phylogenetic relationships among the braconidae (hymenoptera: ichneumonoidea) inferred from partial...

Molecular Phylogenetics and Evolution 37 (2005) 104–116

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Phylogenetic relationships among the Braconidae (Hymenoptera: Ichneumonoidea) inferred from partial 16S rDNA, 28S rDNA D2,

18S rDNA gene sequences and morphological characters

M. Shi a, X.X. Chen a,¤, C. van Achterberg b

a Institute of Applied Entomology, Zhejiang University, 268 Kaixuan Road, Hangzhou 310029, Chinab Department of Hymenoptera, National Museum of Natural History, Leiden, The Netherlands

Received 18 October 2004; revised 8 March 2005Available online 23 May 2005

Abstract

Phylogenetic relationships among the Braconidae were examined using homologous 16S rDNA, 28S rDNA D2 region, and 18SrDNA gene sequences and morphological data using both PAUP* 4.0 and MRBAYES 3.0B4 from 88 in-group taxa representing 35subfamilies. The monophyletic nature of almost all subfamilies, of which multiple representatives are present in this study, is well-supported except for two subfamilies, Cenocoelinae and Neoneurinae that should probably be treated as tribal rank taxa in the sub-family Euphorinae. The topology of the trees generated in the present study supported the existence of three large generally acceptedlineage or groupings of subfamilies: two main entirely endoparasitic lineages of this family, referred to as the “helconoid complex”and the “microgastroid complex,” and the third “the cyclostome.” The Aphidiinae was recovered as a member of the non-cyclosto-mes, probably a sister group of Euphorinae or Euphorinae-complex. The basal position of the microgastroid complex among thenon-cyclostomes has been found in all our analyses. The cyclostomes were resolved as a monophyletic group in all analyses if twoputatively misplaced groups (Mesostoa and Aspilodemon) were excluded from them. Certain well-supported relationships evident inthis family from the previous analyses were recovered, such as a sister-group relationships of Alysiinae + Opiinae, ofBraconinae + Doryctinae, and a close relationship between Macrocentrinae, Xiphozelinae, Homolobinae, and Charmontinae. Therelationships of “Ichneutinae + ((Adeliinae + Cheloninae) + (Miracinae + (Cardiochilinae + Microgastrinae)))” was conWrmed withinthe microgastroid complex. The position of Acampsohelconinae, Blacinae, and Trachypetinae is problematic. 2005 Elsevier Inc. All rights reserved.

Keywords: Braconidae; Phylogenetic relationships; 28S rDNA; 16S rDNA; 18S rDNA; Morphology

1. Introduction

The Braconidae is a very large family of parasitic waspswith about 17,500 valid described species worldwide(TAXAPAD-database; data kindly supplied by Dr. D.S.Yu, Vancouver) and at least Wve times as many remain tobe described. The species are currently classiWed intoabout 40+ subfamilies (van Achterberg, 1993). The precisenumber accepted by braconid workers has not yet stabi-

* Corresponding author.E-mail address: [email protected] (X.X. Chen).

1055-7903/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ympev.2005.03.035

lized but application of cladistic methodology in recentyears has led to the creation of a number of additionalsubfamilies. Generally, the family shows signiWcant speci-Wcity in host relationships at the subfamily level. Forexample, the Microgastrinae parasitize only lepidopteranlarvae (with the exception of one species being a parasit-oid of Trichoptera; van Achterberg, 2002), the Helconinaeattack coleopteran larvae, the Alysiinae and Opiinaeattack cyclorraphous dipteran larvae, whereas the Aphi-diinae parasitize aphids. Thus, the Braconidae representan important model system to examine the evolution ofparasitoid lifestyles (Gauld, 1988; WhitWeld, 1992).

mailto: [email protected]

mailto: [email protected]

M. Shi et al. / Molecular Phylogenetics and Evolution 37 (2005) 104–116 105

The relationships between the braconid subfamilieshave been the subject of considerable discussion overpast couple of decades (van Achterberg, 1976, 1984,1993; Bapek, 1970; Fischer, 1972; Quicke and van Ach-terberg, 1990; Tobias, 1967), but few Wrm conclusionshave been reached, though it has been generally acceptedthere are two major groupings of subfamilies, “cyclosto-mes” and relatives which are predominantly idiobiontectoparasitoids and the remainder consisting of koinobi-ont endoparasitoids (van Achterberg, 1984; Askew andShaw, 1986; Gauld, 1988). However, phylogenetic rela-tionships within the family are controversial, with themost comprehensive morphological study (Quicke andvan Achterberg, 1990) criticized by a number of workers(Wharton et al., 1992; but see van Achterberg andQuicke, 1992). Because morphology-based phylogeniesoften suVer from problems associated with reductionalsynapomorphies, it is diYcult to determine whether loststructures are due to homologous or convergent events(van Achterberg, 1988; Gibson, 1985). So, moleculardata have recently been employed to describe subfamilyrelationships within the family or generic relationshipswithin subfamilies (Belshaw and Quicke, 1997; Chenet al., 2003; Gimeno et al., 1997; Li et al., 2003; WhitWeldet al., 2002). Meanwhile, some other researchers thinkusing a limited set of molecular data alone will not wellreconstruct the phylogenetic relationship within Bracon-idae or other groups, therefore, they suggested that it isbetter to combine molecular data and morphologicalcharacters or other traits to analyze the phylogeneticrelationships (Will and RubinoV, 2004). Belshaw andQuicke (2002) used partial 28S rDNA (2–10 variableregions) and partial 18S rDNA gene sequences, combin-ing lifestyle traits of diVerent parasitic wasps, to estimatethe phylogeny of Braconidae. WhitWeld et al. (2002)combined 16S, COI, and 28S genes and morphology toreconstruct phylogenetic relationships among Microgas-trinae. Dowton et al. (2002) investigated the phylogenyof the Braconidae, employing 16S and 28S rDNA genefragments together with a suite of morphological char-acters, recovering the Aphidiinae as sister group to thecyclostomes and the Ichneutinae as sister group to themicrogastroids. However, the phylogeny of the Braconi-dae is far from resolved. For a better understanding ofthe phylogenetic relationships among the Braconidaeshould be based on more analyses and of more genes.The purpose of the present study is to examine historicalrelationships among the Braconidae using both molecu-lar and morphological data. Both PAUP* 4.0 andMRBAYES 3.0B4 were performed to generate phyloge-netic trees. Three genes are chosen: mitochondrial 16SrDNA coding the large subunit of the mitochondrialribosome, nuclear 28S rDNA D2 coding the secondexpansion segment of the nuclear ribosome subunit andribosomal 18S rDNA partial gene sequence. These threegenes have been extensively used in phylogenetic analy-

sis within Hymenoptera, at both lower and higher taxo-nomic level. We combined 96 characters about adultexternal morphology and other traits of larval male andfemale reproductive systems.

2. Materials and methods

2.1. Sampling of taxa

We examined more than 100 species belonging to 88genera in 35 subfamilies in this study. The species arelisted in Table 1. The subfamily arrangement largely fol-lows van Achterberg (1993) but a modiWed system isused based on morphological and biological characters.Both Acampsohelconinae and Hydragneocolinae aretreated as independent subfamilies in this paper.

2.2. Laboratory protocols

We extracted DNA from single specimens preservedin 100% ethanol. Legs were removed from larger wasps(>5 mm long) and used for exaction whereas the abdo-men and thorax were used for smaller wasps (<5 mmlong). Ethanol was then removed by washing 3 times(15 min each) in 10 mM Tris–HCl (pH 8) containing100 mM NaCl and 1 mM MgCl2. Tissue was then groundin 400�l of 10 mM Tris–HCl (pH 8), 10 mM EDTA, 1%SDS, and then added 100 �g (or 10�l) proteinase K andincubated at 37 °C for 1 h. The homogenate wasextracted with phenol/chloroform/isoamyl alcohol(25:24:1). DNA was resuspended in 60�l TE buVer andstored at ¡20 °C. Double-strand PCR products wereampliWed in an Eppendorf Mastercycler gradient(Eppendorf AG, Hamburg), using 35 cycles [Wrst dena-turation, 4 min at 94 °C (denaturation, 1 min at 94 °C;annealing, 1.5 min at 55 °C; and elongation, 1.5 min at72 °C) £ 35; Wnal elongation, 8 min at 72 °C]. The partialribosomal 18S rDNA was ampliWed using the 18S up1(5�-TGG TTG ATC CTG CCA GTA G-3�) and 18S 58-3 (5�-GAG TCT CGT TCG TTA TCG GA-3�) primers(Sanchis et al., 2000).

2.3. Sequence data and alignment

New sequences in this paper have been deposited inGenBank under accession numbers from AY920270 toAY920277. Most other sequences of three genes in thispaper were retrieved from GenBank with accessionnumbers and references listed in Table 1. Two membersof the Ichneumonidae (Venturia canescens and Xoridespraecatorius) were included as outgroups. The Ichneu-monidae is widely recognized as the sister group to theBraconidae (Belshaw and Quicke, 2002; Sharkey andWahl, 1992). Before alignment some regions wereremoved manually because they were diYcult to align.

106 M. Shi et al. / Molecular Phylogenetics and Evolution 37 (2005) 104–116

Table 1List of taxa examined in this paper

Subfamily Species GenBank Accession Nos.

28S 16S 18S

Cyclostome subfamiliesAlysiinae Alysia soror Marshall Z93635[1] Z93707[1] —

Chorebus sp. UK Z93643[1] Z93712[1] —Aspilota sp. Costa Rica AF173219[2] AF003476[3] —Dacnusa sibirica Telenga Z93644[1] Z93713[1] —Exotela sp. UK Z93647[1] Z93714[1] —Phaenocarpa sp. UK Z93660[1] Z93710[1] —

Betylobraconinae Betylobracon waterhousi Tobias AJ245686[2] AF003479[3] —Mesocentrus sp. Australia Z97968[4] AF003480[3] —

Braconinae Bracon phylacteophagus Austin AF173222[2] AF003481[3] —Callibracon limbatus (Brullé) AJ231532[18] AF003482[3] —Habrobracon hebetor (Say) AJ245691[2] AF003483[3] —Syntomernus sp. UK AJ302925[5] — AJ307456[5]

Doryctinae Jarra maculipennis Marsh and Austin AJ302928[5] AF003485[3] AJ307459[5]

Heterospilus prosopidis Viereck Z83599[6] AF003484[3] —Syngaster lepidus (Brullé) AJ245698[2] AF003487[3] —

Exothecinae Colastes incertus (Wesmael) Z83610[6] Z93720[1] —Gnamptodontinae Gnamptodon pumilio (Nees) Z93662[1] AF003488[3] —Histeromerinae Histeromerus mystacinus Wesmeal Z83601[6] AF003489[3] —Hormiinae Hormius sp. France Z97965[4] AF176049[2] —Hydrangeocolinae Aspilodemon sp. Brazil AJ245685[2] AF176051[2] —Mesostoinae Mesostoa kerri Austin and Wharton Z97972[4] AF003490[3] AJ307460[5]

Opiinae Fopius arisanus (Sonan) AF173218[2] AF003491[3] —Biosteres carbonarius (Nees) Z93639[1] AJ245698 Z93705[1] —Opius caricivorae Fischer AY167649[7] AF003493[3] —Xynobius ssp.

X. maculipes Wesmael Z93659[1] Z93704[1] —X. sp. China — — AY937226

Pambolinae Pambolus sp. Costa Rica AF173224[2] AF003497[3] —Rhyssalinae Dolopsidea ssp.

D. indagator (Haliday) AF029136[8] — —D. sp. UK — AF003494[3] —

Rhyssalus ssp.R. clavator Haliday — AF176052[2] —R. sp. UK Z83603[6] — —Thoracoplites sp. UK AJ302920[5] — AJ307450[5]

Oncophanes sp. Australia and UK Z97973[4] AF176050[2] —Rogadinae Aleiodes spp.

A. nigricornis Wesmael AJ784934[9] — —A. sp. UK and China — AF003496[3] AY920277

Spinaria fuscipennis Brullé Z97964[4] AF176053[2] —

Helconoid subfamiliesAgathidinae Agathiella sp. Australia AJ245682[2] AF003498[3] —

Bassus sp. Australia and China AF173217[2] AF003500[3] AY920270Braunsia sp. UK AJ302919[5] — AJ307449[5]

Earinus elator (Fabricius) Z97944[4] AF176054[2] —Alabagrus stigma Brullé AJ302790[5] AF003499[3] —

Blacinae Blacus sp. Costa Rica and UK Z97950[4] AF003501[3] —Acampsohelconinae Urosigalphus sp. UK AJ302923[5] — AJ307454[5]

Charmontinae Charmon ssp.C. ruWthorax Chen and He — — AY920272C. sp. UK Z97949[4] AF176055[2] —

Cenocoeliinae Cenocoelius analis (Nees) Z83605[6] AF003502[3] —Capitonus sp. UK AJ245687[2] AF176056[2]

Euphorinae Perilitus ssp.P. ruWcephalus Chen and van Achterberg AY291567[11] — —P. pallipes Curtis — AF473538[12]

Rhopalophorus sp. UK AJ302922[5] — AJ307453[5]

Streblocera ssp.S. janus Chen and van Achterberg — — AY920271


chis et al. (2000); [18]Belshaw et al. (2001); [19]Shi and Chen (2005).

Table 1 (continued)

[1]Gimeno et al. (1997); [2]Belshaw et al. (2000); [3]Dowton et al. (1998); [4]Belshaw et al. (1998); [5]Belshaw and Quicke (2002); [6]Belshaw and Quicke(1997); [7]Chen et al. (2003); [8]Dowton and Austin (1998); [9]Mori et al. (unpublished); [10]Kambhampati et al. (2000); [11]Li et al. (2003); [12]Erlandsonet al. (unpublished); [13]WhitWeld (1997); [14]Michel-Salzat and WhitWeld (2004); [15]WhitWeld et al. (2002); [16]Mardulyn and WhitWeld (1999); [17]San-

Subfamily Species GenBank Accession Nos.

28S 16S 18S

S. olivera Quicke and Purvis AJ302831[5] — —Zele ssp.

Z. deceptor Thomson AY291568[11] — AY920273Z. albiditarsis Curtis — AF176061[2] —

Dinocampus coccinellae Schrank AY291565[11] AF174357[10] —Centistes sp. China and UK AJ245688[11] AF003503[3] —Leiophron uniformis (Gahan) AJ245692[11] AF176057[2] —Meteorus ssp.

M. versicolor Wesmael AY291566[11] — —M. pulchricornis (Wesmael) — U68146[13] —

Helconinae Helcon sp. France Z97946[4] AF176059[2] —Schizoprymnus sp. UK AJ302934[5] AF176060[2] AJ307463[5]

Ussurohelcon nigricornis van Achterberg AJ302912[5] — AJ307445[5]

Eubazus semirugosus (Nees) Z83608[6] AF176058[5] —Diospilus sp. Costa Rica AF029134[8] AF003504[3]

Homolobinae Homolobus australiensis (Nixon) Z97955[4] AF003506[3] —Macrocentrinae Macrocentrus ssp.

M. melanogaster He and Chen — — AY920274M. sp. Australia AF029135[8] AF003507[3] —

Orgilinae Stantonia sp. UK AJ302935[5] — AJ307464[5]

Orgilus lepidus Muesebeck AF173221[2] AF003508[3] —Trachypetinae Megalohelcon ichneumonoides Tobias Z97941[4] AF003510[3] AJ307443[5]

Sigalphinae Sigalphus irrorator (Fabricius) Z97942[4] AF003509[3] —Xiphozelinae Xiphozele sp. UK AJ302931[5] — AJ307461[5]

Microgastroid subfamiliesAdeliinae Adelius sp. UK Z97961[4] AF029111[9] —Cardiochilinae Cardiochiles fuscipennis Szepligeti AF029118[8] AF029112[8] —

Toxoneuron nigriceps (Viereck) Z97957[4] U68151[13] —Cheloninae Ascogaster sp. Australia and China AF029121[8] AF029114[8] AY920276

Chelonus inanitus (L.) AJ535956[14] AJ535933[14] AY920275Phanerotomella sp. UK AJ302933[5] — AJ307462[5]

Microgastrinae Cotesia Xavipes (Cameron) AJ535942[14] AJ535922[14] —

Fornicia sp. UK and USA Z97959[4] AY044195[15] —Sathon falcatus (Nees) AF102746[16] AF029116[8] —Apanteles subandinus Blanchard AF029126[8] AF003514[3] —Exoryza schoenobii (Wilkinson) AY592965[19] AY592960[19] —Microgaster canadensis Muesebeck AF102733[16] U68154[13] —Microplitis demolitor Wilkinson AF029129[8] AY044196[15] —

Miracinae Mirax lithocolletidis Ashmead AF029131[8] U68152[13] —Neoneurinae Elasmosoma sp. UK AJ302929[5] — AJ307444[5]

Neoneurus mantis Shaw AF029133[8] U68147[13] —Ichneutinae Ichneutes bicolor Cresson AF029132[8] AF003518[3] —

Paroligoneurus sp. Costa Rica AJ245695[2] AF003519[3] —

Unplaced subfamiliesAphidiinae Aphidius ervi Haliday Z83582[6] AF174310[10] AJ009321[17]

Dyscritulus planiceps (Marshall) Z83594[6] AF174350[10] AJ009340[17]

Ephedrus persicae Froggatt Z83598[6] AF174348[10] AJ009329[17]

Lipolexis gracilis Foerster AJ245693[2] AF176063[2] AJ009334[17]

Praon ssp.P. dorsale Haliday Z83592[6] — AJ009341[17]

P. pequodorum Baker — AF174351[10] —Monoctonia vesicarii Tremblay — AF174341[10] AJ009337[17]

Trioxys pallidus Haliday Z83588[6] AF174336[10] AJ009351[17]

Pseudephedrus chilensis Stary AJ245697[2] AF176064[2] —

OutgroupIchneumonidae Venturia canescens (Gravenhorst) AJ245958[2] U06961[13] —

Xorides praecatorius (F.) Z83612[6] AF003520[3] —

108M

. Shi et al. / M

olecular Phylogenetics and E

volution 37 (2005) 104–116

Table 2Morphol

Note. “?”

Adelinae 000??000 0120111110 10111????1 1100??Agathidi 10001000 0?20110101 10101???11 010001Alysiinae 01101A1 10BA000001 1100?00001 110011Acampso 100??000 0120110111 10001????1 0A0???Aphidiin 00021A0A 012A11A1A2 1100100001 A11111Betylobr 01000101 10100???11 ?????????? ??????Blacinae 100??000 0120110111 10001????1 0A0???Braconin 01000101 01210AA?01 000000010A 0A0000Cardioch 10001000 0120111110 1111101111 010001Cenocoe 10011000 0110110111 10101????1 00000?Charmon 00001000 0110110101 10101????1 01010?Chelonin ?0001000 0110110110 1010111011 010001Doryctin 01000111 A02A0A0001 0000?001?0 000000Euphorin 10011000 01A0110?11 11A01110A1 0AAA1AExotheci 0000011- 1120010011 000000010 0A0000Gnampto ?11?0101 1020110001 00000????1 1100??Helconin ?0011000 0??0110101 1010110?11 000001Histerom 01000111 - - - -000010 00000- - - - - 0000- -Homolob 10001000 0000110111 10101????1 01000?Hormiin ????0101 1120110011 00000????0 01000?Hydrang 0010000 1120011000 ?????????0 010???Ichneutin ?0001101 0110111110 11001????1 1100??Paroligon ???????? ?????????? 11101????1 110???Macroce 00011000 0110110101 1010110011 010101Mesostoi 010????? ?????????? 01001????? ??????Microgas ?0001000 0120111110 1AAAA11011 0101?1Miracina ??0?0000 0120111110 10101?????1 0100??Neoneur 000??000 0110????11 11101????1 011?1?Opiinae 011001?1 A02A011001 11A0110101 110011Orgilinae 00011000 ?110110001 10101???11 01000?Pambolin 000??111 1010000000 000000??0 0A00??Rhyssalin 000A1111 ?01?000000 000000???0 ?000??Rogadin 01000101 10200?0001 0100?0???P 1A00?0Sigalphin 10011000 1020110101 10101???01 01000?Trachype 10011100 0020?????2 ?????????? ??????Xiphozel ?0011000 0000????11 10101????1 1101??Ancestor 00000000 0000000000 000000??00 000?00

ogical character matrix for the subfamilies of Braconidae

means unknown; “-” means missing; A D (01); B D (12); C D (012); D D (02).

1002000100 0000010011 0101100??0 0001111111 1100101000 01020100 00 ?1nae 00?2?B0?30 0?1A?00011 1001101101 110011AA1A A1A0AA1000 0100100000 11

0000020?30 0?01010011 A000A0020A A000A111A1 111000A000 0A0010001? 10helconinae 0002000100 0A001A0011 0001100?0A 0000110111 1110001000 0100100100 11ae 0000000000 1000010011 1A010A010A 1000010111 1110001A00 0100100000 11aconinae 0A00100000 0000000011 0000A00201 0000A11101 1110001000 0000101001 00

0002000100 0A001A0011 0001100?0A 0000110111 1110001000 0100100100 11ae 01??0D0?31 10010A0A11 A000A00101 00000A1111 1110001000 00101010A2 00ilinae 0011?21?3? 1001?00011 010000A211 000A110011 A1A1101011 1101000000 11

liinae 0012100000 0100001021 0000020101 0000000111 11A0A01000 0100101000 11tinae 0002010130 0000000011 000A100111 0000010111 1000000000 0100100000 11ae 10???00??0 0000001011 000AA0021A 0A00A10111 11001A1000 102010000 11ae 01000D0??0 00000A00C1 0000A0020A 0000AA11A1 111000AA00 0A00A0A0A2 0?ae 00?2000??0 0A00100011 00AA11020A A000110111 11A000A100 0100100100 11

nae 01000100P0 0A01000011 000000020A 0000011101 1110001000 0000001011 00dontinae 0A000D0?30 A001010011 A000000201 0000A111A1 1110001000 0010101011 ??ae 00?2000100 01001000CA 000AAA0B01 0000AAA111 AA10001000 01001000A0 11erinae 0100010100 0000000011 0000000200 0000A1A101 1110001000 0100010100 ??inae 0002000100 0000100011 0001010201 0000010a11 1a00001000 0100100100 11

ae 0100000020 0A0A000011 000000020A 0000011101 111000A000 0000101011 ??eocolinae 0100000100 1000010011 0000000200 0000011101 1110001000 0100100001 00ae 0002020?3? 100A0A0011 0A0AA0A211 0AA001A111 11A0001000 0100001000 11eurus 0002?20001 1001010011 0101100?11 0101111111 1110001000 0100?????? ??

ntrinae 0002010?30 0000000001 0001000201 0000010111 1100001000 0100100000 11nae 01??1A0001 1001010011 0001000?A0 0001011111 1110001000 0100101010 ?0trinae 2012?21?30 A001000011 1100100C11 00000A1011 1101AA1011 1101000000 11e 2002000?31 1001000011 0100100?A1 0000111111 1110101010 110201?000 ??inae 2002120?3? 1001000010 1A0010010P 0000111111 110A101000 0100001000 11

0A000C00?0 0?01010011 000000020A A0000111A1 111000A000 0A00100010 100002?10120 0100?00011 0001100?A1 0000010111 1110A11000 0100101000 11

ae 0?00000100 000000A011 0000100201 0000A11101 111A0A1000 0000001010 00ae 0100000100 0000000011 0000000201 0000011101 1110010000 0100001010 00

ae 0100?C0??0 0A00000011 0000P0020A 0000P111A1 1110A0A000 0000101010 00ae 00?2000?00 0110100011 0000001?A1 0P00010110 1100A01100 0100100000 11tinae 0012000100 0000100110 0010011100 0000010110 0100001100 0100100000 11inae 0002010?? 0 0000100001 0000010101 000001011? 1000001100 0100100100 ??

00??000??0 0000000000 0000000?00 000000000 0000000000 0000000000 00


Then sequences were aligned based on distanceapproach using CLUSTAL X version 1.81 (Thompsonet al., 1997) with diVerent gap opening and gap extensionvalues. Because some sequences used in this paper fromthe literature containing both portion D2 and portionD3 or other portion, the manual alignment was followedto remove these portions. These alignments are availableat http://qpm.zju.edu.cn/alignment.htm. The Wnal align-ments of 16S rDNA were 338 bp, of 18S rRNA 724 bp,and of 28S rDNA D2 375 bp long, including gaps.

2.4. Morphological data matrix

We used a suite of 96 morphological charactersdescribed previously (Quicke and van Achterberg, 1990)and scored at the subfamily level. Certain codings wereslightly modiWed (van Achterberg, 1997; van Achterbergand Quicke, 1992 (Table 2); Dowton et al., 2002). The datamatrix is included in the Appendix. The subfamily Mete-orinae is treated as a tribe in the subfamily Euphorinaeand the subfamily Acampsohelconinae as an independentone to include the genus Urosigalphus in this analysis.

2.5. Phylogenetic analysis

Following alignment, maximum parsimony (MP)were performed using PAUP* 4.0 (beta 10 version)(SwoVord, 2001) to Wnd the most parsimonious tree(s),heuristic parsimony search (Hillis et al., 1996) were per-formed using 100 replicates of random additionsequences and TBR option for branch swapping, andfollowed by additional round of branch swapping on theresulting trees with restriction on the number of trees toone. Each base was treated as an unordered characterwith equal weights, with gaps treated as missing data.Where more than one most parsimonious tree wasfound, a strict consensus tree was calculated. Down-weighting transitions or treating gaps as Wfth base didnot markedly aVect the results obtained. Statistical sup-port for each node was evaluated by bootstrap analysis(Felsenstein, 1985) with 1000 replications. The Bayesianapproach to phylogenetic reconstruction (Huelsenbecket al., 2001; Yang and Rannala, 1997) was implementedusing MRBAYES 3.0B4 (Huelsenbeck and Ronquist,2001). Each run was performed using default staringparameters and comprised 5,000,000 generations. Bayes-ian posterior probabilities (Pbay) were calculated frommajority–rule consensus of trees sampled every 100 gen-erations once the Markov chain reached stationary(determined by empirical checking of likelihood values).

3. Results and discussion

We tested alignment using the Clustal X programwith diVerent gap opening and gap extension values, and

resulted in diVerent length of aligned sequences. Theresult was identical to that of Morrison and Ellis (1997).They concluded in their paper that the multiple align-ments from diVerent procedures varied greatly in lengthand the alignments produced from Clustal W programwith diVerent gap weights were at least as diVerent fromeach other as those produced by the diVerent alignmentalgorithms (Morrison and Ellis, 1997). Because thedefault parameters in version 1.81 (gap opening 15, gapextension 6.66) were optimized using the balibase multi-ple alignment in the 142 alignment test in balibase (J.Thompson, pers. comm.), we used the alignments withdefault parameters for analysis in the present paper.

Because the combination of several genes generallyimproves phylogenetic accuracy (Remson and DeSalle,1998), we combined 18S rDNA, 16S rRNA, and 28SrDNA genes, and obtained 1682 characters in total andincluding the gaps. Of 1682 characters, 847 characters(50.35%) were variable and 734 characters (43.64%) wereparsimony informative.

Regarding the base composition, the overall GC con-tent of 16S rDNA is 14.79%, ranging from 10.76 to21.93%, of 18S rDNA 49.23%, ranging from 47.31 to56.16%, and of 28S rDNA D2 44.47%, ranging from31.18 to 56.0%.

3.1. Phylogenetic inference

The trees resulting from PAUP* and MrBayes analysesin this study are present in Figs. 1–4. The topology of thetrees is quite similar. Three large generally acceptedlineage or groupings of subfamilies were well-supported(Figs. 1–4). Two main entirely endoparasitic lineages ofthis family, referred to as the “helconoid complex” andthe “microgastroid complex” as proposed by Quicke andvan Achterberg (1990) and Wharton (1993) are resolved(Figs. 1–4). The “microgastroid complex” includes thegenerally recognized subfamilies Microgastrinae,Ichneutinae, Cardiochilinae, Miracinae, Adeliinae, andCheloninae. The “helconoid complex” consists of Helcon-inae, Macrocentrinae, Agathidinae, Euphorinae, Orgili-nae, and other several smaller subfamilies. We found theAphidiinae to be a member of the non-cyclostomes, prob-ably a sister group of Euphorinae or Euphorinae-com-plex, diVerent from the results of Dowton et al. (2002) whorecovered that the Aphidiinae have a close relationship tothe cyclostomes. The cyclostomes were resolved as amonophyletic group in all analyses, in agreement with tra-ditional views (Belshaw et al., 1998; van Achterberg, 1984;Wharton et al., 1992), although Mesostoa (Mesostoinae)and Aspilodemon (Hydrangeocolinae) were placed outsidethe cyclostomes except in Fig. 1. These two rare andabnormal subfamilies were recovered as a basal clade inthe helconoid complex within the non-cyclostomes (Figs. 2and 3) or occupied even a basal position in the cladeEuphorinae+ Aphidiinae within the helconoid complex

http://qpm.zju.edu.cn/alignment.htm

http://qpm.zju.edu.cn/alignment.htm


(Fig. 4). However, the position of these two subfamilies asbasal clade of the cyclostomes (Fig. 1) as in the most previ-ous studies (Dowton et al., 2002) is probably correct.

The monophyletic nature of almost all subfamilies,of which multiple representatives are present in thisstudy, is well-supported except for two subfamilies,

Fig. 1. Phylogeny of the Braconidae based on three genes and morphological data matrix with equal weights using MP method (PAUP*). Venturia
and Xorides used as outgroups. Numbers at nodes are bootstrap values (%).


Fig. 2. Phylogeny of the Braconidae based on three genes and morphological data matrix with the Wrst set of weights using MP method (PAUP*).
Venturia and Xorides used as outgroups. Numbers at nodes are bootstrap values (%).


Fig. 3. Phylogeny of the Braconidae based on three genes and morphological data matrix with the second set of weights using MP method (PAUP*).
Venturia and Xorides used as outgroups. Numbers at nodes are bootstrap values (%).


Cenocoeliinae (Figs. 1–3) and Neoneurinae (Fig. 4),and the position of the genus Diospilus is problematicin Fig. 1. The subfamilies Cenocoeliinae andNeoneurinae might be treated as tribes of the subfam-ily Euphorinae.

3.2. Relationships among the “braconoid complex” of subfamilies

The topology of the trees generated suggested that thecyclostome (or braconoid) subfamilies were naturally

Fig. 4. Phylogeny of the Braconidae based on three genes and morphological data matrix with equal weights using ML method (MrBayes). Vent-uria and Xorides used as outgroups. Numbers at nodes are Bayesian posterior probabilities.


derived (Fig. 1), but in Figs. 2–4 two genera (Mesostoaand Aspilodemon) were excluded from them. Certainwidely accepted phylogenetic relationships, such as the sis-ter group of Alysiinae +Opiinae, and of Braconinae+Doryctinae, were consistently resolved in all analyses(Figs. 1–4). The Exothecinae was always recovered as thesister group to the clade “Alysiinae+ Opiinae” with theGnamptodontinae occupying a basal position within thisclade (Figs. 1–4). The Rogadinae were recovered as a sis-ter group to “Braconinae + Doryctinae” (Figs. 1 and 4) inthe MP (with equal weights) and ML analyses, while as asister group to Horminae when character weightings wereemployed (Figs. 2 and 3). A group containing the Histero-merinae, Pambolinae, and Rhyssalinae was always foundin the basal grade in braconoid complex of subfamilies,but the exact relationships between these three subfamiliesare not robustly resolved. The Betylobraconinae were par-tially resolved as a basal group of the clade composed ofBraconinae, Doryctinae, and Rogadinae. The position ofthe Hormiinae within the cyclostomes is variable (seeTables 3 and 4).

3.3. Relationships among the “helconoid complex” of subfamilies

The “helconoid complex” of subfamilies was recoveredas a monophyletic group in most of our analyses duringthe present study, in agreement with the previous analysisby Quicke et al. (1992) who considered the “helconoidcomplex” to be a monophyletic lineage based on thederived structure of the sperm. Basal relationshipsbetween the helconoid subfamilies are not well resolved.However, we did Wnd certain well-supported close rela-tionships, such as a group containing Neoneurinae,Euphorinae, Cenocoeliinae, and Aphidiinae, a clade com-posed of “Orgilinae + (Sigalphinae + Agathidinae), and aclose relationship between Macrocentrinae, Xiphozelinae,Homolobinae, and Charmontinae. The position of Orgili-nae in our analyses was apparently diVerent from that byBelshaw and Quicke (2002) who recovered Orgilinae as amember of the microgastroid complex of subfamilies. Werecovered a close relationship between Macrocentrinaeand Charmontinae (Figs. 2 and 3) as already proposed onsimilarities in ovipositor structure (Quicke and vanAchterberg, 1990), DNA sequence (Belshaw et al., 1998)and DNA sequence and morphology combined (Dowton

et al., 2002). The combination of the Euphorinae, Neo-neurinae, and Cenocoeliinae with Aphidiinae as sistergroup present in all trees (Figs. 1–4) was not found byBelshaw and Quicke (2002) and Dowton et al. (2002), butwas proposed long ago by Bapek (1970) because of simi-larities in biology: all groups (except for the Cenocoelii-nae) contain parasitoids of adult insects. The position ofCenocoeliinae within the Euphorinae may be correct con-sidering its morphology; however, its biology indicate amore basal position in the Euphoroid complex.

The Blacinae and Acampsohelconinae were alwaysrecovered as sisters in all analyses (Figs. 1–4), but theposition of this clade within the helconoid complex wasvariable. The position of the Trachypetinae is also prob-lematic since its members possess a highly derived mor-phology and an aberrant composition of the DNA.

In general, the PAUP analysis using equal weightsresulted in the best tree (Fig. 1), only the genus Diospilus(Helconinae) is clearly misplaced and the members ofthe Xiphozele–Macrocentrus-group are better posi-tioned.

3.4. Relationships among the microgastroid complex of subfamilies

We recovered the microgastroid subfamilies as amonophyletic group among the non-cyclostomes, asfound by Dowton et al. (1998, 2002). The basal positionof the microgastroid complex among the non-cyclosto-mes has been found in all our analyses (Figs. 1–4).According to Belshaw and Quicke (2002) the microgast-roid complex is one of the most derived groups of thenon-cyclostomes, sister to the Helconinae-group.

Our analysis supported that the subfamily Ichneuti-nae is a member of the microgastroid complex, in agree-ment with the previous analyses based on morphologicalcharacters (Austin and Wharton, 1992; Quicke and vanAchterberg, 1990), and occupies a basal position in thisclade. Subfamily relationships among the microgastroidswere almost identical to those found by Dowton andAustin (1998). Therefore, we conWrmed a relationshipwithin the microgastroid subfamilies: “Ichneutinae +((Adeliinae + Cheloninae) + (Miracinae + (Cardiochilinae+ Microga strinae))).”

The Adeliinae are often found to be in the sameclade of the Cheloninae with high bootstrap support

Table 3Set 1 of simple weights

Weights 2311111112 1331321111 1221112221 1111111122 1112121131 11111113113222321111 1111321211 2123131122 111111

Table 4Set 2 of simple weights

Weights 2211111111 1221221111 1221111111 1111111122 1111111121 11111112112111221111 1111211211 2122121122 111111


(90–97%), occupying a basal position, and therefore,probably could be treated as a tribe in the subfamilyCheloninae, as indicated by the results of Belshaw andQuicke (2002).

Acknowledgments

We thank Dr. S.A. Belokobyskij (St. Petersberg,Russia) for his advice and discussion and Prof. He Jun-hua (Hangzhou, China) for his review of an early draftof the manuscript. We also thank two anonymousreviewers for their valuable comments. This work waspartly supported by National Natural Science Founda-tion of China (NSFC, No. 30170120) and the Teachingand Research Award Program for Outstanding YoungTeachers in Higher Education Institutions of MOE,China (TRAPOYT) to the second author.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at doi:10.1016/j.ympev.2005.03.035.

References

van Achterberg, C., 1976. A preliminary key to the subfamilies of theBraconidae (Hymenoptera). Tijdschr. Ent. 119, 158–160.

van Achterberg, C., 1984. Essay on the phylogeny of Braconidae(Hymenoptera: Ichneumonoidea). Ent. Tidskr. 105, 41–58.

van Achterberg, C., 1988. Parallelisms in the Braconidae (Hymenop-tera) with special reference to the biology, p. 85–115, Figs. 1–101.In: Gupta, V.K. (Ed.), Advances in Parasitic HymenopteraResearch. Leiden, pp. 1–546.

van Achterberg, C., 1993. Illustrated key to the subfamilies of the Bra-conidae (Hymenoptera: Ichneumonoidea). Zool. Verh. Leiden 83,1–189.

van Achterberg, C., 1997. Braconidae. An illustrated key to all subfam-ilies. ETI World Biodiversity Database CD-ROM Series.

van Achterberg, C., 2002. Apanteles (Choeras) gielisi spec. nov. (Hyme-noptera: Braconidae: Microgastrinae) from The Netherlands andWrst report of Trichoptera as host of Braconidae. Zool. Med. Lei-den 76, 53–60, Wgs 1–20.

van Achterberg, C., Quicke, D.L.J., 1992. Phylogeny of the subfamilies ofthe family Braconidae: a reassessment assessed. Cladistics 8, 1–95.

Askew, R.R., Shaw, M.R., 1986. Parasitoid communities: their size,structure and development. In: Wagge, J., Greathead, D., (Eds.),Insect Parasitoids. 13th Symp. R. Ent. Soc. Lond. Academic Press,London, pp. 225–264.

Austin, A.D., Wharton, R.A., 1992. New records of subfamilies, tribesand genera of Braconidae (Insecta: Hymenoptera) from Australia,with description of seven new species. Trans. R. Soc. S. Austr. 116,41–63.

Belshaw, R., Quicke, D.L.J., 1997. A molecular phylogeny of the Aphi-diinae (Hymenoptera: Braconidae). Mol. Phylogen. Evol. 7, 281–293.

Belshaw, R., Quicke, D.L.J., 2002. Robustness of ancestral state esti-mates: evolution of life history strategy in ichneumonoid parasit-oids. Syst. Biol. 51 (3), 450–477.

Belshaw, R., Fitton, M., Herniou, E., Gimeno, C., Quicke, D.L.J., 1998.A phylogenetic reconstruction of the Ichneumonoidea (Hymenop-tera) based on the D2 variable region of 28S ribosomal RNA. Syst.Entomol. 23, 109–123.

Belshaw, R., Dowton, M., Quicke, D.L.J., Austin, A.D., 2000. Estimat-ing ancestral geographical distributions: a Gondwanan origin foraphid parasitoids?. Proc. R. Soc. Lond., B, Biol. Sci. 267 (1442),491–496.

Belshaw, R., Lopez-Vaamonde, C., Degerli, N., Quicke, D.L.J., 2001.Paraphyletic taxa and taxonomic chaining: evaluating the classiW-cation of braconine wasps (Hymenoptera: Braconidae) using 28SD2-3 rDNA sequences and morphological characters. Biol. J. Linn.Soc. Lond. 73, 411–424.

Bapek, M., 1970. A new classiWcation of the Braconidae (Hymenop-tera) based on the cephalic structures of the Wnal instar larva andbiological evidence. Can. Ent. 102, 846–875.

Chen, X.-x., Piao, M.-h., WhitWeld, J.B., He, J.-h., 2003. A molecularphylogeny of the subfamily Rogadinae (Hymenoptera: Braconidae)based on the D2 variable region of 28S ribosomal RNA. ActaEntomol. Sin. 46 (2), 209–217.

Dowton, M., Austin, A.D., 1998. Phylogenetic relationships among themicrogastroid wasps (Hymenoptera: Braconidae): combined analy-sis of 16S and 28S rDNA genes and morphological data. Mol.Phylogenet. Evol. 10 (3), 354–366.

Dowton, M., Austin, A.D., Antolin, M.F., 1998. Evolutionary relation-ships among the Braconidae (Hymenoptera: Ichneumonoidea)inferred from partial 16S rDNA gene sequences. Insect Mol. Biol. 7,129–150.

Dowton, M., Belshaw, R., Austin, A.D., Quicke, D.L.J., 2002. Simulta-neous molecular and morphological analysis of braconid relation-ships (Insecta: Hymenoptera: Braconidae) indicates independentmt-tRNA gene inversions within a single wasp family. J. Mol. Evol.54, 210–226.

Erlandson, M., Braun, L., Baldwin, D., Soroka, J., Hegedus, D., unpub-lished. Molecular markers for Lygus spp. (Hemiptera: Miridae) andassociated Peristenus spp. (Hymenoptera: Braconidae) parasitoids.

Felsenstein, J., 1985. ConWdence limits on phylogenies: an approachusing the bootstrap. Evolution 39, 783–791.

Fischer, M., 1972. Hymenopetra, Braconidae (Opiinae). Tierreich 91,1–620.

Gauld, I.D., 1988. Evolutionary patterns of host utilization by ichne-umonoid parasitoids (Hymenopetra: Ichneumonidae and Braconi-dae). Biol. J. Linn. Soc. 35, 351–377.

Gibson, G.A.P., 1985. Some pro- and mesothoracic structures impor-tant for phylogenetic analysis of Hymenoptera, with a review ofterms used for the structures. Can. Entomol. 117, 1395–1443.

Gimeno, C., Belshaw, R.D., Quicke, D.L.J., 1997. Phylogenetic relation-ships of the Alysiinae/Opiinae (Hymenoptera: Braconidae) and theutility of cytochrome b, 16S and 28S D2 rRNA. Insect Mol. Biol. 6(3), 273–284.

Hillis, D.M., Moritz, C., Mable, B.K., 1996. Molecular Systematic, sec-ond ed. Sinauer, Sunderland, MA.

Huelsenbeck, J.P., Ronquist, F., 2001. Mr BAYES: Bayesian inferenceof phylogenetic trees. Bioinformatics 17, 754–755.

Huelsenbeck, J.P., Ronquist, F., Nielsen, R., Bollback, J.P., 2001.Bayesian inference of phylogeny and its impact on evolutionarybiology. Science 294, 2310–2314.

Kambhampati, S., Voelkl, W., Mackauer, M., 2000. Phylogenetic rela-tionships among genera of Aphidiinae (Hymenoptera: Braconidae)based on DNA sequence of the mitochondrial 16S rRNA gene.Syst. Entomol. 25 (4), 437–445.

Li, F.-f., Chen, X.-x., Piao, M.-h., He, J.-h., Ma, Y., 2003. Phylogeneticrelationships of the Euphorinae (Hymenoptera: Braconidae) basedon the D2 variable region of 28S ribosomal RNA. Entomotaxono-mia 25 (3), 217–226.

Michel-Salzat, A., WhitWeld, J.B., 2004. Preliminary evolutionary rela-tionships within the parasitoid wasp genus Cotesia (Hymenoptera:

http://dx.doi.org/10.1016/j.ympev.2005.03.035




Braconidae: Micorgastrinae): combined analysis of four molecularmarkers. Syst. Entomol. 29 (3), 371–382.

Mori, M., Shaw, M., Quicke, D.L.J., Unpublished. Molecular phylog-eny of rogadine braconid genus Aleiodes (Insecta, Hymenoptera,Braconidae): biogeography and host relations.

Morrison, D.A., Ellis, J.T., 1997. EVects of nucleotide sequence align-ment on phylogeny estimation: a case study of 18S rDNA of api-complexa. Mol. Biol. Evol. 14 (4), 428–441.

Mardulyn, P., WhitWeld, J.B., 1999. Phylogenetic signal in the COI, 16S,and 28S genes for inferring relationships among genera of Micro-gastrinae (Hymenoptera: Braconidae): evidence of a high diversiW-cation rate in this group of parasitoids. Mol. Phylogenet. Evol. 12(3), 282–294.

Quicke, D.L.J., van Achterberg, C., 1990. Phylogeny of the subfamiliesof the family Braconidae (Hymenoptera: Ichneumonoidea). Zool.Verh. Leiden 258, 1–95.

Quicke, D.L.J., Ingram, S.N., Baillie, H.S., Gaiten, P.V., 1992. Spermstructure and ultrastructure in the Hymenoptera (Insecta). Zool.Scrip. 21, 381–402.

Remson, J.R., DeSalle, R., 1998. Character congruence of multipledatasets and the origin of Hawaiian Drosophilidae. Mol. Phyloge-net. Evol. 9, 225–235.

Sanchis, A., Latorre, A., Gonzalez-Candelas, F., Michelena, J.M., 2000.An 18S rDNA-based molecular phylogeny of aphidiinae (Hyme-noptera: braconidae). Mol. Phylogenet. Evol. 14 (2), 180–194.

Sharkey, M.J., Wahl, D.B., 1992. Cladistics of the Ichneumonoidea(Hymenoptera). J. Hymenop. Res. 1, 15–24.

Shi, M., Chen, X.-x., 2005. Molecular diVerentiation of the microgas-trine species commonly found in paddy Welds from Southeast Asia,with additional data on their phylogeny (Hymenoptera: Braconi-dae). Insect Sci. 12(3), in press.

SwoVord, D.L., 2001. PAUP*: Phylogenetic Analysis Using Parsimony(¤ and Other Methods). Sinauer Associates, Sunderland, MA.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins,D.G., 1997. The ClustalX windows interface: Xexible strategies formultiple sequence alignment aided by quality analysis tools.Nucleic Acids Res. 24, 4876–4882.

Tobias, V.I., 1967. A review of the classiWcation, phylogeny and evolu-tion of the family Braconidae (Hym.). Ent. Obozr. 46, 645–699[Russian; transl.: Ent. Rev. 46: 387–399].

Wharton, R.A., 1993. Bionomics of the Braconidae. Annu. Rev. Ent.38, 121–143.

Wharton, R.A., Shaw, S.R., Sharkey, M.J., Wahl, D.B., Woolley, J.B.,WhitWeld, J.B., Marsh, P.M., Johnson, J.W., 1992. Phylogeny of thesubfamilies of the family Braconidae (Hymenoptera: Ichneumonoi-dea): a reassessment. Cladistics 8, 199–235.

WhitWeld, J.B., 1992. The polyphyletic origin of endoparasitism in thecyclostome lineages of Braconidae (Hymenoptera). Syst. Entomol.17, 273–286.

WhitWeld, J.B., 1997. Molecular and morphological data suggest a sin-gle origin of the polydnaviruses among Braconid wasps. Naturwis-senschaften 84 (11), 502–507.

WhitWeld, J.B., Mardulyn, P., Austin, A.D., Dowton, M., 2002. Phyloge-netic relationships among microgasrinae braconid wasp generabased on data from the 16S, COI and 28S genes and morphology.Syst. Entomol. 27, 1–24.

Will, K.W., RubinoV, D., 2004. Myth of the molecule: DNA barcodesfor species cannot replace morphology for identiWcation and classi-Wcation. Cladistics 20 (1), 47–55.

Yang, Z., Rannala, B., 1997. Bayesian phylogenetic inference usingDNA sequences: a Markov chain Monte Carlo method. Mol. Biol.Evol. 14, 717–724.

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