THE EVOLUTION OF BIOLUMINESCENCE IN CANTHAROIDS (COLEOPTERA: ELATEROIDEA)

Materials and Methods

Eighty-five exemplar taxa were selected to represent a diversity of Lampyridae and outgroup families. Selection of taxa included as many subfamilies and tribes as possible within Lampyridae, based on the classification schemes of Crowson (1972) and Lawrence & Newton (1995), and 11 subfamilies within 9 other families, based on Lawrence & Newton (1995) (Appendix 1). We did not include any members of Elateridae in this analysis as they are too distantly related to the taxa considered here (Lawrence 1988). Seventy four male morphological characters with a total of 212 character states were used in the analysis. Inapplicable characters were coded as "-", while missing characters were coded as "?". All characters were analyzed under equal weights with 20 multistate characters as additive (see Appendix 2, 3). Plastoceridae was designated as the root of the tree based on Lawrence's (1988) phylogenetic analysis of the Elateriformia. The parsimony ratchet (Nixon 1999) (consisting of 100 iterations, weighting 12% of the characters) was implemented in Nona (Goloboff 1993), run within Winclada (Nixon 2000). The most parsimonious trees discovered were used as the starting place for a more exhaustive search using the "max*" command within NONA. The "best" command was then used to eliminate sub-optimal trees. A strict consensus tree was then calculated from these most parsimonious trees. Bremer support (Bremer 1988, 1994) was calculated using NONA, and the search was set to a Bremer support level of 5, with four runs, each with a buffer of 5000 trees.

Results

The parsimony ratchet returned trees of 818 steps. Starting from these 52 trees, "max*" and "best" gave 280 most parsimonious trees of 818 steps. A strict consensus (Fig. 1) of these 280 trees collapsed 13 nodes and produced a consensus tree of 848 steps (ci = 0.16, ri = 0.57). Bremer values listed in Figure 2 indicate the number of steps that are required, up to 5, to find the closest tree that does not contain that particular node. Lampyridae is monophyletic with the exception of a few taxa that have been of controversial affinity (Harmatelia, Drilaster, Pterotus, and Stenocladius.) Two genera currently classified as phengodids (Dioptoma and Diplocladon) were placed with rhagophthalmids in this phylogenetic analysis. The families Drilidae, Omalisidae, Lycidae, Omethidae, Teleguesidae and Phengodidae appear to be monophyletic. The monophyly of Cantharidae is not supported.

Discussion

Testing the Monophyly of Existing Families

Lampyridae.

In view of our phylogeny, Lampyridae as defined by Crowson (1972) and Lawrence and Newton (1995) is not monophyletic. The three synapomorphies that define the base of Lampyridae are: covered head position, oblique attachment of trochanter to femora, and wing vein CuA1 intersecting MP above fork (Kukalova-Peck & Lawrence 1993). The genera Harmatelia, Pterotus, Drilaster (Ototreta) and Stenocladius are currently classified as lampyrids (Lawrence & Newton 1995; after Crowson 1972) though in our analysis they are clearly placed outside of the family Lampyridae. This is not entirely unexpected, as several previous authors (LeConte 1859; McDermott 1964; Crowson 1972) who have examined some of these taxa have viewed them as possessing questionable affinities to existing families. LeConte (1859) placed Pterotus obscuripennis in Drilidae and then later (1881) moved it to Phengodidae. Of the genus Pterotus, LeConte (1859) stated, "A singular genus, which I have described at length from my inability to place it properly. It seems to have a mixture of characters belonging to the Lampyrides, Telephorides and Drilids, but from the small size of the posterior coxae is probably better placed with the latter." McDermott (1964) also mentions the difficulty he encountered in trying to place some of these taxa, "Both Pterotus and Harmatelia share a large degree of similarity between some characters. Also neither fits strictly to the accepted lampyrid characteristics and both have some suggestion of phengodid affinities. Combining these two genera in the sub-family Pterotinae is admittedly arbitrary but nevertheless serves to bring them together as transitional forms." Crowson (1972) wrote that the genera Pterotus and Ototretadrilus were the most phengodid-like and probably the most primitive firefly genera he had studied. (Specimens of Ototretadrilus were not available to us.)

The genus Drilaster was originally described in the family Drilidae by (Kiesenwetter 1879), and Ototreta was originally described in the family Lampyridae. The synonymy between Drilaster and Ototreta was noticed by Nakane (1950) (see also Sato 1968). However, Asian workers continued to use the older name (Drilaster) but moved it to Lampyridae. American and European workers continued using Ototreta as it appeared to be the valid name in McDermott 1964 and 1966. Therefore, not only should American workers discontinue the use of the name "Ototreta," but these taxa should no longer be associated with the family Lampyridae. For a short history of the taxonomic placement of Drilaster and Ototreta, see Table 1.

According to our phylogeny (Fig. 1), if Harmatelia, Pterotus, Drilaster and Stenocladius continue to be considered fireflies, the families Lycidae, Cantharidae, Phengodidae, Omethidae and Teleguesidae would need to be synonymized with Lampyridae. This seems drastic given the peripheral significance of the genera and the traditional affection for the families. Harmatelia, Pterotus, and Drilaster should be removed from Lampyridae and given the taxonomic label of "Elateroidea incertae sedis" awaiting further study to place them properly. Drilaster may not be monophyletic. Our analysis clearly places Stenocladius sp. in the family Phengodidae. Ohba et al. (1996) studied the external morphology of Stenocladius larvae and found that they did not posses an epicranial suture on the dorsal surface of the larval head. This suture is well developed in larvae of Lampyridae. They hypothesized that the fused dorsal surface of the larval head in Stenocladius is more closely allied with Phengodidae than Lampyridae. Our analysis supports this association. Because the clade containing these taxa in our analysis is unresolved, there is no information concerning which subfamily of Phengodidae Stenocladius should be placed within (Fig. 2).

Other Cantharoid families.

The families Plastoceridae, Drilidae, Omalisidae, Rhagophthalmidae, Lycidae, Omethidae, and Teleguesidae are supported as being monophyletic in our analysis including very few representatives. Phengodidae and Cantharidae are not supported as being monophyletic. With the exception of Dioptoma adamsi and Diplocladon sp., the four other phengodid taxa used in this analysis are a monophyletic clade. Our analysis placed Dioptoma and Diplocladon at the base of the clade containing the family Rhagophthalmidae. The Bremer support value for the base of this clade (Fig. 2) is high (>5), indicating strong support. The seven synapomorphies that define this clade are: twelve antennomeres in male antennae, third antennomere long, basal antennal flagellomeres not symmetrical with apical flagellomeres, mandible apices acute (inside angle < 90 degrees), emarginate eyes, eyes posterior-ventrally approximated, and wing vein MP3 not contacting MP1+2. Therefore, we propose moving the genera Dioptoma and Diplocladon out of Phengodidae and into Rhagophthalmidae. On the other hand, Cantharidae does not seem to be supported as monophyletic in this analysis, and none of the four cantharid taxa included in the analysis form a clade. Cantharidae needs to be further examined in relation to other taxa and sampled more thoroughly within a phylogenetic context before a taxonomic change is made.

Phylogenetic Relationship
Between Lampyridae and Phengodidae

The family Phengodidae is composed of bioluminescent species that commonly resemble fireflies in their general appearance and are usually also found to be sympatric with many firefly species. Even though phengodid beetles share aspects of their biology with lampyrids (larviform females, the use of pheromones and luminescence), phengodids have historically been seen as a group taxonomically distinct from Lampyridae. However, it is probably the similarities between Lampyridae and Phengodidae, with bioluminescence being one of the most obvious, which have linked them as closely related taxa in the eyes of many cantharoid workers. "Within Cantharoidea, Phengodidae and Lampyridae appear to be directly related, so that the luminosity of both groups can plausibly be attributed to inheritance from a common ancestor . . ." (Crowson 1972). Our phylogeny provides evidence that Phengodidae is not sister to, or basal to, Lampyridae. In addition, it shows that Rhagophthalmidae and Omalisidae are the bioluminescent families that are sister, or basal to Lampyridae, respectively (Fig. 3).

The Evolution of Bioluminescence
in Non-lampyrid Cantharoids

Bioluminescence in the order Coleoptera is known to occur in Elateridae, Staphilinidae (Costa et al. 1986) and four cantharoid families: Omalisidae, Rhagophthalmidae, Phengodidae and Lampyridae (Lloyd 1978). Several recent studies have provided hypotheses concerning the evolutionary relationships within or around Cantharoidea (Crowson 1972; Potatskaja 1983; Beutel 1995) using a variety of techniques, characters and explemplar taxa. Plotting luminescence (larval or adult) onto these different trees, supports interpretations ranging from three origins, to one origin and three loses. However, these studies treat families as single units. Therefore, if luminescence arises only once in each of the three luminescent families, (Omalisidae, Phengodidae, and Lampyridae, with Rhagophthalmus species treated as phengodids), three separate origins would be the maximum number of steps. Conversely, a single origin would be the minimum number of steps if all families were treated as being monophyletic. Crowson (1972) proposed a dendrogram for the relationships between the cantharoid families (Omethidae, Cantharidae, Plastoceridae, Lycidae, Omalisidae, Drilidae, Telegeusidae, Phengodidae, and Lampyridae). This scheme predicts two character optimizations of three steps each. One optimization poses three origins for bioluminescence, while the second poses two origins and one loss of bioluminescence. Potatskaja (1983) proposed a dendrogram for the relationships between the cantharoid families (Brachyspectridae, Cantharidae, Phengodidae, Drilidae, Omalisidae, Lycidae and Lampyridae) based on larval mouthpart characters. Potatskaja concluded that two lineages, one termed "cantharid" (composed of Phengodidae, Drilidae, Omalisidae, Brachyspectridae, and Cantharidae) and the other "lycid" (composed of Lampyridae and Lycidae) originated from a phengodid ancestral form. No specific taxon was designated as the root. In reference to the origin of bioluminescence, this topology predicts two optimizations of three steps each: three separate origins, or two origins and one loss. In 1995, Beutel proposed a phylogenetic analysis of Elateriformia based on 27 larval characters (33 states). Within this analysis Cantharoidea was represented by seven taxa, one species per each of Brachyspectridae, Cantharidae, Drilidae, Omalisidae, Phengodidae, Lampyridae, and Lycidae. All of the cantharoid taxa were placed in the same clade except for Cantharidae, which was placed close to Elateridae, rather than with the rest of the cantharoids. The clade containing the bioluminescent cantharoid taxa was poorly resolved in the consensus tree and predicts a single topology of one origin and three losses.

Our analysis suggests a single solution, considering a taxon to be luminescent if any life stage is luminescent. There are two origins of bioluminescence with one loss: luminescence arose once basally, early in the evolutionary history of the cantharoid clade, and was subsequently lost and then later regained in the phengodids, see Fig. 3. Taxa in which luminescence was regained under this scenario are currently classified as belonging to the family Phengodidae, (Cenophengus pallisus, Phrixothrix reducticornis, Pseudophengodes pulchella, and Zarhipis integripennis), as well as Stenocladius sp. which we consider to be a phengodid and propose its inclusion in this family. The seven synapomorphies that define Phengodidae are: tibial spurs are absent, bipectinate antennae, distal margin of antennal flagellomeres approximating proximal margin in width, antennal lobes produced from basal region of flagellomere, two elongated antennal lobes per flagellomere, narrow juncture between flagellomere and antennal lobe, and juncture between lateral and hind margins of pronotum are truncate (= 90 degrees). Therefore, all luminescence in the cantharoid lineage is homologous except for that of Phengodidae, which is a reversal to luminous habit. Additionally, all known luminescent cantharoid taxa have luminous larvae, and in Omalisidae the larvae are luminous, but not either adult (Crowson 1972). The fact that Omalisidae is the most basal of all the bioluminescent cantharoids indicates that luminescence arose first in the larvae and then subsequently in the adults (Fig. 4).

Photic Organ Evolution in Non-lampyrid Cantharoids

Larvae.

Only the larval photic organs of Phengodidae and Lampyridae have been studied in detail. The larvae of Omalisidae have never been studied, and the larvae of Rhagophthalmidae have been studied and described only recently (Wittmer & Ohba 1994), though no morphological, physiological or histological work has been published on this group. Therefore, from what is currently known from evidence scattered among the taxa, the pattern of two luminous spots per segment on larvae is the most ancient and common larval photic organ pattern in the cantharoid lineage (Fig. 4). The number of luminous segments varies, but all known luminous cantharoid larvae bear pairs of luminous photic organs. While most lampyrid larvae bear only a single pair of photic organs on the eighth abdominal segment, larvae of the other taxa generally posses a pair of photic organs on each abdominal segment with additional pairs sometimes present on the larval thorax (Table 2). While the larvae of many genera of luminous cantharoids are not yet known, all species known to be luminous as adults are also luminous as larvae. Therefore, while only some larvae are known from the families Omalisidae, Rhagophthalmidae, Phengodidae and Lampyridae, the larvae of all species in these families are hypothesized to be luminous (Fig. 4). Crowson (1972) hypothesized that Barber's (1908) luminous larva from Guatemala, described as Astraptor sp., could have been a large female larva or a larviform female of Telegeusis. Sivinski (1981) points out that in a later unpublished manuscript Schwarz and Barber identified the single specimen as the phengodid Microphenus gorhami. Therefore, as far as is known, the family Teleguesidae does not contain any luminous taxa. Barber (1908) mentions that there was a single photic organ in the head which produced a red light that was thrown directly forward and hence was not easily seen from above. This specimen seemed to have no other photic organs, though it was observed in the daytime and not for long. A red head-light is known only in other phengodid larvae (Viviani & Bechara 1997).

Adult Females.

Females of luminous cantharoid taxa, excluding Lampyridae, generally posses the same photic organ morphology as their larvae, which is generally paired, luminous spots on the post-lateral margins on some of the thoracic and each of the abdominal segments (Table 2). Females, and males of the family Lampyridae vary in photic organ morphology (Lloyd 1978), perhaps due to sexual selection as the females of many firefly species attract mates via a luminescent sexual signal system (McDermott 1917; Schwalb 1960; Lloyd 1978 and 1979; Branham & Greenfield 1996;Vencl & Carlson 1998). While the luminescent sexual signals of fireflies have received considerable attention, pheromones are the dominant sexual signals used in courtship in most cantharoids, including Phengodidae, and are also used by many lampyrids (McDermott 1964; Lloyd 1971). Therefore, (with the exception of the family Omalisidae and the genus Drilaster), the pattern of bioluminescent evolution in the larvae (Fig. 4) and the pattern found in the females of luminous taxa (Fig. 5) is very similar. The fact that Omalisidae is the basal-most luminous cantharoid taxon and both adult males and females are not bioluminescent suggests that bioluminescence first arose in larvae and later in adults.

Rhagophthalmus ohbai females, in addition to retaining the larval pattern of photic organs, also possess a novel photic organ on the eighth ventrite, which is used in courtship, see Table 2 (Ohba et al. 1996a). After using the ventral photic organ on the eighth ventrite for courtship, females curl around their eggs and glow from ten sets of paired photic organs located at the lateral margins of the ten luminous body segments, (see Table 2), which serve as an aposematic warning display (Ohba et al. 1996a; Chen 1999). Rhagopthalmidae appears to be the sister of Lampyridae, which is the only other cantharoid family known to contain females that employ photic signals in courtship. In addition, based on the presence of extremely well developed eyes in rhagopthalmid males and the lack of greatly elaborate bipectinate antennae, such as those found in Phengodidae, we believe that photic signals are the primary mode of sexual signaling.

Crowson (1972) incorrectly cited Green (1912) as reporting that the female of Harmatelia is apterous and larviform. Green (1912) states that, "I have not yet succeeded in determining the female of this beetle, and it remains uncertain whether the other sex is an apterous grub-like creature, or whether it is in the form of another beetle."

Adult Males.

All known bioluminescent adult cantharoid males are restricted to the families Lampyridae, Rhagophthalmidae (as defined here) and Phengodidae (as defined here) as well as the genus Harmatelia. While the exact number and position of the photic organs varies, they are generally found in pairs on one or more of the thoracic segments, and on each of the first eight abdominal segments. Male photic organs, like those of larvae and females, are found near the lateral margins of these body segments (Table 2). The more dorsal position of these lateral photic organs in the adult males versus larval males is probably due to the lateral tergites, found as plates in the dorsal region on the side of the larval abdomen, becoming fused to the larval tergites to form a single large plate covering the entire dorsal surface of the adult male abdomen. One exception is Pseudophengodes pulchella, which bears a large photic organ on the ventral surface of the eighth ventrite that seems to be used in courtship. Until recently, the only phengodid genus that was known to contain adult luminescent males was Pseudophengodes. However, Viviani and Bechara (1997) discovered through rearing experiments that phengodid males in the tribe Mastinocerini (Brasilocerus, Euryopa, Mastinocerus, Mastinomorphus, Phrixothrix, Stenophrixothrix, and Taxinomastinocerus) are luminous throughout the adult stage and that the luminescent emissions seem to serve a defensive rather than courtship function. No adult phengodid males in the North American tribe Phengodini (Phengodes and Zarhipis) are known to be continuously luminescent through the entire stage. Even though there is little variation found in male photic organs outside of Lampyridae, the scattered occurrence of photic organs in males clearly seems to indicate multiple origins (Fig. 6).

J. W. Green's first published observations (1911) of live Harmatelia bilinea males did not include any notice of luminescence, even though Green was specifically looking for evidence that this insect was luminescent. The following year (1912), Green published that he had observed two specimens that "exhibited a distinct light when examined in a dark room." The fact that Green had examined many Harmatelia specimens without noticing any photic emission may be an indication that these males are not luminescent throughout their entire adult life. It is well known (McDermott 1965; Lloyd 1978;Vivani & Bechara 1997; Branham & Archangelsky 2000) that some lampyrid species which are not luminescent as adults retain the ability to glow via larval photic organs for a short time after they have eclosed and are still teneral. McDermott (1965) hypothesized that the males of both Pterotus and Harmatelia might have the ability to produce light only briefly after eclosion as does the firefly Lucidota atra. McDermott also mentioned in this same work that H. S. Barber (unpublished observation, confirmed by J. E. Lloyd) observed that Phengodes males also have the ability to produce light shortly after eclosion. This photic carry-over into the adult, while only temporary in some taxa, is suggestive of a larval origin of the photic organ and its carry over into the adult.

Some Rhagophthalmus ohbai males are known to be luminous from paired spots along the lateral edges of ten body segments, (see Table 2). These males are weakly luminous (Chen 1999) and evidently are not always observed (Ohba et al. 1996a). It seems likely that Rhagophthalmus males have only a temporary ability to produce light immediately following eclosion and luminescence is not used in courtship (Ohba et al. 1996a).

Luminesence and Life Stages.

The phengodid genus Phrixothrix is the only luminous non-lampyrid cantharoid in which larvae, adult males, and adult females are known to be luminescent throughout all life stages (Table 2). Luminescence throughout all life stages of Phrixothrix hirtus is essentially the same. The photic organ morphology appears to be identical between all life stages with the exception that the head lantern is lacking in the adult males. Therefore the lateral lanterns of all stages appear identical and the head lanterns of the larvae and females are identical. In addition, the photic emission spectra is essentially the same for each type of photic organ regardless of life stage (Costa et al. 1999). An additional example of similarity in the emission spectra and a possible connection between larval and adult luminescence was found by Viviani and Bechara (1997) who argued that, "Continuance of the same bioluminescent color in the lateral lanterns of larval, pupal, and adult stages of Mastinomorphus sp.1 and P. heydeni suggests conservation of the same luciferase iso-form throughout its life cycle." The pattern of photic organ morphology appearing more or less identical across life stages along with similar photic emission spectra being emitted from these organs supports the hypothesis by Crowson (1972) that luminescence first evolved in larvae and was then "carried over into adults."

Evolution of Photic Signaling in Non-firefly Cantharoids

Sivinski (1981) provides a detailed synopsis of the various theories that have been proposed for the function of larval luminescence and the evidence supporting each. While it is now generally accepted that lampyrids are chemically defended and larval photic emissions probably function as aposematic displays (Lloyd 1973; Sydow & Lloyd 1975; Eisner et al. 1978, Belt 1985, Underwood et al. 1997; Knight et al. 1999; De Cock 2000), there exists much less information concerning whether other larval cantharoids are distasteful as well, though it appears that at least some phengodids are chemically defended (Burmeister 1873; Harvey 1952; Sivinski 1981). It is interesting that almost all larval photic organs are paired and are located on the sides of the abdomen or on the eighth abdominal ventrite where the photic emissions are readily seen from the side or from above. This is most consistent with the aposematic warning display hypothesis. The exception to this rule is the pair of medial photic organs on the head of some phengodid larvae such as Phrixothrix (Table 1). The photic emissions from these organs were measured by Viviani and Bechara (1997) and were found to be in the range of 574-636nm, well into the red range. Electroretinograms of Phrixothrix larvae showed that these larvae have a spectral sensitivity shifted to the red (V. R. Viviani, E. J. H. Bechara, D. Ventura and A. Lall unpublished data; Viviani & Bechara 1997). Viviani and Bechara (1997) hypothesize that these red-emitting head-mounted photic organs provide an illumination function, which may help in locating prey that do not posses spectral sensitivity shifted to the red, and that the lateral photic organs serve an aposematic defensive function.

As the basal luminescent taxa only possess lateral photic organs, it is probable that the first function of larval luminescence was as an aposematic warning display. Larval photic organs where then lost in Lycidae, Omethidae and Cantharidae, and then reappear in Phengodidae. In some phengodids, a photic organ arose on the head and produced red light which was used for illuminating prey (Viviani & Bechara 1997).

The function of luminescence in the adult phengodids is not well understood. Available data suggest that bioluminescence produced by the lateral organs of the adult males and females in this family, seems to serve an aposematic defensive role rather than mate attraction (Rivers 1886, Tiemann 1967, Sivinski 1981). However, the continuous glow produced by the ventral photic organ on the eight abdominal ventrite in Pseudophengodes is also consistent with use as either illumination of the surroundings during flight or intersexual communication (Viviani & Bechara 1997).

The function of luminescence in Rhagophthalmus ohbai was studied by Ohba et al. (1996a) who provide evidence that the emissions of the lateral photic organs in females serve an aposematic warning function, illumination while the females guards her eggs, while the ventral photic organ on the eighth abdominal segment seems to function exclusively in a courtship context. Hence, in this family the paired lateral and the ventral photic organs are used independently in separate contexts: defense and courtship. Across all luminescent cantharoid taxa, photic organs used to produce sexual signals seem to be exclusively restricted to the ventral regions of the body. It is also interesting to note that the eighth abdominal segment is consistently associated with the location of such photic organs. The reason for this association remains unknown.

Conclusion

Our phylogenetic analysis suggests that bioluminescence arose twice within the cantharoid lineage and was lost once. The first origin of luminescence in the lineage was ancient and luminescence first arose in larvae where it served as an aposematic warning display. Luminescence was retained in the larvae of the Rhagophthalmidae and Lampyridae and was likely carried over through the pupae into the adult stage where it became functional in some taxa and not in others. While photic signals are used in mate attraction in the Rhagophthalmidae, adult photic signals reached their greatest sophistication in the adults of the family Lampyridae, where photic signals are used in intraspecific communication and both photic organs and photic signals became greatly elaborated under the context of sexual selection. The second origin of luminescence occurred in the family Phengodidae where its function in both larvae and adults is as an aposematic warning display. In some phengodid taxa, luminescence has become elaborated to serve possibly as an illumination device for locating prey. In addition, males of the genus Pseudophengodes possess a lampyrid-like photic organ on the eighth abdominal ventrite, which glows continuously and likely serves to either illuminate potential landing sites or functions in courtship. While some researchers have previously hypothesized that the families Lampyridae and Phengodidae were close relatives and shared the charismatic ability to produce bioluminescent signals, these two families are perhaps more interesting than previously thought because they are not closely related and their bioluminescence is convergent.

Acknowledgments

We would like to thank John Sivinski and James Lloyd for the invitation to participate in the Behavioral Ecology Symposium and Jim Lloyd for his help, counsel, encouragement and friendship along the way. We gratefully acknowledge the help and assistance of several friends and colleagues: Ming-Luen Jeng, Rich Miller, Fernando Noll, Kurt Pickett, and Vadim Viviani.

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