﻿Phylogenetic analysis reveals a new net-winged beetle genus of Eurrhacini (Coleoptera, Lycidae) from the Pacific slopes of Central America and Ecuador

﻿Abstract The first phylogenetic inference of Calopterini and Eurrhacini focused on Calocladon and related taxa was carried out. A data matrix composed of 46 species and 51 morphological characters was assembled and analyzed using parsimony and model-based approaches. Eurrhacini were recovered monophyletic. Furthermore, phylogenetic analyses highly supported the Calocladon clade including also Atlanticolycus, Cladocalon, and Gorhamiumgen. nov. as its sister clade. Our trees consistently recovered monophyly of the new genus with two new species: Gorhamiumbidentatumsp. nov. (Panama, Baru Volcano) and G.unidentatumsp. nov. from the Pacific slopes of Ecuador. A revised key to the genera of Eurrhacini is given and illustrations of distinguishing characters are provided. Phylogenetic relationships of Eurrhacini and character evolution are discussed.


Introduction
The Eurrhacini is a Neotropical lineage of Lycidae, which until recently was part of the tribe Calopterini in the broader sense (Bocakova 2003(Bocakova , 2005)).However, the inclusion of Eurrhacini in Calopterini was challenged by the first molecular analysis of Lycidae (Bocak et al. 2008) which showed that Eurrhacus Waterhouse, 1879 is sister to the Oriental Conderis.Consequently, Eurrhacini was excluded from Calopterini (Bocak and Bocakova 2008) and elevated to the tribal rank.Nevertheless, inferring the Eurrhacini sister group is convoluted because DNA analyses proposed several candidates.Recent molecular trees on large data sets (Masek et al. 2018) recovered the Eurrhacini sister is either American Thonalmini or Oriental Lycoprogenthini, thus indicating that Calopterini and Eurrhacini are not sister lineages.
The placement of Eurrhacini in the Calopterini was based on their resemblance, as the two groups often have similar coloration.Eurrhacini, however, are characterized by a very long male terminal sternum, which is twice as long as that of the Calopterini, and a distorted phallus and phallobase.When ZooKeys 1204: 241-259 (2024), DOI: 10.3897/zookeys.1204.114932 Elynton Alves Nascimento & Milada Bocakova: A new lycid genus from Central America and Ecuador established (Bocakova 2005), Eurrhacini included six genera.Of these, Calocladon Gorham, 1881 has a markedly elongated pronotum, Lycoplateros Pic, 1922 is characteristic by a conspicuous protuberance on the posterior margin of the pronotum, and Haplobothris Bourgeois, 1879 is easily distinguishable by the absence of secondary elytral costae.The remaining three genera (Eurrhacus, Emplectus Erichson, 1847, andNeolinoptes Nascimento &Bocakova, 2017) are less distinctive externally, but easily separated by the shape of the male genitalia.
Likewise, species of the recently discovered Cladocalon Nascimento & Bocakova, 2022, Currhaeus Nascimento, Bressan &Bocakova, 2020, andAtlanticolycus Nascimento &Bocakova, 2023 were originally placed in Calocladon, as they are similar to Calocladon and Emplectus (Nascimento et al. 2020;Nascimento and Bocakova 2022).However, showing great male genitalia disparity, they were assigned to generic rank.Recently, an examination of H. S. Gorham's types from Panama and further research on material from Ecuador have revealed another previously hidden generic lineage described below.Here we elucidate phylogenetic relationships of the group and its placement within Eurrhacini by analyzing morphological data.

Materials and methods
The morphological matrix is based on that of Bocakova (2005), updated by Nascimento et al. (2020) and Ferreira et al. (2023).The dataset was expanded by the inclusion of two recently proposed Eurrhacini genera (Atlanticolycus, Cladocalon) and the new one described here (altogether six newly coded species).Our final matrix (Table 1) is composed of 46 species and 51 characters (Suppl.material 1), including five outgroup taxa.Of these, ten characters were coded to multistates, 41 characters as binary.Eight additional characters (#44-51) were newly defined, other characters required the inclusion of new character states, or minor redefinition.Unknown and inapplicable characters were coded by a question mark "?", or a dash "-", respectively.
Phylogenetic analyses were conducted using maximum parsimony (MP), Bayesian (BA), and maximum likelihood (ML) criteria.MP analyses were performed in TNT 1.5 (Goloboff et al. 2008;Goloboff and Catalano 2016) using traditional search with characters treated as unordered.MP trees were evaluated by tree length (TL), consistency (CI), and retention indices (RI), and summarized in strict and majority rule consensus trees.Initial fundamental analyses with equal weights were followed by searches with implied weighted schemes (Goloboff 1993) with concavity constant k = 3-25.Standard bootstrapping (Bootstrap support, BS) and symmetric resampling (SR) with 1000 replicates were applied to the unweighted dataset to assess the branch support.Furthermore, Bremer support values (BrS;Bremer 1994) were calculated in TNT for the clades of the unweighted MP tree.Character optimizations were mapped on the strict consensus tree using unambiguous changes, accelerated (ACCTRAN) and delayed (DELTRAN) transformations in WinClada (Nixon 2002).
Maximum likelihood (ML) searches were applied under IQ-Tree 2 software (Minh et al. 2020) with branch support estimated by ultrafast bootstrapping (UF-Boot) using 1000 replicates.The best-fit model was selected by ModelFinder (Kalyaanamoorthy et al. 2017) according to Bayesian information criterion
Approximately 100 Eurrhacini specimens were examined using an Olympus SZX 12, or Zeiss Stereo Discovery V8 stereoscopic microscopes.Eyes are differentiated into small, medium-sized, and large.In medium-sized eyes, the eye diameter is equal to the interocular distance; in small eyes the eye diameter is less than the interocular distance; in large eyes the eye diameter is greater than the interocular distance.Nine longitudinal elytral costae are distinguished in four strong primary costae and five less elevated alternate secondary costae.Costae and intercostal intervals are numbered from the suture as in other Coleoptera.Dissection of genitalia was made after boiling in 10% KOH solution and followed previous studies (Nascimento and Bocakova 2017).Relative measurements were taken using an ocular micrometer, and dimension measurements (in millimeters) and scale bar insertions were processed by the camera software.Digital photographs were taken using an attached Canon EOS 1100D camera and stacked by QuickPhoto Camera 3.0 microscope software using a Deep Focus 3.3 module.Images were further edited in GIMP 2.10.22 and Adobe Photoshop CS3.
The syntypes of Calocladon chiriquense Gorham, 1884 were borrowed from The Natural History Museum (NHMUK) in London, U.K., while other material is deposited in the collection of Palacky University Olomouc (UPOL), Czech Republic.
Etymology.The genus is named in honor of H. S. Gorham, the author of chapters on Malacodermata in Biologia-Centrali Americana (Gorham 1880(Gorham , 1881(Gorham , 1884)), where he described many genera and species of Eurrhacini and Calopterini.The gender is neuter.
Etymology.Named after the shape of apical portion of parameres.Distribution.Panama.Diagnosis.Pronotum black.Elytra bicolor orange-black with suture, longitudinal median oval spot, and triangular apical spot black.Phallus ball-shaped apically, ventromedial opening widest in basal third.Apex of each paramere fitted with a sharp laterally projected tooth, internal sac with a series of diminutive teeth (e, Fig. 6G).
Etymology.The specific name refers to the single sharp tooth at the apex of each paramere. Distribution.Ecuador.

Discussion
The Calopterini Support for a monophyletic origin of Calopterini and the subtribe Calopterina has been confirmed by previous (Bocakova 2005;Nascimento et al. 2020;Ferreira et al. 2023) and our current (this study) morphology-based analyses.However, the formerly recovered Acroleptina, comprising all neotenic calopterins, is now predominantly paraphyletic (Table 2) and split into two lineages.

The Eurrhacini
Consistent with our results, previous analyses supported the Eurrhacini and showed Haplobothris as the deepest branch.The initial trees (Bocakova 2005;Nascimento et al. 2020) further implied an early separation of Calocladon.However, after the inclusion of Xenomorphon, an enigmatic anelytrous beetle male (Ferreira et al. 2023), Calocladon was recovered as a crown group being sister to Lycoplateros, although support values were low.By contrast, our results (Fig. 1) have indicated the Calocladon clade is sister to Emplectus + Eurrhacus + Currhaeus clade, whereas Lycoplateros is recovered as one of early Eurrhacini branches.

The Calocladon clade
Our updated dataset is the first to include the recently described Atlanticolycus (Brazil), Cladocalon (Mexico, Guatemala, and Panama), and Gorhamium gen.nov.(Panama, Ecuador) proposed here.The analyses show Calocladon and the three closely related genera constitute a highly supported clade (UFBoot = 88, pp = 0.93).Members of the Calocladon clade share two unambiguous synapomorphies (Suppl.materials 3-5), particularly the convergent ventrobasal projections on the parameres that often fuse medially into a ventral bridge (character 45, state 1; Fig. 6G, d 3 ).The character is present in all genera, although the length and thickness of these projections varies.While Calocladon and Atlanticolycus have the strongly developed ventral bridge of the parameres, the ventrobasal parameral projects are often shorter and less pronounced in the Cladocalon + Gorhamium clade.The second unambiguous synapomorphy of the Calocladon clade is the strong, sharply triangular, or inverted mushroom-shaped base of phallus (character 47, state 1).Furthermore, Cladocalon has the characteristic L-shaped parameres.The feature is also present in Gorhamium unidentatum sp.nov.(Fig. 6G), while it is only indicated in G. bidentatum sp.nov.(Fig. 6C, D).Genera Cladocalon, Atlanticolycus, and Gorhamium gen.nov.also share several external characters as flabellate antennae in males, transversely trapezoidal pronotum with lenticular median areola (areola absent, replaced by median longitudinal carina in Lycoplateros and Neolinoptes), and each elytron with nine longitudinal costae (i.e., secondary less elevated alternate costae present).Conversely, secondary costae are absent in Haplobothris (each elytron with only four longitudinal costae).While in Eurrhacus and Lycoplateros primary costae 1 and 3 are strongly elevated, the genera of the Calocladon clade have primary costae 1 and 3 only slightly thicker compared to primary costae 2 and 4.These features are also shared by Calocladon, except for its characteristic elongated pronotum and considerably more slender median areola.(Bocakova 2005;Bocak and Bocakova 2008;this study).Branch support values are based on ultrafast bootstrapping (UFBoot) for maximum likelihood (ML) and posterior probabilities (PP) for Bayesian analyses (BA).For maximum parsimony (MP) analyses, clade percentages on the majority-rule consensus tree (MRCT), standard bootstrapping (BS) and symmetric resampling (SR) values are given.Abbreviations: P -paraphyletic or polyphyletic; NWno weights, IW -implied weights.

Figure 1 .
Figure 1.Maximum likelihood phylogeny of Calopterini and Eurrhacini inferred from the morphological dataset using IQ-Tree 2 and the best-fit MK+FQ+ASC+G4 model selected by ModelFinder.Node labels represent ultrafast bootstrap support values.

Table 1 .
Data matrix of 51 morphology-based characters of Calopterini and Eurrhacini used in phylogenetic analyses.

Table 2 .
Support for major Calopterini and Eurrhacini lineages