Syst. Biol. 51(3):450–477, 2002
DOI: 10.1080/10635150290069896
Robustness of Ancestral State Estimates: Evolution of Life History
Strategy in Ichneumonoid Parasitoids
R OBERT B ELS HAW1 AND D ONALD L. J. Q UICKE2
1
Department of Biological Sciences, Imperial College at Silwood Park, Ascot, Berkshire SL5 7PY, UK;
E-mail: [email protected]
2
Department of Biological Sciences and Centre for Population Biology, Imperial College at Silwood Park,
and Department of Entomology, The Natural History Museum, London SW7 5BD, UK; E-mail: [email protected]
Abstract.— We test hypotheses for the evolution of a life history trait among a group of parasitoid
wasps (Hymenoptera: Ichneumonoidea), namely, the transition among koinobiont parasitoids (parasitoids whose hosts continue development after oviposition) between attacking exposed hosts and
attacking hosts that are concealed within plant tissue. Using a range of phylogeny estimates based
on 28S rDNA sequences, we use maximum parsimony (MP) and maximum likelihood (ML) methods to estimate the ancestral life history traits for the main clades in which both traits occur (using the programs MacClade and Discrete, respectively). We also assess the robustness of these estimates; for MP, we use step matrices in PAUP¤ to Žnd the minimum weight necessary to reverse
estimates or make them ambiguous, and for ML, we measure the differences in likelihood after Žxing the ancestral nodes at the alternative states. We also measure the robustness of the MP ancestral
state estimate against uncertainties in the phylogeny estimate, manipulating the most-parsimonious
tree in MacClade to Žnd the shortest suboptimal tree in which the ancestral state estimate is reversed
or made ambiguous. Using these methods, we Žnd strong evidence supporting two transitions among
koinobiont Ichneumonoidea: (1) to attacking exposed hosts in a clade consisting of the Helconinae
and related subfamilies, and (2) the reverse transition in a clade consisting of the Euphorinae and
related subfamilies. In exploring different methods of analyzing variable-length DNA sequences, we
found that direct optimizatio n with POY gave some clearly erroneous results that had a profound
effect on the overall phylogeny estimate. We also discuss relationships within the superfamily and
expand the Mesostoinae to include all the gall-associated braconids that form the sister group of the
Aphidiinae.
Estimates of ancestral states are subject to
two sources of error: uncertainties in estimating the ancestral state given a phylogeny estimate, and uncertainties in the phylogeny
estimate itself.
Considering the Žrst source of uncertainty,
for a given estimate of phylogeny, we can
use maximum parsimony (MP) or maximum
likelihood (ML) to estimate any ancestral
character state. Cunningham et al. (1998)
compare these two methods and show that
simple MP estimates may be misleading if
rates of change are high or transition rates are
asymmetric. Particularly valuable for molecular phylogenetics is the ability of ML-based
ancestral state estimates to take into account
branch lengths (changes are more likely to occur on long branches if branch length is proportional to time). The robustness of a ML
ancestral state estimate can be assessed by
comparing the likelihoods of models of trait
change in which the trait is Žxed at the alternative states (Pagel, 1999a). As currently
implemented in available software, estimating ancestral states by using MP is a simple
optimization and assumes that, for a binary
character, transitions in either direction are
equally probable (but see Maddison (1995)
and Salisbury and Kim (2001) for discussions of the probability of MP-based ancestral state estimations). We can measure the robustness of a MP estimate to this assumption
by using asymmetric step matrices (Ree and
Donoghue, 1998; Omland, 1999). By applying
increasing weights to one of the two transformations, we can Žnd the minimum additional weight necessary to change the observed result. Several authors (Sperling and
Feeny, 1996; Omland, 1997) have used this
method, generally because they suspect that
gains and losses of complex traits are not
equally probable.
The importance of the second source of uncertainty for comparative methods has been
recognized for some time (Harvey and Pagel,
1991:203). We can gain a crude measure of
the robustness of an ancestral state estimate
by repeating it on estimates of phylogeny
that use different methods and parameters.
However, for a more rigorous examination,
we need to test whether trees supporting
the alternative ancestral state estimate are
450
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
signiŽcantly worse. This general approach is
used for testing a wide range of evolutionary hypotheses that use phylogeny estimates
(Huelsenbeck and Rannala, 1997). For example, to assess the support for different numbers of independent domestications of fungi
by fungus-growing ants, Mueller et al. (1998)
compared optimal trees with trees found by
searches that were constrained to recover certain fungus cultivars as monophyletic. Applied to ancestral state estimates, this approach is used by Dumbacher and Fleischer
(2001) to assess the support for a proposed
convergence in bird plumage. Other studies compare the results obtained by using
optimal trees with those from suboptimal
trees, for example, Donoghue and Ackerly
(1996) examining the evolution of branching architecture in Acer plants and Hibbett
and Donoghue (2001) studying correlations
between life history traits of homobasidiomycete fungi. Although most studies, including the one we present here, pursue
this only within a MP framework, the approach is applicable to other methods, for
example, Bayesian inference (Huelsenbeck
et al., 2000).
In this paper we use the above methods to
analyze the evolution of a particular life history trait among a group of parasitoid wasps.
To summarize, our procedure involves (1) estimating phylogeny, (2) estimating ancestral
states by MP and ML, (3) measuring the robustness of these ancestral states estimates by
using step matrices (for MP) and differences
in likelihood (for ML), and (4) measuring the
robustness of the ancestral state estimates to
uncertainties in the phylogeny (MP only).
Insect parasitoids oviposit onto, or into,
their host, which typically is the immature
stage of another insect. The parasitoid larva
feeds on the host and eventually kills it
(Eggleton and Belshaw, 1992). Perhaps the
most striking feature of life history evolution among parasitoids has been that from
idiobiosis to koinobiosis (Askew and Shaw,
1986), the distinction being that koinobionts
allow the host to continue development after oviposition, whereas idiobionts do not.
Commonly, koinobiont species oviposit in
immature hosts, which the parasitoid larva
kills only after the host has Žnished growing. In contrast, the ovipositing idiobiont female usually permanently paralyzes or kills
the host before laying an egg or eggs on
it. Many other life history traits appear to
451
be correlated with this apparently fundamental dichotomy (Sheehan and Hawkins,
1991; Belshaw, 1994; Quicke, 1997; Mayhew
and Blackburn, 1999). In this paper we examine one group of parasitoids, the Ichneumonoidea (Hymenoptera), to Žnd the direction of the evolutionary transition between
koinobiont parasitoids whose hosts feed
concealed in plant tissue, and koinobiont
parasitoids whose hosts feed exposed on
plants.
Evidence is strong that the common ancestor of the Ichneumonoidea was an idiobiont parasitoid of concealed hosts. On the
basis of morphological characters, this is estimated to be the ancestral biology of the sister group of the Ichneumonoidea, the Aculeata (Hanson and Gauld, 1995), and it is
the biology of most taxa phylogenetically
close to the (Ichneumonoidea C Aculeata)
clade (Ronquist, 1999). Dowton and Austin
(2001), using molecular data, also estimate
this to be the ancestral biology of the Ichneumonoidea (although they do not recover
the Aculeata as the sister group). Within
the Ichneumonoidea transitions to koinobiosis apparently have occurred multiple times
(WhitŽeld, 1998; Belshaw et al., 1998). Commonly, ichneumonoid koinobiont species
oviposit in young hosts that feed exposed
on the surface of plants and kill them only
after the host has Žnished its larval growth
and moved to its pupation site (Shaw, 1983;
Gauld, 1988). Many ichneumonoid koinobionts, however, attack hosts that are concealed in plant tissue. Quicke et al. (1999)
suggested that this latter trait is more likely to
have evolved through an intermediate stage
of being a koinobiont parasitoid of exposed
hosts, rather than directly from the concealed
idiobiont ancestor. They argued that, despite
the selective advantages proposed for koinobiosis in parasitoids attacking exposed hosts
(for example, in killing the host only after it
has moved to a more protected site for pupation), such selective advantages are difŽcult to attribute to koinobiosis when the
host is already concealed. Hence, among
koinobionts, attacking concealed hosts is
more likely to be a reversal to the ancestral habit. Conversely, several authors have
suggested that—at least in certain ichneumonoid clades—koinobiosis in parasitoids
attacking concealed hosts arose directly from
the ancestral idiobiont biology without an intermediate stage of attacking exposed hosts
452
S YSTEMATIC BIOLOGY
(Shaw, 1983; Gauld, 1988; Wharton, 1993). In
the present study we attempt to resolve this
debate by using 28S rDNA sequence data,
having previously shown that many morphological characters are related to the life
history trait investigated here (Quicke and
Belshaw, 1999).
M ETHODS
The Taxa Categorization of and Their Life
History
Our previous molecular phylogenetic
studies of this superfamily showed basal relationships to be poorly resolved (Belshaw
et al., 1998, 2000; Quicke et al., 2000). Therefore, rather than attempting to determine all
ancestral states in the superfamily, we focus on the ancestral states of well-supported
koinobiont clades that display both life history traits: species attacking concealed hosts
and species attacking exposed hosts. Five of
these koinobiont clades are within the noncyclostome Braconidae, and the sixth is within
the ophioniform clade of the Ichneumonidae
(the two families together form the Ichneumonoidea). With one exception, the noncyclostome braconids and ophioniform ichneumonids are also exclusively endoparasitoid
(eggs are laid within the hosts, and the larva
develops internally—at least for the Žrst part
of its life). The exception is the tryphonine
ichneumonids, which are a clade of ectoparasitoid koinobionts. The other ichneumonoid
taxa are in clades dominated by the ancestral idiobiont biology and are not examined
here, namely, the cyclostome clade in the
Braconidae and the (pimpliformes C ichneumoniformes) clade in the Ichneumonidae.
Gauld (1988) provides an overview of the diversity of life history strategies within the entire Ichneumonoidea. We categorize taxa according to whether the host is concealed at
the stage at which it is attacked and we use
the term concealed to include hosts in all degrees of concealment: from within wood to
inside leaf mines or leaf rolls. The depth of
concealment of the host may not automatically reect how difŽcult it is to parasitize:
Parasitoids attacking hosts deeply concealed
in wood often simply thread their ovipositors through cracks, whereas those attacking
miners may have to penetrate plant tissue.
Nonkoinobionts, and taxa whose biology is
unknown or difŽcult to code, were treated
as uncertain for MP estimates and pruned
VOL. 51
from the tree for ML estimates (see below).
Taxa whose biology was difŽcult to code include the tryphonines mentioned above and
species such as the ichneutine Paroligoneurus Muesebeck, which attacks the eggs of
leaf-mining hosts (eggs of leaf-miners can be
pushed into plant tissue to various degrees).
Taxa in which the female enters the chamber
of concealed hosts to attack them were coded
as exposed. The ancestral node of a clade
was treated as the Žrst bifurcation within that
clade, rather than the node linking the clade
to its sister group.
The taxa analyzed are listed in the
Appendix with their EMBL/GenBank accession numbers. Our taxon coverage is
very good at the subfamily level: Among
the noncyclostome Braconidae we lack representatives only of the Amicrocentrinae,
which attack lepidopteran stalk borers and
have long ovipositors (van Achterberg,
1979a); the Ecnomiinae, of which the biology is unknown and the afŽnities are
unclear (Park and van Achterberg, 1994);
and a few small microgastroid subfamilies, namely, the Dirrhopinae, Khoikhoiinae,
and Mendesellinae. We lack representatives for only four ichneumonid subfamilies, and three of these probably belong
in the (pimpliformes C ichneumoniformes)
clade: the Agrioptypinae, Pedunculinae, and
Claseinae. The only omission from the
ophioniformes clade is the Tatogastrinae; the
Townesioninae has recently been shown to
belong to the Banchinae (Gauld and Wahl,
2000a). More detail of the life history of
the noncyclostome Braconidae is given in
Table 1; the composition of the six analyzed
clades is summarized below.
The sigalphoid clade.—This contains the
Sigalphinae and the Disophrini (Agathidinae) braconids, which attack exposed hosts,
and the remaining Agathidinae, all of which
attack concealed hosts.
The macrocentroid clade.—Two macrocentrine genera (Austrozele Roman and
Dolichozele Viereck) attack exposed hosts,
whereas the remaining genera attack
concealed hosts.
The helconoid clade.—The Xiphozelinae
and some Homolobinae attack exposed
hosts, whereas the Helconinae (as previously constituted), Microtypinae, and Charmontinae attack concealed hosts exclusively. Two genera currently placed in
the Helconinae, Canalicephalus Gibson and
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BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
453
TABLE 1. Categorization of life history for noncyclostome braconid taxa. “Semiconcealed” refers to hosts such
as leaf miners and leaf rollers, which are treated as concealed in our analyses; “other” is used for all life histories
that do not Žt our categorization (discussed further in text).
Life history
category
Taxon
Sigalphoid Clade
Agathidinae
Sigalphinae
Helconoid Clade
Helconinae (part)
Agathidini, Earinini, and Microdini Concealed
Disophrini
Exposed
Concealed
Semiconcealed Lepidoptera larvaea
Lepidoptera larvaea
Semiconcealed Lepidoptera larvaea
Brachistini and Diospilini
Concealed
Eadyiini
Exposed
Helconini
Concealed
Seed-feeding Coleopterab, c except
for Brulleia, which has one record
from cerambycid larvaed .
Egg-larval where known
Eadya attacks exposed chrysomelid
larvae h
Cerambycid and other wood-boring
Coleoptera larvaec
One record from an exposed
Lepidoptera larva; both genera have
short ovipositorse
Sequenced species have short
ovipositors and attack caterpillars
that feed exposed at night; other
species have a long ovipositor
Xiphozelinae
Exposed
Homolobinae
Exposed
Macrocentroid Clade
Macrocentrinae
Macrocentrus, Hymenochaonia,
and Aulacocentrum
Austrozele
Dolichozele
Microtypinae
Charmontinae
Euphoroid Clade
Euphorinae
Neoneurinae
Meteorinae
Microgastroid Clade
Cardiochilinae,
Microgastrinae,
and Miracinae
Cheloninae
Concealed
Semiconcealed Lepidoptera larvaeb
Exposed
Host feeds exposed nocturnallyb .
Short ovipositor
One record from an exposed hosta .
Short ovipositor
Semiconcealed Lepidoptera larvaea
Semiconcealed Lepidoptera larvaeb
Exposed
Concealed
Concealed
Exposed
Meteorus
Exposed
Exposed or
concealed
Zele
Exposed
Adult insects in at least six ordersb ;
Rhopalophorus and Cosmophorini
attack scolytid beetles, but at least
in Cosmophorus the female goes into
the host burrow and holds the host
with its mandiblesb
Ants
Lepidoptera and Coleoptera larvae;
exposed and concealed to various
degrees, including leaf miners and
bark borersb
Nocturnal Lepidoptera larvaeb
Exposed,
Wide range of Lepidoptera larvae,
concealed,
both exposed and semiconcealeda ;
or other
some Microgastrinae are egg-larvali
Other
Egg-larval parasitoids of Lepidoptera
(chiey semiconcealed). Degree of
exposure of eggs, and length of
ovipositor, varya
Other
Leaf-mining Lepidoptera larvae,
especially Nepticulidae; assumed
to be egg-larvalb
Adelius
Other taxa
Orgilinae
Further details
Mimagathidini
Concealed
Orgilini
Concealed
Leaf-mining Lepidoptera larvae, but
probe the workings rather than
piercing the plant tissueb
Leaf-mining Lepidoptera larvae
Sources: a D Wharton et al. (1997) , b D Shaw and Huddleston (1991), c D Hanson and Gauld (1995), d D Chen and van Achterberg
(1993), e D van Achterberg (1979b), f D Belshaw et al. (2000) , g D Gibson (1972), h D Huddleston and Short (1978), i D Ruberson and
WhitŽeld (1996) , and j D Oda et al. (2001).
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S YSTEMATIC BIOLOGY
TABLE 1. Continued.
Life history
category
Taxon
Ichneutinae
Helconinae (part)
Cenocoeliinae
Aphidiinae
Mesostoinae
Other
Canalicephalus and
Urosigalphus
Concealed
Concealed
Exposed
Other
Urosigalphus Huddleston, were rarely recovered in this clade. Eadya, previously placed
in the Euphorinae, clearly appears to belong
in the Helconinae and attacks exposed hosts.
The euphoroid clade.—This contains the Euphorinae and Neoneurinae, all of which attack exposed (adult) hosts, plus the Meteorinae, some of which attack concealed hosts.
In some analyses the Cenocoeliinae, which
attack concealed hosts, are also recovered
among this clade.
The microgastroid clade.—This contains
many genera that attack exposed hosts and
others that attack concealed hosts. Unlike in
the above clades, many genera in this clade,
representing both life history states, are not
represented in our data set.
The Campopleginae clade.—This clade consists of the taxa currently placed in the Campopleginae but excluding Chriodes Förster,
Hellwigia Svépligeti, and Nonnus Cresson,
which are misplaced within this subfamily.
Not all of the variation in the Ichneumonidae
is represented by the taxa sequenced here,
and we assumed that life histories are constant within genera. In some cases (Chriodes,
Hellwigia, Nonnus, Oxytorus Förster, tersilochine sp.) the host is unknown, and we have
inferred life history from the length of the
ovipositor and, in the case of Nonnus, the
swollen foretibia often associated with locating concealed hosts (Broad and Quicke,
Further details
Egg-larval parasitoids of sawy larvaea, b except for
Oligoneurus and Paroligoneurus, which attack
leaf-mining Lepidoptera and Diptera but probably
also egg-larvala, b
Urosigalphus attacks seed-feeding Coleopterag ; assume
the same for Canalicephalus
Coleoptera larvae with various degrees of concealmentb
Aphids
Phytophagous, idiobiont, or unknownf, j
2000); Panteles Förster is treated as missing
data, although its host is a gall former, because of its putative close relationship to Stilbops Förster, which oviposits into the host
egg.
Laboratory Protocols
DNA was extracted from single specimens (or parts of specimens weighing no
more than »30 mg) by using the DNeasy kit
(Qiagen) with Žnal elution into 100 ¹l of water. All polymerase chain reactions were carried out in ABI 2400 or 9600 thermal cyclers in
50 ¹l reaction solutions containing 1.0 ¹l of
DNA extract, 20 pmol of primers, 10 nmol
of dNTPs (Amersham Pharmacia Biotech;
APB), 1.5 units of Taq polymerase (Roche),
5.0 ¹l of Taq buffer (containing 1.5 mM
MgCl2 ). Cycling conditions (35 cycles, plus
an initial denaturation for 2 min and a Žnal
extension for 7 min) were 94± C for 30 sec,
50± C for 30 sec, and 72± C for 1 min. Products
were cleaned by using the GFX band puriŽcation kit (APB) and were sequenced directly
with dye terminators on ABI 373 and 3700
automated sequencers (using one-half and
one-fourth the recommended volumes, respectively). The D2–D3 28S rDNA region was
ampliŽed as a single fragment, and D4–D10
28S rDNA was ampliŽed in a further three
fragments. All primer sequences are given in
Table 2.
TABLE 2. Primer sequences. All sequences are written 5’ to 3’. Variable regions in 28S rDNA match those in
Drosophila melanogaster (Hancock et al., 1988). 18S rDNA primers are from Sanchis et al. (2000): forward is V4.up1,
reverse is 18S.lo1. In a few taxa these were used in combination with one of two internal primers (forward is
NS58 C 2; reverse is NS58 ¡ 3) to amplify the gene in two contiguous fragments.
Region
Forward primer
18S
18S internal
28S D2-3
28S D4-5
28S D6-7
28S D8-10
CAG CCG CGG TAA TTC CAG C
TCC GAT AAC GAA CGA GAC TC
GCG AAC AAG TAC CGT GAG GG
CCC GTC TTG AAA CAC GGA CCA AGG
GGA GTG TGT AAC AAC TCA CCT GCC G
CCC ATA TCC GCA GCA GGT CTC C
Reverse primer
CTT CYG CAG GTT CAC CTA C
GAG TCT CGT TCG TTA TCG GA
TAG TTC ACC ATC TTT CGG GTC
GTT ACA CAC TCC TTA GCG GA
GAC TTC CCT TAC CTA CAT
AGT CAA ACT CCC TAC CTG GC
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BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
Phylogeny Estimation
Our phylogeny estimates are based on an
approximately threefold expansion of the D2
28S rDNA molecular data set used in previous studies (Belshaw et al., 1998, 2000;
Dowton et al., 2002). Because this region is
difŽcult to align across the entire superfamily, we therefore analyze the Ichneumonidae
and Braconidae separately and root them by
using the results of a third (“rooting”) phylogenetic analysis. This rooting analysis is
based on a data set comprising representative braconid and ichneumonid taxa plus
several aculeates (the putative sister group
of the Ichneumonoidea; a total of 32 taxa).
We sequenced these for a much larger region
of the 28S gene (spanning the D2–D10 regions; length 2643–3260 bp), which includes
more conserved regions that are alignable
between more distantly related taxa, plus
a 1177–1223 bp region of the 18S rDNA
gene. We also included in this rooting analysis the braconid Megalohelcon Turner, which
we excluded from our analysis of the Braconidae because of its unknown life history
and highly divergent (both in length and base
composition) D2–D3 28S sequence (Belshaw
et al., 1998).
The data sets for the Braconidae and Ichneumonidae consist of 121 and 114 species,
respectively, sequenced for the D2–D3 regions of 28S rDNA; sequence lengths were
593–716 bp (Braconidae; both extremes were
found within the aberrant subfamily Aphidiinae, discussed below) and 625–664 bp (Ichneumonidae). The small D3 region was not
sequenced in 45% of the genera, which are
from earlier studies, mostly our own. In
addition, we repeated some of the analysis with an additional 21 braconid species
(total 142); these species were additional
Aphidiinae, which we included to help
overcome phylogenetic artifacts caused by
the group (see below), plus representatives
of novel taxa obtained near the end of
the project. The Braconidae analysis consisted of noncyclostomes plus six representatives of the well-supported cyclostome clade.
For the Ichneumonidae, where fewer assumptions about relationships can be made,
we included representatives of all available
subfamilies.
Several phylogenetic methods are available for analyzing DNA sequences of such
variable lengths, and the main problem is
455
in choosing between them (Morrison and
Ellis, 1997; Sanchis et al., 2001). One approach is to use congruence as a measure,
preferring alignment methods or alignment parameters that maximize congruence. This congruence can be with a taxonomic classiŽcation, morphological data,
other genes, or even different regions of
the same gene (Wheeler, 1995; Giribet and
Wheeler, 1999; Cognato and Vogler, 2001;
Giribet, 2001). However, in the absence of
independent data, the approach we chose
was to use a wide range of methods and
compare the results. Representative DNA
alignments are available in Nexus format
at http://www.bio.ic.ac.uk/research/data/
Ichneumonoid.
The traditional method for analyzing
variable length sequences is to produce a
multiple alignment, from which a tree is subsequently built. An appealing recent alternative is the program POY by D. Gladstein
and W. Wheeler (Wheeler, 1996), which implements a direct optimization, Žnding the
most-parsimonious tree (MPT) for variable
length sequences, given the cost of inserting a single gap relative to a substitution.
We discuss POY below among the alignment
methods.
Alignment by using POY.—We analyzed
our sequences with POY version 2.6
(obtained from ftp://ftp.amnh.org/pub/
molecular/poy), running in parallel on a
cluster of Žve 486 MHz processors. In addition to the directly optimized phylogeny
estimates, POY can also generate a multiple alignment (using the command, impliedalignment), which we used for subsequent tree-building, treating the gaps as
missing data. The original tree found by POY
can be recovered from this multiple alignment by searching with a step matrix that
gives gaps the same weight as in the original
POY search.
POY was the only method we used with
the D2–D10 28S and 18S rDNA rooting data
set. To speed computation with the 28S data,
and hence to Žnd shorter trees in heuristic
searches, we made initial alignments of the
original four ampliŽed fragments, which
we then subdivided to give a total of 31
homologous regions. These 31 regions were
aligned independently in POY, using 20 random additions with SPR (subtree pruning
and regrafting) and TBR (tree bisection and
456
VOL. 51
S YSTEMATIC BIOLOGY
reconnection) branch swapping on a maximum of 10 trees in each case. The 18S rDNA
sequences were much less variable in length
and so were aligned as single fragments,
using the same search protocol (except for
Homolobus Foerster and Masona van
Achterberg, which we were unable to
amplify for this gene).
Although the braconid D2–D3 region was
ampliŽed as a single fragment, we aligned it
in four data Žles simultaneously. We did this
to avoid obvious alignment artifacts caused
by a deletion of »50 bp found in one genus,
Ephedrus, and because we lacked the D3 region for many of the taxa. Large gaps appear to cause problems for all of the alignment programs we have come across, such
that obviously homologous regions at the
beginning and ends of gaps are not being
aligned together. This problem can be overcome by creating multiple data sets so that
the regions homologous to large gaps are
aligned separately. In addition to the default gap:substitution cost in POY of 2:1, we
also explored the robustness of results to
the alignment protocol by repeating analyses
with two additional gap: substitution cost ratios, 1:1, and 3:1. Each search consisted of 20
random additions followed by branch swapping by SPR and TBR on a maximum of two
trees (we used only the Žrst multiple alignment where two were found). To align the
ichneumonid D2–D3 region in two data Žles
(D2 and D3), we used the default gap cost
only and 50 random additions.
Alignment by using ClustalX.—We also
used the distance-based program ClustalX
(Thompson et al., 1997), again using a range
of gap costs: We used gap opening costs of
20, 10, 5, and 2.5 with gap extension costs
of 10, 5, 1, and 0.5, respectively (contrary to
the default setting, we gave transitions the
same weight as transversions). The D2–D3
sequences were subdivided as for the POY
analysis, but each was aligned individually
and then pasted together into a Nexus Žle for
tree building.
Alignment by using secondary structure.—
The use of the secondary structure of RNA
molecules can improve phylogeny inference,
although the preferred methods are difŽcult to implement (Page, 2000). Here, we
used Page’s alignment editor SSE (obtained from http://darwin.zoology.gla.ac.uk/
»rpage/sse/) to align sequences manually
with reference to the secondary structure
according to Kjer (1995). The initial alignment was by Clustal as implemented in SSE.
The initial folding estimates for all sequences
were the single optimum folding structure
obtained from the program mfold version
3.0 (Zuker et al., 1999; Mathews et al., 1999)
as implemented at http://bioinfo.math.rpi.
edu/»mfold/rna with default settings. We
did not include the smaller D3 region in the
secondary structure–based analyses.
Alignment “by eye”.—We also aligned the
sequences “by eye,” using the primary structure only. This method can give defensible
results (Belshaw and Quicke, 1997; Belshaw
et al., 2000; Sanchis et al., 2001) but is hindered by subjectivity, especially in the order
in which taxa are aligned to each other, and
by not being replicable. It is a useful adjunct
to alignment programs, especially in helping
to spot artifacts.
Tree-Building Methods
Unfortunately, ML searches were not possible, given the computational demands of
our large data set. Using Modeltest version
3.0 (Posada and Crandall, 1998), we found
that incorporating rate heterogeneity into a
model improved the Žt of that model to the
data much more than did incorporating any
other parameter. For example, the difference
in log likelihoods between a Jukes–Cantor
(1969) model (JC) and JC plus a shape parameter (0) was more than twice that between JC and general time reversible (GTR)
models. However, even after setting JC C 0
parameters from the MPT, heuristic searches
consisting of a single random addition without branch swapping took »1 week on a
250 MHz G3 Powermac and failed to Žnd
more likely trees. We therefore used two
methods: unweighted MP, and minimum
evolution (ME).
We built MP trees using the above multiple
alignments using PAUP¤ (Swofford, 1998)
and found the MPT by using 1,000 random
additions with the TBR method of branch
swapping with TBR on a single tree, followed
by an additional round of branch swapping
on the resulting trees with no restriction on
the number of trees held. Searches were made
with gaps treated as missing data.
Within the 28S sequences of our taxa is a
range of compositional biases. In addition to
the general reduction in GC content in Braconidae compared with the Ichneumonidae
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
and other Hymenoptera (Belshaw et al.,
1998), further marked reductions are evident within the family. Therefore, because
of this potential problem of convergent base
composition, we also built ME trees by using LogDet/Paralinear distances in PAUP¤ .
The use of LogDet/Paralinear distances can
make phylogenetic estimates more robust to
changes in base composition (Swofford et al.,
1996) and has reduced this problem in other
studies (Chang and Campbell, 2000). In addition to the problem of compositional biases,
a moderate degree of among-site rate heterogeneity (Yang, 1996) is apparent among our
sequences; for example, the POY alignment
of braconid sequences with gap cost of 2 has
a shape parameter ® of 0.70 (obtained from
MPT with the JC C 0 model; gaps treated
as missing data). The presence of invariable
sites can mislead model-based phylogeny estimation (Lockhart et al., 1996) unless they
are accommodated by the model (Swofford
et al., 1996), so we incorporated an invariable
sites model into our ME search in PAUP¤ .
The proportion of invariable sites (I) was calculated from a MPT and a simple ML model
(JC C I; use of alternative trees and more complex models changed the estimates of I by
<1%). Because of the variation in base composition, sites were removed in proportion to
base frequencies, which were estimated from
constant sites only (Swofford et al., 1996).
We assessed the robustness of branches by
using bootstrapping (Felsenstein, 1985), with
1,000 pseudoreplicates for ME phylogeny
estimates; for MP phylogeny estimates, we
used 100 pseudoreplicates each of 100 random additions with branch swapping on a
single tree.
Some preliminary phylogeny estimates of
the Braconidae recovered the aphidiine tribe
Aphidiini together with two of the ichneutines: Masonbeckia Sharkey and Wharton plus
Proterops Wesmael. This result is clearly an
artifact because, in addition to the morphological evidence (Quicke and Belshaw, 1999),
several studies using a range of genes have
shown the Aphidiini to be a derived clade
within the Aphidiinae (Belshaw and Quicke,
1997; Smith et al., 1999; Sanchis et al., 2000).
This problem may be caused by convergent
GC content, with Masonbeckia having the
lowest CG content in our data set (0.31), outside of the Aphidiinae (where CG content decreases to 0.25). To alleviate this problem, we
457
included more aphidiine sequences in our expanded data set (142 taxa) to reduce branch
lengths among the Aphidiinae. As a measure of the accuracy of the different methods
of phylogeny estimation, we noted which
analyses failed to recover the Aphidiinae as
monophyletic. We also scored the Aphidiinae
as monophyletic when they were (occasionally) recovered as paraphyletic with respect
to their sister group, which we refer to in this
study as the Mesostoinae.
Ancestral State Estimation
Using the phylogeny estimates obtained as
explained above, we estimated the ancestral
states of the main koinobiont clades by both
MP and ML and assessed the robustness of
these estimates by using step matrices and
differences in likelihood, respectively.
First, we estimated ancestral life history
states by using unweighted MP as implemented in MacClade version 4.0 (Maddison
and Maddison, 1992). The character was
treated as binary: either koinobionts with
host concealed (D 0) or host exposed (D 1).
As discussed above, other life history states
were treated as missing data. In analyses
of the main braconid data set (D 121 taxa)
we deliberately miscoded the life history of
one member of the genus Meteorus Haliday
so as to represent the range of life history
states known to occur within this morphologically well-supported genus. Late in the
project we obtained a member of this genus
with the relevant life history, so we could
avoid this artiŽciality in the analyses of the
expanded data set (D 142 taxa). To assess
the robustness of these MP estimates, we
used step matrices as follows. We Žrst created a data set consisting only of the life history character repeated many times plus a
corresponding number of step matrices with
the 0 to 1 transformation increasing in increments of 0.1, holding constant the weight of
the reverse transformation. Using this Žle,
we estimated the state of an internal node
of interest under each step matrix in a single PAUP¤ execution, repeating the procedure for each phylogeny estimate (with both
ACCTRAN and DELTRAN transformations).
We found the weight at which the estimate
of a speciŽc internal node became either ambiguous or reversed by logging the output to
a Žle and using a program written in Python
458
S YSTEMATIC BIOLOGY
VOL. 51
2.1. (PAUP¤ and Python Žles are available
at http://www.bio.ic.ac.uk/research/data/
Ichneumonoid.)
Second, we estimated ancestral life history
states by using ML as implemented in Discrete 4.0 (Pagel, 1994). Rates of transitions
between the two states were not restricted
(estimated values of the transition from concealed to exposed were between 1.1 and 2.7
times greater than the reverse on different
phylogeny estimates). Discrete accepts only
binary data, and taxa coded as missing data
for the MP ancestral state estimates were
pruned from the trees by MacClade. Trees
were then converted to the format required
for Discrete by using the program Moreways
(written by R. Freckleton, Department of Zoology, University of Oxford). This format includes a basal node with two branches, so
one outgroup taxon was left in; after conversion, the branch of the outgroup taxon
was deleted, together with the branch leading to the ingroup. Repeated analyses gave
identical results for all data sets, with no evidence of local optima. To assess the robustness of these ML estimates, we calculated
the likelihood with the node of interest successively set to state 0 and then to state 1,
following Pagel (1999a) in using “local” estimates. We interpreted differences between
the log likelihood values of 2.0 and above as
showing signiŽcant support for the ancestral
state with the greatest likelihood (Schluter
et al., 1997; Pagel, 1999b). All ML ancestral
state estimations were carried out on ME C I
trees, which should provide more accurate
branch lengths.
2. Give the life history character a weight of
0, so the tree length displayed comes from
the DNA characters only.
3. Move branches to make the estimated ancestral state ambiguous or reversed while
adding the least to the tree length. Although one cannot possibly check all
topologies, which even in smaller trees
would be astronomically high, the range
of candidate topologies is actually manageable and can be compared in this way.
We found that only two or three changes
were required, moving taxa with the alternative life history nearer to the base of the
clade, or joining basal clades together.
4. After each change, use the search option in MacClade to try to Žnd moreparsimonious rearrangements both above
and below the altered region of the tree.
5. Save the new suboptimal tree.
6. Repeat for each node of interest.
7. Repeat, using other alignments.
8. Compare the suboptimal trees with the
original MPT from which they were
derived. Several tests are available for assessing the signiŽcance of differences between two trees. We used the Shimodaira–
Hasegawa (Shimodaira and Hasegawa,
1999) as implemented in PAUP¤ . This test
compares the likelihoods of the two trees
and is more appropriate to the a posteriori
hypothesis testing here (Goldman et al.,
2000; Buckley et al., 2001). However, for
consistency of approach with the MP manipulations, we also present the results of
the parsimony-based Kishino–Hasegawa
test (Kishino and Hasegawa, 1989), also
implemented in PAUP¤ .
Robustness of MP Estimate to Uncertainties in
Phylogeny
The above manipulations in MacClade
are very laborious, so here we present the
results of manipulating only a single MPT
from each alignment. Only minor differences
were found when other different MPT were
analyzed; for example, in preliminary analyses (not shown) we compared 10 MPT with
their individual suboptimal trees. In 12 of 13
comparisons, the length differences between
the optimal and suboptimal trees were all
within 20% of the minimum difference
observed.
When this method found trees that were
not signiŽcantly longer, in which the ancestral state estimate had been reversed or
ambiguous, then clearly the null hypothesis
cannot be rejected. When the differences are
As mentioned above, the consistency of ancestral state estimates on different estimates
of phylogeny gives us a crude measure of
their robustness. In addition, we manipulated MP trees to Žnd the shortest tree in
which the ancestral state estimate for a speciŽc mode was reversed or rendered ambiguous. We could then test whether this
suboptimal tree was signiŽcantly worse. As
illustrated in Figure 1, our procedure was as
follows:
1. Map unambiguous changes in life history
onto a single MPT in MacClade.
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
459
FIGURE 1. Method of assessing robustness of MP ancestral state estimate to uncertainties in phylogeny estimation. (a) Optimal tree: one MPT from the expanded noncyclostome data set aligned “by eye” (length 5,683). For
clarity, this tree has been pruned of additional branches within clades of uniform life history. Note that the estimated
ancestral state of the helconoid clade is unambiguously concealed. (b) Shortest suboptimal tree found by branch
manipulatio n in MacClade in which the estimated ancestral state of the helconoid clade was not unambiguously
concealed (length 5,704). Two branches were moved to Žnd this suboptimal tree: First, outside the helconoid clade,
the Canalicephalus branch was moved one node nearer to the base of tree; second, inside the helconoid clade, the
Homolobinae branch was moved one node nearer to the base of the clade. These two changes lengthen the tree by
23 steps; use of the searching tool in MacClade then found a tree only 21 steps longer (length 5,704). (Note: The
branch labeled Canalicephalus includes Urosigalphus.)
460
S YSTEMATIC BIOLOGY
signiŽcant, however, the possibility still remains that better suboptimal solutions exist that have been missed by our manual
searching strategy. One approach is to search
through many sub-optimal trees to Žnd such
trees. We explored this approach, but the
large numbers of trees involved make this a
problematic procedure. By keeping a single
tree from each random addition in PAUP¤
and restricting the branch swapping, we obtained a wide distribution of tree lengths (using TBR, SPR, nearest neighbor interchange,
or no branch swapping progressively lengthens the mean lengths of the trees found). We
then, using a Python 2.1 program (mentioned
above), found the shortest of these on which
the ancestral state estimation at a speciŽc
node was reversed or made ambiguous. In
the example illustrated in Figure 1, by manipulation in MacClade we found a tree only
21 steps longer in which the ancestral state
estimate for the helconoid clade was ambiguous. In an examination of 30,000 suboptimal
trees, the shortest we found by using the automated method was 102 steps longer! Although we can focus the search by excluding large regions of tree space through the
use of constraint commands, the automated
method then starts to resemble the manual
one.
R ES ULTS
Phylogeny Estimation
The lengths of the aligned data sets were
3,463, 1,302, and 971 bp for the D2–D10 28S
rDNA, Braconidae D2–D3 (121 taxa), and
VOL. 51
Ichneumonidae D2–D3, respectively (all
from POY multiple alignments with gap
cost of 2). The braconid D2–D3 sequences
were highly variable, with 39% of characters
being parsimony-informative, but perhaps
surprisingly homoplasy was not high: data
decisiveness (DD; Goloboff, 1991) D 0.61
(with 10,000 random trees; RI D 0.67).
The ichneumonid D2–D3 sequences were
more conserved, with 30% of characters
parsimony-informative, but homoplasy was
greater (DD D 0.54; RI D 0.60). The data sets
used for the rooting were more conserved
but at the same time more homoplastic: The
complete D2–D10 28S alignment had 23%
of characters parsimony-informative with
DD D 0.35 (RI D 0.45), and the 1,302 bp POY
(gap cost 2) alignment of 18S sequences
had only 8.1% of characters parsimonyinformative and DD D 0.49 (RI D 0.55). Note
that gaps were treated as missing data for all
the above measurements.
Direct optimization of the rooting data set
by using POY gave some clearly erroneous
results. Both Megalohelcon and Ussurohelcon
Belokobylskij have long insertions into a similar region of the 28S gene (Fig. 2). These
insertions lack the sequence similarity that
would indicate they were homologous, and
the repeated simple motifs suggest a series
of unrelated slippage events in DNA replication (Hancock and Dover, 1988); nevertheless, presumably because of the length similarity, POY always recovers these two taxa
together (Fig. 3). This greatly affects the
overall phylogeny estimate because Megalohelcon is a basal braconid (Belshaw et al.,
1998, 2000) and this erroneous linkage causes
FIGURE 2. Part of the D7 region of the 28S subunit in selected species showing apparently homoplastic insertions
into Megalohelcon and Ussurohelcon. Sequences aligned by POY (see text). Positions relative to start of aligned D2–D10
region are shown.
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
FIGURE 3. Estimated phylogeny in Ichneumonoidea
using direct optimization of D2–D10 28S rDNA sequences in POY (length 8,141). Searching protocol:
100 random additions with SPR and TBR swapping
on 10 trees, and gap:substitution cost of 2:1 (default).
Branches are thickened if they are also recovered with
gap:substitution costs of 1:1, 3:2, and 3:1; robustness of
these branches is shown by jackboot values obtained
from POY (the four estimates show gap:substitution
costs increasing from 1:1 to 3:1, each calculated by using
100 random additions with SPR and TBR swapping on
10 trees).
461
Ussurohelcon and other helconoids to be
pulled to the base of the tree, forming a grade.
Another artifact shown in Figure 3 is that the
agathidine Braunsia Kriechbaumer and the
aphidiine Lysiphlebus Foerster were always
recovered together—despite the presence of
another aphidiine Ephedrus Haliday and the
related Mesostoa van Achterberg in the data
set. We suspect this result is caused by localized convergence in length and base composition (discussed further below).
Our preferred rooting analysis is shown
in Figure 4, obtained by using the POY
multiple alignment of the D2–D10 28S
sequences followed by ME C I tree building (note that gaps are treated as missing
data in such distance-based tree building).
Addition of the 18S gene had little effect:
All branches with bootstrap values >60%
in the preferred analysis of the 28S gene
alone were also recovered when this gene
was included. All analyses of the complete
D2–D10 28S region found Megalohelcon to
be the most basal Braconidae (although
sometimes as the sister taxon to a helconoid), but as discussed above, we did not
include this species in subsequent analyses
of the Braconidae. Instead, we placed
the root of the Braconidae between
the cyclostomes and the noncyclostomes.
FIGURE 4. Estimated phylogeny in Ichneumonoidea used for rooting the separate Braconidae and Ichneumonidae phylogenetic estimates. ME (LogDet) C I tree from POY multiple alignment of D2–D10 28S rDNA sequences (gap cost, 2). Bootstrap values >50% shown.
462
S YSTEMATIC BIOLOGY
VOL. 51
FIGURE 5. Estimated phylogeny in the noncyclostome Braconidae. ME (LogDet) C I tree from ClustalX multiple
alignment of D2–D3 28S rDNA sequences (gap opening and extension costs of 5 and 1, respectively). Ancestral MP
life history estimates are shown for main clades. See Figures 6 and 7 for species names. Extended dataset (142 taxa).
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
In the Ichneumonidae, the root appears
between the ophioniformes and the (pimpliformes C icheumoniformes) clade. The
only other taxa for which placement was
uncertain with respect to these rootings were
the Xoridinae, Labeninae, and Hybrizon in
the Ichneumonidae and Masona in the
Braconidae. However, these uncertainties
do not affect our later analyses because
xoridines and labenines are idiobionts, and
the life histories of Hybrizon and Masona are
unknown.
In Figures 5–7 we present the estimated
phylogenetic relationships within the
Braconidae, and Figure 8 illustrates the relationships within the Ichneumoninae, both
rooted as described above. Differences in the
463
recovery of the clades of interest caused by
different alignment and tree-building methods are summarized in Table 3. Regarding
the recovery of the Aphidiinae test clade, we
Žnd reasons to suspect only those alignments
obtained when using POY with default and
higher gap costs. This increases our concern
over this method. Failure to recover the
Aphidiinae as monophyletic in one of the
analyses with the “by eye” alignment was
rectiŽed when more aphidiine sequences
were included. The only other problem
was with the ClustalX alignment with very
low gap costs. We also found some striking
branch length differences, for example, at
the base of sigalphoid clade (Fig. 5). Some
of this may result from the greater lineage
FIGURE 6. Expansion of Figure 5 to show composition of helconoid and euphoroid clades in the noncyclostome
Braconidae. Bootstrap values >50% are shown. For MP ancestral life history codings, see Figure 5.
464
S YSTEMATIC BIOLOGY
VOL. 51
FIGURE 7. Expansion of Figure 5 to show composition of macrocentroid, microgastroid, and sigalphoid clades
in the noncyclostome Braconidae. Bootstrap values >50% are shown. For MP ancestral life history codings, see
Figure 5.
sampling involved (more sequence variation
will be represented in better-sampled clades;
Page and Holmes, 1998), but this clade also
apparently has an increased rate of molecular evolution (the base composition in this
clade is similar to that in, for example, the
microgastroid and ichneutine clades). Similar changes have been observed in this gene
in other insect lineages (Friedrich and Tautz,
1997).
Differences in the recovery of taxa of uncertain afŽnities are summarized in Table 4.
We commonly recover Masona close to the
sigalphoid clade and the Ichneutinae close
to the microgastroid clade, whereas the
afŽnities of the Orgilinae and Cenocoeliinae remain unresolved. Also, in all analyses, Proavga Belokobylskij, currently placed
in the outgroup (Hormiinae), is recovered in
the sister clade to the Aphidiinae (Fig. 5).
The placement of the clade consisting of the
Aphidiinae plus its sister group is sensitive
to the method of phylogeny estimation, although it is always recovered near the base
of the Braconidae.
Ancestral State Estimates and Their Robustness
In Table 3 we present the MP estimates
of ancestral life history under all our
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
465
FIGURE 8. Estimated phylogeny in the Ichneumonidae, showing Campopleginae clade. Single MPT length 3,590
(gap D 2) from POY alignment of D2–D3 28S rDNA sequences. For MP ancestral life history coding, see Figure 5.
Bootstrap values >50% are shown.
phylogenetic estimates. We Žnd no clear
difference in the ancestral state estimates
based on the different methods of phylogeny
estimation, even including those that failed
to recover the test clade as monophyletic.
We consistently Žnd that the estimate for the
ancestral node of the helconoid clade is to
attack concealed hosts and for the ancestral
node of the euphoroid clade is to attack
exposed hosts. Results are ambiguous for the
other four clades, with weak tendencies towards attacking concealed hosts in the
macrocentroid clade (although usually
this was because it was recovered within the
466
NA
Secondary
structure
p
3
2
ME C I
ME C I ext
MP
MP ext
ME C I
MP
MP
ME C I
ME C I
ME C I ext
ME C I
ME C I
MP
MP
ME C I
MP
MP
ME C I
MP
MP
ME C I
Tree building
method
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
2
0
0
3
0
0
Gap
cost
No
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
Aphidiinae
monophyletic
1
?
1
1
1
?
1
1
1
1
?
?
?
?
?
1
?
1
1
?
1
Sigalphoid
recovered as paraphyletic; c clade recovered excluding Charmon; m clade recovered excluding Mirax.
POY
2.5 (0.5)
5 (1)
5 (1)
10 (5)
20 (5)
1
NA
“By eye”
Clustal
Gap cost
(extension)
Alignment
method
Method of phylogeny estimation
0
?/?
0p /?
0p /?
0p
0p
0p
0p
0p
0
0
0
0p
0p /?
0p
0p
0p
0
0p
0p
0p
Helconoid
1
1
1p
1
1
1p
1
1
1
1
1
1
1p
1p
1
1p
1
1p
1p
1
1
Euphoroid
0
?
0
0
1c
0
0
?
?
?
?
?
?
?
0
0
0
?
0
0
?
Macrocentroid
Clade
?
?
0
?
1
0
?
?
?
?
?
?
0
?
?
1
1
?
1m
?
?
Microgastroid
?
NA
1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
?
1
1
NA
NA
NA
Campopleginae
TABLE 3. Maximum parsimony estimates of ancestral life history states in the six clades of interest. States: 0 D Host concealed. 1 D Host exposed. Tree building methods:
MP D maximum parsimony; ME C I D minimum evolution with invariable sites; all analyses of the Braconidae were with the 121 taxa data set except those marked “ext,”
in which the extended (142 taxa) data set was used. In some analyses the Diospilini was recovered separate from the main helconoid clade, and in those cases the ancestral
state of the Diospilini is shown after a forward slash. NA D not applicable (analysis not performed).
2002
467
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
TABLE 4. AfŽnities of taxa for which placement is sensitive to method of phylogeny estimation. Shown are the
clades or taxa the phylogeny-sensitive taxa are recovered within, or as sister groups to. See Table 3 for explanation of abbrevations of tree building methods. Other abbreviations: Cen D Cenocoeliinae, eph D euphoroid clade,
helc D helconoid clade, Ich D Ichneutinae, Org D Orgilinae, mac D macrocentroid clade, mic D microgastroid clade,
out D outgroup, sig D sigalphoid clade.
Method of phylogeny estimation
Alignment
method
Gap cost
(extension)
“By eye”
NA
Secondary
structure
NA
Clustal
POY
2.5 (0.5)
5 (1)
5 (1)
10 (5)
20(5)
1
2
3
Tree building
method
ME C I
ME C I ext
MP
MP ext
ME C I
MP
MP
ME C I
ME C I
ME C I ext
ME C I
ME C I
MP
MP
ME C I
MP
MP
ME C I
MP
MP
ME C I
Recovery of taxa
Gap
cost
Cenocoeliinae
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
2
0
0
3
0
0
mic
Org
euph
euph
euph
euph
—
mic
mac
mac
Org
mic
euph
euph
mic
euph
—
euph
euph
—
—
helconoid clade) and towards attacking exposed hosts in the sigalphoid and Campopleginae clades.
The ML ancestral state estimates are consistent with the above MP estimates (Table 5):
We Žnd conict only where the MP estimate is also sensitive to the method of phylogeny estimation; hence, we have little conŽdence in it anyway. There is also a strong
(and expected) correlation between the measures of robustness used with the two types
of estimates: step matrices (MP) and likelihood differences (ML) shown in Figure 9.
Only in two clades, therefore, the helconoid
and euphoroid, do we have a signiŽcant
level of support for their ancestral state
estimate, given our phylogeny estimates. In
Table 6 we show the robustness of the ancestral state estimates in these two clades
to the uncertainties in the phylogeny estimate. All trees on which the MP-based ancestral state estimate was either reversed or
ambiguous were signiŽcantly longer—or almost so (both Shimodaira–Hasegawa and
Kishino–Hasegawa tests). Furthermore, as
expected, performing the same analyses on
less well supported ancestral state estimates
found insigniŽcant amounts of difference
Orgilinae
—
Ich C mic
Ich C mic
Ich C mic
helc
none
none
Cen C mic
Ich C mic
Ich C mic
Cen
Cen C mic
Ich C mic
Ich C mic
Ich C mic
—
—
mic
helc C mac
—
Ich C mic
Ichneutinae
Masona
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
mic
sig
mic
sig
sig
sig
sig
sig
sig
sig
sig
out
sig
sig
sig
out
out
out
out
out
—
out
out
—
(not shown). For example, the ancestral state
of the Campopleginae (Ichneumonidae) was
either exposed or ambiguous (Table 3), but
in none of the three analyses where it was
exposed was the suboptimal tree on which
it was ambiguous >3 steps longer than the
MPT (not signiŽcant).
D ISCUSS ION AND CONCLUSIONS
Within this group of parasitoid wasps, we
are able to estimate with conŽdence the ancestral life history for two clades. These estimates, however, support life history transitions in different directions.
First, the robust estimate of the ancestral life history of the helconoid clade as a
koinobiont attacking concealed hosts means
there have been one or more transitions to
attacking exposed hosts within this clade.
This supports the more traditional view that
koinobiosis in the Helconinae evolved directly from the ancestral idiobiont biology
without an intermediate stage of attacking
exposed hosts (Gauld, 1988; Wharton, 1993)
rather than the hypothesis of Quicke et al.
(1999). We are surprised by this because helconoids lack the drilling ovipositors used
468
5 (1)
NA
Clustal
“By eye”
1
5 (1)
Clustal
POY
NA
Alignment gap
cost (extension)
Secondary structure
Alignment
method
ME C I
ME C I
ME C I
ME C I
ME C I
Tree building
method
142
142
121
121
121
Number of
taxa
MP
ML
MP
ML
MP
ML
MP
ML
MP
ML
Method of estimating
ancestral state
1
0
1
1
?
1
1
0
?
1
Sigalphoid
0
0¤
0
0¤
0
0¤
0
0
?
0
Helconoid
1
1¤
1
1¤
1
1¤
1
1¤
1
1¤
Euphoroid
Clade
1
0
?
0
?
0
?
0
?
1
Macrocentroid
1
0
?
1
?
0
?
0
?
0
Microgastroid
TABLE 5. Comparison of MP and ML ancestral state estimates for the Žve main clades in the noncyclostome braconids. See Table 3 for explanation of abbrevations of
tree building methods. ML estimate treated as binary, showing state with highest likelihood. 0 D host concealed, 1 D host exposed. ML estimates are marked with an asterisk
where the differences are signiŽcant (difference between the log. likelihoods for the alternative states is ¸2.0).
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
469
TABLE 6. Comparisons between MPT and shortest
trees found by manipulation in MacClade on which the
ancestral state estimation is ambiguous or reversed. The
ancestral state estimates based on the MPT are for the
helconoid clade to attack concealed hosts and for the
euphoroid clade to attack exposed hosts. The results of
two tests for comparing trees are shown: Shimodaira–
Hasegawa (SH) test (JC C0 model), indicating difference
in log likelihood, and the Kishino–Hasegawa (KH) test,
showing difference in tree length. (Note: KH test is twotailed). All tree building was with gaps treated as missing data.
FIGURE 9. Relationship between robustness of MP
and ML ancestral state estimates. Data points are from
Table 5, with clade names abbreviated. The x-axis shows
the weight given to the transition necessary to make the
MP estimate either ambiguous or reversed (weight of
reverse transition held at 1.0); the y-axis shows the difference in log likelihoods when the ancestral nodes are
Žxed sequentially at one of the alternative states.
Tree-building
method
Test
Helconoid
clade
Euphoroid
clade
“By eye”
(142 taxa)
Secondary
structure
POY
(gap cost 1:1)
POY
(gap cost 2:1)
POY
(gap cost 3:1)
SH
KH
SH
KH
SH
KH
SH
KH
SH
KH
37 ¤
21¤ ¤ ¤
25 (P D 0:065)
19¤ ¤ ¤
51 ¤
18 ¤
41 ¤¤
24¤ ¤ ¤
44 ¤
15 ¤¤
43¤
14¤
31¤
15¤ ¤
50¤
22 ¤ ¤ ¤
41¤ ¤
20 ¤ ¤ ¤
44¤
10 (P D 0:059)
¤P
by most idiobionts to gain access to their
hosts; instead, they have a morphologically
different ovipositor with an elaborate steering mechanism, which enables them to follow natural cracks or host-made openings in
the plant tissue (Quicke et al., 1995; Edwards
and Hopper, 1999).
Second, the robust estimate of the ancestral life history of the euphoroid clade as a
koinobiont attacking exposed hosts means
that there have been one or more transitions
to attacking concealed hosts within this
clade. This supports the hypothesis of Quicke
et al. (1999). It may be important that in this
clade the concealed hosts tend to be less concealed than in the helconoids—leaf miners
rather than seed, stem, and bark borers—and
hence the reversal from attacking exposed
hosts may be easier to make (requiring fewer
adaptations).
Considering the superfamily as a whole,
although analysis of related taxa strongly indicates a concealed idiobiont ancestral state
for the superfamily Ichneumonoidea, it does
not allow us to infer more about the degree of concealment. Many of the taxa related to the Ichneumonoidea attack hosts that
are only weakly concealed, and Gauld and
Wahl (2000b) suggest that drilling into solid
wood, as opposed to dead wood or bark, is
a derived trait within at least some ichneumonoid lineages. Another potential transi-
< 0:05, ¤ ¤ P < 0:01,
¤¤ ¤ P
< 0:001.
tion that emerges from our analyses is that
among the orgilines, to attack less concealed
hosts. The tribe Orgilini, whose ovipositors
are as long as the metasoma, is commonly
recovered as paraphyletic with respect to
the single other traditionally recognized
tribe, the Mimagathidini, whose ovipositors
are no longer than the height of the
metasoma.
For several other groups of koinobionts
in the Ichneumonoidea we cannot estimate their ancestral state because of the
lack of the necessary phylogenetic data.
In the Ichneumonidae, the (plimpliformes C
ichneumoniformes) clade has some endoparasitoid koinobionts of exposed hosts:
Diplazon Nees attacks aphidophagous syrphid eggs or larvae, and Euceros Grav. has
planidial larvae. The other endoparasitoid
koinobionts of exposed hosts occur within
subfamilies (Ichneumoninae and Cryptinae)
that typically attack weakly concealed hosts,
including pupae, and our analyses contain
insufŽcient examples to allow us to infer
transitions in these cases. Similarly, within
the cyclostome Braconidae is one major
clade of koinobiont parasitoids of exposed
hosts (Rogadinae: Rogadini). Shaw (1983) argued that these koinobiont parasitoids of exposed hosts evolved via koinobiont parasitoids of concealed hosts, but phylogenetic
470
S YSTEMATIC BIOLOGY
relationships to the latter group have yet
to be resolved. We are also unable to test
Gauld’s (1988) hypothesis that the transition
to koinobiont parasitoids of exposed hosts in
the ophioniform ichneumonids was by way
of ectoparasitic koinobiosis; in this clade, the
latter biology is today found only in the Tryphoninae, the placement of which is poorly
resolved by our analyses and which are commonly recovered as monophyletic (although
often at the base of the ophioniform clade).
Proposed Taxonomic Change
Proavga clearly does not belong in the
Hormiinae but rather in the Aphidiinae’s
sister group: a gall-associated Southern
Hemisphere clade, the members of which
have previously been scattered taxonomically through the Braconidae (Belshaw et al.,
2000; Oda et al., 2001). We also anticipate
that further DNA sequencing will reveal
more morphologically aberrant braconids
to belong in this clade. To raise each of
them to subfamily status would be unsatisfactory; therefore, we formally synonymize
the group name Hydrangeocolinae WhitŽeld
with Mesostoinae Achterberg, and transfer
Camberriini Belokobylskij to it (Wharton and
van Achterberg, 2000). All species found to
belong in this clade should now be placed
in the Mesostoinae (unless an older available
family-level name is involved).
ACKNOWLEDGMENTS
This work was funded by the NERC. We thank Rob
Freckleton, Rod Page, Mark Pagel, and Ward Wheeler
for their programs Moreways, SSE, Discrete, and POY,
respectively (and for help in running them). Richard
Grenyer and Adam Lewin installed and have valiantly
maintained our POY cluster. We are also grateful to
Gavin Broad, Konrad Dolphin, and Andrea Webster for
advice, and to the editors and reviewers of Systematic Biology for very helpful comments on an earlier draft of the
manuscript. For specimens we thank C. van Achterberg,
Y. Braet, G. Broad, C. Lopez-Vaamonde, M. Macedo, P.
Mayhew, K. Ryall, M. Sharkey, M. R. Shaw, S. R. Shaw,
R. Ubaidillah, G. Vick, R. A. Wharton, and D. Yanega.
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BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
473
APPENDIX. S PECIES US ED IN THE ANALYSES
Subfamily classiŽcation follows Quicke and van Achterberg (1990). Tribal classiŽcation, given only where relevant
to biology, follows Austin et al. (1993) and Sharkey (1992). One Chelonus sequence is courtesy of Jim WhitŽeld.
Accession numbers refer to D2–D3 28S rDNA sequences unless otherwise indicated: a D D2 28S region only,
b D D2–D10 28S region, c D 18S rDNA, d D taxa used in expanded data set only. Silwood D Silwood Park, Ascot,
Berkshire, UK.
ClassiŽcation
Braconidae (noncyclostomes)
Adeliinae
Agathidinae
Microdini
Cremnoptini
Disophrini
Earinini
Aphidiinae
Species
Adelius sp., Silwood
Z97961 a
Aerophilus sp.
Agathiella sp., Mt. Barker, Australia
Agathis montana Shestakov, Turkey
Alabagrus haenschi (Enderlein), Colombia
A. pachamama Sharkey, Colombia
A. parvifaciatus (Cameron), Colombia
A. stigma Brullé, Colombia
A. sp., Surinam
Bassus sp. 1, Silwood
B. sp. 2, Mt. Barker, Australia
B. sp. 3, Sarawak, Malaysia
Braunsia sp. 1, Sabah, Malaysia
AJ302785 a
AJ245682 a
AJ302786
AJ302787
AJ302788 a
AJ302789 a
AJ245683 a
AJ302790 a
Z97943
AF173217
AJ302793
AJ302919 b
AJ307449 c
AJ302797
AJ302804
AJ302805
AJ302802 a
AJ302806
AJ302826
AJ302810
AJ302811
AJ302826
Z97944 a
AJ245684
Z83587 a,d
AJ245689
AJ245690
Z83594
AJ302938 b
AJ009329 c
Z83598 a,d
AJ245693
AJ302910 b
AJ009332 c
Z83589 a,d
AJ245694 a, d
Z83585 a,d
AJ245696 a, d
AJ245697 a
Z83593 a,d
Z83588 a,d
Z97950 a
AJ302796 a
AF029120 a
AF029118 a
Z83605 a
Z97945 a
AJ245687 a
Z97949 a
B. sp. 2, Sao Tome, Africa
Cremnops sp. 1, Sulawesi, Indonesia
C. sp. 2. Seram, Indonesia
Coccygidium sp.
Dichelosus sp., French Guyana
Zelomorpha sp., Peru
Euagathis sp. 1
E. sp. 2
Pseudocremnops sp., Sulawesi, Indonesia
Earinus elator (Fabricius), Silwood
Archaphidius sp., Bangladesh
Binodoxys acalephae (Marshall), Silwood
Choreopraon totarae ms name, New Zealand
Diaeretus leucopterus Haliday, Germany
Dyscritulus planiceps (Marshall), Silwood
Ephedrus californicus Baker, USA
Ephedrus persicae Froggatt, Silwood
Lipolexis gracilis Foerster, Germany
Lysiphlebus fabarum (Marshall), Silwood
Blacinae
Cardiochilinae
Cenocoeliinae
Charmontinae
EMBL/GenBank
accession number
Monoctonus pseudoplatani (Marshall), Silwood
Paralipsis inervis Nees, Germany
Pauesia juniperorum Stary, CABI culture
Praon pequodorum Baker, USA
Pseudephedrus chilensis Stary, Chile
Pseudopraon mindariphagum Stary, USA
Trioxys pallidus (Halliday), Silwood
Blacus sp. 1, Silwood
B. sp. 2
Toxoneuron nigriceps (Viereck), culture
Cardiochiles fuscipennis Szepligeti
Cenocoelius analis (Nees), Silwood
C. artseni, Silwood
Capitonius sp., Colombia
Charmon sp., Silwood
474
S YSTEMATIC BIOLOGY
VOL. 51
APPENDIX . Continued
ClassiŽcation
Species
Cheloninae
Ascogaster sp. Queensland, Australia
Chelonus sp.1, Silwood
C. sp. 2, Fayetteville, Arkansas, USA
C. sp. 3, Lenswood, Australia
Phanerotoma sp., Kakadu, N. Territory, Australia
Phanerotomella sp., Java
Euphorinae
Chrysopophthorus sp.
Cosmophorus cembrae Ruschka, Switzerland
Centistes gasseni Shaw, Brazil
C. sp., Costa Rica
Dinocampus coccinellae (Schrank)
Falcosyntretus sp., USA
Lecythodella sp., Costa Rica
Leiophron uniformis (Gahan)
Mannokeraia gibsoni MS name, Australia
Microctonus aethiopiodes Loan
Orionis eximius Shaw
Peristenus sp., Silwood
Pygostolus sp., Silwood
Rhopalophorus sp., Canada
Helconinae
Eadyini
Brachistini
Streblocera (Eutanycerus) sp.
S. (Asiastreblocera) olivera Quicke and Purvis, Thailand
Wesmaelia lizanoi Shaw
Eadya sp. (or new genus near Eadya), Australia
Brulleia sp., Sarawak, Malaysia
Canalicephalus sp., Sabah, Malaysia
Eubazus semirugosus (Nees), Switzerland
Foersteria sp.
Schizoprymnus sp., Silwood and Israel
Triaspis sp., Turkey
Urosigalphus sp., Texas A&M culture
Diospilini
Helconini
Homolobinae
Ichneutinae
Macrocentrinae
Diospilus sp. 1, Costa Rica
D. sp. 2, Brazil
D. sp. 3, South Africa
Taphaeus sp., Turkey
Vadum sp., Brazil
Austrohelcon sp. 1, Australia
A. sp. 2, Australia
Helcon sp. 1, Ardenne, France, and Bola, Turkey
H. sp. 2, Sabah, Malaysia
Ussurohelcon nigricornis van Achterberg,
Sabah, Malaysia
Wroughtonia sp., Canada
Homolobus australiensis (Nixon), Australia
H. truncator (Say), Ankara, Turkey
Ichneutes bicolor Cresson
I. sp., Ascot, UK
Masonbeckia sp., French Guyana
Oligoneurus sp., Cameroun
Paroligoneurus sp., Costa Rica
Proterops sp., Belize
Aulacocentrum sp.
Austrozele sp.
Dolichozele sp.
Hymenochaonia sp., Costa Rica
Macrocentrus sp. 1, Mt. Barker, Australia
M. sp. 2, New Zealand
EMBL/GenBank
accession number
AF029121
Z83607 a
See legenda
AF029123 a
Z97960 a
AJ302933 b
AJ307462 c
AJ302801
AJ302803
AJ245688
AJ302800 a
AJ302807
AJ302812
AJ302818
AJ245692
AJ416968 d
AJ302821
AJ302824
Z97952 a
AJ302828
AJ302922 b
AJ307453 c
AJ302830
AJ302831
AJ302835
AJ302814
AJ302798
AJ302799
Z83608 a
AJ302813
AJ302934 b
AJ307463 c
AJ302833
AJ302923 b
AJ307454 c
AF029134
AJ302808
AJ416973 d
AJ302832
AJ302834
AJ416970 d
AJ416971 d
Z97946
AJ302815
AJ302912 b
AJ307445 c
AJ416972 d
AJ302915 b
AJ302816
Z97956 a
AF173223 a
AJ302819
Z97962 a
AJ245695 a
AJ302825
AJ302791 a
AJ302792 a
AJ302809
AJ302817
AF029135
AJ416969 d
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
475
APPEND IX. Continued
ClassiŽcation
Masoninae
Mesostoinae
Meteorideinae
Meteorinae
Microgastrinae
Microtypinae
Miracinae
Neoneurinae
Orgilinae
Mimagathidini
Species
Masona sp., Australia
Aspilodemon sp., Brazil
Mesostoa kerri Austin and Wharton, Australia
Proavga sp., Australia
Meteoridea sp. 1, Thailand
M. sp. 2, Thailand
M. sp. 3, Thailand
Meteorus rogerblancoi Zitani, Costa Rica
M. sp. 1, Silwood
M. sp. 2, Taiwan
Zele albiditarsis Curtis, Silwood
Apanteles subandinus Blanchard
Cotesia glomerata (L.)
Diolcogaster schizurae (Muesebeck)
Fornicia sp., Uganda
Microplitis demolitor Wilkinson
Sathon falcatus (Nees)
Microtypus wesmaelii Ratzeburg
Mirax lithocolletidis Ashmead
Elasmosoma sp., USA
Neoneurus mantis Shaw
Bentonia sp. 1
B. sp. 2
Trachypetinae
B. sp. 3
Stantonia sp., Sumatra
Orgilonia sp., Malaysia
Orgilus lepidus Muesebeck
O. sp.1, Silwood
O. sp.2, Sulawesi, Indonesia
Acampsis alternipes (Nees), Silwood
Sigalphus irrorator (Fabricius), Dordogne, France
S. gyrodontus He and Chen, Vietnam
Megalohelcon ichneumonoides Tobias, Australia
Xiphozelinae
Xiphozele sp., Java, Indonesia
Braconidae (cyclostomes)
Acanthormiinae
Braconinae
Acanthormius sp.
Syntomernus sp., Sabah, Malaysia
Orgilini
Sigalphinae
Doryctinae
Syngaster lepidus Brullé
Jarra maculipennis Marsh and Austin
Gnamptodontinae
Histeromerinae
Pseudognamptodon sp.
Thoracoplites sp.
Rogadinae
Ichneumonidae
Acaenitinae
Adelognathinae
Alomyinae
Anomaloninae
Rogas sp.
Banchinae
Atrophini
Phaenolobus sp., Dordogne, France
Adelognathus sp., Silwood
Alomya debellator (Fabricius), Silwood
Agrypon varitarsum (Wesmael), Silwood
A. sp., Silwood
Anomalon sp., Sabah, Malaysia
Brachynervus sp., Sabah, Malaysia
Trichomma sp., Sabah, Malaysia
Diradops sp., Peru
Leptobatopsis sp., Sabah, Malaysia
EMBL/GenBank
accession number
AJ302914 b
AJ245685
AJ302930 b
AJ307460 c
AJ416977 d
AJ416974 d
AJ416975 d
AJ416976 d
AJ302820
Z97953 a
AJ416967
Z97954 a
AF029126 a
AF029127 a
AF102741 a
Z97959 a
AF029129
AF029130
AJ302822 a
AF029131 a
AJ302929 b
AJ307444 c
AF029133
AJ302794
AJ302935 b
AJ307464 c
AJ302795
AJ302829 a
AJ416965 d
AF173221 a
Z97951 a
AJ302823
Z83609 a
Z97942 a
AJ416966 d
AJ302911 b
AJ307443 c
AJ302931 b
AJ307461
AJ302883
AJ296042 b
AJ307456 c
AJ245698 b
AJ302928 b
AJ307459 c
AJ296059
AJ302920 b
AJ307450 c
AJ296058
Z97928 a
Z97918 a
Z83645 a
AJ302927 b
AJ307458 c
Z97896
AJ302838
AJ302843
AJ302878
AJ302860
AJ302918 b
AJ307448 c
476
S YSTEMATIC BIOLOGY
VOL. 51
APPENDIX . Continued
ClassiŽcation
Banchini
Glyptini
Brachycyrtinae
Campopleginae
Collyriinae
Cremastinae
Cryptinae
Ctenopelmatinae
Cylloceriinae
Diacritinae
Diplazontinae
Eucerotinae
Ichneumoninae
Labeninae
Species
Lissonota sp. 1, Turkey
L. sp. 2, Silwood
Banchus volutatorius (L.) Hilbre Island, UK
Exetastes sp., Silwood
Apophua bipunctoria (Thunberg), Silwood
Brachycyrtus sp.
Charops sp., Sabah, Malaysia
Chriodes sp.
Diadegma mollipla (Holmgren)
D. sp., Brazil
Dusona sp., Silwood
Hellwigia obscura Gravenhorst, France
Lathrostizus sp., Silwood
Nonnus sp., Costa Rica
Rhimphoctona sp., Turkey
Venturia canescens (Gravenhorst),
Texas A&M Culture
Xanthocampoplex sp., Australia
Collyria coxator (Villers), Silwood
gen. sp. indeterminate , Kenya
Pristomerus vulnerator (Panzer), Silwood
Arthula sp., Australia
Ateleute sp., Sabah, Malaysia
Cisaris sp., Sabah, Malaysia
Demopheles corruptor (Taschenberg), Silwood
Dichrogaster sp., Silwood
Hadrocryptus sp., Silwood
Nematopodius debilis (Ratzeberg), Silwood
Paraphylax sp., Sabah, Malaysia
Phygadeuon sp., Silwood
Xanthocryptus sp., Sabah, Malaysia
Absyrtus sp., Silwood
Alexeter sp., Silwood
Pion sp., Dordogne, France
Perilissus sp., Silwood
Xenoschesis sp., Silwood
Cylloceria sp., Silwood
Diacritus aciculatus (Voll.),
Chippenham Fen, UK
Diplazon laetatorius (Fabricius), Silwood
Euceros sp., Silwood
CryptefŽgies albilarvatus (Gravenhorst), Silwood
Lusius sp., Sabah, Malaysia
Apechoneura sp., Belize
Grotea sp., Belize
Labena sp.1, Costa Rica
L. sp. 2, Belize
Poecilocryptus nigromaculatus Cameron, Australia
Lycorinae
Mesochorinae
Metopiinae
Microleptinae
Neorhacodinae
Lycorina sp. (apicalis gp)
Mesochorus sp., Silwood
Colpotrochia cincta (Scopoli), Silwood
Exochus sp., Silwood
Hypsicera sp., Silwood
Microleptes aquisgranensis (Först.),
Chippenham Fen, UK
Neorhacodes enslini (Ruschka),
Chippenham Fen, UK
EMBL/GenBank
accession number
AJ302859
Z97906 a
AJ302842
Z97907 a
AJ302840
Y18585 a
AJ302844
AJ302845
AJ302851
AJ302852
Z97891 a
AJ302858
Z97892 a
Z97893 a
AJ302872
AJ245958 a
AJ302917 b
AJ307447 c
Z97923 a
Z97895 a
Z97894 a
AJ302841
AJ302926 b
AJ307457 c
AJ302846
AJ302850
Z97920 a
AJ302857 a
Z97921 a
AJ302867
AJ292080
AJ302880
Z97901 a
Z97902 a
Z97904 a
Z97903 a
Z97905 a
AJ302847
Z97929 a
Z97925 a
Z97917 a
Z97919 a
AJ302913 b
AJ307446 c
AJ302839
AJ302856
Y18583 a
AJ302932 b
AJ307467 c
AJ302921 b
AJ307452 c
AJ302861 a
Z97900 a
Z97897 a
Z97898 a
Z97899 a
Z97924 a
Z97910 a
2002
BELSHAW AND QUICKE—ROBUSTNESS OF ANCESTRAL S TATE ESTIMATES
477
APPENDIX . Continued
ClassiŽcation
Ophioninae
Orthocentrinae
Orthopelmatinae
Oxytorinae
Paxylommatinae
Phrudinae
Pimplinae
Poemeniinae
Rhyssinae
Stilbopinae
Tersilochinae
Tryphoninae
Xoridinae
Species
Enicospilus ramidulus (L.), Silwood
Eremotylus sp., Dordogne, France
Euryophion latipennis (Kirby), Togo, Africa
Ophion ventricosus Gravenhorst, Silwood
O. obscuratus Fabricius
Thyreodon laticinctus Cresson, Belize
Proclitus sp., Silwood
Orthocentrus sp., Silwood
Megastylus sp., Belize
Picrostigeus recticauda (Thomson), Silwood
Orthopelma mediator (Thunberg),
Chippenham Fen, UK
Oxytorus sp., Silwood
Hybrizon buccatus Brebisson, Silwood
Phrudus badensis Hilpert, Silwood
Acropimpla sp., Sabah, Malaysia
Apechthis sp., Silwood
Delomerista mandibularis (Gravenhorst), Silwood
Dolichomitus imperator (Kriechbaumer), Silwood
Ephialtes manifestator (L.), Silwood
Exeristes sp., Turkey
Itoplectis sp., Silwood
Nomosphecia melanosoma (Morley), Australia
Parema sp., Sabah, Malaysia
Schizopyga sp., Silwood
Tromatobia oculatoria (Fabricius), Silwood
Zaglyptus sp., Florida, USA
Neoxorides nitens (Gravenhorst), Dordogne, France
Poemenia hectica (Gravenhorst), Silwood
Pseudorhyssa alpestris (Holmgren), Henley, UK
Cyrtorhyssa sp., Sabah, Malaysia
Epirhyssa sp., Sabah, Malaysia
Megarhyssa sp., Canada
Myllenyxis sp., Sabah, Malaysia
Rhyssella approximato r (Fabricius),
Chippenham Fen, UK
Triancyra sp., Sabah, Malaysia
Panteles schnetzeanus (Roman), Ascot, UK
Stilbops vetula (Gravenhorst), Dordogne, France
gen. sp. indeterminate, UK
Probles sp.
Stethantyx sp., Belize
Cosmoconus sp., Silwood
Dyspetes praerogator (Thomson), Silwood
Grypocentrus sp., Silwood
Monoblastus sp., Silwood
Netelia sp., Silwood
Oedemopsis scabricula (Gravenhorst), Silwood
Polyblastus sp., Silwood
Ischnoceros caligatus (Gravenhorst), Silwood
Odontocolon dentipes (Gmelin), Silwood
Aculeata
Dryinidae
Xorides praecatorius (Fabricius), Silwood
Xorides (Cyanoxorides) sp., Sabah, Malaysia
Lonchodryinus ruŽcornis (Dalman)
Bethylidae
Cephalonomia stephanoderis Betrem
Apidae
Apis mellifera L.
EMBL/GenBank
accession number
Z97887 a
Z97886 a
AJ302854
Z97888 a
Z97889 a
AJ302876
Z97926 a
Z97927 a
AJ302862
AJ302870
Z97922 a
AJ302865 a
AJ302908 b
AJ307441 c
Z97908 a
AJ302837
Z97934 a
AJ302849
Z97935 a
AJ302909 b
AJ307442 c
AJ302855
Z97937 a
AJ302864
AJ302868
Z97938 a
Z97939 a
AJ302882
Z97930 a
Z97931 a
Z97932 a
AJ302848
AJ302875
Z97933 a
AJ302863
AJ302873
AJ302877
AJ302866
Z97909 a
Z97890 a
AJ302871
AJ302874
Z97911 a
AJ302853
Z97912 a
Z97913 a
Z97914 a
Z97915 a
Z97916 a
AJ302916 b
AJ307451 c
AJ302924 b
AJ307455 c
Z83612 a
AJ302881
AJ302907 b
AJ307440 c
AJ302937 b
AJ307466 c
AJ302936
AJ307465 c