Evolutionary relationships of courtship songs in the parasitic wasp genus, Cotesia (Hymenoptera: Braconidae)

Abstract

Acoustic signals play an important role in premating isolation based on sexual selection within many taxa. Many male parasitic wasps produce characteristic courtship songs used by females in mate selection. In Cotesia (Hymenoptera: Braconidae: Microgastrinae), courtship songs are generated by wing fanning with repetitive pulses in stereotypical patterns. Our objectives were to sample the diversity of courtship songs within Cotesia and to identify e underlying patterns of differentiation. We compared songs among 12 of ca. 80 Cotesia species in North America, including ten species that have not been recorded previously. For Cotesia congregata, we compared songs of wasps originating from six different host-foodplant sources, two of which are considered incipient species. Songs of emergent males from wild caterpillar hosts in five different families were recorded, and pattern, frequency, and duration of song elements analyzed. Principal component analysis converted the seven elements characterized into four uncorrelated components used in a hierarchical cluster analysis and grouped species by similarity of song structure. Species songs varied significantly in duration of repeating pulse and buzz elements and/or in fundamental frequency. Cluster analysis resolved similar species groups in agreement with the most recent molecular phylogeny for Cotesia spp., indicating the potential for using courtship songs as a predictor of genetic relatedness. Courtship song analysis may aid in identifying closely related cryptic species that overlap spatially, and provide insight into the evolution of this highly diverse and agriculturally important taxon.

Fig 1. Courtship songs of Cotesia species divided into acoustic elements based on relative amplitude and position for use in analysis and comparisons.

Represented are typical waveform segments of (A) C. congregata and (B) C. flavichonchae.

Fig 2. Waveforms and spectrograms of typical courtship songs of twelve species of Cotesia.

Species are labeled by the first two letters of the species name.

Fig 3. (A) Mean (± SE) duration of interpulse interval divided into pause (bottom), pulse (middle), and terminal buzz (top) elements and (B) fundamental frequency for four Cotesia species with similar patterns of song structure in the “rubecula” phylogenetic group.

Letters indicate significant differences (ANOVA followed by Tukey’s post-hoc test, p < 0.05). Species with similar time elements differ in frequency.

Fig 4. Scatterplot of fundamental frequency (Hz) of adjacent pulse and terminal buzz elements in courtship songs across ten Cotesia species and six host-foodplant sources of C. congregata.

Pulses and buzzes were generally correlated in song sections containing both elements (all species, linear regression: r² = 0.65, p < 0.0001; Pulse = -31.0 + 1.1*TBuzz).

Fig 5. Scatterplots of factors of a principal components analysis of seven Cotesia courtship song elements.

(A) PC1 vs. PC2, (B) PC1 vs PC3, (C) PC3 vs PC2. Each data point represents a single individual coded by species. (D) 3-dimensional plot of the mean PC of each species color-coded by dominant courtship song pattern (red—rapid pulses; green—pulse-buzz; blue—long pauses; orange—long buzz-pulse). Letter codes indicate species name. Cotesia congregata is represented by the MsT host-foodplant complex. Some species are separated from the others by one or more PCs; most species form a close cluster with some overlap.

Fig 6. Hierarchical clustering using the first four principal components of seven courtship song elements to group Cotesia species.

Each species is represented by a 2-second waveform section.

Fig 7. Cotesia courtship songs of representative species overlaid on a maximum likelihood tree from the analysis of four genes [44].

Recorded species are in ovals and colors match those in the dendrogram produced by hierarchical cluster analysis using principal components of song elements. Cotesia diacrisiae has been added as a sister species of C. empretiae based on analysis of the NADH1 gene. Nodes have bootstrap values (above, 100 replicates) and Baysian posterior probabilities (below). Non-Cotesia outgroups have been removed for clarity (rooted with Chelonus inanitus). Courtship song grouping generally follows the genetic phylogeny with the exception of C. rubecula. The molecular phylogeny was used and modified with permission of J. Whitfield, University of Illinois, USA.

Fig 8. Scatterplots of principal components resulting from principal components analysis of courtship song elements produced by six host-foodplant complex sources of Cotesia congregata.

(A) PC1 vs. PC2, (B) PC1 vs PC3, (C) PC3 vs PC2. Songs from different host-plant complex sources cannot be reliable distinguished from each other.

Fig 9. Scatterplots of principal components resulting from principal components analysis of courtship song elements produced by MsT and CcC host-foodplant complex sources of Cotesia congregata.

(A) PC1 vs. PC2, (B) PC1 vs PC3, (C) PC3 vs PC2. MsT wasps originated from two sources in Virginia and Indiana. Songs from different host-plant complex sources or different locations cannot be reliably distinguished from each other.

Fig 10. Scatterplots of principal components resulting from principal components analysis of courtship song elements produced by Cotesia nr. phobetri originating in Arizona and Virginia.

(A) PC1 vs. PC2, (B) PC1 vs PC3, (C) PC3 vs PC2. Songs from these two locations can be reliably distinguished from each other based on PC1 (active song element durations), but not PC2 or PC3 (frequency and pause duration). Mean pulse-buzz duration is 0.13 s longer in songs of wasps from Arizona vs. Virginia (t₁₃ = -8.6, p < 0.0001).