A new European species of Ceratophysella (Collembola, Hypogastruridae) revealed by morphological data and DNA barcodes

Abstract A new species, Ceratophysella stachi, from Denmark, Germany, Luxembourg, Norway, Poland, and Ukraine is described based on morphological data and DNA barcodes. It belongs to a small European group of species with type B chaetotaxy and strong tegumentary granulation with distinct fields of coarse granules: C. granulata Stach, 1949, C. lawrencei (Gisin, 1963), C. neomeridionalis (Nosek & Červek, 1970), C. scotica (Carpenter & Evans, 1899), and C. silvatica Rusek, 1964. It differs from all of them in the chaetotaxy of lateral parts of thoracic terga II–III (setae m6 present and one additional seta outside lateral sensillum m7 present or absent) that is exceptional within the whole C. armata-group. Notes on closely related species C. granulata are also given.


Introduction
Ceratophysella Börner, 1932, comprising 140 species (Bellinger et al. 1996(Bellinger et al. -2021, is one of the largest collembolan genera within the family Hypogastruridae. Although the genus is considered cosmopolitan, the vast majority of species live in the temperate climatic zone of the northern hemisphere. Unfortunately, some of these species are insufficiently known, and there are doubts concerning their taxonomic status. One of these is Ceratophysella granulata Stach, 1949. This species was described from the Tatra Mountains (Polish Carpathians) by Stach (1949) and also reported by him from Slovakia, Ukraine, the former Yugoslavia (Slovenia), and France. Then, it has been frequently recorded from various European countries: Austria (Christian 1987), Bosnia and Herzegovina (Bogojevič 1968), Denmark (Fjellberg 1998), Germany (Eckert and Palissa 1999), Great Britain (Goto 1955a, b), Hungary (Danyi and Traser 2008), Norway (Fjellberg 1998), Poland (Stach 1949, Weiner 1981, Sterzyńska and Kaprus' 2000, Smolis and Skarżyński 2003, 2006, Romania (Danyi et al. 2006, Popa 2012, Slovakia (Nosek 1958, 1969, Kováč et al. 2016, the former Soviet Union (Grinbergs 1960), Switzerland (Gisin 1949), and Ukraine (Kaprus' et al. 2006). However, the reliability of these data is questioned. For example, Hopkin (2007) found that British specimens from the collection of Natural History Museum in London refer to Ceratophysella denticulata (Bagnall, 1941). Babenko et al. (1994) came to a similar conclusion after examination of specimens identified as C. granulata from the area of the former Soviet Union. They found that most of them referred to other species, usually of the C. denticulata group. Moreover, the comparison of the morphology of the topotypic population (Skarżyński 2004a) and the populations from Denmark and Norway (Fjellberg 1998) showed subtle differences in chaetotaxy, which indicates that C. granulata may be a complex of species. In order to establish the taxonomic status of the forms included in this complex, a classical taxonomic analysis of materials identified as C. granulata from several European scientific collections and DNA barcoding were performed.

Species/populations studied
Morphological analysis of available specimens designated as C. granulata (C. cf. granulata, Hypogastrura granulata) from Denmark, Germany, Hungary, Luxembourg, Norway, Poland (including syntypes and topotypes), Romania, Slovakia, Switzerland, and Ukraine from the collections of eight institutions (Table 1) was performed. Unfortunately, specimens of C. granulata mentioned in the original description (Stach 1949) from Slovakia (Podlužany, orig. "Dobó-Berekalja"), West Ukraine (Czarnohora Range: Zaroślak and Breskuł), former Yugoslavia (Slovenia, Škocjan Caves, orig. "St. Canzian cave"), and France (Arles) could not be found in the ISEZ collection (in litt. Wanda M. Weiner); therefore, they were not examined. The search for materials of this species mentioned in the "Catalogus faunae Austriae" (Christian 1987) in the collection of Natural History Museum Vienna also did not bring any results (in litt. Harald Bruckner). In addition, the sequences from 21 specimens from five species were analysed to assess the status of C. granulata forms in the context of the genetic divergence within the genus (Table 2).

Morphology
Specimens stored in alcohol were cleared in Nesbitt's fluid (chloral hydrate, concentrated hydrochloric acid, distilled water), slide-mounted in a mixed medium (distilled water, gum arabic, glycerol, chloral hydrate), and studied using a Nikon Eclipse E600 phase contrast microscope. Figures were drawn using a camera lucida. A set of characters commonly used in the taxonomy of the genus (Fjellberg 1984(Fjellberg , 1998(Fjellberg , 1999Babenko et al. 1994;Thibaud et al. 2004) was analysed.

DNA barcoding
Lysis of the tissues was carried out in 50 µl volume of lysis buffer and proteinase K incubated at 56 °C overnight. DNA extraction followed a standard automated protocol on 96-well glass fibre plates (Ivanova et al. 2006), and during this DNA extraction, a voucher recovery specially designed for high-throughput workflow (Porco et al. 2010) was used. The 5' region of COI used as a standard DNA barcode was amplified using M13 tailed primers LCO1490 and HCO2198 (Folmer et al. 1994). A standard PCR reaction protocol was used for PCR amplifications and products were checked on a 2% E-gel 96Agarose (Invitrogen). Unpurified PCR amplicons were sequenced in both directions using M13 tails as primers. The sequencing reactions followed standard protocols of the Canadian Centre for DNA Barcoding (Hajibabaei et al. 2005), with products subsequently purified using Agencourt CleanSEQ protocol (Agencourt) and processed using BigDye v. 3.1 on an ABI 3730 DNA Analyzer (Applied Biosystems). Sequences were assembled with Sequencer v. 4.5 (GeneCode Corporation, Ann Arbor, MI, USA) and aligned by eye using BIOEDIT v. 7.0.5.3 (Hall 1999); we observed no indels in this coding region of the mitochondrial genome, and therefore, all base positions were aligned with confidence in positional homology. Sequences are publicly available on BOLD (Ratnasingham and Hebert 2007; http://www.barcodinglife. org) within the public dataset NCERAT and in GenBank (HM398990-HM399010, JX261875, MW471668). Distance analyses were conducted with MEGA7 (Tamura et al. 2007) using a Neighbor-Joining (Saitou and Nei 1987) algorithm and distances corrected with the Kimura-2 parameter (Kimura 1980). Kimura-2 parameter is the best metric when distances are low (Nei and Kumar 2000). The robustness of nodes was evaluated through bootstrap re-analysis of 1000 pseudoreplicates.

Morphology
The material under study appeared to be taxonomically heterogeneous. Hungarian specimens (HNHM, "Hypogastrura granulata" det. Loksa: 27 spp., Sz(N.) 1975/76, Szentbékkála, 1975.05.09, leg. Loksa, coll-1868and 2 spp., Bátorliget, 1989-90, coll-1008 were identified as C. denticulata and C. silvatica (Rusek, 1964 1946-1948 were assigned to C. armata (Nicolet, 1842) and C. attenuata Cassagnau, 1959. The original designations of some specimens from Luxembourg also proved to be erroneous, six specimens from Obereisenbach (5 juv., Holzbichsbaach, oaks, litter and mosses, 5.VII.1991, leg. Among the examined specimens that can be referred to C. granulata, two morphotypes were found, differing in the chaetotaxy of the lateral part of thoracic terga II-III, the size and shape of accessory boss near the postantennal organ, and the shape of mucro. Considering their clear morphological differentiation, both forms are treated as separate species. Thus, the form from the northwestern part of the Carpathians (Poland and Slovakia) was recognized (based on syntypes and topotypes) as C. granulata, whereas the form distributed in Norway through Denmark, Germany, Luxembourg, and the eastern part of Polish Carpathians to Ukraine ( Fig. 1) was considered to be a species new to science-C. stachi sp. nov. Notes on the former and description of the later are given below.

DNA barcoding
The mean genetic divergence among the four Ceratophysella species included into the analysis was 24.2% (ranging from 19% to 28.2%), and their mean intraspecific variation was 0.5% (ranging from 0% to 0.6%). Similar values were found for both interspecific (mean 23% ranging from 21% to 24.4%) and intraspecific (1%) in C. stachi sp. nov., thus supporting the status of the new species (Table 3, Fig. 2). Moreover, these values are in line with the usual 'barcoding gap' described so far in the family Hypogastruridae (e.g. Nakamori 2013; Skarżyński et al. 2018) but also more generally in Collembola (e.g. Porco et al. 2014;Nilsai et al. 2017).    Etymology. Dedicated to Jan Stach, the excellent specialist in Collembola. Description. Body length 1-2 mm. Colour (in alcohol) bluish-gray to bluishblack. Tegumental granulation strong, with fields of especially coarse granules on head (large uniform field covering whole dorsal side except antennal bases), thoracic terga II-III (two large subaxial fields and two lateral ones of medium size, Fig. 3), abdominal terga I-III (variable distribution: from four - Fig. 4 to seven fields of medium size as in C. granulata; see Skarżyński 2004a: fig. 8), abdominal tergum IV (medium axial field and two lateral large ones, Fig. 4) and abdominal terga V-VI (large uniform fields covering almost whole dorsal side, Fig. 4). 6-9 granules between macrosetae p 1 on abdominal tergum V.
Ocelli: 8 + 8. Postantennal organ 1.8-2.3 times as large as single ocellus; the former with four lobes, its anterior pair larger than posterior pair. Accessory boss large (equal to or only slightly smaller than posterior lobes of postantennal organ), often granulated (Fig. 7).
Anal spines yellowish, slightly curved, situated on high basal papillae, 1.1-1.7 times as long as inner edge of claws III (Fig. 4).
Distribution and ecology. The range of distribution of C. stachi sp. nov. appears to be relatively wide. It is known from Denmark (Jutland, Funen), Germany (Brandenburg, Hesse, Mecklenburg-West Pomerania, Saxony), Luxembourg, southern Norway (Akershus, Vestfold), Poland (Carpathians: Beskid Niski, Beskid Sądecki, Bieszczady mountains) and Ukraine (Lviv District) (Fig. 1). Probably this species is distributed much more widely in Europe, but additional research is needed to prove this thesis. Ceratophysella stachi sp. nov. lives in lowlands and in the mountains (up to ca. 1000 m a.s.l) where it inhabits litter and mosses in different types of forests, and also heathlands and bogs.
Remarks. Ceratophysella stachi sp. nov. belongs to a small European branch of species of the C. armata-group, which have strong tegumentary granulation, with distinct fields of coarse granules: C. granulata, C. lawrencei (Gisin, 1963), C. neomeridionalis (Nosek & Červek, 1970), C. scotica (Carpenter & Evans, 1899), and C. silvatica. It differs from all of them in the chaetotaxy of the lateral parts of the thoracic terga II-III (setae m 6 present and one additional seta outside lateral sensillum m 7 present or absent vs setae m 6 and additional setae absent) which is exceptional within the whole C. armata-group (both characters are found in the genus, but in other groups of species: formerly classified as Mitchellania Wray, 1953 and C. denticulata). The remaining differences between C. stachi sp. nov. and related species mentioned above are summarized in Table 4.
gorges (litter and mosses), and caves (mosses, litter, and rotten wood at cave the entrance, and bat guano and cave sediments even 100 m from the entrance) in mountain forests zone. At the end of the Pleistocene, this psychro-and hygrophilous species was probably more common in the periglacial region, and due to the warming Holocene climate, its range became limited to scattered, high-mountain refuges and cold caves and other subterranean habitats at lower elevations. Based on the current distribution data, it is concluded that C. granulata is endemic to the Western Carpathians. However, to verify this thesis, additional research should be undertaken covering the rest of the Carpathians, the Alps, and other mountainous areas of central Europe.

Discussion
Traditionally, the most commonly used method in the taxonomy of the genus Ceratophysella, as in other Collembola, is the analysis of morphological features (Gisin 1949;Stach 1949;Cassagnau 1959;Yosii 1960;Bourgeois and Cassagnau 1972;Babenko et al. 1994;Jordana et al. 1997;Christiansen and Bellinger 1998;Fjellberg 1998;Thibaud et al. 2004). Recently, hybridization in laboratory conditions and DNA barcoding have also been used, although on a small scale (Skarżyński 2004a(Skarżyński , b, c, 2005Porco et al. 2012;Nakamori 2013). Research on the Xenylla maritima complex (Skarżyński et al. 2018) and Ceratophysella comosa Nakamori, 2013 showed that combined use of the morphological and genetic criteria may bring good results in establishing species status and support in the family Hypogastruridae. The use of integrative taxonomy methods in this study allowed for the revision of "C. granulata" status and the description of a species new to science. Considering its effectiveness and its relative low cost, this method has the potential to bring a significant contribution in the field of taxonomic revision.