A New Genus and Two New Species of Fireﬂies from South America (Lampyridae: Lampyrinae: Photinini) †

: Lampyridae taxonomy has traditionally relied on a few characters now deemed to be highly homoplastic, and their classiﬁcation—especially at the genus level—is yet to be consolidated based on rigorous phylogenetic analyses. Recent studies highlighted the value of genitalic trait variation in the evolution in Lampyridae, particularly for the rich and poorly known South American Photinini fauna. Here, we describe a new genus, with a new species from the Cerrado and another one from the Atlantic Forest. Phylogenetic analyses based on Bayesian and Maximum Parsimony approaches recovered these two species as sister to each other, which we place here in Zoiudo gen. nov. Males of this new lineage of ﬁreﬂies are overall strikingly similar to Photinus Laporte 1833, but can be readily distinguished by traits heretofore neglected, including the structure of tibial spurs and many genitalic traits. Instead, Zoiudo gen. nov. is strongly supported as sister to Ybytyramoan Silveira and Mermudes, 2014, supported by eight synapomorphies, the most conspicuous being the sternum VIII with lateral margins divergent up to basal 1/5, then convergent posteriorly, and the rudimentary ventral plate of phallus. Our study conﬁrms the value of extensive character and taxon sampling towards a revised classiﬁcation of Photinini taxa and highlights the need for a continued sampling and protection of South American biomes.


Introduction
Fireflies (Coleoptera: Lampyridae) are a charismatic group of beetles with a surprisingly confused taxonomy. Lampyridae taxonomy-especially at the genus-level-is traditionally reliant on few characters recurrently shown to be homoplastic (e.g., [1][2][3][4][5]), which complicates their classification and identification. Most genera of fireflies across subfamilies were last comprehensively reviewed over a century ago, and their diagnoses must be updated upon a phylogenetic framework.
Photinini LeConte, 1881, is the largest Lampyrinae tribe, with around 750 species and over 30 genera [6,7]. Despite the description of several new genera and species (e.g., [8][9][10]), many taxa in this tribe are difficult to identify due to overlapping genus-level diagnoses in this tribe. Recent phylogenetic studies have highlighted the value of previously overlooked morphological characters, mainly in the male terminalia and genitalia, to unravel the evolution and relationship of Photinini taxa [2,4,5]. The study and phylogenetic assessment of these characters has helped to improve and stabilize the tribe's classification, benefiting both taxonomists and non-taxonomists.
More than half of the world's Lampyridae (ca 2200 species) are housed in the Neotropics [11]. In the Neotropical realm, the South American fauna stands out due to its high diversity and endemicity [11]. Much of this diversity is, however, known only by their original descriptions and illustrations, which are usually insufficient to inform identification (e.g., [6]). Past work on the firefly fauna of the Atlantic Forest (e.g., [4]) and, more recently, Amazonia (e.g., [5]), led to the discovery of dozens of new species, but most other South American biomes remain unexplored, with their firefly fauna almost completely unknown. The Cerrado stands out as one of the most species-rich South American biomes, with a high level of endemicity [12]. This biome is highly threatened by human activities, with 55% of its area already deforested [13]. Sadly, most of its firefly fauna remain unstudied and many taxa unknown to science may be extinct even before their description.
Recent examination of museum entomological collections allowed us to identify two undescribed species-one from Cerrado and other from an unexplored area of the Atlantic Forest-which resemble Photinus Laporte, 1833 and Ybytyramoan Silveira and Mermudes, 2014. In order to explore the phylogenetic relationships of these two new species and improve the standing diagnoses of Photinini genera, we performed a phylogenetic analysis including most of the tribe genera. Our phylogenetic analyses based on Bayesian and maximum parsimony criteria recovered these two new species as sisters to each other, in a new genus described and illustrated herein.

Morphology, Terminology, and Illustrations
We examined specimens from the following institutions: Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil (MZUSP); Coleção de Entomologia Prof. José Alfredo Pinheiro Dutra, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil (DZRJ); and the Seção de Entomologia da Coleção Zoológica, Universidade Federal do Mato Grosso, Mato Grosso, Brazil (CEMT). Whole specimens or only their dissected abdomens were soaked in 10% KOH for 24-48 h. Photographs were made with a Leica DFC5400 and Leica Application Suite CV3 multi-focus software or with a Leica M205 C stereomicroscope and Leica Application Suite X Automontage Software.
We recorded label data for all type specimens using the following conventions: double quotes (" ") for label data quoted verbatim, single forward slashes to separate label lines (/), double backslashes (\\) to separate labels; and brackets [ ] to enclose our comments or notes. All labels are typed unless otherwise noted.
The classification follows [14] and terminology follows [9], except for wing terminology, which follows [15]. A distribution map of the species was plotted on the biogeographical map of the Neotropical region provided in [16] using R packages "sf", "dplyr", and "ggplot2".

Taxon Sampling
The new genus, with the two new species, could be confidently placed in Photinini based on their morphology. To determine the placement of the two new species, we expanded the taxon sampling of [7], which included taxa from all four Photinini subtribes, by adding the two new species and two Ybytyramoan Silveira and Mermudes, 2017 species (total = 25 species). Data for the material examined of the new species in the new genus are provided below (see Results), and additional material used in the phylogenetic analyses is provided in Supplementary Material S1. The root was set on the Lampyris noctiluca (Linnaeus, 1758) (Lampyridae: Lampyrini), which was never placed in Photinini. However, we also tested on the other non-Photinini taxon, Vesta thoracica (Olivier, 1790) (Lampyridae: Incertae sedis).

Phylogenetic Analysis
Our matrix expanded on the 93 morphological characters listed in [7], by adding four new characters (total = 97). Character coding followed the logical basis of [17]. The matrix was constructed in Mesquite v3.2 [18]. The character matrix is provided in Supplementary Material S2. The proposition of homologies was based on direct observations of adult males and females.
We ran phylogenetic analyses based on maximum parsimony (MP) and Bayesian inference (BI). We ran the MP analyses on TNT [19], using new technology heuristic searches with tree bisection and reconnection, with equal and implied weights (EW and IW, respectively), unevenly scaling the k parameter to investigate how homoplasy impacts tree topology [20,21]. Node support was assessed using the Bremer index (for MP-EW) and symmetric resampling (for both MP-EW and MP-IW) with 1000 replicates.

Characters
Our matrix included 97 characters from male and female specimens, spanning the three tagmata: head (10), thorax (19), and abdomen (68, 41 of which from the aedeagus). For each character, the following is indicated for the MP-EW analysis: the number of steps (L); the consistency index (CI); and the retention index (RI).

Phylogenetic Analyses
Maximum parsimony (MP) applied with equal weights found three most parsimonious trees (L = 339; CI = 43; RI = 60), which were largely congruent with the Bayesian inference (BI) majority consensus (Figure 1 Zoiudo gen. nov. was consistently recovered with high support as sister to Ybytyramoan (Figure 1, Supplementary Material S5). This relationship is supported by eight synapomorphies (seven homoplastic), the most conspicuous ones being the sternum VIII with lateral margins convergent posteriorly (36:1, homoplastic), and the rudimentary ventral plate of phallus (83:3, not homoplastic). Ybytyramoan monophyly was recovered with moderate support in MP (both EW and IW) but with poor support in BI. The latter genus was supported by three homoplastic synapomorphies (Figure 2). Lucidota banoni Laporte, 1833 was consistently found sister to Ybytyramoan + Zoiudo, with moderate support, in all analyses (except the EW, which recovered this node in a polytomy). The most remarkable synapomorphy of Lucidota banoni with (Ybytyramoan + Zoiudo) is the blunt apex of the mandible. Overall, the backbone of Photinini was poorly resolved in all analyses (Figure 1, Supplementary Material S5). When topologies were found with finer resolution on the implied weights maximum parsimony analyses, the nodes were overall poorly supported (Supplementary Material S5).                 Figure  4D); antennae filiform, antennomeres III-X progressively shorter toward apex (Figure 4F); pronotum with posterior angle slightly projected posteriorly ( Figure 5A), anterior expansion strongly convex ( Figure 5A), hypomeron extremely short in lateral view (slightly longer than deep) ( Figure 5E); tibial spurs absent ( Figure 5N); elytra slightly dehiscent ( Figure 5L); abdominal sterna VI and VII with lanterns medially constricted or split in two ( Figure 3B); sternum VIII with lateral margins convergent posteriad, posterior margin truncate, not covered by VII ( Figure 6C); aedeagus with phallobase asymmetrical, dorsal plate of phallus with subapical lateral keels, with a Diagnosis. Head moderately depressed at vertex, antennal socket subtriangular ( Figure 4D); antennae filiform, antennomeres III-X progressively shorter toward apex ( Figure 4F); pronotum with posterior angle slightly projected posteriorly ( Figure 5A), anterior expansion strongly convex ( Figure 5A), hypomeron extremely short in lateral view (slightly longer than deep) ( Figure 5E); tibial spurs absent ( Figure 5N); elytra slightly dehiscent ( Figure 5L); abdominal sterna VI and VII with lanterns medially constricted or split in two ( Figure 3B); sternum VIII with lateral margins convergent posteriad, posterior margin truncate, not covered by VII ( Figure 6C); aedeagus with phallobase asymmetrical, dorsal plate of phallus with subapical lateral keels, with a subapical spike, paramerers coplanar to phallus, with a subapical spike, with a ventral lobe ( Figure 6E-I).
Etymology. "Zoiudo" is a Latinized form of a vernacular Portuguese word meaning "with large eyes". Gender neutral.
Coloration ( Figure 3A,B). Body overall brown to dark-brown, except for: antennal scape light brown; light brown pronotal expansions, and elytral inner and outer margin (outlined in light brown, outer stripe sometimes reaching elytron mid-width); legs light brown up to basal 1/3 of tibia, then dark brown, with brown tibia and tarsus; abdominal segments VI and VII variably translucent; pygidium light brown, often with a darker stripe at central 1/3 to 1/4. Sternum VIII with translucent lateral spots.
Immature stages and female. Unknown. Distribution. The genus is known from Chapada dos Guimarães, Mato Grosso State (Cerrado Biome), and São Bento do Sul, Santa Catarina State (Araucaria Forest in Atlantic Rainforest Biome), Brazil ( Figure 10). The distribution and diversity of this genus is likely underestimated due to lack of targeted sampling, at least between these two largely distant sites.
Biology. Zoiudo rosae sp. nov. males were collected at night using light traps (white sheet). Along with the species overall morphology, with bulgy eyes, short antennae, and the presence of lanterns, males might be following light signals from females. Given the striking similarity between the two species of Zoiudo gen. nov., it is likely that the Zoiudo araucariorum sp. nov. has a similar biology.
Remarks. Zoiudo gen. nov. can be distinguished from all other Photinini genera by the combination of medially constricted or split lanterns on sterna VI and VII, absence of tibial spurs, and the presence of lateral keels on the dorsal plate of phallus. Among Photinini genera, Zoiudo gen. nov. is morphologically similar to Ybytyramoan, with which it shares the large eyes, housed under pronotal bulges, pronotum without a notch on the posterior angle, sternum VIII with lateral margins convergent posteriorly, parameres with a subapical spike. However, Zoiudo gen. nov. has the pronotal posterior angles slightly projected (obtuse in Ybytyramoan), elytra dehiscent (elytra widest at the posterior 1/3 in Ybytyramoan), tibial spurs absent (each leg with a pair of tibial spurs in Ybytyramoan), rounded and medially constricted to completely separate and divided lanterns (rounded or billycock-shaped in Ybytyramoan), pygidium somewhat heart-shaped (with sides rounded in Ybytyramoan), dorsal plate of aedeagus without a subapical dorsal keel (present in Ybytyramoan), and with subapical lateral keels (absent in Ybytyramoan). Zoiudo gen. nov. is also similar to Photinus in outline, from which it can be easily differentiated by the absence of a pronotal notch on the posterior angle, absence of tibial spurs (tibial spur formula 1-2-2 or 2-2-2 in Photinus), the sternum VIII coplanar with VII (usually partially retracted under VII in Photinus), and the lack of phallic ventrobasal processes (present in Photinus). Biology. Zoiudo rosae sp. nov. males were collected at night using light traps (white sheet). Along with the species overall morphology, with bulgy eyes, short antennae, and the presence of lanterns, males might be following light signals from females. Given the striking similarity between the two species of Zoiudo gen. nov., it is likely that the Zoiudo araucariorum sp. nov. has a similar biology.
Remarks. Zoiudo gen. nov. can be distinguished from all other Photinini genera by the combination of medially constricted or split lanterns on sterna VI and VII, absence of tibial spurs, and the presence of lateral keels on the dorsal plate of phallus. Among Photinini genera, Zoiudo gen. nov. is morphologically similar to Ybytyramoan, with which it shares the large eyes, housed under pronotal bulges, pronotum without a notch on the posterior angle, sternum VIII with lateral margins convergent posteriorly, parameres with a subapical spike. However, Zoiudo gen. nov. has the pronotal posterior angles slightly projected (obtuse in Ybytyramoan), elytra dehiscent (elytra widest at the posterior 1/3 in Ybytyramoan), tibial spurs absent (each leg with a pair of tibial spurs in Ybytyramoan), rounded and medially constricted to completely separate and divided lanterns (rounded or billycock-shaped in Ybytyramoan), pygidium somewhat heart-shaped (with sides rounded in Ybytyramoan), dorsal plate of aedeagus without a subapical dorsal keel (present in Ybytyramoan), and with subapical lateral keels (absent in Ybytyramoan). Zoiudo gen. nov. is also similar to Photinus in outline, from which it can be easily differentiated by the absence of a pronotal notch on the posterior angle, absence of tibial spurs (tibial spur formula 1-2-2 or 2-2-2 in Photinus), the sternum VIII coplanar with VII (usually partially retracted under VII in Photinus), and the lack of phallic ventrobasal processes (present in Photinus).
Key to Zoiudo gen. nov. species 1 Body length around 18.8 mm, phallus with lateral keel of dorsal plate smooth (except for subapical spike, Figure 6E-I), surface of parameres almost smooth (with few dome-shaped sensillae), Zoiudo rosae sp. nov. Key to Zoiudo gen. nov. species 1 Body length around 18.8 mm, phallus with lateral keel of dorsal plate smooth (except for subapical spike, Figure 6E-I), surface of parameres almost smooth (with few dome-shaped sensillae), Zoiudo rosae sp. nov.
Etymology. "rosae" is a noun in the genitive case. This species is named in honor of the Brazilian entomologist Simone Policena Rosa, a leading expert on Elateroid groups, with a special emphasis on their immature stages. Simone encouraged and auxiliated us to study fireflies. She was also among the collectors of the specimens of this new species.
Etymology. "araucariorum" is a noun in the genitive case, after the Araucaria trees, which are abundant on the type locality of this new species.

Phylogeny and Classification of Photinini
Our study used morphological characters to address the phylogeny of Photinini to inform the taxonomy of Zoiudo gen. nov. Our study found Zoiudo gen. nov. to be the sister of Ybytyramoan, with high support (see above). This node has been consistently found sister to Lucidota banoni, the type species of its genus. The other Lucidota species in our sampling, L. atra Olivier 1790, was recovered at another branch, consistent with the artificial definition of this genus [6].
A few other clusters are recurrent in recent phylogenies. For example, Scissicauda + Haplocauda, Alychnus + Photinus, Costalampys + Dadophora, and Luciuranus (Lucidota atra (Phosphaenus + Phosphaenopterus) are all well-supported nodes. In contrast, the backbone of Photinini was largely unresolved or resolved without support, a recurrent pattern in published morphology-based phylogenies of the Lampyridae [4,5,7]. In addition, the finer resolution of the analyses with implied weights is sensitive to K value. The lack of resolution might suggest a relatively fast radiation of this lineage, consistent with the greater species diversity of this branch.
Even though nearly half of Photinini genera are represented in our study, the sampling within genera is still rudimentary to subsidize a comprehensive taxonomic review of this tribe. Future studies should focus on covering more taxa and morphological characters and combine with other data sources when available.

Hypothesized Habits of Zoiudo
Individuals of Zoiudo gen. nov. are yet to be observed alive. However, comparing its morphology with other known Photinini could provide insight on its biology. Ybytyramoan-the sister taxon of Zoiudo gen. nov.-is known from three nocturnal species in which males produce long single-flashes ( [9], Silveira, pers. ob.), like some Photinus spp. [29]. However, the relatively larger eyes and shorter antennae of male Zoiudo gen. nov. are closer to those Magnoculus McDermott, 1964, Lamprohiza Motschulsky, 1853, and Phausis LeConte, 1851-rather than Ybytyramoan-suggesting that they might be after glows, not flashes, as courting signals [30].

Conclusions
Here we described two new species of Photinini, recovered as a new monophyletic genus close to Ybytyramoan. Our study confirms the value of extensive character and taxon sampling towards a revised classification of Photinini taxa and highlights the need for the continued sampling and protection of South American biomes before they are gone.