Corresponding author: Adrien Perrard (
Academic editor: W. Pulawski
The only previous comprehensive phylogenetic analysis of the 22 species of the genus
The genus
The first cladistic study of
Archer’s study was based mostly on male characters, which are known to be reliable phylogenetic characters (e. g.
This study presents results from an ongoing project on the evolution of vespine wasps, focusing on the genus
Phylogeny of the genus
Phylogenetic relationships were inferred for the 22 species of the genus
The morphological matrix was scored from specimens in the following natural history collections: American Museum of Natural History, Muséum National d’Histoire Naturelle, Nationaal Natuurhistorisch Museum and United States National Museum of Natural History. Specimens for molecular study were collected by J.K. and J.M.C. and preserved in 95 - 99% ethanol.
DNA was extracted from one leg and one antenna per specimen using QIAGEN “DNeasy tissue Kit”. Genes were amplified using PCR with PuReTaq Ready-To-Go beads in a total volume of 25µL including primers (Appendix 1) and DNA. Amplification cycles were specific to genes (Appendix 2). AMPures and CleanSEQ procedures were used for DNA purification and sequencing was performed on ABI PRISM 3730xl machines by Agencourt Biosciences (Beverly, USA). One missing gene fragment of
The analyses are based on 45 morphological characters and multiple nuclear and mitochondrial loci, comprising: 374 sites of 12S, 528 sites of 16S, 2231 sites of 28S (sequenced in 4 fragments), 1442 sites of CO1 (sequenced in 2 fragments), 880 sites of elongation factor 1α (EF1α), and 328 sites of H3. Each of these genes was aligned separately using the MAFFT software with the L-INS-i algorithm (
Phylogenetic analyses were performed in a parsimony framework using TNT (
Analysis of the 45 morphological characters (Appendix 3) resulted in a single most parsimonious tree (not shown; Length: 107, Consistency Index (CI): 0.617, Retention Index (RI): 0.784). The support tree is shown in
Of the 27 studied species, specimens relatively recently collected were available for 17 species (including the outgroups). Due to low quality DNA templates and the use of non-specific primers, molecular data are not homogeneous across the genus. The monophyly of the genus
Support tree for relationships among the 22
Results of phylogenetic analyses of molecular data for the genus
|
|
|
|
|
|
|
|
|
12S | 5 | 261 | 0.709 | 0.651 | yes | yes | yes | - |
16S | 2 | 332 | 0.654 | 0.545 | yes | yes | yes | yes |
28S | >1000 | 145 | 0.909 | 0.750 | no | yes | no | no |
CO1 | 1 | 1431 | 0.508 | 0.369 | yes | yes | yes | - |
EF1a | 1 | 219 | 0.886 | 0.819 | yes | yes | yes | - |
H3 | 1 | 81 | 0.889 | 0.690 | no | yes | - | - |
Multi-locus | 207 | 2494 | 0.620 | 0.468 | yes | yes | yes | yes |
Support tree for the relationships among 13
The combined analysis of morphological and molecular data returned eight equally parsimonious trees all showing
The monophyly of the genus is supported by five synapomorphies: the long prestigma, the developed vertex, the strongly elevated interantennal space, the presence of a carina on the hindcoxa, and the projection at the apex of the digitus in males.
The addition of molecular data to the morphological matrix resulted in stronger support of the
In the combined analysis tree, most of the clades of
Within the main
The two species of the
Support tree for the relationships within the genus
Our analyses of both morphological and molecular characters confirm the monophyly of the genus
While our molecular sample is incomplete, it nonetheless confirms the monophyly of two of Archer’s species groups within
Our results are also consistent with previous authors regarding the close relationships of
Archer’s finding of a main clade of
Our extended morphological matrix and the molecular sequences partly support Archer’s results, and this analysis confirms that male characters such as the shape of the last metasomal sterna and the genitalia are reliable phylogenetic characters in
We thank B. Baliff, and M. Cunningham from the University of Vermont and F. Jacquet from the Muséum National d’Histoire Naturelle for assistance with obtaining the molecular data. This work benefited from NSF grant DEB-0843505. The first author benefited from an Annette Kade Fellowship from the American Museum of Natural History.
Primers used for sequencing the six genes.
|
|
|
|
12S | F | 12S AI | AAACTAGGATTAGATACCCTATTAT |
12S | R | 12S BI | AAGAGCGACGGGCGATGTGT |
16S | F | 16S A | CGCCTGTTTATCAAAAACAT |
16S | R | 16S B | CTCCGGTTTGAACTCAGATCA |
28S-1 | F | 28S 1A | CCCSCGTAAYTTAGGCATAT |
28S-1 | R | 28S 4 BR | CCTTGGTCCGTGTTTCAAGAC |
28S-2 | F | 28S 3.2a | AGTACGTGAAACCGTTCASGGGT |
28S-2 | R | 28S Br | TCGGAAGGAACCAGCTACTA |
28S-3 | F | 28S 4a | GGAGTCTAGCATGTGYGCAAGTC |
28S-3 | R | 28S 5b | CCACAGCGCCGATTCTGCTTACC |
28S-4 | F | 28S 4.8a | ACCTATTCTCAAACTTTAAATGG |
28S-4 | R | 28S 7b1 | GACTTCCCTTACCTACAT |
CO1-1 | F | LCO | GGTCAACAAATCATAAAGATATTGG |
CO1-1 | R | HCO out out | GTAAATATATGRTGDGCTC |
CO1-2 | F | Jerry | CAACATTTATTTTGATTTTTTGG |
CO1-2 | R | Pat | TCCAATGCACTAATCTGCCATATTA |
EF1α | F | HaF2For | GGGYAAAGGWTCCTTCAARTATGC |
EF1α | R | F2Rev1 | AATCAGCAGCACCTTTAGGTGG |
H3 | F | H3-AF | ATGGCTCGTACCAAGCAGACVGC |
H3 | R | H3-AR | GTCACYATYATGCCYAAGGATAT |
PCR program of each marker with temperature and time (°C – minute). Den. = Denaturing phase. Anneal. = Annealing phase. Elong. = Elongation phase. N = number of cycles of Denaturing + Annealing + Elongation phases.
Marker | Den. | Anneal. | Elong. | N |
---|---|---|---|---|
12S | 97 – 0.5 | 42 – 0.75 | 68 – 0.5 | 40 |
16S | 94 – 0.5 | 42 – 0.5 | 72 – 0.5 | 40 |
28S-1 | 94 – 1 | 43.5 – 0.5 | 72 – 1 | 40 |
28S-2 | 94 – 1 | 43.5 – 0.5 | 72 – 1 | 40 |
28S-3 | 94 – 1 | 43.5 – 0.5 | 72 – 1 | 40 |
28S-4 | 94 – 1 | 40 – 0.5 | 72 – 1 | 40 |
CO1-1 | 94 – 0.5 | 36 / 51 – 0.5 | 72 – 0.5 | 5 / 35 |
CO1-2 | 94 – 0.5 | 36 / 48 – 1 | 72 – 1 | 5 / 35 |
EF1α | 94 – 1 | 54 – 1 | 72 – 1.5 | 35 |
H3 | 94 – 0.4 | 51 – 0.5 | 72 – 0.75 | 40 |
Matrix of morphological characters. Outgroup species are in grey.
|
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 4 | 4 | 4 | 4 | 4 | 4 |
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 0 | 1 | 2 | 3 | 4 | 5 | |
|
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
|
1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 2 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 1 | 0 | 1 | 0 | 2 | 0 | 5 | 0 | 2 | 0 | 0 | 0 |
|
1 | 1 | 1 | 1 | 1 | 1 | 2 | 0 | 0 | 0 | 1 | 1 | 2 | 0 | 0 | 0 | 0 | 1 | 1 | 3 | - | - | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 6 | 0 | 1 | 0 | 0 | 0 |
|
1 | 1 | 1 | 1 | 1 | 1 | 2 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | - | 0 | 2 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 7 | 1 | 1 | 0 | 0 | 0 |
|
1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | - | 0 | 2 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 4 | 0 | 1 | 0 | 0 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 1 | 1 | 0/1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 0 | 2 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 3 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 1 | 0/1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 0 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 0 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 0 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 2 | 0 | 1 | 1 | 1 | 0 | 1 | 1 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 0 | 1 | 2 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 2 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 3 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 0/1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 1 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 2 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 1 | 0 | 3 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 2 | 0 | 1 | 1 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 1 | 1 | 1 | 1 | 1 | 2 | 1 | 1 | 1 | 0 | 0 | 1 | 0 | 1 | 1 | 1 | 0 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 2 | 1 | 1 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
|
2 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0 | 0 | 3 | 1 | 1 | 0 | 1 | 0 | 0 | 1 | 0 | 0 | 1 | 1 | 1 | 0 | 0 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 1 | 1 | 1 | 0 |
Morphological codes
(F / M): character restricted to Females / Males
1. Prestigma length: (0) prestigma shorter than pterostigma, (1) prestigma longer than pterostigma, (2) prestigma length 3X pterostigma length.
2. Base of second submarginal cell: (0) M obliquely oriented with respect to m-cu1, (1) M vertically oriented (angle at M noticeable).
3. Placement of forewing m-cu2: (0) close to r-m2, (1) far from r-m2.
4. Hamuli placement: (0) beginning basad of fork of R1&Rs, (1) beginning at fork R1&Rs.
5. Hindwing jugal lobe: (0) present, (1) absent.
6. Hindwing axillary incision: (0) present, (1) absent.
7. (M) Tyloides: (0) two on apical flagellomeres, (1) one on apical flagellomeres, (2) absent.
8. Vertex length: (0) ocelloccipital distance short, < length of ocellar triangle, (1) ocelloccipital distance long, > length of ocellar triangle, (2) ocelloccipital distance long, gena produced behind eye.
9. Vertex indentation: (0) absent, (1) present.
10. Ocelli diameter: (0) less than distance between posterior ocellus and eye, (1) greater than this distance.
11. Interantennal space: (0) broad, rounded, (1) defined triangular area, (2) strongly elevated, with rounded edges, (3) defined triangular area strongly elevated, with sharp edges.
12. Clypeus dorsum: (0) straight, (1) bisinuate.
13. (F) Apex of clypeus: (0) pointed, (1) shallowly emarginated, anterior angles blunt, broad, (2) shallowly emarginated, anterior angles triangular.
14. (F) Mesal clypeal tooth: (0) absent, (1) present.
15. (F) Clypeal punctures: (0) sparse, superficial mesally, (1) dense mesally.
16. (M) Clypeal-eye contact: (0) touching, (1) gap.
17. (F) Malar space: (0) short, (1) long, > length of penultimate flagellomere.
18. Mandibular teeth: (0) pointed, (1) with elongate cutting edge, twice length of apical part.
19. Labial palpus third segment: (0) with strong seta, (1) without this seta, but with hairs.
20. Pronotal carina: (0) present, (1) dorsally reduced, (2) lateral remnants, (3) absent.
21. Pronotal carina dorsally: (0) largely transverse before scutum, (1) deeply U-shaped before scutum.
22. Pronotal carina laterally: (0) little interrupted by the pronotal fovea, (1) widely interrupted by the pronotal fovea.
23. Pretegular carina: (0) complete, (1) ventrally effaced, (2) absent.
24. (F) Pronotal striae: (0) absent, (1) few ventral striae, (2) dense ventral and dorsal striae.
25. Scutal lamella: (0) present, (1) absent.
26. Scutal punctures: (0) dense micropunctures, (1) superficial, (2) dense and deep micropunctures.
27. Epicnemial carina: (0) present, (1) absent.
28. Dorsal groove: (0) present, (1) absent.
29. Scutellum profile in lateral view: (0) bulging, (1) largely flat.
30. Metanotal orientation: (0) partly vertical, (1) largely vertical (dorsal surface reduced).
31. Metanotal lobe: (0) absent, (1) posteromedial lobe present.
32. Metapleural sculpture: (0) striae, (1) superficial punctures ventrally, (2) well-defined punctures ventrally.
33. Hind coxa carina: (0) absent, (1) present.
34. Metasomal segment I: (0) rounded in lateral view, (1) sharply angled between anterior and dorsal faces.
35. Metasomal segment I length: (0) short, (1) long.
36. Metasomal tergum II lateral macropunctures: (0) superficial to sparse, (1) dense, well defined.
37. (M) Apical margin of metasomal sternum VI: (0) almost straight, (1) shallowly emarginated, (2) deeply emarginated.
38. (M) Apical margin of metqasomal sternum VII: (0) convex, (1) shallowly emarginated, (2) deeply emarginated.
39. (M) Median process of metasomal sternum VII: (0) absent, (1) present.
40. (M) Aedeagal apex: (0) little differentiated, (1) transverse, projecting laterally, (2) rounded with subapical processes, (3) spade-shaped, (4) elongate, (5) narrow, (6) subcircular, (7) triangular.
41. (M) Aedeagal apical lobes: (0) absent, (1) apex forming expanded lobes.
42. (M) Aedeagus width: (0) narrow throughout, (1) as wide or wider apically as medially, (2) narrower apically than medially.
43. (M) Aedeagal shaft: (0) non-bulbous, (1) bulbous.
44. (M) Digital apical processes: (0) absent, (1) present.
45. (M) Inner apical margin of paramere: (0) obtusely angled with aedeagus, (1) forming right angle to aedeagus.