The genus Pheggomisetes Knirsch, 1923 (Coleoptera: Carabidae: Trechinae) in Serbia: taxonomy, morphology and molecular phylogeny

The genus Pheggomisetes Knirsch, 1923 (Coleoptera: Carabidae: Trechinae) in Serbia: taxonomy,... Abstract The genus Pheggomisetes Knirsch, 1923 consists of stenoendemic troglobitic ground beetles restricted to underground habitats in both Western Bulgaria and Southeast Serbia. A review of the genus in Serbia is given in this article. The following new taxa are described from three caves and pits on slopes of the Stara Planina Mountains in Southeast Serbia: Pheggomisetes serbicus sp. nov., P. serbicus belensis subsp. nov. and P. globiceps ciniglavcensis subsp. nov. Also, Pheggomisetes ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004, originally described as an independent species, is downgraded to a subspecies of P. globiceps Buresch, 1925 – P. globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. All the important morphological features of the taxa are listed in the article. The diagnoses of taxa are based on the characters ascertained by bright-field microscopy and nonlinear microscopy (NLM). The use of NLM in investigating Pheggomisetes anatomy is explained, and it is shown to be superior to classical microscopy in observing minute details of different structures (e.g. genitalia) on cross and longitudinal sections. A key to the species of Pheggomisetes (including the Serbian taxa) is given. In addition, we have included morphometric and molecular analyses of all Serbian Pheggomisetes taxa. ground beetles, molecular systematics, morphometrics, new species, new subspecies, Trechini, troglobites INTRODUCTION The genus Pheggomisetes Knirsch, 1923 includes four species and 12 subspecies of supposedly archaic troglobitic ground beetles, which have been highly modified during their evolution (Beron, 1994; Moravec, Uéno & Belousov, 2003; Ćurčić et al., 2004). The genus occupies an isolated position in the tribe Trechini, alone forming a distinct phyletic series (Jeannel, 1928; Casale & Laneyrie, 1982; Casale, Vigna Taglianti & Juberthie, 1998). It is considered to be related to the Caucasian aphaenopsoid genus Taniatrechus Belousov & Dolzhansky, 1994 based on the supernumerary supraorbital and submentum setae, two widened male protarsomeres and male genital structure (Belousov & Dolzhansky, 1994; Belousov & Koval, 2009). All representatives of Pheggomisetes are stenoendemics and restricted to certain caves and pits in Western Bulgaria and Southeast Serbia. The currently known species of the genus are Pheggomisetes buresi (Knirsch, 1923); Pheggomisetes globiceps Buresch, 1925; Pheggomisetes radevi Knirsch, 1924; and Pheggomisetes ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 (Casale & Laneyrie, 1982; Moravec et al., 2003; Ćurčić et al., 2004). One species (P. ninae) and one subspecies (P. globiceps ilandjievi V. Guéorguiev, 1964 according to Pretner, 1970) have been recorded in Serbia so far (Guéorguiev, 1964; Ćurčić et al., 2004; Ćurčić, Brajković & Ćurčić, 2007; Nešić et al., 2010). The morphology and anatomy of Pheggomisetes taxa have been investigated by several authors (Jeannel, 1928; Decou & Botosaneanu, 1964; Juberthie & Decu, 1968). Jeannel (1928) was the first to investigate features of the mouthparts, the dorsal habitus and male genital structures. Other aspects of the nervous, digestive, genital and pygidial gland systems were analysed in P. buresi (Decou & Botosaneanu, 1964; Juberthie & Decu, 1968). Most recently, thanks to two-photon excitation autofluorescence (auto-TPEF) of the chitin, certain features of the mouthparts and male genitalia were presented for P. ninae (Rabasović et al., 2015). Troglobitic insects are depigmented or transparent, with a thin cuticle consisting mostly of homogeneous chitin (Christiansen, 2012). The use of TPEF microscopy seems to be a highly promising way to study troglobitic taxa like Pheggomisetes since it enables the investigator to avoid the fluorescence of pigments and other cuticle components. The fluorescent signal of chitin is dominant here, in contrast to the situation with strongly coloured insects. Troglobitic beetles, including trechine ground beetles, are regarded as good models for deliberations about both biogeography and evolution since the reduced dispersal out of subterranean environments produces phylogenetic patterns of area distribution that largely match the geological history of mountain ranges and underground habitats (Ribera et al., 2010; Faille et al., 2014). The molecular phylogeny of trechine ground beetles (especially the troglobites) is largely unknown in spite of the fact that these are among the best-studied and widespread groups of beetles (Faille et al., 2013). Within the Trechinae, the molecular phylogeny of Trechus species from Spain and subterranean Pyrenean taxa (Faille, Casale & Ribera, 2010a; Faille et al., 2010b) and that of Alpine taxa (Faille et al., 2013) have been more or less thoroughly treated in recently published articles (Contreras-Díaz et al., 2007; Faille, Bourdeau & Fresneda, 2012; Faille et al., 2014). Among other groups of ground beetles, representatives of the Carabinae and Harpalinae have been somewhat better studied with respect to molecular biology (Ober, 2002; Osawa, Su & Imura, 2004; Ober & Maddison, 2008; Ruiz, Jordal & Serrano, 2009; Ober & Heider, 2010; Andújar et al., 2012; Deuve et al., 2012; Šerić Jelaska et al., 2014). Phylogenetic relationships of the highly diverse trechine fauna of the Balkan Peninsula are almost unknown. One study only concluded that the genera Neotrechus Müller, 1913 and Adriaphaenops Noesske, 1928 are related to certain Alpine or Pyrenean taxa (Faille et al., 2013). Sparse molecular data on the genus Pheggomisetes are mentioned solely by Faille et al. (2013). Organized by the Institute of Zoology, University of Belgrade – Faculty of Biology, several field trips were carried out in Southeast Serbia from 2012 until 2014. They resulted in the discovery of one new species and two new subspecies of Pheggomisetes. The aims of this article were as follows: (1) to describe and diagnose the new trechine taxa; (2) to review the taxonomic status of Pheggomisetes taxa from Serbia; (3) to show the benefits of nonlinear microscopy (NLM) in investigating morphology of the beetles’ internal chitinous structures (genitalia and some sclerites); (4) to conduct a morphometric study of all the investigated Pheggomisetes taxa; and (5) to perform a phylogenetic analysis of all the Serbian taxa using molecular data. MATERIAL AND METHODS Sampling information Ground beetle specimens were collected by hand or by pitfall trapping (the traps contained salt-saturated water/alcoholic vinegar and rotten meat as bait) (Faille et al., 2012) in a number of underground habitats from Southeast Serbia and Western Bulgaria belonging to the Stara Planina (Balkan) Mountain system. The traps were placed on both the floor and walls in dark parts of the explored caves and pits, where the level of humidity was high. All the analysed Pheggomisetes taxa are troglobitic and live in underground habitats (caves and pits) in the Stara Planina (Balkan) Mountains of Southeast Serbia and Western Bulgaria. Collected specimens were transferred to 70% ethanol at room temperature, except in the case of specimens designated for molecular analysis, which were transferred to 96% ethanol at −20 °C. They were analysed in laboratories of the Institute of Zoology, University of Belgrade – Faculty of Biology, Belgrade, Serbia; the Department of Crop Science, University of Belgrade – Faculty of Agriculture, Belgrade, Serbia; and the Photonics Centre, Institute of Physics, University of Belgrade, Belgrade, Serbia. Measurements AL – maximum length of antennae including the scape AL/TL – ratio of maximum length of antennae including the scape to total body length (from the anterior margin of the clypeus to the elytral apex) BW/PW – ratio of the elytral base width to maximum pronotum width as the greatest transverse distance EL – elytral length (as the linear distance along the suture from the elytral base to the apex) EL/EW – ratio of elytral length (as the linear distance along the suture from the elytral base to the apex) to maximum elytral width EL/TL – ratio of elytral length (as the linear distance along the suture from the elytral base to the apex) to total body length (from the anterior margin of the clypeus to the elytral apex) EW – maximum elytral width EWP – position of maximum elytral width (percentage of length) FL – length of frontal furrows FL/HL – ratio of frontal furrow length to head length HL – head length HL/AL – ratio of head length to maximum length of antennae including the scape HL/HW – ratio of head length to maximum head width HL/PL – ratio of head length to pronotum length (along the median line) HL/TL – ratio of head length to total body length (from the anterior margin of the clypeus to the elytral apex) HW – maximum head width HW/EW – ratio of maximum head width to maximum elytral width HW/NW – ratio of maximum head width to maximum neck width HW/PW – ratio of maximum head width to maximum pronotum width as the greatest transverse distance HWP – position of maximum head width (percentage of length) M – mean value for certain measurements NW – maximum neck width PaW – width of pronotal apex between tips of the anterior angles PaW/PbW – ratio of pronotal apex width between tips of the anterior angles to pronotal base width between tips of the posterior angles PaW/PW – ratio of pronotal apex width between tips of the anterior angles to maximum pronotum width as the greatest transverse distance PbW – pronotal base width between tips of the posterior angles PbW/PW – ratio of pronotal base width between tips of the posterior angles to maximum pronotum width as the greatest transverse distance PL – pronotum length (along the median line) PL/PW – ratio of pronotum length (along the median line) to maximum pronotum width as the greatest transverse distance PL/TL – ratio of pronotum length (along the median line) to total body length (from the anterior margin of the clypeus to the elytral apex) PW – maximum pronotum width as the greatest transverse distance PW/EW – ratio of maximum pronotum width as the greatest transverse distance to maximum elytral width PWP – position of maximum pronotum width (percentage of length) R – range of total measurements performed TL – total body length (from the anterior margin of the clypeus to the elytral apex) Collections HT – holotype IZFB – collection of the Institute of Zoology, University of Belgrade – Faculty of Biology, Belgrade, Serbia NMNH – collection of the National Museum of Natural History, Sofia, Bulgaria Other examined taxa Pheggomisetes buresi (Knirsch, 1923): one topotype male, Bulgaria, Balkan Mts., Ledenika Cave, 830 m a.s.l., near Vratsa, 19.VII.1963, leg. E. Pretner (IZFB). Pheggomisetes globiceps globiceps Buresch, 1925: syntype male, Bulgaria, West Balkan range, Mt. Ponor Planina, Sofia district, a cave near Iskrets (= Dushnika Cave), village of Iskrets, 580 m a.s.l., 10.XII.1924, leg. D. Iltchev (NMNH); two topotype females, idem, 08.VII.1925, leg. N. Radev (NMNH); one topotype female, idem, 17.IX.1943, collector unknown (NMNH); one topotype male, idem, 15.II.1992, leg. I. Pandurski (NMNH); two topotype males and one topotype female, idem, 16.X–04.XI.2016, from pitfall traps, leg. B. Guéorguiev & S. Goranov (IZFB); one female, Bulgaria, West Balkan range, Mt. Ponor Planina, Sofia district, Otechestvo Cave, village of Iskrets, 720 m a.s.l., 24.V.1959, leg. A. Popov (NMNH); two males, idem, 16.X.2016, leg. B. Guéorguiev & S. Goranov (NMNH). Pheggomisetes globiceps ilandjievi V. Guéorguiev, 1964: two topotype males and three topotype females, Bulgaria, Balkan Mts., Golyama Balabanova Dupka Cave, 1100 m a.s.l., village of Komshtitsa, near Godech, 12.X.1995, leg. B. Guéorguiev & V. Beshkov (IZFB); two topotype males and three topotype females, idem, 30.V.2015, leg. S. Goranov (IZFB). Taxonomic and morphological analyses The traditional method of studying insect morphology by bright-field microscopy was mainly used in the study. Apart from this, 3D images and clips obtained by NLM provided additional details that were used for descriptions of the genital structures and diagnoses of the studied taxa. Bright-field microscopy The genitalia were removed from the bodies, preserved in clove oil for a few days and subsequently fixed on microscope slides in Canada balsam. The beetles were then glued onto rectangular paper labels and analysed as dry specimens. Stemi 2000 and Stereo Discovery.V8 binocular stereomicroscopes (Carl Zeiss, Jena, Germany) with AxioCam MRc and Axio Cam ICc 1 digital cameras (Carl Zeiss, Jena, Germany) attached were used to photograph whole specimens, while a DMLS light microscope (Leica, Wetzlar, Germany) with a DC 300 camera (Leica, Wetzlar, Germany) attached was used to photograph the genitalia. Nonlinear microscopy Bright-field microscopy is most often used to study insect morphology, but recently a few articles have appeared treating the use of confocal fluorescence microscopy to observe certain structures of insects and crustaceans (Klaus, Kulasekera & Schawaroch, 2003; Michels, 2007; de Campos Vidal, 2011). NLM has also been introduced as a method offering unique insight into a variety of biological structures. This technique is similar to confocal microscopy (in employing localized laser excitation and scanning), but is characterized by higher penetration depth, reduced photodamage and photobleaching, and no need for specimen staining in most cases (Denk, Strickler & Webb, 1990; Williams, Zipfel & Webb, 2001; Mertz, 2004; Masters & So, 2008). Tissues and individual cells can be observed with high resolution of volume details. Up until now, NLM has been used extensively in biomedical research, but only marginally in entomology (Lin et al., 2008; Chien et al., 2011). It was recently confirmed that NLM can be used for deep imaging of chitinous structures (both chemically purified chitin and chitin originating from insect integument) (Rabasović et al., 2015; Reinhardt, Breunig & König, 2017). On the basis of the autofluorescence properties of chitin, the TPEF modality of NLM was mainly used in the latter study. In addition, even a second harmonic generation signal originating from chitinous structures was detected, but this signal is unsuitable for imaging since it is weak and hindered by the much stronger auto-TPEF. Before analysis, specimens were stored in 70% ethanol at room temperature. The genitalia were removed from the bodies and preserved in clove oil for 2 weeks. Genital structures were placed on double-sided adhesive tape on microscope slides in glycerin as the medium, with or without a cover slip, depending on the microscope objective used. Statistical analysis All variables that entered the statistical analysis were tested for normality using the Shapiro-Wilk test. A multivariate test of significance (one-way MANOVA) of normally distributed data, followed by a univariate test of significance (one-way ANOVA) for each variable, was used to identify which data sets (between groups) differ significantly. Non-normally distributed variables were compared between taxon samples using the Mann–Whitney U test (P ≤ 0.05). MANOVA allows comparison of population means of all variables of interest at the same time (multivariate response) rather than considering multiple responses as a suite of univariate responses (Zar, 1999). The statistical test most often used in biology, Wilks’ lambda, was applied (Zar, 1999). One-way MANOVA was used to examine the differences in morphological variation among Serbian Pheggomisetes taxa (species and subspecies). To describe and interpret effects from MANOVA, multivariate discriminant analysis (DA) was used only on normally distributed variables to determine the relative importance of characters as discriminators between a priori groups and the relative positions of centroids of the groups (Manly, 1986). The distance matrix for subspecies was calculated based on the squared Mahalanobis distance between subspecies centroids, and a dendrogram was generated using UPGMA (unweighted pair group method with arithmetic mean) clustering. This was used to evaluate the phenetic relationships between subspecies. Statistical analyses were conducted using the Statistica 6 software package (StatSoft, Inc., 2001). Molecular analysis DNA extraction, PCR amplification and sequencing We used nine Pheggomisetes specimens (belonging to all Serbian taxa) for molecular analyses (Table 1). DNA was extracted from one hind leg of each specimen using the KAPA Express Extract Kit (Kapa Biosystems Inc., Boston, MA, USA) and following the manufacturer’s instructions. The primers used to amplify the barcoding region of cytochrome c oxidase subunit I (COI) gene were Jerry [(CI-J-2183)5′-CAACATTTATTTTGATTTTTTGG-3′] and Pat [(TL2-N-3014)5′-TCCAAAGCA CTAATCTGCCATATTA-3′] (Simon et al., 1994). Each PCR was carried out in a volume of 25 µL containing 1 µL of extracted DNA, 9 µL of H2O, 1.25 µL of each primer and 12.5 µL of KAPA2GtmHotStart ReadyMix. All PCRs were conducted in an Eppendorf Mastercycler (Hamburg, Germany) using the following thermal profile: initial denaturation (95 °C for 5 min); amplification (35 cycles consisting of 60 s at 95 °C, 60 s at 51 °C and 120 s at 72 °C); and final extension (72 °C for 7 min). Table 1. Trechine specimens used for molecular analyses with GenBank accession numbers Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 *Outgroup. View Large Table 1. Trechine specimens used for molecular analyses with GenBank accession numbers Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 *Outgroup. View Large The PCR products were purified using the QIAquick Purification Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s instructions. DNA sequencing was performed using automated equipment (Macrogen Inc., Seoul, South Korea). Sequences were manually edited in FinchTV (Geospiza Inc., Seattle, WA, USA) and aligned using the ClustalW program integrated in MEGA5 (Tamura et al., 2011). Genetic divergence was estimated using Kimura’s two-parameter (K2P) method of base substitution (Kimura, 1980). Phylogenetic reconstruction was performed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ) incorporated in the MEGA5 software package. One thousand bootstrap replicates were performed in every analysis to assess robustness of the trees. Tamura’s three-parameter model (T92 + G) was identified as the best-fitting model of sequence evolution based on the Bayesian information criterion and corrected Akaike information criterion (Nei & Kumar, 2000) for the ML method of phylogenetic reconstruction. The subspecies Duvalius (Paraduvalius) stankovitchi georgevitchi (Jeannel, 1924) was used as an outgroup. The nucleotide sequence data were deposited in the GenBank database under accession numbers KY351542–KY351551 (Table 1). K2P model was used to estimate genetic divergence of the analysed taxa. RESULTS AND DISCUSSION Taxonomy Family Carabidae Latreille, 1802 Subfamily Trechinae Bonelli, 1810 Tribe Trechini Bonelli, 1810 Genus Pheggomisetes Knirsch, 1923 Pheggomisetes serbicus Ćurčić, Vrbica & B. Guéorguiev sp. nov. (Figs 1 and 2A–H) Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Pež Dupka Cave, 43°13′17.77″N 22°47′17.08″E, village of Dojkinci, 869 m a.s.l., near Pirot, 11.VII–10.X.2013, from pitfall traps, leg. D. Antić & M. Petković’ (white label, printed)/‘Holotypus Pheggomisetes serbicus sp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: six males and eight females, same data as for holotype (IZFB); three males and four females labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Pež Dupka Cave, village of Dojkinci, 869 m a.s.l., near Pirot, 11.VII.2013, leg. D. Antić & M. Petković’ (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes serbicus sp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 5.55–6.675 mm (M 6.11 mm) (HT 6.30 mm). Head oval, HL/HW R 1.18–1.34 (M 1.26) (HT 1.27), widest somewhat before its middle, scarcely wider than pronotum (Fig. 1). Frontal furrows long, slightly exceeding mid head level, deeply impressed anteriorly and sigmoidally curved. Neck narrow, HW/NW R 1.97–2.75 (M 2.50) (HT 2.19). Antennae long, around the same length as TL in males, while shorter than TL in females. Figure 1. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 1. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Pronotum widest somewhat after the anterior third, almost as long as wide (Fig. 1). Anterior pronotal margin concave, shorter than pronotal base. Lateral pronotal margins rounded anteriorly and slightly concave posteriorly. Pronotal base somewhat concave in the middle. Fore pronotal angles obtuse, rounded. Hind pronotal angles acute, almost right. Elytra relatively long, oval, convex, with the lateral sides rounded anteriorly, widest slightly after the mid level, EL/EW R 1.61–1.83 (M 1.725) (HT 1.81). Elytral base slightly narrower than pronotum (Fig. 1). Humeral angles obtuse, rounded and quite elevated. Elytral apex rounded. Legs and claws long and thin (Fig. 1). Median lobe of the aedeagus in lateral view curved, with a rounded somewhat elevated apex (Fig. 2A, E). Basal bulb small, rounded. Parameres with three apical setae each. Median lobe in dorsal view straight, with a rounded apex, narrowing towards basal bulb (Fig. 1B). Gutter-shaped copulatory piece covered with numerous spines (Fig. 2B, F), wide at its basal three fifths and markedly narrowed at its apical two fifths. Figure 2. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 2. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Male abdominal sternite IX (urite) subtriangular, slightly elongate, slightly longer than aedeagus (Fig. 2C, G). Apophysis narrow, constricted proximally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 2D, H. Gonocoxites IX of moderate length, slightly curved, apically rounded, basally completely jointed with massive gonosubcoxites IX (Fig. 2D, H). Chaetotaxy. Frons with six to seven (HT – 7) setae on each side. Pronotum with normal chaetotaxy (two pairs of setae). Five to seven setae on third interstria (HT – 7) on each elytron (Fig. 1). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, while between pores 3 and 4 longest; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 about as long as distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 1). Differential diagnosis: The new species is compared here with the other known Pheggomisetes species (Casale & Laneyrie, 1982; Moravec et al., 2003). A comparison of the new species with P. ninae is not provided here since the latter taxon is regarded as a subspecies of P. globiceps (see below). The new species differs from P. globiceps in having a smaller value of TL M (6.11 vs. ≥ 6.295 mm), a smaller value of HL M (1.39 vs. ≥ 1.40 mm), smaller values of AL M (5.91, males 6.01, females 5.70 vs. ≥ 6.525 mm, males ≥ 6.56 mm, females ≥ 6.30 mm), smaller values of AL/TL M (0.97, males 0.99, females 0.92 vs. ≥ 1.01, males ≥ 1.05, females ≥ 0.97), a greater value of HL/AL M (0.235 vs. ≤ 0.225), a greater value of FL M (0.75 vs. ≤ 0.68 mm), a greater value of FL/HL M (0.54 vs. ≤ 0.48), different shape of the humeral angles (more rounded, quite elevated vs. more obtuse, less elevated), a greater value of EL/TL M (0.55 vs. ≤ 0.54) and different shape of the copulatory piece in dorsal aspect (more markedly narrowed apically vs. gradually narrowed apically) (Tables 2 and 3; Figs 1–13; Supporting Information, Table S1) (Jeannel, 1928; Guéorguiev, 1964; this study). Even though the differences obtained between the mean values of certain measurements (HL) and ratios (HL/AL, EL/TL) are very small (Supporting Information, Table S1), the distributions of ranges in the two species show statistically significant differences (Table 3). Table 2. Results of ANOVA for each variable between P. serbicus sp. nov. and P. globiceps (exact significance level P ≤ 0.05, marked in bold) Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 View Large Table 2. Results of ANOVA for each variable between P. serbicus sp. nov. and P. globiceps (exact significance level P ≤ 0.05, marked in bold) Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 View Large Table 3. Results of Mann–Whitney U test between P. serbicus sp. nov. (n = 24) and P. globiceps (n = 44) (exact significance level P ≤ 0.05, marked in bold) Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 View Large Table 3. Results of Mann–Whitney U test between P. serbicus sp. nov. (n = 24) and P. globiceps (n = 44) (exact significance level P ≤ 0.05, marked in bold) Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 View Large Figure 3. View largeDownload slide Shape of the shoulders in the Pheggomisetes subspecies analysed. A, P. serbicus serbicus subsp. nov. B, P. globiceps ciniglavcensis subsp. nov. C, P. globiceps ilandjievi. D, P. serbicus belensis subsp. nov. E, P. globiceps ninaecomb. & stat. nov. F, P. globiceps globiceps. Scales = 0.5 mm. Figure 3. View largeDownload slide Shape of the shoulders in the Pheggomisetes subspecies analysed. A, P. serbicus serbicus subsp. nov. B, P. globiceps ciniglavcensis subsp. nov. C, P. globiceps ilandjievi. D, P. serbicus belensis subsp. nov. E, P. globiceps ninaecomb. & stat. nov. F, P. globiceps globiceps. Scales = 0.5 mm. Figure 4. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 4. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 5. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 5. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 6. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 6. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 7. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 7. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 8. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 8. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 9. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, topotype male, aedeagus (lateral view). B, F, topotype male, aedeagus (dorsal view). C, G, topotype male, abdominal sternite IX (urite). D, H, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 9. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, topotype male, aedeagus (lateral view). B, F, topotype male, aedeagus (dorsal view). C, G, topotype male, abdominal sternite IX (urite). D, H, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 10. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 10. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 11. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 11. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 12. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 12. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 13. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 13. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. The new species differs from P. radevi in having a smaller value of TL R (5.55–6.675 vs. 7–8 mm), a greater value of HL/HW M (1.26 vs. 1.00), different shape of the head (widest slightly before its mid part, posteriorly somewhat convex vs. widest around its mid part, posteriorly very convex), a smaller value of HW/NW M (2.50 vs. c. 3.00), different shape of the pronotum (weakly narrowed basally, strongly rounded anteriorly, well sinuate in back vs. strongly narrowed basally, weakly rounded anteriorly, strongly sinuate in back), a different value of PaW/PbW (pronotal apex between tips of the anterior angles narrower than pronotal base between tips of the posterior angles vs. pronotal apex between tips of the anterior angles wider than pronotal base between tips of the posterior angles), different shape of the hind pronotal angles (almost right, not prominent vs. acute, protruding backwards and outwards), different form of the elytra (less elongate, with more prominent shoulders vs. more elongate, with less prominent shoulders), different shape of the median lobe (less bent vs. more bent) and different shape of the basal bulb (relatively small, rounded vs. medium-sized, relatively elongate) (Jeannel, 1928; Guéorguiev, 1964; this study). The new species differs from P. buresi in having a smaller value of TL R (5.55–6.675 vs. 7.20–9.00 mm), different shape of the head (less elongate, posteriorly more convex, abruptly narrowing towards the neck vs. more elongate, posteriorly less convex, gradually narrowing towards the neck), a different position of maximum head width (slightly in front of the middle vs. anteriorly), a greater value of HW/NW M (2.50 vs. c. 2.00), different form of the lateral pronotal margins (a little rounded anteriorly, slightly sinuate posteriorly vs. somewhat arcuate), different shape of the hind pronotal angles (almost right, not prominent vs. acute, protruding backwards and outwards), different shape of the elytra (less elongate, with more pronounced shoulders vs. more elongate, with less pronounced shoulders), different shape of the median lobe (less elongate, less bent vs. more elongate, more bent) and different shape of the basal bulb (relatively small, rounded vs. relatively massive, elongate) (Jeannel, 1928; Guéorguiev, 1964; this study). Among the three known species of the genus, P. globiceps is the smallest one and the only species that has right hind angles of the pronotum (Jeannel, 1928; Guéorguiev, 1964). It has a pronotum that is weakly narrowed basally, with lateral margins moderately rounded anteriorly and weakly sinuate posteriorly, in addition to a relatively narrow elytral base (Jeannel, 1928; Guéorguiev, 1964). Thus, it is quite clear that P. serbicus sp. nov. shares the same character states and is closely related to it. In addition, P. buresi differs from all other congeners in having a rather thick neck. No significant differences within the genus are evident in the male genitalia (especially in regard to shape of the median lobe), even at the species level (Jeannel, 1928; Guéorguiev, 1964; this article). Variability: The number of setae on both frons (six to seven on each side) and elytra can vary (five to seven on each side). Etymology: The new species is named after Serbia, its terra typica. Distribution: The type locality is the Pež Dupka Cave in the village of Dojkinci (near Pirot) in the Stara Planina Mountains of Southeast Serbia. The new species inhabits a few caves in the villages of Dojkinci and Bela in the Stara Planina Mountains of Southeast Serbia; P. buresi and P. radevi live in caves near the town of Vratsa and villages of Chiren, Eliseyna, Chelopek, Druzhevo and Milanovo in the West Stara Planina Mountains of Western Bulgaria; and P. globiceps inhabits numerous caves in the West Stara Planina Mountains and Pre-Balkan region of Western Bulgaria, as well as a few caves in the villages of Petrlaš and Činiglavci in the Stara Planina Mountains of Southeast Serbia (Guéorguiev & Guéorguiev, 1995; this study). Pheggomisetes serbicus belensis Ćurčić, Vrbica & B. Guéorguiev subsp. nov. (Figs 4 and 5A–H) Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Suva Dupka Cave, 43°14′40.9″N 22°44′16.5″E, village of Bela, 801 m a.s.l., near Pirot, 25.V.2014, leg. S. Ćurčić, D. Antić & I. Petrović’ (white label, printed)/‘Holotypus Pheggomisetes serbicus belensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: three males and two females, same data as for holotype (IZFB); four males and eight females labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Suva Dupka Cave, village of Bela, 801 m a.s.l., near Pirot, 25.V-05.VII.2014, from pitfall traps, leg. D. Antić’ (IZFB); one female labelled as follows: ‘Southeast Serbia, Stara Planina Mts., a cave in the vicinity of the Suva Dupka Cave, 43°14′30.84″N 22°44′9.40″E, village of Bela, 793 m a.s.l., near Pirot, 25.V-05.VII.2014, from pitfall traps, leg. D. Antić’ (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes serbicus belensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 5.70–6.60 mm (M 6.045 mm) (HT 5.925 mm). HL/HW R 1.23–1.33 (M 1.27) (HT 1.24) (Fig. 4). Frontal furrows reaching mid head level. HW/NW R 2.09–2.50 (M 2.24) (HT 2.25). Anterior pronotal margin clearly (in males) or slightly (in females) concave, shorter than pronotal base (Fig. 3). Lateral pronotal margins very slightly concave posteriorly. Elytra with lateral sides almost straight in the anterior half, EL/EW R 1.62–1.92 (M 1.77) (HT 1.71) (Fig. 4). Median lobe of the aedeagus in lateral view slightly convex dorsally at around the level of two fifths, having an almost straight apex (Fig. 5A, E). Median lobe in dorsal view as presented in Figure 5B, F and inner sac as presented in Figure 5A, B, E, F. Male abdominal sternite IX (urite) as presented in Figure 5C, G, subtriangular, slightly elongate, slightly longer than aedeagus. Apophysis narrow, constricted proximally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 5D, H. Chaetotaxy. Frons with six to eight (HT – 6) setae on each side. Pronotum with normal chaetotaxy (two pairs of setae). Five to seven setae on third interstria (HT – 6–7) on each elytron (Fig. 4). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 1 and 2 approximately the same as between pores 3 and 4; median series at around the middle of the elytra, two setae being somewhat distanced from the gutter, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 4). Differential diagnosis: The new subspecies is compared with the nominal subspecies, P. serbicus serbicus subsp. nov. The new subspecies clearly differs from P. serbicus serbicus subsp. nov. in having a smaller value of TL M (6.045 vs. 6.11 mm), a different value of FL/HL M (frontal furrows reaching mid head level vs. frontal furrows somewhat exceeding mid head level), a smaller value of HW/NW M (2.24 vs. 2.50), a smaller value of FL M (0.68 vs. 0.75 mm), a greater value of AL M (5.99 vs. 5.91 mm), different shape of the anterior pronotal margin in females (less concave vs. more concave), different shape of the pronotal base in the middle (less concave vs. more concave), different shape of the lateral margins of the elytra anteriorly (more straight vs. rounded), a greater value of PaW/PbW M (0.78 vs. 0.74), a greater value of EL/EW M (1.77 vs. 1.725), a different position of certain humeral and median setae (distance between pores 2 and 3 shortest, distance between pores 1 and 2 approximately the same as between pores 3 and 4, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3 vs. distance between pores 2 and 3 shortest, distance between pores 3 and 4 longest, distance between pores 5 and 6 about as long as distance between pores 2 and 3) belonging to the elytral umbilicate series, different shape of the median lobe apex in lateral aspect (almost straight vs. somewhat elevated) and different shape of the median lobe’s basal bulb in lateral aspect (more curved vs. less curved) (Supporting Information, Table S1) (this study). Variability: The number of setae on both frons (six to eight on each side) and elytra can vary (five to seven on each side). Etymology: The new subspecies is named after the village of Bela, in which the type locality is situated. Distribution: It inhabits two caves in the village of Bela (near Pirot) in the Stara Planina Mountains of Southeast Serbia – the Suva Dupka Cave and a cave in its vicinity. Pheggomisetes globiceps Buresch, 1925 Pheggomisetes globiceps ciniglavcensis Ćurčić & Vrbica, subsp. nov. (Figs 6 and 7A–H) Pheggomisetes globiceps ilandjievi:Gajović et al. (2011: 80). Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Propas Pit, 43°04′05.7″N 22°44′18.5″E, village of Činiglavci, 714 m a.s.l., near Pirot, 29.V-08.VII.2013, from pitfall traps, leg. Đ. Marković & M. Petković’ (white label, printed)/‘Holotypus Pheggomisetes globiceps ciniglavcensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: 29 males and 20 females, same data as for holotype (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes globiceps ciniglavcensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 6.15–6.825 mm (M 6.46 mm) (HT 6.525 mm). Head oval, HL/HW R 1.19–1.355 (M 1.275) (HT 1.19), widest somewhat before its mid part, scarcely wider than pronotum (Fig. 6). Frontal furrows almost reaching mid head level, deeply impressed anteriorly and sigmoidally curved. Neck narrow, HW/NW R 2.17–2.61 (M 2.405) (HT 2.50). Antennae long, longer (in males) or slightly shorter (in females) than TL. Pronotum widest somewhat after the anterior third, almost as long as wide (Fig. 6). Anterior pronotal margin slightly concave, shorter than pronotal base. Lateral pronotal margins rounded anteriorly and slightly concave posteriorly. Pronotal base very slightly concave in the middle. Fore pronotal angles obtuse, rounded. Hind pronotal angles acute, almost right. Elytra relatively long, oval, convex, widest somewhat after the mid level, EL/EW R 1.53–1.73 (M 1.64) (HT 1.68). Elytral base slightly wider than pronotum (BW/PW R 0.85–1.10, M 1.03, HT 1.00) (Fig. 6). Humeral angles obtuse, rounded and relatively elevated. Elytral apex rounded. Legs and claws long and thin (Fig. 6). Median lobe of the aedeagus curved, slightly convex dorsally around the basal fourth, with a rounded apex (Fig. 7A, B, E, F). Basal bulb small, rounded. Parameres with three setae each, of which two are apically positioned. Triangular gutter-shaped copulatory piece covered with numerous thorns (Fig. 7B, F), gradually narrowed apically in dorsal aspect. Male abdominal sternite IX (urite) subtriangular, slightly elongate, somewhat longer than aedeagus (Fig. 7C, G). Apophysis narrow, gradually narrowing distally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 7D, H. Gonocoxites IX slightly elongate, somewhat curved, apically rounded, basally completely jointed with massive gonosubcoxites IX (Fig. 7D, H). Chaetotaxy. Frons with five to seven setae (HT – 6–7) on each side. Pronotum with normal chaetotaxy (two pairs of setae). Six to eight setae on third interstria (HT – 6–7) on each elytron (Fig. 6). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 1 and 2 is approximately the same as between pores 3 and 4; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 about as long as distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 6). Differential diagnosis: The known subspecies of P. globiceps differ in shape of the head, length and depth of the frontal furrows, shape of the hind pronotal angles, lateral margins of the head and pronotum, the HL/PL and shape of the humeral angles (Guéorguiev, 1964). Some new characters should also be taken into account in separating Pheggomisetes taxa (e.g. TL, HL, HL/HW, HL/AL, HL/TL, HW/NW, HW/PW, HW/EW, AL, AL/TL, PL, PL/PW, PL/TL, PaW, PbW, PW, PW/EW, PWP, EW, EL/EW, BW/PW, elytral umbilicate series position, and aedeagus and copulatory piece shapes) (Tables 2 and 3; Supporting Information, Table S1). The new subspecies is compared here with the morphologically and geographically closest subspecies of P. globiceps and the nominotypical subspecies. The former are P. globiceps ilandjievi (Figs 10, 11A–D) and P. globiceps ninae comb. & stat. nov. (with the head elongately ovoid, lateral margins of the head moderately rounded, head slightly rounded both anteriorly and posteriorly as well, acute/right posterior pronotal angles, pronotum basally constricted, head slightly broader than pronotum and humeral angles slightly elevated) (Guéorguiev, 1964; Ćurčić et al., 2004; this article). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps ilandjievi in having a smaller value of TL M (6.46 vs. 6.60 mm), different shape of the head (widest at 2/5 of its length vs. widest slightly before the middle), a smaller value of HL/HW M (1.275 vs. 1.32), a greater value of AL M (6.775 vs. 6.64 mm), a greater value of AL/TL M (1.05 vs. 1.01), different shape of the lateral pronotal margins (less rounded anteriorly, more concave posteriorly vs. more rounded anteriorly, less concave posteriorly), greater values of PaW (R 0.63–0.68, M 0.66 vs. R 0.53–0.595 mm, M 0.57 mm), a greater value of PbW M (0.83 vs. 0.75 mm), a greater value of PaW/PW M (0.64 vs. 0.57), different shape of the humeral angles (less rounded and less elevated vs. more rounded and more elevated), a smaller value of EL/EW M (1.64 vs. 1.83), a greater value of BW/PW M (1.03 vs. 0.95), different shape of the median lobe (slightly convex dorsally around the basal fourth, with a narrower apex in dorsal view vs. not convex dorsally around the basal fourth, with a wider apex in dorsal view) and different size of the basal bulb (smaller vs. bigger) (Supporting Information, Table S1) (Guéorguiev, 1964; this study). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps ninae comb. & stat. nov. in having a greater value of TL M (6.46 vs. 6.295 mm), a smaller value of HL/HW M (1.275 vs. 1.30), a greater value of FL M (0.68 vs. 0.63 mm), greater values of AL M (6.775, males 6.84, females 6.50 vs. 6.525 mm, males 6.56 mm, females 6.30 mm), a greater value of HW/NW M (2.405 vs. 2.26), a greater value of EL M (3.50 vs. 3.36 mm), a greater value of EW M (2.135 vs. 1.99 mm), a smaller value of EL/EW M (1.64 vs. 1.69), a greater value of BW/PW M (1.03 vs. 0.91), different shape of the median lobe (narrower, somewhat more curved basally, then regularly curved, slightly convex dorsally around the basal fourth, with a narrow anterior part in dorsal view vs. thicker, regularly curved, somewhat convex dorsally in the middle, with a wide anterior part in dorsal view) and a different number of parameral setae (three, two of them apical vs. five, three of them apical) (Supporting Information, Table S1) (Ćurčić et al., 2004; this study). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps globiceps (Figs 12, 13A–D) in having different shape of the head (widest at the anterior 2/5 of its length vs. widest somewhat after the middle), a greater value of HW/NW M (2.405 vs. 2.24), greater values of AL M (6.775, males 6.84, females 6.50 vs. 6.585 mm, males 6.775 mm, females 6.30 mm), a smaller value of HL/PL M (1.51 vs. 1.63), a greater value of PL/PW M (0.91 vs. 0.88), a greater value of EW M (2.135 vs. 2.02 mm), a smaller value of EL/EW M (1.64 vs. 1.725), a greater value of BW/PW M (1.03 vs. 0.885), different shape of the median lobe (narrower, elongate, with more elongate basal bulb vs. wider, stout, especially basally, with a short stout basal bulb) and different shape of the copulatory piece in dorsal aspect (gradually narrowing towards the apex vs. wide at the basal 3/5 and markedly narrowed at the apical 2/5) (Supporting Information, Table S1) (Guéorguiev, 1964; this study). Variability: The number of setae on both frons (five to seven on each side) and elytra can vary (six to eight on each side). Etymology: The subspecies is named after the village of Činiglavci, in which the type locality is situated. Distribution: It lives solely in the Propas Pit in the village of Činiglavci (near Pirot) in the Stara Planina Mountains of Southeast Serbia. Remarks: The new subspecies was originally treated as P. globiceps ilandjievi by Gajović et al. (2011), who collected a sample several years ago. Pheggomisetes globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. (Figs 8 and 9A–H) Material examined: Sixty topotype males and 75 topotype females, Southeast Serbia, Stara Planina Mts., Hodžina Dupka Pit, 43°04′27.9″N 22°47′48.5″E, 692 m a.s.l., village of Petrlaš, near Dimitrovgrad, 26.VI-24.IX.2012, from pitfall traps, leg. Đ. Marković & D. Dragulović (IZFB); nine males and 25 females, Southeast Serbia, Mt. Stara Planina Mts., Petrlaška (= Velika) Pećina Cave, 43°04′27.81″N 22°47′46.50″E, 701 m a.s.l., village of Petrlaš, near Dimitrovgrad, 26.VI-24.IX.2012, both collected by hand and from pitfall traps, leg. D. Antić & S. Ćurčić (IZFB); one male and one female, idem, 03.XII.2012, leg. D. Antić & S. Ćurčić (IZFB); one female, idem, 19.IX.2013, leg. P. Beron (IZFB); two females, Southeast Serbia, Stara Planina Mts., Džemanska Propast Pit, 43°07′44.2″N 22°79′15.9″E, 738 m a.s.l., village of Petrlaš, near Dimitrovgrad, 24.IX.2012, leg. M. Petković & D. Dragulović (IZFB); one male, Southeast Serbia, Stara Planina Mts., Tmna Dupka Cave, 43°04′42.0″N 22°47′24.5″E, 720 m a.s.l., village of Petrlaš, near Dimitrovgrad, 24.IX.2012, leg. S. Ćurčić (IZFB); two males and ten females, idem, 24.IX-03.XII.2012, from pitfall traps, leg. D. Antić & S. Ćurčić (IZFB). Description: The description has been already presented by Ćurčić et al. (2004). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 3 and 4 longest; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 shorter than distance between pores 3 and 4 (Fig. 8). Differential diagnosis: The subspecies is compared here with both the morphologically and geographically closest subspecies of P. globiceps and the nominotypical subspecies. The former are P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov. (Guéorguiev, 1964; this study). Pheggomisetes globiceps ninae comb. & stat. nov. differs clearly from P. globiceps ilandjievi in having a smaller value of TL M (6.295 vs. 6.60 mm), smaller values of AL M (6.525, males 6.56, females 6.30 vs. 6.64 mm, males 6.675 mm, females 6.60 mm), a smaller value of HW/NW M (2.26 vs. 2.35), a greater value of PaW M (0.61 vs. 0.57 mm), a greater value of PbW M (0.79 vs. 0.75 mm), different shape of the pronotal base in males (straight vs. concave), different shape of the hind pronotal angles (almost right vs. acute, rarely right), a smaller value of EL M (3.36 vs. 3.555 mm), a greater value of EW M (1.99 vs. 1.905 mm), a smaller value of EL/EW M (1.69 vs. 1.83), different shape of the median lobe (thicker, with a wider anterior part in dorsal view vs. more elongate, with a narrower anterior part in dorsal view) and a different number of parameral setae (five vs. three to four) (Supporting Information, Table S1) (Guéorguiev, 1964; Ćurčić et al., 2004; this study). All morphological differences between P. globiceps ninae comb. & stat. nov. and P. globiceps ciniglavcensis subsp. nov. are mentioned above (see the Differential diagnosis of P. globiceps ciniglavcensis subsp. nov.) (Figs 6, 7A–H, 8, 9A–H; Supporting Information, Table S1) (Ćurčić et al., 2004; this study). P. globiceps ninae comb. & stat. nov. can be easily distinguished from P. globiceps globiceps on the basis of having a smaller value of TL M (6.295 vs. 6.405 mm), a smaller value of HL M (1.40 vs. 1.47 mm), a smaller value of HW M (1.08 vs. 1.17 mm), a greater value of HL/HW M (1.30 vs. 1.26), different shape of the head (widest at around the anterior 2/5 of its length vs. widest somewhat after the middle), a smaller value of HL/PL M (1.52 vs. 1.63), a smaller value of HW/PW M (1.07 vs. 1.14), a greater value of PL/PW M (0.92 vs. 0.88), a smaller value of EL M (3.36 vs. 3.47 mm), a different number of parameral setae (five vs. three to four) and different shape of the median lobe (somewhat convex dorsally around the middle, with a somewhat elongate basal bulb vs. somewhat convex dorsally around the basal third, with a stout, relatively small basal bulb) (Supporting Information, Table S1) (Guéorguiev, 1964; Ćurčić et al., 2004; this study). Distribution: It lives in a few caves and pits on the western border of the Odorovačko Polje (692–738 m a.s.l.) in the village of Petrlaš (near Dimitrovgrad) in the Stara Planina Mountains of Southeast Serbia. Remarks: Interestingly, the taxon was originally treated as P. globiceps ilandjievi by Pretner (1970), who collected the first specimens from the Hodžina Dupka Pit with P. R. Deeleman. A similar opinion was expressed by Nešić et al. (2010) in a recent contribution. After the performed morphological and molecular analyses, we found that there is no difference between Pheggomisetes specimens from the Hodžina Dupka Pit, the Petrlaška (= Velika) Pećina Cave, the Džemanska Propast Pit and the Tmna Dupka Cave, all situated in the village of Petrlaš near Dimitrovgrad in the Stara Planina Mountains of Southeast Serbia. They all belong to the same taxon, which was previously described under the name P. ninae. After a thorough morphological analysis supported by molecular data, we established that the existing differences between P. ninae and other Pheggomisetes species are not great enough to treat it as a distinct species. The taxon in question deserves a subspecies rank within P. globiceps since certain smaller differences (both morphological and phylogenetical) were proved to exist between it and the geographically nearest subspecies of P. globiceps, but these were not significant enough to convince us of the need to separate it as a species. To be specific, certain morphological differences were observed in regard to TL, AL, HW/NW, FL, FL/HL, PaW, PbW, PaW/PW, shape of the lateral pronotal margins, pronotal base shape, EL, EW, EL/EW, BW/PW, shape of the humeral angles and position of the elytral umbilicate series (Supporting Information, Table S1), but shapes of the aedeagi and copulatory pieces are quite similar, indicating that the above-mentioned differences are in reality interpopulational, not interspecific (an assertion supported by small genetic differences recorded between the given taxon and its closest relatives, 0.5 and 1.3%, respectively). We therefore suggest that the taxonomic status of P. ninae be changed to P. globiceps ninae comb. & stat. nov. Key to Species of the Genus Pheggomisetes Knirsch, 1923 (FIG. 14) 1 Neck constriction very broad and not abrupt in dorsal view, while flat in lateral view. Head elliptical, cheeks less rounded (Northwest Bulgaria) …………………….……......………….……...…… P. buresi (Knirsch, 1923) – Neck constriction narrow and abrupt in dorsal view, while deeper in lateral view. Head circular or ovoid, cheeks more rounded ………………………..……………...……………...………..……………...……………......... 2 2 Pronotum clearly narrowed in front of base. Head very broad in posterior third, with frontal furrows very deep (Northwest Bulgaria) ………………………….……….……….……….……….….…. P. radevi Knirsch, 1924 – Pronotum not narrowed in front of base, sometimes with lateral margins more or less sinuate in posterior third. Head narrower in posterior third, with frontal furrows less deep 3 3 Longer TL M (≥ 6.295 mm), antennae longer (M ≥ 6.525 mm), longer than body, frontal furrows not reaching middle of the head, humeral angles more obtuse, less elevated, copulatory piece gradually narrowed apically (Western Bulgaria and Southeast Serbia) [P. globiceps Buresch, 1925] 4 – Smaller TL M (≤ 6.11 mm), antennae shorter (M ≤ 5.99 mm), slightly shorter than body, frontal furrows exceeding/reaching middle of the head, humeral angles more rounded, quite elevated, copulatory piece more markedly narrowed apically (Southeast Serbia) [P. serbicus Ćurčić, Vrbica & B. Guéorguiev, sp. nov.] 5 4 TL M 6.295 mm, antennae shorter (M 6.525 mm), humeral angles more elevated, HW/NW M 2.26, EL M 3.36 mm, EW M 1.99 mm, elytra at the base narrower than pronotum, median lobe thicker, regularly curved, somewhat convex dorsally in the middle, with a wide anterior part in dorsal view (Southeast Serbia) …………….. P. globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. –  TL M 6.46 mm, antennae longer (M 6.775 mm), humeral angles less elevated, HW/NW M 2.405, EL M 3.50 mm, EW M 2.135 mm, elytra at the base slightly wider than pronotum, median lobe narrower, somewhat more curved basally, then regularly curved, slightly convex dorsally around the basal fourth, with a narrow anterior part in dorsal view (Southeast Serbia) ……………...………………………………………………………………………………………………………….....…..…… P. globiceps ciniglavcensis Ćurčić & Vrbica, subsp. nov. 5 TL M 6.11 mm, frontal furrows somewhat exceeding mid head level, HW/NW M 2.50, FL M 0.75 mm, AL M 5.91 mm, anterior pronotal margin more concave in females, pronotal base more concave in the middle, PaW/PbW M 0.74, EL/EW M 1.725, lateral margins of elytra rounded anteriorly, median lobe apex somewhat elevated, basal bulb and basal part of median lobe narrower (Southeast Serbia) ……………. .……………...……………...……………...…… P. serbicus serbicus Ćurčić, Vrbica & B. Guéorguiev, subsp. nov. – TL M 6.045 mm, frontal furrows reaching mid head level, HW/NW M 2.24, FL M 0.68 mm, AL M 5.99 mm, anterior pronotal margin less concave in females, pronotal base less concave in the middle, PaW/PbW M 0.78, EL/EW M 1.77, lateral margins of elytra more straight anteriorly, median lobe apex almost straight, basal bulb and basal part of median lobe wider (Southeast Serbia) ………...……………...………….….….….….….….….….….….….….….….….….….….…. P. serbicus belensis Ćurčić, Vrbica & B. Guéorguiev, subsp. nov. Figure 14. View largeDownload slide Distribution of Pheggomisetes taxa in Serbia and the immediate surroundings. Scale = 10 km. Figure 14. View largeDownload slide Distribution of Pheggomisetes taxa in Serbia and the immediate surroundings. Scale = 10 km. TPEF microscopy of the internal structures of Pheggomisetes Certain well-chitinized internal morphological structures of Pheggomisetes ssp. were observed by two-photon excited autofluorescence microscopy. The samples were not fluorescently labelled, so autofluorescence was detected and used for imaging. For the study, we used a 25× numerical aperture 0.8 water/glycerin immersion objective and 930-nm excitation wavelength. This somewhat longer wavelength was utilized to avoid the autofluorescence of residual tissues remaining after beetle dissection. In addition, it was possible to penetrate deeper (up to 200 µm for the studied sample) through the chitinous cuticle due to the reduced two-photon absorption of chitin (Rabasović et al., 2015). We present TPEF 3D images of the male (aedeagus) and female (gonocoxites IX and gonosubcoxites IX) genitalia and the male abdominal sternite IX (urite) of all Pheggomisetes taxa from Serbia. The images revealed morphological details similar to those observed using classical bright-field microscopy (Figs 2E–H, 5E–H, 7E–H, 9E–H). In addition, selected 3D video clips of the three morphological structures are included, showing them in rotation around the longitudinal, lateral and vertical axes (Supporting Information, Appendices S1–S6). This makes it possible for the structures to be observed in every direction, which provides better insight into the shape and spatial relations of internal structures. The aedeagus is observed both laterally and dorsally (Figs 2E, F, 5E, F, 7E, F, 9E, F). The structure of both the surface (the fine relief) and the inner part (the copulatory piece composed of numerous tooth-like structures and the inner sac) of the median lobe (Fig. 15) is clearly distinguished. Both strongly (e.g. the copulatory piece) and weakly (e.g. the inner sac) chitinized parts of the aedeagus are visible (Figs 2E, F, 5E, F, 7E, F, 9E, F). All parts of the aedeagus (median lobe, basal bulb, parameres and their setae, copulatory piece and inner sac) are sharply delimited from each other (Figs 2E, F, 5E, F, 7E, F, 9E, F), as in the case of the images recorded earlier by bright-field microscopy (Figs 2A, B, 5A, B, 7A, B, 9A, B). Figure 15. View largeDownload slide TPEF microscopy image of part of the tooth-like copulatory piece of P. globiceps ninae comb. & stat. nov., showing fine details of the structure. Figure 15. View largeDownload slide TPEF microscopy image of part of the tooth-like copulatory piece of P. globiceps ninae comb. & stat. nov., showing fine details of the structure. One of the male internal sclerites, abdominal sternite IX (urite), is clearly visible and can be imaged by TPEF microscopy since it is well chitinized (Figs 2G, 5G, 7G, 9G). The shape and thickness of the structure are as visible as in the photographs obtained by bright-field microscopy (Figs 2C, 5C, 7C, 9C). Similarly, the parts of the female genitalia, which are highly sclerotized (gonocoxites IX and gonosubcoxites IX), were also observed. Sharply delimited parts of the aforementioned female genital structures are visible. The setation and fine relief are distinctly discernible on the surface, while the internal structure can also be observed (Figs 2H, 5H, 7H, 9H). The images of both cross and longitudinal sections of Pheggomisetes male genitalia (Fig. 16C, D) show the clear advantage of NLM vs. traditional classical microscopy in investigating anatomical features. To be specific, all features of the internal structures (e.g. shape and position of copulatory piece) are discernible on any section of the genitalia using this method (Fig. 16A–D). Apart from internal characteristics of the structures, their thickness can be ascertained and measured as well. One more benefit of using TPEF is that it provides additional data on the shapes of certain structures (e.g. median lobe, parameres, parameral setae) on cross sections (any level) (Fig. 16D), which cannot be detected by classical light microscopy. The images can be further used to calculate data on the structure’s surface, shape and volume. The female genitalia can be observed in a similar manner as well. Figure 16. View largeDownload slide TPEF microscopy images of the aedeagus of P. globiceps ninae comb. & stat. nov. A, lateral view with longitudinal section plane (red square). B, lateral view with cross-sectional plane (red line). C, a corresponding longitudinal section. D, a corresponding cross section. Scales = 0.10 mm (A–C) and 0.05 mm (D). Figure 16. View largeDownload slide TPEF microscopy images of the aedeagus of P. globiceps ninae comb. & stat. nov. A, lateral view with longitudinal section plane (red square). B, lateral view with cross-sectional plane (red line). C, a corresponding longitudinal section. D, a corresponding cross section. Scales = 0.10 mm (A–C) and 0.05 mm (D). Statistical morphometric analysis Only 20 variables (eight commonly used morphological trait measurements and 12 ratio variables) passed the Shapiro-Wilk normality test (HL, HW, FL, PW, PaW, PbW, EW, EWP, HL/HW, HL/AL, HL/PL, HL/TL, HW/NW, HW/PW, AL/TL, PL/PW, PaW/PW, PbW/PW, PaW/PbW and BW/PW) and were further used for parametric analyses. Normality tests were also performed on log-transformed data, but they resulted in the same 20 variables. Descriptive statistics of the quantitative traits and ratio variables of P. globiceps and P. serbicus sp. nov. subspecies from Serbia are given in Supporting Information, Table S1. One-way MANOVA of samples of the two Pheggomisetes species revealed significant differences between the species [Wilks’ Λ = 0.095, F (20, 40) = 19, P < 0.001]. Post hoc pairwise comparison using Scheffe’s test indicated that seven variables are statistically significant (Table 2). The HL/AL and HL/TL variables have the most distinct discriminative power. AL/TL, FL, EW, PaW and HL are statistically less important for distinguishing the two species. Table 3 presents the results of non-parametric comparisons between samples of the two species (for non-normally distributed variables) using the Mann–Whitney U test. Ten variables are recognized as statistically significant, but AL and TL are most important, while EL/TL and PL/TL are somewhat less important, followed by PWP, PW/EW, FL/HL, HW/EW, PL and EL/EW. One-way MANOVA of Pheggomisetes taxa (populations belonging to two species and six subspecies) revealed significant differences in the variation of eight commonly used morphological trait measurements and 12 ratios [Wilks’ Λ = 0.095, F (20, 40) = 18.941, P < 0.001 and Wilks’ Λ = 0.003, F (90, 222) = 5.890, P < 0.001, respectively]. The results of linear DA of 20 variables showed that the total correct percentage of the classification matrix of all six Pheggomisetes subspecies was very high (95.59%). Only one specimen from the P. serbicus belensis group is classified into the P. serbicus serbicus group, and one specimen from the P. globiceps ciniglavcensis group is classified into the P. globiceps ninae group. All pairwise squared Mahalanobis distances between the taxa were significant at a level of 99%. UPGMA cluster analysis of the squared Mahalanobis distances clustered both P. serbicus sp. nov. subspecies in the same branch and all the analysed subspecies of P. globiceps together in another branch, indicating that the two species are clearly separate (Fig. 17). Figure 17. View largeDownload slide UPGMA tree diagram of two Pheggomisetes species and six subspecies based on squared Mahalanobis distances (scale shown) obtained from eight analysed morphological trait measurements and 12 ratio variables. Figure 17. View largeDownload slide UPGMA tree diagram of two Pheggomisetes species and six subspecies based on squared Mahalanobis distances (scale shown) obtained from eight analysed morphological trait measurements and 12 ratio variables. On the basis of morphometric study, it can be asserted that the phenetically closest subspecies within P. globiceps are P. globiceps ninae comb. & stat. nov., P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov., while P. globiceps globiceps is morphologically somewhat separate (Fig. 17). Unquestionably, there is a need for a comprehensive morphometric analysis within the genus, including all currently existing taxa and more numerous samples of specimens, to obtain the most precise results possible. Molecular and phylogenetic analyses Since the taxonomy of Pheggomisetes is not well settled (Guéorguiev, 1964; Ćurčić et al., 2004), a substantial molecular analysis performed on the taxa could help us to solve some taxonomic problems. An appreciable interspecific, intraspecific and individual variability of characters (number of supraorbital, elytral and parameral setae; dorsal outlines of the head, pronotum and elytra) is evident (Guéorguiev, 1964; Nešić et al., 2010) within this morphologically isolated genus (Jeannel, 1928; Guéorguiev, 1977). For these reasons, we performed a molecular analysis of the Serbian taxa and their closest Bulgarian relatives that were available to us. Phylogenetic reconstruction of Pheggomisetes taxa was performed using three different methods, and all of them resulted in trees with the same topology (Fig. 18). Specimens were grouped into two distinct, well-supported clades. The mean genetic distance between clades was 3.6%. Figure 18. View largeDownload slide Phylogenetic tree of Pheggomisetes taxa based on COI sequences obtained using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. Bootstrap values are indicated above/below branches in the following order: NJ (black)/MP (red)/ML (blue). Duvalius stankovitchi georgevitchi was used as the outgroup taxon. Specimen codes are listed in parentheses. Figure 18. View largeDownload slide Phylogenetic tree of Pheggomisetes taxa based on COI sequences obtained using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. Bootstrap values are indicated above/below branches in the following order: NJ (black)/MP (red)/ML (blue). Duvalius stankovitchi georgevitchi was used as the outgroup taxon. Specimen codes are listed in parentheses. The taxa grouped within the first clade belong to P. globiceps. Four recognized subspecies are clustered separately with high bootstrap support. Pheggomisetes globiceps globiceps and P. globiceps ilandjievi are separate from the subclade consisting of P. globiceps ciniglavcensis subsp. nov. and P. globiceps ninae comb. & stat. nov. The genetic distances between subspecies range from 0.5% between P. globiceps ciniglavcensis subsp. nov. and P. globiceps ninae comb. & stat. nov. up to 1.8% between P. globiceps globiceps and P. globiceps ilandjievi. The distance between P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov. was 1.5%, while distance between P. globiceps ilandjievi and P. globiceps ninae comb. & stat. nov. was 1.3%. Conversely, the distance between P. globiceps globiceps and P. globiceps ciniglavcensis subsp. nov. was 1.5%, while distance between P. globiceps globiceps and P. globiceps ninae comb. & stat. nov. was 1.3%. The taxa grouped within the second clade belong to the newly described P. serbicus sp. nov., which clearly differentiates into two subspecies, viz., P. serbicus serbicus subsp. nov. and P. serbicus belensis subsp. nov., with a mean genetic distance of 1.1% between them. The obtained levels of sequence divergence between the species (> 3.5%) and subspecies (0.5–1.8%) are significant at species/subspecies levels (Hebert, Ratnasingham & de Waard, 2003), as was recently shown for the trechine genus Paraphaenops Jeannel, 1916 (Ortuño et al., 2016), as well as for other animal models (Hebert et al., 2003). The recorded molecular data are in agreement with the results achieved by classical taxonomic analysis (based on morphological characters and their variations) of Serbian Pheggomisetes taxa, thus confirming the correctness of erecting three taxa new to science (a species and two subspecies) and assigning a new status (subspecific within P. globiceps) to a taxon previously treated as a species. On the basis of two analysed Pheggomisetes taxa (P. globiceps globiceps Buresch, 1925 and P. globiceps ninae comb. & stat. nov., the latter being treated as P. globiceps ilandjievi), Faille et al. (2013) hypothesized that the genus is most likely an adelphotaxon of a clade containing isotopic species of the largely paraphyletic Duvalius Delarouzée, 1859 and five other subterranean genera. More genera inhabiting both Dinaric and Balkan mountain ranges need to be included in a comprehensive phylogenetic analysis to establish the true relationships of subterranean trechines in the region and disclose the origin and paths of colonization of different lineages on the Balkan Peninsula (Faille et al., 2013). CONCLUSIONS On the basis of the results of taxonomic, morphological and molecular analyses, we were able to identify one new trechine ground beetle species (P. serbicus sp. nov.) and two new subspecies (P. serbicus belensis subsp. nov. and P. globiceps ciniglavcensis subsp. nov.), in addition to which we propose a change in the status of one taxon (P. globiceps ninae comb. & stat. nov.). The new trechine taxa belong to an isolated and probably ancient phyletic lineage that most likely originated in the Oligocene (Guéorguiev, 1977; Ćurčić et al., 2004; Faille et al., 2013). The aforementioned new taxa are all relicts whose current distribution is limited to confined underground localities in Southeast Serbia. The use of TPEF microscopy in this study has provided better knowledge and additional information about the morphology and anatomy of Pheggomisetes taxa. It is one more tool that taxonomists can use to define more easily the taxonomic status of lower taxa, especially ones whose morphology is difficult to examine using classical light microscopy. NLM images and 3D models enable investigators to achieve deeper penetration into chitinized tissues, thereby revealing in-volume details that represent additional information useful in the determination of taxa. In analysing partial sequences of the COI gene, we confirmed our taxonomic findings. In this study, we show that the COI gene can be used for molecular identification of Pheggomisetes taxa. It would be of importance in the future to arrange a comprehensive morphological and molecular analysis of Pheggomisetes specimens from all known sites in both Bulgaria and Serbia to find out whether they belong to the taxa and species groups already known or whether a new classification would be more appropriate. In addition, a detailed molecular study of all Pheggomisetes subspecies (especially those subordinate to P. globiceps) with analysis of various morphological characteristics is needed to define their true taxonomic position. SUPPORTING INFORMATION Additional Supporting Information can be found in the online version of this article at the publisher’s web-site: Table S1. Measurements, morphometric ratios, and qualitative characteristics of Pheggomisetes taxa analysed in the current paper. Numerical unbolded values out of parentheses represent mean values, the bold ones are standard deviations (SD), while the ones in parentheses are ranges. The most important characters for distinction of taxa are underlined (* - values in mm). Appendix S1. TPEF microscopy video clip showing rotation of the aedeagus of P. globiceps ninae comb. & stat. nov. around the vertical axis. Appendix S2. TPEF microscopy video clip showing rotation of the aedeagus of P. globiceps ninae comb. & stat. nov. around the lateral axis. Appendix S3. TPEF microscopy video clip showing rotation of the male abdominal sternite IX (urite) of P. globiceps ninae comb. & stat. nov. around the longitudinal axis. Appendix S4. TPEF microscopy video clip showing rotation of the male abdominal sternite IX (urite) of P. globiceps ninae comb. & stat. nov. around the lateral axis. Appendix S5. TPEF microscopy video clip showing rotation of gonocoxites IX and gonosubcoxites IX of P. globiceps ciniglavcensis subsp. nov. around the longitudinal axis. Appendix S6. TPEF microscopy video clip showing rotation of gonocoxites IX and gonosubcoxites IX of P. globiceps ciniglavcensis subsp. nov. around the lateral axis. [Version of Record, published online 13 December 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:85900D92-A76D-4781-8829-CBED73A49334] ACKNOWLEDGEMENTS This study was financially supported by the Serbian Ministry of Education, Science, and Technological Development (Grants Nos. ON173038, III43001, ON171038 and III45016). We are grateful for the support of the EU Commission Project AREA (Grant No. 316004). In addition, we owe many thanks to Prof. Dr Zora Dajić-Stevanović and Mr Radenko Radošević (University of Belgrade – Faculty of Agriculture, Belgrade, Serbia) for helping us with imaging. Finally, we also thank Mr Darko Dragulović (Podgorac Timok, Serbia), Mr Ivo Petrović (Pirot, Serbia) and Prof. Dr Petar Beron (Sofia, Bulgaria), who helped us in collecting some of the beetle specimens analysed in this article. REFERENCES Andújar C , Gómez-Zurita J , Rasplus JY , Serrano J . 2012 . Molecular systematic and evolution of the subgenus Mesocarabus Thomson, 1875 (Coleoptera: Carabidae: Carabus), based on mitochondrial and nuclear DNA . Zoological Journal of the Linnean Society 166 : 787 – 804 . Google Scholar CrossRef Search ADS Belousov IA , Dolzhansky VY . 1994 . A new aphaenopsoid genus of the tribe Trechini from the Caucasus . Mitteilungen der Münchner Entomologischen Gesellschaft 84 : 59 – 63 . Belousov IA , Koval AG . 2009 . To the knowledge on the aphaenopsoid trechine beetles (Coleoptera: Carabidae: Trechini) of the Caucasus . Caucasian Entomological Bulletin 5 : 163 – 173 . Beron P . 1994 . Résultats des recherches biospéléologiques en Bulgarie de 1971 à 1994 et liste des animaux cavernicoles Bulgares . Tranteeva 1 : 1 – 137 . Casale A , Laneyrie R . 1982 . Trechodinae et Trechinae du Mónde. Tableau dés sous-familles, tribus, séries phylétiques, genres, et catalogue général des espèces . Mémoires de Biospéologie 9 : 1 – 226 . Casale A , Vigna Taglianti A , Juberthie C . 1998 . Coleoptera Carabidae . In: Juberthie C , Decu V , eds. Encyclopaedia biospeologica. Tome II . Moulis-Bucharest : Société de Biospéologie & Académie Roumaine , 1047 – 1081 . Chien CH , Chen WW , Wu JT , Chang TC . 2011 . Label-free imaging of Drosophila in vivo by coherent anti-Stokes Raman scattering and two-photon excitation autofluorescence microscopy . Journal of Biomedical Optics 16 : 016012 . Google Scholar CrossRef Search ADS PubMed Christiansen K . 2012 . Morphological adaptations . In: White WB , Culver DC , eds. Encyclopedia of caves, 2nd edn . Amsterdam : Elsevier , 517 – 528 . Google Scholar CrossRef Search ADS Contreras-Díaz HG , Moya O , Oromí P , Juan C . 2007 . Evolution and diversification of the forest and hypogean ground-beetle genus Trechus in the Canary Islands . Molecular Phylogenetics and Evolution 42 : 687 – 699 . Google Scholar CrossRef Search ADS PubMed Ćurčić SB , Brajković MM , Ćurčić BPM . 2007 . The carabids of Serbia . Belgrade–Vienna : Institute of Zoology, Faculty of Biology, University of Belgrade, Committee for Karst and Speleology, Serbian Academy of Sciences and Arts, Department of Conservation Biology, Vegetation- and Landscape Ecology, Faculty of Life Sciences, University of Vienna & UNESCO MAB Committee of Serbia . Ćurčić SB , Schönmann H , Brajković MM , Ćurčić BPM , Tomić VT . 2004 . On a new cave-dwelling beetle (Trechinae, Carabidae) from Serbia . Archives of Biological Sciences, Belgrade 56 : 109 – 113 . Google Scholar CrossRef Search ADS de Campos Vidal B . 2011 . Butterfly scale form birefringence related to photonics . Micron 42 : 801 – 807 . Google Scholar CrossRef Search ADS PubMed Decou V , Botosaneanu L . 1964 . Quelques données relatives a l’anatomie de Pheggomisetes bureschi Knirsch (Coleoptera, Trechinae) . Annales de Spéléologie 19 : 759 – 768 . Denk W , Strickler JH , Webb WW . 1990 . Two-photon laser scanning fluorescence microscopy . Science 248 : 73 – 76 . Google Scholar CrossRef Search ADS PubMed Deuve T , Cruaud A , Genson G , Rasplus JY . 2012 . Molecular systematics and evolutionary history of the genus Carabus (Col. Carabidae) . Molecular Phylogenetics and Evolution 65 : 259 – 275 . Google Scholar CrossRef Search ADS PubMed Faille A , Andújar C , Fadrique F , Ribera I . 2014 . Late Miocene origin of a Ibero-Maghrebian clade of ground beetles with multiple colonisations of the subterranean environment . Journal of Biogeography 41 : 1979 – 1990 . Google Scholar CrossRef Search ADS Faille A , Bourdeau C , Fresneda J . 2012 . Molecular phylogeny of the Trechus brucki group, with description of two new species from the Pyreneo-Cantabrian area (France, Spain) (Coleoptera, Carabidae, Trechinae) . ZooKeys 217 : 11 – 51 . Google Scholar CrossRef Search ADS Faille A , Casale A , Balke M , Ribera I . 2013 . A molecular phylogeny of Alpine subterranean Trechini (Coleoptera: Carabidae) . BMC Evolutionary Biology 13 : 248 . Google Scholar CrossRef Search ADS PubMed Faille A , Casale A , Ribera I . 2010a . Phylogenetic relationships of Western Mediterranean subterranean Trechini groundbeetles (Coleoptera: Carabidae) . Zoologica Scripta 40 : 282 – 295 . Google Scholar CrossRef Search ADS Faille A , Ribera I , Deharveng L , Bourdeau C , Garnery L , Quéinnec E , Deuve T . 2010b . A molecular phylogeny shows the single origin of the Pyrenean subterranean Trechini ground beetles (Coleoptera: Carabidae) . Molecular Phylogenetics and Evolution 54 : 97 – 106 . Google Scholar CrossRef Search ADS Gajović V , Mandić M , Njunjić I , Pavićević D . 2011 . Kompleksna speleološka istraživanja jame Propas’ u Činiglavcima . In: Ćalić J , ed. Conference Proceedings of the 7th Symposium on Karst Protection , 21–22 May 2011 , Bela Palanka, Serbia . Belgrade : Academic Speleological Alpinistic Club , 71 – 81 . Guéorguiev VB . 1964 . Révision du genre Pheggomisetes Knirsch (Coleoptera, Carabidae) . Časopis Československé Společnosti Entomologické 61 : 265 – 278 . Guéorguiev VB . 1977 . La faune troglobie terrestre de la péninsule Balkanique. Origine, formation et zoogéographie, special edn . Sofia : Bulgarian Academy of Sciences . Guéorguiev VB , Guéorguiev BV . 1995 . Catalogue of the ground-beetles of Bulgaria (Coleoptera: Carabidae) . Sofia-Moscow : Pensoft Publishers . Hebert PDN , Ratnasingham S , de Waard JR . 2003 . Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species . Proceedings of the Royal Society of London B: Biological Sciences 270 : 596 – 599 . Google Scholar CrossRef Search ADS Jeannel R . 1928 . Monographie des Trechinae. Morphologie comparée et distribution géographique d’un groupe de Coléoptères (Troisième Livraison). Les Trechini cavernicoles . L’Abeille 35 : 1 – 808 . Juberthie C , Decu V . 1968 . Les glandes pygidiales de quelques Trechitae cavernicoles . Annales de Spéléologie 23 : 195 – 210 . Kimura M . 1980 . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences . Journal of Molecular Evolution 16 : 111 – 120 . Google Scholar CrossRef Search ADS PubMed Klaus AV , Kulasekera VL , Schawaroch V . 2003 . Three-dimensional visualization of insect morphology using confocal laser scanning microscopy . Journal of Microscopy 212 : 107 – 121 . Google Scholar CrossRef Search ADS PubMed Lin CY , Hovhannisyan V , Wu JT , Lin CW , Chen JH , Lin SJ , Dong CY . 2008 . Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy . Journal of Biomedical Optics 13 : 050502 . Google Scholar CrossRef Search ADS PubMed Manly FJB . 1986 . Multivariate statistical methods – a primer . New York : Chapman and Hall . Masters BR , So PTC . 2008 . Handbook of biomedical nonlinear optical microscopy . Oxford : Oxford University Press . Mertz J . 2004 . Nonlinear microscopy: new techniques and applications . Current Opinion in Neurobiology 14 : 610 – 616 . Google Scholar CrossRef Search ADS PubMed Michels J . 2007 . Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans . Journal of Microscopy 227 : 1 – 7 . Google Scholar CrossRef Search ADS PubMed Moravec P , Uéno S-I , Belousov IA . 2003 . Tribe Trechini . In: Löbl I , Smetana A , eds. Catalogue of Palaearctic Coleoptera. Vol. 1. Archostemata – Myxophaga – Adephaga . Stenstrup : Apollo Books , 288 – 346 . Nei M , Kumar S . 2000 . Molecular evolution and phylogenetics . Oxford : Oxford University Press . Nešić D , Kličković M , Pavićević D , Mijatović M , Ognjenović S . 2010 . Rezultati novijih istraživanja Petrlaških pećina . Zaštita prirode 61 : 117 – 142 . Ober KA . 2002 . Phylogenetic relationships of the carabid subfamily Harpalinae (Coleoptera) based on molecular sequence data . Molecular Phylogenetics and Evolution 24 : 228 – 248 . Google Scholar CrossRef Search ADS PubMed Ober KA , Heider TN . 2010 . Phylogenetic diversification patterns and divergence times in ground beetles (Coleoptera: Carabidae: Harpalinae) . BMC Evolutionary Biology 10 : 262 . Google Scholar CrossRef Search ADS PubMed Ober KA , Maddison DR . 2008 . Phylogenetic relationships of tribes within Harpalinae (Coleoptera: Carabidae) as inferred from 28S ribosomal DNA and the wingless gene . Journal of Insect Science 8 : 63 . Google Scholar CrossRef Search ADS PubMed Ortuño VM , Sendra A , Reboleira ASPS , Fadrique F , Faille A . 2016 . The Iberian genus Paraphaenops Jeannel, 1916 (Coleoptera: Carabidae: Trechini): morphology, phylogeny and geographical distribution . Zoologischer Anzeiger 266 : 71 – 88 . Google Scholar CrossRef Search ADS Osawa S , Su Z-H , Imura Y . 2004 . Molecular phylogeny and evolution of carabid ground beetles . Tokyo : Springer Verlag . Google Scholar CrossRef Search ADS Pretner E . 1970 . Antrosedes longicollis sp. n. iz Bosne, razprostranjenost vrste Blattodromus herculeus Reitter in rod Pheggomisetes v Srbiji (Coleoptera: Bathysciinae in Trechinae) . Razprave IV razreda SAZU 13 : 153 – 164 . Rabasović MD , Pantelić DV , Jelenković BM , Ćurčić SB , Rabasović MS , Vrbica MD , Lazović VM , Ćurčić BPM , Krmpot AJ . 2015 . Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects . Journal of Biomedical Optics 20 : 016010 . Google Scholar CrossRef Search ADS PubMed Reinhardt K , Breunig HG , König K . 2017 . Autofluorescence lifetime variation in the cuticle of the bedbug Cimex lectularius . Arthropod Structure & Development 46 : 56 – 62 . Google Scholar CrossRef Search ADS PubMed Ribera I , Fresneda J , Bucur R , Izquierdo A , Vogler AP , Salgado JM , Cieslak A . 2010 . Ancient origin of a Western Mediterranean radiation of subterranean beetles . BMC Evolutionary Biology 10 : 29 . Google Scholar CrossRef Search ADS PubMed Ruiz C , Jordal B , Serrano J . 2009 . Molecular phylogeny of the tribe Sphodrini (Coleoptera: Carabidae) based on mitochondrial and nuclear markers . Molecular Phylogenetics and Evolution 50 : 44 – 58 . Google Scholar CrossRef Search ADS PubMed Simon C , Frati F , Beckenbach A , Crespi B , Liu H , Flook P . 1994 . Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers . Annals of the Entomological Society of America 87 : 651 – 701 . Google Scholar CrossRef Search ADS StatSoft, Inc . 2001 . STATISTICA (data analysis software system), version 6 . Available at: www.statsoft.com Šerić Jelaska L , Jambrošić Vladić Ž , Radovanović H , Franjević D . 2014 . Comparison of molecular and morphological systematics of Carabus species (Coleoptera: Carabidae) with special emphasis on species from Dinaric karst . Periodicum Biologorum 116 : 249 – 257 . Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S . 2011 . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Molecular Biology and Evolution 28 : 2731 – 2739 . Google Scholar CrossRef Search ADS PubMed Williams RM , Zipfel WR , Webb WW . 2001 . Multiphoton microscopy in biological research . Current Opinion in Chemical Biology 5 : 603 – 608 . Google Scholar CrossRef Search ADS PubMed Zar J . 1999 . Biostatistical analysis, 4th edn . New Jersey : Prentice Hall . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Zoological Journal of the Linnean Society Oxford University Press

Loading next page...
 
/lp/ou_press/the-genus-pheggomisetes-knirsch-1923-coleoptera-carabidae-trechinae-in-xwtgN7sa6d
Publisher
Oxford University Press
Copyright
© 2017 The Linnean Society of London, Zoological Journal of the Linnean Society
ISSN
0024-4082
eISSN
1096-3642
D.O.I.
10.1093/zoolinnean/zlx078
Publisher site
See Article on Publisher Site

Abstract

Abstract The genus Pheggomisetes Knirsch, 1923 consists of stenoendemic troglobitic ground beetles restricted to underground habitats in both Western Bulgaria and Southeast Serbia. A review of the genus in Serbia is given in this article. The following new taxa are described from three caves and pits on slopes of the Stara Planina Mountains in Southeast Serbia: Pheggomisetes serbicus sp. nov., P. serbicus belensis subsp. nov. and P. globiceps ciniglavcensis subsp. nov. Also, Pheggomisetes ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004, originally described as an independent species, is downgraded to a subspecies of P. globiceps Buresch, 1925 – P. globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. All the important morphological features of the taxa are listed in the article. The diagnoses of taxa are based on the characters ascertained by bright-field microscopy and nonlinear microscopy (NLM). The use of NLM in investigating Pheggomisetes anatomy is explained, and it is shown to be superior to classical microscopy in observing minute details of different structures (e.g. genitalia) on cross and longitudinal sections. A key to the species of Pheggomisetes (including the Serbian taxa) is given. In addition, we have included morphometric and molecular analyses of all Serbian Pheggomisetes taxa. ground beetles, molecular systematics, morphometrics, new species, new subspecies, Trechini, troglobites INTRODUCTION The genus Pheggomisetes Knirsch, 1923 includes four species and 12 subspecies of supposedly archaic troglobitic ground beetles, which have been highly modified during their evolution (Beron, 1994; Moravec, Uéno & Belousov, 2003; Ćurčić et al., 2004). The genus occupies an isolated position in the tribe Trechini, alone forming a distinct phyletic series (Jeannel, 1928; Casale & Laneyrie, 1982; Casale, Vigna Taglianti & Juberthie, 1998). It is considered to be related to the Caucasian aphaenopsoid genus Taniatrechus Belousov & Dolzhansky, 1994 based on the supernumerary supraorbital and submentum setae, two widened male protarsomeres and male genital structure (Belousov & Dolzhansky, 1994; Belousov & Koval, 2009). All representatives of Pheggomisetes are stenoendemics and restricted to certain caves and pits in Western Bulgaria and Southeast Serbia. The currently known species of the genus are Pheggomisetes buresi (Knirsch, 1923); Pheggomisetes globiceps Buresch, 1925; Pheggomisetes radevi Knirsch, 1924; and Pheggomisetes ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 (Casale & Laneyrie, 1982; Moravec et al., 2003; Ćurčić et al., 2004). One species (P. ninae) and one subspecies (P. globiceps ilandjievi V. Guéorguiev, 1964 according to Pretner, 1970) have been recorded in Serbia so far (Guéorguiev, 1964; Ćurčić et al., 2004; Ćurčić, Brajković & Ćurčić, 2007; Nešić et al., 2010). The morphology and anatomy of Pheggomisetes taxa have been investigated by several authors (Jeannel, 1928; Decou & Botosaneanu, 1964; Juberthie & Decu, 1968). Jeannel (1928) was the first to investigate features of the mouthparts, the dorsal habitus and male genital structures. Other aspects of the nervous, digestive, genital and pygidial gland systems were analysed in P. buresi (Decou & Botosaneanu, 1964; Juberthie & Decu, 1968). Most recently, thanks to two-photon excitation autofluorescence (auto-TPEF) of the chitin, certain features of the mouthparts and male genitalia were presented for P. ninae (Rabasović et al., 2015). Troglobitic insects are depigmented or transparent, with a thin cuticle consisting mostly of homogeneous chitin (Christiansen, 2012). The use of TPEF microscopy seems to be a highly promising way to study troglobitic taxa like Pheggomisetes since it enables the investigator to avoid the fluorescence of pigments and other cuticle components. The fluorescent signal of chitin is dominant here, in contrast to the situation with strongly coloured insects. Troglobitic beetles, including trechine ground beetles, are regarded as good models for deliberations about both biogeography and evolution since the reduced dispersal out of subterranean environments produces phylogenetic patterns of area distribution that largely match the geological history of mountain ranges and underground habitats (Ribera et al., 2010; Faille et al., 2014). The molecular phylogeny of trechine ground beetles (especially the troglobites) is largely unknown in spite of the fact that these are among the best-studied and widespread groups of beetles (Faille et al., 2013). Within the Trechinae, the molecular phylogeny of Trechus species from Spain and subterranean Pyrenean taxa (Faille, Casale & Ribera, 2010a; Faille et al., 2010b) and that of Alpine taxa (Faille et al., 2013) have been more or less thoroughly treated in recently published articles (Contreras-Díaz et al., 2007; Faille, Bourdeau & Fresneda, 2012; Faille et al., 2014). Among other groups of ground beetles, representatives of the Carabinae and Harpalinae have been somewhat better studied with respect to molecular biology (Ober, 2002; Osawa, Su & Imura, 2004; Ober & Maddison, 2008; Ruiz, Jordal & Serrano, 2009; Ober & Heider, 2010; Andújar et al., 2012; Deuve et al., 2012; Šerić Jelaska et al., 2014). Phylogenetic relationships of the highly diverse trechine fauna of the Balkan Peninsula are almost unknown. One study only concluded that the genera Neotrechus Müller, 1913 and Adriaphaenops Noesske, 1928 are related to certain Alpine or Pyrenean taxa (Faille et al., 2013). Sparse molecular data on the genus Pheggomisetes are mentioned solely by Faille et al. (2013). Organized by the Institute of Zoology, University of Belgrade – Faculty of Biology, several field trips were carried out in Southeast Serbia from 2012 until 2014. They resulted in the discovery of one new species and two new subspecies of Pheggomisetes. The aims of this article were as follows: (1) to describe and diagnose the new trechine taxa; (2) to review the taxonomic status of Pheggomisetes taxa from Serbia; (3) to show the benefits of nonlinear microscopy (NLM) in investigating morphology of the beetles’ internal chitinous structures (genitalia and some sclerites); (4) to conduct a morphometric study of all the investigated Pheggomisetes taxa; and (5) to perform a phylogenetic analysis of all the Serbian taxa using molecular data. MATERIAL AND METHODS Sampling information Ground beetle specimens were collected by hand or by pitfall trapping (the traps contained salt-saturated water/alcoholic vinegar and rotten meat as bait) (Faille et al., 2012) in a number of underground habitats from Southeast Serbia and Western Bulgaria belonging to the Stara Planina (Balkan) Mountain system. The traps were placed on both the floor and walls in dark parts of the explored caves and pits, where the level of humidity was high. All the analysed Pheggomisetes taxa are troglobitic and live in underground habitats (caves and pits) in the Stara Planina (Balkan) Mountains of Southeast Serbia and Western Bulgaria. Collected specimens were transferred to 70% ethanol at room temperature, except in the case of specimens designated for molecular analysis, which were transferred to 96% ethanol at −20 °C. They were analysed in laboratories of the Institute of Zoology, University of Belgrade – Faculty of Biology, Belgrade, Serbia; the Department of Crop Science, University of Belgrade – Faculty of Agriculture, Belgrade, Serbia; and the Photonics Centre, Institute of Physics, University of Belgrade, Belgrade, Serbia. Measurements AL – maximum length of antennae including the scape AL/TL – ratio of maximum length of antennae including the scape to total body length (from the anterior margin of the clypeus to the elytral apex) BW/PW – ratio of the elytral base width to maximum pronotum width as the greatest transverse distance EL – elytral length (as the linear distance along the suture from the elytral base to the apex) EL/EW – ratio of elytral length (as the linear distance along the suture from the elytral base to the apex) to maximum elytral width EL/TL – ratio of elytral length (as the linear distance along the suture from the elytral base to the apex) to total body length (from the anterior margin of the clypeus to the elytral apex) EW – maximum elytral width EWP – position of maximum elytral width (percentage of length) FL – length of frontal furrows FL/HL – ratio of frontal furrow length to head length HL – head length HL/AL – ratio of head length to maximum length of antennae including the scape HL/HW – ratio of head length to maximum head width HL/PL – ratio of head length to pronotum length (along the median line) HL/TL – ratio of head length to total body length (from the anterior margin of the clypeus to the elytral apex) HW – maximum head width HW/EW – ratio of maximum head width to maximum elytral width HW/NW – ratio of maximum head width to maximum neck width HW/PW – ratio of maximum head width to maximum pronotum width as the greatest transverse distance HWP – position of maximum head width (percentage of length) M – mean value for certain measurements NW – maximum neck width PaW – width of pronotal apex between tips of the anterior angles PaW/PbW – ratio of pronotal apex width between tips of the anterior angles to pronotal base width between tips of the posterior angles PaW/PW – ratio of pronotal apex width between tips of the anterior angles to maximum pronotum width as the greatest transverse distance PbW – pronotal base width between tips of the posterior angles PbW/PW – ratio of pronotal base width between tips of the posterior angles to maximum pronotum width as the greatest transverse distance PL – pronotum length (along the median line) PL/PW – ratio of pronotum length (along the median line) to maximum pronotum width as the greatest transverse distance PL/TL – ratio of pronotum length (along the median line) to total body length (from the anterior margin of the clypeus to the elytral apex) PW – maximum pronotum width as the greatest transverse distance PW/EW – ratio of maximum pronotum width as the greatest transverse distance to maximum elytral width PWP – position of maximum pronotum width (percentage of length) R – range of total measurements performed TL – total body length (from the anterior margin of the clypeus to the elytral apex) Collections HT – holotype IZFB – collection of the Institute of Zoology, University of Belgrade – Faculty of Biology, Belgrade, Serbia NMNH – collection of the National Museum of Natural History, Sofia, Bulgaria Other examined taxa Pheggomisetes buresi (Knirsch, 1923): one topotype male, Bulgaria, Balkan Mts., Ledenika Cave, 830 m a.s.l., near Vratsa, 19.VII.1963, leg. E. Pretner (IZFB). Pheggomisetes globiceps globiceps Buresch, 1925: syntype male, Bulgaria, West Balkan range, Mt. Ponor Planina, Sofia district, a cave near Iskrets (= Dushnika Cave), village of Iskrets, 580 m a.s.l., 10.XII.1924, leg. D. Iltchev (NMNH); two topotype females, idem, 08.VII.1925, leg. N. Radev (NMNH); one topotype female, idem, 17.IX.1943, collector unknown (NMNH); one topotype male, idem, 15.II.1992, leg. I. Pandurski (NMNH); two topotype males and one topotype female, idem, 16.X–04.XI.2016, from pitfall traps, leg. B. Guéorguiev & S. Goranov (IZFB); one female, Bulgaria, West Balkan range, Mt. Ponor Planina, Sofia district, Otechestvo Cave, village of Iskrets, 720 m a.s.l., 24.V.1959, leg. A. Popov (NMNH); two males, idem, 16.X.2016, leg. B. Guéorguiev & S. Goranov (NMNH). Pheggomisetes globiceps ilandjievi V. Guéorguiev, 1964: two topotype males and three topotype females, Bulgaria, Balkan Mts., Golyama Balabanova Dupka Cave, 1100 m a.s.l., village of Komshtitsa, near Godech, 12.X.1995, leg. B. Guéorguiev & V. Beshkov (IZFB); two topotype males and three topotype females, idem, 30.V.2015, leg. S. Goranov (IZFB). Taxonomic and morphological analyses The traditional method of studying insect morphology by bright-field microscopy was mainly used in the study. Apart from this, 3D images and clips obtained by NLM provided additional details that were used for descriptions of the genital structures and diagnoses of the studied taxa. Bright-field microscopy The genitalia were removed from the bodies, preserved in clove oil for a few days and subsequently fixed on microscope slides in Canada balsam. The beetles were then glued onto rectangular paper labels and analysed as dry specimens. Stemi 2000 and Stereo Discovery.V8 binocular stereomicroscopes (Carl Zeiss, Jena, Germany) with AxioCam MRc and Axio Cam ICc 1 digital cameras (Carl Zeiss, Jena, Germany) attached were used to photograph whole specimens, while a DMLS light microscope (Leica, Wetzlar, Germany) with a DC 300 camera (Leica, Wetzlar, Germany) attached was used to photograph the genitalia. Nonlinear microscopy Bright-field microscopy is most often used to study insect morphology, but recently a few articles have appeared treating the use of confocal fluorescence microscopy to observe certain structures of insects and crustaceans (Klaus, Kulasekera & Schawaroch, 2003; Michels, 2007; de Campos Vidal, 2011). NLM has also been introduced as a method offering unique insight into a variety of biological structures. This technique is similar to confocal microscopy (in employing localized laser excitation and scanning), but is characterized by higher penetration depth, reduced photodamage and photobleaching, and no need for specimen staining in most cases (Denk, Strickler & Webb, 1990; Williams, Zipfel & Webb, 2001; Mertz, 2004; Masters & So, 2008). Tissues and individual cells can be observed with high resolution of volume details. Up until now, NLM has been used extensively in biomedical research, but only marginally in entomology (Lin et al., 2008; Chien et al., 2011). It was recently confirmed that NLM can be used for deep imaging of chitinous structures (both chemically purified chitin and chitin originating from insect integument) (Rabasović et al., 2015; Reinhardt, Breunig & König, 2017). On the basis of the autofluorescence properties of chitin, the TPEF modality of NLM was mainly used in the latter study. In addition, even a second harmonic generation signal originating from chitinous structures was detected, but this signal is unsuitable for imaging since it is weak and hindered by the much stronger auto-TPEF. Before analysis, specimens were stored in 70% ethanol at room temperature. The genitalia were removed from the bodies and preserved in clove oil for 2 weeks. Genital structures were placed on double-sided adhesive tape on microscope slides in glycerin as the medium, with or without a cover slip, depending on the microscope objective used. Statistical analysis All variables that entered the statistical analysis were tested for normality using the Shapiro-Wilk test. A multivariate test of significance (one-way MANOVA) of normally distributed data, followed by a univariate test of significance (one-way ANOVA) for each variable, was used to identify which data sets (between groups) differ significantly. Non-normally distributed variables were compared between taxon samples using the Mann–Whitney U test (P ≤ 0.05). MANOVA allows comparison of population means of all variables of interest at the same time (multivariate response) rather than considering multiple responses as a suite of univariate responses (Zar, 1999). The statistical test most often used in biology, Wilks’ lambda, was applied (Zar, 1999). One-way MANOVA was used to examine the differences in morphological variation among Serbian Pheggomisetes taxa (species and subspecies). To describe and interpret effects from MANOVA, multivariate discriminant analysis (DA) was used only on normally distributed variables to determine the relative importance of characters as discriminators between a priori groups and the relative positions of centroids of the groups (Manly, 1986). The distance matrix for subspecies was calculated based on the squared Mahalanobis distance between subspecies centroids, and a dendrogram was generated using UPGMA (unweighted pair group method with arithmetic mean) clustering. This was used to evaluate the phenetic relationships between subspecies. Statistical analyses were conducted using the Statistica 6 software package (StatSoft, Inc., 2001). Molecular analysis DNA extraction, PCR amplification and sequencing We used nine Pheggomisetes specimens (belonging to all Serbian taxa) for molecular analyses (Table 1). DNA was extracted from one hind leg of each specimen using the KAPA Express Extract Kit (Kapa Biosystems Inc., Boston, MA, USA) and following the manufacturer’s instructions. The primers used to amplify the barcoding region of cytochrome c oxidase subunit I (COI) gene were Jerry [(CI-J-2183)5′-CAACATTTATTTTGATTTTTTGG-3′] and Pat [(TL2-N-3014)5′-TCCAAAGCA CTAATCTGCCATATTA-3′] (Simon et al., 1994). Each PCR was carried out in a volume of 25 µL containing 1 µL of extracted DNA, 9 µL of H2O, 1.25 µL of each primer and 12.5 µL of KAPA2GtmHotStart ReadyMix. All PCRs were conducted in an Eppendorf Mastercycler (Hamburg, Germany) using the following thermal profile: initial denaturation (95 °C for 5 min); amplification (35 cycles consisting of 60 s at 95 °C, 60 s at 51 °C and 120 s at 72 °C); and final extension (72 °C for 7 min). Table 1. Trechine specimens used for molecular analyses with GenBank accession numbers Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 *Outgroup. View Large Table 1. Trechine specimens used for molecular analyses with GenBank accession numbers Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 Code Locality Taxon Accession number S3 Hodžina Dupka Pit Pheggomisetes globiceps ninae comb. & stat. nov. KY351544 S12 Tmna Dupka Cave P. globiceps ninae comb. & stat. nov. KY351545 S28 Petrlaška Pećina Cave P. globiceps ninae comb. & stat. nov. KY351546 S19 Propas Pit P. globiceps ciniglavcensissubsp. nov. KY351547 S21 Pež Dupka Cave P. serbicus serbicus subsp. nov. KY351548 S26 Suva Dupka Cave P. serbicus belensis subsp. nov. KY351549 S27 A cave in the vicinity of the Suva Dupka Cave P. serbicus belensis subsp. nov. KY351542 S41 Golyama Balabanova Dupka Cave P. globiceps ilandjievi KY351543 S66 Otechestvo Cave P. globiceps globiceps KY351550 S1 Zlotska (= Lazareva) Pećina Cave Duvalius (Paraduvalius) stankovitchi georgevitchi* KY351551 *Outgroup. View Large The PCR products were purified using the QIAquick Purification Kit (QIAGEN Inc., Valencia, CA, USA) according to the manufacturer’s instructions. DNA sequencing was performed using automated equipment (Macrogen Inc., Seoul, South Korea). Sequences were manually edited in FinchTV (Geospiza Inc., Seattle, WA, USA) and aligned using the ClustalW program integrated in MEGA5 (Tamura et al., 2011). Genetic divergence was estimated using Kimura’s two-parameter (K2P) method of base substitution (Kimura, 1980). Phylogenetic reconstruction was performed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ) incorporated in the MEGA5 software package. One thousand bootstrap replicates were performed in every analysis to assess robustness of the trees. Tamura’s three-parameter model (T92 + G) was identified as the best-fitting model of sequence evolution based on the Bayesian information criterion and corrected Akaike information criterion (Nei & Kumar, 2000) for the ML method of phylogenetic reconstruction. The subspecies Duvalius (Paraduvalius) stankovitchi georgevitchi (Jeannel, 1924) was used as an outgroup. The nucleotide sequence data were deposited in the GenBank database under accession numbers KY351542–KY351551 (Table 1). K2P model was used to estimate genetic divergence of the analysed taxa. RESULTS AND DISCUSSION Taxonomy Family Carabidae Latreille, 1802 Subfamily Trechinae Bonelli, 1810 Tribe Trechini Bonelli, 1810 Genus Pheggomisetes Knirsch, 1923 Pheggomisetes serbicus Ćurčić, Vrbica & B. Guéorguiev sp. nov. (Figs 1 and 2A–H) Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Pež Dupka Cave, 43°13′17.77″N 22°47′17.08″E, village of Dojkinci, 869 m a.s.l., near Pirot, 11.VII–10.X.2013, from pitfall traps, leg. D. Antić & M. Petković’ (white label, printed)/‘Holotypus Pheggomisetes serbicus sp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: six males and eight females, same data as for holotype (IZFB); three males and four females labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Pež Dupka Cave, village of Dojkinci, 869 m a.s.l., near Pirot, 11.VII.2013, leg. D. Antić & M. Petković’ (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes serbicus sp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 5.55–6.675 mm (M 6.11 mm) (HT 6.30 mm). Head oval, HL/HW R 1.18–1.34 (M 1.26) (HT 1.27), widest somewhat before its middle, scarcely wider than pronotum (Fig. 1). Frontal furrows long, slightly exceeding mid head level, deeply impressed anteriorly and sigmoidally curved. Neck narrow, HW/NW R 1.97–2.75 (M 2.50) (HT 2.19). Antennae long, around the same length as TL in males, while shorter than TL in females. Figure 1. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 1. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Pronotum widest somewhat after the anterior third, almost as long as wide (Fig. 1). Anterior pronotal margin concave, shorter than pronotal base. Lateral pronotal margins rounded anteriorly and slightly concave posteriorly. Pronotal base somewhat concave in the middle. Fore pronotal angles obtuse, rounded. Hind pronotal angles acute, almost right. Elytra relatively long, oval, convex, with the lateral sides rounded anteriorly, widest slightly after the mid level, EL/EW R 1.61–1.83 (M 1.725) (HT 1.81). Elytral base slightly narrower than pronotum (Fig. 1). Humeral angles obtuse, rounded and quite elevated. Elytral apex rounded. Legs and claws long and thin (Fig. 1). Median lobe of the aedeagus in lateral view curved, with a rounded somewhat elevated apex (Fig. 2A, E). Basal bulb small, rounded. Parameres with three apical setae each. Median lobe in dorsal view straight, with a rounded apex, narrowing towards basal bulb (Fig. 1B). Gutter-shaped copulatory piece covered with numerous spines (Fig. 2B, F), wide at its basal three fifths and markedly narrowed at its apical two fifths. Figure 2. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 2. View largeDownload slide Pheggomisetes serbicus sp. nov. from the Pež Dupka Cave, village of Dojkinci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Male abdominal sternite IX (urite) subtriangular, slightly elongate, slightly longer than aedeagus (Fig. 2C, G). Apophysis narrow, constricted proximally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 2D, H. Gonocoxites IX of moderate length, slightly curved, apically rounded, basally completely jointed with massive gonosubcoxites IX (Fig. 2D, H). Chaetotaxy. Frons with six to seven (HT – 7) setae on each side. Pronotum with normal chaetotaxy (two pairs of setae). Five to seven setae on third interstria (HT – 7) on each elytron (Fig. 1). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, while between pores 3 and 4 longest; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 about as long as distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 1). Differential diagnosis: The new species is compared here with the other known Pheggomisetes species (Casale & Laneyrie, 1982; Moravec et al., 2003). A comparison of the new species with P. ninae is not provided here since the latter taxon is regarded as a subspecies of P. globiceps (see below). The new species differs from P. globiceps in having a smaller value of TL M (6.11 vs. ≥ 6.295 mm), a smaller value of HL M (1.39 vs. ≥ 1.40 mm), smaller values of AL M (5.91, males 6.01, females 5.70 vs. ≥ 6.525 mm, males ≥ 6.56 mm, females ≥ 6.30 mm), smaller values of AL/TL M (0.97, males 0.99, females 0.92 vs. ≥ 1.01, males ≥ 1.05, females ≥ 0.97), a greater value of HL/AL M (0.235 vs. ≤ 0.225), a greater value of FL M (0.75 vs. ≤ 0.68 mm), a greater value of FL/HL M (0.54 vs. ≤ 0.48), different shape of the humeral angles (more rounded, quite elevated vs. more obtuse, less elevated), a greater value of EL/TL M (0.55 vs. ≤ 0.54) and different shape of the copulatory piece in dorsal aspect (more markedly narrowed apically vs. gradually narrowed apically) (Tables 2 and 3; Figs 1–13; Supporting Information, Table S1) (Jeannel, 1928; Guéorguiev, 1964; this study). Even though the differences obtained between the mean values of certain measurements (HL) and ratios (HL/AL, EL/TL) are very small (Supporting Information, Table S1), the distributions of ranges in the two species show statistically significant differences (Table 3). Table 2. Results of ANOVA for each variable between P. serbicus sp. nov. and P. globiceps (exact significance level P ≤ 0.05, marked in bold) Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 View Large Table 2. Results of ANOVA for each variable between P. serbicus sp. nov. and P. globiceps (exact significance level P ≤ 0.05, marked in bold) Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 Variable F P-value HL 4.760 0.033 HW 0.273 0.603 FL 11.519 0.001 HL/HW 1.346 0.251 HL/AL 54.485 < 0.001 HL/PL 0.559 0.458 HL/TL 22.596 < 0.001 HW/NW 1.875 0.176 HW/PW 0.040 0.843 AL/TL 17.269 < 0.001 PW 1.092 0.300 PL/PW 3.788 0.056 PaW 5.207 0.026 PbW 0.287 0.594 PaW/PW 3.582 0.063 PbW/PW 0.047 0.829 PaW/PbW 3.744 0.058 EW 11.428 0.001 EWP 0.673 0.415 BW/PW 0.643 0.426 View Large Table 3. Results of Mann–Whitney U test between P. serbicus sp. nov. (n = 24) and P. globiceps (n = 44) (exact significance level P ≤ 0.05, marked in bold) Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 View Large Table 3. Results of Mann–Whitney U test between P. serbicus sp. nov. (n = 24) and P. globiceps (n = 44) (exact significance level P ≤ 0.05, marked in bold) Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 Variable U P-value TL 116.5 < 0.001 HWP 447.5 0.305 AL 1.5 < 0.001 FL/HL 250.0 < 0.001 HW/EW 320.0 0.014 PL 345.5 0.026 PWP 233.5 < 0.001 PL/TL 224.5 < 0.001 PW/EW 245.0 0.001 EL 431.0 0.325 EL/EW 351.5 0.042 EL/TL 206.0 < 0.001 View Large Figure 3. View largeDownload slide Shape of the shoulders in the Pheggomisetes subspecies analysed. A, P. serbicus serbicus subsp. nov. B, P. globiceps ciniglavcensis subsp. nov. C, P. globiceps ilandjievi. D, P. serbicus belensis subsp. nov. E, P. globiceps ninaecomb. & stat. nov. F, P. globiceps globiceps. Scales = 0.5 mm. Figure 3. View largeDownload slide Shape of the shoulders in the Pheggomisetes subspecies analysed. A, P. serbicus serbicus subsp. nov. B, P. globiceps ciniglavcensis subsp. nov. C, P. globiceps ilandjievi. D, P. serbicus belensis subsp. nov. E, P. globiceps ninaecomb. & stat. nov. F, P. globiceps globiceps. Scales = 0.5 mm. Figure 4. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 4. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 5. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 5. View largeDownload slide Pheggomisetes serbicus belensis subsp. nov. from the Suva Dupka Cave, village of Bela (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 6. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 6. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Holotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 7. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 7. View largeDownload slide Pheggomisetes globiceps ciniglavcensis subsp. nov. from the Propas Pit, village of Činiglavci (near Pirot), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, holotype male, aedeagus (lateral view). B, F, holotype male, aedeagus (dorsal view). C, G, holotype male, abdominal sternite IX (urite). D, H, paratype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 8. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 8. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 9. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, topotype male, aedeagus (lateral view). B, F, topotype male, aedeagus (dorsal view). C, G, topotype male, abdominal sternite IX (urite). D, H, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 9. View largeDownload slide Pheggomisetes globiceps ninae comb. & stat. nov. from the Hodžina Dupka Pit, village of Petrlaš (near Dimitrovgrad), Stara Planina Mts., Southeast Serbia. Bright-field (A–D) and TPEF (E–H) microscopy images. A, E, topotype male, aedeagus (lateral view). B, F, topotype male, aedeagus (dorsal view). C, G, topotype male, abdominal sternite IX (urite). D, H, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 10. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 10. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 11. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 11. View largeDownload slide Pheggomisetes globiceps ilandjievi from the Golyama Balabanova Dupka Cave, village of Komshtitsa (near Sofia), Stara Planina Mts., Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 12. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 12. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. Topotype male, habitus (dorsal view). Scale = 5.0 mm. Figure 13. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. Figure 13. View largeDownload slide Pheggomisetes globiceps globiceps from the Dushnika Cave, village of Iskrets (near Sofia), Mt. Ponor Planina, Western Bulgaria. A, topotype male, aedeagus (lateral view). B, topotype male, aedeagus (dorsal view). C, topotype male, abdominal sternite IX (urite). D, topotype female, gonocoxites IX and gonosubcoxites IX. Scales = 0.1 mm. The new species differs from P. radevi in having a smaller value of TL R (5.55–6.675 vs. 7–8 mm), a greater value of HL/HW M (1.26 vs. 1.00), different shape of the head (widest slightly before its mid part, posteriorly somewhat convex vs. widest around its mid part, posteriorly very convex), a smaller value of HW/NW M (2.50 vs. c. 3.00), different shape of the pronotum (weakly narrowed basally, strongly rounded anteriorly, well sinuate in back vs. strongly narrowed basally, weakly rounded anteriorly, strongly sinuate in back), a different value of PaW/PbW (pronotal apex between tips of the anterior angles narrower than pronotal base between tips of the posterior angles vs. pronotal apex between tips of the anterior angles wider than pronotal base between tips of the posterior angles), different shape of the hind pronotal angles (almost right, not prominent vs. acute, protruding backwards and outwards), different form of the elytra (less elongate, with more prominent shoulders vs. more elongate, with less prominent shoulders), different shape of the median lobe (less bent vs. more bent) and different shape of the basal bulb (relatively small, rounded vs. medium-sized, relatively elongate) (Jeannel, 1928; Guéorguiev, 1964; this study). The new species differs from P. buresi in having a smaller value of TL R (5.55–6.675 vs. 7.20–9.00 mm), different shape of the head (less elongate, posteriorly more convex, abruptly narrowing towards the neck vs. more elongate, posteriorly less convex, gradually narrowing towards the neck), a different position of maximum head width (slightly in front of the middle vs. anteriorly), a greater value of HW/NW M (2.50 vs. c. 2.00), different form of the lateral pronotal margins (a little rounded anteriorly, slightly sinuate posteriorly vs. somewhat arcuate), different shape of the hind pronotal angles (almost right, not prominent vs. acute, protruding backwards and outwards), different shape of the elytra (less elongate, with more pronounced shoulders vs. more elongate, with less pronounced shoulders), different shape of the median lobe (less elongate, less bent vs. more elongate, more bent) and different shape of the basal bulb (relatively small, rounded vs. relatively massive, elongate) (Jeannel, 1928; Guéorguiev, 1964; this study). Among the three known species of the genus, P. globiceps is the smallest one and the only species that has right hind angles of the pronotum (Jeannel, 1928; Guéorguiev, 1964). It has a pronotum that is weakly narrowed basally, with lateral margins moderately rounded anteriorly and weakly sinuate posteriorly, in addition to a relatively narrow elytral base (Jeannel, 1928; Guéorguiev, 1964). Thus, it is quite clear that P. serbicus sp. nov. shares the same character states and is closely related to it. In addition, P. buresi differs from all other congeners in having a rather thick neck. No significant differences within the genus are evident in the male genitalia (especially in regard to shape of the median lobe), even at the species level (Jeannel, 1928; Guéorguiev, 1964; this article). Variability: The number of setae on both frons (six to seven on each side) and elytra can vary (five to seven on each side). Etymology: The new species is named after Serbia, its terra typica. Distribution: The type locality is the Pež Dupka Cave in the village of Dojkinci (near Pirot) in the Stara Planina Mountains of Southeast Serbia. The new species inhabits a few caves in the villages of Dojkinci and Bela in the Stara Planina Mountains of Southeast Serbia; P. buresi and P. radevi live in caves near the town of Vratsa and villages of Chiren, Eliseyna, Chelopek, Druzhevo and Milanovo in the West Stara Planina Mountains of Western Bulgaria; and P. globiceps inhabits numerous caves in the West Stara Planina Mountains and Pre-Balkan region of Western Bulgaria, as well as a few caves in the villages of Petrlaš and Činiglavci in the Stara Planina Mountains of Southeast Serbia (Guéorguiev & Guéorguiev, 1995; this study). Pheggomisetes serbicus belensis Ćurčić, Vrbica & B. Guéorguiev subsp. nov. (Figs 4 and 5A–H) Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Suva Dupka Cave, 43°14′40.9″N 22°44′16.5″E, village of Bela, 801 m a.s.l., near Pirot, 25.V.2014, leg. S. Ćurčić, D. Antić & I. Petrović’ (white label, printed)/‘Holotypus Pheggomisetes serbicus belensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: three males and two females, same data as for holotype (IZFB); four males and eight females labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Suva Dupka Cave, village of Bela, 801 m a.s.l., near Pirot, 25.V-05.VII.2014, from pitfall traps, leg. D. Antić’ (IZFB); one female labelled as follows: ‘Southeast Serbia, Stara Planina Mts., a cave in the vicinity of the Suva Dupka Cave, 43°14′30.84″N 22°44′9.40″E, village of Bela, 793 m a.s.l., near Pirot, 25.V-05.VII.2014, from pitfall traps, leg. D. Antić’ (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes serbicus belensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 5.70–6.60 mm (M 6.045 mm) (HT 5.925 mm). HL/HW R 1.23–1.33 (M 1.27) (HT 1.24) (Fig. 4). Frontal furrows reaching mid head level. HW/NW R 2.09–2.50 (M 2.24) (HT 2.25). Anterior pronotal margin clearly (in males) or slightly (in females) concave, shorter than pronotal base (Fig. 3). Lateral pronotal margins very slightly concave posteriorly. Elytra with lateral sides almost straight in the anterior half, EL/EW R 1.62–1.92 (M 1.77) (HT 1.71) (Fig. 4). Median lobe of the aedeagus in lateral view slightly convex dorsally at around the level of two fifths, having an almost straight apex (Fig. 5A, E). Median lobe in dorsal view as presented in Figure 5B, F and inner sac as presented in Figure 5A, B, E, F. Male abdominal sternite IX (urite) as presented in Figure 5C, G, subtriangular, slightly elongate, slightly longer than aedeagus. Apophysis narrow, constricted proximally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 5D, H. Chaetotaxy. Frons with six to eight (HT – 6) setae on each side. Pronotum with normal chaetotaxy (two pairs of setae). Five to seven setae on third interstria (HT – 6–7) on each elytron (Fig. 4). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 1 and 2 approximately the same as between pores 3 and 4; median series at around the middle of the elytra, two setae being somewhat distanced from the gutter, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 4). Differential diagnosis: The new subspecies is compared with the nominal subspecies, P. serbicus serbicus subsp. nov. The new subspecies clearly differs from P. serbicus serbicus subsp. nov. in having a smaller value of TL M (6.045 vs. 6.11 mm), a different value of FL/HL M (frontal furrows reaching mid head level vs. frontal furrows somewhat exceeding mid head level), a smaller value of HW/NW M (2.24 vs. 2.50), a smaller value of FL M (0.68 vs. 0.75 mm), a greater value of AL M (5.99 vs. 5.91 mm), different shape of the anterior pronotal margin in females (less concave vs. more concave), different shape of the pronotal base in the middle (less concave vs. more concave), different shape of the lateral margins of the elytra anteriorly (more straight vs. rounded), a greater value of PaW/PbW M (0.78 vs. 0.74), a greater value of EL/EW M (1.77 vs. 1.725), a different position of certain humeral and median setae (distance between pores 2 and 3 shortest, distance between pores 1 and 2 approximately the same as between pores 3 and 4, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3 vs. distance between pores 2 and 3 shortest, distance between pores 3 and 4 longest, distance between pores 5 and 6 about as long as distance between pores 2 and 3) belonging to the elytral umbilicate series, different shape of the median lobe apex in lateral aspect (almost straight vs. somewhat elevated) and different shape of the median lobe’s basal bulb in lateral aspect (more curved vs. less curved) (Supporting Information, Table S1) (this study). Variability: The number of setae on both frons (six to eight on each side) and elytra can vary (five to seven on each side). Etymology: The new subspecies is named after the village of Bela, in which the type locality is situated. Distribution: It inhabits two caves in the village of Bela (near Pirot) in the Stara Planina Mountains of Southeast Serbia – the Suva Dupka Cave and a cave in its vicinity. Pheggomisetes globiceps Buresch, 1925 Pheggomisetes globiceps ciniglavcensis Ćurčić & Vrbica, subsp. nov. (Figs 6 and 7A–H) Pheggomisetes globiceps ilandjievi:Gajović et al. (2011: 80). Material examined: Holotype male labelled as follows: ‘Southeast Serbia, Stara Planina Mts., Propas Pit, 43°04′05.7″N 22°44′18.5″E, village of Činiglavci, 714 m a.s.l., near Pirot, 29.V-08.VII.2013, from pitfall traps, leg. Đ. Marković & M. Petković’ (white label, printed)/‘Holotypus Pheggomisetes globiceps ciniglavcensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’ (red label, printed) (IZFB). Paratypes: 29 males and 20 females, same data as for holotype (IZFB). All paratypes are labelled with white printed locality labels and with red printed labels ‘Paratypus Pheggomisetes globiceps ciniglavcensis subsp. nov. Ćurčić, Vrbica & Guéorguiev det. 2016’. Description: TL R 6.15–6.825 mm (M 6.46 mm) (HT 6.525 mm). Head oval, HL/HW R 1.19–1.355 (M 1.275) (HT 1.19), widest somewhat before its mid part, scarcely wider than pronotum (Fig. 6). Frontal furrows almost reaching mid head level, deeply impressed anteriorly and sigmoidally curved. Neck narrow, HW/NW R 2.17–2.61 (M 2.405) (HT 2.50). Antennae long, longer (in males) or slightly shorter (in females) than TL. Pronotum widest somewhat after the anterior third, almost as long as wide (Fig. 6). Anterior pronotal margin slightly concave, shorter than pronotal base. Lateral pronotal margins rounded anteriorly and slightly concave posteriorly. Pronotal base very slightly concave in the middle. Fore pronotal angles obtuse, rounded. Hind pronotal angles acute, almost right. Elytra relatively long, oval, convex, widest somewhat after the mid level, EL/EW R 1.53–1.73 (M 1.64) (HT 1.68). Elytral base slightly wider than pronotum (BW/PW R 0.85–1.10, M 1.03, HT 1.00) (Fig. 6). Humeral angles obtuse, rounded and relatively elevated. Elytral apex rounded. Legs and claws long and thin (Fig. 6). Median lobe of the aedeagus curved, slightly convex dorsally around the basal fourth, with a rounded apex (Fig. 7A, B, E, F). Basal bulb small, rounded. Parameres with three setae each, of which two are apically positioned. Triangular gutter-shaped copulatory piece covered with numerous thorns (Fig. 7B, F), gradually narrowed apically in dorsal aspect. Male abdominal sternite IX (urite) subtriangular, slightly elongate, somewhat longer than aedeagus (Fig. 7C, G). Apophysis narrow, gradually narrowing distally. Both gonocoxites IX and gonosubcoxites IX as presented in Figure 7D, H. Gonocoxites IX slightly elongate, somewhat curved, apically rounded, basally completely jointed with massive gonosubcoxites IX (Fig. 7D, H). Chaetotaxy. Frons with five to seven setae (HT – 6–7) on each side. Pronotum with normal chaetotaxy (two pairs of setae). Six to eight setae on third interstria (HT – 6–7) on each elytron (Fig. 6). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 1 and 2 is approximately the same as between pores 3 and 4; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 about as long as distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 longer than distance between pores 3 and 4 (Fig. 6). Differential diagnosis: The known subspecies of P. globiceps differ in shape of the head, length and depth of the frontal furrows, shape of the hind pronotal angles, lateral margins of the head and pronotum, the HL/PL and shape of the humeral angles (Guéorguiev, 1964). Some new characters should also be taken into account in separating Pheggomisetes taxa (e.g. TL, HL, HL/HW, HL/AL, HL/TL, HW/NW, HW/PW, HW/EW, AL, AL/TL, PL, PL/PW, PL/TL, PaW, PbW, PW, PW/EW, PWP, EW, EL/EW, BW/PW, elytral umbilicate series position, and aedeagus and copulatory piece shapes) (Tables 2 and 3; Supporting Information, Table S1). The new subspecies is compared here with the morphologically and geographically closest subspecies of P. globiceps and the nominotypical subspecies. The former are P. globiceps ilandjievi (Figs 10, 11A–D) and P. globiceps ninae comb. & stat. nov. (with the head elongately ovoid, lateral margins of the head moderately rounded, head slightly rounded both anteriorly and posteriorly as well, acute/right posterior pronotal angles, pronotum basally constricted, head slightly broader than pronotum and humeral angles slightly elevated) (Guéorguiev, 1964; Ćurčić et al., 2004; this article). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps ilandjievi in having a smaller value of TL M (6.46 vs. 6.60 mm), different shape of the head (widest at 2/5 of its length vs. widest slightly before the middle), a smaller value of HL/HW M (1.275 vs. 1.32), a greater value of AL M (6.775 vs. 6.64 mm), a greater value of AL/TL M (1.05 vs. 1.01), different shape of the lateral pronotal margins (less rounded anteriorly, more concave posteriorly vs. more rounded anteriorly, less concave posteriorly), greater values of PaW (R 0.63–0.68, M 0.66 vs. R 0.53–0.595 mm, M 0.57 mm), a greater value of PbW M (0.83 vs. 0.75 mm), a greater value of PaW/PW M (0.64 vs. 0.57), different shape of the humeral angles (less rounded and less elevated vs. more rounded and more elevated), a smaller value of EL/EW M (1.64 vs. 1.83), a greater value of BW/PW M (1.03 vs. 0.95), different shape of the median lobe (slightly convex dorsally around the basal fourth, with a narrower apex in dorsal view vs. not convex dorsally around the basal fourth, with a wider apex in dorsal view) and different size of the basal bulb (smaller vs. bigger) (Supporting Information, Table S1) (Guéorguiev, 1964; this study). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps ninae comb. & stat. nov. in having a greater value of TL M (6.46 vs. 6.295 mm), a smaller value of HL/HW M (1.275 vs. 1.30), a greater value of FL M (0.68 vs. 0.63 mm), greater values of AL M (6.775, males 6.84, females 6.50 vs. 6.525 mm, males 6.56 mm, females 6.30 mm), a greater value of HW/NW M (2.405 vs. 2.26), a greater value of EL M (3.50 vs. 3.36 mm), a greater value of EW M (2.135 vs. 1.99 mm), a smaller value of EL/EW M (1.64 vs. 1.69), a greater value of BW/PW M (1.03 vs. 0.91), different shape of the median lobe (narrower, somewhat more curved basally, then regularly curved, slightly convex dorsally around the basal fourth, with a narrow anterior part in dorsal view vs. thicker, regularly curved, somewhat convex dorsally in the middle, with a wide anterior part in dorsal view) and a different number of parameral setae (three, two of them apical vs. five, three of them apical) (Supporting Information, Table S1) (Ćurčić et al., 2004; this study). Pheggomisetes globiceps ciniglavcensis subsp. nov. differs from P. globiceps globiceps (Figs 12, 13A–D) in having different shape of the head (widest at the anterior 2/5 of its length vs. widest somewhat after the middle), a greater value of HW/NW M (2.405 vs. 2.24), greater values of AL M (6.775, males 6.84, females 6.50 vs. 6.585 mm, males 6.775 mm, females 6.30 mm), a smaller value of HL/PL M (1.51 vs. 1.63), a greater value of PL/PW M (0.91 vs. 0.88), a greater value of EW M (2.135 vs. 2.02 mm), a smaller value of EL/EW M (1.64 vs. 1.725), a greater value of BW/PW M (1.03 vs. 0.885), different shape of the median lobe (narrower, elongate, with more elongate basal bulb vs. wider, stout, especially basally, with a short stout basal bulb) and different shape of the copulatory piece in dorsal aspect (gradually narrowing towards the apex vs. wide at the basal 3/5 and markedly narrowed at the apical 2/5) (Supporting Information, Table S1) (Guéorguiev, 1964; this study). Variability: The number of setae on both frons (five to seven on each side) and elytra can vary (six to eight on each side). Etymology: The subspecies is named after the village of Činiglavci, in which the type locality is situated. Distribution: It lives solely in the Propas Pit in the village of Činiglavci (near Pirot) in the Stara Planina Mountains of Southeast Serbia. Remarks: The new subspecies was originally treated as P. globiceps ilandjievi by Gajović et al. (2011), who collected a sample several years ago. Pheggomisetes globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. (Figs 8 and 9A–H) Material examined: Sixty topotype males and 75 topotype females, Southeast Serbia, Stara Planina Mts., Hodžina Dupka Pit, 43°04′27.9″N 22°47′48.5″E, 692 m a.s.l., village of Petrlaš, near Dimitrovgrad, 26.VI-24.IX.2012, from pitfall traps, leg. Đ. Marković & D. Dragulović (IZFB); nine males and 25 females, Southeast Serbia, Mt. Stara Planina Mts., Petrlaška (= Velika) Pećina Cave, 43°04′27.81″N 22°47′46.50″E, 701 m a.s.l., village of Petrlaš, near Dimitrovgrad, 26.VI-24.IX.2012, both collected by hand and from pitfall traps, leg. D. Antić & S. Ćurčić (IZFB); one male and one female, idem, 03.XII.2012, leg. D. Antić & S. Ćurčić (IZFB); one female, idem, 19.IX.2013, leg. P. Beron (IZFB); two females, Southeast Serbia, Stara Planina Mts., Džemanska Propast Pit, 43°07′44.2″N 22°79′15.9″E, 738 m a.s.l., village of Petrlaš, near Dimitrovgrad, 24.IX.2012, leg. M. Petković & D. Dragulović (IZFB); one male, Southeast Serbia, Stara Planina Mts., Tmna Dupka Cave, 43°04′42.0″N 22°47′24.5″E, 720 m a.s.l., village of Petrlaš, near Dimitrovgrad, 24.IX.2012, leg. S. Ćurčić (IZFB); two males and ten females, idem, 24.IX-03.XII.2012, from pitfall traps, leg. D. Antić & S. Ćurčić (IZFB). Description: The description has been already presented by Ćurčić et al. (2004). Elytral umbilicate series: First three humeral setae close to marginal gutter, fourth being somewhat farther from the gutter, distance between umbilicate pores 2 and 3 shortest, distance between pores 3 and 4 longest; median series at around the middle of the elytra, two setae being somewhat distanced from marginal gutter, distance between pores 5 and 6 somewhat shorter than distance between pores 2 and 3; apical series: setae 7 and 8 being somewhat distanced from marginal gutter, distance between pores 7 and 8 shorter than distance between pores 3 and 4 (Fig. 8). Differential diagnosis: The subspecies is compared here with both the morphologically and geographically closest subspecies of P. globiceps and the nominotypical subspecies. The former are P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov. (Guéorguiev, 1964; this study). Pheggomisetes globiceps ninae comb. & stat. nov. differs clearly from P. globiceps ilandjievi in having a smaller value of TL M (6.295 vs. 6.60 mm), smaller values of AL M (6.525, males 6.56, females 6.30 vs. 6.64 mm, males 6.675 mm, females 6.60 mm), a smaller value of HW/NW M (2.26 vs. 2.35), a greater value of PaW M (0.61 vs. 0.57 mm), a greater value of PbW M (0.79 vs. 0.75 mm), different shape of the pronotal base in males (straight vs. concave), different shape of the hind pronotal angles (almost right vs. acute, rarely right), a smaller value of EL M (3.36 vs. 3.555 mm), a greater value of EW M (1.99 vs. 1.905 mm), a smaller value of EL/EW M (1.69 vs. 1.83), different shape of the median lobe (thicker, with a wider anterior part in dorsal view vs. more elongate, with a narrower anterior part in dorsal view) and a different number of parameral setae (five vs. three to four) (Supporting Information, Table S1) (Guéorguiev, 1964; Ćurčić et al., 2004; this study). All morphological differences between P. globiceps ninae comb. & stat. nov. and P. globiceps ciniglavcensis subsp. nov. are mentioned above (see the Differential diagnosis of P. globiceps ciniglavcensis subsp. nov.) (Figs 6, 7A–H, 8, 9A–H; Supporting Information, Table S1) (Ćurčić et al., 2004; this study). P. globiceps ninae comb. & stat. nov. can be easily distinguished from P. globiceps globiceps on the basis of having a smaller value of TL M (6.295 vs. 6.405 mm), a smaller value of HL M (1.40 vs. 1.47 mm), a smaller value of HW M (1.08 vs. 1.17 mm), a greater value of HL/HW M (1.30 vs. 1.26), different shape of the head (widest at around the anterior 2/5 of its length vs. widest somewhat after the middle), a smaller value of HL/PL M (1.52 vs. 1.63), a smaller value of HW/PW M (1.07 vs. 1.14), a greater value of PL/PW M (0.92 vs. 0.88), a smaller value of EL M (3.36 vs. 3.47 mm), a different number of parameral setae (five vs. three to four) and different shape of the median lobe (somewhat convex dorsally around the middle, with a somewhat elongate basal bulb vs. somewhat convex dorsally around the basal third, with a stout, relatively small basal bulb) (Supporting Information, Table S1) (Guéorguiev, 1964; Ćurčić et al., 2004; this study). Distribution: It lives in a few caves and pits on the western border of the Odorovačko Polje (692–738 m a.s.l.) in the village of Petrlaš (near Dimitrovgrad) in the Stara Planina Mountains of Southeast Serbia. Remarks: Interestingly, the taxon was originally treated as P. globiceps ilandjievi by Pretner (1970), who collected the first specimens from the Hodžina Dupka Pit with P. R. Deeleman. A similar opinion was expressed by Nešić et al. (2010) in a recent contribution. After the performed morphological and molecular analyses, we found that there is no difference between Pheggomisetes specimens from the Hodžina Dupka Pit, the Petrlaška (= Velika) Pećina Cave, the Džemanska Propast Pit and the Tmna Dupka Cave, all situated in the village of Petrlaš near Dimitrovgrad in the Stara Planina Mountains of Southeast Serbia. They all belong to the same taxon, which was previously described under the name P. ninae. After a thorough morphological analysis supported by molecular data, we established that the existing differences between P. ninae and other Pheggomisetes species are not great enough to treat it as a distinct species. The taxon in question deserves a subspecies rank within P. globiceps since certain smaller differences (both morphological and phylogenetical) were proved to exist between it and the geographically nearest subspecies of P. globiceps, but these were not significant enough to convince us of the need to separate it as a species. To be specific, certain morphological differences were observed in regard to TL, AL, HW/NW, FL, FL/HL, PaW, PbW, PaW/PW, shape of the lateral pronotal margins, pronotal base shape, EL, EW, EL/EW, BW/PW, shape of the humeral angles and position of the elytral umbilicate series (Supporting Information, Table S1), but shapes of the aedeagi and copulatory pieces are quite similar, indicating that the above-mentioned differences are in reality interpopulational, not interspecific (an assertion supported by small genetic differences recorded between the given taxon and its closest relatives, 0.5 and 1.3%, respectively). We therefore suggest that the taxonomic status of P. ninae be changed to P. globiceps ninae comb. & stat. nov. Key to Species of the Genus Pheggomisetes Knirsch, 1923 (FIG. 14) 1 Neck constriction very broad and not abrupt in dorsal view, while flat in lateral view. Head elliptical, cheeks less rounded (Northwest Bulgaria) …………………….……......………….……...…… P. buresi (Knirsch, 1923) – Neck constriction narrow and abrupt in dorsal view, while deeper in lateral view. Head circular or ovoid, cheeks more rounded ………………………..……………...……………...………..……………...……………......... 2 2 Pronotum clearly narrowed in front of base. Head very broad in posterior third, with frontal furrows very deep (Northwest Bulgaria) ………………………….……….……….……….……….….…. P. radevi Knirsch, 1924 – Pronotum not narrowed in front of base, sometimes with lateral margins more or less sinuate in posterior third. Head narrower in posterior third, with frontal furrows less deep 3 3 Longer TL M (≥ 6.295 mm), antennae longer (M ≥ 6.525 mm), longer than body, frontal furrows not reaching middle of the head, humeral angles more obtuse, less elevated, copulatory piece gradually narrowed apically (Western Bulgaria and Southeast Serbia) [P. globiceps Buresch, 1925] 4 – Smaller TL M (≤ 6.11 mm), antennae shorter (M ≤ 5.99 mm), slightly shorter than body, frontal furrows exceeding/reaching middle of the head, humeral angles more rounded, quite elevated, copulatory piece more markedly narrowed apically (Southeast Serbia) [P. serbicus Ćurčić, Vrbica & B. Guéorguiev, sp. nov.] 5 4 TL M 6.295 mm, antennae shorter (M 6.525 mm), humeral angles more elevated, HW/NW M 2.26, EL M 3.36 mm, EW M 1.99 mm, elytra at the base narrower than pronotum, median lobe thicker, regularly curved, somewhat convex dorsally in the middle, with a wide anterior part in dorsal view (Southeast Serbia) …………….. P. globiceps ninae S. Ćurčić, Schönmann, Brajković, B. Ćurčić & Tomić, 2004 comb. & stat. nov. –  TL M 6.46 mm, antennae longer (M 6.775 mm), humeral angles less elevated, HW/NW M 2.405, EL M 3.50 mm, EW M 2.135 mm, elytra at the base slightly wider than pronotum, median lobe narrower, somewhat more curved basally, then regularly curved, slightly convex dorsally around the basal fourth, with a narrow anterior part in dorsal view (Southeast Serbia) ……………...………………………………………………………………………………………………………….....…..…… P. globiceps ciniglavcensis Ćurčić & Vrbica, subsp. nov. 5 TL M 6.11 mm, frontal furrows somewhat exceeding mid head level, HW/NW M 2.50, FL M 0.75 mm, AL M 5.91 mm, anterior pronotal margin more concave in females, pronotal base more concave in the middle, PaW/PbW M 0.74, EL/EW M 1.725, lateral margins of elytra rounded anteriorly, median lobe apex somewhat elevated, basal bulb and basal part of median lobe narrower (Southeast Serbia) ……………. .……………...……………...……………...…… P. serbicus serbicus Ćurčić, Vrbica & B. Guéorguiev, subsp. nov. – TL M 6.045 mm, frontal furrows reaching mid head level, HW/NW M 2.24, FL M 0.68 mm, AL M 5.99 mm, anterior pronotal margin less concave in females, pronotal base less concave in the middle, PaW/PbW M 0.78, EL/EW M 1.77, lateral margins of elytra more straight anteriorly, median lobe apex almost straight, basal bulb and basal part of median lobe wider (Southeast Serbia) ………...……………...………….….….….….….….….….….….….….….….….….….….…. P. serbicus belensis Ćurčić, Vrbica & B. Guéorguiev, subsp. nov. Figure 14. View largeDownload slide Distribution of Pheggomisetes taxa in Serbia and the immediate surroundings. Scale = 10 km. Figure 14. View largeDownload slide Distribution of Pheggomisetes taxa in Serbia and the immediate surroundings. Scale = 10 km. TPEF microscopy of the internal structures of Pheggomisetes Certain well-chitinized internal morphological structures of Pheggomisetes ssp. were observed by two-photon excited autofluorescence microscopy. The samples were not fluorescently labelled, so autofluorescence was detected and used for imaging. For the study, we used a 25× numerical aperture 0.8 water/glycerin immersion objective and 930-nm excitation wavelength. This somewhat longer wavelength was utilized to avoid the autofluorescence of residual tissues remaining after beetle dissection. In addition, it was possible to penetrate deeper (up to 200 µm for the studied sample) through the chitinous cuticle due to the reduced two-photon absorption of chitin (Rabasović et al., 2015). We present TPEF 3D images of the male (aedeagus) and female (gonocoxites IX and gonosubcoxites IX) genitalia and the male abdominal sternite IX (urite) of all Pheggomisetes taxa from Serbia. The images revealed morphological details similar to those observed using classical bright-field microscopy (Figs 2E–H, 5E–H, 7E–H, 9E–H). In addition, selected 3D video clips of the three morphological structures are included, showing them in rotation around the longitudinal, lateral and vertical axes (Supporting Information, Appendices S1–S6). This makes it possible for the structures to be observed in every direction, which provides better insight into the shape and spatial relations of internal structures. The aedeagus is observed both laterally and dorsally (Figs 2E, F, 5E, F, 7E, F, 9E, F). The structure of both the surface (the fine relief) and the inner part (the copulatory piece composed of numerous tooth-like structures and the inner sac) of the median lobe (Fig. 15) is clearly distinguished. Both strongly (e.g. the copulatory piece) and weakly (e.g. the inner sac) chitinized parts of the aedeagus are visible (Figs 2E, F, 5E, F, 7E, F, 9E, F). All parts of the aedeagus (median lobe, basal bulb, parameres and their setae, copulatory piece and inner sac) are sharply delimited from each other (Figs 2E, F, 5E, F, 7E, F, 9E, F), as in the case of the images recorded earlier by bright-field microscopy (Figs 2A, B, 5A, B, 7A, B, 9A, B). Figure 15. View largeDownload slide TPEF microscopy image of part of the tooth-like copulatory piece of P. globiceps ninae comb. & stat. nov., showing fine details of the structure. Figure 15. View largeDownload slide TPEF microscopy image of part of the tooth-like copulatory piece of P. globiceps ninae comb. & stat. nov., showing fine details of the structure. One of the male internal sclerites, abdominal sternite IX (urite), is clearly visible and can be imaged by TPEF microscopy since it is well chitinized (Figs 2G, 5G, 7G, 9G). The shape and thickness of the structure are as visible as in the photographs obtained by bright-field microscopy (Figs 2C, 5C, 7C, 9C). Similarly, the parts of the female genitalia, which are highly sclerotized (gonocoxites IX and gonosubcoxites IX), were also observed. Sharply delimited parts of the aforementioned female genital structures are visible. The setation and fine relief are distinctly discernible on the surface, while the internal structure can also be observed (Figs 2H, 5H, 7H, 9H). The images of both cross and longitudinal sections of Pheggomisetes male genitalia (Fig. 16C, D) show the clear advantage of NLM vs. traditional classical microscopy in investigating anatomical features. To be specific, all features of the internal structures (e.g. shape and position of copulatory piece) are discernible on any section of the genitalia using this method (Fig. 16A–D). Apart from internal characteristics of the structures, their thickness can be ascertained and measured as well. One more benefit of using TPEF is that it provides additional data on the shapes of certain structures (e.g. median lobe, parameres, parameral setae) on cross sections (any level) (Fig. 16D), which cannot be detected by classical light microscopy. The images can be further used to calculate data on the structure’s surface, shape and volume. The female genitalia can be observed in a similar manner as well. Figure 16. View largeDownload slide TPEF microscopy images of the aedeagus of P. globiceps ninae comb. & stat. nov. A, lateral view with longitudinal section plane (red square). B, lateral view with cross-sectional plane (red line). C, a corresponding longitudinal section. D, a corresponding cross section. Scales = 0.10 mm (A–C) and 0.05 mm (D). Figure 16. View largeDownload slide TPEF microscopy images of the aedeagus of P. globiceps ninae comb. & stat. nov. A, lateral view with longitudinal section plane (red square). B, lateral view with cross-sectional plane (red line). C, a corresponding longitudinal section. D, a corresponding cross section. Scales = 0.10 mm (A–C) and 0.05 mm (D). Statistical morphometric analysis Only 20 variables (eight commonly used morphological trait measurements and 12 ratio variables) passed the Shapiro-Wilk normality test (HL, HW, FL, PW, PaW, PbW, EW, EWP, HL/HW, HL/AL, HL/PL, HL/TL, HW/NW, HW/PW, AL/TL, PL/PW, PaW/PW, PbW/PW, PaW/PbW and BW/PW) and were further used for parametric analyses. Normality tests were also performed on log-transformed data, but they resulted in the same 20 variables. Descriptive statistics of the quantitative traits and ratio variables of P. globiceps and P. serbicus sp. nov. subspecies from Serbia are given in Supporting Information, Table S1. One-way MANOVA of samples of the two Pheggomisetes species revealed significant differences between the species [Wilks’ Λ = 0.095, F (20, 40) = 19, P < 0.001]. Post hoc pairwise comparison using Scheffe’s test indicated that seven variables are statistically significant (Table 2). The HL/AL and HL/TL variables have the most distinct discriminative power. AL/TL, FL, EW, PaW and HL are statistically less important for distinguishing the two species. Table 3 presents the results of non-parametric comparisons between samples of the two species (for non-normally distributed variables) using the Mann–Whitney U test. Ten variables are recognized as statistically significant, but AL and TL are most important, while EL/TL and PL/TL are somewhat less important, followed by PWP, PW/EW, FL/HL, HW/EW, PL and EL/EW. One-way MANOVA of Pheggomisetes taxa (populations belonging to two species and six subspecies) revealed significant differences in the variation of eight commonly used morphological trait measurements and 12 ratios [Wilks’ Λ = 0.095, F (20, 40) = 18.941, P < 0.001 and Wilks’ Λ = 0.003, F (90, 222) = 5.890, P < 0.001, respectively]. The results of linear DA of 20 variables showed that the total correct percentage of the classification matrix of all six Pheggomisetes subspecies was very high (95.59%). Only one specimen from the P. serbicus belensis group is classified into the P. serbicus serbicus group, and one specimen from the P. globiceps ciniglavcensis group is classified into the P. globiceps ninae group. All pairwise squared Mahalanobis distances between the taxa were significant at a level of 99%. UPGMA cluster analysis of the squared Mahalanobis distances clustered both P. serbicus sp. nov. subspecies in the same branch and all the analysed subspecies of P. globiceps together in another branch, indicating that the two species are clearly separate (Fig. 17). Figure 17. View largeDownload slide UPGMA tree diagram of two Pheggomisetes species and six subspecies based on squared Mahalanobis distances (scale shown) obtained from eight analysed morphological trait measurements and 12 ratio variables. Figure 17. View largeDownload slide UPGMA tree diagram of two Pheggomisetes species and six subspecies based on squared Mahalanobis distances (scale shown) obtained from eight analysed morphological trait measurements and 12 ratio variables. On the basis of morphometric study, it can be asserted that the phenetically closest subspecies within P. globiceps are P. globiceps ninae comb. & stat. nov., P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov., while P. globiceps globiceps is morphologically somewhat separate (Fig. 17). Unquestionably, there is a need for a comprehensive morphometric analysis within the genus, including all currently existing taxa and more numerous samples of specimens, to obtain the most precise results possible. Molecular and phylogenetic analyses Since the taxonomy of Pheggomisetes is not well settled (Guéorguiev, 1964; Ćurčić et al., 2004), a substantial molecular analysis performed on the taxa could help us to solve some taxonomic problems. An appreciable interspecific, intraspecific and individual variability of characters (number of supraorbital, elytral and parameral setae; dorsal outlines of the head, pronotum and elytra) is evident (Guéorguiev, 1964; Nešić et al., 2010) within this morphologically isolated genus (Jeannel, 1928; Guéorguiev, 1977). For these reasons, we performed a molecular analysis of the Serbian taxa and their closest Bulgarian relatives that were available to us. Phylogenetic reconstruction of Pheggomisetes taxa was performed using three different methods, and all of them resulted in trees with the same topology (Fig. 18). Specimens were grouped into two distinct, well-supported clades. The mean genetic distance between clades was 3.6%. Figure 18. View largeDownload slide Phylogenetic tree of Pheggomisetes taxa based on COI sequences obtained using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. Bootstrap values are indicated above/below branches in the following order: NJ (black)/MP (red)/ML (blue). Duvalius stankovitchi georgevitchi was used as the outgroup taxon. Specimen codes are listed in parentheses. Figure 18. View largeDownload slide Phylogenetic tree of Pheggomisetes taxa based on COI sequences obtained using the neighbor-joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. Bootstrap values are indicated above/below branches in the following order: NJ (black)/MP (red)/ML (blue). Duvalius stankovitchi georgevitchi was used as the outgroup taxon. Specimen codes are listed in parentheses. The taxa grouped within the first clade belong to P. globiceps. Four recognized subspecies are clustered separately with high bootstrap support. Pheggomisetes globiceps globiceps and P. globiceps ilandjievi are separate from the subclade consisting of P. globiceps ciniglavcensis subsp. nov. and P. globiceps ninae comb. & stat. nov. The genetic distances between subspecies range from 0.5% between P. globiceps ciniglavcensis subsp. nov. and P. globiceps ninae comb. & stat. nov. up to 1.8% between P. globiceps globiceps and P. globiceps ilandjievi. The distance between P. globiceps ilandjievi and P. globiceps ciniglavcensis subsp. nov. was 1.5%, while distance between P. globiceps ilandjievi and P. globiceps ninae comb. & stat. nov. was 1.3%. Conversely, the distance between P. globiceps globiceps and P. globiceps ciniglavcensis subsp. nov. was 1.5%, while distance between P. globiceps globiceps and P. globiceps ninae comb. & stat. nov. was 1.3%. The taxa grouped within the second clade belong to the newly described P. serbicus sp. nov., which clearly differentiates into two subspecies, viz., P. serbicus serbicus subsp. nov. and P. serbicus belensis subsp. nov., with a mean genetic distance of 1.1% between them. The obtained levels of sequence divergence between the species (> 3.5%) and subspecies (0.5–1.8%) are significant at species/subspecies levels (Hebert, Ratnasingham & de Waard, 2003), as was recently shown for the trechine genus Paraphaenops Jeannel, 1916 (Ortuño et al., 2016), as well as for other animal models (Hebert et al., 2003). The recorded molecular data are in agreement with the results achieved by classical taxonomic analysis (based on morphological characters and their variations) of Serbian Pheggomisetes taxa, thus confirming the correctness of erecting three taxa new to science (a species and two subspecies) and assigning a new status (subspecific within P. globiceps) to a taxon previously treated as a species. On the basis of two analysed Pheggomisetes taxa (P. globiceps globiceps Buresch, 1925 and P. globiceps ninae comb. & stat. nov., the latter being treated as P. globiceps ilandjievi), Faille et al. (2013) hypothesized that the genus is most likely an adelphotaxon of a clade containing isotopic species of the largely paraphyletic Duvalius Delarouzée, 1859 and five other subterranean genera. More genera inhabiting both Dinaric and Balkan mountain ranges need to be included in a comprehensive phylogenetic analysis to establish the true relationships of subterranean trechines in the region and disclose the origin and paths of colonization of different lineages on the Balkan Peninsula (Faille et al., 2013). CONCLUSIONS On the basis of the results of taxonomic, morphological and molecular analyses, we were able to identify one new trechine ground beetle species (P. serbicus sp. nov.) and two new subspecies (P. serbicus belensis subsp. nov. and P. globiceps ciniglavcensis subsp. nov.), in addition to which we propose a change in the status of one taxon (P. globiceps ninae comb. & stat. nov.). The new trechine taxa belong to an isolated and probably ancient phyletic lineage that most likely originated in the Oligocene (Guéorguiev, 1977; Ćurčić et al., 2004; Faille et al., 2013). The aforementioned new taxa are all relicts whose current distribution is limited to confined underground localities in Southeast Serbia. The use of TPEF microscopy in this study has provided better knowledge and additional information about the morphology and anatomy of Pheggomisetes taxa. It is one more tool that taxonomists can use to define more easily the taxonomic status of lower taxa, especially ones whose morphology is difficult to examine using classical light microscopy. NLM images and 3D models enable investigators to achieve deeper penetration into chitinized tissues, thereby revealing in-volume details that represent additional information useful in the determination of taxa. In analysing partial sequences of the COI gene, we confirmed our taxonomic findings. In this study, we show that the COI gene can be used for molecular identification of Pheggomisetes taxa. It would be of importance in the future to arrange a comprehensive morphological and molecular analysis of Pheggomisetes specimens from all known sites in both Bulgaria and Serbia to find out whether they belong to the taxa and species groups already known or whether a new classification would be more appropriate. In addition, a detailed molecular study of all Pheggomisetes subspecies (especially those subordinate to P. globiceps) with analysis of various morphological characteristics is needed to define their true taxonomic position. SUPPORTING INFORMATION Additional Supporting Information can be found in the online version of this article at the publisher’s web-site: Table S1. Measurements, morphometric ratios, and qualitative characteristics of Pheggomisetes taxa analysed in the current paper. Numerical unbolded values out of parentheses represent mean values, the bold ones are standard deviations (SD), while the ones in parentheses are ranges. The most important characters for distinction of taxa are underlined (* - values in mm). Appendix S1. TPEF microscopy video clip showing rotation of the aedeagus of P. globiceps ninae comb. & stat. nov. around the vertical axis. Appendix S2. TPEF microscopy video clip showing rotation of the aedeagus of P. globiceps ninae comb. & stat. nov. around the lateral axis. Appendix S3. TPEF microscopy video clip showing rotation of the male abdominal sternite IX (urite) of P. globiceps ninae comb. & stat. nov. around the longitudinal axis. Appendix S4. TPEF microscopy video clip showing rotation of the male abdominal sternite IX (urite) of P. globiceps ninae comb. & stat. nov. around the lateral axis. Appendix S5. TPEF microscopy video clip showing rotation of gonocoxites IX and gonosubcoxites IX of P. globiceps ciniglavcensis subsp. nov. around the longitudinal axis. Appendix S6. TPEF microscopy video clip showing rotation of gonocoxites IX and gonosubcoxites IX of P. globiceps ciniglavcensis subsp. nov. around the lateral axis. [Version of Record, published online 13 December 2017; http://zoobank.org/urn:lsid:zoobank.org:pub:85900D92-A76D-4781-8829-CBED73A49334] ACKNOWLEDGEMENTS This study was financially supported by the Serbian Ministry of Education, Science, and Technological Development (Grants Nos. ON173038, III43001, ON171038 and III45016). We are grateful for the support of the EU Commission Project AREA (Grant No. 316004). In addition, we owe many thanks to Prof. Dr Zora Dajić-Stevanović and Mr Radenko Radošević (University of Belgrade – Faculty of Agriculture, Belgrade, Serbia) for helping us with imaging. Finally, we also thank Mr Darko Dragulović (Podgorac Timok, Serbia), Mr Ivo Petrović (Pirot, Serbia) and Prof. Dr Petar Beron (Sofia, Bulgaria), who helped us in collecting some of the beetle specimens analysed in this article. REFERENCES Andújar C , Gómez-Zurita J , Rasplus JY , Serrano J . 2012 . Molecular systematic and evolution of the subgenus Mesocarabus Thomson, 1875 (Coleoptera: Carabidae: Carabus), based on mitochondrial and nuclear DNA . Zoological Journal of the Linnean Society 166 : 787 – 804 . Google Scholar CrossRef Search ADS Belousov IA , Dolzhansky VY . 1994 . A new aphaenopsoid genus of the tribe Trechini from the Caucasus . Mitteilungen der Münchner Entomologischen Gesellschaft 84 : 59 – 63 . Belousov IA , Koval AG . 2009 . To the knowledge on the aphaenopsoid trechine beetles (Coleoptera: Carabidae: Trechini) of the Caucasus . Caucasian Entomological Bulletin 5 : 163 – 173 . Beron P . 1994 . Résultats des recherches biospéléologiques en Bulgarie de 1971 à 1994 et liste des animaux cavernicoles Bulgares . Tranteeva 1 : 1 – 137 . Casale A , Laneyrie R . 1982 . Trechodinae et Trechinae du Mónde. Tableau dés sous-familles, tribus, séries phylétiques, genres, et catalogue général des espèces . Mémoires de Biospéologie 9 : 1 – 226 . Casale A , Vigna Taglianti A , Juberthie C . 1998 . Coleoptera Carabidae . In: Juberthie C , Decu V , eds. Encyclopaedia biospeologica. Tome II . Moulis-Bucharest : Société de Biospéologie & Académie Roumaine , 1047 – 1081 . Chien CH , Chen WW , Wu JT , Chang TC . 2011 . Label-free imaging of Drosophila in vivo by coherent anti-Stokes Raman scattering and two-photon excitation autofluorescence microscopy . Journal of Biomedical Optics 16 : 016012 . Google Scholar CrossRef Search ADS PubMed Christiansen K . 2012 . Morphological adaptations . In: White WB , Culver DC , eds. Encyclopedia of caves, 2nd edn . Amsterdam : Elsevier , 517 – 528 . Google Scholar CrossRef Search ADS Contreras-Díaz HG , Moya O , Oromí P , Juan C . 2007 . Evolution and diversification of the forest and hypogean ground-beetle genus Trechus in the Canary Islands . Molecular Phylogenetics and Evolution 42 : 687 – 699 . Google Scholar CrossRef Search ADS PubMed Ćurčić SB , Brajković MM , Ćurčić BPM . 2007 . The carabids of Serbia . Belgrade–Vienna : Institute of Zoology, Faculty of Biology, University of Belgrade, Committee for Karst and Speleology, Serbian Academy of Sciences and Arts, Department of Conservation Biology, Vegetation- and Landscape Ecology, Faculty of Life Sciences, University of Vienna & UNESCO MAB Committee of Serbia . Ćurčić SB , Schönmann H , Brajković MM , Ćurčić BPM , Tomić VT . 2004 . On a new cave-dwelling beetle (Trechinae, Carabidae) from Serbia . Archives of Biological Sciences, Belgrade 56 : 109 – 113 . Google Scholar CrossRef Search ADS de Campos Vidal B . 2011 . Butterfly scale form birefringence related to photonics . Micron 42 : 801 – 807 . Google Scholar CrossRef Search ADS PubMed Decou V , Botosaneanu L . 1964 . Quelques données relatives a l’anatomie de Pheggomisetes bureschi Knirsch (Coleoptera, Trechinae) . Annales de Spéléologie 19 : 759 – 768 . Denk W , Strickler JH , Webb WW . 1990 . Two-photon laser scanning fluorescence microscopy . Science 248 : 73 – 76 . Google Scholar CrossRef Search ADS PubMed Deuve T , Cruaud A , Genson G , Rasplus JY . 2012 . Molecular systematics and evolutionary history of the genus Carabus (Col. Carabidae) . Molecular Phylogenetics and Evolution 65 : 259 – 275 . Google Scholar CrossRef Search ADS PubMed Faille A , Andújar C , Fadrique F , Ribera I . 2014 . Late Miocene origin of a Ibero-Maghrebian clade of ground beetles with multiple colonisations of the subterranean environment . Journal of Biogeography 41 : 1979 – 1990 . Google Scholar CrossRef Search ADS Faille A , Bourdeau C , Fresneda J . 2012 . Molecular phylogeny of the Trechus brucki group, with description of two new species from the Pyreneo-Cantabrian area (France, Spain) (Coleoptera, Carabidae, Trechinae) . ZooKeys 217 : 11 – 51 . Google Scholar CrossRef Search ADS Faille A , Casale A , Balke M , Ribera I . 2013 . A molecular phylogeny of Alpine subterranean Trechini (Coleoptera: Carabidae) . BMC Evolutionary Biology 13 : 248 . Google Scholar CrossRef Search ADS PubMed Faille A , Casale A , Ribera I . 2010a . Phylogenetic relationships of Western Mediterranean subterranean Trechini groundbeetles (Coleoptera: Carabidae) . Zoologica Scripta 40 : 282 – 295 . Google Scholar CrossRef Search ADS Faille A , Ribera I , Deharveng L , Bourdeau C , Garnery L , Quéinnec E , Deuve T . 2010b . A molecular phylogeny shows the single origin of the Pyrenean subterranean Trechini ground beetles (Coleoptera: Carabidae) . Molecular Phylogenetics and Evolution 54 : 97 – 106 . Google Scholar CrossRef Search ADS Gajović V , Mandić M , Njunjić I , Pavićević D . 2011 . Kompleksna speleološka istraživanja jame Propas’ u Činiglavcima . In: Ćalić J , ed. Conference Proceedings of the 7th Symposium on Karst Protection , 21–22 May 2011 , Bela Palanka, Serbia . Belgrade : Academic Speleological Alpinistic Club , 71 – 81 . Guéorguiev VB . 1964 . Révision du genre Pheggomisetes Knirsch (Coleoptera, Carabidae) . Časopis Československé Společnosti Entomologické 61 : 265 – 278 . Guéorguiev VB . 1977 . La faune troglobie terrestre de la péninsule Balkanique. Origine, formation et zoogéographie, special edn . Sofia : Bulgarian Academy of Sciences . Guéorguiev VB , Guéorguiev BV . 1995 . Catalogue of the ground-beetles of Bulgaria (Coleoptera: Carabidae) . Sofia-Moscow : Pensoft Publishers . Hebert PDN , Ratnasingham S , de Waard JR . 2003 . Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species . Proceedings of the Royal Society of London B: Biological Sciences 270 : 596 – 599 . Google Scholar CrossRef Search ADS Jeannel R . 1928 . Monographie des Trechinae. Morphologie comparée et distribution géographique d’un groupe de Coléoptères (Troisième Livraison). Les Trechini cavernicoles . L’Abeille 35 : 1 – 808 . Juberthie C , Decu V . 1968 . Les glandes pygidiales de quelques Trechitae cavernicoles . Annales de Spéléologie 23 : 195 – 210 . Kimura M . 1980 . A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences . Journal of Molecular Evolution 16 : 111 – 120 . Google Scholar CrossRef Search ADS PubMed Klaus AV , Kulasekera VL , Schawaroch V . 2003 . Three-dimensional visualization of insect morphology using confocal laser scanning microscopy . Journal of Microscopy 212 : 107 – 121 . Google Scholar CrossRef Search ADS PubMed Lin CY , Hovhannisyan V , Wu JT , Lin CW , Chen JH , Lin SJ , Dong CY . 2008 . Label-free imaging of Drosophila larva by multiphoton autofluorescence and second harmonic generation microscopy . Journal of Biomedical Optics 13 : 050502 . Google Scholar CrossRef Search ADS PubMed Manly FJB . 1986 . Multivariate statistical methods – a primer . New York : Chapman and Hall . Masters BR , So PTC . 2008 . Handbook of biomedical nonlinear optical microscopy . Oxford : Oxford University Press . Mertz J . 2004 . Nonlinear microscopy: new techniques and applications . Current Opinion in Neurobiology 14 : 610 – 616 . Google Scholar CrossRef Search ADS PubMed Michels J . 2007 . Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans . Journal of Microscopy 227 : 1 – 7 . Google Scholar CrossRef Search ADS PubMed Moravec P , Uéno S-I , Belousov IA . 2003 . Tribe Trechini . In: Löbl I , Smetana A , eds. Catalogue of Palaearctic Coleoptera. Vol. 1. Archostemata – Myxophaga – Adephaga . Stenstrup : Apollo Books , 288 – 346 . Nei M , Kumar S . 2000 . Molecular evolution and phylogenetics . Oxford : Oxford University Press . Nešić D , Kličković M , Pavićević D , Mijatović M , Ognjenović S . 2010 . Rezultati novijih istraživanja Petrlaških pećina . Zaštita prirode 61 : 117 – 142 . Ober KA . 2002 . Phylogenetic relationships of the carabid subfamily Harpalinae (Coleoptera) based on molecular sequence data . Molecular Phylogenetics and Evolution 24 : 228 – 248 . Google Scholar CrossRef Search ADS PubMed Ober KA , Heider TN . 2010 . Phylogenetic diversification patterns and divergence times in ground beetles (Coleoptera: Carabidae: Harpalinae) . BMC Evolutionary Biology 10 : 262 . Google Scholar CrossRef Search ADS PubMed Ober KA , Maddison DR . 2008 . Phylogenetic relationships of tribes within Harpalinae (Coleoptera: Carabidae) as inferred from 28S ribosomal DNA and the wingless gene . Journal of Insect Science 8 : 63 . Google Scholar CrossRef Search ADS PubMed Ortuño VM , Sendra A , Reboleira ASPS , Fadrique F , Faille A . 2016 . The Iberian genus Paraphaenops Jeannel, 1916 (Coleoptera: Carabidae: Trechini): morphology, phylogeny and geographical distribution . Zoologischer Anzeiger 266 : 71 – 88 . Google Scholar CrossRef Search ADS Osawa S , Su Z-H , Imura Y . 2004 . Molecular phylogeny and evolution of carabid ground beetles . Tokyo : Springer Verlag . Google Scholar CrossRef Search ADS Pretner E . 1970 . Antrosedes longicollis sp. n. iz Bosne, razprostranjenost vrste Blattodromus herculeus Reitter in rod Pheggomisetes v Srbiji (Coleoptera: Bathysciinae in Trechinae) . Razprave IV razreda SAZU 13 : 153 – 164 . Rabasović MD , Pantelić DV , Jelenković BM , Ćurčić SB , Rabasović MS , Vrbica MD , Lazović VM , Ćurčić BPM , Krmpot AJ . 2015 . Nonlinear microscopy of chitin and chitinous structures: a case study of two cave-dwelling insects . Journal of Biomedical Optics 20 : 016010 . Google Scholar CrossRef Search ADS PubMed Reinhardt K , Breunig HG , König K . 2017 . Autofluorescence lifetime variation in the cuticle of the bedbug Cimex lectularius . Arthropod Structure & Development 46 : 56 – 62 . Google Scholar CrossRef Search ADS PubMed Ribera I , Fresneda J , Bucur R , Izquierdo A , Vogler AP , Salgado JM , Cieslak A . 2010 . Ancient origin of a Western Mediterranean radiation of subterranean beetles . BMC Evolutionary Biology 10 : 29 . Google Scholar CrossRef Search ADS PubMed Ruiz C , Jordal B , Serrano J . 2009 . Molecular phylogeny of the tribe Sphodrini (Coleoptera: Carabidae) based on mitochondrial and nuclear markers . Molecular Phylogenetics and Evolution 50 : 44 – 58 . Google Scholar CrossRef Search ADS PubMed Simon C , Frati F , Beckenbach A , Crespi B , Liu H , Flook P . 1994 . Evolution, weighting and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers . Annals of the Entomological Society of America 87 : 651 – 701 . Google Scholar CrossRef Search ADS StatSoft, Inc . 2001 . STATISTICA (data analysis software system), version 6 . Available at: www.statsoft.com Šerić Jelaska L , Jambrošić Vladić Ž , Radovanović H , Franjević D . 2014 . Comparison of molecular and morphological systematics of Carabus species (Coleoptera: Carabidae) with special emphasis on species from Dinaric karst . Periodicum Biologorum 116 : 249 – 257 . Tamura K , Peterson D , Peterson N , Stecher G , Nei M , Kumar S . 2011 . MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods . Molecular Biology and Evolution 28 : 2731 – 2739 . Google Scholar CrossRef Search ADS PubMed Williams RM , Zipfel WR , Webb WW . 2001 . Multiphoton microscopy in biological research . Current Opinion in Chemical Biology 5 : 603 – 608 . Google Scholar CrossRef Search ADS PubMed Zar J . 1999 . Biostatistical analysis, 4th edn . New Jersey : Prentice Hall . © 2017 The Linnean Society of London, Zoological Journal of the Linnean Society This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Zoological Journal of the Linnean SocietyOxford University Press

Published: Dec 13, 2017

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off