Interspecific Courtship Between Two Endemic Fireflies

Abstract

Reproductive interactions between species could have negative effects on the fitness of the species involved, which can have important ecological and evolutionary consequences, such as population declines (including local extinction) or character divergence. Here, we report the courtship and attempted mating between two congeneric species of fireflies endemic to Mexico. The interactions involved males of the synchronous firefly Photinus palaciosi and females of the much larger, non-synchronous P. extensus. In the study site, the population density of P. palaciosi is much higher than that of P. extensus. Observations of marked P. extensus females throughout most of the mating season showed that 37.8% of their interactions with males were with P. palaciosi males. Although interspecific interactions were usually of shorter length, they frequently consumed a significant portion of the nightly mate-locating/courting period. These interspecific interactions are probably facilitated by the similarities in the mate location and courtship behavior of both species, which also share female brachyptery (elytra and wing reduction that makes females unable to fly). The simplest hypothesis to explain our behavioral observations is that P. palaciosi males mistakenly courted P. extensus females. The available evidence suggests that the operational sex ratio (OSR) of P. palaciosi is male-biased, as it seems to be the case in all synchronous fireflies studied to date. We hypothesize that the intense male competition for mates resulting from a male-biased OSR explains, at least in part, the “indiscriminate” sexual responses of P. palaciosi males. Another still not studied factor that could contribute to the frequent interspecific sexual interactions observed is the degree of similitude of the mating signals. The relatively high frequency of interspecific interactions and the significant amount of time invested in many of them (relative to the duration of the nightly mating period) indicate that the study of the potential fitness costs (and benefits?) of these interactions is a promising line of research.

1. Introduction

Reproductive interactions (e.g., courtship and copulation) between members of different species have been observed in several taxa [1,2]. Their potential evolutionary consequences depend on the sign and magnitude of their effects on the fitness of the interacting species. In some cases, the effects could be negligible, for example when the interactions occur very rarely. In other cases, one of the species is selected to induce these interactions against the interests of the other species, as is the case of predatory female Photuris fireflies that respond to the mating signals of the males of their firefly prey species to attract them and feed on them. When these interactions have a negative fitness effect on one or more of the interacting species, the interaction is called reproductive interference [1,2]. Reproductive interference is a potentially powerful selective pressure that can affect the evolution of mating signals and genitalia and promote isolation mechanisms, but it could also result in hybridization [1,2,3,4,5]. Stanger-Hall and Lloyd [6] performed a phylogeny-controlled comparative study of the mating signals of 34 North American Photinus firefly species, finding that reproductive character displacement explained male flash duration divergence among sympatric species and suggesting that reproductive interference has been an important selective pressure in this insect group. However, we were not able to find published reports of naturally occurring interspecific sexual interactions between firefly species other than those induced by the aggressive mimicry of the predatory Photuris spp. females [7].

The existence of synchronous fireflies is an intriguing problem for scientists [8,9,10] and a fascinating phenomenon for non-scientists [11,12]. This magnificent spectacle is produced by thousands of mostly male fireflies signaling synchronously to prospective mates during a relatively short period of time every night. Why these fireflies congregate in such large numbers and why they synchronize their luminous signals is not understood for any particular species, although there are hypotheses on the mechanisms responsible for the synchronization and on the potential reproductive advantages, e.g., [7,8,10]. Evidence indicates that the operational sex ratio (OSR: the number of sexually receptive females divided by the number of sexually active males) is male-biased in synchronous fireflies of North America and Asia, and this kind of bias is known to produce strong competition for mates between males [13,14]. This bias is present in synchronous fireflies of North America and Asia despite their behavioral differences: in North America, males mostly signal in flight while females remain perched, while in most (but not all [8]) Asian species, the males signal while perching on trees where females are also perched but do not participate in the synchronous flashing. We are aware of direct measurements of OSRs for only three synchronic species from Asia, and the values observed in each species were 60%, 70% and 74% of males in mating “congregations” (data summarized in [8]). Direct evidence of strong competition for females, most likely resulting from a male-biased OSR, is the formation of “mating clusters”, groups consisting of one female and two or more courting males [15]. In these clusters, the males walk or fly very close to the female, frequently sending signals, and in many cases, they are in physical contact with the female and with each other. Copeland et al.’s [16] description of male behavior in clusters of the synchronous P. carolinus clearly indicates the competitive character of this behavior: “males…vigorously mount each other or the courting female or both. Piles of grappling males formed and engaged in extensive grappling, walking, attempted coupling”. Faust [9], p. 216 also mentions that “Direct interference competition among males appears intense in P. carolinus, and the mating clusters described [for this species] are similar to…[the] “love knots” described in P. pyralis” (P. pyralis is considered a synchronic species by Copeland et al. [16]). Copeland et al. [16], p. 20, also mention that mating males in “stage I” (this stage is when the male, at the beginning of copulation, is on top of and looking in the same direction as the female; stage II is when the copulating pair is in the tail-to-tail position, looking in more or less opposite directions) can be displaced by other males. Copeland et al. [16], p. 111 say that cluster formation is “only regularly found” in synchronous fireflies in North America (they mention four more Photinus species including P. carolinus) and in Southeast Asia. Wing et al. [15], p.89, mention that the copulation of Pteroptyx valida, a synchronous firefly from Thailand, “occurs amidst high densities of intensely competing males” and suggest that the male mating “clamp” formed by the terminal part of the abdomen and the posterior tip of the elytra could have evolved to “prevent take-over of the female by other males during copulation”.

The very large numbers of males competing for the attention of the relatively scant receptive females makes synchronous fireflies a potential problem for the non-synchronous, much less abundant, sympatric firefly species. The male-biased OSR observed in synchronous fireflies results in intense competition for mates that could select for decreased male choosiness and increased willingness to approach, court, and attempt mating with any signaling female (or even male) firefly including heterospecific individuals [1,2,17]. The similitude of the mating signals of the interacting species could also affect the occurrence and frequency of interspecific sexual interactions [17]. This situation could result in mistaken approaches and courtship or mating attempts with females of other sympatric firefly species that also employ bioluminescent signals. If these interactions have a negative effect on female and/or male fitness, we have a case of reproductive interference [1,2,4,5], a potentially powerful selective pressure that could affect the evolution of bioluminescent signals and other elements of the mating sequence [18]. However, as mentioned above, we could not find reports of naturally occurring interspecific sexual interactions between firefly species, with the exception of those induced by the aggressive mimicry of Photuris spp. females [7]. Lloyd [17] explored the possibility of interspecific sexual interactions between Photinus species of the USA by employing an experimental approach; we consider this work in the discussion.

In this paper, we report the results of an observational field study of the interactions between males of the synchronous firefly Photinus palaciosi Zaragoza-Caballero attempting to court and mate with females of the much larger, and much less abundant, non-synchronous P. extensus Gorham (Figure 1A,B), two endemic species of Central Mexico [19,20]. Our investigation was inspired by the similarities in the mate location and courtship behavior (together, the mating sequence) of both species (although we have not yet studied their luminous signals), which also share female brachyptery (elytra and wing reduction that makes females unable to fly), and by three serendipitous observations of this type of interaction in a locality where both species coexist (see Section 3.1).

2. Materials and Methods

2.1. Fireflies’ Natural History

We identified the fireflies with the keys provided by Zaragoza-Caballero et al. [21] for the fireflies of Central Mexico. Our study was made in the pine–oak forest of a private ecotourism ranch (“Rancho del Valle”) in the village of Santiago Cuahutenco, municipality of Amecameca, Estado de México, Mexico. We made voice notes, filmed videos and took photographs of the interactions with cell phone cameras. To minimize the risk of perturbing the fireflies, the brightness of the cell phones was set to the minimum and we made efforts to conceal the screen from the fireflies with our own bodies. Fireflies are a tourist attraction in the study area, although no population estimates have been made. However, our non-quantitative observations made during several reproductive seasons led us to guess that at the peak of the P. palaciosi reproductive season, there are several hundreds of individuals signaling per night, while P. extensus is much less abundant, with probably a few dozen per night. Although we do not have quantitative estimates, it was obvious that, as expected for a synchronous firefly (see Section 1), the operational sex ratio (the ratio of sexually active males to sexually receptive females) of P. palaciosi was male-biased. Flying, signaling males are easy to observe and, although females are harder to detect, after several field seasons, we have developed a good “search image” for signaling females.

The mating season of the two species starts at slightly different times in June. Normally, P. palaciosi adults are first observed the first half of June and finished by the end of July (although in some years, a few males have been observed in August), whereas P. extensus is observed one or two weeks after the first P. palaciosi and finished approximately by the third week of July. However, in 2023, the rains started very late and the mating seasons of both firefly species were delayed (see Section 2.2). Our previous observations indicated that both species are protandrous. The courtship activity of P. extensus is mostly observed in areas of low vegetation, such as lawns, near the facilities of “Rancho del Valle”, whereas P. palaciosi tends to prefer forested areas. However, despite these differences, there is considerable spatial overlap between these two species in their courting areas. The nightly mating period of P. extensus starts at twilight, about 19:45 h, and finishes at about 21:00 h, while that of P. palaciosi starts approximately at 20:15 in the night and finishes at about 21:45 h; in both species, it is possible to find some isolated signaling males and females later in the night. The males and females of P. extensus are substantially larger than those of P. palaciosi (

Table 1. Body size ranges of females and males of P. extensus and P. palaciosi. The area covered by the body in dorsal view is used as an estimate of body size.

	Photinus palaciosi a	Photinus extensus b
Females
Body area (mm2)	11.7–40.7 N = 93	35.5–132 N = 14
Males
Body area (mm2)	19.7–53.6 N = 113	59.6–85.3 N = 70

^a The area covered by the body of P. palaciosi was taken from López-Palafox et al. [21] and does not include the area covered by the head, so the differences with P. extensus are somewhat smaller. ^b The area covered by the body of P. extensus is an approximation obtained by multiplying body length by body width (data taken from Zurita-García et al. [22]) and by assuming that the firefly with the smallest body length also had the smallest body width and that the firefly with the largest body length also had the largest body width.

; Figure 1A), and the females of both species have reduced wings and elytra (i.e., they are brachypterous; Figure 1A); the male genitalia are, accordingly, much smaller in P. palaciosi (Figure 2).

2.2. Field Study

Inspired by the preliminary observations made in 2021 and 2022 and described in the first section of our Results, in 2023, we performed a systematic field study of the interactions between P. extensus females and P. extensus and P. palaciosi males. In 2023, the rains started late and the mating seasons of both firefly species were delayed. The first P. extensus males were observed on 28 June. We started looking for P. extensus females on 30 June (on this day, we observed a male P. palaciosi apparently courting a male P. extensus; see below) and finished our observations on 23 July, when there were very few females and males of both species and after several nights without observations of interspecific interactions. On 4 July, we observed the first female P. extensus (she was mating); the first interaction between a P. extensus female and a P. palaciosi male occurred on 10 , and the last interspecific interaction was observed on 19 July.

During the observation period, between two and four observers looked for P. extensus females that were perched on the ground or on vegetation at heights ≤ 1.2 m (usually ≤30 cm). We detected females when they were sending bioluminescent signals or when they were in copula (although during copulation, no light is emitted by the pair; with practice, the lantern can be detected if it is at least partially exposed). When an observer detected a female, they made focal observations of the females during the rest of the mating period. Observers sometimes observed more than one female simultaneously, but once a female started to interact with one male the observer remained observing this female until her interaction finished. Each observer remained in the dark at about one meter from the female(s) and registered her/their interactions with males in voice notes in a cell phone; we also made videos of several interactions. Once the nightly observation period finished, the females were individually marked with a code of white dots of corrector on the reduced elytra (Kores Aqua™), painted with a metal pin.

The recorded activities corresponded to most of the mating sequence [18], which is roughly similar in both species and consists of the following behaviors: (1) Communication in flight: the flying male and the perched female exchange bioluminescent signals (hereafter, signals); the male/female emits a signal, the female/male answers with a signal, and, in turn, the male/female responds. (2) Approaching: the male flies toward the female, sometimes still exchanging signals. (3) Landing: the male lands on the ground or vegetation relatively close to the female (usually ≤50 cm). (4) Communication on the ground: the male standing on the ground (or vegetation) and the perched female exchange signals; the male walks toward the female while exchanging signals; at some point, the female can stop sending signals, but the male is still able to locate her. (5) Tapping: once the male contacts the female, he starts tapping her body with his antennae and forelegs and soon climbs on the dorsal or ventral side of the female and walks over her body while continuing his tapping. (6) Mounting: the male stands on the dorsum of the female, with the tip of his abdomen near the tip of her abdomen and his head pointing in the same direction as that of the female, as if preparing to try to copulate; tapping with antennae and legs continues during this phase; no signals are produced by either the male or the female during this phase. (7) Mating attempt: the male everts his aedeagus and touches the tip of the posterior end of the female with it, giving the impression of trying to find the female genitalia; tapping with antennae and legs continues during this phase; no signals are produced by either the male or the female during this phase; the female could twist up the tip of her abdomen towards the male, a behavior suggesting that the female is consenting to mate. (8) Copulation (Figure 1B): the intromission of the aedeagus in the female genitalia is accomplished while tapping gradually diminishes until the pair remains motionless. No signals are produced by either the male or the female from phase 5 on.

3. Results

3.1. Preliminary Observations

Our preliminary observations were made in 2021 and 2022 while studying the interaction between P. palaciosi males and the predatory females of the Photuris lugubris Gorham firefly [23,24]. All observations started when the interactions were in progress, with the fireflies already in physical contact, and finished before the end of the interaction. The date, hour and approximate amount of time we observed each interaction was: Observation #1 (Ob#1): 29 June 2021, 21:00 h, 20 min; Ob#2: 6 July 2022, 21:11 h, 25 min; and Ob#3: 11 July 2022, 21:51 h, 30 min. We did not observe light emission during these interactions. In one case (Ob#2), there were two males in contact with the female; one male was on the ventral side of the female’s abdomen, near the posterior end (as in Figure 1C), and the other was on the same leaf where the female was perched, tapping her left elytrum with his antennae and first pair of legs (this male separated from the female at the beginning of our observation and remained on the leaf for some time before leaving). In the other two cases, the male was on the back of the female, in one case near the posterior end of the abdomen (Ob#1; Figure 1C) and in the other case on the reduced female elytra (Ob#3; Figure 1D). In all cases, the male tapped the body of the female with his antennae and his first pair of legs. In Ob#2, there was a mating attempt. We did not observe any female behavior suggesting attempts to repel the male or escape from him. The females remained motionless most of the time, with occasional movements of the antennae. In one case (Ob#1), the female twisted up the tip of her abdomen towards the heterospecific male, as they do when accepting copulation with a male of their own species.

3.2. Field Study of Interspecific Sexual Interactions

In 7 of the 10 nights of our field study, we observed interspecific interactions. We studied 22 P. extensus individually marked females; 10 of them were observed for more than one night (between two and seven). We observed 74 interactions with conspecific males and 45 with P. palaciosi males, i.e., 37.8% of the observed interactions were interspecific and involved at least one of the behaviors of the mating sequence described in the Materials and Methods section. The percentage of interactions that started with an exchange of signals with a flying male (communication in flight) was 29.7% (22/74) in the intraspecific case and 35.6% (16/45) when the male was P. palaciosi (Figure 3); in other cases, for example, the male approached a female without signaling just after she emitted a glow or the male approached a group of two or more males that were signaling to a female that was not emitting signals. Both intra- and interspecific interactions finished at different moments of the mating sequence, but interactions with heterospecific males tended to finish earlier in the sequence (Chi square = 40.8, df = 7, p < 0.0001; Figure 3).

The duration of most intra- and interspecific interactions was less than 20 min (70.6% and 73.3% of intra- and interspecific interactions, respectively), and the interactions that lasted more than one hour (15.6%) were exclusively with P. extensus males (Figure 4). Pooling the durations of interactions in three categories 1–19, 20–59 and ≥60 min, there was a significant between-species difference in the distribution of durations (Chi square = 9.1, df = 2, p < 0.01). Five out of the eight interactions that lasted more than 80 min corresponded to intraspecific copulations (median copula duration was 101 min; minimum–maximum: 58–166, N = 6). We observed one possible interspecific copula (included in Figure 3 and Figure 4) in which the couple exhibited behavior and a position that strongly suggested copulation and that lasted 40 min. However, we did not confirm if the male had introduced his aedeagus into the female.

Between 30 June and 19 July, we observed five perched P. extensus males (since males were not marked, we cannot be sure they were different, but they were observed on different days and in different places) being approached at different times by 14 P. palaciosi males. The behavior exhibited by the P. palaciosi males looked similar to the behavior shown when approaching (con- and heterospecific) females. In fact, in three of these cases, the P. palaciosi male managed to climb on the heterospecific male while tapping his body with their antennae. In contrast to the females of their own species, P. extensus males tried to move away from the approaching P. palaciosi males. None of the P. extensus males were observed emitting signals; in fact, we never observed perched P. extensus males emitting signals.

4. Discussion

To the best of our knowledge, this is the first report of spontaneous interspecific courtship and mating attempts in fireflies under natural conditions. Of course, predatory females of many Photuris species rely on sexual deception to attract male prey from other firefly species [7], but in the case we report here, males and females emitted and/or responded to signals produced to obtain mates. Lloyd [17] explored the possibility of interspecific sexual interactions between Photinus species of the USA (he did not include the synchronous P. carolinus) with an experimental approach by exposing, in “glass cages” or petri dishes, females of one species in places where males of a different species were actively looking for females. Lloyd tested ten different pairs of species using 12 different species. Sample sizes were usually small (in six species pairs, the number of females tested was six or less), although in two cases, he tested “numerous”/“several” females. In four species pairs, Lloyd [17] observed interactions similar to courtship, and in three of them, there was physical contact between males and females, including one copula between the only tested female of P. granulatus and a P. tenuicinctus male. It is interesting that the case that resulted in copulation involved species with brachypterous females, whereas in two species pairs that interacted but did not attempt copulation, the females of only one of the species were brachypterous. The overlap of mating sites also did not explain the patterns: although five species pairs in which no interactions were observed were sympatric, two of the four species pairs in which males and females interacted were also sympatric. Differences between signals do not explain the results because in eight pairs of species, the signals of each species were different. However, it is interesting that the case in which a copula was observed involved allopatric species with similar signals. In our opinion, Lloyd’s [17] experimental observations of interactions similar to courtship in four out of the ten tested species pairs (including one interspecific copulation) suggest that interspecific courtship in nature is probably more common than we know. Furthermore, we suggest that this could be especially true when one of the involved species is a synchronous firefly, as explained below.

We think that the simplest explanation for our behavioral observations is that P. palaciosi males mistakenly courted P. extensus females. These females were sexually receptive because they were actively emitting luminous signals during the mating period. We suggest that the P. extensus females mistakenly responded to the bioluminescent signals of P. palaciosi, which, in turn, mistakenly interpreted these responses as produced by females of their own species and courted the heterospecific females. The males of P. palaciosi, as all synchronous fireflies, experience intense competition for females as a result of the typically large number of males looking for females every night (see [15,16]). Although we have not measured the operational sex ratio (OSR: the number of sexually receptive females divided by the number of sexually active males) in P. palaciosi, our experience in the studied population, as well as in other populations of Central Mexico, indicates that the OSR is biased, probably heavily, towards males, which could increase the intensity of male competition for mates. As discussed in the Introduction, male-biased OSRs are probably typical of synchronous fireflies. This intense competition for females could have selected for males with enhanced willingness to approach, court, and attempt mating with any firefly roughly resembling a female of their own species [1,2], and this could explain the male mistakes reported in this paper. Furthermore, factors such as a preference for large females could facilitate even more of this type of mistake. Although a previous study failed to detect an effect of female size on the probability of mating in P. palaciosi, that study was correlative and based on the natural variation in female size observed in the field [21]. Thus, we cannot discard the possibility that the large females of P. extensus (and their signals) are a supernormal stimulus [25] for the mate searching males of P. palaciosi. The observations that some P. palaciosi males approached and courted perched males of P. extensus provides further support to the idea that P. palaciosi males are somewhat indiscriminate in their responses. What is more difficult to explain is the behavior of P. extensus females. Their behavior during interactions was similar to that exhibited when interacting with conspecific males. If the females do not pay any fitness cost from these interactions, no discriminatory behavior is expected to evolve. However, there are reasons to think that these interactions are costly for them.

We do not know for certain if the interspecific courtship between P. palaciosi males and P. extensus females results in reproductive interference because we have no data on the effects of these interactions on the fitness of males and females of P. palaciosi and P. extensus. However, our observations led us to suggest that it is possible that the fitness of P. extensus females is negatively affected, even though their “passivity” does not suggest a large investment of energy in these interactions. The large disparity in the density of mate-locating males of both species and the possibly male-biased OSR of P. palaciosi are, very probably, responsible for the high frequency of interspecific courtship interactions [1,2,4,5]. Considering that the P. extensus females courted by P. palaciosi males stop signaling and responding to their own males and that a significant proportion of the interspecific interactions encompasses most of the nightly P. extensus mate location and courtship period (Figure 4), it is possible that the females courted by heterospecific males will mate days later than other females or, in the worst case, fail to find a mate, which could result in a fitness decrease. Additionally, we have evidence that P. extensus females are polyandrous (of the 10 females observed more than one night, two mated two times and one three times) and that males transfer spermatophores during copulation (AV, YM and CC, unpublished observations). Since spermatophores have been shown to contain valuable resources for females beyond sperm [26,27,28,29], interspecific courtship could reduce the amount of spermatophore resources obtained by the females. Furthermore, possible negative effects of interspecific courtship on future intraspecific courtships, such as the impaired receptivity observed in female Nasonia longicornis wasps courted by males of a N. vitripennis [30], must also be investigated. In the case of P. palaciosi males, they obviously spent more energy in the interspecific interactions than P. extensus females. However, we think that due to the male-biased OSR, many (probably most) male P. palaciosi do not mate in any given night, thus reducing the negative impact of these interactions on male fitness. Of course, this is a hypothesis that also needs to be tested. On the other hand, although the benefits of interspecific sexual interactions seem unlikely in this case, until experimental investigations are conducted, we cannot discard the possibility. Two hypothetical benefits (without any empirical support) help us to illustrate this possibility: females courted by heterospecific males could improve their mate choice abilities or males courting heterospecific females could improve their courting ability. Although these potential benefits could sound far-fetched, examples of unexpected benefits of other kinds of non-reproductive sexual behaviors (see, for example, ref. [31] for a reproductive benefit of receiving homosexual courtship when immature for male Drosophila melanogaster) suggest we should not hurry in discarding ideas without experimental tests.

If our suspicions about the costs involved are true, reproductive interference is acting as a selective pressure on the fireflies we studied. The consequences of this interaction could affect the evolution of the sexual communication systems (i.e., the signals and responses) of both species [6] and even have an impact on the diversity of fireflies. For example, if the negative effects on P. extensus are strong, the populations interacting with the synchronous P. palaciosi could become extinct. Since P. extensus is a species endemic to Central Mexico, an area heavily impacted by human activity, its risk of extinction could increase. Alternatively, if the communication system of P. extensus evolves to prevent reproductive interference, populations interacting with P. palaciosi could diverge from those not suffering reproductive interference, possibly initiating a speciation process. Thus, further study of reproductive interference by synchronous fireflies seems a promising avenue of research.