Phylogeny of pteromalid parasitic wasps (Hymenoptera: Pteromalidae): Initial evidence from four protein-coding nuclear genes

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Molecular Phylogenetics and Evolution 45 (2007) 454–469 www.elsevier.com/locate/ympev

Phylogeny of pteromalid parasitic wasps (Hymenoptera: Pteromalidae): Initial evidence from four protein-coding nuclear genes Christopher A. Desjardins b

a,* ,

Jerome C. Regier b, Charles Mitter

a

a Department of Entomology, University of Maryland, College Park, MD 20742, USA Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742, USA

Received 8 November 2006; revised 10 August 2007; accepted 15 August 2007 Available online 26 August 2007

Abstract Chalcidoidea (approximately 22,000 described species) is the most ecologically diverse superfamily of parasitic Hymenoptera and plays a major role in the biological control of insect pests. However, phylogenetic relationships both within and between chalcidoid families have been poorly understood, particularly for the large family Pteromalidae and relatives. Forty-two taxa, broadly representing Chalcidoidea but concentrated in the ‘pteromalid lineage,’ were sequenced for 4620 bp of protein-coding sequence from four nuclear genes for which we present new primers. These are: CAD (1719 bp) DDC (708 bp), enolase (1149 bp), and PEPCK (1044 bp). The combined data set was analyzed using parsimony, maximum likelihood, and Bayesian methods. Statistical significance of the apparent nonmonophyly of some taxonomic groups on our trees was evaluated using the approximately unbiased test of Shimodaira [Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51(3), 492–508]. In accord with previous studies, we find moderate to strong support for monophyly of Chalcidoidea, a sister-group relationship of Mymaridae to the remainder of Chalcidoidea, and a relatively basal placement of Encarsia (Aphelinidae) within the latter. The ’pteromalid lineage’ of families is generally recovered as monophyletic, but the hypothesis of monophyly for Pteromalidae, which appear paraphyletic with respect to all other families sampled in that lineage, is decisively rejected (P < 1014). Within Pteromalidae, monophyly was strongly supported for nearly all tribes represented by multiple exemplars, and for two subfamilies. All other multiply-represented subfamilies appeared para- or polyphyletic in our trees, although monophyly was significantly rejected only for Miscogasterinae, Ormocerinae, and Colotrechninae. The limited resolution obtained in the analyses presented here reinforces the idea that reconstruction of pteromalid phylogeny is a difficult problem, possibly due to rapid radiation of many chalcidoid taxa. Initial phylogenetic comparisons of life history traits suggest that the ancestral chalcidoid was small-bodied and parasitized insect eggs.  2007 Elsevier Inc. All rights reserved. Keywords: Hymenoptera; Chalcidoidea; Pteromalidae; Phylogenetics; Approximately unbiased test

1. Introduction Chalcidoidea, one of the largest superfamilies of parasitic Hymenoptera, includes about 22,000 described species (Noyes, 2003). The majority of chalcidoids, commonly called chalcids, are parasitoids and have major importance in the biological control of insect pests (Greathead, 1986; Debach and Rose, 1991). Chalcidoids show a diversity of *

Corresponding author. Present address: Department of Biology, University of Rochester, RC Box 270211, Rochester, NY 14627, USA. E-mail address: [email protected] (C.A. Desjardins). 1055-7903/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.08.004

parasitic habits comparable to that of all other parasitic Hymenoptera combined (Grissell and Schauff, 1997), attacking 13 orders of insects as well as a variety of arachnids and even nematodes. This host diversity is mirrored by their broad array of life history strategies. Chalcidoids may be endo- or ectoparasitic (developing within or outside of their host), idio- or koinobionts (arresting host development immediately or at a later life stage), arrheno- or thelytokous (unfertilized eggs develop into males or parthenogenetically into females), and show tremendous variety in other life history features as well. Reversion to the phytophagy ancestral for Hymenoptera, primarily in

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the form of gall forming and seed feeding, has also occurred in Chalcidoidea. Numerous hypotheses, often conflicting, have been offered about the evolution of chalcid life histories. For example, both egg parasitism (Dowton and Austin, 2001) and ectoparasitism of wood-boring beetles (Boucek, 1988b) have been argued to be the ancestral chalcidoid habit. Chalcidoid evolution offers a prominent example of diversification associated with shifts in ecological roles, awaiting reconstruction and deconstruction by phylogenetic analysis. Monophyly for Chalcidoidea, which currently contains 20 families (Grissell and Schauff, 1997), is well established (Gibson, 1986), and a basal position for Mymaridae is supported by both morphological (Heraty et al., 1997; Quicke et al., 1994) and molecular evidence (Campbell et al., 2000). Despite the ecological and economic importance of Chalcidoidea, however, phylogenetic relationships both within and between chalcidoid families are still largely obscure. Noyes (1990) offered an intuitive scheme of family relatedness, and recent morphological cladistic studies have treated several individual families or small complexes of families (e.g. Heraty and Darling, 1984; Woolley, 1988; Gibson, 1989, 2003; Wijesekara, 1997; Heraty, 2002), but broad analyses of among-family relationships have been few. A central difficulty in chalcidoid systematics is the status of the problematic family Pteromalidae, one of the three largest in the superfamily (3506 described species; Noyes, 2003). Pteromalidae is defined only by the absence of features defining other families and has often been hypothesized to be paraphyletic with respect to a number of these. However, definitive evidence for this assertion has been lacking. A group of putatively related families including Pteromalidae and relatives are often referred to collectively as the ‘pteromalid lineage’, distinguished from ‘mymarid’ and ‘eulophid lineages’ of chalcids. The ‘pteromalid lineage’ is defined here, following Gibson et al. (1999), as chalcidoids with five tarsal segments and, to a lesser degree, with at least eight flagellar segments and a recurved protibial spur. It is comprised of Agaonidae, Chalcididae, Encyrtidae, Eucharitidae, Eupelmidae, Eurytomidae, Leucospidae, Perilampidae, Pteromalidae, Tanaostigmatidae, and Torymidae (Campbell et al., 2000). Within Pteromalidae 31 subfamilies are currently recognized (Noyes, 2003), although inclusion and exclusion of many subfamilies is still highly uncertain. Numerous hypotheses have been advanced about relationships among subfamilies (e.g. Graham, 1969; Boucek, 1974; Boucek, 1988a,b; Darling, 1988), but none have been subjected to rigorous phylogenetic tests. A subfamily-level phylogeny for the pteromalids and other associated chalcidoids, testing monophyly for Pteromalidae, would therefore be a significant step toward better understanding of chalcidoid phylogeny and evolution. To¨ro¨k and Abraham (2001) conducted the only broadranging morphological phylogenetic study of Pteromalidae. Their data set consisted of 90 characters coded for

455

38 exemplars, representing seven families all belonging to the ‘pteromalid lineage.’ Eleven subfamilies of Pteromalidae were represented, although the sampling was heavily focused on Pteromalinae and Miscogasterinae (20 taxa). ‘Pteromalid lineage’ families Chalcididae and Eurytomidae were used as outgroups, and the authors report that Pteromalidae emerged as polyphyletic, although no support values were given. Previous molecular studies of relationships within and among chalcid families, based mainly on mitochondrial and nuclear ribosomal markers, have sometimes, but not always, produced strong resolution. Analyses of nuclear ribosomal RNA have supported placement of Elasmidae within Eulophidae (Gauthier et al., 2000), polyphyly of Agaonidae sensu lato (Rasplus et al., 1998), and nonmonophyly of Eurytomidae (Chen et al., 2004). Analysis of 28S rRNA has discerned subfamily-level relationships within Eucharitidae (Heraty et al., 2004). A broader study of Chalcidoidea utilizing 28S rRNA by Campbell et al. (2000), sampling multiple chalcid families and seven pteromalid subfamilies, provided strong resolution for basal chalcidoid familes, but little resolution within Pteromalidae. Mitochondrial gene studies of hymenopteran phylogeny have often proved problematic. Chen et al. (2004) found that the phylogeny generated for Eurytomidae from 16S and COI was largely incongruent with the one generated from nuclear ribosomal genes 18S and 28S. In studies of higher-level hymenopteran relationships, Dowton and Austin (1995, 2001) found that resolution was limited by extreme base compositional heterogeneity across taxa. In a broad survey across insect studies, Lin and Danforth (2003) found that nuclear genes had more homogenous patterns of among-site rate variation, less biased base compositions, and more symmetrical rate matrices than mitochondrial genes. Given this evidence, it seems clear that additional markers are needed to solve problems of chalcidoid phylogeny, particularly pteromalid relationships. Protein-coding nuclear genes are an especially promising source of evidence for insects generally, and have begun to be applied in Hymenoptera. Several studies of this order have included sequences of elongation factor-1a (EF-1a; Belshaw and Quicke, 1997; Belshaw et al., 2000; Dowton and Austin, 1998, 2001; Danforth et al., 2004). Danforth et al. (2004) used wingless and long-wavelength opsin, in addition to EF-1a, to resolve Cretaceous-age divergences in bees. Rokas et al. (2002) examined the phylogenetic utility of eight genes in cynipid wasps, including both EF-1a and long-wavelength opsin, and concluded that the latter show promise for resolving intra-familial divergences within parasitic Hymenoptera. This paper presents an initial attempt to bring multiple protein-coding nuclear genes to bear on the problem of pteromalid phylogeny. The 42 exemplars analyzed represent a broad array of ‘pteromalid lineage’ families and pteromalid subfamilies, treated provisionally as the ingroup,

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plus multiple outgroups. These taxa were sequenced for a total of 4620 bp of sequence from four nuclear protein-coding genes which have been applied rarely, if ever, to hymenopteran phylogenetics, but are known to be highly useful in other insects. The genes studied were: CAD (also known as rudimentary; 1.8 kbp; Moulton and Wiegmann, 2004); dopa decarboxylase (DDC; 700 bp; Fang et al., 1997, 2000), enolase (1.1 kbp; Farrell et al., 2001), and phosphoenolpyruvate carboxykinase (PEPCK; 1.0 kbp; Friedlander et al., 1996). These genes were selected largely on criteria discussed by Friedlander et al. (1992), including probable phylogenetic utility, low apparent copy number, extensive coding regions, and a lack of obvious nucleotide bias and internal repeats. They were also chosen for their complementary rates of divergence in order to collectively provide resolution across the varying depth that pteromalid/chalcid phylogeny probably represents. Our chief goals were to assess the potential of these genes for resolving pteromalid relationships, and to provide a first quantitative test of the long-held postulate that Pteromalidae are paraphyletic with respect to other chalcid families. We also hoped to make these genes more widely available, by developing primers for their use in chalcidoid phylogenetics. 2. Materials and methods 2.1. Taxon sampling The taxon sample (Table 1) included all the tribes and subfamilies of Pteromalidae for which we were able to obtain material that proved suitable for sequencing. The 28 pteromalid genera analyzed represented 14 subfamilies, two tribes each of Colotrechninae, Eunotinae, Miscogasterinae, and Ormocerinae, and three tribes of Cleonyminae. While the exemplars chosen represent less than half of the 31 pteromalid subfamilies, the 14 subfamilies sampled encompass 95% of the genera (559/588) and 97% of the species (3405/3506; Noyes, 2003), thus representing the bulk of the diversity in Pteromalidae. The five species of Diparinae included formerly represented five genera, reduced to two by a new reclassification (Desjardins, in press). Also included were six exemplars representing five of the other ten ‘pteromalid lineage’ families (Chalcididae, Eurytomidae, Eucharitidae, Perilampidae, and Torymidae), providing a test for paraphyly of Pteromalidae. Putative outgroup chalcidoids, i.e. outside the ‘pteromalid lineage,’ included single exemplars from three of the remaining eight families: Mymaridae, the basal position of which in the superfamily is strongly supported (Quicke et al., 1994; Heraty et al., 1997; Campbell et al., 2000); Eulophidae, for which the ‘eulophid lineage’ of Noyes (1990) is named; and Encarsia, which belongs to the probably polyphyletic family Aphelinidae (Gibson et al., 1999). Encarsia, resolved as a basal chalcidoid in the Campbell et al. (2000) analysis of 28S rDNA, was added to break up a long branch separating the mymarid from the remainder of Chalcidoidea in preliminary analyses. Finally, three

non-chalcidoid outgroups were included, one from Ceraphronidae (Ceraphronoidea) and one each from Scelionidae and Platygasteridae (Platygastroidea). Both superfamilies have been previously hypothesized to be close relatives of Chalcidoidea (e.g., Dowton and Austin, 2001). 2.2. Gene sampling CAD is a multienzymatic protein, composed of carbamoyl-phosphate synthetase II (CPSase), aspartate transcarbamylase, and dihydro-orotase, that catalyses multiple steps in the de novo synthesis of pyrimidines. In the first application of CAD to systematics, Moulton and Wiegmann (2004) used 4 kbp of CAD (particularly the CPSase domain) to resolve relationships within eremoneuran Diptera, recovering trees strongly concordant with those from morphology and 28S rDNA. Danforth et al. (2006) utilized a 3 kbp fragment of the CPSase domain of CAD in a phylogenetic study in combination with RNA polymerase II, obtaining strong support for family- and subfamily-level relationships within bees. In this study we sequenced 1719 bp of the CPSase domain, which corresponds roughly to the 50 -end of Moulton and Wiegmann’s 4 kbp fragment. DDC catalyzes the conversion of tyrosine to dopamine, and of tryptophan to serotonin. Both synonymous and non-synonymous changes in DDC have proven useful for resolving relationships within and between families and superfamilies of Lepidoptera (Fang et al., 1997, 2000; Friedlander et al., 1998, 2000), and in Diptera, within Simuliidae (Moulton, 2000) and Drosophila (Tatarenkov et al., 1999). In this study, we sequenced 708 bp of DDC, which lies approximately in the center of previously studied fragments. Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway, and catalyzes the reverse reaction during gluconeogenesis. Enolase was judged to be more slowly evolving than PEPCK or DDC by Friedlander et al. (1992), and has been recently utilized to resolve Lower-Mesozoic-aged relationships within curculionid beetles (Farrell et al., 2001; Sequeira and Farrell, 2001). Two copies of enolase were discovered in beetles by Sequeira and Farrell (2001), but these were easily distinguishable by intron structure. Although only one copy amplified in most taxa in this study, two copies were found in a few. Intron structure could not be examined because the gene was amplified by RT-PCR, but the two copies within species were much more divergent from each other than from the apparently corresponding copies in other taxa. Thus, establishment of orthology was not problematic. PEPCK catalyzes the conversion of oxaloacetate to phosphoenolpyruvate during gluconeogenesis. Both PEPCK and DDC were judged to be comparably intermediate in rate (Friedlander et al., 1992). Non-synonymous changes in PEPCK recovered Mesozoic-aged subordinal divergences in Lepidoptera (Friedlander et al., 1996), but the

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Table 1 Taxa sampled in this study, and their higher classification within Hymenoptera. Genbank accession numbers for each gene are listed to the right of each taxon Higher Taxon Ceraphronoidea Ceraphronidae Platygastroidea Platygasteridae Scelionidae Chalcidoidea ‘mymarid lineage’ Mymaridae ‘eulophid lineage’ Aphelinidae Eulophidae ‘pteromalid lineage’ Chalcididae Dirhinae Haltichellinae Eurytomidae Perilampidae Pteromalidae Asaphinae Cerocephalinae Cleonyminae Chalcedectini Cleonymini Lyiscini Coelycobinae Colotrechninae Colotrechnini Hetreulophini Diparinaea

Eunotinae Eunotini Moranilini Eutrichosomatinae Herbertiinae Miscogasterinae Trigonodorini Sphegigastrini Ormocerinae Melanosomellini Systasini Pireninae Pteromalinae Spalangiinae Torymidae

Genus

PEPCK

DDC

Enolase

CAD

Aphanogmus sp.

DQ924418

DQ990739

DQ990780

DQ999898

Platygaster sp. Scelio sp.

DQ924442 DQ924446

DQ990766 DQ990769

DQ990807 DQ990811

DQ999924 DQ999928

Gonatocerus sp.

DQ924437

DQ990760

DQ990801

Encarsia sp. Baryscapus sp.

DQ924424 DQ924426

DQ990745 DQ990749

DQ990786 DQ990790

DQ999904 DQ999908

Dirhinus sp. Hockeria sp. Heimbra opaca Steffanolampus sp.

DQ924423 DQ924434 DQ924430 DQ924450

DQ990744 DQ990757 DQ990753 DQ990773

DQ990785 DQ990798 DQ990794 DQ990815

DQ999903 DQ999916 DQ999912 DQ999932

Enoggera sp. Neocalosoter sp. Theocolax sp.

DQ924439 DQ924419

DQ990746 DQ990762 DQ990740

DQ990787 DQ990803 DQ990781

DQ999905 DQ999920 DQ999899

DQ990777 DQ990782 DQ990788 DQ990816 DQ990805

DQ999900 DQ999906 DQ999933 DQ999922

Chalcedectus sp. Cleonymus sp. Epistenia sp. Thaumasura sp. Ormyromorpha sp.

DQ924415 DQ924420 DQ924451 DQ924441

DQ990737 DQ990741 DQ990747 DQ990774 DQ990764

Colotrechnus sp. Hetreulophus sp.

DQ924421 DQ924432

DQ990742 DQ990755

DQ990783 DQ990796

DQ999901 DQ999914

Neapterolelaps sp. 1 Neapterolelaps sp. 2 Dipara trilineatus Dipara sp. 1 Dipara sp. 2 Lelaps sp.

DQ924416 DQ924438 DQ924414 DQ924422 DQ924435

DQ990738 DQ990761 DQ990736 DQ990743 DQ990765 DQ990758

DQ990778 DQ990802 DQ990776 DQ990784 DQ990806 DQ990799

DQ999896 DQ999919 DQ999895 DQ999902 DQ999923 DQ999917

Eunotus sp. Scutellista sp. Moranila sp. Ophelosia sp. Eutrichosoma sp. Herbertia sp.

DQ924427 DQ924447 DQ924436 DQ924440 DQ924428 DQ924431

DQ990750 DQ990770 DQ990759 DQ990763 DQ990751 DQ990754

DQ990791 DQ990812 DQ990800 DQ990804 DQ990792 DQ990795

DQ999909 DQ999929 DQ999918 DQ999921 DQ999910 DQ999913

Plutothrix sp. Polstonia sp.

DQ924443 DQ924444

DQ990767

DQ990808 DQ990809

DQ999925 DQ999926

Hemadas sp. Semiotellus sp. Gastrancistrus sp. Brachycaudonia sp. Psilocera sp. Spalangia cameroni Torymus sp.

DQ924433 DQ924448 DQ924429 DQ924417 DQ924445 DQ924449 DQ924452

DQ990756 DQ990771 DQ990752

DQ990797 DQ990813 DQ990793 DQ990779 DQ990810 DQ990814 DQ990817

DQ999915 DQ999930 DQ999911 DQ999897 DQ999927 DQ999931 DQ999934

DQ990768 DQ990772 DQ990775

a Classification of Diparinae follows Desjardins (in press). Here, Neapterolelaps sp. 1 = Australolaelaps sp. in the prior classification scheme, Dipara sp. 2 = Parurios sp., and Dipara trilineatus = Alloterra trilineatus.

amino acid sequence was deemed by the authors to be too invariant at lower taxonomic levels. PEPCK has also been utilized to study the phylogenetics of simuliid flies (Moulton, 2000), xylocopine bees (Leys et al., 2002), and

carabid beetles (Zhang et al., 2005; Sota et al., 2005). In this study 1044 bp of PEPCK were sequenced, of which the Friedlander et al. fragment correspond roughly to the 30 -half.

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2.3. Sample collection and storage Most specimens were collected directly into 100% EtOH at ambient temperatures and transferred within a month to storage at 80 C. Specimens collected in malaise traps in 95% EtOH proved satisfactory as long as they were placed in 100% EtOH at 80 C within a month of collection. Voucher specimens are stored at 80 C at the Center for Biosystems Research, University of Maryland Biotechnology Institute. Since extractions were conducted on entire specimens, voucher specimens represent individuals from the same collecting event judged to be the same species based on morphological examination. 2.4. Extraction and amplification Total nucleic acid (TNA) extractions were performed using the SV Total RNA Isolation System (Promega). Extractions were conducted on entire specimens due to the minute size of these insects (most are <2 mm). The extractions were subsequently lyophilyzed and rehydrated in a smaller volume (20 ll, 1/5 of initial volume) to concentrate the nucleic acids. All genes were amplified by RT-PCR to avoid introns. Reverse transcription (RT) was performed in 10 ll of a solution including 2 ll 25 mM MgCl2, 1 ll 10· PCR buffer II (Applied Biosystems), 4 l 2.5 mM dNTPs, 0.5 ll RNase inhibitor (Applied Biosystems), 0.5 ll RTase (Applied Biosystems), 1 ll reverse complement (RC) primer (20pm/ll), and 1 ll of RNA template. RT reactions which used oligo-dT as the RC primer replaced the typical 1 ll of gene-specific RC primer with 0.5 ll oligo-dT (20 pm/ll) and 0.5 ll H2O. RT protocol was 42 C for 35 min followed by 99 C for 5 min. Most RT reactions used the gene-specific RC primer. However, oligo-dT was used to prime reverse transcription of enolase RNA because the gene-specific RC primer (344R, in combination with 23F) preferentially yielded an undesired DNA fragment in the subsequent PCR. PCR was performed in a 50 ll solution which included the 10 ll solution from the RT Step, 2 ll 25 mM MgCl2, 4 ll 10· PCR buffer II (Applied Biosystems), 30.5 ll H2O, 0.5 ll Taq solution (1 part Amplitaq (5 u/ll, Applied Biosystems), and 1 part TaqStart Ab (7 lM, Clonetech)), 2 ll forward (F) primer (20 pm/ll), and 1 ll RC primer (20pm/ll). When the general dT primer was used during the RT step, 1 ll H2O was replaced with one additional ll of RC primer. PCR thermocycling followed a 3-phase touchdown protocol: (1) Denaturation: 38 cycles at 94 C for 30 s. (2) Primer annealing: 25 cycles starting at 50 C for 30 s and changing at 0.4 C/cycle, followed by 13 cycles at 45 C for 30 s. (3) Chain extension: 25 cycles at 72 for 1 min and changing at +2 sec/cycle (cycles 1–25), followed by 13 cycles at 72 C for 2 min and changing at +3 s/cycle (cycles 26–38), followed by 72 C for 10 min. In the case of DDC, enolase, and PEPCK, double-stranded RT-PCR amplification products were isolated from 1.0% low-melting-point agarose gels. Primers and low-molecular-weight salts were removed from CAD RT-PCR reactions without

gel isolation, due to the lack of visible product. All fragments were then reamplified using standard PCR with nested or hemi-nested primers to both improve product yield and ensure clean products. Reamplifications were performed in 50 ll solutions using similar reagent concentrations to those in the PCR described in the above RT-PCR step, plus 1 ll of the isolated RT-PCR product. The PCR thermocycler protocol was as follows: 94 C for 30 s, 50 C for 30 s, 21 cycles at 72 C starting at 1 min and increasing +2 s/cycle, followed by 72 C for 10 min. All products were then gel isolated a second time. In cases where concentration of the isolated product was not high enough for sequencing, a second reamplification was performed using M13REV and M13(21) primers, followed by gel isolation. See Regier (2006) for a detailed guide to using these methods for amplification of nuclear protein-coding genes in arthropods. 2.5. Primer development All primers are listed in Table 2. Primers with a C following the number are designed to be specific to Chalcidoidea, while the primer with an M following the number is designed to be specific to Gonatocerus (Mymaridae). CAD was amplified in two fragments: 46F/350RC reamplified with M13REV/309RC, and 295F/673RC reamplified with M13REV/606RC. In cases where these steps resulted in no visible bands on agarose gels, various combinations of other listed primers were attempted (e.g. 46F/350RC reamplified with 61CF/ M13(21)). DDC was initially amplified using 1.7F/4RC. Nested PCR reamplification used M13REV/3.3RC and 1.9CF/M13(21) (1.9F for outgroups) to produce two overlapping fragments. In taxa where the fragment was not amplified by this method, 1.6F was used in placed of 1.7F, and the nested reamplifications described above were used. Enolase was amplified in two fragments: 23F/344RC reamplified with M13REV/ 241RC, and 167F/407RC reamplified with M13REV/ 406RC. For PEPCK, 2–3 fragments were amplified: 159CF/351RC (155F for outgroups) reamplified with M13REV/335RC and 291F/510RC reamplified with 292F/M13(21). In many cases 292F/510RC did not produce a clean band, and in these cases the RT-PCR product was reamplified using both 344CF/M13(21) (344F for outgroups) and M13REV/501RC to produce two overlapping fragments. Gonatocerus (Mymaridae) had a single amino acid insertion near the 501RC primer, which significantly altered its amino acid sequence through the primer region. A taxon-specific primer (501MRC) was developed specifically for this amplification. 2.6. Sequencing and assembly Products were isolated a final time and directly sequenced (both strands) from M13 primers. Sequence chromatograms were checked for accuracy, and contigs were assembled using the software package (Staden, 1999). Alignments were straightforward due to sequence

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Table 2 Primers used in this study Gene

Primer

Primer sequence (50 –30 )

Original Publication

CAD

46F 61CF 295F 309R 309CF 350R 606R 673R

GTN GTN TTY CAR ACN GGN ATG GT GAY CCN TCN TAY TGY GAR CAR AT TAY GGY AAY MGN GGN CAY AA TC NAC NGC RAA NCC RTG RTT YTG CAR AAY CAY GGN TTY GCN ATH GA RTG YTC NGG RTG RAA YTG AC NAC YTC RTA YTC NAY YTC YTT CCA GC RTA YTG NAY RTT RCA YTC

See Regier (2006)a This paper See Regier (2006) see Regier (2006) This paper See Regier (2006) See Regier (2006) See Regier (2006)

DDC

1.6F 1.7F 1.9F 1.9CF 3.3R 4R

TTY CAY GCN TAY TTY CC GCH TGY ATY GGN TTY WCN TGG AT ATG HTN GAY TGG YTV GGY CAR ATG ATG YTN GAY TGG YTN GGN AAR ATG CCA YTT RTG NGG RTT RAA RTT RAA GG DAT YTG CCA RTG HCK RTA RTC

This paper Fang et al. (1997) Fang et al. (1997) This paper Friedlander et al. (1998) Fang et al. (1997)

Enolase

23F 167F 241R 344R 406R 407R

AAY CCN ACN GTN GAR GT GCN ATG CAR GAR TTY ATG GC NAC RTC CAT NCC DAT CC DAT YTG RTT NAC YAA YTG RTT RTA YTT NGC TC YTC DAT NCG NAR NAD YTG RTT RTA YTT

See Regier (2006)b This paperb See Regier (2006) See Regier (2006) See Regier (2006) See Regier (2006)

PEPCK

155F 159CF 291F 292F 344F 335R 344CF 351R 501R 501CR 501MR 510R

CGN TTC CCN GGN TGY ATG TGY ATG AAR GGN CGN ACN GAR GGN TGG YTN GCN GAR CA GAR GGN TGG YTN GCN GAR CAY ATG GAY GAY ATH GCN TGG ATG ARR TT AA YYT CAT CCA NGC DAT RTC RTC GAY GAY ATH GCN TGG ATG CGN TT CC RAA RAA NCC RTT YTC NGG RTT DAT CAT NGC RAA NGG RTC RTG CAT CAT NGC RAA NGG RTC RTT CAT CAT TGC RAA NGG RTC RTG CC RAA RTT RTA NCC RAA RAA

This paper This paper This paperc Friedlander et al. (1996) This paperc This paperc This paper This paper This paperc This paper This paper This paper

F denotes a forward primer, R denotes a reverse primer. Primers labeled with C are specific to Chalcidoidea, while the primer labeled with M specific to Gonatocerus (Mymaridae). All primers contained an M13 sequence at the 50 -end—F primers contained an M13REV sequence (CAGGAAACAGC TATGACC) and R primers contained an M13(21) sequence (TGTAAAACGACGGCCAGT). M13 sequences are not shown on the primers below for brevity. a Also matches primer 54F from Moulton and Wiegmann (2004). b Also partially overlaps primers from Farrell et al. (2001). c Partially overlaps primers from Friedlander et al. (1996).

and length conservation, and were done manually in GDE (Smith et al., 1994). All sequences were deposited in Genbank (see Table 1 for accession numbers).

2.7. Phylogenetic analysis The data were partitioned by gene and codon position, and each partition was examined for base compositional heterogeneity using the v2 test as implemented in PAUP* version 4 (Swofford, 2002). Relative rates by gene and codon position were estimated from the partitioned data set using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) utilizing a GTR + SSR model as described by Danforth et al. (2005). Four simultaneous chains were run for 2 · 106 generations, and trees were sampled every 1000 generations. Likelihood scores were plotted against generation time and the first 1000 trees were discarded as burn-in.

Mean and 95% confidence intervals were then calculated from the remaining 1001 trees. All parsimony and likelihood analyses were conducted using PAUP* version 4 (Swofford, 2002). Parsimony analyses were performed on both all nucleotides and first and second codon positions only, using equal weights and a heuristic search with 1000 random addition replicates. Parsimony bootstrap values were calculated using 1000 replicates and 10 random addition sequences per replicate. Maximum likelihood analyses were performed using a GTR + C + I model on both all nucleotides and first and second codon positions only. The model was selected using a generalized likelihood ratio test as implemented in MODELTEST (Posada and Crandall, 1998). Likelihoods for the test were calculated in PAUP* on a neighbor-joining tree from J-C, F81, HKY, GTR, GTR + C, GTR + I, and GTR + C + I models. Maximum likelihood analyses were performed as follows: (1) One of the best trees from

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the parsimony analysis was used as a starting tree. (2) Likelihood parameters were estimated from the parsimony tree, and a likelihood analysis was performed using the tree from step 1, the estimated parameters, and nearest-neighbor interchange. (3) Likelihood parameters were estimated from the final step-2 tree, and this tree and parameter set were used for a second likelihood analysis using tree bisection and reconnection. (4) The final tree from step 3 was saved and its likelihood parameters were estimated. (5) The likelihood parameters were set to those estimated in step 4, a heuristic search with 100 random addition replicates and TBR branch swapping was performed, and the most likely tree from step 5 was also saved. Two different types of searches (steps 1–4 and 5) were performed in order to maximize the chance of actually finding the most likely tree, although in all analyses the tree from step 4 topologically matched the step 5 tree. Bootstrap values were calculated using a GTR + C + I model with parameters set to those estimated in step 4, using 1000 replicates and 10 random addition sequences per replicate. Bayesian analyses were conducted using MrBayes 3.1 (Huelsenbeck and Ronquist, 2001) utilizing a single GTR + C + I model. Two independent runs, consisting of four simultaneous chains each, were run for 1 · 107 generations, and trees were sampled every 1000 generations. Likelihood scores were plotted against generation time and trees from the first 9 · 106 generations were discarded as burn-in. Posterior probabilities were then calculated from the remaining 1001 trees. Combinability of the individual gene data sets was tested using the ILD test (Farris et al., 1994) as implemented in PAUP* version 4 (Swofford, 2002). The ILD test was based on 100 replicates and 10 random addition sequences per replicate, and an analysis was run for all genes and for all 2-gene combinations. Existing taxonomic groups which appeared to be paraor polyphyletic by our analyses were tested against the null hypothesis of monophyly using Shimodaira’s approximately unbiased (AU) test (2002), as implemented in the CONSEL package (Shimodaira and Hasegawa, 2001). These tests ask whether the best tree possible under the constraint of monophyly, no matter what its topology may be otherwise, is a significantly worse fit to the data than the best tree without that constraint. The test is especially useful for assessing the confidence we can place in broad conclusions based on partial resolution of relationships, in cases where the data do not unambiguously resolve most or all individual nodes on the tree. Constraint trees used for the AU test were generated as in steps 1–4 above, except that in all searches the group being tested was constrained as monophyletic.

Fig. 1. Overall, enolase evolves most slowly followed by PEPCK, then DDC, with CAD being the most rapidly evolving, although at nt2 PEPCK evolves faster than DDC. For all genes, nt3 had a much higher substitution rates than nt1 or nt2, and even the slowest evolving nt3 partition (enolase) has a substitution rate over three times that of the fastest evolving nt1 or 2 partition (CAD nt1). Base composition (Table 3) was not significantly heterogeneous across taxa at nt1 and nt2 for any gene. However, nt3 was significantly heterogeneous for all genes. The ILD tests (Table 4) showed minimal evidence of discordant 3.5 3 Relative Substitution Rate

460

2.5 2 1.5 1 0.5 0 eno eno eno pep pep pep ddc ddc ddc cad cad cad nt1 nt2 nt3 nt1 nt2 nt3 nt1 nt2 nt3 nt1 nt2 nt3

Fig. 1. Relative substitution rates partitioned by codon position and gene (eno = enolase, pep = PEPCK).

Table 3 Base composition partitioned by gene and codon position Gene

Codon position

A

C

G

T

v2 P-value

PEPCK

nt1 nt2 nt3

0.30 0.26 0.16

0.20 0.26 0.37

0.35 0.22 0.27

0.15 0.26 0.20

1.00 1.00 <0.001*

DDC

nt1 nt2 nt3

0.24 0.31 0.28

0.19 0.22 0.22

0.37 0.19 0.20

0.20 0.28 0.30

1.00 1.00 <0.001*

Enolase

nt1 nt2 nt3

0.32 0.34 0.19

0.15 0.25 0.31

0.38 0.14 0.20

0.15 0.27 0.30

1.00 1.00 <0.001*

CAD

nt1 nt2 nt3

0.28 0.32 0.19

0.23 0.23 0.30

0.32 0.18 0.27

0.17 0.27 0.24

0.72 1.00 <0.001*

P-values are listed for the v2 test against a null hypothesis of base composition homogeneity across taxa.

Table 4 P-values for pairwise comparisons of gene combinability using the ILD test

3. Results

DDC

Enolase

CAD

0.08 — —

0.03* 0.05* —

0.28 0.07 0.02*

3.1. Characteristics of genes

PEPCK DDC Enolase

As predicted, the four genes showed complementary rates of divergence; their relative rates are shown in

When all genes were included, the null hypothesis of homogeneity was rejected at the 0.05 level (P = 0.02).

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phylogenetic signal among genes. When all genes were analyzed together, congruence of signal was rejected at a modest level (p = 0.02). However, among the twelve pairwise combinations of genes, congruence was rejected only in the comparisons involving enolase (p = 0.02–0.05). 3.2. Phylogeny estimation Maximum likelihood (ML) and parsimony (MP) analyses were conducted using nt1 and nt2 only as well as all nucleotides, to investigate the potential impact of base compositional heterogeneity at nt3. In both ML and MP analyses with nt1 + nt2 only (not shown), significantly fewer clades were resolved with bootstrap support (BP) >50% than when all nucleotides were included. Moreover, the differences between the nt1 + nt2 and the all nucleotides analyses were weakly supported. For this reason, the nt1 + nt2 only analyses are not considered further here. The best score ML tree using all nucleotides under the GTR + C + I model is shown in Fig. 2, along with support values for each branch under each kind of analysis (bootstrap support >50% for MP and ML, posterior probabilities >90% for Bayesian). Of the 41 nodes on this tree, 23 have ML bootstrap support (BP) >50%, 16 have BP >70%, and 10 have BP >90%. Branch lengths for the ML analysis are shown in Fig. 3, along with ML bootstrap support >50%. The Bayesian and ML trees were identical in topology. Unweighted parsimony analysis resulted in two most parsimonious trees, one of which is shown in Fig. 4 (20805 steps, CI = 0.24, RI = 0.31, RCI = 0.07), also with along with MP bootstrap support >50%. These trees differed only in the relationships between the outgroup taxa Aphanogmus (Ceraphronoidea) and Platygaster (Playgastroidea). In the tree shown, Aphanogmus and Platygaster are sister taxon, while in the alternate tree, Platygaster is sister-group to Chalcidoidea–Mymaridae. Bootstrap support on the MP tree under parsimony was somewhat lower than for ML on the ML tree. Of 41 nodes, 16 have BP >50%, 12 have BP >70%, and 9 have BP >90%. The MP and ML trees were largely similar in areas which were well supported in the separate analyses. Thus, 15 of the 16 nodes which were supported by BP >50% in the parsimony analysis were also recovered in the ML analysis, and 14 of these 15 also had BP >50% under ML. The groupings that conflict were, for the most part, unsupported in either the parsimony analysis alone or in both parsimony and likelihood analyses. The one striking exception to the foregoing generalization is as follows. In the ML and Bayesian analyses, monophyly of Chalcidoidea is supported (71% ML BP, 97% PP), as is the basal placement therein of Mymaridae (96% ML BP, 100% PP). The two exemplars of Platygastroidea are also strongly grouped (99% ML BP, 100% PP). In contrast, under MP, Mymaridae are grouped strongly (99% BP) with Scelio (Platygastroidea: Scelionidae), breaking up both superfamilies. Within Chalcidoidea (with or without

461

Mymaridae), Encarsia (Aphelinidae) branches off next in all analyses (89% MP BP, 77% ML BP, 100% PP). Also in all analyses, the next divergence separates a monophyletic ‘pteromalid lineage’ from the exemplar of Eulophidae, albeit with less support (53% MP BP, <50% ML BP, 99% PP). The basal branches within the ‘pteromalid lineage’ are all subfamilies of Pteromalidae, namely, Herbertinae, Cerocephalinae, Spalangiinae, and Cleonyminae: Cleonymini. Therefore, Pteromalidae appears to be paraphyletic with respect to all the other ‘pteromalid lineage’ families sampled, namely, Eurytomidae, Torymidae, Chalcididae, Eucharitidae, and Perilampidae. Chalcididae itself is strongly recovered as monophyletic in the likelihood and Bayesian analyses (95% ML BP, 100% PP). While Chalcididae is not monophyletic in the parsimony analysis, no nodes with BP >50% separate the two exemplars. Some relationships of individual families to sub-groups of Pteromalidae are moderate to strongly supported. The strongest single example (70% MP BP, 82% ML BP, 100% PP) of pteromalid paraphyly is the grouping of Eucharitidae + Perilampidae with Eutrichosomatinae (Pteromalidae). Both Eurytomidae and Torymidae fall (separately) within a moderately well-supported clade (recovered in best MP tree, 73% ML BP, 100% PP) consisting otherwise of the pteromalid groups Pteromalinae, Miscogasterinae, Colotrechnini, Asaphinae, Pireninae, Systasini (Ormocerinae), and Coelocybinae. The overall hypothesis of monophyly for Pteromalidae is rejected with a P value of 7 · 1015 by the approximately unbiased (AU) test of Shimodaira (2002); Table 5), using ML under the GTR + C + I model as the criterion of fit for trees resulting from monophyly-constrained versus unconstrained tree searches. Within Pteromalidae, monophyly is nearly always strongly supported for tribes represented by multiple exemplars, including Lyscini (78% MP BP, 98% ML BP, 100% PP) of Cleonyminae and Eunotini (100% MP BP, ML BP, and PP) and Moranilini (100% MP BP, ML BP, and PP) of Eunotinae. Monophyly of one subfamily, Cerocephalinae, is strongly supported (100% MP BP, ML BP, and PP). All the other multiply-represented subfamilies appeared para- or polyphyletic in all analyses, including Cleonyminae, Colotrechninae, Diparinae, Eunotinae, Miscogasterinae, Ormocerinae, and Pteromalinae, but support for these conclusions was generally weak. The AU test significantly rejected monophyly only for Miscogasterinae, Ormocerinae, and Colotrechninae (Table 5; P = 0.02, P = 0.002, and P = 0.0002, respectively). A number of other relationships among and within pteromalid subfamilies (and other families) are suggested by this analysis, some novel, although the deeper divergences are mostly weakly supported. Colotrechnini and Asaphinae are strongly supported as sister groups (96% MP BP, 90% ML BP, 100% PP) and are grouped in turn with Eurytomidae in all analyses. Miscogasterinae and Pteromalinae form a clade in all analyses (recovered in best MP tree,

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*/*/99

Colotrechninae: Colotrechnini Colotrechnus 96/90/100 Asaphinae Enoggera EURYTOMIDAE

[*]/53/100

Heimbra Ormocerinae: Systasini Semiotellus 77/73/100 Pireninae Gastrancistrus

[*]/*/99 TORYMIDAE

Torymus Miscogasterinae: Sphegigastrini Polstonia */*/* Pteromalinae */*/100 Psilocera Pteromalinae */55/100 Brachycaudonia */73/100 Miscogasterinae: Trigonodorini Plutothrix [*]/*/* Coelocybinae Ormyromorpha Colotrechninae: Hetreulophini [*]/*/* Hetreulophus Eunotinae: Moranilini Moranila 100/100/100 [*]/*/* Ophelosia Diparinae Neapterolelaps sp. 2 100/100/100 Neapterolelaps sp. 1 */*/100 Cleonyminae: Lyscini Epistenia 78/98/100 [*]/63/* [*]/*/98

Thaumasura Dipara trilineatus Dipara sp. 1

100/100/100 [*]/*/*

100/100/100 Diparinae

[*]/*/*

[*]/*/99

*/54/100

Cleonyminae: Cleonymini

[*]/58/100

Dipara sp. 2 Lelaps Eunotinae: Eunotini Eunotus 100/100/100 62/55/100 Scutellista Ormocerinae: Melanosomellini Hemadas CHALCIDIDAE Dirhinus [*]/95/100 [*]/*/93 Hokeria Cleonyminae: Chalcedectini Chalcedectus 59/59/*

EUCHARITIDAE

70/82/100 [*]/*/99 'pteromalid lineage' 53/*/99

PERILAMPIDAE

Eutrichosomatinae Spalangiinae Cerocephalinae

Cleonymus Chalcura Steffanolampus

Eutrichosoma Spalangia Theocolax Neocalosoter

100/100/100

89/77/100

Herbertinae 98/96/100 EULOPHIDAE CHALCIDOIDEA [*]/71/97 APHELINIDAE

Herbertia Baryscapus 99/[*]/[*]

MYMARIDAE [*]/99/100

PLATYGASTROIDEA CERAPHRONOIDEA

Encarsia Gonatocerus Scelio Platygaster Aphanogmus

Fig. 2. Maximum likelihood cladogram based on a GTR + C + I model including all nucleotides. Branches depicted by solid lines represent pteromalids, and the subfamily name is listed above the branch. Branches depicted by striped lines represent the higher classification (family or superfamily) of nonpteromalid taxa. Support values are listed above all nodes as MP BP >50%/ ML BP >50%/PP >90%. Asterisks indicate nodes which were supported by <50% BP or <90% PP, and asterisks in brackets indicate the node was not recovered in the respective analysis. In the one instance where analyses provided a conflicting, well-supported topology, an arrow indicates the alternate topology and support values. Nodes supported by morphology are indicated by open circles.

55% ML BP, 100% PP), though neither subfamily is resolved as monophyletic. The internal phylogeny of the larger of the two clades of Diparinae is identical in the likelihood and Bayesian analyses as Lelaps + (Dipara sp. 2 + (Dipara sp. 1 + Dipara trilineatus)) and, for the most part, strongly supported. Under parsimony Lelaps does not group with Dipara, although this result is without support.

4. Discussion 4.1. Chalcidoid and pteromalid phylogeny Gibson (1986) hypothesized three synapomorphies for Chalcidoidea: (1) presence of multiporous plate sensillae on the antennal flagellum, (2) unique position of mesothoracic spiracle, and (3) prepectus visible externally. Our like-

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463

100

Dipara trilineatus Dipara sp. 1 58 Dipara sp. 2 Lelaps 100 Neapterolelaps sp. 2 Neapterolelaps sp. 1 98 Epistenia 63 Thaumasura Brachycaudonia Polstonia 55 Psilocera Plutothrix 90 Colotrechnus 99 Enoggera 53 Heimbra 73 73 Gastrancistrus Semiotellus Torymus Ormyromorpha Hetreulophus 100 Moranila Ophelosia 55 100 Eunotus Scutellista Hemadas Chalcedectus Dirhinus 95 Hokeria Cleonymus Chalcura 59 82 Steffanolampus Eutrichosoma Spalangia Theocolax 100 Neocalosoter Herbertia Baryscapus Encarsia Gonatocerus Platygaster Scelio 100

54

'pteromalid lineage'

77

Chalcidoidea 96 71 99

Aphanogmus

0.1 substitutions/site Fig. 3. Maximum likelihood phylogram based on a GTR + C + I model including all nucleotides. Likelihood bootstrap values are listed above nodes which had >50% support. Branches depicted by solid lines represent pteromalids, while branches depicted by striped lines represent non-pteromalid taxa.

lihood and Bayesian analyses provide further support for chalcidoid monophyly (71% BP, 97% PP) with respect to the two other superfamilies sampled. The grouping of all other chalcidoids to the exclusion of Mymaridae is also strongly supported by our likelihood and Bayesian results (96% BP, 100% PP), corroborating morphological evidence for the basal placement of Mymaridae, including unique features of the ovipositor (Quicke et al., 1994) and thoracic musculature (Heraty et al., 1997). Analysis of 28S rDNA (Campbell et al., 2000) similarly recovered a monophyletic Chalcidoidea with Mymaridae as the basal-most taxon, although without strong bootstrap support. Our parsimony analysis, in contrast, strongly grouped the mymarid with Scelio (Scelionidae, Platygastroidea) (99% BP). This result contradicts the morphological evidence for monophyly of both Platygasteroidea and Chalcidoidea including Mymaridae. Given in addition the long branch leading to Scelio, surpassed only by the exceedingly long branch joining Mymaridae to the remainder of Chalcidoidea (see

Fig. 2), we hypothesize that this placement of Mymaridae is the result of long-branch attraction (Felsenstein, 1978; Anderson and Swofford, 2004). Our results further accord with Campbell et al. (2000) in placing Encarsia (Aphelinidae) basal to all chalcidoids we sampled except for Mymaridae (89% MP BP, 77% ML BP, 100% PP). A number of groups within the ‘pteromalid lineage’ supported by our results are also concordant with previous morphological evidence. Recovery of a monophyletic Chalcididae (Hockeria + Dirhinus; 95% ML BP, 100% PP) accords with strong morphological synapomorphies for this family (Wijesekara, 1997), and Eucharitidae + Perilampidae (59% MP BP, 59% ML BP, recovered in Bayesian analysis) is supported by characteristics of their planidiaform larvae (Heraty and Darling, 1984). The prevailing concordance of our trees with previous evidence lends credence to several groupings which appear here for the first time. An example is the strongly supported sister-group relationship between Colotrechnus (Colotre-

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C.A. Desjardins et al. / Molecular Phylogenetics and Evolution 45 (2007) 454–469 Dipara trilineatus Dipara sp. 1 Dipara sp. 2 Brachycaudonia Polstonia Psilocera Plutothrix 96 Colotrechnus Enoggera Heimbra 77 Gastrancistrus Semiotellus Ormyromorpha Torymus 100 Neapterolelaps sp. 2 Neapterolelaps sp. 1 78 Epistenia Thaumasura Hetreulophus 100 Moranila Ophelosia Eunotus 100 62 Scutellista Hemadas Lelaps Spalangia Chalcedectus Hokeria Cleonymus Dirhinus Chalcura 59 70 Steffanolampus Eutrichosoma Herbertia Theocolax Neocalosoter 100

'pteromalid lineage' 53 89

100

98

99

100

Baryscapus Encarsia Gonatocerus Scelio

Aphanogmus Platygaster

100 substitutions Fig. 4. One of two most parsimonious trees including all nucleotides and using equal weights (20805 steps, CI = 0.24, RI = 0.31, RCI = 0.07). Parsimony bootstrap values are listed above nodes which had >50% support. Branches depicted by solid lines represent pteromalids, while branches depicted by striped lines represent non-pteromalid taxa.

Table 5 P-values from the A.U. test of non-monophyly Taxonomic group

P-value

Eunotinae Pteromalinae Diparinae Cleonyminae Miscogasterinae Ormocerinae Colotrechninae Pteromalidae

0.412 0.376 0.225 0.143 0.017 0.002 2 · 104 7 · 1015

Taxonomic groups which did not appear monophyletic in the ML tree were subjected to the test.

chini) and Enoggera (Asaphinae; 96% MP BP, 90% ML BP, 100% PP), not previously postulated or defended by morphology. Similarly novel is the pairing of Eutrichosomatinae with Perilampidae + Eucharitidae (70% MP BP, 82% ML BP, 100% PP). Although Boucek (1988a) consid-

ered Eutrichosomatinae to be one of the most primitive members of Pteromalidae, no hypotheses have been put forth regarding its sister-group relationships. In a morphological study of the chalcidoid labrum, Darling (1988) found that Eutrichosoma had a similarly structured labrum to that of Perilampidae and Eucharitidae, but attributed this resemblance to convergence. Our results suggest that it might represent synapomorphy. Our analyses also support the monophyly of the three previously proposed tribal-level entities for which multiple exemplars were sequenced, i.e. Lyscini (Cleonyminae), Eunotini (Eunotinae), and Moranilini (supported as monophyletic by morphological evidence (Berry, 1994); Eunotinae), plus the two major lineages of Diparinae indicated by morphology (Desjardins, in press). By contrast, our trees fail to recover a number of pteromalid subfamilies. However, in only one such instance, the strong rejection of monophyly for Colotrechninae (P = 0.0002; Table 5), is there clear conflict with morphol-

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ogy. Colotrechninae is defined by anteriorly projecting axillae and a scutellum with two submedian, parallel longitudinal grooves; our analysis suggests that these characters are independently derived in the two tribes represented. The other apparent conflicts can reasonably be attributed to non-robust signal from sequences, morphology, or both. While the four taxa sampled from Pteromalinae and Miscogasterinae formed a clade together (recovered in best MP tree, 55% ML BP, 100% PP), neither subfamily was monophyletic with respect to the other. The AU test results showed that while monophyly of Pteromalinae was not significantly less likely than non-monophyly (P = 0.38; Table 5), monophyly of Miscogasterinae was significantly rejected (P = 0.02). No morphological synapomorphies are known for either subfamily. This suggests, at least, that Miscogasterinae are paraphyletic with respect to Pteromalinae, even if the converse, also suggested by our tree, is not true. Our two representatives of Miscogasterinae belong to different tribes, Polstonia to Sphegigastrini and Plutothrix to Trigonoderini. Thus, Trigonoderini may represent the basal lineage of the complex, with Sphegigastrini derived from within Pteromalinae. Ormocerinae was the third subfamily for which monophyly was rejected by the AU test (P = 0.002; Table 5). The subfamily appears polyphyletic, as our exemplars of the tribes Systasini and Melanosomellini are separated by several moderately to strongly supported nodes. While Graham (1969) considered Melanosomellini to be its own subfamily (at that time called Brachyscelidiphaginae), Boucek (1988a) considered it to be a tribe of Ormocerinae. Our results suggest that either Graham’s classification was more phylogenetically accurate, or that Hemadas (Melanosomellini), which has been treated by some authors as unplaced within Pteromalidae, actually does not belong within Melanosomellini. The remaining three subfamilies which appeared polyphyletic in the tree but for which monophyly was not rejected by the AU test are Cleonyminae, Diparinae, and Eunotinae. The lack of definitive support for, or rejection of, monophyly for Cleonyminae by our data mirrors the morphological study of Gibson (2003), who found no synapomorphies arguing for or against recognition of this subfamily. Synapomorphies are likewise unknown for Eunotinae. Diparinae appear to be one of the few pteromaline subfamilies strongly supported by morphological synapomorphies (Desjardins, in press), although fewer outgroups were sampled in the morphological analysis than here. A central finding of this study is that all ‘pteromalid lineage’ families sampled render Pteromalidae paraphyletic in all analyses. While most of the individual positions of these families among the pteromalid subfamilies are not strongly resolved, the overall conclusion of pteromalid non-monophyly is exceedingly robust (P <1014, AU test; Table 5). This finding suggests that classification within the ‘pteromalid lineage’ will need extensive revision once relationships are established in more detail. Either the majority of other families in the ‘pteromalid lineage’ will

465

need to be classified as subordinate groups within Pteromalidae, or Pteromalidae will need to be divided into many smaller families. While the nuclear gene phylogeny presented here is not robust at all levels, it appears to be the best-supported hypothesis about pteromalid phylogeny to date. Most groups strongly corroborated by morphological evidence are recovered by most analyses, the major exception being strong support for non-monophyly of Chalcidoidea (and Platygastroidea) in the parsimony analysis, which we provisionally attribute to long-branch attraction. Our analyses also corroborate other basal chalcidoid groupings that were recovered in a previous analysis of 28S rDNA (Campbell et al., 2000), generally with stronger support. Additionally, a number of groupings within the ‘pteromalid lineage’ which appeared para- or polyphyletic in previous molecular studies were recovered here, mostly with strong support, including Chalcididae (ML and Bayesian analyses only), Eunotini (all analyses), and Eucharitidae + Perilampidae (all analyses). The limited resolution of among-family and among-subfamily relationships we obtained within the pteromalid lineage, despite a data set of over 4.6 kbp, was initially surprising. Similar data sets, with mostly the same genes, have been much more effective at solving problems at similar taxonomic level in other groups (e.g. bombycoid and noctuoid Lepidoptera; Mitchell et al., 2006; J. Regier and others, unpublished). However, our experience accords with the previous history of pteromalid systematics, reinforcing the conclusion that pteromalid phylogeny is a difficult problem characterized by short internal branches. Progress is likely to remain incremental, with much further accumulation of both molecular and morphological data required to fully reconstruct the radiation of pteromalids and other Chalcidoidea. It is encouraging to note that relationships among some families of bombycoid moths, very weakly supported by five genes including the four used here (6.7 kb total; J. Regier and others, unpublished), appear to be strongly resolved when the sample of sequence is tripled (J. Regier and others, unpublished). Thus, there is hope that apparent rapid radiations such as the ‘pteromalid lineage’ can eventually be solved. 4.2. Evolution of parasitic habits One reason to study chalcidoid phylogeny is to understand the origin and evolution of the exceptional diversity of parasitic habits within this superfamily. A necessary step toward that goal will be reconstruction of evolutionary transitions in trophic habits, coupled with quantification of the degree to which different dimensions of the feeding niche are phylogenetically conserved (see review in Winkler and Mitter, 2007 for analogous issues in herbivorous insects). The distribution of the dominant habits of the taxa sampled on our trees (Fig. 5) suggests rapid evolution: adjacent taxa most frequently have different habits, and host diversity is high within many subfamilies. Reconstruc-

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C.A. Desjardins et al. / Molecular Phylogenetics and Evolution 45 (2007) 454–469 primary hosts 1,2

Colotrechninae: Colotrechnini

Torymidae

Colotrechnus Asaphinae Enoggera Eurytomidae Heimbra Ormocerinae: Systasini Semiotellus Pireninae Gastrancistrus Torymus

Miscogasterinae: Sphegigastrini Polstonia Pteromalinae Psilocera Pteromalinae Brachycaudonia Miscogasterinae: Trigonodorini Plutothrix Coelocybinae Ormyromorpha Colotrechninae: Hetreulophini Hetreulophus Eunotinae: Moranilini Moranila Ophelosia

associated with galls, hosts unknown Sternorrhyncha stem-boring Coleoptera 3 gall formers or galls (phytophagous) gall-associated Diptera gall formers or galls (phytophagous) leaf-mining and stem-boring Diptera Holometabola leaf-mining and stem-boring Diptera Holometabola associated with galls, hosts unknown hosts unknown Sternorrhyncha

Neapterolelaps sp. 2 Coleoptera, but mostly unknown Neapterolelaps sp. 1 Cleonyminae: Lyscini wood-boring Coleoptera and Epistenia Diparinae

Thaumasura Dipara trilineatus Dipara sp. 1 Diparinae

Dipara sp. 2 Lelaps Eunotus Scutellista

Eunotinae: Eunotini Ormocerinae: Melanosomellini Chalcididae

Hemadas Dirhinus

twig-nesting Hymenoptera Coleoptera, but mostly unknown

Sternorrhyncha galls (phytophagous)3 Holometabola

Hokeria Cleonyminae: Chalcedectini Cleonyminae: Cleonymini

'pteromalid lineage'

Cerocephalinae

Cleonymus Eucharitidae Chalcura Perilampidae Steffanolampus Eutrichosomatinae Eutrichosoma Spalangiinae Spalangia Theocolax Neocalosoter

Herbertinae Eulophidae Chalcidoidea

Chalcedectus

Aphelinidae Mymaridae

Platygastroidea Ceraphronoidea

wood-boring Coleoptera wood-boring Coleoptera ants (Formicidae), planidiaform larvae4 varied, planidiaform larvae 4 Coleoptera in flower heads dipteran pupae wood-boring Coleoptera

Herbertia Baryscapus

leaf-mining Diptera

Encarsia

Sternorrhyncha3

Gonatocerus

heteropteran eggs

Scelio Platygaster Aphanogmus

eggs, hosts diverse 5

leaf-mining and stem-boring Holometabola

hosts diverse, largely unknown

Fig. 5. Life history strategies of chalcidoids mapped onto the maximum likelihood tree based on a GTR + C + I model including all nucleotides. 1As most chalcidoid groups have a diverse host range, the listed ‘‘primary hosts’’ indicate the most common hosts for that group. 2Host information compiled from Boucek (1988a), Gibson (1993), and Grissell and Schauff (1997). 3As these taxa are not necessarily representative of their higher classification, the host information listed pertains specifically to the genus rather than subfamily or subfamily. 4Perilampids and eucharitids have planidiaform larvae, i.e., their larvae actively search for hosts rather than being laid on or in hosts. 5While the majority of platygastrids are not egg parasitoids, egg parasitism is considered the ancestral state for both Scelionidae and Platygasteridae (Austin et al., 2005).

tion of chalcidoid life history evolution will therefore require a densely-sampled phylogeny that is well resolved to the subfamily level and below. It will also require that variation in parasitic habits be coded into a series of characters plausibly viewed as homologous. This will be challenging, given the many partly-independent axes along which host use can differ (e.g., host taxon, host life stage, host mobility, etc.). While neither of the foregoing conditions is met in this study, our results do permit some initial inferences about

the evolution of chalcidoid parasitism. Parasitism of wood-boring beetles has commonly been thought to be ancestral for chalcidoids (e.g., Boucek, 1988b), presumably because the symphytan family Orussidae, the sister-group to all apocritan Hymenoptera, has this trait, as do basal members of several other parasitoid superfamilies (Whitfield, 1998). Within Chalcidoidea, moreover, parasitism of wood-boring beetles is found in several groups argued to have primitive morphology (e.g., Pteromalidae: Cleonyminae; Boucek, 1988b; see Gibson et al., 1999). Our

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phylogeny, however (Fig. 4), provides little apparent support for ancestral wood-boring beetle parasitism within Chalcidoidea. The most basal extant chalcidoids, mymarids, are parasitoids of insect eggs (of many orders), while the next-most basal taxon, Encarsia, parasitizes scale insects and whiteflies. Wood-boring beetle parasitoids first appear in Cerocephalinae, at least four nodes (three in the parsimony analysis) removed from the chalcidoid ancestor. If the habits of Cerocephalinae and all taxa below it on Fig. 4 are treated as states of a single character, the most parsimonious reconstruction would assign egg parasitism to the chalcidoid ancestor. Additional evidence supports this conclusion (Dowton and Austin, 2001; see Gibson et al., 1999), albeit not yet decisively. Life histories are not known for the probable sister group to Chalcidoidea (Gibson, 1986), the rarely collected Mymarommatoidea (not sampled here), but the extremely small size of these wasps (typically 0.3 mm) strongly suggests that they are egg parasites, like the comparably tiny Mymaridae. Small size (0.5–2.5 mm) and egg parasitism also characterize the Platygastroidea, which is supported as sistergroup to Mymarommatoidea + Chalcidoidea by several morphological and molecular studies (Dowton and Austin, 2001; Ronquist et al., 1999), though not in all analyses therein. If Encarsia and related aphelinids are indeed relatively basal within Chalcidoidea, as current evidence suggests, a plausible scenario suggests itself regarding early life history evolution in chalcidoids. Species of Aphelinidae most commonly parasitize sternorrhynchans such as scale insects and whiteflies, though some parasitize eggs of various insect orders, and others attack puparia of cecidomyiid flies. One can thus envision ancestral egg parasitism giving rise in a small evolutionary step to parasitism of other small, immobile hosts. On this view, large, robust-bodied parasitoids of active hosts such as wood-boring beetles (e.g., many cleonymine pteromalids) are derived relative to small-bodied, morphologically simplified parasitoids of immobile hosts. The foregoing scenario, finally, points toward a broader hypothesis about chalcidoid life history evolution: the most conserved aspects of host use may most often reflect broad adaptive syndromes of behavioral and morphological traits (including body size), rather than fidelity to particular host taxa. All of the foregoing postulates await rigorous phylogenetic test. Acknowledgments This study was funded in part by a Dissertation Improvement Grant from the National Science Foundation (DEB-0206696) to the authors. We thank Simon van Noort and Chris Burwell for assistance in collecting specimens, and John Heraty, Michael Gates, Bruce Campbell, and Sinthya Penn for providing specimens used in this study. We also thank Zaile Du for laboratory assistance and the following people for commenting on an earlier version of this manuscript: E. Eric Grissell, Charles Delwiche,

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