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Insect Molecular Biology (2002) 11(5), 453 – 465
Elongation Factor 1 α resolves the monophyly of
the haplodiploid ambrosia beetles Xyleborini
(Coleoptera: Curculionidae)
Blackwell Science, Ltd
B. H. Jordal
Department of Zoology, University of Bergen, Allegt 41,
N-5007 Bergen, Norway
Abstract
Elongation Factor 1-α was used to test the monophyly
of the wood boring beetle tribe Xyleborini, where
all species are haplodiploid and perform regular
inbreeding by brother–sister mating. Due to their
feeding requirements, being highly dependent on
ophiostomatoid fungi which they cultivate in wood
tunnels, monophyly may be expected due to nutritional
constraints. During the course of analyses, two copies
α were amplified in these beetles, differing in
of EF-1α
intron structure. The high similarity between paralogous amino acid sequences (93–94%) indicates a
rather recent duplication in beetles, but phylogenetic
analyses of different copies in insects rejected this
hypothesis. Subsequent phylogenetic analyses of
eighty orthologous sequences from Xyleborini and
allied taxa, using the single-intron bearing copy, were
greatly improved in resolution and node support by
including the intron sequences (c. 60 bp). Most analyses resulted in a monophyletic Xyleborini, implying
one origin of fungus feeding in this tribe. However,
clear evidence for a polyphyletic Xyleborus and three
more xyleborine genera calls for further revision of
xyleborine classification.
Keywords: haplodiploidy, sib-mating, gene duplication,
intron sequences, weevils.
Introduction
One of the largest radiations in wood-boring phytophagous
beetles coincided with the evolution of sib-mating, haplodiploidy and fungus feeding in scolytine weevils of the
Received 12 February 2002; accepted after revision 10 June 2002. Correspondence and present address: School of Biological Sciences, University of
East Anglia, Norwich NR4 7TJ, U.K. E-mail: [email protected]
© 2002 The Royal Entomological Society
pantropical Xyleborini (Normark et al., 1999; Farrell et al.,
2001; Jordal et al., 2002a). While the origin of sib-mating
and haplodiploidy occurred in an ancestor shared by three
closely related sib-mating genera of Dryocoetini (160 spp.),
the obligate feeding on ophiostomatoid fungi (cultivated
in wood tunnels) occurs only in the much more species rich
Xyleborini (c. 1300 spp.). Altogether, these sib-mating taxa
constitute a monophyletic clade containing nearly onequarter of the 6000 described species of scolytine weevils
world-wide (Wood & Bright, 1992; Bright & Skidmore,
1997). Their ecological significance is most prominent in
lowland tropical forests, where these insects constitute
more than half the species or insect body mass in guilds of
early stage wood-decomposers (Schedl, 1956; Browne,
1961). Many of the haplodiploid, sib-mating species are
also extremely widespread in the tropics and an increasing
number of species have recently been introduced and
established outside their native ranges, perhaps promoted
by their genetic system in conjunction with sib-mating
(Jordal et al., 2001). The wood boring and fungus cultivating
Xyleborini pose a significant problem to the timber trade,
and much attention has been directed towards suppression
of the spread and growth of these beetles. On a brighter
side, these beetles have proven extremely informative in
studies on a wide range of general biological and ecological
systems, which in turn informs management of their impact
on diverse forest products.
The great potential for culturing lineages of xyleborine
species in the laboratory, using artificial diets (Norris &
Baker, 1967; Norris & Chu, 1970), creates a rich model
system to study the developmental and evolutionary genetics
of the group. However, comparative studies on variation
within the inbreeding mating system or haplodiploid genetic
system, including microbial function in haploid male
development from unfertilized eggs (cf. Peleg & Norris,
1972), calls for a reliable hypothesis on their genealogical
history. Recent phylogenetic studies have demonstrated
difficulties with resolving the monophyly of the fungus
feeding Xyleborini, with respect to the sib-mating dryocoetine
genera (Normark et al., 1999; Jordal et al., 2000; Farrell
et al., 2001; Jordal et al., 2002a). Based on these previous
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B. H. Jordal
results, two hypotheses remain: either (a) obligate fungus
feeding is a purely derived feature, implying monophyly
of the Xyleborini; or (b) Xyleborini gave rise to some or all
of the sib-mating dryocoetines, implying one or several
reversals to the ancestral phloem feeding habit. It has been
argued (Jordal et al., 2000) that the latter hypothesis is the
least probable of the two due to the assumed irreversibility
of fungus feeding. Under the latter scenario, fungus feeders
are dependent on ergosterol and other steroid components
from the fungi to initiate egg hatching and complete development (Kok et al., 1970), a dependence not observed in
true bark beetles (e.g. Kukor & Martin, 1989; Six & Paine,
1998).
Partial sequences from the nuclear protein encoding
gene Elongation Factor 1α (EF-1α) were used to test the
proposed hypotheses of xyleborine phylogeny. This is a
low-copy gene that promotes the GTP-dependent binding
of aminoacyl-tRNA to ribosomes, and shows considerable
conservatism in amino acid substitution rate (e.g. Uetsuki
et al., 1989). EF-1α has also proven very informative in a
wide range of phylogenetic analyses of arthropods (e.g.
Shultz & Regier, 2000) and insects (e.g. Cho et al., 1995;
Mitchell et al., 1997; Cryan et al., 2000; Cruickshank et al.,
2001; Rokas et al., 2001; Danforth, 2002). Used in combination with other nuclear and mitochondrial genes in
analyses of other scolytine beetle groups, EF-1α provided crucial data for obtaining well resolved topologies
(Sequeira et al., 2000; Cognato & Vogler, 2001; Jordal
et al., 2002b; Sequeira & Farrell, 2001). The major radiation
in haplodiploid beetles probably occurred rapidly as far back
as in the early Miocene – a hypothesis based on a general
high level of sequence divergence in combination with
the absence of such beetles in Oligocene amber (Jordal
et al., 2000; Farrell et al., 2001). The relatively low substitution rate in EF-1α may help to resolve the shallow
internodes characterizing the early stages of this group’s
phylogenesis.
Multiple copies of EF-1α have been described from
Drosophila flies (Hovemann et al., 1988) and Apis bees
(Danforth & Ji, 1998), and this is probably a universal
feature throughout the two insect orders. Normark (1994)
found no evidence for multiple copies of EF-1α in his study
on Aramigus weevils, but a second putative copy has more
recently been suggested for scolytine weevils (Normark
et al., 1999). This second copy has not yet been characterized and compared to the commonly used copy. Because
‘cryptic’ paralogous sequences would interfere in phylogeny reconstruction, efforts should be made to ensure that
only orthologous sequences are included in a phylogenetic
analysis. Hence, DNA sequences from each of the two
putative EF-1α copies were obtained from four scolytine
species, to test the monophyly of each copy (and shared
intron structure) in a phylogenetic framework. Eighty orthologous sequences with identical intron structure (921
aligned characters) were used in the final analyses to test
the monophyly of Xyleborini.
Results and discussion
Multiple copies of EF-1α
A majority (fifty-two out of eighty) of the PCR amplified
products obtained from Xyleborini and sib-mating Dryocoetini revealed two bands that were clearly separable on
a low melting agarose gel. Four of these double-banded
products were selected for further comparison between
the two putative copies. After sequencing and removal of
introns, none of the four sequences from each of the two
different-length fragments contained indels or stop codons,
and all sequences aligned perfectly with other insect
sequences obtained from GenBank. This may suggest that
both copies are functional although 498 bp of the coding
region (1392 bp in bees and flies) were not sequenced.
The inferred amino acid sequences for the two copies
showed considerable similarity with each other as well as
to other published insect EF-1α sequences. Amino acid
distances between the two copies in scolytine beetles
ranged between 5.9 and 7.7%, well below the differences
found between the two Apis copies and between the two
Drosophila copies (9.0%). It is also well below the smallest
difference measured between homologous EF-1α copies
from different arthropod orders, including apterygote
insects (Regier & Shultz, 2001). The corresponding nucleotide sequence divergence between the two copies in
beetles ranged between 22.0 and 27.1%.
The two copies in beetles differ by the number of introns;
the shortest fragment (C1) had one intron and the longest
fragment (C2) had three introns in the region sequenced
(149–1043, cf. Fig. 1C). All four introns were short, ranging
between 50 and 90 base pairs. Both beetle copies shared
the placement of the intron in position 753/754, which
is equal to the placement of intron 2 in the bee F2 copy
(Danforth & Ji, 1998) and in aphids (Normark, 1999). C2 had
a second intron in position 1029/1030, equal to the bee and
fly F2 copies, and in aphids and booklice [but not lice
(Cruickshank et al., 2001)], and a third intron not observed
in any other insect, in position 430/431. Because so many
of the scolytine beetles did amplify two copies, it is not likely
that any of these copies contain another intron adjacent to
the primer site (149), as observed in position 143/144 in the
bee F2 copy and in Protura (Carapelli et al., 2000).
Given the fixed differences in intron structure between
the two beetle copies, and the much lower divergence
levels within than between the copies at both the nucleotide
(10.6 vs. 22%) and amino acid (2.9 vs. 5.9%) levels, it
seems likely that EF-1α copies with shared intron structure
in beetles are orthologous. However, a phylogenetic analysis of the different copies in insects can more precisely
determine the homology of shared intron structure and
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Elongation Factor 1-α resolves the monophyly of Xyleborini
455
Figure 1. Phylogeny of EF-1α sequences from various insects and placement of introns in different copies. (A) Strict consensus of the two most parsimonious
trees (CI = 0.38, RI = 0.61) resulting from the analysis of 864 bp of EF-1α with first and second positions weighted ten times third positions. Bootstrap support
values higher than 50% are shown above internodes; values in bold mark the monophyly of the haplodiploid beetles, for each copy. The tree was rooted by the
remiped crustacean Speleonectes. (B) Same as A, but with the long branched paralogs Apis F1, Drosophila F2 and the beetle C2 sequences removed, resulting
in one most parsimonious tree (CI = 0.43, RI = 0.61). (C) An overview of known intron positions (triangles) for some well studied insect groups. The stippled
vertical lines indicate positional homology among introns and the length of horizontal lines shows the maximum length sequenced within each insect order.
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B. H. Jordal
the timing of duplication events. Thus, one crustacean
and twenty-three additional insect EF-1α sequences were
added to a phylogeny matrix and subjected to a variety of
phylogenetic analyses (see methods). All parsimony and
likelihood analyses of nucleotides (or amino acid parsimony) resulted in monophyly of each beetle copy, and the
two beetle copies were not grouping together in any of
the analyses (Fig. 1A). Given these results, duplication of
EF-1α in beetles most likely predates the origin of beetles.
The likelihood analysis using the GTR+Γ+I model was
largely consistent with some of the parsimony analyses
(unweighted, 5:10:2, 5:10:1), placing the C2 copy closest to
the Apis F1 copy and nested within the Diptera clade, which
also contained the Drosophila F2 copy and the Metajapyx
sequence. All other weighted parsimony analyses (10:10:5,
10:10:2, 10:10:1) placed the beetle C2 + Apis F1 as sister
clade to the Hymenoptera F2 clade (Fig. 1A). When the
paralogs with the longest branches (beetle C2, Apis F1 and
Drosophila F2 sequences) were removed, some weighting
schemes (10:10:1, 5:10:1, 5:10:2) successfully recovered a
topology (Fig. 1B) consistent with current phylogeny of insect
orders (Wheeler et al., 2001). Replacing the C1 sequences
with the C2 paralogs did not recover this topology in any of
the analyses, disfavouring C2 as a phylogenetic marker.
Although the basal relationships among the different
insect EF-1α copies were ambiguously resolved, all phylogenetic analyses suggest frequent intron gains and losses
in insects (Fig. 1A,C), consistent with Danforth & Ji’s (1998)
previous analysis of insect EF-1α sequences. Intron loss
in EF-1α has also been invoked for a variety of apterygote
insects (Carapelli et al., 2000), as well as in lice vs. thrips,
aphids and booklice (e.g. Cruickshank et al., 2001). In
addition, deuterostome taxa show a similar trend of
frequent loss of positional identical introns in the same
gene (Wada et al., 2002). Also, frequent intron loss has
been observed in other nuclear genes, for instance the
dipteran white gene (Krzywinski & Besansky, 2002), and
recent origins of introns have been detected in the
xanthine dehydrogenase gene in Drosophila, favouring
the ‘intron-late’ hypothesis (Tarrìo et al., 1998). These
findings all argue against the traditional conservative view
of intron evolution (e.g. Rokas et al., 1999; Venkatesh et al.,
1999).
To conclude, intron structure then may not be a good
indicator of deep homology in insect EF-1α phylogenies.
However, the strongly supported monophyly of each of the
two putative beetle copies (Fig. 1A,B) suggest that intron
structure is a sufficient orthology criterion in beetles. In this
study, eighty C1 sequences of shared single-intron structure
were obtained for further analyses of xyleborine relationships. Given the congruent placement of C1 within the
Holometabola (Fig. 1B), and the lower substitution rate in
C1 than in C2 (average 8.0 vs. 10.6% in four scolytine spp.),
it seems prudent to also select the C1 copy in further work
on EF-1α beetle phylogenies.
C1 sequence evolution in Xyleborini
The base composition of the C1 sequences (Table 1) had
a distinct T-bias in third positions and in the intron, a bias
also observed in the C2 sequences. Base frequencies
were also very similar to those measured in bees (Danforth
et al., 1999; Danforth & Ji, 2001), but less so to butterflies
(Reed & Sperling, 1999), a seemingly phylogenetic bias
in accord with recent holometabolan phylogeny (Wheeler
et al., 2001; cf. Fig. 1B).
The maximum nucleotide divergence in the coding
region for the haplodiploid ingroup was 11.4% (12.6% HKY
corrected) between Cnestus suturalis and Cyclorhipidion
pruinosum. However, most pair-wise comparisons were below
10% (cf. Fig. 2A), well below the minimum nucleotide divergence between the two different copies (22.0%). The rather
low substitution rate in C1 seems advantageous judged by
the lack of saturation in exon transversions and transitions
for the ingroup (Fig. 2A). More surprisingly perhaps was
the similar properties revealed by the single C1 intron.
Although the divergence scatter was more dispersed, the
C1 intron clearly mimics the C1 exon by showing a steady
increase in its uncorrected distances plotted against the
HKY corrected exon distances (Fig. 2B). Despite the much
higher substitution rate and considerable AT bias in the
intron (Table 1), the ratio of transitions to transversions is
still higher than unity, suggesting a far from arbitrary substitution pattern. This may seem contrary to the commonly
held opinion that intron sequences evolve so rapidly that
they should be excluded in phylogenetic studies due to
ambiguities with alignments. However, introns have been
Table 1. Properties of the different subpartitions of the EF-1α C1 fragment in scolytine beetles (n = 80). Ti/tv ratios are averaged over the most likely tree
topology (see Fig. 5). None of the χ2 values exceeded the critical value in the homogeneity test of base frequencies across taxa
Partition
Characters
Variable
Informative
ti/tv ratio
Adenine
Cytosine
Guanine
Thymine
χ2
Pos 1
Pos 2
Pos 3
Intron*
All
282
282
281
76
921
40
19
261
72
392
26
12
240
68
346
3.55
0.77
4.12
1.27
–
0.29
0.31
0.21
0.26
0.27
0.17
0.24
0.25
0.15
0.22
0.39
0.16
0.17
0.12
0.23
0.15
0.29
0.37
0.47
0.28
9.8 ns
5.8 ns
157.3 ns
113.0 ns
64.9 ns
*Includes 6 gap-length coded characters.
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Elongation Factor 1-α resolves the monophyly of Xyleborini
Figure 2. (A) Pair-wise uncorrected exon distances (all positions) between
the eighty C1 sequences (ti = transitions and tv = transversions), plotted
against HKY corrected C1 exon distances. (B) Pair-wise uncorrected
distances of the C1 intron plotted against HKY corrected C1 exon distances.
used successfully in aphids (Normark, 1999) and proved
crucial to resolve certain relationships in halictid bees
(Danforth et al., 1999; Danforth & Ji, 2001; Danforth, 2002).
Based on the useful properties of the intron in this EF-1α
study, these data are included in the phylogenetic study of
the C1 sequences.
Phylogeny estimation of xyleborine C1 sequences
Incorporation of the intron greatly improved the maximum
parsimony (MP) and minimum evolution (ME) trees in
Table 2. Number of bootstrap supported (BP) nodes with and without the
intron included, under the MP and ME optimality criteria. Increase per node
was calculated over nodes with more than 50% bootstrap support in the
exon analyses
Maximum parsimony
Minimum evolution
Partition / BP
50%
90%
Increase
per node
50%
90%
Increase
per node
Exon
Exon + intron
40
46
21
31
6.4%
43
48
26
30
3.3%
457
terms of an increased number of nodes supported and by
an average increase in support per node (Table 2). All of
the majority bootstrap supported nodes from the exon
analysis were also supported in the analyses of all data
combined; hence, I only report details from the latter analyses. It also led me to analyse only the full data set in the
much more computationally intensive maximum likelihood
(ML) analysis.
Given the large sequence divergences and many
sequences included, it is not surprising to find some
discrepancies between the different phylogenetic methods.
However, the large proportion of identical clades is striking
and indicates secondary support for many of the weakly supported and shallow nodes. Moreover, the longest branches,
which could cause long branch attraction of unrelated
taxa, did not seem to introduce additional ambiguity.
Judged by the similar groupings found in the long branch
correcting ML analysis and in the MP analysis, for instance
by Eccoptopterus, Webbia and allied taxa (Figs 3–5), long
branch problems seem minor. On the other hand, the ME
and ML analyses did manage to remove the obviously
misplaced Xyleborus and Cyclorhipidion taxa from a paraphyletic Xylosandrus in the MP analysis (Fig. 3).
All analyses supported the sistergroup relationship
between Dryocoetes and the sib-mating clade, and
between Ozopemon and the remaining sib-mating genera.
This is in accordance with former results (Jordal et al.,
2000; Jordal et al., 2002a), but the more extensive sampling
of xyleborine species in this study demonstrated increased
resolution in accordance with morphological classification
(Wood, 1986). First of all, the Coccotrypes plus Dryocoetiops
were monophyletic in the MP and ML analyses and were
parapatrically distributed basally in the sib-mating clade in
the ME analyses. Species of these two genera combined
had identical internal topologies in all analyses, which also
were identical to a recent analysis of Coccotrypes based
on three genes and morphology (Jordal et al., 2002b). This
indicates great reliability of EF-1α at this level of phylogenetic
analysis. Second, the Xyleborini appeared monophyletic in
the ML analysis, nearly so in the ME analysis (ex. Coptodryas),
and monophyly was not contradicted by the MP analyses.
Hence, this is the first molecular study to support the simultaneous monophyly of each of the Xyleborini and Coccotrypes (including Dryocoetiops). However, neither of these
two clades were supported by synapomorphic amino acid
or base substitutions – as would be expected given the
shallow divergence between the two groups. Substitutional
reversals are also an expected outcome in analyses of
this many sequences of Miocene age (Jordal et al., 2000;
Farrell et al., 2001), potentially obscuring the synapomorphic
signature from ancient cladogenesis.
All analyses also supported the monophyly of the
following genera: Xyleborinus (related to Coptodryas),
Webbia (related to Dryoxylon) and Theoborus (related to
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458
B. H. Jordal
Figure 3. Strict consensus tree of twenty-one most parsimonious trees produced with all characters equally weighted (length 2456, CI = 0.29, RI = 0.47).
Bootstrap majority support values are written above the internodes. The tree was rooted by the dryocoetine outbreeding genera Thamnurgus, Lymantor and
Dryocoetes. Capital letters indicate country of origin for the two species with multiple samples: CR, Costa Rica; J, Japan; U, Uganda; PNG, Papua New Guinea.
Coptoborus). The ME analysis resulted in a monophyletic
Xylosandrus (provided that mutilatus should be placed in
Cnestus) and nearly so in the ML analysis (Figs 4, 5). More
characters may help to consistently group this morpholo-
gically characteristic genus. Ambrosiodmus and Euwallacea
appeared polyphyletic with respect to Xyleborus, within
which these were formerly included (Wood, 1986). Although
species of the first two genera are sometimes difficult to
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Elongation Factor 1-α resolves the monophyly of Xyleborini
459
Figure 4. Single tree with lowest score (3.083) resulting from the Minimum Evolution analysis of maximum likelihood distances. A GTR+Γ+I model was selected
by the procedure described and implemented in ‘Modeltest’ (Posada & Crandall, 1998). Parameters estimated from the Neighbour Joining tree in ‘Modeltest’
were refined on an initial ME tree, with marginal changes. The final parameters were: estimated base frequencies, I = 0.554, Γ = 1.346 with four rate categories,
and six substitution types with the following estimated frequencies: A↔C 1.07, A↔G 8.20, A↔T 1.56, C↔G 1.30, C↔T 9.78, G↔T 1.00.
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460
B. H. Jordal
Figure 5. The most likely tree topology resulting from the Maximum Likelihood analysis using five random additions of heuristic searches (score 12246.130).
Model and parameter settings were similar to the ME analysis (see Figure 4). Arrows point to evolutionary changes as follows: sib-mating, all species have
strongly female biased broods or male is unknown; haplodiploidy confirmed, all examined species in Coccotrypes, Xylosandrus and Xyleborus are haplodiploid;
fungus feeding, all species in the tribe Xyleborini feed upon the ambrosia fungi they cultivate in wood tunnels. Three independent origins of horned males are
indicated by ‘H’.
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Elongation Factor 1-α resolves the monophyly of Xyleborini
separate morphologically, the putatively unrelated Ambrosiodmus colossus is unique by having males with a large
horn-like projection on the anterior edge of pronotum. This
character seems phylogenetically conservative with only
three origins traced on the various topologies (Figs 3–5).
That A. colossus groups exclusively with Xyleborus species
also having this feature (see Fig. 5), may suggest that this
species is more correctly placed by the EF-1α data than in
current classification (Wood & Bright, 1992). Furthermore,
the EF-1α data clearly rejected any relationship of Ambrosiodmus and Euwallacea to the putative close genera
Amasa and Cyclorhipidion (according to Wood, 1986). On
the other hand, most species of the latter two genera defined
a well supported clade also containing Arixyleborus and
one species of Xyleborus. This clade was the largest
strongly supported clade in the Xyleborini and occurred
in all analyses. All these species have rather narrow protibiae
with more than ten socketed lateral teeth and strongly reticulate
and dull elytral declivity, characters that may provide some
clues towards finding good synapomorphies for this group.
The clear evidence for a polyphyletic Xyleborus,
Cyclorhipidion, Ambrosiodmus and Euwallacea may be
expected based on previous extensive reports on specific
as well as generic synonyms, and recent work has emphasized the preliminary nature of the current classification
(Wood, 1986; Wood & Bright, 1992; Bright & Skidmore,
1997). Thus, rather than claiming the inadequacy of the EF1α data, it seems more productive to argue for an extensive
revision of all genera in Xyleborini. In this regard, the EF1α data point to several presumably misclassified species
in addition to A. colossus. One of these, Xylosandrus mutilatus,
groups morphologically with Cnestus by the short anterior
segment 1 of the antennal club, contiguous procoxae and
similarly shaped pronotum and protibiae, and groups with
strong support with Cnestus suturalis in all analyses.
Another example is Dryoxylon onoharaensum, tentatively
placed in Dryocoetini by Bright & Rabaglia (1999), but
molecular as well as biological data suggest otherwise (see
also Jordal et al., 2000). The single species of this genus is
wood boring and possibly cultivate ambrosia fungi for food
(Bright & Rabaglia, 1999). More importantly, the suggested
dryocoetine diagnostic characters (Wood, 1986) do not
exclude membership of the Xyleborini insofar as there are
more than twenty species of Xyleborus that also match the
tribal description of dryocoetine females (R. A. Beaver, pers.
comm.). For instance, most species related to X. dolosus, X.
subdentatus and X. fallax (cf. Figs 3–5) do not have depressed
pregula and have narrow meso- and metatibiae with few
socketed teeth. It is particularly interesting to observe in the
ML analysis the basal position of these taxa in Xyleborini
(Fig. 5). Thus, it seems conceivable that the shallow internodes distinguishing the sib-mating Dryocoetini and Xyleborini
simply reflect the small and gradual morphological differences observed between the two biologically distinct groups.
461
Conclusion
Neither molecular nor morphological markers provide clear
cut results to distinguish xyleborine beetles from their
sib-mating dryocoetine sister group, despite their fundamental differences in feeding behaviour. Although the phloem
based diet of true bark beetles sometimes also contain
ophiostomatoid (ambrosia) fungi, the exclusive and
obligate feeding upon such domesticated asexual fungi
(Beaver, 1989) has probably made the Xyleborini developmentally dependent upon this food resource (Kok et al.,
1970). Reversal of fungus feeding under such circumstances
seems quite unlikely, if not impossible. Thus, the molecular
support for a monophyletic Xyleborini, albeit weak, must be
viewed in this light. Taken together with these arguments,
the EF-1α data thus provide a reasonably good estimate of
haplodiploid beetle phylogeny and xyleborine monophyly.
That several mitochondrial and nuclear genes have proven
insufficient in previous analyses of the haplodiploid clade
(Normark et al., 1999; Farrell et al., 2001; Jordal et al.,
2002b), further points to the inherent difficulties in resolving
the phylogeny of this group. Nonetheless, the improvements
achieved by increased taxon sampling in this study not only
shows the importance of the latter, but also demonstrates
great properties of EF-1α as a phylogenetic marker for
Miocene radiations (see Jordal et al., 2000). Finally, the
inclusion of intron sequences proved very informative, and
arguments for excluding such partitions in future may not
be well founded.
Experimental procedures
Sampling
All sequenced taxa are listed in Table 3, including their GenBank
accession numbers. Among these, twenty-six sequences were
determined in this study. Twenty-one of the twenty-six described
genera nested in the haplodiploid clade sensu (Normark et al.,
1999) have been sampled excluding Premnobius, which does not
belong to this clade, and Mesoscolytus, which is a synonym of
Xyleborus (see Beaver, 1998). For two morphologically variable
species, Eccoptopterus spinosus and Coccotrypes advena, two
sequences per species were included for intraspecific comparisons. Voucher specimens taken from the same brood as the
sequenced specimens were pinned or kept in ice cold ethanol at
the Museum of Comparative Zoology, Harvard University, or in the
Department of Zoology, University of Bergen.
PCR amplification and DNA sequencing
Genomic DNA was extracted from single or half (thorax) specimens by the Qiagen DNeasy kit™. Partial EF-1α gene
fragments were amplified by the polymerase chain reaction (PCR)
technique using the following primers designed by Normark
et al. (1999): efs149, 5′-ATCGAGAAGTTCGAGAAGGAGGCYCARGAAATGGG-3′ (forward); efa1043, 5′-GTATATCCATTGGAAAT T TGACCNGGRTGRTT-3′ (reverse); efa923, 5′ACGTTCTTCACGTTGAARCCAA-3′ (reverse). Amplification
© 2002 The Royal Entomological Society, Insect Molecular Biology, 11, 453 – 465
IMB_354.fm Page 462 Wednesday, August 28, 2002 10:49 AM
462
B. H. Jordal
Table 3. Taxa sampled, including biological features and GenBank accession numbers. Possibly new or unidentified species are denoted by ‘cf.’ which indicates
affiliation with the closely related species given by the specific epithet
Tribe
Copy 1
Dryocoetini
Dryocoetini?
Xyleborini
Species
Mating
system
Breeding
site
Feeding
tissue
GeneBank
accession no.
Lymantor coryli (Perris)
Thamnurgus senecionis Schedl
Thamnurgus lobeliae Eggers
Dryocoetes affaber (Mannerhein)
Dryocoetes autographus (Ratzeburg)
Ozopemon uniseriatus Eggers
Ozopemon brownei Schedl
Coccotrypes advena Blandford – Japan
Coccotrypes advena Blandford – Costa Rica
Coccotrypes cardamomi Schaufuss
Coccotrypes cf. cardamomi Schaufuss
Coccotrypes carpophagus (Hornung)
Coccotrypes cyperi (Beeson)
Coccotrypes dactyliperda (Fabricius)
Coccotrypes cf. distinctus (Motschusky)
Coccotrypes gedeanus (Eggers)
Coccotrypes graniceps (Eichhoff)
Coccotrypes impressus Eggers
Coccotrypes litoralis (Beeson)
Coccotrypes longior (Eggers)
Coccotrypes marginatus (Browne)
Coccotrypes medius (Eggers)
Coccotrypes petioli Blandford
Coccotrypes cf. rhizophorae (Hopkins)
Coccotrypes variabilis (Beeson)
Dryocoetiops coffeae (Eggers)
Dryocoetiops cf. eugeniae (Schedl)
Dryoxylon onoharaensum (Murayama)
Amasa versicolor (Sampson)
Ambrosiodmus aegir (Eggers)
Ambrosiodmus compressus (Lea)
Ambrosiodmus colossus (Blandford)
Arixyleborus cf. granifer (Eichhoff)
Arixyleborus medius (Eggers)
Arixyleborus cf. sus (Schedl)
Cnestus suturalis (Eggers)
Coptoborus pseudotenuis (Schedl)
Coptodryas eucalyptica (Schedl)
Cyclorhipidion agnatum (Eggers)
Cyclorhipidion dentatulus (Browne)
Cyclorhipidion pruinosum (Blandford)
Cyclorhipidion cf. sexspinatum (Schedl)
Dryocoetoides cristatus (Fabricius)
Eccoptopterus spinosus (Olivier) – Uganda
Eccoptopterus spinosus (Olivier) – PNG
Euwallacea validus (Eichhoff)
Euwallacea wallacei (Blandford)
Euwallacea xanthopus (Eichhoff)
Leptoxyleborus concisus (Blandford)
Sampsonius dampfi Schedl
Theoborus ricini (Eggers)
Theoborus theobromae Hopkins
Webbia bicornis (Schedl)
Webbia cf. platypoides Eggers
Webbia quattuordecimspinatus Sampson
Xyleborinus intersetosus (Blandford)
Xyleborinus saxeni (Ratzeburg)
Xyleborus affinis Eichhoff
Xyleborus annexus Schedl
Xyleborus biuncus Browne
Xyleborus dolosus Blandford
Xyleborus fallax Eichhoff
Xyleborus insulindicus Eggers
Xyleborus justus Schedl
Xyleborus meritus Wood
Outbreeding
Outbreeding
Outbreeding
Outbreeding
Outbreeding
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
bark
leaf
inflorescence
bark
bark
bark
bark
bark, seed, petiole
bark, seed, petiole
bark, seed, petiole
bark, seed, petiole
seed
bark, seed, petiole
seed
seed
bark, seed, petiole
seed
seed
mangrove radicle
bark (+ petiole bark)
petiole
bark, seed, petiole
petiole
petiole
bark, seed, petiole
twig
twig
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
phloem
leaf
inflorescence
phloem
phloem
phloem
phloem
many
many
many
many
endosperm
many
endosperm
endosperm
many
endosperm
endosperm
‘endosperm’
phloem
petiole
many
petiole
petiole
many
pith
pith
fungus?
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
AF439743
AF186662
AF186665
AF186661
AF259873
AF439740
AF259870
AF186668
AF444076
AF259869
AF444072
AF259872
AF444074
AF444078
AF444075
AF259867
AF259866
AF259874
AF259864
AF259871
AF186669
AF259875
AF444077
AF444071
AF259865
AF186670
AF439741
AF186660
AF186696
AF259877
AF508869
AF508868
AF508874
AF186695
AF508875
AF186694
AF508880
AF508878
AF259892
AF508877
AF259883
AF508867
AF186687
AF186686
AF508881
AF259878
AF508885
AF259893
AF259886
AF259885
AF186691
AF259881
AF259884
AF508882
AF259882
AF186684
AF259876
AF186688
AF508870
AF508871
AF259887
AF508873
AF508884
AF508876
AF508883
© 2002 The Royal Entomological Society, Insect Molecular Biology, 11, 453 – 465
IMB_354.fm Page 463 Wednesday, August 28, 2002 10:49 AM
Elongation Factor 1-α resolves the monophyly of Xyleborini
463
Table 3. continued
Tribe
Species
Mating
system
Breeding
site
Feeding
tissue
GeneBank
accession no.
Xyleborus metacuneolus Eggers
Xyleborus multispinatus Eggers
Xyleborus perforans (Wollaston)
Xyleborus pfeili (Ratzeburg)
Xyleborus pseudopilifer Schedl
Xyleborus semipunctatus Eggers
Xyleborus spathipennis Eichhoff
Xyleborus sphenos Sampson
Xyleborus cf. subdentatus Browne
Xylosandrus crassiusculus (Motschulsky)
Xylosandrus mancus (Blandford)
Xylosandrus morigerus (Blandford)
Xylosandrus mutilatus (Blandford)
Xylosandrus cf. zimmermanni (Hopkins)
Xylosandrus sp. n., related to ursa (Eggers)
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
Sib-mating
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
wood
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
fungus
AF259891
AF186690
AF259880
AF259879
AF259888
AF508887
AF508879
AF186692
AF259889
AF259890
AF186693
AF508866
AF508872
AF186685
AF508886
Copy 2
Coccotrypes advena (A)
Coccotrypes impressus (B)
AF508928
AF508923
AF508924
AF508927
AF508925
AF508926
Theoborus ricini
Xyleborus sphenos
Additional sequences used to produce Fig. 1 are found under the following GenBank accession numbers: U20125, U90054, U90056, X06869, X06870, X52884,
AF015267, AF044815, AF044826, AF063405, AF063416, AF068466, AF068477, AF137388, AF137389, AF137390, AF140324, AF140336, AF147820,
AF151624, AF163887, AF264861, AY048510, AY048536.
cycles for the new sequences consisted of 30 s initial denaturation
at 94 °C, then forty cycles consisting of 48 °C annealing for 45 s,
extension at 72 °C for 60 s and 94 °C denaturing for 30 s. PCR
amplifications were performed in a 25 µl volume containing 0.2 M
of each primer, 0.2 mM of each dNTP, 2.5 µl 10× Applied Biosystems buffer with additional MgCl2 to a final concentration of
2.0 mM, and 0.75 unit Applied Biosystems AmpliTaq DNA polymerase. PCR products were gel purified using Qiagen gel purification
kit. Sequencing reactions followed Perkin Elmer’s recommended
thermal profile (but with 10 s annealing at 50 °C) and analysed on
a ninety-six well capillary sequencer.
Sequence alignment
Sequences were assembled, edited and preliminary alignments
performed using Sequencher 3.1 (Gene Codes Corporation).
Intron positions were identified by GT (5′) and AG (3′) intron terminals and compared to intron positions in published insect
sequences. The putative coding regions were validated by inferred
amino acid translations. The intron used in the phylogenetic
analysis was aligned in ClustalX (Thompson et al., 1997) under ten
different parameter settings: gap cost of 2, 4, 8, 16 and 32, with
gap extension cost half or equal to gap cost, and transitions equal
to transversions. Consistently misaligned taxa were removed one
after another to detect taxon specific regions disturbing the global
alignment. Three such sequences were found and putative
autapomorphic insertions were deleted to accommodate a less
ambiguous alignment: Xylosandrus crassiusculus (4 bases
deleted), Coccotrypes litoralis (14 bases [repeats] deleted) and
Cyclorhipidion cf. sexpinatum (28 bases deleted). Based on
careful comparison of closely related taxa determined from the
phylogenetic analysis of the coding region, the final alignment
parameters were selected to minimize topological conflict with
the exon analysis. The final alignment was based on gap and
extension costs of 16, and the alignment of some sequences were
manually corrected to fit separate alignments of close relatives.
Gaps were treated as missing data, but gap lengths were coded
and used in the parsimony analysis as described by Danforth
et al. (1999).
Phylogenetic analyses
PAUP* 4.0b4a (Swofford, 1999) was used to calculate distances
and perform phylogenetic analyses of the two data sets. In the
analyses of different insect EF-1α copies, introns were removed
and the remaining 864 coding nucleotides subjected to weighted
and unweighted MP analyses and ML analyses. The following
weighting scheme was used in the parsimony analyses
(pos1:pos2:pos3): 10:10:5, 10:10:2, 10:10:1, 5:10:2, 5:10:1, and
amino acid coding. All analyses consisted of heuristic searches
with 100 random additions. Parameters for the ML nucleotide
analysis were estimated directly during heuristic searches with ten
random additions, using a GTR+Γ+I model.
In the final analysis of eighty beetle sequences of 845 coding
nucleotides and one intron of 70 aligned base pairs, I also used
ME analysis of maximum likelihood distances in addition to
unweighted MP and ML. In the MP and ME analyses, heuristic
searches with 500 random additions was used, all characters
weighted equally. Node support was estimated via the bootstrap
method (Felsenstein, 1985) with 200 replicates and ten random
additions each. Maximum likelihood analysis of eighty taxa is
computationally intensive and only five random additions were
performed. Parameter settings for these ML and ME searches
were estimated using the ‘Modeltest’ software (Posada & Crandall,
1998) in conjunction with PAUP*. The initial parameter settings
estimated by ‘Modeltest’ (by Neighbour Joining) were refined over
© 2002 The Royal Entomological Society, Insect Molecular Biology, 11, 453 – 465
IMB_354.fm Page 464 Wednesday, August 28, 2002 10:49 AM
464
B. H. Jordal
the ME topology, with marginal differences in estimates (slightly
increased C-T substitution rate).
Acknowledgements
I want to thank R. A. Beaver for identifying many of the
specimens, B. B. Normark and D. J. Rees for helpful
discussions and comments on the manuscript, and B. D.
Farrell and L. R. Kirkendall for access to lab and computer
facilities. This paper was supported by a Norwegian
Research Council grant 123588/410, and a Marie Curie
fellowship HPMF-CT2001-01323.
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