Contribution to the weevil fauna of Montenegro with the description of a new Echinodera Wollaston, 1863 species, and lectotype designations in the genus Sciaphobus K. Daniel, 1904 (Coleoptera, Curculionoidea)

We provide first records for 108 weevils species sensu lato (Curculionoidea) to the fauna of Montenegro. During a field trip 203 weevil species were recorded and are listed here. The records of Archarius anatoliensis Voss, 1962 are the first ones from the Balkans and document a further spreading of this species towards Northwest. Echinodera bryneri spec. nov. is described and compared with the sympatrically occurring, morphologically similar species E. romanboroveci Stüben, 1998. The lectotypes of Sciaphobus paliuri Apfelbeck, 1908, and S. polydrosinus Apfel-beck, 1922 conserved in the Georg Frey collection are designated, and illustrated comments on their differentiation are provided. For Leiosoma komovicum Pedroni, 2018 habitat, images of both sexes, a description of the female genitalia, and the presumable host plants are presented.


Introduction
One important goal of the excursion reported here was to contribute to the extraordinarily poorly explored weevil fauna of Montenegro. Only little was published on Curculionoidea after several expeditions by the ancient specialists: Victor Apfelbeck to Czerna Gora (Apfelbeck 1895(Apfelbeck , 1906(Apfelbeck , 1907(Apfelbeck , 1908(Apfelbeck , 1915(Apfelbeck , 1919a(Apfelbeck , 1919b(Apfelbeck , 1919c(Apfelbeck , 1920(Apfelbeck , 1922(Apfelbeck , 1927(Apfelbeck , 1928 who described several endemic weevil species and subspecies in the subfamily Entiminae, and very recently Pedroni described Liosoma komovicum Pedroni, 2018 based on specimens from Montenegro (Pedroni 2018). But apart from these few examples, faunistic data from Montenegro are scarce (Pešić 2006) or largely lacking in recent catalogues (Löbl & Smetana 2011, 2013, Alonso-Zarazaga et al. 2017. And even when primary literature is searched, only exceptionally data specifically from Montenegro can be found (e.g. Caldara 1990 for Tychius Germar, 1817, Meregalli 1985 for Plinthus Germar, 1817, or more often Stüben 2018Stüben , 2019 for Cryptorhynchinae), and more recent records from the Balkans by Szénási (2017). Hence the weevil fauna of Montenegro still awaits being discovered. In the following we give a first insight in the rich fauna, and provide data for 204 Curculionoidea species from the families Apionidae (18 species), Attelabidae (2 species), Curculionidae (122 species), Erirhinidae (2 species), Rhynchitidae (3 species) and Nanophyidae (8 species). The recent study of Echinodera from Western Balkans, including already a small part of the material from Montenegro based on an integrative taxonomical approach (Germann & Schütte 2021) allowed to discover another new species of this genus, which is compared, described and discussed in this paper. Within the attempt to determine the collected species of the genus Sciaphobus, we found a series of type material in the Georg Frey collection at the NMB, which helped decisively in understanding the descriptions within that genus by Apfelbeck (1908Apfelbeck ( , 1922, therefore a current insight into this genus is given here.

Localities
The following 28 localities were visited from 21st April to 1st May 2018 (see Fig. 1): 28 Budva Mun., 3.5 km SE Petrovac, N 42.1811E 18.9695, 30 m, 1.5.2018 The collected weevils are deposited in the Naturhistorisches Museum Basel (NMB), except for selected specimens which are conserved in the author's collections. Label data are given as written, different labels are separated by a double slash (//). Additional remarks are given in square brackets ([]

Molecular analyses
For the molecular part of this study, a new CO1 barcoding sequence was generated and combined with 37 already published ones. All sequences originate from the Curculio Institute's joint projects, either with the Research Museum Alexander Koenig (ZFMK) or the SDEI/NMB. Due to the profound determination work by recognized specialists before the molecular processing of the specimens, a highly reliable dataset can be guaranteed. In many cases, additional specimens or remaining tissue samples from the same finding spot are available in frozen Ethanol. In most cases, the extracted specimens were recovered after the lysis step and stored in pinned dry collections at ZFMK or NMB.
This additional laboratory step facilitates the possibility of verifying the previous determination.
For all sequences, two Cryptorhynchinae adopted primer sets with degenerated nucleotides are used in ZFMK and SDEI laboratory routines. For LCO1490-JJ / HCO2198-JJ see Astrin & Stüben 2008 and for LCO1490-JJ2/ HCO2198-JJ2 see Astrin et al. 2016. Both target the same binding sites as the well-known primer set from Folmer et al. 1994 does, delivering 658 nucleotides in weevils. Thus, the derived COI barcodes are compatible with Folmer primer generated ones and meet the requirement of BOLD (Ratnasingham & Hebert 2007, www. barcodinglife.org).
A nucleotide alignment with 37 sequences is created with Muscle-Plugin in Geneious 6.1.8 (Kearse et al. 2012). All sequences comprise 658 nucleotides (full-length barcode). From the nucleotide alignment, a Jukes-Cantor (Jukes & Cantor 1969) corrected Neighbor-Joining (NJ) tree is built with Genious, Acalles camelus is designated as outgroup species.
The finding spots associated with the sequences are plotted on an ArcGIS map base layer with GPS Visualizer (www.gpsvisualizer.com). The p-distance values are provided in the map with connection lines between the new species Echinodera bryneri spec. nov. and its four closest congeners.

General comments
As specific faunistic contributions to Montenegro are largely missing, especially even more so in recent times, it is not a big surprise that out of 203 recorded species, 108 -more than half of them -are new to the fauna of Montenegro. Most of these new records concern species which are generally widespread, but some of them are worth being discussed more in detail, as they were rarely found, narrowly distributed (endemic) or unexpected and thus surprising.

Sampled habitats and excerpt of typical weevil species
Montenegro has a broad variety of interesting landscapes (Figs 2A-H). We mainly focused on the coastal region with limestone hills and low growing summergreen deciduous forests (Carpinus-Fraxinus-Quercus forests ( Fig. 2A) (Figs 4A-B). Head: eyes flattened, large and oval, upper margin above rostral groove, visible from above. Rostrum deeply punctuate-striate; densely covered with small oval scales and hairs from middle on towards rostral apex. Antennae reddish brown, antennal scape: 5 times longer than wide, segments of antennal funiculus: 1st thicker than the following, 1st and 2nd: 2 x longer than wide, 3rd to 5th: as long as wide, 6th and 7th transverse, club oval, 2.5 x thicker than last segments of funiculus. Pronotum: transverse (length/width: 0.7); maximal width before base in the first third, slightly rounded towards base; strongly narrowed, cone shaped towards front margin (Fig. 4A). Coarsely punctured, larger punctures towards hind margin. Integument and vestiture: colour patterns varying with darker and lighter brown scales; spotty placed light brownish to whitish scales. The integument consists of loosely standing, appressed, almost circular scales. Longer (2 x longer than wide), broadly oval, clubbed and nearly vertically raised bristles arise from punctures. Elytra: globular (length/width: 1.0-1.1); widest in middle; without shoulders; base straight, elytral decline in lateral view rounded, vertical towards apex. Integument and vestiture: colour patterns varying with darker and lighter brown scales; spotty placed light brown or whitish scales forming irregular transversal bandings. Consisting of almost circular appressed scales not entirely covering the intervals. Striae narrow, about half the span of intervals, visible through integument; punctures with adjacent thin, scalelike bristles. On intervals long (3.5 to 4 x longer than wide), scale-like, clubbed, vertically raised bristles. Legs: brown, strong, densely covered with elongated brown and light brown scales. Spine at tip of hind tibia regularly bowed inwards in males. Penis: tip of medianlobus broadly pointed, in lateral view bent, S-shaped waved (Figs 5A-D). Female genitalia (Figs 5E-G). Spermatheca with cornu c-shaped, ramus and nodulus not visible (Fig. 5E). Spiculum ventrale with shovel-shaped plate, basal edges of plate inclined in an angle of 40°, and apodeme more than twice as long, tip of apodeme thickened (Fig. 5F). Gonocoxite consisting of two sclerotized segments, stylus cylindrical ( Fig. 5G).

Variability:
The width of the erect bristles on the elytra varies to a certain extend, as well as the body size.

Etymology:
The new species Echinodera bryneri spec. nov. (Fig. 3) is named after our dear friend and renowned expert in Microlepidoptera (especially Adelidae) Ruedi Bryner (Biel), who is furthermore a gifted breeder of various insects including occasionally weevils as unintended guests together with his target organisms.
Differential diagnosis: Echinodera bryneri spec. nov. is morphologically similar to E. romanboroveci Stüben, 1998, which was also found at five more northwestern localities within the sampled area ( Fig. 1). E. bryneri spec. nov. was found at three localities in the southeast and one locality (3) harboured both species.
Echinodera bryneri spec. nov. has longer raised setae on elytra and scales on pronotum, the shape of the elytra is more rounded, the whole body is more robust and broader. The penis is broader, its tip wider with apex not rather regularly attenuated and pointed as in E. romanboroveci, but more abrubtly and more obtuseangled pointed. The tip of the penis in lateral view with s-shaped wave ventrally less bowed than in E. roman boroveci. The female genitalia differ in the shape of the spermatheca (Fig. 5E, L), the shape of the plate of the spiculum ventrale, where the basal edges are rectangular in E. romanboroveci instead of inclined as in E. bryneri spec. nov. (Fig. 5F, M), and the shape of the gonocoxite (Fig. 5G, N). With its long and raised setae E. bryneri spec. nov. is furthermore superficially similar to E. capiomonti (Brisout de Barneville, 1865) occurring in Italy, Croatia and Bosnia and Herzegovina, but the distinctly shorter setae (8-10 times longer than wide in E. capiomonti) and the more oval elytra of E. bryneri spec. nov. already allow a reliable differentiation. E. bryneri spec. nov. is also similar to E. brisouti (Reitter, 1885) from Greece, but differs by the more erect bristles on elytra, and the different shape of the penis, which is attenuated before the tip in E. brisouti, and broadly rounded in E. bryneri spec. nov.
Taxonomical comments on the genus Sciaphobus K. Daniel, 1904 based on specimens from Victor Apfelbeck in the Georg Frey collection Part of the genus Sciaphobus was revised by Borovec & Skuhrovec (2015). They redefined the subgenera Sciaphobus s. str. K. Daniel, 1904 andNeosciaphobus Apfelbeck, 1922, and mentioned difficulties that remain concerning those species where at that time no type material was available. As a small part of the Apfelbeck collection (especially Curculionidae) was included in the Georg Frey collection in 1935 (conserved in   the Naturhistorisches Museum Basel), including type specimens collected by Kurt Hermann Gustav Otto Noesske  as written by Apfelbeck (1922) in his first descriptions, the opportunity was given to revise these specimens when a series of an undetermined Sciaphobus was collected during the present excursion.
The examination of the available specimens showed, that specimens from all three somewhat enigmatic taxa were present. According to article 74.7.3 of the Code of Zoological Nomenclature (ICZN 1999), the lectotypes and paralectotypes of Sciaphobus paliuri and S. polydrosi nus are selected based on the specimens and characters indicated and shown below.
The syntypes of S. paliuri Apfelbeck, 1908  Studying all specimens of the three species allowed to confirm the value of the given characters by Apfelbeck (1922), namely: i) the absence of any femoral teeth in the case of Sciaphobus paliuri; ii) all femora are conspicuously teethed in S. curvimanus; iii) tiny tooth on profemora and mostly stronger teeth on meso-and metafemora in S. polydrosinus, furthermore in that latter species the more elongate antennomeres and the more protruding eyes are reliable characters. The dorsal and lateral shapes of the peneses, and the shape of their tips, are furthermore suitable for discrimination. There the provided Figs 6A-L illustrate these differences partly already drawn by Apfelbeck (1922), and again included here (Figs 6J-L). In S. paliuri the medianlobus is parallel sided in dorsal view, weakly bowed in lateral view and the tip more regularly attenuated with a hook -like tip. In S. curvimanus the medianlobus is more triangularly shaped, stronger bowed with a more abruptly attenuated, rounded tip. In S. polydrosinus the medianlobus is constricted in its middle, and broadly rounded before the tip, in lateral view thick with an almost pointed tip. On the other side, some characters used for species discrimination did not proof to be suitable for a reliable differentiation, namely i) the rather variable shape of pronotum and body, these vary within the sexes, but even more between them; more than between the three species investigated; ii) the same with the scales which show the whole spectre from greenish to pearllike lustre, these furthermore are varying in shape from narrow setiform to broader and almost circular.

Plinthus squalidus gerlii Boheman, 1842
At present recorded from Croatia and Bosnia-Herzegovina (Kippenberg 1981, 1982, Meregalli 1985. The characteristic vestiture of broad scales on elytra, pronotum and sternites can be used for a reliable determination (Kippenberg 1981). Based on the appressed broad scale-like hairs on the sternites, the other subspecies P. squalidus purkynei Kippenberg, 1981 with narrow and detached hairs, which is known from the Durmitor massif in Montenegro, can be excluded. The single specimen (Fig. 7A) was found under a stone at the margin of a Fagus forest on Lovcén (locality nr 6). As the Lovcén massif is a continuation of the narrow mountain chain close to the coast reaching from Croatia to Montenegro, and P. squalidus gerlii seems to follow this chain, this present discovery fits well in the hitherto known distribution and enlarges it towards the Southeast. Pedroni, 2018 This species was recently described based on one couple from the Komovi mountains in the Durmitor massif in Montenegro (Pedroni 2018). The species derives in its habitus strongly from all other species of the Leiosoma cribrum-group, which was also mentioned by the author. We collected three specimens of L. komovicum, in a Fagus forest. The exact finding place (FO 19) was in close vicinity of old Fagus sylvatica trees and consisted of leaf litter between flowering Anemone nemorosa and A. apennina (Figs 8A-B) which might be both suitable candidates of host plants, as mostly Ranunculaceae are mentioned as host plants, and are the only ones where larvae were observed up to now (Ranunculus spp., Anemone nemorosa in the case of L. deflexum, Scherf 1964). As the female is not yet described in detail, we provide as a supplement to the description good photos of both sexes (Figs 9A-H), additional photos of the penis (Figs 9C-E) and give here a description of the sclerotized female genitalia. The spiculum ventrale (or sternite VIII) (Fig. 9F) shows a biforked plate, the apodeme is 1.5 times longer than the plate, with its base broadened. The spermatheca (Fig. 9G) has a long, almost straight cornu, the corpus is bell-shaped with both, nodulus and ramus reduced. The gonocoxite is weakly sclerotized, simply bow-shaped with rather robust, somewhat flattened stylus at apex (Fig. 9H).
A remarkable number of six species of these mostly underwater living species could be collected along the rather unspectacular shore of a pond at locality 16 (Fig. 2D) Péricart, 1989, andB. subcarinatus (Gyllenhal, 1836). The quickly dropping water level of this small lake allowed to beat against mud and drying water plants, which kept hanging on shrubs and branches towards the shore.

Molecular analyses
The molecular analysis clearly confirms the species status of Echinodera bryneri spec. nov. from the molecular perspective. The Neighbor-Joining (NJ) tree is provided in Fig. 11. The distribution map with E. bryneri and its congeners in focus is provided in Fig. 12, the genetic distances listed were taken from the distance matrix (Appendix 5), uncorrected p-distance values are listed.
These findings regarding the interspecific genetic distances are in concordance with the MWI dataset: The average p-distance value for continental endemic Echinodera species is ~11 % (Schütte et al. 2022, in preparation). The smallest currently known interspecific p-distance value for continental endemic Echinodera species is 5.8 % for E. cognita from Spain (e-490-cog/ GU213684) vs. E. incognita from Spain (e-594-inc/ GU213709), both not included in the NJ tree. With 5.9-6.2 % genetic distance between E. bryneri and E. romanboroveci is clearly on the lower end of the scale, but the morphological characters leave no doubt about its species status as well. For all currently known closely related species COI sequence data is available and was used in the molecular analyses.

Discussion
Our present contribution adds the remarkable number of 108 further species to the fauna of Montenegro, among them one species new to science (Echinodera bryneri spec. nov.). This discovery is not unusual. Despite of the thorough dedication by Stüben (2018) to this group, the same author and some of us just recently went on finding new species in Greece (Stüben & Kramp 2019, Germann & Schütte 2021, therefore future investigations will certainly lead to more such discoveries. This present effort in continuing the exploration of the remarkably rich biodiversity in the Balkans follows recent additions by Pešić (2006) and Szénási (2017). The determination of several species collected during the present excursion was difficult, and in the case of Sciaphobus only the direct comparison with specimen from the museum collection was successful, which led furthermore to the designation of lectotypes, and the re-evaluation of the morphological characters used for species discrimination. For future studies, the species presented here in the photo galleries might be helpful, as in general only old keys with scarce illustrations are available.

Appendix 1 -species list
Species list of all 203 species of Curculionoidea recorded from Montenegro. Locality no. = refers to the numbered localities given; ME = the exclamation mark (!) indicates a new record for the fauna of Montenegro.