First Phylogeny of Pseudolychnuris Reveals Its Polyphyly and a Staggering Case of Convergence at the Andean Paramos (Lampyridae: Lampyrini)

Simple Summary Two species of Andean-endemic fireflies are herein revised, and their evolutionary relationships addressed for the first time. We show that despite their similarities, these two species are distantly related. We provide original reports of these species interacting with Andean-endemic flowers for the first time, and we propose that their similarities are due to participation in mimicry rings. Abstract South America is likely the cradle of several New World firefly lineages but remains largely understudied. Despite several advances in firefly systematics in the Neotropical region, the Andean region has been largely unstudied for over a century. The Colombian Páramos are a critically threatened biodiversity hotspot that houses several endemic species, including the firefly genus Pseudolychnuris, with two species—P. vittata and P. suturalis. Here, by analyzing the phylogenetic relationships of Pseudolychnuris, we found that this genus is polyphyletic. Pseudolychnuris vittata and P. suturalis were found to be distantly related despite the striking similarity in outline and color pattern of males and females. We redescribe Pseudolychnuris and its type species P. vittata. Moreover, we revalidate Alychnus Kirsch, 1865 stat. rev. to accommodate A. suturalis comb. nov., also redescribed here. We provide updated distribution maps and report field observations for both monotypic genera. Since adults visit flowers and interact with pollen and nectar, Pseudolychnuris and Alychnus may be occasional pollinators of Andean-endemic plants, a phenomenon previously neglected. Our findings reveal an interesting case of convergence between Pseudolychnuris and Alychnus—probably associated with life in the Páramos—and shed light on character evolution in the Photinini lineage of fireflies.


Introduction
Roughly a fourth of the 2200 firefly species are endemic to South America [1], the hypothesized cradle of several lampyrid lineages [2]. In fact, South America is home to several endemic genera of fireflies across its biomes. For example, recent systematic efforts shed light on several lineages of fireflies endemic to the Atlantic Rainforest [3][4][5][6][7][8], a rather common trend in soft-bodied beetle families [9][10][11][12]. Likewise, endemic genera were recently identified in the Amazon Rainforest [13] and in the Chilean matorral (Cladodes s.s. sensu [8]). Narrow endemicity is also widespread at the species level in South American fireflies [14,15]. Despite their singularity, a historical lack of targeted sampling, as well Insects 2022, 13, 697 3 of 33 stereomicroscope, using the Leica Application Suite X Automontage software. We follow the classification of Martin et al. [41] and the anatomical terminology of Silveira et al. [13].
We recorded label data for all type specimens using the following conventions: double quotes (" ") for label data quoted verbatim, double forward slashes (//) to separate labels; and brackets [ ] to enclose our comments or notes. All labels are typed unless otherwise noted.

Phylogenetic Analyses
To test the monophyly of Pseudolychnuris in the context of its tribe, Photinini, we ran phylogenetic analyses comparing two approaches: maximum parsimony analysis and Bayesian inference. Our taxon sampling included 20 Lampyrinae taxa, in addition to one incertae sedis taxon (Vesta thoracica Olivier, 1790) morphologically close to one of our ingroup taxa, Dilychnia guttula (Fabricius, 1801) (see [40]). The tree was rooted in the lampyrini Lampyris noctiluca Linnaeus, 1758. The ingroup included the remaining 20 taxa were all Photinini, including three incertae sedis taxa (Ethra marginata, Haplocauda albertinoi Silveira, Lima, and McHugh, 2022, Scissicauda disjuncta (Olivier, 1896) The material examined for this study is given below (see Taxonomy, below), and additional material used in the phylogenetic analyses is provided in Supplemental Material File S1.
We coded 93 morphological characters for 21 taxa, using MESQUITE 3.61 [46]. We drew or modified 73 characters from Silveira et al. [13], following the guidelines of Sereno [47]. Measurement-based characters were taken at the longest or the widest point of the respective structure. Key character states are labeled in figures, abbreviated as C:S, where C and S indicate character and state number, respectively.
We implemented a maximum parsimony analysis using implied weights, a method where characters are weighted according to their homoplasy and, thus, characters with less homoplasy have more weight [48,49]. The K-value determines how strongly the analyses will weigh against homoplasy; however, there are no well-justified criteria to choose some particular value of K, and this decision is probably matrix-dependent [48]. To choose the value of K used in our analysis, we used the methodology proposed by Mirande [50], which divides at regular intervals the mean fit values of each character in the most parsimonious trees obtained under different values of K and for which the main criterion for choosing among these trees is their stability. This method was applied using the script for TNT developed by Mirande [50] (available at: http://phylo.wikidot.com/tntwiki, accessed on 1 January 2022). The most stable trees are those that share the largest number of nodes with the remaining trees, as measured by the SPR distance [49]. The best K range for the data matrix presented was in the intervals five to nine (Supplementary Materials  Table S1); this range was chosen based on comparison and selection of the highest similarity coefficient (SPR). In each of these intervals, a single tree was obtained, and in all the intervals the tree obtained was the same, (see Supplementary Materials Table S1). We implemented symmetric resampling with default settings and 1000 replicates to estimate node support [51]. Consistency [52] and retention [53] indices are given for each character.

Distribution (based on P. vittata):
Pseudolychnuris is endemic to the Colombian Andes, present in municipalities of the departments of Cundinamarca, Valle del Cauca, Meta, and Caquetá. Its altitude range goes from 550 to 3400 m.a.s.l. (Figure 10). Its location contrasted with data from Colombian ecosystems [67] suggests that Pseudolychnuris is found in humid mountainous and sub-humid high Andean landscapes, as well as in landscapes of erosional alluvial plains of Andean rivers in the department of Caquetá. Considering the material examined, adults of Pseudolychnuris are more commonly found between November and December and are mostly absent from March through July. posteriorly, spiculum ventrale almost 1/3 as long as sternum, with posterior margin emarginate medially, deeper in sternite VIII. One spermatheca and one accessory gland present. Ovipositor with coxital rods 2× longer than core coxite.

Distribution (based on P. vittata):
Pseudolychnuris is endemic to the Colombian Andes, present in municipalities of the departments of Cundinamarca, Valle del Cauca, Meta, and Caquetá. Its altitude range goes from 550 to 3400 m.a.s.l. (Figure 10). Its location contrasted with data from Colombian ecosystems [67] suggests that Pseudolychnuris is found in humid mountainous and sub-humid high Andean landscapes, as well as in landscapes of erosional alluvial plains of Andean rivers in the department of Caquetá. Considering the material examined, adults of Pseudolychnuris are more commonly found between November and December and are mostly absent from March through July.  Remarks: Pseudolychnuris originally comprised two species: P. vittata and P. suturalis. Here, we showed that P. suturalis is more distantly related, and revalidated Alychnus Kirsch, 1865, to accommodate A. suturalis (Motschulsky, 1854) comb. nov. (see above). The phylogenetic affinities of Pseudolychnuris are still poorly known, since in our analyses (Figure 1), this genus appears in a polytomy.
Pseudolychnuris is unique among Photinini by the presence of a lightly sclerotized longitudinal window on the dorsal plate of the phallus (79:1). Even though this window Remarks: Pseudolychnuris originally comprised two species: P. vittata and P. suturalis.
Here, we showed that P. suturalis is more distantly related, and revalidated Alychnus Kirsch, 1865, to accommodate A. suturalis (Motschulsky, 1854) comb. nov. (see above). The phylogenetic affinities of Pseudolychnuris are still poorly known, since in our analyses (Figure 1), this genus appears in a polytomy.
Pseudolychnuris is unique among Photinini by the presence of a lightly sclerotized longitudinal window on the dorsal plate of the phallus (79:1). Even though this window resembles the cleft on the dorsal plate of other Photinini, including Alychnus and Photinus species (e.g., Figure 15G), in Pseudolychnuris, the ejaculatory duct runs ventral to the dorsal plate. In contrast, the ejaculatory duct runs through the dorsal plate in Alychnus and Photinus species. Among the Photinini, Pseudolychnuris is most similar to Alychnus and "dark" (i.e., lantern-less) species of Photinus. However, Pseudolychnuris has a lack of ventrobasal processes on the dorsal plate of the phallus (65:0), typical of Photinus and Alychnus (65:1), and has spikes on the parameres (85:1), which are lacking on Photinus and Alychnus (85:0). Pseudolychnuris can be further distinguished from Alychnus by the following combination of traits: a proleg bearing one tibial spur (25:1; vs. zero (25:0) in Alychnus); and the sternum VIII without a median posterior projection (38:0; present (38:1) in Alychnus).
Pseudolychnuris is also superficially similar to many species currently listed under Lucidota, a "wastebin" genus in need of revision [7]. While it is beyond the scope of this work to elucidate the taxonomy of Lucidota, we provide a comparison to the type species, L. banoni: the dorsal plate of the phallus in L. banoni is entire (i.e., lacks the longitudinal window typical of Pseudolychnuris) and lacks the well-developed struts (64:0) seen in Pseudolychnuris (64:1); the phallobase is as long as the phallus in L. banoni (58:1), but a 1/4 shorter than the phallus in Pseudolychnuris (58:0); the subapical spike on the paramere of Pseudolychnuris is well-developed and basally elongate, while in L. banoni the spike is rudimentary. Together, these traits clearly separate Pseudolychnuris and L. banoni.
The spermatophore digesting gland and proctiger could not be determined in the specimen examined, but poor preservation cannot be ruled out.

Diagnosis:
Color pattern variable (Figures 3 and 10A,B): overall black, except for pronotum, from entirely black to black with expansions pale yellow to orange and elytron, from entirely black to black with an oblique, elongate pale-yellow to orange line. Antennomere III nearly 2× longer than pedicel ( Figure 4G,H). Antennal sockets separated by 1/3 labrum greatest width ( Figure 4A). Eye small, nearly 1/5 head width ( Figure 4A-F).
Biology: P. vittata is a diurnal species commonly seen on flowers of angiosperms native to the Andean paramos, including the "frailejones" (Espeletia spp.) ( Figure 11E-G). They have been found in copula on these plants ( Figure 11E). Interestingly, P. vittata has been seen in relatively urban areas of the Meta department, in addition to conservation units on the Paramo. Remarks: P. vittata is here regarded as the single species of its genus. P. vittata has a remarkably widespread distribution for a species with flightless females. However, other firefly species with similarly flightless females also have widespread distributions, including the type species of the family, Lampyris noctuluca L., 1758 (cf. Kazantsev, 2010).       Figure 13G,H). Labrum with anterior margin straight ( Figure 13A), connected to frons by membrane. Pronotum bearing a notch by the posterior angle ( Figure 14A). Tibial formula 0-1-1 ( Figure 14R-T). Lanterns absent ( Figure 15A). Sternum VIII with posterior margin mucronate ( Figure 15D). Sternum IX with lateral rods separated ( Figures 15F and 16E), basally biconcave. Phallobase mildly emarginate ( Figure  15G-L). Dorsal plate hollowed, through which runs the ejaculatory duct, sides bearing spikes ( Figure 15G-L). Paramere robust, lacking spikes, inner margin excavate ( Figure  15G-L). Female brachypterous ( Figure 17A,B), sternum VI with posterior margin emarginate ( Figure 17H).
Female. Elytron brachypterous ( Figure 17A,E), nearly 3.5 longer than wide, leaving exposed at least the pygidium, sometimes tergite VI. Hind wing 2× wider than long. metascutellum 1/2 as long as alinotum ( Figure 17F). Pygidium 2× longer than wide, sides convergent posteriorly, posterior margin straight ( Figure 17I). Sternum VI deeply emarginate ( Figure 17H), VII with posterior margin straight, VIII with posterior margin deeply emarginate ( Figure 17J), sides convergent posteriorly, spiculum ventrale almost 1/4 as long as sternum. One spermatheca and one accessory gland present, spermatophore digesting gland slightly smaller than spermatheca ( Figure 17K). Bursa with a commashaped sclerite ( Figure 17M). Ovipositor with coxital rods 2× longer than core coxite ( Figure 17L The fact that Pseudolychnuris and Alychnus have heretofore been synonyms comes at no surprise. These taxa are indeed strikingly similar in general outline, size, and color patterns, and have largely overlapping distributions. What is more, females of both genera are similarly brachypterous, an observation that has caught the attention of taxonomists from relatively early on [70][71][72]. A rather uncommon phenomenon among diurnal Photinini, female brachyptery was previously reported only for Pyropyga nigricans (Say, 1823) [77], Lucidota luteicollis (LeConte, 1878) [78], Phosphaenus Laporte, 1833, and Phosphaenopterus Schaufuss, 1870 [79]. In contrast, brachyptery is somewhat more common in nocturnal Photinus species, especially at higher-elevation sites (e.g., Photinus extensus Gorham, 1881; [80]), although not unheard of in lower-elevation species (e.g., P. collustrans LeConte, 1878 and Photinus brimleyi Green, 1956;[81]). In fact, firefly female brachyptery is widespread in the Andean Paramos, including several Photinus species, particularly those previously listed under its junior synonym Macrolampis Motschulsky, 1853 (e.g., [82]). While the causes for such a trend are yet to be tested, the widespread flightlessness on highelevation endemics is seen as an adaptation to the strong winds associated with thinner air and lower pressure [83,84]. Therefore, it is likely that Pseudolychnuris and Alychnus have convergently evolved female brachyptery due to the occurrence in higher elevation sites on the Paramos. However, a sound phylogenetic test for this hypothesis is pending on a better resolved phylogeny, in addition to a broader sampling of female traits across the Photinini. In addition, the similarity in color patterns of these two genera could be the outcome of their participation in similar mimicry rings, although thermal melanism and protection against UV radiation-recurrent themes at higher elevation sites-may also have played a role in shaping these phenotypes. The finding of both species on the same plants (see above) provides a further indication that they participate in the same mimicry ring.

Genitalic Traits and Systematics of the Photinini
The evolution of Photinini seems to be heavily impacted by shifts in signal use [85][86][87][88]. In short, organs involved in emitting (i.e., lanterns) or perceiving (i.e., eyes, antennae) sexual signals have been proposed to be evolutionarily labile, sometimes being significantly diverged among closely related species (e.g., [87]). The present work is congruent with this observation (see Figures 1 and 2).
Unfortunately, the classification and definition of Photinini genera have been largely built upon differences in these very organs (e.g., [2]), putting in check their definition and calling for a revised classification of this tribe. Meanwhile, other structures involved in reproduction, namely male genitalia, have yielded important characters for discrimination across species and genera [39,60,89,90]. Genitalic traits are of special interest since these are expected to be largely unrelated to changes in the sexual signaling mode, which makes their use especially useful for Photinini taxonomy and the study of their evolution. Female genitalic traits were rarely studied, but also proved successful to discriminate Photinini taxa (e.g., [6,40,79,91]). Nevertheless, the use of genitalic traits to investigate the phylogeny of the Photinini is still in its infancy, although they have yielded important results ( [7,13,36]; this work). In fact, nearly half of the characters in this study came from the male genitalia. In contrast, the use of female traits in phylogenetic analyses of Photinini has been hampered by a lack of female data.
A few interesting patterns emerge from the present work, which might impact ongoing and future taxonomic work in Photinini. For example, we found out that both Alychnus and Photinus share the presence of ventrobasal processes on the dorsal plate of the phallus (char. 65:1; see Taxonomy above for a more thorough comparison). The presence of these aedeagal processes has led Zaragoza-Caballero et al. [61] to synonymize Ellychnia and Macrolampis with Photinus in the absence of a phylogenetic treatment, a position kept in the phylogeny of Zaragoza-Caballero et al., [36]. We point out that, despite sharing these ventrobasal processes with Photinus, Alychnus differs in many other noteworthy traits here, including the shape of these processes themselves, but also: a different configuration of tibial spurs (chars. 25,27,28); the unique spiked phallic dorsal plate sides (char. 78); and the phallobase-to-dorsal plate ratio (char. 57), among other traits that have been missing from other phylogenetic treatments. Considering our study, a new look to these particular cases of synonymy is warranted, pending on a more comprehensive dataset-including the type-species of all names involved-analyzed upon a phylogenetic framework. Finally, the function of these ventrobasal processes during copulation is to our knowledge unknown. Observational studies are needed to raise and test hypotheses about the function of these mysterious processes.

Conclusions
We reviewed and explored the phylogenetic relationships of two Lampyrinae: Photinini genera, Pseudolychnuris and Alychnus stat. rev., endemic to the Colombian Andes. We found Alychnus to be close to Photinus, whereas Pseudolychnuris affinities remain poorly resolved. Our work provided new characters to diagnose these genera, and also updated distribution maps.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/insects13080697/s1, Supplementary Materials File S1: additional material used in the phylogenetic analyses; Supplementary Materials