Methods:
Male encounter rates and reactions to conspecifics
Starting around 10:00 hrs. on sunny days when individuals become active at Lark’s Lake in a field 150 m from the shore, we will follow males originally caught in tandem with a female, noting her color morph before releasing her. We will mark the male by writing a number on his wing, and adding a small white dot of paint to his thorax, which helps us locate him should he fly more than a few meters away from the observer. Observers follow at a distance a meter way from the male, while documenting the most extreme interaction he has with a conspecific. Interactions are scored as: 1) fly-by – the male flies within 10 cm of a conspecific without reacting to it, 2) hover from above or behind a conspecific 3) face-off, hovering in front of the conspecific, often with a bouncing up and down motion, 4) chase, which typically follows a face-off 5) grab 6) hit, sometimes knocking an individual to the ground 7) tandem attempt when the male tries to engage his claspers with a conspecific’s thorax, 8) tandem in which the male clasps the conspecific by its mesostigmal plates on the thorax, and 9) copula in which a female raises her abdomen to engage her genital opening with the male’s penis structure (wheel position). Sexual reactions (6-8 above) are those that most accurately differentiate sex and female sexual maturity (Piersanti et al., 2021).
Males will be followed for as long as possible until lost from view. Given my past experience with this method (Piersanti et al., 2021), I anticipate that at least five males per observer per day can be followed for 20 min. or more.
Statistical tests
To understand the realized landscape of female morphs encountered by focal males, while accounting for differences in male activity, we will examine partial correlations between focal male encounters with each female morph.
We will use an open source software Markovian behavioral model on line, Behatrix v 0.92, (https://github.com/olierfiard/behavtrix) only to identify the behavioral transition states of males that had at least two interactions (i.e. representing one transition) with a given conspecific type. The expected null for learned recognition is that males encountering a given morph type only once during observations are as likely to react sexually to it as males encountering that morph more than once. If males form a search image of a mature female morph, we expect him to exhibit more sexual to sexual transitions than sexual to non-sexual transitions. To test whether males learned to avoid inappropriate sexual reactions towards immature females and males, the expected null is that transitions from sexual to nonsexual behaviors (including fly-bys), are as likely as those from non-sexual to sexual responses. Results will be analyzed using likelihood ratio tests and Clopper-Pearson exact tests )
I have already followed individual females of both morphs in the Larks Lake population to measure harassment rates in the wild (Fincke 2015). On a subset of days, we will additionally do short surveys at hourly intervals to document the time frame over which receptive females are mating. This is important because in our earlier study of the European species, Ischnura elegans, we found that the opportunities that mate-searching males have to encounter receptive females is highly constrained. Thus, in retrospect, it might not have been surprising to find that sexually mature males do not learn to recognize female morphs; they lack the time to do so.