Morphometric changes in wings of bess beetles (Coleoptera: Passalidae) related to elevation: a case of study in the Colombian Andes

ABSTRACT Flight is a crucial evolutionary novelty for insect diversification. Despite its importance, some insects lose this ability. Flight loss is related to the reduction in the veins and membrane of hind wings (brachypterism), often related to high elevations. Brachypterism evolved independently in some lineages of beetles, like in the family Passalidae. Due to the double function of the passalid hind wings (dispersion and acoustic communication), the wing reduction process is partial and apparently constrained by the stridulatory apparatus. The tribe Proculini is a good model to study brachypterism due to the number of flightless species and its distribution between low land and high montane ecosystems. We evaluated the relationship between wing reduction and elevation in the elytra and hind wings of 25 Proculini species distributed in Colombia, using linear measurements and geom-25etric morphometry. Specimens were categorized in three elevation ranges: low (0–999 m), middle (1000–1999 m), and high (<2000 m). In conclusion, this study demonstrates that elevation is most likely the factor that has driven size and shape modifications of the Passalidae hind wings and elytra. The present work is the first study that tests the relationship between brachypterism and elevation using Proculini species belonging to different genera.


Introduction
One of the most remarkable evolutionary novelties for insect diversification is the evolution of wings and the ability of flight (Kingsolver & Koehl 1994;Labandeira et al. 1994;Grimaldi & Engel 2005;Rainford et al. 2014). However, some groups have a partial or complete reduction of wings, leading to the loss of flight. This evolutionary process knowning as brachypterism consists of the reduction or loss of veins and membranes (Chapman 1982). Brachypterism occurs in different groups of insects, causing variations in the size, shape, and functional flight restrictions of the wings (Roff 1990;Wootton 1992). Their evolution is associated with food availability and habitat stability (Roff 1975(Roff , 1990. Brachypterism has evolved independently in different families of Coleoptera being a factor that may promote beetle diversification (Ikeda et al. 2012;Tänzler et al. 2016), given the fast-molecular evolution of flightless species (Mitterboeck & Adamowicz 2013). Wing reduction is coupled with a decrease in the flight nerves and muscles, modification of size and shape in the metathorax, and elytral fusion (Jackson 1928). The flight loss occurs principally in species distributed at a high elevation (Darlington 1970;Brandmayr 1983;Ball 1992;Brühl 1997;Riedel et al. 2010;McCulloch et al. 2019), and this relationship has been suggested but not tested in the families Lucanidae (Endrödi-Younga 1986;Grossi & Almeida 2010) and Passalidae (Reyes-Castillo 1970a;MacVean & Schuster 1981;Moreno-Fonseca & Amat-García 2016).
Passalidae is a family of saproxylophagous beetles with a pantropical distribution, characterized by acoustic communication between individuals (Reyes-Castillo 2000; Boucher 2005). Their hind wings play two roles in both sexes: dispersion and acoustic communication. The stridulatory apparatus is abdomino-alary, composed of spines located in two lateral tubercles in the sixth abdominal tergum (pars stridens) and spines located in the ventral side of the radial cell in hinds wings (plectrum) (Reyes-Castillo & Jarman 1983). Therefore, the functional role of hind wings for acoustic communication causes that no species has a total loss of the hind wings (Reyes-Castillo 1970a;Boucher 2005). The brachypterism is associated with morphological modification in the eyes, antennae, elytra, and metathorax of bess beetles (Hincks 1933;Reyes-Castillo 1970a). Boucher (2005) proposed three types of hind wings: macropterous, hemibrachypterous, and brachypterous, most species have only one type of wing. Passalidae has two subfamilies: Aulacocyclinae and Passalina. Both subfamilies have genera with macropterous (well-developed wings) and brachypterous (reduced wings) species. Passalinae is distributed in the neotropics and has two tribes: Passalini and Proculini (Reyes-Castillo 1970a). The tribe Proculini is a useful model for studying the relationship between brachypterism and elevation due to numerous flightless species and the montane distribution of its genera (Reyes-Castillo 1970a;Boucher 2005). Proculini had a wing plan named Staphyliniformia, as in other families of Scarabaeoidea, characterized by having the maximum width in the middle zone, a small jugal lobe, and a wavy posterior margin (Fedorenko 2009). Intraspecific wing polymorphism in Proculini is rare, just Spurius halffteri (Reyes-Castillo 1970b) and Veturius hincksi have wing polymorphism both with macropterous and hemibrachypterous wings, the latter with an incomplete evolutionary process from macropterism to brachypterism in montane habits (Salazar & Boucher 2018).
Due to the montane distribution of brachypterous species, it was expected that the elytra and hind wings would decrease their size and change their shape when elevation increases. This work aimed to evaluate the morphological changes in the hind wings and elytra (size and shape) related to altitudinal ranges in species of Proculini distributed in the Colombian Andes and other close mountain ranges.

Selection of specimens, extraction, and photographs of wings of passalids
We selected 25 species of six of the seven genera of Proculini with distribution in Colombia (Jiménez-Ferbans et al. 2018) to study the relationship between brachypterism and elevation. A total of 71 specimens were used from the Entomological Collection of the Instituto de Ciencias Naturales, Universidad Nacional de Colombia (supplementary material, Table S1). The sample size depended on the number of specimens deposited in the entomological collection per species.
Specimens were hydrated with hot water for one hour to extract and straighten the left hind wing. Hind wings were spread out and dried between two glass plates with an additional half a pound weight for 36 hours to avoid wing folding. Hind wings were kept in a plastic envelope, built with transparent plastic acetate, and sealed with adhesive tape. Hind wing samples were deposited in the Coleoptera reference collection of the Entomological Collection of the Instituto de Ciencias Naturales of the Universidad Nacional de Colombia, Bogotá. Three labels were added to each wing sample: taxonomic identification, collection information, and an ID of the specimen. Photographs of the ventral side of the hind wing and the dorsal side of the left elytra were taken at a resolution of 96 dpi, with a camera CANON EOS 5D MARK III attached to a fixed camera holder and adding a scale.

Hind wing description
The hind wings in Coleoptera have veins, cells, folding elements, and articulation sclerite. The presence, topography, and morphology of these elements were used to make evolutionary and systematic interpretations (Kukalová-Peck & Lawrence 1993). We used the wing nomenclature proposed by Fedorenko (2009) for the superfamily Scarabaeoidea to describe changes in the configuration of hind wing veins among welldeveloped and reduced wings ( Figure 1).

Linear morphometric data
We took five measurements using a digital electronic caliper to describe the size of the hind wing and the elytra. Hind wing measurements included the total length (lw) and the length between the base and the top of the stridulatory region (lsr) (distance between points 1 and 2, Figure 1). The elytra measurements included the length at the base (le) and the width at the metacoxal level (we). We eliminated the body size effect in the hind wing measurements using ratios with the length of pronotum (lw/lp; lsr/lp) due to the length of the body could be affected by the elytral modification. We calculated elytral aspect ratio (ratio between width and length) to determine elytral variation in its relative length (we/le); values vary between 0 to 1, high ratios mean short elytra ( Figure 2).

Geometric morphometric data
We quantified variation in the shape of elytra and hind wings. The shape of the elytra was sampled with a combination of two landmarks and 28 semilandmarks ( Figure 2). The shape of the hind wing was sampled with seven landmarks, recognizing from the wing nomenclature proposed by Fedorenko (2009): two in the articular sclerites, four in the apex of veins, and one in the top of the radial cell (stridulatory region) ( Figure 3). We registered cartesian coordinates of both wings with tpsDig (Rohlf 2007). Elytra contour was sampled with 30 equidistant points between the base and the apex of elytra, using the 'draw background curves' tool and resampling by its length in tpsDig. Elytral curves were appended as a collection of points with tpsUtil software (Rohlf 2008). We adjusted both configurations of coordinates with a generalized Procrustes analysis using CoordGen8 (Sheets 2005a). Semilandmarks of elytra configuration were aligned with Semiland8, defining each point on the curve as landmark or semilandmark (Sheets 2002). Semilandmarks are used when landmarks are insufficient to characterize shape variation, and they are appropriate for analyzing homologous and smooth curves (Gunz & Mitteroecker 2013) like the elytral outer edge. Finally, partial warps were used in multivariate analyses to compare amongst the three altitudinal ranges.

Statistical analyses
Morphological variation of the wing ratios related to the elevation was tested with linear models for lw/lp and we/le and a generalized linear model with gamma distribution to lsr/lp, using R 4.1.1. We performed orthogonal contrast using the 'gmodels' package (Warnes et al. 2018) to test significant differences between altitudinal groups.
Wing shape variations related to elevation were examined with a canonical variate analysis (CVA) without PCA Reduction for the hind wings and with PCA Reduction for the elytra (7 PC: 95% variance), using CVAGen8 (Sheets 2005b). To visualize changes in the shape, we performed Thin-plate Spline analyses for the significant canonical axes of both wings in CVAGen8. To validate the canonical axes, we performed a Jackknife grouping test with 1000 replicates in CVAGen8; if the percentage of correctly assigned were higher than expected by random assignment, the axes were valid.   To determine which group was discriminated against by each significant canonical axis (CVs), we performed an ANOVA test with CVs scores and orthogonal contrasts using the 'gmodels' package (Warnes et al. 2018). To illustrate the similarity in the shape of the wings between altitudinal ranges, we performed a cluster analysis based on the UPGMA algorithm with a matrix of Procrustes distance among the average of groups, using the 'cluster' package (Rousseeuw et al. 2019).

Wing variations related to elevation
The size and shape of the hind wings were different among the three altitudinal groups. Hind wings reduce their size as elevation increases ( Table 2). Specimens of low elevation were significantly different from the other ranges, having the longest hind wings (ls/lp, Figure 5a) and a greater distance between the base of the wing and the apical joint (lsr/lp, Figure 5b). Regarding the hind wing shape, canonical variate analysis (CVA) showed that specimens of high elevation were the most different, discriminated from other ranges by one significant canonical axis (CV) (Wilk's λ 0.6292, X2 31.043, P < 0.001) (Figure 6a). The Jackknife grouping test validated the CV; the percentage of correct assignments (50.7%) was higher than that expected by random assignment (35.57%). The CVA plot showed two groups (Figure 7), discriminated by the first canonical axis (CV1). The first one had macropterous species, and it was formed principally by specimens from the low and middle elevation range. The second had brachypterous and hemibrachypterous species, composed mainly of specimens from the high elevation range (Figure 7). Discrimination in the shape of both groups was in the relative position of two landmarks; the apex of the radial cell (landmark 2) was closer to the top of the wing (landmark 3), and the length of the vein MP3 + 4 (landmark 5) was short in the hemibrachypterous and brachypterous wings (Figure 8a).
The shape and size of the elytra changed with elevation. The elytral aspect ratio (we/le) was different among elevation ranges (Table 2), increasing with elevation. Elytra in high elevation were shorter and significantly different from the other ranges ( Figure 5c). Canonical variate analysis (CVA) for the elytra shape showed that the elytra shape of the high range is the most different. However, only the low and middle elevational ranges were discriminated against by one significant canonical axis (CV) (Wilk's λ 0.7447, X2 19.452, P = 0.003) (Figure 6b). The Jackknife grouping test validated the CV, the percentage of correct assignments (50%) was higher than expected by random assignment (41.67%).
The CVA plot had two groups, one located in the positive values of CV1 and the other in the negative values. The first group had macropterous species; it was formed mainly by specimens from the low elevation range. The second had brachypterous and hemibrachypterous species distributed in the middle and high elevation ranges (Figure 9). Discrimination in   Figure 5. Error bars of the mean of each ratio measurements and standard error. a) Length of the wing ratio (lw/lp). b) Length to the top of the stridulation zone ratio (lsr/lp). c) Elytral aspect ratio (we/le). Different letters above the bars indicate significant differences found with orthogonal contrasts. shape between groups was in the relative position of the semilanmarks located in the humeri and the poster-ior half of the elytra. Specimens of the high elevation had rounded humeri, and broader (semilandmarks in  the middle part of the edge) and shorter elytra (semilandmarks and landmark in the apex) (Figure 8b).

Discussion
The process of wing reduction in beetles could occur in one pair of wings or simultaneously in both pairs of wings. For example, species of the family Trogidae reduce or lose their hind wings (Scholtz 1986), whereas, in the subfamily Atractocerinae (Lymexylidae), only the elytra reduce its size (Yamamoto 2019). Our study reports a simultaneous process of reduction in both wings, like in some species of the families Lycidae (Bocak et al. 2013). The wing reduction related to elevation increases had been observed in Plecoptera, Grylloblattodea, Neuroptera, and other families of Coleoptera (Carabidae) (Sømme et al. 1996;Hodkinson 2005;McCulloch et al. 2019). Our results support the idea that changes in one pair of wings affect the other (Jackson 1928;Goczał et al. 2018), which can be attributed to the fact that elytra and fore membranous wings have the same genetic identity (Clark-Hachtel et al. 2013). Our study is the first that characterize hind wing and elytral modification related to elevation in passalids.
In bess beetles, the hind wings have a double function: dispersion (flight) and acoustic communication (Reyes-Castillo 1970a;Reyes-Castillo & Jarman 1983). For that reason, it would be expected that wing reduction will be constrained by the stridulatory apparatus located in the radial cell known as plectrum (Reyes-Castillo & Jarman 1983;Schuster 1983). Although plectrum spines are sexually dimorphic at a microscopical level (Ariza-Marín & De Luna in review), wing development is not sexually dimorphic, and both sexes produce sounds. Despite the plectrum function, its location changed with elevation, the ratio of the length between the base of the wing and the apex of radial cell or apical joint (lsr/lp) had a reduction with elevation (Figure 5b), and the landmark at the end of the radial cell was closer to the base of the hind wing (Figure 8a, landmark 2). This change in the position of the radial cell could be due to the joint effect of the stridulatory apparatus (abodmino-alary) and the reduction of metathorax size related to brachypterism proposed by Jackson (1928). Adults make sounds by rubbing their hind wings with two tubercles of the sixth abdominal tergum (Reyes-Castillo & Jarman 1983). To maintain the functionality of the stridulatory apparatus, we suggest that hind wings may have a parallel reduction in two distance: between the base of the wing and the apex of the radial cell (points 1-2, Figure 2) and between the base of the wing and the abdominal tubercles. On the other hand, hind wings had a reduction in the membrane of the distal region and posterior region due to the apical joint approaches to the wing apex (landmarks 2 and 3), and the reduction of MP3 + 4 vein (landmark 5) produces a decrease in the width of the wing (Figure 8a).
Regarding the elytral modification, the reduction of the elytral aspect ratio (we/le) and its rounded shape were related to an increase in the elevation. These modifications support the results of Moreno-Fonseca and Amat-García (2016), which found that the elytra were shorter and round in high elevation in a passalid community located in the eastern range of the Colombian Andes. The elytra are reduced in their aspect ratio and have a rounded shape, as suggested by Reyes-Castillo (1970a) and Boucher (2005). The pattern of elevational differences in the shape of hind wings and elytra was similar (Figure 9), suggesting that elevation produces a simultaneous change in both wings in passalids. According to Goczał et al. (2018), the elytra reduction leads to hind wing shape modification. It is necessary to prove if the elevation directly affects one or both wings.
Brachypterism could be a driver in the passalids speciation, decreasing the dispersion capacity of montane species. According to Boucher (2005), the reduction of hind wings evolved independently in different lineages from the macropterous wings. Additionally, the loss of flight is associated with faster molecular evolution in beetles (Mitterboeck & Adamowicz 2013), and tropical mountains have local endemic species due to geographically restricted species (Menéndez-Guerrero et al. 2020). However, the adaptative significance of brachypterism in passalids and other insects remains unstudied (Wooton 2001). According to Brühl (1997), the evolution of flightless insects in montane habitats is due to the extreme environmental characteristics of high montane ecosystems, which caused the increase of environmental stability and the decrease of the dispersion capacity. Indeed, the flight capability in tenebrionid beetles decreases with low temperature associated with high elevations (Perez-Mendoza et al. 2014). It is necessary to study the dispersal capacity of a species distributed in the three elevation ranges and compare climatic stability among elevational gradients. Studied species were not restricted to one elevational range; two species with wing reduced are distributed in the middle and high elevational range, nine macropterous species in two ranges, and one macropterous in the three ranges (supplementary material, Table S1). For example, Odontotaenius striatopunctatus are from lowland to high lands, distributed to a wide range of ecosystems and distributed from Mexico to Colombia (Schuster 1994;Reyes-Castillo 2003), probably its high environmental tolerance probably allows it to be widely dispersed and prevents the wing reduction process.
In conclusion, this study demonstrates that elevation is most likely the factor that has driven size reduction and shape modifications of the passalids hind wings and elytra. The macropterous species were present throughout the elevational gradient, hemibrachypterous were between 500 and 2000 m, and brachypterous over 2000 m. It is essential to clarify that the effect of phylogeny was not evaluated in our study. Brachypterism evolved independently in many passalids genera and species (Cano et al. 2018). The main problem for testing the effect of the evolutionary history on brachypterism is that phylogenetic relationships within studied genera are poorly studied, just Veturius had a published phylogeny. According to Boucher (2005) Veturius is divided into three subgenera, being the subgenus Publius a completely brachypterous subgenus discriminated from others by brachypterism. However, the monophyly of subgenera was not recovered in recent molecular phylogenetic analyses, and brachypterous species did not have a common ancestor (Beza-Beza et al. 2020). Indeed, the wing reduction is not enough for grouping species, and more synapomorphies must support clades. For example, the monophyly of brachypterous genus Ogyges was supported by characteristics on the front, mandibular teeth, and pronotum (Cano et al. 2018), whereas the brachypterous genera Proculejus probably is not a monophyletic genus (Reyes-Castillo 1970a). Previous information suggests that a brachypterous ancestor is unlikely in the brachypterous species studied. Future work should study changes in the wings related to elevation, considering the evolutionary history of a genus using geographically close species to prevent latitudinal effects. It could be interesting to determine if the elevation causes a morphological modification in the hind wings and elytra in a focal species with a wide range of altitudinal distribution.