Conceived and designed the experiments: JMH JBM. Performed the experiments: JMH JBM RAB DH JM AC J-YR PJ. Analyzed the data: JMH JBM. Wrote the paper: JMH JBM. Designed voucher database: JM.
The authors have declared that no competing interests exist.
Chalcidoidea (Hymenoptera) are extremely diverse with more than 23,000 species described and over 500,000 species estimated to exist. This is the first comprehensive phylogenetic analysis of the superfamily based on a molecular analysis of 18S and 28S ribosomal gene regions for 19 families, 72 subfamilies, 343 genera and 649 species. The 56 outgroups are comprised of Ceraphronoidea and most proctotrupomorph families, including Mymarommatidae. Data alignment and the impact of ambiguous regions are explored using a secondary structure analysis and automated (MAFFT) alignments of the core and pairing regions and regions of ambiguous alignment. Both likelihood and parsimony approaches are used to analyze the data. Overall there is no impact of alignment method, and few but substantial differences between likelihood and parsimony approaches. Monophyly of Chalcidoidea and a sister group relationship between Mymaridae and the remaining Chalcidoidea is strongly supported in all analyses. Either Mymarommatoidea or Diaprioidea are the sister group of Chalcidoidea depending on the analysis. Likelihood analyses place Rotoitidae as the sister group of the remaining Chalcidoidea after Mymaridae, whereas parsimony nests them within Chalcidoidea. Some traditional family groups are supported as monophyletic (Agaonidae, Eucharitidae, Encyrtidae, Eulophidae, Leucospidae, Mymaridae, Ormyridae, Signiphoridae, Tanaostigmatidae and Trichogrammatidae). Several other families are paraphyletic (Perilampidae) or polyphyletic (Aphelinidae, Chalcididae, Eupelmidae, Eurytomidae, Pteromalidae, Tetracampidae and Torymidae). Evolutionary scenarios discussed for Chalcidoidea include the evolution of phytophagy, egg parasitism, sternorrhynchan parasitism, hypermetamorphic development and heteronomy.
Chalcidoidea (Hymenoptera) are minute wasps that generally range in size from 1-4 mm, with the smallest only 0.11 mm and the largest up to 45 mm. With an estimated diversity of up to 500,000 morphologically distinct species and an even larger number of cryptic species possible
Despite their importance, our understanding of their taxonomy and evolutionary relationships is clearly wanting. Partly because of their small size, they are difficult to collect and study, and only about 23,000 species have been described
Chalcidoidea and their proposed sister group Mymarommatoidea first appear in mid Cretaceous amber deposits (Mymaridae)
Synapomorphies uniting most of the members of Chalcidoidea include an exposed prepectus, positioning of the mesothoracic spiracle on the lateral margin of the mesoscutum, wing venation reduced to submarginal, marginal, stigmal, and postmarginal veins, and the presence of multiporous plate sensilla on one or more of the antennal flagellomeres
Both morphological and molecular evidence place Mymaridae as the sister group of the rest of Chalcidoidea
Herein we present the first comprehensive phylogenetic analysis of relationships within the Chalcidoidea using 18S rDNA and the 28S rDNA D2–D5 expansion regions sampled across 722 taxa. The diversity of the superfamily is addressed by the inclusion of 72 subfamilies and 343 genera. Data were aligned according to a secondary structural model, which allows for the unambiguous partitioning of data into conserved regions and regions of ambiguous alignment
Sequences were obtained for 722 taxa, with 56 outgroups and 666 ingroups (
The majority of taxa were sequenced and vouchered at the University of California Riverside (UCR). Additional sequences were provided by co-authors (AC and JYR: Agaonidae and some Pteromalidae; PJ: Torymidae), the HymAToL project (various outgroup taxa), Matt Yoder (NC State University; various outgroup taxa), and Andy Austin (University of Adelaide; various outgroup taxa). See
Genomic DNA extraction at UCR followed a modified version of the Chelex® protocol
Primer Name | Primer Sequence | Reference |
28S D2-3551 F | 5′ - |
|
28S D3-4046 F | 5′ - GAC |
|
28S D2-4057 R | 5′ - TCA |
|
28S D3-4413 R | 5′ - TCG |
|
28S D5-4625 R | 5′ - |
|
18Sa-1 F | 5′ - TAC |
|
18Sb-441 F | 5′ |
|
18Sa-591 R | 5′- G |
|
18Si-673 F | 5′- |
|
18Si-905 R | 5′- |
|
18Sc-1204 F | 5′ - AT |
|
18Sb-1299 R | 5′ |
|
18Sc-1991 R | 5′ - GAT |
28S primers are named for the relative structural position of the primer (next expansion region in direction of primer), for 18S and 28S their complementary 5′ start position in
Sequences were manually aligned using secondary structure models following Deans et al.
Two important aspects of the dataset led us to compare the results obtained with various alignment strategies. First, we are confident of the alignment in the conserved stem-based and core regions; however vagaries of the secondary structure model lead to some local alignments that might not be optimal based on exact pairing of compensatory base changes. Second, distribution and size of RAAs are variable across Chalcidoidea. For such a large matrix, by-eye alignment of these highly-variable ambiguous regions from distantly related taxa is hard to justify. However, these RAAs can be locally informative
The core secondary structure-derived (SS) submatrix was created by manually removing regions of ambiguous alignment (RAAs), leaving only the structurally aligned helices, core regions, and conserved blocks. As alluded to previously, not all loops are ‘highly variable’ and conserved non-pairing regions, including some loops found in the core, were retained in the SS submatrix.
The second submatrix (RAAs) included the regions of ambiguous alignment
Sixteen datasets were constructed from these submatrices (
dataset | core/stem | RAA | length | inform. | uninfo. | 18Sa | 18Sb | 18Sc | 28S | 28S | 28S | RAxML | No. of steps |
alignment | alignment | D2 | D3 | D4-5 | best score | SSME data | |||||||
SSNR | SS | no RAA | 2996 | 853 | 356 | 500 | 757 | 633 | 591 | 333 | 182 | -85277.62 | 32461 |
SSGE | SS | guide tree+E-INS-i | 4369 | 1675 | 566 | 507 | 969 | 701 | 1302 | 519 | 371 | -144234.60 | 32236 |
SSGL | SS | guide tree+L-INS-i | 4369 | 1676 | 565 | 507 | 969 | 701 | 1302 | 519 | 371 | -144255.37 | 32223 |
SSGG | SS | guide tree+G-INS-i | 4536 | 1773 | 550 | 507 | 963 | 697 | 1451 | 531 | 387 | -144123.77 | 32220 |
SSME | SS | no guide+E-INS-i | 3917 | 1408 | 483 | 506 | 906 | 693 | 993 | 450 | 369 | -150220.93 | 31951 |
SSML | SS | no guide+L-INS-i | 3917 | 1408 | 487 | 506 | 906 | 693 | 993 | 450 | 369 | -150223.77 | 31957 |
SSMG | SS | no guide+G-INS-i | 3906 | 1433 | 468 | 506 | 906 | 694 | 1023 | 450 | 327 | -147954.87 | 31951 |
MENR | E-INS-i | no RAA | 3024 | 861 | 375 | 507 | 758 | 634 | 605 | 337 | 183 | -85889.86 | 32522 |
MLNR | L-INS-i | no RAA | 3024 | 861 | 374 | 507 | 758 | 634 | 605 | 337 | 183 | -85852.51 | 32483 |
MGNR | G-INS-i | no RAA | 3025 | 859 | 380 | 507 | 758 | 634 | 606 | 337 | 183 | -85953.75 | 32527 |
MEME | E-INS-i | no guide+E-INS-i | 3944 | 1415 | 502 | 513 | 907 | 694 | 1007 | 453 | 370 | -150774.64 | 32247 |
MLML | L-INS-i | no guide+L-INS-i | 3944 | 1415 | 501 | 513 | 907 | 694 | 1007 | 453 | 370 | -150775.39 | 32236 |
MGMG | G-INS-i | no guide+G-INS-i | 3934 | 1438 | 492 | 513 | 907 | 695 | 1038 | 453 | 328 | -148553.26 | 32254 |
MESR | E-INS-i (all data by partition) | 4133 | 1536 | 553 | 506 | 901 | 693 | 1196 | 531 | 306 | -145056.78 | 31983 | |
MLSR | L-INS-i (all data by partition) | 4099 | 1507 | 545 | 506 | 901 | 693 | 1162 | 531 | 306 | -145084.06 | 32187 | |
MGSR | G-INS-i (all data by partition) | 4139 | 1519 | 551 | 506 | 901 | 694 | 1201 | 531 | 306 | -145293.59 | 31997 |
The guide tree was generated from a RAxML analysis of the SSNR dataset (no RAA). Except for the all data alignments (no submatrix partition), each of the 55 RAA blocks were aligned independently and reinserted into the appropriate gene partition for analysis. E-INS-i, G-INS-i and L-LINS-i are MAFFT alignment options. The RAxML best score was obtained from 10 independent runs using CIPRES v.2.0. The number of informative and uninformative sites and parsimony steps were calculated in PAUP 4.0* for each resulting tree using the SSME dataset.
Automated alignments were performed with MAFFT
The RAAs were aligned both with and without a guide tree that was generated using the SSNR (core with no RAA) dataset. Our purpose for using a guide tree was to optimize local alignments for each of the RAAs within terminal clusters of independently recognized taxa grouped through analysis of the SSNR, thus aligning nearest neighbors, as opposed to aligning disparate taxa across the entire dataset without any prior grouping. Maximum likelihood (ML) analyses of this dataset were conducted with RAxML v.7.2.7 using a partitioned GTR+Γ model
The secondary structure-derived matrix with MAFFT-aligned RAA regions (SSME) is deposited on Texas A&M's Parasitic Hymenoptera Research Labs' jRNA Secondary Structure and its Phylogenetic Implications website (available through
Sequences were partitioned into six gene regions 18Sa, 18Sb, 18Sc, D2, D3, and D4+D5, with each partition including their respective aligned RAA regions. The 18Sa-c partitions were defined simply as the region sequenced, inclusive of the primers used. The 28S rDNA expansion regions are also contiguous, being bounded on either side by core sequence, which was amplified in the PCR reaction. The decision as to where to define the end of D2 and start of D3 and likewise, the end of D3 and start of D4+D5, was arbitrarily made to fall within the core regions between the expansion regions. The helix H1a' (UUUCAGG), was assigned to mark the end of D2; while the un-named, non-pairing block of sequence (AC), which follows helix H1a' and proceeds helix H563 (CCGU) marked the start of D3. Helix H812 (CCCUCC) was assigned to mark the end of D3, while the un-named, non-pairing block of sequence (GAAG), which follows helix H812 and precedes helix H822 (UUUCC), marks the start of D4+D5.
Maximum Likelihood (ML) analyses and associated bootstrapping (BS) were conducted on the 16 datasets with RAxML v.7.2.7 using a partitioned GTR+Γ model
The parsimony analysis of the SSME dataset was conducted with TNT v.1.1
To be consistent with our interpretation of bootstrap percentage (BP), we use the following scale: a bootstrap percentage of ≥90% is considered very strong, 80–89% means strong, 70–79% means moderate, and 50–70% means low bootstrap support.
To better track relationships, each taxon includes a prefix which is an abbreviation of it family-group (c.f.
core only | core and RAA | RAxML | TNT | ||||||||||
Code | Taxonomy | gen | spp | SSNR | MENR | SSGE | SSME | MGMG | MGSR | MJR* | SSME | ||
AG | 19 | 104 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 97 | |||
AGA | ‘Agaoninae’ |
12 | 48 | – | – | – | – | – | – | – | – | ||
AG4 | ‘Agaonidae group 4′ | 2 | 3 | – | 70 | 75 | 86 | 92 | 75 | – | |||
AGB | ‘Blastophaginae’ | 3 | 24 | – | – | – | – | – | – | – | – | ||
AGK | Kradibiinae | 2 | 25 | – | – | – | – | – | – | – | |||
AGT | Tetrapusinae | 1 | 4 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
AP | 21 | 87 | – | – | – | – | – | – | – | – | |||
API | Aphelinidae |
4 | 4 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
APA | Aphelininae | 7 | 22 | 88 |
88 |
97 |
96 |
91 |
86 |
100 |
56 |
||
APAY | Aphytini | 3 | 12 | 53 | + | ||||||||
APZ | Azotinae | 1 | 12 | 99 | 100 | 100 | 100 | 100 | 100 | 100 | 99 | ||
APC | Coccophaginae | 6 | 43 | + | + | 81 | + | + | + | 94 | – | ||
APCP | Pteroptricini | 5 | 31 | par | par | par | par | par | par | par | – | ||
APE | Eretmocerinae | 1 | 5 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
APR | Euryischiinae | 2 | 2 | 100 | 100 | 100 | 89 | 100 | 100 | 100 | 100 | ||
CAL | 1 | 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | |||
CH | 20 | 37 | – | – | – | – | – | – | – | – | |||
CHC | Chalcidinae | 8 | 19 | – | – | – | – | – | – | – | – | ||
CHCB | Brachymeriini | 1 | 6 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
CHCC | Chalcidini | 2 | 8 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
CHCR | Cratocentrini | 3 | 3 | – | – | – | – | – | – | – | – | ||
CHCP | Phasgonophorini | 2 | 2 | 98 | 100 | 100 | 100 | 100 | 99 | 100 | 100 | ||
CHD | Dirhininae | 1 | 5 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
CHE | Epitranininae | 1 | 3 | + | 90 | 99 | 95 | 94 | 98 | 100 | 56 | ||
CHH | Haltichellinae | 8 | 12 | 88 | 90 | 100 | 98 | 98 | 97 | 100 | + | ||
CHHA | Haltichellini | 5 | 9 | + | + | + | – | 56 | + | – | |||
CHHY | Hybothoracini | 3 | 3 | 93 | – | ||||||||
CHS | Smicromorphinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
EN | 12 | 14 | + | 50 | 81 | 72 | 73 | 78 | 100 | + | |||
ENE | Encyrtinae | 8 | 9 | + | 72 | + | 89 | + | |||||
ENT | Tetracneminae | 4 | 5 | 72 | 69 | 87 | 77 | 97 | par | 65 | + | ||
EU | 22 | 46 | 100 |
100 |
100 |
100 |
100 |
100 |
100 |
100 |
|||
EUE | Eucharitinae | 16 | 27 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 96 | ||
EUG | Gollumiellinae | 2 | 3 | 80 | 93 | 98 | 76 | 86 | 99 | 100 | par | ||
EUO | Oraseminae | 4 | 16 | par | + | 71 | + | + | + | 75 | + | ||
EL | 27 | 28 | 89 |
92 |
99 |
98 |
97 |
98 |
100 |
+ |
|||
ELI | Eulophidae |
1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
ELE | Entedoninae | 8 | 8 | – | + | 50 | + | 74 | 59 | 88 | + | ||
ELN | Entiinae | 5 | 6 | – | – | 67 | + | 58 | 81 | + | |||
ELU | Eulophinae | 9 | 10 | 66 | + | 96 | 95 | 91 | 85 | 100 | – | ||
ELO | Opheliminae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
ELT | Tetrastichinae | 3 | 3 | 98 | 98 | 100 | 100 | 100 | 100 | 100 | 99 | ||
EP | 19 | 25 | – | – | – | – | – | – | – | – | |||
EPC | Calosotinae | 5 | 7 | – | – | – | – | – | – | – | – | ||
EPE | Eupelminae | 12 | 14 | + | + | + | – | + | – | – | – | ||
EPN | Neanastatinae | 2 | 4 | – | – | – | + | – | – | – | – | ||
EY | 14 | 28 | – | – | – | – | – | – | – | – | |||
EYE | Eurytominae | 9 | 14 | 100 |
99e | 100 |
100 |
100 |
100 |
100 |
100 |
||
EYH | Heimbrinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
EYR | Rileyinae | 2 | 7 | + | + | 97 | 90 | 87 | 87 | 100 | + | ||
LEU | 2 | 6 | 98 | 90 | 100 | 100 | 98 | 98 | 100 | 98 | |||
MY | 13 | 15 | 98 | 95 | 100 | 99 | 98 | 97 | 100 | 61 | |||
MYI | Mymaridae |
1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
MYA | Alaptinae | 3 | 3 | – | – | – | – | – | – | – | – | ||
MYE | Eubronchinae | 1 | 2 | 99 | 100 | 98 | 99 | 100 | 87 | 100 | 84 | ||
MYM | Mymarinae | 8 | 9 | – | – | – | – | – | – | – | – | ||
ORM | 2 | 3 | 66 | 56 | 67 | + | 61 | 52 | 100 | + | |||
PE | 14 | 34 | + |
+ |
– | – | – | – | – | – | |||
PEI | Perilampidae |
1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PEA | Akapalinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PEM | Philomidinae | 3 | 3 | 99 | 98 | 100 | 100 | 100 | 100 | 100 | 97 | ||
PEC | Chrysolampinae | 4 | 9 | 73 | 67 | 88 | 72 | 68 | 80 | 100 | – | ||
PEP | Perilampinae | 5 | 20 | 96 | 98 | 100 | 100 | 100 | 99 | 100 | 76 | ||
PT | 111 | 130 | – | – | – | – | – | – | – | – | |||
PTI | Pteromalidae |
2 | 2 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT01 | Asaphinae | 3 | 3 | – | – | – | – | – | – | + | – | ||
PT02 | Ceinae | 1 | 2 | 93 | 93 | 100 | 98 | 98 | 99 | 100 | 98 | ||
PT03 | Cerocephalinae | 3 | 3 | 99 | 99 | 100 | 100 | 100 | 100 | 100 | 100 | ||
PT04 | Chromeurytominae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT05 | Cleonyminae | 10 | 10 | – | – | – | – | – | – | – | – | ||
PT05D | Chalcedectini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT05C | Cleonymini | 3 | 3 | 68 | 56 | 84 | 54 | + | 52 | 100 | + | ||
PT05L | Lyciscini | 5 | 5 | + | + | 92 | 55 | + | + | 100 | + | ||
PT05O | Ooderini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT06 | Coelocybinae | 4 | 4 | – | – | – | – | – | – | – | – | ||
PT07 | Colotrechninae | 2 | 2 | – | – | – | – | – | – | – | – | ||
PT08 | Cratominae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT09 | Diparinae | 6 | 8 | – | – | – | – | – | – | – | – | ||
PT09D | Diparini | 4 | 4 | – | – | – | – | – | – | – | – | ||
PT09N | Neapterolelapini | 1 | 2 | 57 | 55 | 96 | 73 | 63 | + | 81 | – | ||
PT10 | Epichrysomallinae | 16 | 28 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 93 | ||
PT11 | Eunotinae | 6 | 7 | – | – | – | – | – | – | – | – | ||
PT11E | Eunotini | 4 | 5 | 52 |
75 |
90 |
86 |
93 |
98 |
100 |
61 |
||
PT11M | Moranilini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT11T | Tomocerodini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT12 | Eutrichosomatinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT13 | Herbertiinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT14 | Leptofoeninae | 2 | 3 | – | – | – | – | – | – | – | – | ||
PT15 | Macromesinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT16 | Miscogasterinae | 9 | 10 | – | – | – | – | – | – | – | – | ||
PT16M | Miscogasterini | 5 | 6 | – | – | – | – | – | – | – | – | ||
PT16S | Sphegigasterini | 2 | 2 | – | – | – | – | – | – | – | – | ||
PT16T | Trigonoderini | 2 | 2 | – | – | – | – | – | – | – | – | ||
PT17 | Ormocerinae | 6 | 5 | – | – | – | – | – | – | – | – | ||
PT17M | Melanosomellini | 3 | 3 | – | – | – | + | – | – | – | |||
PT17S | Systasini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT18 | Otitesellinae | 3 | 4 | par | – | – | – | – | – | – | – | ||
PT19 | Panstenoninae | 1 | 2 | 96 | 89 | 98 | 98 | 84 | 77 | 100 | 96 | ||
PT20 | Pireninae | 4 | 4 | – | – | – | – | – | – | – | – | ||
PT21 | Pteromalinae | 17 | 18 | – | – | – | – | – | – | – | – | ||
PT21P | Pteromalini | 4 | 4 | – | – | – | – | – | – | – | |||
PT22 | Spalangiinae | 1 | 3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
PT23 | Sycoecinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
PT24 | Sycophaginae | 5 | 6 | 82 | 94 | 91 | 81 | 77 | 91 | 100 | + | ||
PT25 | Sycoryctinae | 2 | 2 | – | – | – | – | – | – | – | – | ||
ROT | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | |||
SI | 8 | 26 | 81 | 80 | 95 | 98 | 97 | 97 | 100 | 52 | |||
SIS | Signiphorinae | 1 | 9 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 99 | ||
SIT | Thysaninae | 3 | 12 | ||||||||||
TAN | 4 | 5 | 98 |
95 |
99 |
100 |
99 |
100 |
100 |
77 |
|||
TE | 6 | 7 | – | – | – | – | – | – | – | ||||
TEM | Mongolocampinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TEP | Platynocheilinae | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TET | Tetracampinae | 4 | 5 | 100 |
100 |
100 |
100 |
100 |
100 |
100 |
97 |
||
TO | 29 | 41 | – | – | – | – | – | – | – | – | |||
TOM | Megastigminae | 3 | 6 | 66 | 67 | 99 | 99 | 97 | 97 | 100 | 92 | ||
TOT | Toryminae | 28 | 37 | – | + | 67 | + | + | 62 | 86 | + | ||
TOTI | Toryminae |
3 | 4 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TOTM | Microdonteromerini | 6 | 8 | – | – | – | |||||||
TOTN | Monodontomerini | 6 | 8 | 80 | 100 | 91 | 89 | 81 | 100 | 97 | |||
TOTP | Palachiini | 2 | 2 | – | – | – | – | – | – | – | – | ||
TOTO | Podagrionini | 4 | 4 | 57 | 90 | 55 | 62 | + | |||||
TOTT | Torymini | 3 | 6 | 75 | 74 | 66 | 87 | 68 | 66 | 100 | – | ||
TOTY | Torymoidini | 4 | 5 | – | – | – | – | – | 88 | – | |||
TR | 12 | 21 | – | + | 61 | 65 | 64 | + | 94 | + | |||
TRO | Oligositinae | 9 | 10 | 98 | 100 | 97 | 96 | 95 | 93 | 100 | + | ||
TROI | Oligositinae |
3 | 4 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TROC | Chaeotostrichini | 2 | 3 | 99 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
TROO | Oligositini | 1 | 2 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
TROP | Paracentrobiini | 1 | 1 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TRT | Trichogrammatinae | 3 | 11 | + | |||||||||
TRTI | Trichogrammatinae |
3 | 5 | n/a | n/a | n/a | n/a | n/a | n/a | n/a | n/a | ||
TRTT | Trichogrammatini | 2 | 6 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | ||
Dataset abbreviations explained in
= without Agaonidae Group 4 (
= without Azotinae or
= excluding Akapalinae and Philomidinae;
= without
= excluding
= including
= excluding
= not including
= excluding
Summaries of the 16 datasets generated from the two submatrices are presented in
A summary of supported clades across six of the 16 analyses is presented in
RAxML analysis with seed 38652 and 1000 rbs bootstrap replicates (support >50% above branches). Phylogram of entire tree on left colored to match inset. Taxon names with prefix indicating classification (see
core only | core and RAA | RAxML | TNT | |||||
Group Relationships | SSNR | MENR | SSME | SSGE | MGSR | MGMG | MJR | SSME |
− | + | − | + | − | + | 62 | − | |
Diaprioidea (part) + Chalcidoidea | − | − | + |
− | − | − | 56 | − |
‘Diapriidae’ + Chalcidoidea | + | − | − | − | − | − | − | − |
Mymarommatoidea + Chalcidoidea | − | − | − | − | + | − | − | − |
(Proctotrupoidea + Diaprioidea) sister to Chalcidoidea | − | − | − | − | − | − | − | + |
Chalcidoidea | 99 | 95 | 100 | 100 | 98 | 98 | 100 | 100 |
remaining Chalcidoidea minus Mymaridae | 91 | 55 | 97 | 95 | 55 | 85 | 94 | + |
remaining Chalcidoidea minus Rotoitidae and Mymaridae | + | + | + | 76 | + | + | 94 | − |
Mymaridae: 4−segmented taxa | 74 | 78 | 75 | 87 | 57 | 80 | 88 | + |
Mymaridae: 5-segmented taxa | + | + | 76 | 62 | 83 | + | 88 | + |
Eulophidae: (Opheliminae + |
− | − | + | + | − | − | 56 | + |
Eucharitidae + Perilampidae | − | − | + | + | + | + | − | + |
Perilampidae (with Akapalinae, Philomidinae and |
+ | + | + | + | + | − | − | |
− | − | + | + | + | + | − | + | |
− | + | − | − | − | − | − | − | |
pteromaloid complex |
+ | + | + | + | + | + | − | + |
Spalangiinae + Agaonidae | − | − | + | − | − | − | − | − |
Sycophaginae + Agaonidae | + | − | − | − | − | − | − | − |
remaining Agaonidae minus Tetrapusinae | + | 55 | + | − | − | + | − | + |
Aphelininae + Coccophaginae | + |
− | − | − | − | − | − | − |
Azotinae + Trichogrammatidae | + | + | + | − | + | + | 62 | − |
Azotinae + Signiphoridae | − | − | − | − | − | − | − | + |
Agaoninae + Blastophaginae (excluding group 4) | + | + | 65 | 61 | + | + | 62 | + |
a = Monomachidae + Diapriidae as sister groups;
b = includes Cratominae, Miscogastrinae, Otitesellinae, Panstenoninae, Pteromalinae and Sycoryctinae;
c = without
d = including
Dataset abbreviations explained in
Interestingly, the automated (MAFFT) alignments of all data were comparable in clade support to any of the divided alignment strategies based on recognizing the core and stem data. There was slightly better clade support using G-INS-i when applied to data that included RAAs.
Within 28S and 18S, distinct structural differences occur between RAA regions for the outgroups, Mymaridae, and the remaining Chalcidoidea taxa. For example, RAA(11) shows a pattern of increase in the number of bases and an associated decrease in degree of conservation for Chalcidoidea in comparison to the outgroup taxa (
In all Chalcidoidea excluding Mymaridae, RAA(15) undergoes a dramatic decrease to either 1 or no nucleotides and RAA(4) shows a slight increase in size. The bordering alignment around RAA(15) demonstrates compensatory changes in helices 3m, 3n and 3o.
Inclusion of the RAAs contributed to the monophyly of Encyrtinae, Entedoninae and Entiinae (
Relationships across the 16 ML analyses overall were the same regardless of alignment method or the inclusion or exclusion of RAAs (
Outgroup relationships generally favored a paraphyletic Diaprioidea as sister group to Chalcidoidea (
Chalcidoidea were always monophyletic with strong support, as was a sister group relationship between Mymaridae and the remaining Chalcidoidea (
Relationships within Chalcidoidea were highly variable along the backbone of the tree and should be regarded as a broad polytomy, but with consistent and sometimes strong support for many traditional taxon groupings at the family, subfamily, and tribe levels (
Overall, there was little impact of the application of different MAFFT alignments to either the RAA regions, the core secondary structure data, or to the different gene regions without reference to secondary structure. This is optimistic for the future inclusion of new taxa to our data set where we can avoid the labor-intensive approach of having to align new taxa to our existing secondary structure model. Inclusion of the RAAs contributed to monophyly and clade support for a number of taxa, and also increased support at higher levels. Furthermore, structural differences found in various RAAs (
We found either Mymarommatoidea or Diaprioidea as the sister group of Chalcidoidea. These equivocal results were similar to results from a recent analysis of Hymenoptera that used more extensive molecular data from four gene regions and nearly complete 28S and 18S data
Chalcidoidea are well supported as monophyletic. Mymaridae are strongly supported as monophyletic and the sister group of the remaining Chalcidoidea. This hypothesis was first proposed by Gibson
After Rotoitidae, the relationships within Chalcidoidea become vague. The backbone of the chalcidoid tree has little support, with taxonomic groups shifting in different analyses from the base to somewhere more apical in the topology. As well, there are few consistent sister group relationships supported among the higher-level groups. One of the few relationships that can be substantiated based on larval morphology, Eucharitidae + Perilampidae
We found some taxa that could not be placed within any traditional higher-level group. There were also a few singleton taxa that defied placement, including
For the discussions below, some historical information on relationships is presented for each family group followed by the results of the current study. A more detailed review of classification history and biology can be found in Gibson et al.
Agaoninae and Sycophaginae (as Idarninae), once included in Torymidae, were moved to Agaonidae by Bouček
Agaonidae (
None of the other subfamilies previously placed in Agaonidae were placed near to Agaonidae, although in the SSNR dataset (core only), Sycophaginae were placed as the sister group of Agaonidae but without bootstrap support.
Woolley
Our results lend support to the idea that Aphelinidae are not monophyletic (
There was no consistent or plausible sister group taxon for Aphelininae or Coccophaginae. In the majority of analyses, Euryischiinae is sister to
Calesinae were monophyletic with 100% BS support in all analyses (
Bouček and Halstead
Chalcididae were not monophyletic in any of our analyses. The MENR analysis produced the closest approximation to a monophyletic Chalcididae, with a grouping of
Our results do not offer much resolution for the relationships within Chalcididae, but do offer support for recognition of Brachymeriinae, Dirhininae, Epitraninae, Chalcidinae (as Chalcidini), Haltichellinae and Smicromorphinae. Both Phasgonophorini and Cratocentrini are less easily placed, and we could not recover the monophyly of the Cratocentrini (
The monophyly of Encyrtidae is not questioned and there is strong morphological evidence to support this family
Encyrtidae were monophyletic across all analyses, with moderate to very strong BS support from the likelihood analyses with RAAs included (
Several morphological features support the monophyly of Eucharitidae
Eucharitidae
While Eucharitinae were always very strongly supported, Oraseminae was occasionally paraphyletic to Eucharitinae. Gollumiellinae was paraphyletic only in the parsimony analysis. Monophyly of Psilocharitini (
A Eucharitidae + Perilampidae sister group was retrieved in most of the likelihood analyses that included RAAs, and also in the parsimony analysis (
Monophyly of Eulophidae generally has not been challenged, although morphological support is based almost entirely on character reduction
Eulophidae were monophyletic with strong to very strong support in all of our analyses (
The exclusion of
Importantly, there was no relationship supported for Eulophidae with any of the aphelinid subfamilies, including Calesinae, which have many similar reductive features
While there is strong morphological support for the monophyly of each of the three subfamilies of Eupelmidae, it has been proposed that the family might represent a grade rather than a clade
Eupelmidae were never monophyletic. Also, its subfamilies Calosotinae, Eupelminae, and Neanastatinae were almost never monophyletic. The SSME dataset was one of the rare instances in which Neanastatinae were monophyletic (
The monophyly of Eurytomidae was recently questioned as no synapomorphies defining the family are known
Eurytomidae was never recovered as monophyletic in any of our analyses. However, Eurytominae (excluding
Leucospidae are generally recognized as a monophyletic group of four genera closely related to Chalcididae
Leucospidae were monophyletic and had greater than 90% support across all analyses. Our one species of
Although there was some early doubt about the monophyly of Mymaridae
Mymaridae were found to be monophyletic in all analyses with very strong support (
Hanson
The two genera,
The limits of Perilampidae are not clear, with variable inclusion of the subfamilies Chrysolampinae, Philomidinae and Perilampinae, and treatment of each or all groups as a separate family or subfamily of Pteromalidae
In likelihood results, Perilampidae
Pteromalidae are essentially a dumping-ground for presumably monophyletic groups that cannot be assigned to established families and which lack family status in their own right
Pteromalidae were expected to be polyphyletic
Coelocybinae, Ormocerinae, Pireninae and Pteromalinae were never monophyletic. Cleonyminae were polyphyletic. In all analyses, Cleonymini and Lyciscini were each monophyletic with low support in all analyses, with Lyciscini gaining increased support from the inclusion of RAAs. Chalcedectini (
For Pteromalidae, our results are similar to those of Desjardins et al.
In their description of the family, Bouček and Noyes
Rotoitidae were represented by one species,
There is little doubt over the monophyly of Signiphoridae; however, Thysaninae may be paraphyletic with respect to Signiphorinae
Signiphoridae and Signiphorinae (
Signiphoridae were not placed with Azotinae, or any logical outgroup, in any of the likelihood analyses. In these analyses, Azotinae was consistently the sister group of Trichogrammatidae. However, in the parsimony analysis, Azotinae and Signiphoridae were monophyletic and did not group with Trichogrammatidae.
Tanaostigmatidae
Tanaostigmatidae
Tetracampidae probably represents a polyphyletic assemblage with three extant subfamilies
Tetracampidae were never monophyletic in our analyses. Excluding
Placement of Torymidae is uncertain, and it was proposed that the family arose from within the pteromalid lineage
Torymidae were never monophyletic, but Megastigminae and Toryminae were each monophyletic with very strong support (
Trichogrammatidae are well defined and according to Bouček and Noyes
Trichogrammatidae were monophyletic in nearly all of our analyses (94% of the MJR consensus trees), but with low BS support in likelihood analyses only after the inclusion of RAAs.
Is the diverse and unsupported backbone of Chalcidoidea the product of a rapid radiation event
Using an array of nuclear protein coding genes but with fewer taxa, Desjardins et al.
What is the ancestral mode of host association for Chalcidoidea? Bouček
Noyes
Mymaridae are virtually all egg parasitoids, primarily of Auchenorrhyncha, Heteroptera and Coleoptera
Associated with an extreme diversity of host use, larval morphology is extremely diverse in Chalcidoidea
Another important trait is the use of sessile Sternorrhyncha as hosts within Chalcidoidea, which ultimately leads to their importance in biological control programs. Mapping sternorrhynchan parasitism, either as primary parasitoids or hyperparasitoids, onto our current ‘best’ hypothesis shows a general scattering of host use that suggests multiple independent host shifts to this group. Probably most significant is the lack of grouping in any of our analyses of Encyrtidae and the aphelinid subfamilies Aphelininae, Azotinae, Coccophaginae, Eretmocerinae and Euryischiinae, which have in the past been treated as a single family
Our results present the most comprehensive phylogenetic analysis of relationships Chalcidoidea based only on molecular data.. While not robust across the backbone of relationships within Chalcidoidea, they offer some firm insights into the origin and evolution of this important and highly diverse group of insects. Monophyly of many of the traditional groups is supported, and the secondary structure alignment and data set will be useful for future studies. Many changes in the higher classification of taxa within Chalcidoidea are suggested by these results. However, we reserve any judgment on these changes until our combined morphological and molecular analyses are complete.
Parsimony analysis of SSME dataset using TNT (31,607 steps; r.i. 0.62, strict consensus of >10,000 trees). Bootstrap values plotted to nodes with values greater than 95% represented by dot.
(PDF)
Specimens sequenced and deposition information for specimen data and genebank accession numbers.
(XLS)
Chalcidoidea SSME dataset.
(NEX)
We thank Andrew Carmichael, Jan Kostecki, Andrew Ernst, Elizabeth Murray and Albert Owen for sequencing taxa. Help with imaging specimens was provided by Lisa Gonzalez, Jessica Ortiz, Christine Martinez, Maria Saleh, and Jasmine Soto. Sequence data for outgroups were provided by Andy Austin, Matt Buffington and Matt Yoder. Specimens were obtained from various sources, but in particular we would like to thank Chris Burwell, Terry Erwin, Lisa Foerster, Michael Gates, Gary Gibson, Tony van Harten, Yoshimitsu Higashiura, John Huber, Jung-Wook Kim, John LaSalle, Robert Luck, Lubomir Masner, John Pinto, Alain Roques, Mike Sharkey, Richard Stouthamer, Serguei Triapitsyn, Doug Yanega and Bob Zuparko. Help with identifications was provided by Chris Darling, Gérard Delvare, Gary Gibson, Michael Gates, John Huber and John Pinto.