the basal ichneumonidae (insecta, hymenoptera): 28s d2 rdna considerations of the brachycyrtinae,...

The basal Ichneumonidae (Insecta, Hymenoptera): 28S D2 rDNAconsiderations of the Brachycyrtinae, Labeninae,Paxylommatinae and XoridinaeDONALD L. J. QUICKE, CARLOS LOPEZ-VAAMONDE & ROBERT BELSHAW

Accepted 8 January 1999 Quicke, D. L. J., Lopez-Vaamonde, C. & Belshaw, R. (1999) The basal Ichneumonidae

(Insecta, Hymenoptera): 28S D2 rDNA considerations of the Brachycyrtinae, Labeninae,

Paxylommatinae and Xoridinae. Ð Zoologica Scripta 28, 203±210.

Determining groundplan morphological and biological features can depend on knowing

which groups are derived most basally within that group. Within the Ichneumonidae,

several subfamilies variously have been postulated as occupying a basal phylogenetic

position or possessing largely plesiomorphic morphologies and biologies (e.g. Labeninae),

and therefore potentially exemplifying ancestral life-history stategies. Sequence data from

the D2 variable region of the nuclear 28S rDNA gene have been used to examine basal

relationships in the family. Paxylommatinae and Xoridinae consistently appear as basal

within the family under both maximum parsimony and neighbour-joining analyses.

Labeninae sensu Wahl (1993) often appear near the base of the Ichneumonidae but never as

the sister group of the remainder of the family. The Brachycyrtinae, recently removed

from the Labeninae by Wahl (1993), appear as part of the informal ichneumonoid group

of subfamilies in the most parsimonious trees but this was not statistically significant using

the likelihood-based, Kishino-Hasegawa test compared with a monophyletic Labeninae +

Brachycyrtinae. Biologies of labenines and brachycyrtines are discussed in relation to those

of putative relatives.

D. L. J. Quicke, Unit of Parasitoid Systematics, CABI Bioscience UK Centre (Ascot), Department

of Biology, Imperial College at Silwood Park, Ascot, Berkshire, SL5 7PY UK, and Department of

Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD UK. E-mail:

[email protected]

C. Lopez-Vaamonde and R.Belshaw, Unit of Parasitoid Systematics, CABI Bioscience UK Centre

(Ascot), Department of Biology, Imperial College at Silwood Park, Ascot, Berkshire, SL5 7PY UK.

IntroductionIn phylogenetic studies of relationships between higher

taxa it is frequently necessary to hypothesize groundplan

states, especially for large groups that encompass high

levels of internal homoplasy. Further, even if groundplans

are not the issue, studies are still better based on the

widest possible representation of taxa, and so knowledge of

most basal extant lineages helps ful®l this aim (Mishler

1994). Determination of groundplans should be indepen-

dent of those characters being considered for higher level

relationships so as to avoid potential circularity in the

argumentation. The parasitic wasp family Ichneumonidae

is not only one of the largest families of organisms, it is

also one typi®ed by very high levels of homoplasy (Gauld

& Mound 1982), and this makes interpretation of subfam-

ily level relationships particularly dif®cult. Therefore, we

have attempted to identify the most basal members of the

Ichneumonidae on the basis of gene sequence data and

have included representatives of the putatively least derived

groups together with a small but diverse range of other

ichneumonid and braconid species.

We recently presented analyses of the relationships

suggested by the D2 expansion region of the 28S rDNA

gene in a wide range of ichneumonid and braconid wasps

(Belshaw & Quicke 1997; Gimeno et al. 1997; Belshaw

et al. 1998), and found evidence that the subfamilies Xori-

dinae and Paxylommatinae occupy relatively basal posi-

tions in the Ichneumonidae, and that the Trachypetinae

occupied a relatively basal position in the Braconidae.

Unfortunately, we were not at that time able to obtain

suitable specimens for sequencing of any members of the

subfamily Labeninae. This subfamily has a Gondwanan

Q The Norwegian Academy of Science and Letters . Zoologica Scripta, 28, 1±2, 1999, pp203±210 203

distribution and comprises species that are plesiomorphic

for many characters of both adult and larval morphology

(Gauld 1983; Gauld & Holloway 1986; Wahl 1993), and

which are therefore putatively basal members of the

family. We have now obtained 28S D2 gene fragment

sequences from three representatives of Labeninae sensu

lato, representing three of the tribes recognized by Gauld

(1983), viz. Brachycyrtini, Labenini and Poecilocryptinae,

though we have not managed to obtain suitable material of

the Groteini, nor of the more recently recognized

Xenothyrini (Wahl 1996). Monophyly of the Labeninae

sensu Authors has been debated by Wahl (1993) who raised

the Brachycyrtini to subfamily status and, on the basis of a

putatively derived feature of the larval head capsule,

suggested that this group was more closely related to the

Ichneumoninae + Cryptinae group of subfamilies. Wahl

(1993) did not comment on the relationships of the

remaining Labeninae though he placed them with an unre-

solved, putatively basal grouping including the `Pimpli-

formes' (Wahl 1990) group of subfamilies, together with

the Orthocentrinae, Collyriinae and the Agriotypinae.

Material and methodsWe selected a representative sample of ichneumonid and

braconid taxa for analysis. The total number was limited to

28 species to allow reasonably thorough computerized

alignments to be constructed within a practical time frame.

The ichneumonids represented the informal pimpliformes,

ichneumoniformes, tryphoniformes and ophioniformes

groups (Gauld 1991) as well as the Xoridinae, Paxylomma-

tinae, Brachycyrtinae (Brachycyrtus sp.) and Labeninae

(Poecilocryptus sp. ex galls on Banksia in South Australia;

Labena sp. Costa Rica). The Braconidae was represented

by the Rhyssalus group, other cyclostomes, helconoid and

microgastroid groups of noncyclostomes and the Trachy-

petinae which previous analyses had suggested as being a

basal member of the family (see also Quicke et al., 1999).

The aculeates, Vespa crabro (Vespoidea) and Apis mellifera

(Apoidea) were used as outgroups; their sequences were

retrieved from GENBANK where all our sequences are

also deposited (Y1853-5).

DNA was extracted from single individuals by crush-

ing and incubation at 378C for approximately 18 h in

Proteinase K, followed by sodium acetate/ethanol preci-

pitation and re-suspension in 15 mL Tris/EDTA buffer

(pH 7.5). PCRs were then carried out in 50 mL volumes

containing 0.5 mL DNA extract, 0.5 mL Boehringer Taq

polymerase, 1.25 mL 20 mm primer, 1.25 mL 10 mm

dNTPs and 5 mL buffer (1.5 mM MgCl2). PCR products

were cleaned using QIAquick PCR puri®cation kits

(Qiagen) and then sequenced in both directions using

Thermosequenase (Amersham) at half recommended

volumes on an ABI 373 automated sequencer. The

reverse primer of Campbell et al. (1993) was used with

the forward primer of Belshaw & Quicke (1997). PCR

conditions were 30 cycles of 988C denaturation (15 s),

488C annealing (30 s) and 728C extension (40 s) with an

initial denaturation of 3 min at 938C and a ®nal exten-

sion of 728C for 3 min.

Sequences were aligned using the parsimony-based

program MALIGN (version 2.7 for UNIX) (Wheeler &

Gladstein 1994) with the following parameters: Gap to

substitution ratio 2 : 1, alignaddswap, alignswap, randor-

ders = 10, build, score = 4. Subsequent tree building was

performed using a prerelease PAUP version 4.0d63 (writ-

ten by David L. Swofford) treating gaps as missing data

with 100 random additions followed by TBR. Two of the

loop regions in Megalohelcon are much longer than those of

other braconids or ichneumonids, and excess bases were

trimmed prior to alignment (the full sequence of Megalo-

helcon is presented in Fig. 1 of Belshaw et al. 1998). These

were excluded from the alignment procedure as their

inclusion led to clearly non-sensical output. The aligned

sequences are shown in Appendix I.

A major consideration in interpreting the present

sequence analysis results is the possibility that Xoridinae,

Paxylommatinae, and Trachypetinae form a link between

the other ichneumonids and braconids because each of

these taxa are on long branches and so their placements

may therefore be an effect of long branch attraction (the

Felsenstein zone). This may be further exacerbated by the

considerable base compositional differences between

Ichneumonidae and Braconidae (excluding Trachypetinae).

In addition to using maximum parsimony, we have used

neighbour joining to construct dendrograms from our data

as this method is less susceptible to the effects of long

branch attraction.

The systematic placement of taxa included in the

analyses is shown in Appendix II.

Results and discussionBoth maximum parsimony analysis and neighbour joining

produced similar trees (Fig. 1A,B, respectively), with iden-

tical ichneumonoid backbone stucture, viz. (other Ichneu-

monidae ± Xoridinae - Paxylommatinae - Trachypetinae -

other Braconidae), and this is identical to that found by

Belshaw et al. (1998) which was based on manually aligned

sequences (gaps treated as 5th base or as missing data) and

which did not include any representatives of the Labeni-

nae. However, the rooting of the two trees shown here

differs from that found by Belshaw et al. (1998) in which

the representative aculeates joined between Paxylommati-

nae and Trachypetinae rather than between Xoridinae and

Paxylommatinae. Thus, rooting of trees from this gene

Basal Ichneumonidae ± molecular evidence . Quicke et al.

204 Zoologica Scripta, 28, 1±2, 1999, pp203±210 . Q The Norwegian Academy of Science and Letters

fragment is not stable to variations in taxonomic represen-

tation and alignment and should not therefore be relied

upon in making taxonomic decisions. Thus, although

present analyses showed high bootstrap support for the

Paxylommatinae + Braconidae clade suggesting that the

Paxylommatinae may be better considered as basal braco-

nids, until additional gene regions have been analysed and

sensitivity analyses conducted we do not recommend any

formal changes in classi®cation. Incorporation of addi-

tional species of Xoridinae, Paxylommatinae and Trachy-

petinae are also highly desirable. Further, it should be

noted that some fossils attributed to the Paxylommatinae

possess forewing vein 2 m-cu and so more closely resemble

extant Ichneumonidae sensu stricto. Interestingly, Paxylom-

matinae appeared basal to the Braconidae in some of the

analyses of Quicke et al. (1999) in which they were not

scored for the presence or absence of vein 2 m-cu because

of its presence in some fossil taxa.

Excluding the Paxylommatinae, the next most basal of

the ichneumonids, according to present analyses, is the

Xoridinae, in agreement with recent molecular analyses

that did not include labenines (Belshaw et al. 1998). The

Labeninae sensu stricto (Labena, Poecilocryptus) are placed at

the base of the Ophioniformes clade in the maximum

parsimony tree (Fig. 1A) and at the base of the Pimpli-

formes + Ichneumoniformes clade in the neighbour joining

tree (Fig. 1B) but in neither case was there strong boot-

strap support.

In neither of the analyses shown here, nor in many

others not shown, did Brachycyrtus form a clade with the

two Labeninae sensu stricto, instead in both it appeared as

the sister group of the Eucerotinae and together these

were placed within the group of taxa that we selected to

represent the ichneumoniformes group of subfamilies as

recovered by Belshaw et al. (1998) (most parsimonious

tree; Fig. 1A) or at the base of an ichneumoniformes +

pimpliformes clade (neighbour joining tree; Fig. 1B). The

®rst of these ®ndings is largely in agreement with Wahl

(1993) who considered the derived U-shaped larval salivary

ori®ce to be a character uniting a large group of ichneu-

Fig. 1 Phylogenetic af®nities of the Brachycyrtinae and labenine species (shown boxed) based on 28S D2 rRNA sequences (othersequences are from EMBL/GenBank/DDBJ databases). Ð A. Phylogram is one of four shortest trees length 1215 (CI = 0.498,RC = 0.254), a strict consensus of which merely collapses the nodes marked with an asterisk; Ð B. Neighbour joining tree (based onuncorrected distance matrix). (Bootstrap values above 50% are shown).

Quicke et al. . Basal Ichneumonidae ± molecular evidence


monid subfamilies (i.e. most ichneumonids except for the

Labeninae sensu stricto, the pimpliformes, Collyriinae,

Orthopelmatinae and Agriotypinae), and thus the presence

of this apomorphy in Brachycyrtus indicates that this genus

should be removed from the Labeninae sensu stricto, and

consequently Wahl (loc. cit.) elevated the Brachycyrtini to

subfamily status. Wahl (1993) further inferred that the

Brachycyrtinae may be the sister group of the Ichneumoni-

nae + Cryptinae (Ichneumoniformes) on the basis of the

elongate 1st metasomal sternite and pentagonal forewing

areolet. Although in our analyses, Brachycyrtinae came out

next to Eucerotinae, these together formed the sister

group of the cryptine, Dichrogaster, within a paraphyletic

Cryptinae. Thus the most parsimonious tree (Fig. 1A)

supports Wahl's (1993) association of Brachycyrtinae with

the Ichneumoniformes. However, constraining the three

species that had in the past been placed in the Labeninae

(Labena, Poecilocryptus and Brachycyrtus) to form a mono-

phyletic group, resulted in two most parsimonious trees

that are only four steps longer than the unconstrained one

(1219 vs. 1215 steps) and this difference was not signi®cant

in the Kishino-Hasegawa test as performed in paup version

4.0d63. Further molecular data are therefore desirable

before reaching ®rm conclusions about the relationships of

the Brachycyrtinae.

Wahl (1993) proposed a number of characters in support

of the monophyly of the Labeninae (excluding the Brachy-

cyrtinae), viz. the form of the notauli and the lower ovipo-

sitor valves which partly enclose the upper valve near the

apex (though a similar condition also occurs in some

Pimplinae, e.g. Delomerista). Present analyses which consis-

tently place Labena and Poecilocryptus as sister taxa therefore

supports Wahl's views at least to some extent.

The ancestral Ichneumonoidea have been postulated as

being most likely to have been idiobiont ectoparasitoids of

wood-boring insect larvae (Gauld 1988). This biology is

exhibited by the Xoridinae and many members of the

pimpliformes group of Ichneumonidae and by the Rhyssalus

group and many cyclostome braconids. The biologies of

Hybrizon (Paxylommatinae) and Megalohelcon (Trachypeti-

nae) are not known but the former are associated with ants

and the short curved ovipositor of the latter show that

neither of these have that biology. Their basal positions in

the results of these analyses and morphological data

presented in a companion paper (Quicke et al. 1999) there-

fore either suggests that generally accepted views about

ichneumonoid ancestral biology are wrong or shows that

these putatively basal lineages have very specialized biolo-

gies and do not represent the ancestral condition at all.

The Labeninae sensu lato display a range of biologies:

Labena species are idiobiont ectoparasitoids of xylophagous

coleopteran and symphytan larvae, Poecilocryptus attacks

gall formers and may be partly phytophagous. Unlike

these, Brachycyrtus attacks loosely concealed pupae of chry-

sopid lacewings and the closely related genus, Adelphion,

attacks spider egg cocoons. The biology of the Brachycyr-

tinae is thus more similar to that of typical cryptines than

to Labeninae sensu stricto, and further supports ®ndings

based on morphology (Wahl 1993) and DNA.

AcknowledgementsWe would like to thank Ian Gauld and Andy Austin for

providing specimens of brachycyrtines and labenines for

DNA extraction, and Mark Dowton for supplying the

sequence data for Toxoneuron. This work was supported by

the NERC (Natural Environment Research Council)

Initiative in Taxonomy, and prompted by the excellent

Workshop on Hymenoptera Phylogenetics organized by

Fredrik Ronquist in Uppsala in March 1998.

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rDNA. Insect Molecular Biology, 6, 273±284.

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Appendix I Malign alignment of D2 region of 28S rDNA of taxa studied. Two long excised loop regions of the Megalohelcon sequence notincluded in the alignment are not shown, their positions are indicated by `[insert 1]' and `[insert 2]' and the full sequence for this taxon isshown in Figure 1 of Belshaw et al. (1998).



Appendix IÐContinued



Appendix II Classi®cation of Ichneumonoidea taxa included in study (same order as in Appendix I).

Family Subfamily Genus and species

Ichneumonidae Labeninae Poecilocryptus sp.

Labena sp.

Brachycyrtinae Brachycyrtus sp.

Ophioninae Eremotylus sp.

Cremastinae Pristomerus vulnerator

Metopiinae Exochus sp.

Ctenopelmatinae Perilissus sp.

Neorhacodinae Neorhacodes enslini

Tryphoninae Polyblastus sp.

Adelognathinae Adelognathus sp.

Eucerotinae Euceros sp.

Cryptinae Dichrogaster sp.

Nematopodius debilis

Ichneumoninae Cryptef®gies albilarvatus

Rhyssinae Megarhyssa sp.

Pimplinae Ephialtes manifestator

Xoridinae Xorides praecatorius

Paxylommatinae Hybrizon buccata

Braconidae Trachypetinae Megalohelcon ichneumonoides

Aphidiinae Praon volucre

Helconinae Helcon sp.

Cardiochilinae Toxoneuron nigriceps

Doryctinae Polystenus rugosus

Histeromerinae Histeromerus mystacinus



the basal ichneumonidae (insecta, hymenoptera): 28s d2 rdna considerations of the brachycyrtinae,...

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