Morphological variations in external genitalia do not explain the interspecific reproductive isolation in Nasonia species complex (Hymenoptera: Pteromalidae)

Why I highlight this preprint

Prezygotic isolation is an important barrier that ensures species preservation and prevents hybridization with other species. It describes all behavioral and functional morphological characteristics of a species that serve to prevent successful copulation between two species in advance.
A cryptic species complex refers to very closely related, yet distinct species that even experienced specialists can often not readily differentiate from one another. In these species, as for ecample a cryptic group of parasitoid wasps termed Nasonia, speciation events are often relatively recent and species boundaries are more unstable.
I found this preprinted study, which investigates the morphological and behavioral causes of prezygotic isolation observed in four species of genus Nasonia, despite the occurrence of hybridization, interesting from an evolutionary perspective. In insects, specifically, I would expect the morphological compatibility of the reproductive organs to be significant in the context of prezygotic isolation. This preprint points out that this does not appear to be the case for Nasonia species, despite particularly thorough and methodologically complex morphological and statistical analyses. I consider this very noteworthy from both an evolutionary and a behavioral biology point of view.

Background

Nasonia (Pteromalidae) is a genus of ectoparasitic small wasps. Four similar-looking species (Fig. 1) are currently known, most of them native to North America: N. vitripennis, N. longicornis, N. giraulti, and N. oneida. The females lay their eggs in the pupae of flesh flies and blowflies after injecting a venom into the pupa. The Nasonia larvae consume the pupa from its outside within the puparium (Fig. 2). This life cycle makes these wasp species a potentially suitable biocontrol agent against so-called pest flies.

In the past, multidisciplinary studies have been performed looking at the Nasonia complex, for example focused on host-parasite interactions, but also on speciation, e.g. by S. R. Bordenstein et al. (2001), who found indications for a bacterial Wolbachia-induced incompatibility between two species of Nasonia, thus contributing to postmating isolation.
In order to better understand prezygotic isolation in four species of genus Nasonia, Babita Rahul Baisla and colleagues set out to determine whether the male reproductive organs are suitable as distinguishing characteristics and thus may explain prezygotic isolation mechanisms. In addition, they conducted behavioral studies to find further or alternative evidence for prezygotic isolation mechanisms.

Main methods

The authors studied the four species of genus Nasonia under controlled temperature and humidity and reared them repeatedly due to the short life cycle durations of about 14 days. As host they used Sarcophaga dux (Diptera: Sarcophagidae) pupae. For morphological studies they prepared virgin females and males of a defined age of 12 hours, which was important to avoid morphological variation based on aging processes.
The aedeagus length was measured using a specialized Fiji software, while the aedeagus aperture lengthwas determined using scanning electron microscopy. To measure sperm sizes, the authors dissected the seminal vesicles and fixed sperm individuals.
Mating acceptance within the same species and between partners of different species was observed in virgin males and females for a defined duration. Statistical calculations were performed using the Kruskal-Wallis and the pairwise Dunn’s post hoc tests.

Main results

• The morphological structure of the male genital organ was found to be strikingly similar in all four species: two testes with a single lobe structure were located beneath the digestive system. Round accessory glands lay behind the seminal vesicles. The two vas deferens then merged to form the common ejaculatory duct.
• The external components of the male genital system, namely the aedeagus itself as the copulatory organ, was also deceptively similar. They include the ejaculatory duct and three pairs of cuticular sclerites, the digitus, volsellae, and parameres. Also the number of spines on the parameres was not found to be a suitable criterion for distinguishing species, as it can vary even within a single species.
• The aedeagus length of N. vitripennis and N. oneida was statistically the same. The lengths of N. longicornis (longest aedeagus) and N. giraulti (shortest aedeagus) were significantly different. The aedeagus aperture length was statistically similar in N. vitripennis and N. longicornis, but both were significantly different from N. giraulti and N. oneida. Additionally, the aperture showed a variation within species but also between species.
•The teeth-like spines on each digitus or the sclerite on either side of the aedeagus varied only in N. giraulti (3-5 spines). In some specimens, the spine number also varied between the two sclerites. All other species always had 4 spines.
•The length of sperm cells varied in the four species. But N. longicornis had the longest sperm cells, which statistically differed from all other species.
•In behavioral tests in which a male and a female were placed together, the complete mating behaviour happend significantly most often between conspecifics. In interspecific mating pairs, N. longicornis and N. giraulti females accepted statistically more heterospecific males than N. vitripennis and N. oneida females. But the authors found no statistical correlation between female acceptance and the morphological parameters of aegeagal length and aperture length.

Future perspectives

Since the correlation between aedeagus morphology and the inhibition of the mating procedure with interspecific partners remained unclear, the authors suggest chemisensory or acoustic signals as putatively crucial for prezygotic isolation. A theory that will need to be tested in future studies.
In their discussion chapter, the authors discuss possible functions of the spines. Future research may provide more insight into whether the spines play a more significant role in stabilizing prezygotic isolation.
The ultrastructure of the aedeagus and the aedeagal aperture showed variation in the four species. The authors suggest these found parameters to be used in future studies to, for example, determine local variations in populations of different geographic distribution.

Questions to the authors

• How do different species interact in their natural range? Are there ecological limitations that prevent partners of two species from encountering each other frequently enough to experience a mating stimulus?
• S. R. Bordenstein et al. (2001) report postmating islation in Nasonia giraulti and N. longicornis due to cytoplasmic incompatibility caused by Wolbachia bacteria. Do the authors know whether this postmating effect as hybridization barrier also concerns the two other Nasonia species? The study above also found hybrids between N. giraulti and N. longicornis as being completely viable and fertile in the F1 and F2 generations. Is this also true for N. vitripennis and N. oneida? And can fertile hybrids compete equally with purebred competitors in mate selection?
•The authors determined that there is no correlation between the morphology of the male genital tract, the mating success and the female choice. However, the rounded proportions of the male genitals represent novel findings, but appear to be similar in all species. Is this rounded structure of single genital compartments a striking new innovation of the stem species of Nasonia, or are these genital proportions not unusual within the higher-level taxon Pteromalinae? If the former is true, is there any idea what selection pressure might have favored these special Nasonia male genital proportions?
•Is the function of the spines on the external parts of the male genitalia – in species that show no variability in numbers – possibly different in a mechanical context, for example regarding their elasticity conditions, so that different functions during the mating procedure would still be imaginable?

Reference

Bordenstein, S. R., O’Hara, F. P., Werren, J.H. (2001) Wolbachia-induced incompatibility precedes other hybrid incompatibilities in Nasonia. Nature, 409(6821), pp. 707–710. Available at: https://doi.org/10.1038/246170a0.

Tags: aedeagus, behavior, diptera, entomology, evolution, genital morphology, mating, morphology, nasonia, prezygotic isolation, species complex, zoology

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