A Single Origin of Gall Association in a Group of Parasitic Wasps with Disparate Morphologies

Molecular Phylogenetics and Evolution 44 (2007) 981–992 www.elsevier.com/locate/ympev

A single origin of gall association in a group of parasitic wasps with disparate morphologies

Alejandro Zaldivar-Riveroń a,*, Sergey A. Belokobylskij b,c, Virginia Leoń-Regagnon a, Juan Jose´ Martıńez d, Rosa Bricenõ e, Donald L.J. Quicke f,g

a Departamento de Zoologıá, Instituto de Biologıá, Universidad Nacional Autońoma de Me´xico, 3er. Circuito Exterior, Cd. Universitaria, Ap. Postal 70-153, C. P. 04510, Me´xico D. F., Mexico b Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia c Museum and Institute of Zoology, PAN, Wilcza 64, Warsaw 00-679, Poland d Divisioń de Entomologıá, Museo Argentino de Ciencias Naturales ‘‘Bernardino Rivadavia’’, Av. Angel Gallardo 470, C1405DJR, Buenos Aires, Argentina e Universidad Centroccidental ‘‘Lisandro Alvarado’’, Decanato de Agronomıá, Depto. de Ciencias Biolo´gicas, Seccioń de Entomologıá, Cabudare, Estado Lara, Venezuela f Division of Biology and Centre for Population Biology, Imperial College London, Silwood Park Campus, Ascot, Berkshire, SL5 7PY, UK g Department of Entomology, Natural History Museum, London SW7 5BD, UK

Received 17 August 2006; revised 10 May 2007; accepted 17 May 2007 Available online 9 June 2007

Abstract

The braconid wasp subfamily Doryctinae mainly comprises idiobiont ectoparasitoids of other insect larvae. In recent years, however, members of a few genera have been discovered to be associated with galls from various unrelated host plant families, with some of these being gall inducers whereas others are suspected as being predators of gallers. Because of their considerable morphological differences, these gall-associated taxa traditionally have been placed in separate tribes or even in other subfamilies. In this study, we investigate the phylogenetic relationships among representatives of a number of different doryctine genera, including five of its seven gall-associated genera using two genetic markers. Here we analyzed the length-variable 28S sequence data based on secondary structure both excising the unalignable regions and recoding them according to indel length. In addition, multiple alignments were carried out with a range of gap-opening and extension parameters. The combined (28S + CO1) phylogenetic hypotheses obtained, both excluding and recoding the unalignable regions, recover a clade comprising the five gall-associated genera, and most of the analyses using multiple alignments also support this relationship. These results support a scenario in which secondary phytophagy evolves from initially attacking primary gall- forming hosts. The relationships recovered are also more congruent with a model that explains the macroevolution of insect plant association in the Doryctinae as reflecting geographic proximity rather than host plant relationships. Further, our phylogenetic hypotheses consistently show that one of the main morphological features employed in the higher level classification of the Doryctinae is actually highly homoplastic. Ó 2007 Elsevier Inc. All rights reserved.

Keywords: Braconidae; Doryctinae; Cyclostomes; Character evolution, Molecular phylogeny; Secondary structure alignment; Multiple alignment

1. Introduction

In the Hymenoptera, gall formation, predation of gall- inducers and inquilinism are related phenomena that occur

* in several lineages, principally in the Chalcidoidea and Corresponding author. Address: Museo Nacional de Ciencias Naut- Cynipoidea, but which have also been discovered recently rales, Departamento de Biodiversidad y Biologı´a Evolutiva, C/Jose´ Gutie´rrez Abascal 2, 28006 Madrid, Spain. in a few members of the cyclostome group of the otherwise E-mail address: [email protected] (A. Zaldivar-Rivero´n). almost entirely parasitic family Braconidae (see Wharton

1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.05.016 982 A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 and Hanson, 2005 for a review). Starting from the first shown by the different gall-associated doryctine genera. report of phytophagy in Allorhogas Gahan almost 20 years For this reason, these taxa have been placed in separate ago (Maceˆdo and Monteiro, 1989), cecidogenesis (i.e., gall and in some cases exclusive tribes (e.g., Labanini, Perc- development) in the Braconidae has subsequently been nobraconini) or even in other subfamilies (e.g., Monitoriel- confirmed only in other two cyclostome genera, Monitoriel- la: Wharton, 1993; but see Zaldivar-Riveroń et al., 2006). It la Hedqvist (Infante et al., 1995) and Mesostoa van Achter- has been argued, however, that the external morphology of berg (Austin and Dangerfield, 1998). Some other braconid doryctine wasps is quite homoplastic (Belokobylskij et al., wasps have only ever been reared from galls, though some 2004b), and thus several features employed to differentiate of these appear to display various forms of gall association its higher taxa could have actually evolved separately in rather than being the primary gall-formers themselves. repeated occasions. Most of these gall-associated species belong to the large Recently, a simultaneous molecular and morphological and cosmopolitan subfamily Doryctinae, though this fea- phylogenetic analysis among representatives of the cyclo- ture is also displayed by species of three genera of the Mes- stome braconid subfamilies strongly supported the gall- ostoinae (sensu Zaldivar-Riveroń et al., 2006)(Mesostoa, associated Labania and Monitoriella as being sister taxa Aspilodemon Fischer, and Hydrangeocola Bre´thes; Bre`thes, (Zaldivar-Riveroń et al., 2006). That study, however, did 1927; Austin and Dangerfield, 1998; Oda et al., 2001). not sample other gall-associated doryctine genera and only Doryctine wasps are mainly idiobiont (i.e., they arrest included a scarce number of members of this group. Thus, the host development at the time of parasitization) ectopar- relationships both within the subfamily and among the asitoids of xylophagous Coleoptera larvae (Belokobylskij gall-associated genera were far from conclusive. et al., 2004a,b). However, gall association is exhibited by Different strategies have been proposed for aligning species of seven Neotropical doryctine genera, most of length-variable rDNA sequence data, though unalignable which have been reared from different unrelated host plant regions present in rDNA sequence alignments still are com- families. In Allorhogas, several species are known to induce monly excluded and therefore the results obtained are galls in seeds of species of Melastomataceae, Bignoniaceae based exclusively on regions that appear to be length-con- and at least four legume (Fabaceae) genera, though some served. In the case of secondary structure alignment other species are thought to be parasitoids of gall inhabit- (Gillespie, 2004), strict criteria of matched base-pairing ants (Maceˆdo and Monteiro, 1989; Maceˆdo et al., 1998; are used to delimit putatively homologiseable stem regions Marsh, 2002; Marsh et al., 2000). Moreover, the type spe- from ambiguously alignable loops and expansion zones. cies of Percnobracon Kieffer, Pe. stenopterus Kieffer, was However, coding approaches also exist that align length- reared from cecidomyiid (Diptera) galls on the legume variable regions conditionally based on indel length (e.g., Prosopis strombulifera (Lam.) Benth (Kieffer, 1910), and Simmons and Ochoterena, 2000; Lutzoni et al., 2000; recently this and two other species of the genus have been Zaldivar-Riveroń et al., 2006). Alternatively, computer reared from the Argentinian endemic Pr. caldenia Burkart algorithms are often employed to find best matches (Martıńez, 2006). Species of Monitoriella, on the other between bases in regions of ambiguous alignment, and hand, induce leaf galls on Philodendron Schott (Araceae) the incorporated variation may contribute to the phyloge- (Infante et al., 1995), whereas some members of Labania netic analysis performed. These include multiple alignment Hedqvist and Psenobolus Reinhard have been observed to (e.g., Clustal X, Thompson et al., 1997; MALIGN, develop in galls located on aerial roots and leaves, and Wheeler and Gladstein, 1994) and dynamic-based align- on the syconia of Ficus L. (Moraceae), respectively (Marsh, ments (e.g., POY, Wheeler, 1996; Gladstein and Wheeler, 2002; Ramirez and Marsh, 1996). Finally, the only 1996), and when using them it is increasingly common to described species of Donquickeia Marsh and Mononeuron use a ‘sensitivity’ approach, where the stability of the Fischer were reported to be reared from cecidomyiid galls results is assessed across a range of user-defined parame- of Mikania sp. (Asteraceae) and Eugenia rotundifolia Casar ters. Here we utilize three different alignment strategies, (Myrtaceae) (Penteado-Dias, 2000) and from a species of secondary structure alignment excluding unalignable Duguetia A.St.-Hil (Annonaceae) (Fischer, 1981), regions, secondary structure alignment with conditional respectively. alignment of indel regions, and multiple alignment using Since the process of host shift in phytophagous insects a range of parameters to assess the sensitivity of our results involves a number of genetic and ecological factors (e.g., to alignment procedure. secondary metabolites, competitors, predators; Thompson, In this study, we report a close relationship among most 1999), this is normally expected to occur more between clo- of the currently known gall-associated doryctine wasps sely related host plant species rather than in distant, unre- based on a molecular phylogenetic analysis using two gene lated families (e.g., Becerra, 1997; Ko¨pf et al., 1998). The fragments. We discuss the implications of this finding on different host plant families inhabited by the above doryc- the origin of cecidogeny and on the evolution of host plant tine genera would therefore suggest that at least the con- shifts displayed among these taxa. Moreover, we assess the firmed cecidogenic Allorhogas and Monitoriella are evolution of one of the key features employed to distin- distantly related. This idea has been generally supported guish among supraspecific taxa within the group, the length by the considerable external morphological differences of acrosternite (strongly sclerotised basal sternal plate) of A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 983 the first metasomal segment, and consider the conse- (RAAs), regions of slipped-strand compensation (RSCs), quences that our results will have in the higher level classi- and regions of expansion and contraction (RECs)]. RAAs fication of the Doryctinae. and their adjacent RECs were then combined into single unalignable regions for subsequent analyses using condi- 2. Materials and methods tional alignment (see below). The secondary structure alignment excluding these ambiguous regions is termed 2.1. Taxon sampling 28SN.

DNA sequence data were generated for a total of 51 2.2.2. Indel coding and conditional alignment taxa belonging to 50 different doryctine genera. The taxon Regions of the 28S data set whose positional homology sampling comprised representative genera from the Afro- could not be confidently assigned were recoded following tropical, Australian, Neotropical, Oriental, and Palaearctic the approach used by Zaldivar-Riveroń et al. (2006). This regions, which belong to most of the tribes recognized by approach offers an objective criterion for preserving all Belokobylskij (1992). Monophyly of the Doryctinae, previ- the nucleotide variation that is not in conflict due to ously supported by relatively little convincing morpholog- sequence length variation and additionally includes infor- ical evidence (see Quicke et al., 1992), was tested by mation on indel length by attributing a ‘morphological’ including members of seven cyclostome subfamilies, with character state to indels of identical length regardless to the rhyssaline Rhyssalus Haliday used for rooting all trees: their base composition. This data set, comprising the sec- previous studies have consistently recovered the Rhyssali- ondary structure alignable regions (28SN) together with nae as the sister group of the remaining cyclostome sub- the unalignable regions of identical length and the ‘mor- families except the Aphidiinae + Mesostoinae s.l. clade, phological’ states referring to indel lengths is referred to which appeared as the sister group of all of them (Belshaw as 28SA. et al., 1998, 2000; Dowton et al., 2002; Zaldivar-Riveroń et al., 2006). Details of the included material, voucher 2.2.3. Multiple alignment information, and GenBank/EMBL Accession Numbers Multiple alignments of the 28S data were created with are given in Table 1. Clustal X (Thompson et al., 1997) with each of the following range of gap opening-gap extension penalties: 1:1, 2:1, 2.2. DNA sequence data and alignment 2:2, 4:1, 4:2, 4:4, 6:1, 6:2, 6:4, 6:6, 8:1, 8:2, 8:4, 8:6, 8:8, 10:1, 10:2, 10:4, 10:6, 10:8, 10:10. All of these used a DNA tran- The genetic markers examined included 650 bp of the sition: transversion weight ratio of 1.0: 1.0, and the default second and third domains of the nuclear 28S rDNA gene pairwise alignment parameters. and 603 bp of the COI mtDNA gene. Genomic DNA was extracted from ethanol preserved and up to 15 years 2.3. Phylogenetic analysis old dry pinned specimens. The DNA extraction and amplification procedures employed were those described in Bayesian MCMC analyses were performed for the sepa- Zaldivar-Riveroń et al. (2006). 28S sequences were rate and combined data sets (with 28SN and 28SA matri- obtained using the primers designed by Belshaw and ces) using MrBayes version 3.1.2 (Ronquist and Quicke (1997) (fwd: 50-GCG AAC AAG TAC CGT Huelsenbeck, 2003). Two independent analyses were run GAG GG-30) and Mardulyn and Whitfield (1999) (rev: simultaneously for each data set, each consisting of 2 mil- 50-TAG TTC ACC ATC TTT CGG GTC CC0-30). COI lion generations, sampling trees every 1000 generations, sequences were amplified using the primers designed by and using four chains and uniform priors on three topolo- Folmer et al. (1994) (LCO 50-GGT CAA CAA ATC gies, with the latter implying non-uniform prior probabili- ATA AAG ATA TTG G-30 HCO 50-0TAA ACT TCA ties of clades due to these are dependent on the number of GGG TGA CCA AAA AAT CA-30). taxa in a clade as well as the number of taxa in the analysis COI sequences were unambiguously aligned based on (Pickett and Randle, 2005). The GTR + I + C model of their translated amino acids; however, alignment of the sequence evolution was the model selected for the two gene length-variable 28S gene fragment was more problematic markers examined according to the likelihood ratio test and here we employ three different strategies and compare implemented with MrModeltest version 2.2 (Nylander, the results to assess the sensitivity of our findings to choice 2004). There is no reason to assume a priori that the states of alignment method. derived from the 28SA indel characters, whose states are coded as ‘morphological’ (i.e., with numerical coding), 2.2.1. Secondary structure alignment could have different frequencies. The Mk + C (Markov k; Alignment of the 28S sequences followed the 28S braco- Lewis, 2001) model of evolution was therefore used for nid secondary structure alignment proposed by Gillespie the above characters. This model was originally designed et al. (2005). Length variable regions were identified and to deal with morphological characters and considers equal initially characterized according to the three categories pro- base frequencies, equal mutation rates and makes the like- posed by Gillespie (2004) [regions of ambiguous alignment lihood conditional on characters being variable (Lewis, 984 A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992

Table 1 Provenances and voucher and EMBL/GenBank Accession Numbers of the taxa included Voucher No. 28S COI Taxon Acanthorhogas sp., Costa Ricaa Jo-713 DQ498942 DQ498975 Acrophasmus exilis Marsh, Costa Ricad IB-02 DQ498943 DQ498976 Aivalykus arawak, Costa Ricaa Jo-725 AY935471* AY935398* Allorhogas sp., La Pampa, Argentinad IB-18 DQ498927 — Allorhogas sp., Cartago, Turrialba, Costa Ricad IB-20 — DQ498959 Aphelopsia striata Braet & Barbalho, French Guyana (holotype)b Jo-838 DQ498941 DQ498974 Barbalhoa contophleba Marsh, Costa Ricaa Jo-723 DQ498935 — Barbalhoa licina Marsh, Costa Ricaa Jo-739 — DQ498968 Caenopachys hartigii (Ratzeburg), Corsica, Francea Jo-793 AY935474* AY935401* Caenophanes sp., Africaa Jo-781 AY935465* DQ498961 Curtisella hansoni, Costa Ricad IB-03 DQ498944 DQ498977 Doryctes heydenii Reinhard, Palaearcticd — DQ498913 DQ498945 Doryctes leucogaster (Nees), Israela Jo-876 AY935472* DQ498946 Ecphylus sp., South Americae — AJ302894* — Euscelinus sarawacus Westwood, Thailanda Jo-871 DQ498920 DQ498952 Hansonorum sp. nov. aff. pauli, Costa Ricaa Jo-728 DQ498934 DQ498967 Hecabolus sulcatus Curtis, Ascot, UKa Jo-650 AY935473* AY935400* Hemidoryctes carbonarius (Ashmead), Diego Suarez Province, Madagascara Jo-592 DQ498921 DQ498953 Heterospilus prosopidis Viereck, Silwood culture, UKe — AY935469* AY935396* Heterospilus sp., Costa Ricaa IB-01 DQ498926 DQ498958 Histeromeroides sp., Rio Napo, Ecuadora Jo-866 DQ498940 DQ498973 Hypodoryctes sibiricus Kokujev, no datae — AJ302895* — Hypodoryctes sibiricus kokujev, Finlandd Jo-981 — DQ498965 Ipodoryctes sp., St. Phillippe, Reunion Islanda Jo-929 DQ498916 DQ498949 Jarra maculipennis Marsh & Austin, Australiaa — DQ498925 DQ498957 Johnsonius sp., Costa Ricaa Jo-719 DQ498939 DQ498972 Labania sp., Costa Ricae — DQ498928 AY935397* Lamquetia marshi Braet & Barbalho, French Guyana (holotype)b Jo-833 DQ498938 DQ498971 Leptorhaconotus sp. (1), Madagascara Jo-655 AY935479* AY935406* Liobracon sp., Costa Ricae Jo-714 AY935467* AY935394* Masonius sp., Cuyagua, Venezuelab Jo-578 DQ498933 DQ498966 Megaloproctus sp., Colombiaa Jo-654 AY935466* AY935393* Monitoriella sp., Costa Ricaa Jo-733 AY935457* AY935387* Notiospathius sp., Caucagua, Venezuelab Jo-596 AY935477* AY935404* Odontobracon sp., Costa Ricaa Jo-712 AY935468* AY935395* Ondigus bicolor Braet, Barbalho & van Achterberg, French Guyana (holotype)b Jo-834 DQ498937 DQ498970 Ontsira imperator (Haliday), Honshu Ibaraki, Japana Jo-971 DQ498919 DQ498951 Pedinotus sp., Costa Ricaa Jo-742 DQ498936 DQ498969 Percnobracon cf. Stenopterus Kieffer, Santa Rosa, La Pampa, Argentinad IB-19 DQ498930 DQ498960 Platyspathius sp., Benina AL-183 DQ498915 DQ498948 Psenobolus sp., Camboto, Venezuelad Jo-666 DQ498929 — Pseudodoryctes sp., Malawia Jo-679 DQ498922 DQ498954 Rhaconotus emarginatus Marsh, Costa Ricaa Jo-720 DQ498914 DQ498947 Rhacontsira sp., Hoa Binh Province, Vietnama Jo-923 DQ498917 DQ498950 Rinamba opacicollis Cameron, Madagascara Jo-588 DQ498923 DQ498955 Schlettereriella sp., Kibale, Ugandaa Jo-673 AY935478* AY935405* Semirhytus sp., Munipality of Lara, Yacambu´, Venezuelac Jo-628 DQ498931 — Semirhytus sp., no datad Jo-983 — DQ498962 Spathiomorpha sp. aff.longipalpis, Japana Jo-711 DQ498924 DQ498956 Spathius agrili Yang, China — AY920299* AY920285* Spathius sp., Tollara Province, Madagascarb Jo-591 AY935476* AY935403* Spathius (Antespathius) sp., Madagascara Jo-910 DQ498918 — Stenocorse sp., Belizea Jo-750 AY935475* AY935402* Syngaster lepidus Brulle´, Australiae — AJ245698* DQ498963 Tarasco sp., Camboto, Venezuelab Jo-926 DQ498932 — Tarasco sp., Camboto, Venezuelab Jo-587 — DQ498964 Outgroups Cystomastax sp., Caucagua, Venezuelab Jo-574 AY935445* AY935369* Hormius sp., Mahajanga Province, Madagascarb Jo-582 AY935455* AY935385* Oncophanes sp., Ascot, UKe — AY935481* AY935407* Pambolus sp., Choroni, Venezuelab Jo-597 AY935458* AY935388* (continued on next page) A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 985

Table 1 (continued) Voucher No. 28S COI Pseudorhysipolis sp., Costa Ricab Jo-758 AY935450* AY935377* Rhyssalus clavator Haliday, Kazimierz, Polanda Jo-890 AY935482* AY935409* Stiropius sp., Costa Ricaa Jo-730 AJ784961* AY935373*

*Sequences obtained from previous works. Voucher specimens: aZoological Institute, St. Petersburg, Russia; bNationaal Natuurhistorisch Museum, Leiden, Netherlands; cUniversidad Centrooccidental, Tabudare, Venezuela; dInstituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico, Me´xico; enot retained/none remaining. Specimens whose sequences were used to form a single terminal taxon.

2001). The 28S nucleotide and indel characters and the given alternative phylogenetic hypothesis as statistically three COI codon positions were each treated as unlinked rejected when this was absent in all of the credible set of partitions. Likelihoods reached their plateau ranges before trees. The presence of the alternative hypotheses within 100,000 generations in all the analyses; therefore, their first the 95% posterior intervals was detected by creating the 100 sampled trees were discarded as burn-in. Relationships selected topological constraint with MacClade 4.06 (Madd- derived from the postburn-in samples were similar in the ison and Maddison, 2003) and then using this to filter the two analyses run for each data set and thus they were set of credible trees using the option ‘compatible with con- pooled and used to produce a 50% majority rule consensus straints’ included in PAUP* version 4.0b10 (Swofford, tree with posterior probabilities of clades. 1998). The analyses for the combined data sets derived from The Shimodaira-Hasegawa (SH; Shimodaira and Hase- the 28S multiple alignments used the same search strategies gawa, 1999) test was also performed with PAUP* version and parameters as above, except for the number of run 4.0b10 (Swofford, 1998) to test for significant differences generations, which was of 1 million. Burn-in in all the anal- between the likelihood of the 28SN + COI Bayesian phy- yses was determined to be completed by 100,000 genera- logeny and the likelihood of alternative Bayesian topolo- tions. We also carried out maximum parsimony (MP) gies that enforced the above tribes each as monophyletic. analyses for the combined and separate data sets that The SH tests were carried out using the full optimization resulted from the 28S multiple alignments with PAUP* ver- sampling with 1000 replicates. The alternative Bayesian sion 4.0b10 (Swofford, 1998). Alignments from each topologies were obtained with the constraint option avail- parameter combination were analyzed using 10,000 ran- able in MrBayes version 3.1.2, using the same strategies dom additions followed by TBR branch-swapping and and parameters employed in the unconstrained analyses. holding no more than one most parsimonious tree (MPT) Burn-in in these analyses was determined occur after for swapping at any one time. The resulting MPTs from 300,000 generations. each search were then used as starting trees for further TBR searches with unlimited maxtrees. Application of 2.5. Analysis of character evolution reweighting on these using the maximum value of the reten- tion index as the reweighting function, followed by The evolution of gall-association and of one morpholog- reweighting characters back to unity (Quicke et al., 2001) ical feature used to distinguish higher-level taxa in the Dor- failed to recover any more parsimonious trees. yctinae, the length of acrosternite of the first metasomal The matrices of the separate and combined data sets tergite, were investigated mapping their selected character with 28SN and 28SA and their topologies reconstructed states onto the 2SN + COI Bayesian phylogeny employing can be downloaded from the TreeBase web page (accession maximum likelihood character optimizations using Mes- no. S1800). The matrices with the multiple alignments ana- quite 1.6 (Maddison and Maddison, 2004). The morpho- lyzed and their resulting topologies can be obtained upon logical feature was defined to have three states [(0) 0.2– request to AZR. 0.25 times as long as tergite, petiole absent; (1) 0.3–0.5 times as long as tergite, petiole present but short; (2) 0.6– 2.4. Test of alternative hypotheses 0.85 times as long as tergite, petiole present and long]. The gall association trait was defined as binary [(0) A Bayesian approach (Buckley et al., 2002; Reeder, absent/unknown; (1) present]. Ecological information was 2003; Brandley et al., 2004) was implemented for hypothe- scored from literature and personal observations, and mor- sis testing of alternative topologies not present in our phological data was obtained from the sequenced speci- majority consensus trees derived from the two simulta- mens and from literature. The character states scored for neous analyses. In particular, we examined the monophyly the two features examined can be retrieved from the elec- of each of the large tribes Doryctini, Hecabolini and Spath- tronic supplementary material. The Markov k-state one iini (sensu Belokobylskij, 1992). Of these, the gall-associ- parameter model (Mk1; Lewis, 2001) was used for the like- ated Allorhogas and Psenobolus were placed in the lihood character optimizations, which assumes a single rate Hecabolini and Spathiini, respectively. In this approach, for transitions between character states. A likelihood ratio a 95% credible set of trees sampled after burn-in was test was carried out to discriminate for the best estimate of assembled for each simultaneous analysis, considering a ancestral state at each node following Pagel (1994). In this 986 A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 test, a state is considered as the best estimate for a given ciated genera examined in the COI Bayesian analysis, three branch if it differs by P2.0 from one or more states with of them (Allorhogas, Labania, and Monitoriella) were sig- higher negative log-likelihood values, and considers the nificantly recovered as monophyletic (BPP = 1.0). None ancestral reconstruction as ambiguous when the difference of the separate analyses recovered the taxa with elongated between states is 62.0. first metasomal tergite to be monophyletic. The full hierarchical Bayesian method for inferring The 28SN + COI Bayesian topology with its posterior ancestral states (Huelsenbeck and Bollback, 2001) was also probabilities and the posterior probabilities of similar performed for the gall association character with MrBayes clades recovered in the 28SA + COI Bayesian topology version 3.1.2 (Ronquist and Huelsenbeck, 2003) using the are shown in Fig. 3. The number of significant posterior 28SN + COI data set. This method incorporates uncer- probabilities of clades considerably increased in the two tainty in the tree, branch lengths, and substitution model combined analyses with respect to the separate ones. A parameters, approximating the posterior probability of total of 22 clades were similar and significantly supported ancestral states employing Markov Chain Monte Carlo in the two combined analyses, but two clades were only sig- (Huelsenbeck and Bollback, 2001). The Bayesian analysis nificantly supported in the analysis with 28SN and four performed to infer the ancestral state for the above charac- clades increased their BPP to significant values in the anal- ter was run using the same parameters and settings as the ysis with 28SA (Fig. 3). original phylogenetic analyses, except by including the gall The two simultaneous analyses both show a non-mono- association character in the matrix analyzed and constrain- phyletic Doryctinae, though the clades implicated are not ing the node with the five gall associated genera examined significantly supported and involve the morphologically (see Section 3). less derived genera Doryctes Haliday and Ontsira Cam- eron. In both topologies, a clade with the species of the lat- 3. Results ter two genera (BPP: 28SN, 28SA = 1.0) appears as the sister group of a clade with two divisions, one containing 3.1. Phylogenetic reconstructions all the non-doryctine subfamilies except rhyssalines, and the other one with the remaining doryctine genera. This Despite several attempts were made to obtain both 28S major doryctine clade (BPP: 28SN, 28SA = 1.0) shows an and COI sequences for all the ethanol preserved and dry apparent geographic structure that is composed of two pinned specimens examined, COI sequences could not be main clades. One of these contains genera mainly distrib- obtained for three taxa [Psenobolus, Spathius Nees (Antes- uted in the Palaearctic, Oriental, and Afrotropical regions pathius Belokobylskij), and Ecphylus Foerster]. Moreover, (BPP: 28SN = 0.62; 28SA = 0.56). The second clade has in five genera (Tarasco Marsh, Hypodoryctes Kokujev, the Neotropical Megaloproctus Schulz at the base (only Barbalhoa Marsh, Semirhytus Szepligeti, and Allorhogas) with 28SN; BPP = 0.58), followed by a clade with two spe- sequences of the two genetic fragments were obtained from cies of Spathius + Caenophanes Foerster (BPP: 28SN, different specimens, but in the case of Tarasco sp. and Hyp- 28SA = 1.0), then by a clade with the two genera of the odoryctes sibiricus Kokujev, the sequences were from differ- cosmopolitan Holcobraconini, Liobracon Sze´pligeti and ent individuals of the same species. The 28S data set Odontobracon Cameron (BPP: 28SN, 28SA = 1.0), a clade consisted of 538 unambiguously aligned positions and 15 with two endemic Australian genera, Jarra Marsh & Aus- unalignable regions. Inclusion of the recoded unalignable tin and Syngaster Brulle´ (BPP: 28SN, 28SA = 1.0), and regions added 263 characters to the data set, of which finally by a large, significantly supported clade represented 249 were from the nucleotide clusters created and 14 from by doryctine genera that are mainly distributed in the Neo- the ‘morphological’ indel characters. tropics (BPP: 28SN = 0.69; 28SA = 0.98). The majority rule consensus trees that resulted from the The five gall-associated doryctine genera were recovered separate 28SN and COI Bayesian analyses performed are together in the two simultaneous analyses in a single clade shown in Figs. 1 and 2, respectively. The COI Bayesian within the large ‘Neotropical’ clade, and recoding of the phylogeny recovered the highest number of significantly unalignable regions increased its BPP, though not to a sig- supported clades in comparison to the analyses with the nificant value (BPP: 28SN = 0.49; 28SA = 0.7). Moreover, 28SN and 28SA data sets (COI = 21; 28SN = 18; a clade with all the gall associated genera excluding Perc- 28SA = 19). The topologies reconstructed from the 28SN nobracon appeared significantly supported in the and 28SA data sets were mainly similar, with no conflicting 28SA + COI topology (BPP = 0.95). The clade with the significantly supported clades among them. The three sep- five gall-associated genera was recovered as the sister group arate analyses performed recovered a clade with gall-asso- of a clade with the two included species of the widely dis- ciated genera. A clade with Heterospilus Haliday and the tributed and polyphagous Heterospilus, and this relation- gall-associated genera included except Labania was recov- ship increased its BPP to a significant value when the ered in the 28SN topology (BPP = 0.66), whereas in the unalignable regions were recoded (BPP: 28SN + COI = 28SA topology the five gall-associated genera were recov- 0.69; 28SA + COI = 0.99). ered in a single clade together with the above species of Most of the topologies derived from the MP and Bayes- Heterospilus (BPP = 0.89). Moreover, of the four gall-asso- ian analyses using the multiple alignments for the 28S with A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 987

Rhaconotus Platyspathius Leptorhaconotus Ipodoryctes Rhacontsira Spathius (Antespathius) Megaloproctus Caenopachys Euscelinus Hemidoryctes Caenophanes Liobracon Odontobracon Schlettereriella Pseudodoryctes Rinamba Cystomastax Pseudorhysipolis Pambolus Spathius sp. Spathius agrili Syngaster Jarra Heterospilus prosopidis Heterospilus sp. Allorhogas Psenobolus Monitoriella Percnobracon Semirhytus Johnsonius Pedinotus Lamquetia Acanthorogas Hecabolus Stenocorse Aphelopsia Barbalhoa Curtisella Histeromeroides Labania Ecphylus Aivalykus Ondigus Acrophasmus Notiospathius Hansonorum Masonius Tarasco Stiropius * Spathiomorpha * Hypodoryctes Hormius Doryctes leucogaster Doryctes heydeni Ontsira 0.01 substitutions/site Rhyssalus Oncophanes

Fig. 1. 28SN Bayesian phylogram. Black circles above branches indicate Bayesian posterior probabilities P0.95 recovered in both the 28SN and 28SA Bayesian analyses. Asterisks indicate Bayesian posterior probabilities P0.95 only recovered in the separate 28SN Bayesian analysis. Hollow circles indicate P0.95 only recovered in the 28SA Bayesian analysis. Names of the gall-associated genera are in bold. various gap opening-gap extension penalties recovered a gall-associated genera in at least some of their MPTs clade with at least four of the gall-associated genera obtained, whereas 19 of the 50% majority rule consensus included (Fig. 4). Moreover, all these analyses placed the trees derived from the simultaneous Bayesian analyses also taxa with elongated first metasomal tergite in separate recovered this relationship. clades along the tree. Six of the 21 MP analyses with the 28S alone recovered MPTs containing a group with the 3.2. Tests of alternative hypotheses gall-associated genera except Labania, and 13 analyses reconstructed MPTs that placed them together with the The Bayesian test of alternative phylogenetic hypotheses two species of Heterospilus or with the latter and the Aus- for the two combined analyses showed that the topologies tralian Jarra and Syngaster. Fourteen of the combined recovering the tribes Doryctini, Hecabolini and Spathiini analyses using MP recovered the monophyly of the five (sensu Belokobylskij, 1992) each as monophyletic are statis- 988 A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992

Rhaconotus Platyspathius Rhacontsira Leptorhaconotus Ipodoryctes Caenopachys Euscelinus Hemidoryctes Rinamba Schlettereriella Pseudodoryctes Hypodoryctes Spathiomorpha Megaloproctus Spathius sp. Spathius agrili Caenophanes Syngaster Jarra Heterospilus prosopidis Heterospilus sp. Pedinotus Lamquetia Curtisella Hecabolus Notiospathius Masonius Hansonorium Histeromeroides Tarasco Aphelopsia Stenocorse Percnobracon Acrophasmus Barbalhoa Ondigus Allorhogas Labania Monitoriella Semirhytus Johnosonius Aivalykus Acantorhogas Liobracon Odontobracon Doryctes Cystomastax Hormius Pseudorhysipolis Pambolus 0.01 substitutions/site Stiropius Doryctes heydoni Ontsira Oncophanes Rhyssalus

Fig. 2. COI Bayesian phylogram. Black circles above branches indicate Bayesian posterior probabilities P0.95. Names of the gall-associated genera are in bold. tically rejected (P < 0.05). The SH tests also show that the Origin of gall association appears significantly supported three Bayesian topologies derived from the 28SN + COI once in the Percnobracon + Psenobolus + Allorho- data set with the above tribes forced to be monophyletic gas + Labania + Monitoriella clade. On the other hand, are significantly worse explanations of the data than the at least eight origins are shown for a moderately to consid- unconstrained Bayesian topology (P < 0.0001). The Bayes- erably elongated acrosternite in the first tergite. This fea- ian alternative topologies used for the latter test can be ture is shown to have evolved three times within the retrieved from the electronic supplementary material. Rhaconotini clade [Platyspathius Viereck, Leptorhaconotus Granger and Spathius (Antespathius)], twice within the 3.3. Character evolution remaining members of the ‘Palaearctic-Oriental-Afrotropi- cal’ clade with Schlettereriella Sze´pligeti and Spathiomor- Fig. 3 shows the ancestral states of the two examined pha Tobias, once with the species of Spathius (Spathius features that were mapped onto the 28SN + COI Bayesian Belokoblyskij), and two times within the ‘Neotropical’ phylogeny based on maximum likelihood optimizations. clade, one with the gall-associated Percnobracon and Pse- A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 989

Rhaconotus Platyspathius Leptorhaconotus Ipodoryctes Rhacontsira Spathius (Antespathius) Caenopachys Euscelinus Non gall associated Hemidoryctes Acrosternite 0.2-0.25 x as long as tergite Rinamba 0.3-0.5 x as long as tergite Schlettereriella Pseudodoryctes 0.6-0.85 x as long as tergite Spathiomorpha Gall associated Hypodoryctes Uncertain length of acrosternite Megaloproctus Spathius sp. Spathius agrili Caenophanes Liobracon Odontobracon Syngaster Jarra Heterospilus prosopidis Heterospilus sp. Allorhogas Labania * Monitoriella Gall associated Percnobracon genera Psenobolus Semirhytus Johnsonius Pedinotus Lamquetia Acanthorogas Curtisella Hecabolus Stenocorse Ecphylus Aivalykus Barbalhoa Aphelopsia Histeromeroides Notiospathius Masonius Hansonorum Tarasco Ondigus Acrophasmus Cystomastax Pseudorhysipolis Pambolus Stiropius Hormius Doryctes leucogaster Doryctes heydeni Ontsira 0.01 sustitutions/site Rhyssalus Oncophanes

Fig. 3. Character evolution of gall association and length of acrosternite of ﬁrst metasomal tergite on the Bayesian 28SN + COI phylogram for doryctine genera using maximum likelihood optimizations. Black circles indicate clades supported by BPP P 0.95 in both the 28SN + COI and 28SA + COI Bayesian phylogenies. Open circles indicate clades supported by BPP P 0.95 only in the 28SA + COI Bayesian phylogeny. The asterisk indicates the signiﬁcantly supported clade (BPP = 0.95) recovered in the 28SA + COI Bayesian analysis, but containing Psenobolus instead Percnobracon.

nobolus, and the remaining one in the Notiospathius Mat- ciated wasps within the mainly parasitic wasp subfamily thews & Marsh + Masonius Marsh + Tarasco + Hansono- Doryctinae. This relationship was recovered in our two rum Marsh clade. simultaneous phylogenetic analyses, though the clade The full hierarchical Bayesian method shows that the involved was only signiﬁcantly supported in the 28SA + ancestral state being gall associated has a probability of COI Bayesian topology. The result obtained was surprising 0.93 for the clade involved. because the taxa involved display disparate external morphologies and are associated with distantly related plant 4. Discussion families. The exclusive presence of cecidogenic and presumed Our study shows phylogenetic evidence that suggests a galler-predating taxa within a same clade agrees with close evolutionary relationship among a group of gall-asso- the generally accepted scenario about the origin of sec- 990 A. Zaldivar-Rivero´n et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992

28S parsimony 28S + COI parsimony

Gap extension penalty Gap extension penalty

1246810 1246810

10 *a 50d 27d 39d 23d *a 10 * * 50b *

8 *d 33a 19a 38a 8 * * 50 *b 88

6 66d 57d *d 94d 6 75 61 *

4 *e 13a 47d 4 *b 80 *

2 29d 2 *b 33 Gap opening penalty Gap opening penalty 1 *e 1 *

28S + COI Bayesian

Gap opening penalty

1246810

10 38 39 51 51 56 80

8 62 41c 36 79 50

6 49 57 36 44c

4 61 57 35

2 59 57 Gap opening penalty 1 82

Fig. 4. Summary of groups recovered in the separate and combined analyses based on Clustal X alignments of the 28S with different gap opening and gap extension penalties. Black squares are analyses that recovered the five gall-associated genera included as monophyletic in any of the MPTs obtained and in the 50% majority rule consensus tree derived from the Bayesian method. Grey squares are analyses that recovered at least four of the above genera in a single clade. Numbers in the Bayesian analyses refer to posterior probabilities of clades. Numbers in the parsimony analyses are percentages of MPTs that grouped gall-associated genera (*, 100% of MPTs). (a) Excluding Labania; (b) excluding Percnobracon; (c) excluding Psenobolus; (d) excluding Labania and including the two species of Heterospilus; (e) excluding Labania and including the two species of Heterospilus, Syngaster, and Jarra. ondary phytophagy in parasitic wasps, in which phyto- tantly related plant families is more congruent with a phagy is hypothesized to have arisen via attacking of model that explains the macroevolution of insect plant cone-boring, galler or seed predator insect larvae in liv- associations as primarily reflecting geographical proximity ing plant tissues, with the subsequent shift to feeding (Bernays and Chapman, 1994; Dobler et al., 1996), though on the associated particularly nutritious plant material chemical similarities between the host plants involved (Ronquist, 1995; Quicke, 1997). This hypothesis is also (Futuyma and McCafferty, 1990; Becerra and Venable, supported by the presence of both gall-formers and pre- 1999) cannot be ruled out. dators of gallers within Allorhogas (Maceˆdo et al., 1998; Similar to the separate and combined molecular anal- Wharton and Hanson, 2005). In addition, Allorhogas and yses performed by Zaldivar-Riveroń et al. (2006, Figs. Psenobolus, the latter probably a parasitoid of gallers in 2a, b and 3a) with the same two markers, our phyloge- figs (P. Hanson, pers. comm.), are recovered as sister nies do not recover the Doryctinae as monophyletic, with taxa in the 28SA + COI Bayesian phylogeny (though the support of the clades involved being weak. In fact, a with low BPP), whereas the cecidogenic Monitoriella monophyletic Doryctinae was only recovered with a sig- and the presumed gall former Labania are significantly nificant BPP when morphological data were included. supported as sister taxa in both analyses. Despite this, the relationships recovered here reveal a The fact that all the known gall-forming doryctines are geographic pattern that is congruent with the sequential from the Neotropics where they attack a range of only dis- break up of Gondwanaland with three defined groups: A. Zaldivar-Riveroń et al. / Molecular Phylogenetics and Evolution 44 (2007) 981–992 991 one mainly composed of taxa from the Palaearctic, References Oriental, and Afrotropical regions, a second one of Aus- tralian, and the remaining one of Neotropical taxa, with Achterberg, C. van., 1988. Parallelisms in the Braconidae (Hymenoptera) the latter two being significantly supported as sister with special reference to the biology. In: Gupta, V.K. (Ed.), Advances in Parasitic Hymenoptera Research. E.J. Brill, Leiden, pp. 85–115. groups. Austin, A.D., Dangerfield, P., 1998. 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