Systematics and Biogeography of the New World Genus Plumolepilius (Coleoptera: Curculionidae)

: Plumolepilius Barrios-Iz á s & Anderson, 2016 is a leaf litter weevil genus that inhabits montane broadleaf forests from southern Mexico to northern Panama. The genus consists of 27 species, 22 distributed in the Chiapas Highlands province (Mexican Transition Zone) and 5 found in the Paciﬁc dominion (Neotropical region) in Costa Rica and Panam á . Here, we analyze the phylogenetic relationships of the species of Plumolepilius based on 20 external body characters and 9 characters from the genitalia. The ﬁrst dichotomy of the cladogram separates two species from the Paciﬁc dominion from the remaining species of Plumolepilius from the Chiapas Highlands province and three species restricted to the Paciﬁc dominion. We hypothesize that redundant distributions in the taxon-area cladogram of the genus may be due to dispersal events, probably during the Pleistocene glaciations. Author Contributions: Conceptualization, M.A.B.-I. and J.J.M.; formal analysis, M.A.B.-I. and J.J.M.; investigation, M.A.B.-I.; writing—original draft preparation, M.A.B.-I. J.J.M.; writing—review and editing, M.A.B.-I.


Introduction
Plumolepilius Barrios-Izás & Anderson, 2016 (Curculionidae: Molytinae: Conotrachelini) is a leaf litter weevil genus from southern Mexico and Central America. It comprises at least 27 known species and a preliminary phylogenetic analysis has shown that it is monophyletic [1,2]. The distribution of the species of Plumolepilius ranges from southern Mexico through Central America reaching northern Panama, within the Mexican Transition Zone (Chiapas Highlands province) and the Pacific dominion of the Neotropical region (Puntarenas-Chiriquí province) [3], through a range of about 8 • latitude. Species of Plumolepilius are mostly found in montane broadleaf humid forests, approximately between 1400 and 2800 m.a.s.l. All species have low dispersal capacities and show troglobiomorphic adaptations, such as the absence of hind wings and scutellum, fused elytra and reduced metasternum.
The dispersal-vicariance model has been proposed as one of the main hypotheses for explaining the evolution of the montane biota of Central America [4,5]. Pleistocene glaciations have played an important role in the distribution of biodiversity by expanding and contracting species' and ecosystems' distribution range. It has been demonstrated that during glacial periods, humid montane forest migrated to lower altitudes allowing the migration of species, while in warmer periods, humid forests contracted to upper altitudes, leaving species isolated.
Our aims are to analyze the phylogenetic relationships of the species of Plumolepilius and to propose a biogeographic hypothesis to help explain its diversification.

Materials and Methods
The phylogenetic analysis included all known species of Plumolepilius. Specimens were obtained on loan from the Insect Collection of the Canadian Museum of Nature (CMNC). As outgroups, we used Lepilius pulchellus Champion, 1905 (Charlie W. O'Brien Collection), L. chisosensis Anderson 2010 (Museo de Zoología "Alfonso L. Herrera") and two undescribed species of Lepilius from Honduras and El Salvador (CMNC).
Specimens were relaxed by immersion in hot water with a solution of soap for 15-20 min. Abdominal ventrites were removed from the abdomen together with the alimentary canal, genitalia and fat and muscular tissues. The body was washed with water, then in a solution of ethanol (95%), air dried and double pin mounted. Fat and muscular tissues from abdominal content were cleared by immersion in a warm solution of KOH (10%) for 15-30 min. After clearing, abdominal structures were washed in water and ethanol (95%) to remove the KOH. Ventrites were mounted in a card and pin mounted together with the specimen. Male and female genitalia and associated sternites were mounted in slides using 5,5-dimethyl hydantoin formaldehyde (DMHF) [6].
For constructing the data matrix (Table 1), we used 20 external body characters and 9 characters from male and female genitalia. Multistate characters 10, 11, 19, and 26-28 were treated as additive. Characters and character states are described as follows (0 = plesiomorphic; 1-7 = apomorphic): The cladograms were constructed using TNT software [7]. We conducted an implied weights analysis [8], with the constant of concavity (k) set to 6.48, selecting this value with the script setk [9].

Results
The analysis under implied weights with a concavity of 6.48 led to three cladograms (Figure 1), with 177 steps, a consistency index of 0.31 and a retention index of 0.57. Barrios-Izás et al. (2016) identified as synapomorphies of Plumolepilius the presence of plumose setae in the prosternal channel, the tubercle at the beginning of the elytral declivity on interval 4 and the swollen basal area of pronotum. Lepilius Champion was identified as the sister genus of Plumolepilius; here, we identified 12 morphological characters in Plumolepilius that support its separation from Lepilius. Some of these characters are the transverse pronotum, the presence of plumose scales, asymmetry of the genital and thinner spermatheca in Plumolepilius; and the ovate shape of the body, absence of tubercles or protuberances and absence of erect or suberect elytral scales in Lepilius.
Within Plumolepilius, we recovered two main clades. The first clade (clade A) is constituted by P. maesi and P. obrienorum, and the second clade (clade B) contains the remaining 25 species (Figure 1). Species in clade A inhabit the Pacific dominion, whereas those of clade B inhabit both the Pacific dominion and the Chiapas Highlands province (Mexican Transition Zone). Within clade B, P. canoi and P. schusteri are recovered as sister taxa (clade B1); and P. andersoni, P. daryi, P. camuna, P. hectori and P. molinai constitute a monophyletic group (clade B2). Plumolepilius antonioi, P. yolnabajensis, P. branstetteri, P. solisi, P. zarazagai, P. genieri, P. linaresi, P. velizi, P. cortesi, P. longinoi, P. monzoni, P. guaimacaensis and P. macalajauensis also constitute a monophyletic group (clade B3). The rounded shape of the elytral declivity supports clade A, whereas clade B has an oblique shape. Species within clade B3 are characterized by the females having a well-developed tubercle on interstice 4 at the beginning of the elytral declivity. Within clade B3, females of P. branstetteri, P. solisi and P. zarazagai have an acute angulate shape of the humeral region. We were unable to find synapomorphies for clades B1 and B2. The presence of elytral tubercles in the females shows a nested distribution along the branches of the cladogram, from the absence of tubercles to the presence of small tubercles, two large tubercles on interstice 4 and two large convergent tubercle on interstice 4.  Based on the cladogram of Plumolepilius species, it is possible to obtain a taxon-area cladogram (Figure 2). We hypothesize that the redundant distributions of species of both main clades in the Pacific dominion are due to dispersal events, likely during the Pleistocene glaciations.

Discussion
The species of Plumolepilius show patterns of endemism similar to other weevil genera that inhabit leaf litter (e.g., Theognete [10], Tylodinus [11], Lepilius [12]) or soil (e.g., Neoubychia [13]). With some exceptions, most of the species are allopatric and restricted to one or few localities. Species with a wider range of distribution are found on the Quaternary volcanic terranes of the northern side of the Central American Volcanic Arc, such as P. trifiniensis, recorded from the Atitlan Volcano in Guatemala to the east up to Santa Ana volcano in El Salvador; and P. canoi, recorded from four localities ranging from the cloud forest of La Union in Guatemala south to El Güisayote in Honduras [1,2]. All other species of Plumolepilius have very narrow distributional ranges; some of them are known only from the type locality.
Species distribution is constrained by a combination of species tolerance to physical variables, biotic interactions, historical processes and the dispersal abilities of the species. The species of Plumolepilius are associated with montane broad leaf forest, mainly oak and cloud forests between 1200 m and 2500 m. The main characters that might limit the dispersal abilities of Plumolepilius species are the lack of flight, which is associated with troglobiomorphic characters such as the reduced size of the metasternum, the absence of hind wings, the fused elytra and the absence of scutellum. It is well known for species inhabiting caves or soil that the development of characters (e.g., longer appendices and overdevelopment of some brain areas) for life in the darkness selects against traits that are not useful in these types of habitats (e.g., cuticle pigmentation, eyes and brain associated areas). Troglobiomorphism, either intermediate or extreme, is common in several subfamilies of the Curculionidae, namely, Brachycerinae, Molytinae and Cossoninae [14].
The high number of endemic species in the Mexican Transition Zones and other montane provinces of Central America has been explained under dispersal-vicariance models [4,5] during the Pleistocene climatic oscillations. Lower elevation ecosystems during warmer periods function as biogeographic barriers and montane ecosystems function as land islands where populations remain isolated for several thousands of years that may cause speciation processes; during colder periods, montane habitats descend and expand their distribution in lowlands, allowing the migration of species. We assume, based on the genetic time calibration of the diversification of other montane taxa, that the same processes might have acted in the speciation of Plumolepilius.
Along the Mexican Transition Zone, the Isthmus of Tehuantepec and the Lake Nicaragua depression have been recognized as important biogeographic barriers for species inhabiting montane ecosystems [15,16]; however, these are not universal barriers for species that have similar dispersal abilities, such as those of the genera Lepilius Champion, Plumolepilius, Theognete Champion, Tylodinus Champion as well as other leaf litter weevil genera. Lepilius has been recorded from samples collected from Texas to Costa Rica (Robert Anderson, pers. comm.), Plumolepilius has been recorded from southern Mexico to Panama, and Tylodinus has been recorded from northern Mexico to Panama [11].
The main barriers constraining the distribution of Plumolepilius species distribution are the Isthmus of Tehuantepec and the Chocó lowland forests; specimens of Plumolepilius have not been found beyond these limits until now. This area is considered to be within the Mesoamerican biodiversity hotspot [15,17]. The high number of endemisms and diversification of Plumolepilius within this area show the importance of conservation of the montane forests of Mexico and Central America, most of which have been legally protected since the 1950s. With some exceptions, most of these protected areas, especially in northern Central America, lack proper management to ensure their long-term conservation. The loss of montane habitats at the current rates of deforestation will drive to an accelerated rate of extinction of diverse groups of arthropods that remain unknown at species and molecular levels.