Phylogenetics of Ogyges Kaup and the biogeography of Nuclear Central America (Coleoptera, Passalidae)

Abstract A phylogenetic morphological analysis of the genus Ogyges Kaup, distributed in Nuclear Central America, from Chiapas, Mexico, to northwestern Nicaragua was undertaken. Five species of Proculejus Kaup, distributed north of the Isthmus of Tehuantepec in Mexico, were selected as outgroup. Ogyges was recovered as monophyletic with three species groups: championi, laevissimus, and crassulus. Each species group shows a distinct, generally allopatric distribution. The O. championi species group, with ten species, is distributed in the Maya block, more specifically in the mountainous system north of the Motozintla-Comaltitlán fault in Chiapas, and north of the dry valleys of the Cuilco and Motagua rivers in Guatemala. The two remaining species groups are distributed in the Chortis block. The O. laevissimus species group, including seven species, ranges mostly along the Pacific Volcanic Chain from Guatemala to El Salvador, and from southeastern Honduras to the northwestern area of Nicaragua. The O. crassulus species group, with ten species, is distributed from northeastern Guatemala (Merendón) to northern Honduras. The Isthmus of Tehuantepec in Mexico, the Motagua-Cuilco and Motozintla-Comaltitlán sutures zones in Chiapas and Guatemala, the lowland valleys of Colón and Comalí rivers between Nicaragua and Honduras (or, perhaps, the northern suture of the Siuna Terrane in Nicaragua), the Guayape fault system in Honduras, and the intricate dry valleys of Ulúa-Chamelecón-Olancho in Honduras, are hypothesized to have acted as barriers that affected the geographical distribution of Ogyges, as well as probably other montane organisms.


Introduction
Nuclear Central America (Schuchert 1935), the mountainous region comprising Chiapas (Mexico), Guatemala, Belize, El Salvador, Honduras, and northern Nicaragua, is characterized by several large and high mountain and volcanic ranges reaching an elevation of 4222 m, separated by deep and dry valleys, with the consequent isolation and independent evolution of populations. With few exceptions (e.g. Wake and Lynch 1976, Johnson 1989, Campbell and Frost 1993, Townsend 2014, Pérez-Consuegra and Vásquez-Domínguez 2015, Hofmann and Townsend 2017, its biotic relevance has been overlooked by biogeographers, and phylogenetic analyses of taxa endemic to this area are scarce. The biota has been studied as part of North American, Neotropical, Mexican, Middle American, Mesoamerican or Central American regions, and often is considered a "mixture" of North and South American elements, obscuring the in situ diversification of supraspecific taxa. Nuclear Central America is particularly speciose in endemic taxa such as plethodontid salamanders (Campbell et al. 2010, Townsend 2014, Rovito et al. 2015, cricetid mice (Conroy et al. 2001;Vásquez-Domínguez 2012, 2013;Ordóñez-Garza et al. 2014;Pérez-Consuegra and Vásquez-Domínguez 2015), squamates (Campbell and Frost 1993, Campbell and Brodie 1999, Castoe et al. 2003, Hasbún et al. 2005, Townsend et al. 2013, Hofmann and Townsend 2017 and beetles (Schuster 1993, Micó et al. 2006, Cano 2014, Sokolov and Kavanaugh 2014.
Ogyges Kaup, a flightless genus of the saproxylophagous family Passalidae, consists of 25 described species restricted to the cloud forests of Chiapas to northern Nicaragua (Cano 2014(Cano , 2017. A phylogenetic morphological analysis recovered Ogyges as monophyletic and closely related to the also flightless genera Proculus and Proculejus (Boucher 2006). Cano (2014) showed that the shape of the suprainternal mandibular tooth represents an exclusive synapomorphy for the species of Ogyges. Proculus includes seven species, all gigantic (50-80 mm) and with many autapomorphies, distributed in Nuclear Central America from the Chimalapas region, Oaxaca (Delgado and Mora-Aguilar 2014), to northern Honduras, being also probably distributed in the Chocó area in Colombia (Schuster et al. 2003). Proculejus is found in Mexico north of the Isthmus of Tehuantepec (Reyes-Castillo 1970, Boucher 2006, it includes at least 10 species and is rather similar to Ogyges, except for the presence of a frontoclypeal suture and a different form of suprainternal mandibular tooth. The recent discovery of a new Honduran species with a clearly marked suture on the frontoclypeus makes it difficult to place it in either genus. A phylogenetic morphological analysis was undertaken to test the monophyly of Ogyges, including the new Honduran species, and using Oileus sargi (Kaup) and five species of Proculejus as outgroups. Based on the resulting cladogram, we conducted a biogeographical analysis to describe the areas of distribution and possible barriers, applying the results of the analysis of the biogeography of Nuclear Central America in an evolutionary framework.

Methods
1073 adult specimens were examined (see Appendix 1), belonging to 33 species, deposited in the following collections: For terminology of the head Boucher (2006) is followed, which is based on wellsupported homologies; however, instead of the terms central tubercle, orbital canthus, and inner tubercles, we use center horn, ocular canthus, and internal tubercles, respectively. For terminology of the rest of the body we follow Reyes-Castillo (1970). Measurements were taken with a digital vernier caliper except for the diameter of punctures and the antennal and femoral proportions, which were taken with an ocular micrometer in a Wild Heerbrugg M3B stereomicroscope. Total length was measured from the tip of the open mandibles to the terminal tip of the elytra. Drawings were made using a drawing tube in a Wild Heerbrugg M3B stereomicroscope. Images were taken with a Nikon D5100 camera with macro lens, except those of teeth of mandible, taken with a camera DP12 adapted to a SZX12 Olympus stereomicroscope. All images were processed with the Microsoft Digital Image Pro software.

Outgroup selection
Although Boucher (2006: 346, 364) recovered Proculus as the sister group of Ogyges, he also encountered more than 20 autapomorphies (i.e. uninformative characters) in Proculus. Proculus, the giant passalid beetles, have more autapomorphies than any known passalid, perhaps associated with its greater size. According to Maddison et al. (1984) and Nixon and Carpenter (1994), plesiomorphic-synapomorphic states should be estimated from the outgroup, however, as Lyons-Weiler et al. (1998) state, if rates of evolutionary change vary among lineages, the sister taxon (as apparently occurs in Proculus) may not have the shortest evolutionary distance to the ingroup, reducing the chance that it is the optimal candidate for estimating the ingroup. In addition, outgroup choice can affect ingroup topology, even for nodes far removed from the presumed root placement (Milinkovitch andLyons-Weiler 1998, Tarrio et al. 2000). On the other hand, the suprainternal teeth of mandibles of Ogyges and several species of Proculejus seem to be very similar, suggesting common ancestry and the possibility that both genera can be merged. For these reasons, and, in order to evaluate the monophyly of Ogyges, we selected five species of Proculejus as the outgroup to infer and select the synapomorphies and plesiomorphies of Ogyges, rejecting Proculus as a second outgroup. Additionally, due to the homoplasy involved in flightlessness, we selected the flying species Oileus sargi Kaup to root the resulting cladograms.

Character analysis
A total of 53 morphological characters was used, including both external structures (48) and male genitalia (5). The distribution of character states is shown in Table 1. All multistate characters (1, 2, 7, 8, 14, 17, 19, 22, 26, 30, 32, 33, 34, 36, 39, 40, 45, and 52) were treated as non-additive. Inapplicable characters, those that describe variation with respect to the shape of some feature that is entirely absent in some taxon (Harris et al. 2003: 249), were avoided, except for characters 1, 9 and 46. Alar reduction is widely present in several unrelated genera and species of Passalidae. Brachypterism, together with the associated morphological modifications, shared by all species of Ogyges, is a potential synapomorphy of this taxon. Nevertheless, in order to clarify the relationships with Proculejus, which is a primarily brachypterous genus, we selected only one character, the humeral callus of elytra (character 43), to distinguish brachypterous species from the flighted outgroup species Oileus sargi and Proculejus nudicostis. The bluish iridescence (character 42), when present, may appear on various areas of the body; to avoid overweighting this character, we considered it only once in the analysis.

List of selected characters
0. Frontoclypeal suture: (0) clearly present; (1) absent. 1. Clypeus: (0) delimitated from frons by a complete strong, transverse impression; (1) with a shallow and incomplete or insinuated delimitation with some granulations; (2) flat, without any indication of separation (although an abrupt change in plane is present in species with a vertical clypeus). 2. Clypeus: (0) very thick, forming a transversal and convex tumosity; (1) thin, tapering as a razor blade towards the apex; (2) same thickness all along, not thinned or thickened at apex. 3. Clypeus: (0) inclined; (1) vertical. (1) present, scarce (almost smooth); (2) present, densely abundant. When present, granulations are distributed on areas of the frons and/or vertex, particularly around the epicranial sutures (Boucher 2006). We assume covariation in this character and, in order to avoid double weight we only considered granulations on   (2) present, defined or diffused; origin ("angle") at level or surpassing the eyes (laevissimus type; Figure 4b). Although in the original description Schuster and Reyes-Castillo (1990) indicate that O. furcillatus lacks posterofrontal ridges, the assumption of presence of internal tubercles by the authors suggest a fusion of both characters. We assume the presence (as hyperthelic) in O. furcillatus. In O. quichensis, the development of the posteriorly massive center horn, at the level where the junction ("angle") of the lateroposterior tubercles should be, obscures the presence of the character; nevertheless, most specimens have a ridge very posterior to the level of the eyes and we consider that, although not linear (due to a modification in the center horn, as in O. furcillatus) to be the posterofrontal ridge, corresponding to the championi type. In P. pubicostis, P. nudicostis and O. cavei the ridge is anterior (laevissimus type). 9. Posterofrontal ridges: (0) linear, clearly marked; (1) tumid on each side of the center horn, and then forming a clear (or diffuse) keel running to the sides of frons, marking the anterior margin of the lateropostfrontal areas (frontal fossae). State (1) has not been considered by Cano (2014) and Schuster and Reyes-Castillo (1990) as presence of the posterofrontal ridges ("quillas frontales" or frontal ridges). After a careful examination of tenerals and specimens cleared with KOH we conclude that state (1) of this character is homologous but distinct from character state (0) of the typical O. championi. 10. Area between laterofrontal tubercles and epicranial suture: (0) not shagreened; (1) shagreened. 11. Dorsal groove of center horn: (0) absent or indistinct (Figures 4b, 5a); (1) present, clearly marked (Figures 1b, 4a). 12. Length of center horn (base to tip): (0) short, not surpassing the level of eyes; (1) long, surpassing the level of eyes. 13. Sides of postfrontal groove: (0) shallow, at the same depth as lateropostfrontal areas; (1) deep, more than depth than the lateropostfrontal areas. 14. Supraocular fossae: (0) absent or, at most, a barely indicated impression, less than the length of half of an eye (  (1) setose only on intervals 8-10.  length as a discrete variable (small/medium/large), based on average body length (error bars) of at least three specimens (one or two in species only known from these number of specimens).
The cladograms were constructed using TNT software (Goloboff et al. 2008). A preliminary analysis was conducted assigning all characters equal weights. We then tested the effect of homoplasy on the results by conducting different implied weights analyses (Goloboff 1993), with the constant of concavity (k) set to integer values from 1-12, where 1 was weighted most severely against homoplastic characters. Implied weights analyses were conducted using the heuristic "traditional search" algorithm of TNT, with 1000 replications and tree-bisection-reconnection branch-swapping (TBR), holding 1000 trees during each replication.

Biogeographical analysis
The distribution of individuals of all species of Ogyges were plotted on a map, using ArcGIS 9.2. After the phylogenetic analysis, the range of each well-supported clade (but not of individual species) was colored. Barriers were hypothesized in relation to the dry (to moist) lowland valleys (principally below 1000 m in elevation) and major fault systems separating mountainous/volcanic ranges, and were analyzed and defined. The distributions of the individual species have been previously mapped by Schuster and Reyes-Castillo (1990: 15, 24, 30, 40), Schuster et al. (2005: 117), and Cano (2014: 25).

Phylogenetic analysis
The analysis of the data matrix (Table 1) under equal weights led to six cladograms, with the constant of concavity (k) set at 3 led to three cladograms, and with k = 12 led to a single cladogram with 181 steps, CI of 0.403 and RI of 0.754 ( Figure 6). In all the analyses, the 27 species of Ogyges were recovered as a monophyletic group, as generally occurred with the five species of the outgroup Proculejus. We recognize three main clades within Ogyges, named O. laevissimus species group, O. championi species group, and O. crassulus species group (Figure 6).

Biogeographical analysis
Based on the cladogram ( Figure 6) and the ranges of the species, Ogyges and its three consistent clades show clear distributional patterns (Figure 7). Ogyges is separated from  (Figure 7), with 10 species, is distributed in Chiapas, Mexico, in the northern mountain system from San Cristóbal de las Casas to Lagunas de Montebello, and in the southern system of mountains in the "El Triunfo" Biosphere Reserve; and in Guatemala, in the Sierra de los Cuchumatanes, Montaña Cuilco, Sierra de las Minas and Sierra de Santa Cruz. The distribution of this species group corresponds to the Maya block (Dengo 1969) whereas the other two species groups are endemic to the Chortis block (Dengo 1969). Apparently dryness (now and in past geological times) of the Motagua-Cuilco and Motozintla-Comaltitlán suture zones is the barrier separating it from the O. crassulus and O. laevissimus species groups, and corresponds (partially) to the subhumid corridor delineated by Stuart (1954).
The distribution of the O. laevissimus species group (Figure 7) extends mostly along the Pacific Volcanic Chain from Guatemala to El Salvador, to the north in Guatemala (Zacapa Department), and then to southeastern Honduras and northern Nicaragua, where the distribution corresponds approximately to the Southern Cordillera of the Honduran Chortis highlands as defined by Weyl (1980: 93-94) and highlighted by Townsend (2014: 214). This species group is separated from the majority of species of the O. crassulus species group by a series of intricate lowland (about 300-700m elevation) dry forests between the Central and Southern Cordilleras, perhaps related to the Ulúa-Chamelecón-Olancho system. However, two species of the O. laevissimus species group [O. cavei at Sierra de Agalta (Cerro La Picucha), and O. adamsi at Montaña Santa Bárbara] are sympatric with species of the O. crassulus species group, making the limits unclear. The eastern limit to the distribution of the O. laevissimus species group, and also of the genus Ogyges, appears to be the lowland moist to dry valleys of the Colón river in Nicaragua (0-700m) and its tributary, the Comalí river in Honduras (730-950m), or possibly the northern suture of the Siuna Terrane (Venable 1994) in Nicaragua (Figure 7). Alternatively, the moist (Atlantic) to dry (Central and Pacific) lowland (0-800m) Guayape fault system (Finch and Ritchie 1991) could be considered as a major barrier (Figure 7).
The O. crassulus species group, with 10 species, is distributed almost exclusively in northern Honduras, slightly extending to Guatemala at the Sierra del Merendón (Figure 7). It corresponds well with the Northern and Central Cordilleras of the Honduran Chortis highlands (Weyl 1980: 92-94, Townsend 2014. The Guayape fault system (Figure 7) represents the eastern distributional limit of this species group.

Phylogeny
The high homoplasy levels (CI=0.403) could be explained by the covariation of characters associated with flightlessness in taxa of Passalidae (reduced eyes, very narrow wings, and oval and fused elytra), but also because they have similar ecological niches (interior of rotten logs in humid forests). Flightlessness appears to have evolved several times in montane passalids, occurring in unrelated genera (e.g., Passalus, Chondrocephalus, Veturius, Arrox, etc.) and the body shape of passalids living in sapwood/heartwood tends to be convex (Johki and Kon 1987, Lobo and Castillo 1997, Kon et al. 2002. In addition to the character used traditionally to separate Ogyges from Proculejus, the frontoclypeal suture, we consider the shape of the internal teeth of the mandibles, the punctate border of the pronotum, the sculpture of the prepimeron, and the lateral setation of the elytra to be the most relevant. Of these, until now, only the form of the internal teeth has proven to be stable and autapomorphic in Ogyges (also see Cano 2014). Nevertheless, a clearly marked frontoclypeal suture appeared only once in a terminal species (a reversal) of the O. crassulus species group, suggesting that the character is homoplastic in Passalidae.
The genus Proculejus urgently needs to be revised. At least two species, P. nudicostis Bates and P. obesus (Bates), do not share with the other species in the genus the bidentate mandibles, the laterally setose elytra and the shape of the internal teeth, characters traditionally used to diagnose the genus. Additionally, in one of our phylogenetic analyses (concavity k = 3, strict consensus), P. nudicostis was recovered as basal and excluded from Proculejus, bringing into question the monophyly of the genus.
Based on the phylogeny and distributions of more than twice as many species as were available to Schuster and Reyes-Castillo (1990: 40-45), we reject some of their groupings within Ogyges that were not based on phylogenetic analyses, and we suggest others that appear more natural and well-supported ( Figure 6).

Biogeography
Ogyges belongs to the Mesoamerican Montane cenocron (Morrone 2015). According to Halffter (1987), taxa belonging to it evolved in Nuclear Central America and then dispersed northwest and southeast from there. They have ancient South American affinities and are distributed mainly in montane cloud forests, although they penetrate occasionally into pine-oak forests. In the Oligocene-Miocene they dispersed from Central America northward (Halffter and Morrone 2017).
Regarding the vicariance between Proculejus and Ogyges, the Isthmus of Tehuantepec has been considered as a biogeographic break for several taxa (Marshall andLiebherr 2000, Morrone andMárquez 2001). A vicariant event during the Pliocene has been suggested as responsible for the divergence of several taxa, although an earlier vicariance at the end of the Miocene may have also occurred (Daza et al. 2010).
The Motagua-Polochic-Jocotán fault has been invoked as a sharp biogeographic break for vertebrate taxa (Castoe et al. 2009, Daza et al. 2010, Pérez-Consuegra and Vásquez-Domínguez 2015. We suspect that, for flightless passalids, although low elevation areas may be barriers, they are more effective when they are dry, at least at present. The Polochic suture zone valley (parallel to the north of the Motagua suture valley), is moister than the Motagua valley and does not separate species of passalids as well as does the Motagua; for example, three species of the O. championi species group are found on both sides of the Polochic suture zone (O. tzutuhili, O. kekchii, and O. championi). Here we recognize the Motagua-Cuilco (0-2000 m) system of dry valleys and the Motozintla-Comaltitlán suture zones (0-1900 m), as the major biogeographic barrier involved in the vicariance between the O. championi species group of the Maya block and the rest of the genus distributed in the Chortis block. The Motagua suture zone (although, together with the Polochic suture zone, according to authors) has been proposed as a barrier for several lowland and highland vertebrates from ~3-8 mybp (Daza et al. 2010: 351), or from ~4-5.5 mybp (Castoe et al. 2009: 95).
The distributional barriers between the O. crassulus and O. laevissimus species groups are unclear. Species of the O. laevissimus species group are distributed in the Quaternary Volcanic Chain of Guatemala and El Salvador, and the Tertiary Volcanic Southern Cordillera of the Chortis highlands in Honduras. But, again, the lowland dry valleys, such as the labyrinthic systems between the Ulúa and Chamelecón rivers and the Olancho Department in Central Honduras, merge as barriers. As to the timing of taxon divergence, Townsend (2014) suggests that most of the biota of Honduras could not have survived the mid-Miocene volcanic eruptions, when over 5,000 km 3 of ignimbrites up to 2,000 m thick were de posited on top of the low-relief surface of the southern and western Chortis block, and tens of thousands of square kilometers were covered repeatedly in thick layers of ash (Townsend 2014). Thus, we assume that Honduran species of Ogyges would have to have originated after this event (~11-16 mybp). The southern limit of distribution of Ogyges falls in the Sierra of Dipilto and Jalapa, Department of Nueva Segovia, in Northwestern Nicaragua, where mountains exceed 1500 m elevation. We (EBC, JCS) have collected passalids extensively in the cloud forests further south in the mountains (between 1200-1500 m) surrounding Jinotega and Matagalpa (Selva Negra, El Quetzal, Peñas Blancas, La Dalia and Datanlí-El Diablo) and in Granada at Mombacho volcano (1300 m), without finding a trace of Ogyges.