Host specificity in ACG Microgastrinae. (A) Frequency histogram illustrating the number of host species of caterpillars used by each provisional species of wasp. (B) The number of provisional species of wasp using barcodes that are contained within each morphospecies of wasp. (C) CO1 NJ tree for 8 provisional Apanteles wasp species that are morphologically Apanteles leucostigmus (species 17 through 28). Accessions for the wasp and host species imaged are detailed in SI Appendix, Section 2. The 3 cases marked by an asterisk indicate a wasp whose host records include more than 1 species of host, but these species are all closely related species with nearly identical ecology. (D) NJ tree for 28S for same species presented in C and using a consensus sequence for each species (the number of identical sequences used is in parentheses).

NJ tree (K2P) with 1 representative for each of the 53 provisional barcode species encountered in the sample of Cotesia. Branches are labeled with provisional wasp species name, host species (sample size in parentheses), and host family. Colored circles on the tree link species-pairs or -triplets that would not have been differentiated with confidence using only morphology (See SI Appendix, Section 8). Red text flags a species whose host-breadth suggests that it contains further cryptic species that may have been missed by CO1 barcoding; however, no variation in 28S or ITS1 supported this hypothesis, and therefore we treat it as 1 provisional species. (A) Representative cocoon structure and usual size for parasitoid and host caterpillar (See SI Appendix, Section 4). (B) Cotesia Whitfield01 hosts (Top) and Cotesia Whitfield02 host (Bottom) are from different families that feed on the same species of plant in the same way, yet their barcodes differ by only 1 bp. Despite the extremely small barcode variation, there are polymorphisms within 28S at 3 loci that agree with host family, barcode, and morphology in supporting the hypothesis of 2 species. Numbers refer to the positions of polymorphic loci when aligned with the complete Drosophila melanogaster rRNA gene (M21017). (C) Species complex of Cotesia parasitizing Opsiphanes and having very similar but distinct CO1 barcodes (SI Appendix, Section 1). In this example, the combination of barcode, host, and 28S variation support the hypothesis that there are least 4 species of wasp. Within Cotesia Whitfield77, there are 28S polymorphisms that co-vary with host use and are suggestive of a species-pair that may have been missed by CO1 barcoding alone (as in Aphelinus, 28). Triangles represent polymorphic 28S loci. Base-pair composition is color-coded, and the upper left triangle is the dominant allele. See text for further explanation and SI Appendix, Section 4 for specimen and sequence accessions for individuals sequenced and pictured here.