral h abitat. Alt ernatively, do-
rylaims may have evolved as a group with diversifying
feeding strategies utilising a protrusible spear to pierce
the food organism and ingest its liquid contents, whilst
the mononchid ancestor secondarily developed a captur-
ing apparatus allowing it to sei ze and swallow all or parts
of its prey, thus resulting in a complete reduction of the
spear. Other lineages, among them mermithids, which are
inevitably placed as sister taxon to mononchids in mole-
cular phylogenies (Blaxter et al., 1998), lost the primary
spear function in adults upon shifting to parasitism, yet
still retain its rudiment. Some dorylaims (e.g.,Actino-
laimus) are known to possess both spear and teeth in the
stoma (Jairajpu ri & Ah mad, 199 2). Althoug h the tee th are
not likely to be homologous to those in mononchids,this
case illustrates the possibility of spear-bearing forms ac-
quiring teeth.
Another important morphologicalcharacter,position of
all pharyngeal gland outlets posterior to the nerve ring,
does not t the fact that dioctophymidsare known to have
the glan d ope nings situa ted with in the bu ccal cavit y (viz.,
at the very bottom of the mouth sucker). This peculiar
arrangement of the pharynx glandular structure may be
interpreted as an indication of their closer relationships
with Enoplida. Homology of these systems throughout
Nematoda is not questioned and thus, following topology
B (Fig. 3), will require at least two evolutionary events
of independent shifting of the gland ori ces from the
stoma down into pharynx to t the data, unless one
assumes alternative C, in which case the character would
have changed its status only once in the evolution of
Dorylaimia at the level of the common ancestor of
(Mononchida, Mermithida) and Dorylaimida. On the
basis of molecular evidence (Blaxter et al., 2000), we
assume that the pharynx in Trichinellida and Mermithida
evolved into the stichosome independently.
In all Dory laimia with a c onventional, non-mo di ed
pharynx (i.e., except for Trichinellida, Mermithida and
Muspiceida, the latter not being studied with molecu-
lar methods to date), the pharyngeal gland ori ces open
close to one other into the lumen, either anteriorly in
Diocto phymida, o r po sterior to th e ne rve rin g i n th e mid -
dle part of the pharynx in Dorylaimida and Mononchida.
The anterior position of the ori ces also occurs in out-
groups (Enoplia), which may suggest the ancestral condi-
tion of this character in Dioctophymida. Dioctophymids
are also known to possess a spear-like protractile tooth
(onchiostyle), which is formed ventrally in the anterior
part of the pharynx at earlier stages of postembryonic
development (J1) and disintegrates later. Interestingly, in
common Dorylaimina (at J2-J4 stages) the replacement
tooth is formed posterior to the functional odontostyle
close to the level of gland outlets, while in its putative
sister taxon, Nygolaimina, it is formed almost immedi-
ately posterior in the head region (Coomans & van der
Heiden, 1978). This may suggest that the position of the
spear and pharyngealgland outletsactually marks the bor-
der of the a ncestral stoma i n Dorylaim ia. So far, all ocation
624 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
Fig. 6. Tentative scheme of the evolution of the head region in
Dorylaimia. A: Hypothetical ancestor of the Dorylaimia; B: Re-
cent Dioctophymida; C: Hypothetical transition stage between
the ancestral form and the common lineage of Dorylaimida,
Mononchida and Mermithida; D: Recent Dorylaimida; E: Re-
cent Mononchida.
of all pharyn geal gland ori c es po sterior to t he ne rve ring
is r eported exc lusively for Doryla imida and Mo nonchida.
It cannot be excluded that the stoma in the common ances-
tor of these groups has differentiated to form a functional
stoma and its posterior part (oesophastoma) has merged
with the pharynx and adopted a new function. As a re-
sult, the gland openings became displaced posteriorly in
the pharynx lumen (Fig. 6). An interesting morphological
evidence from pre-parasitic stages (J1) in mermithids fur-
ther supports this idea. The juvenile mermithid pharynx,
which is not yet modi ed into a stichosom e,is clearl y sub-
divided into two zones, the dorsal gland outlet being lo-
cated exactly at the junction of the zones (Poinar & Hess,
1974; Rubtsov, 1977). In many recent Dorylaimina, the
de nitive pharynx is bottle-shaped, its anterior and poste-
rior portions have different musculature arrangement and
the pharyngealglands also open at the junctionlevel. This
hypothesisexplainsthe peculiarpostneuralpositionof the
gland openings in Mononchida by postulating the sec-
ondary loss of the spear at later stages of the posterior
elongationof the oesophastoma.
Thus, adaptive radiation of Dorylaimia gave rise to a
variety of forms, which differ from each other in nerve
ring position, arrangement of glandular ducts and struc-
ture and armature of the mouth cavity. Transformation
of the pharynx into the stichosome and reduction of the
caudal glands and spear were, probably recurring events
in nematode evolution. The common ancestor of Dory-
laimia is likely to have possessed a spear with the pha-
ryngeal gland outlets inside the stoma, a feature gen-
erally lost in recent Dorylaimia with the single excep-
tion of a rare group of Dioctophymida. Thus, the only
non-molecular diagnostic character of Dorylaimia avail-
able remains the unique pattern of early embryogene-
sis, when the endodermal precursor is strictly associated
with the anterior blastomere of the egg. This is charac-
teristic of Mononchida, Dorylaimida (Drozdovskii, 1969,
1975) and Mermithida, Dioctophymida and Trichinellida
(Malakhov & Spiridonov, 1981, 1983; Malakhov et al.,
1984). Variability in cell fate determination in species of
marine Enoplidadistinguishesthis group from both Chro-
madoria and Dorylaimia (Malakhov,1986; Voronovet al.,
1998). Fresh water Triplonchida is a group most proba-
bly linked to Enoplida (Blaxter et al., 1998). According
to molecular evidence, Tobrilus gracilis (Bastian, 1865)
Andrássy, 1959 belongs in the Triplonchida (De Ley &
Blaxter, 2002 ) and is the only representative of this group
for which embryogenesis has been studied so far (Droz-
dovsk ii, 1977). Th e highly varia ble early c ell divisio n in T.
gracilis resembles that of marine Enoplida, the endoder-
mal precursor sometimes arising from the anterior blas-
tomere. Further studies of triplonchids employing tech-
niqu es of uo rescent cell la belling may allow p olarisation
of the state of this character in nematode evolution. Em-
bryologicaldata can distinguish between major nematode
lineages (Drozdovskii, 1975; Voronov et al., 1998; Lahl
et al., 2003), though their interrelationships remain un-
known. Using 18S rDNA sequence data we have de ned
the contents of Dorylaimia. There are few unequivocal in-
dividualmolecular signaturesthat can be used to de ne its
intern al structure . A sing le reliabl e molecula r synapomo r-
phy for the entire Dorylaimia was detected at the 50end
of predicted helix 23 of the18S rRNA secondary struc-
ture model. The frequency of its occurrence beyond Do-
rylaimia is an estimate of 10¡2on the dataset of all ne-
matode sequences currently available. The scantiness of
reliable molecular signatures for major lineages of Do-
rylaimia resembles the situation with marine Enoplida,
where their representatives can only be distinguished by
the presen ce of two low homop lastic substi tutions in ele-
ments of t he 18S rR NA mol ecule primary struc ture (Rusin
et al., 2001).
To concl ude, analysis of se quence da ta combin edwit h a
cladisti c approac h for ana lysing molec ular characte rs pro-
vides support for monophylyof Dorylaimia and de nes its
major internal structure. Comparative analysis of alterna-
Vol. 5(4), 2003 625
L.Y. Rusin et al.
tive phylogenies shows that some of the classic characters
previou sly assign ed sig ni cant value in phy logenetic re-
constructions may not be parsimonious,something which
forces us to reconsider conventionalschemes of morpho-
logical and ecologicalchange in nematode evolution.
Acknowledgements
The authors are greatly indebted to Drs E.M. Droz-
dovskii, V.V. Malakhov, S.E. Spiridonov and M. Blax-
ter for their help and invaluable contribution to the dis-
cussion. We are also thankful to Drs D. Hunt and R.
Cook, and two anonymous reviewers whose comments
were very useful in preparing the manuscript for pub-
lication. Research was supported by the Russian Foun-
dation for Basic Research grants 01-04-48832, 02-04-
48958, and the Department of Industry and Science grant
SS-1712.2003.4.
References
AGU I NA LD O , A. M., TURB E VI L LE , J .M., LIN F ORD , L. S. ,
RIV E RA, M.C ., GA REY, J.R., RAFF, R. & LAK E , J.A.
(1997) . Evidence for a c lade nematod es, arthropods an d other
moulting animals. Nature 387, 489-493.
ALE S HI N , V.V., KE DR OVA, O.S., MILY UTI NA , I.A., VLA DY-
CH E NS KAYA, N. S. & PETROV, N.B. (1998a). Relationships
among nematodes based on the analysis of 18S rRNA gene
sequences: molecular evidence for monophyly of chromado-
rian and secernentian nematodes. Russian Journal of Nema-
tology 6, 175-184.
ALE S HI N , V.V., KE D ROVA, O.S., MILY U TI NA , I.A., VL A-
DYC H EN S KAYA, N. S. & P ETROV, N.B. (1998b). Secondary
structure of some elements of 18S rRNA suggests that
strongylid and a part of rhabditid nematodes are mono-
phyletic. FEBS Letters 429, 4-8.
AND E RS ON, R.C. (2000). Nematode parasites of vertebrates:
their development and transmission
. 2nd edition. Walling-
ford, UK, CABI Publi shing, 672 pp.
BEAV ER , P.C. & THE IS, J.H. (1979). Dioctophymatid larval
nematode in a subcutaneous nodule from man in California.
American Jour nal of Tropical Medi cine and Hygien e 28, 206-
212.
BLA X TE R , M.L., DELE Y, P., GAR EY, J.R ., LIU, L.X.,
SCH E LDE M AN , P., V IERS TAE TE, A., VANFL ETER EN, J.R.,
MAC KEY, L.Y., DORR IS , M., FR IS SE, L.M., VIDA, J.T.
& TH OMA S , W.K . (1998) . A molecular evolutionary fr ame-
work for the phylum Nematoda. Nature 392, 71-75.
BLA X TE R , M.L., DO RRIS , M. & DELE Y, P. (2000). Patterns
and processes in evolution of animal parasitic nematodes.
Nematology 2, 43-55.
CHI TWO OD, B .G. (1933 ). A revise d classi cation of the Nema-
toda. Journa l of Parasitolog y 20, 131.
CHI TWO OD, B.G. & CHITWO O D, M.B. (1977). Introduction
to Nematology. Baltimore, London & Tokyo, University Park
Press, 334 pp.
COO M ANS , A . (2 000). Nematode sys tematics: past, pr esent and
future. Nematology 2, 3-7.
COO M ANS , A. & VAN D E R HEIDEN, A. (1978). The systema-
tic position of the family Ironidae and its relation to the Dory-
laimida. Annales de la Soci été Royal e Zoologique de Belg ique
108, 5-11.
CUN N IN G HA M , C. W., OML A ND, K.E. & OA KLE Y, T.H.
(1998). Reconstructing ancestral character states: a critical
reapprai sal. Trends i n Ecology and Evolution 13, 361-366.
DELEY, P. (2000). Lost in worm space: phylogeny and
morpholo gy as road maps to nemato de diversity. Nematology
2, 9-16.
DELEY, P. & BL AXT ER, M. L. (2 002). Systemat ic position and
phylogeny. In: Lee, D.L. (Ed.). The biology of nematodes.
London, UK, Taylor & Francis, pp. 1-30.
DERIJK, P., WUY TS, J. & DEWACH TER, R. (2003). RnaViz
2: an improved representation of RNA secondary structure.
Bioinforma tics 19, 299-300.
DRO Z DOVS KII , E.M. (1969). [On the analysis of embryogene-
sis in select ed Adenophor ea (Nematoda ).] Doklady Akademii
Nauk SSSR 186, 720-723.
DRO Z DOVS KII , E.M. (1975). [Egg cleavage in the species of
Eudoryaimu s and Mesodoryla imus (Nematoda, Dorylaimida)
and its importance for the assessment of composition of
nematode subclasses.] Doklady Akademii Nauk SSSR 222,
1005-1008.
DRO Z DOVS KII , E .M . (1 977). [On pecu liarities of egg cleavage
and sig ni cance of pr eblastula in the emb ryogenesis of
Nematoda.] Archive s of Anatomy, Histology and Embryology
73, Part 9, 88-94.
EBE R HAR D , M.L. , HURWI T Z, H., SUN, A.M. & C OLE T TA,
D. (1989). Intestinal perforation caused by larval Eu-
strongylid es (Nematoda: Dioc tophymatoida e) in New Jers ey.
American Journ al of Tropical Medici ne and Hy giene 40, 648-
50.
FELS E NS TEI N, J. (1978). Cases in which parsimony or com-
patibility methods will be positively misleading. Systematic
Zoology 27, 401-410.
FELS E NS TEI N, J . ( 1985). Con d ence limit s on p hylogenies: an
approach u sing the b ootstrap. Evolution 39, 783-791.
FELS E NS TEI N, J. (1993). PHYLIP – Phyl ogeny Inference Pack-
age, version 3.5. University of Washington, Seattle, USA.
FIL IPJE V, I.N. (1934). [Nematodes benecial and harmful to
agriculture
.] Moscow-Leningrad, Izdatel’stvo kolhoznoy i
sovhoznoy lit eratury, 440 pp .
HAS E GAWA, M., KIS H IN O , H. & YANO, T. (1985). Dating of
the h uman-ape splitting by a molecular cloc k of mito chondr-
ial DNA. Journal of Molec ular Evolution 22, 160-174.
626 Nematology
The 18S ribosomal RNA gene of Soboliphyme baturini
HER LY N, H., P ISK U RE K , O., SCH M IT Z , J., EHL E RS, R.- U.
& ZI SC H L ER , H. (2003). The syndermatan phylogeny and
the evolution of acanthocephalan endoparasitism as inferred
from 18S rDNA sequences. Molecular Phylogenetics and
Evolutio n 26, 155-164.
HUE L SE N BE C K, J.P. & RO NQ UIS T, F. (2001). MRBAYES:
Bayesian inference of phylogenetic trees. Bioinformatics 17,
754-755.
JAI RAJ PUR I, M.S. & A H MA D , W. (1992). Doryla imida – free-
living, predaceous and plant-parasitic nematodes. Leiden,
The Net herlands, E.J . Brill, 4 58 pp.
KAR M AN OVA, E.M. (1968). [Dioctophymidea of animals and
man and diseases caused by them.] Fundamentals of Nema-
tology 20, 1-383.
KISHINO, H. & HASE GAWA, M . (198 9). Evaluation of th e max-
imum likeli hood estima te of the evoluti onary tree topol ogies
from DNA sequence data, and the branching order in homi-
noidea. Journal of Molecular Evolution 29, 170-179.
KOR B ER , B., MUL D OO N , M. , TH E ILE R , J., GA O, F., GU P TA,
R., LAP E DES , A., HAHN , B.H., WOLINSK Y, S. & BHAT-
TACH A RYA, T. (2 000). Timing th e ancestor of the HIV-1 pan-
demic st rains. Science 288, 1789-1796.
LAH L , V., HA LAMA , C. & SCH EIR E NB E RG, E. (2003). Com-
parative and experimental embryogenesis of Plectidae (Ne-
matoda). Deve lopment, Genes and Evo lution 213, 18-27.
LOR ENZE N, S. (1981). Entwurf eines phylogenetischen Sys-
tems der freilebenden Nematoden. Veröffentlich ungen des In-
stituts für Me eresfoschung in Bremerhave n, Supplement 7, 1-
472. Translated to Engli sh by J. Gr eenwood and pu blished in
1994 as The phylogenetics of freeliving nematodes. London,
The Ray Society, 383 pp.
MAL A KH OV, V.V. (1986). [Nematodes: structure, develop-
ment, classi cation and phylogeny.] Moscow, Nauka, 215 pp.
Translated to English in 1994 and published by Smithsonian
Institution Press (W.D. Hope, Ed.). Washington & London,
286 pp.
MAL A KH OV, V.V. & SP IR ID O NOV, S.E. (1981). [Embryonic
development of Gastromermis sp.] Zoologichesky Zhurnal
60, 1574-1577.
MAL A KH OV, V.V. & SP IR ID O NOV, S.E. (1983). [Embryonic
development of Eustrongylides excisus (Nematoda, Diocto-
phymida). ] Zoologichesky Zhur nal 62, 113-117.
MAL A KH OV, V.V., RO MA S HEV, B. V. & SP I RI DON OV, S. E.
(1984) . [Embryonic developmen t of Trichocephalus tr ichuris
and Eucoleus oesophagicola (Nematoda, Trichocephalida).]
Parazitologiy a 18, 286-290.
MEA S UR E S, L. ( 1985). Centra rchid sh as parat enic hosts of the
giant kidney worm, Dioctophyme renale.Journal of Wildlife
Diseases 26, 11-19.
MED L IN , L., ELWO OD, H.J., STI C KEL, S. & SO GIN , M.L.
(1988) . The charact erization of enzymatically ampli ed eu-
karyoti c 16S-like rRNA-coding r egions. Gene 71, 491-499.
NEE F S, J .-M., VAN D E PE ER , Y., DERIJ K, P., C H AP ELLE , S.
& DEWAC H TE R , R. (1 993). Compilatio n of small ri bosomal
subunit RNA structures. Nucleic Acids Research 21, 3025-
3049.
OLSE N, G.J. , MATS UDA, H., HAGSTROM, R. & OVERB E EK ,
R. (1994). fastDNAml: a tool for construction of phyloge-
netic trees of DNA sequences using maximum likelihood.
Computer Applicati ons in t he Biosci ences 10, 41-48.
PAVLIN OV, I.I. (1990). [Conception of the outgroup in cladis-
tics.] Zhurnal Obshchei Biologii 51, 304-315.
PETR OW, A.M. (1930). Zur Characteristic des Nematodes aus
Kamtchatken Zobelen Soboliphyme baturini nov. gen., nov.
sp. Zoologis cher Anzeiger 86, 265-271.
PHI LIP P E, H. (2000). Opinion: long branch attraction and
protist p hylogeny. Protist 151, 307-316.
POINAR, G.O., JR& HESS , R. (1974). Structure of the pre-
parasitic juveniles of Filipjevimermis lepisandra and some
other Me rmithidae (Nematoda). Nematologica 20, 163-173.
RUB TSOV, I.A. (1977). [Mermithids. Origin, biology and geo-
graphical di stribution
.] Len ingrad, Nauka, 1 91 pp.
RUS IN, L.Y U., AL ESH IN, V.V., VLA DY CHE N SK AYA, N.S.,
MILY UTINA , I.A., KEDR OVA, O.S. & PET R OV, N.B.
(2001). Trefusiidae are a subtaxon of marine Enoplida (Ne-
matoda): evidence from primary structure of hairpin 35 and
48 loops of SSU rRNA gene. Mol ecular Biology 35, 778-784.
RYZ H IKOV, K. M. & SO N IN , M. D. (1 981). [Class i cation of
nematode parasit es of verteb rate animals.] Parazit ologyia 15,
510-518.
SCH MID T-RH AES A , A. (2000). Scanning electron microscopy
of the anterior end of Hystri chis tricolor (Nematoda: Diocto-
phymida) . Russian Journal of Nematology 8, 133-138.
SPAS SKY, A.A. (1956). [On the systematics of aphasmidian
nematodes.] Annals of the Helminthological Laboratory of
the Academy of Science 8, 159-164.
STRI MM E R , K. & VO N HAE SEL E R, A. (1996). Quartet puz-
zling: a quartet maximum likelihood method for reconstruct-
ing tree topologies. Molecular Biology and Evolution 13,
964-969.
SUD H AU S, W. & FIT C H, D . (200 1). Comparative studi es on the
phylogeny and systematics of the Rhabditidae (Nematoda).
Journal of Nematology 33, 1-70.
VAN D E PEER , Y., DERIJ K, P., WU Y TS, J., WINKELMANS,
T. & DEWAC H TE R , R . (2 000). The Eur opean Small Subuni t
Ribosomal RNA database. Nucleic Acids Research 28, 175-
176.
VOR ON OV, D.A., PANCH IN, Y.V. & SPIRI D ON OV, S.E.
(1998). Nematode phylogeny and embryology. Nature 305,
28.
WHE L AN , S., LI Ò, P. & GOLDM AN, N. (2001). Molecular
phylogenetics: state-of-the-art methods for looking into the
past. Trends in Genetics 17, 262-271.
WOE S E, C.R. , GUT ELL , R., GUP TA , R. & NOL LER , H.F.
(1983). Detai led analysis o f the hig her-order structu re of 16S-
like rib osomal ribonu cleic acids. Microbiologi calRevi ews 47,
621-669.
Vol. 5(4), 2003 627
L.Y. Rusin et al.
WUY T S, J., DERI JK, P., VAN DE PE E R, Y., PI SO N, G.,
ROU S SE EUW, P. & DEWACH T ER , R. (2000). Comparative
analysis of more than 3000 sequences reveals the existence
of two pseudoknots in area V4 of eukaryotic small subunit
ribosomal RNA. Nucleic Acids Research 28, 4698-4708.
YANG, Z. (199 4). Maximum likel ihood phylogen etic estimation
from DNA sequences with variable rates over sites: approxi-
mate methods. Journal o f Molecular Evolution 39, 306-314.
YANG, Z. (1996). Among-site rate variation and its impact on
phylogenetic analyses. Trends in Ecology and Evolution 11,
367-370.
ZUK E R, M., MATHEW S, D.H. & TUR NER , D .H. ( 1999). Algo-
rithms and ther modynamics for RNA secon daryst ructure pre-
diction : a practi cal guide. In: Bar ciszewski, J. & Clark, B.F.C.
(Eds). RNA biochemistry and biotechnology
. NATO ASI Se-
ries, Klu wer Academic Pub lishers, pp. 1 1-43.
628 Nematology
