The differences in the vibrational signals between male O. bicornis from three countries in Europe

Abstract

Research into vibrational communication is becoming increasingly important as we discover more species using vibrational signals in different types of behavior. Males of the solitary red mason bee, Osmia bicornis, are known to use vibrations in female choice, where the females not only evaluate a male’s fitness through their vibrational signal but also use them to distinguish between males of different origins. This was shown conclusively via bioassays, in which females from Germany rejected English males unless they were imbued with an artificial German signal and vice versa. However, an investigation into which parameters of the signal might differ between species and populations has been lacking so far. We therefore recorded O. bicornis as well as O. cornuta males from Germany, England, and Denmark using a laser vibrometer and analyzed the recordings using the software Spike to measure frequency, modulation range, and average pulse duration in each signal. Our results clearly showed significant differences in the signal between O. bicornis and O. cornuta males in all three parameters measured. O. bicornis populations from Germany and Denmark surprisingly also differed significantly in frequency and modulation range, with England lying in between the two. We believe that the females probably use another signal parameter that we have not evaluated yet to make their mate choice. This leaves us with the question of why the observed signal parameters differ between Germany and Denmark. From our knowledge about the system to date, we believe that we are looking at the first steps of speciation in this system and further study could help us with new insights into evolutionary processes in the future.

Keywords

vibrational communication biotremology population divergence red mason bee

Introduction

Vibrational signals are widespread throughout the animal kingdom, can be found in almost every class of animals,^1,2 and are estimated to be found in 92% of the described insect species.¹ However, we have only very recently begun to investigate vibrations more thoroughly, probably due to the fact that we, as humans, mostly rely on vision and acoustic signals for communication and do not really use that sensory modality.^1–3 Therefore, most research into vibrational communication looks into those instances where there are combinations of vibrations and acoustic signals as is the case in this study. Vibrational communication may indeed be one of the most primary signaling channels even predating the ear mechanism² and is used in various different functions as in predator-prey interactions,^2,4 foraging,⁵ mating⁶ and many other functions.² Most of the signals that have been investigated to date are related to sexual behavior,³ as is the case in many Araneae,⁷ Orthoptera,⁸ Homoptera,⁹ Coleoptera,¹⁰ Diptera¹¹ and Hemiptera.¹²

In many cases, female choice is based on these vibrational signals, for example in planthoppers¹² and treehoppers.⁹ In the treehopper Enchenopa binotata, the female chooses its male based on substrate-borne vibrations. Various parts of the male signal are responsible for the female choice such as base frequency, length of the whine section, pulse rate and number of pulses.⁹ There are also many reports of intraspecific differences in these vibrational signals between populations and their influence in mate choice.^13–17 It seems clear today that vibrational signals are often species-specific and can be used for species-recognition as well as to discriminate between individuals of different populations. Slight signal differences involved in the mating process have already been recognized as a possible first-step towards reproductive isolation and can play a role in speciation,^18–22 especially when female choice leads to the selection of male traits. It is therefore very likely that vibrational communication can play a part in speciation by sexual selection.

In bees, vibrations and sound are produced through rhythmic oscillations of the thorax and the underlying muscles²³ and consequently are a form of tremulation.²⁴ Vibrations and sound can be transmitted through the substrate²³ or directly from bee to bee,^23,25–27 are used in foraging behaviour^28,29 and, unlike previously thought, in mating behavior.^25,26 Even though it is known for many bee species that they emit vibrational signals during mating, there are very few studies on the function of these vibrations.³⁰ So far research has mainly focused on odor, which plays a dominant role in insect communication and has a key function in the mating of many bee species.^{25,27,31–33}

The reproductive biology of the red mason bee O. bicornis has been intensively studied.^34,35 It is a strongly proterandric species in which the males emerge first in spring to be present in great numbers before and during the emergence of the females.³⁵ The males either wait in front of the nests or around flowers in order to mate with females.³² Since the gender ratio is shifted in favor of the males, the females choose a suitable mating partner from all the males.²⁷

During precopulatory courtship, the male embraces the female by sitting on her back and engaging in a series of behaviors in order to persuade the female to mate. The male may vibrate his thorax, rub himself against the female and pass his antennae repeatedly over those of the female and his forelegs over the female’s compound eyes.³⁵ The female may then reject the male by physically pushing him off her back. In a previous study, we were able to show that the male’s vibrations play an important role in the female’s choice. The males that vibrate the longest without breaks are the ones that the female will choose – probably because they are the healthiest.²⁷ We were also able to show that females prefer mating with males from their own country and use the vibrational signals in order to distinguish between males from different populations.²⁶ However, although this was shown conclusively in bioassays, investigations and results on the actual differences between the signals are lacking so far. This study aims to investigate the parameters of vibrational signals and discover differences between different populations and species.

Materials and methods

Study animals

Both species of mason bees, O. bicornis and the closely related Osmia cornuta, are widespread solitary bees that are common in Europe and they can easily be reared in trap nests (e.g. bamboo canes³⁵). We used O. bicornis from natural populations in Germany (Regensburg, Constance, Halle), England (Kent, Tonebridge), and Denmark (Mön, Copenhagen, Vejle) (see Figure S1 in Conrad 2015). Osmia bicornis in Central and Southern Europe is also commonly found together with its sister species, O. cornuta,³⁶ which it closely resembles in its behavior. We therefore used O. cornuta from Ulm, Germany, to serve as an interspecies out-group. Bees were collected as pupae or pharate adults in cocoons in 2008, 2009 and 2010 and emerged in separate flight cages (ca. 29 cm × 29 cm × 29 cm) the following spring. They were provided ad libitum with a 50% sugar solution of APIInvert® (Südzucker AG, Germany; 1 g citric acid and 3 g potassium sorbate were added per liter API-Invert solution) and kept at room temperature under natural light. One female at a time was introduced into the male flight cage and once a mating pair had established, the pair was taken out and further observed and recorded. Male and female bees were emerged in different flight cages and one female at a time was introduced into the male flight cage. After a bit of scramble competition, one male will establish itself on the females back and begin its precopulatory behavior. The pair can then be taken out of the cage and observed, as they hardly move during this phase. After the observations and experiments, the bees were frozen in liquid nitrogen and kept in the freezer at −20°C.

Recording of the vibrations

The thoracic vibrations produced by males during the precopulatory phase were recorded with a laser vibrometer (Polytec PDV-100, Waldbronn) connected to a computer using a 32-bit sound card and Soundforge 9.0 software (SonicFoundry) at a sampling rate of 44.1 kHz. The files were later analyzed using Spike 2 (Cambridge Electronic Design). All males had been marked with a white spot on their thorax to enhance the reflection of the laser beam. During the evaluation of the data, we compared frequency, average pulse duration and modulation ranges (Figure 1). Since measurements differed greatly in time (from ten seconds up to an hour of recordings), main frequency was acquired by setting a cursor at the end of the vibrational signal (right before copulation or rejection) and another one 10 s prior. Using a power spectrum over that time period with 16384 FFT and a Hanning window, we then identified the main frequency. For average pulse duration, we measured the length of at least 10 clearly visible pulses (within 10 s before the copulation/rejection event) and averaged the results. The modulation range was acquired by measuring the maximum height of the modulations with the 10 s window prior to the copulation/rejection event. We were able to record 215 O. bicornis males (133 from Germany, 43 from England and 39 from Denmark), as well as 39 O. cornuta males from Germany.

Figure 1.

An example of a male signal with frequency and amplitude over time as shown in Spike. We measured main frequency, modulation range (a) and the average pulse duration of at least 10 pulses (b).

Statistics

For statistical analysis of the data, we used the software SigmaStat 3.1 (Systat Software, Chicago, IL, USA) and Sigma Plot 9 (Systat Software). All data were checked for deviation from a normal distribution using a Kolmogorov–Smirnov test. Because the laser vibrometry data was not normally distributed, frequency, average pulse duration and modulation ranges of males from different populations of O. bicornis and O. cornuta were compared using Mann–Whitney U-tests with subsequent Benjamini-Hochberg corrections.

Results

Our analysis of the main frequency, modulation range and average pulse duration showed a significant difference in main frequency and modulation range between all populations of O. bicornis and O. cornuta (Figure 2).

Figure 2.

Comparison of main frequency of males from various O. bicornis populations and O. cornuta. The medians, quartiles, outliers (circles) and sample sizes (numbers) are shown. Significant differences are shown by different letters (Mann–Whitney U, Benjamini–Hochberg, P < 0.05).

O. bicornis from Germany and O. bicornis from Denmark also differed significantly in their main frequency (Figure 3), but there was no significant difference between the main frequency of English males and those of German or Danish males (Mann–Whitney U-Test, Benjamini–Hochberg P < 0.005). Additionally Danish and English males as well as Danish and German males differed significantly in their modulation range (Figure 3)(Mann–Whitney U-Test, Benjamini–Hochberg P < 0.005), but German and English males did not. The comparison of the average burst lengths showed no significant differences within the populations of O. bicornis only at the species level between all O. bicornis and O. cornuta (Figure 4) (Mann–Whitney U-Test, Benjamini–Hochberg P < 0.005). All standard deviations can be found in Table 1.

Figure 3.

Comparison of modulation ranges of males from various O. bicornis populations and O. cornuta. The medians, quartiles, outliers (circles) and sample sizes (numbers) are shown. Significant differences are shown by different letters (Mann–Whitney U, Benjamini–Hochberg, P < 0.05).

Figure 4.

Comparison of average burst lengths of males from various O. bicornis populations and O. cornuta. The medians, quartiles, outliers (circles) and sample sizes (numbers) are shown. Significant differences are shown by different letters (Mann–Whitney U, Benjamini–Hochberg, P < 0.05).

Table 1.

Standard deviations for parameters evaluated.

	Standard deviation
	Main frequency	Modulation range	Average burst length
Germany	84.285	74.454	0.086
England	88.329	97.241	0.0894
Denmark	93.968	86.501	0.0703
Osmia cornuta	62.124	63.745	0.0407

Discussion

The clear difference between vibrational signals of O. bicornis and those of O. cornuta confirm that vibrational signals not only contain information on the physical strength of a male,²⁷ but can also contain information about species affiliation and kin. This is in concordance with the result of our bioassays in which a change in the signal of English and German bees led to a change in female choice.²⁶ So far, bees were suggested to mostly use odor as a base for recognizing kin or origin.^32,37 However, our results in this study combined with our results from previous studies let us assume that vibrational signals do have an additional or even primary function to distinguish between individuals of different populations, as has already been shown in many other insects.^1,2

We also found differences in the vibrations of males from Germany and Denmark, with England lying in between the two and not differing significantly from either one. As the variance of the signals is quite high, we believe that with a much higher sample size differences between all three regions would be detectable. Regardless, these results are surprising. Based on the different subspecies described by Peters,³⁶ one would expect Germany and England to differ significantly while the bees from Denmark, which include both subspecies, should be somewhere in between England and Germany. This would also be in concordance with our previous study in which females from Germany rejected males from England and vice versa whereas males from Denmark got to mate with males from Germany and England, although at a lower rate than native males.²⁶

Our current findings could be due to “reproductive character displacement” or “reinforcement” occurring in Denmark. Populations occurring in sympatry sometimes differ more in various traits than the same populations in allopatry. This is thought to be a result of selective pressure against hybrid formation and loss or suppression of genes required for adaptation to a particular habitat.^38,39 Consequently, populations in sympatry have to avoid hybridization by diverging certain traits involved in mating.^40–43 This has already been shown in various animal species,^44–47 amongst them periodical cicadas in the USA, that show character displacement in male call pitch and female call pitch preference. Magicicada neotredecim males have a higher dominant pitch in sympatry with M. tredecim than when they occur alone. Respectively, females were more responsive to a higher pitch in sympatry, while in allopatry they prefer a lower pitch.⁴⁸

In conclusion, character displacement could explain the surprising result in the differences of vibrations of males from Germany and Denmark. That would imply, however, that all the bees from Denmark belonged to the same subspecies as the ones from England. More experiments and especially genetic analyses would be needed to prove this hypothesis.

Regardless, the results of our previous studies using bioassays clearly show that females are able to detect differences in the vibrations of males from England and Germany and do indeed use them to discriminate between different males. However, our bioassays from the previous study²⁶ showed that females distinguish between English and German males when, in this study, we found no significant difference between the vibrational signals – only a trend. This is not the only case in which animals are able to perceive differences in signals that researchers are unable to detect. There are many examples, however, few of them are published (pers. comm.). In experiments with the elm leaf beetle, the beetle can distinguish between the odors of two differently treated elms. However, the difference was undetectable for the researchers.⁴⁹

We believe that it is more likely though that the signal parameters we have analyzed so far are actually not the ones used by the female to distinguish between males of different origins and that another parameter we haven’t considered is responsible here. However, so far no other parameter (number of pulses, pattern of pulses, etc.) we have looked at has shown any results. Since female choice is a strong force for sexual selection, we would expect the part of the signal used in female choice to show strong differences between populations of England and Germany. It seems that this “simple” signal is able to encode much more than we expected.

Our current study clearly shows that populations of O. bicornis found in Germany, England, and Denmark differ in their thorax vibrations and from our previous studies we know that these differences do have a function in female choice.²⁶ Our results can be explained by an ongoing process of separation between populations based on their premating behavior. Female choice can be a strong driving force of speciation,²¹ as can be seen in the unusual species richness of cichlid fish in the great lakes of eastern Africa.⁵⁰ Another important aspect lies in the question of hybrid fitness and fertility to see if post-zygotic isolation is already in place or in the process of developing. It might very well be that a separation between the populations is already further advanced than we know, due to hybrid sterility or because the hybrids are less adapted to their microhabitat.¹⁹ From our combined knowledge about the system to date,^26,27,51 we believe that we are looking at the first steps of speciation through female choice and female preference in this system and further research could help us with new insights into evolutionary processes in the future.

Footnotes

Acknowledgements

We would like to thank everyone who has provided us with bees from different countries, namely Robin Dean, Dr Karsten Seidelmann, Dr Erhard Strohm, Sabine Rademacher, Mike Hermann, Henrik F Brødsgard, Anja Amtoft Wynns, and Adam Bates and Dr Sebastian Menke for some help with the analysis.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: We thank the German Environmental Foundation (DBU) for funding of TC.

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