Morphological and Genetic Analysis of the Acerentomon doderoi Group (Protura: Acerentomidae) with Description of A. christiani sp. nov

Acerentomon christiani sp. nov. is described from Vienna, Austria. The new species is a member of the “doderoi” group, characterized by the presence of seta x on tergite VII. It is most similar to A. gallicum, A. brevisetosum and A. tenuisetosum, but differs from these species in the length of foretarsal sensillum c and certain other chaetotaxic measurements and indices. In addition to the morphological description, the DNA barcoding region of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) and the 28S ribosomal RNA of the new species are provided. The morphological characters and the barcode of the new species are discussed in comparison to those of other Acerentomon species. An identification key to all known Acerentomon spp. of the “doderoi” group is given.


Introduction
Until recently, the taxonomy of Protura was based exclusively on morphology. Many characters are inconspicuous and difficult to recognize, often making a reliable species determination problematic even for long-experienced experts (for review see [1]). However, it has been convincingly demonstrated [2] that molecular barcodes are an additional important and useful tool in the taxonomy of these minute soil arthropods. Use of barcodes has revealed that morphological taxonomy is reflected very closely by the molecular data. This result is of special importance since the reproductive biology of proturans remains enigmatic to date (for review see [3]). As a consequence it is not possible to check the described morphospecies from a biospecies perspective. Therefore, whenever possible each new species description of Protura should include a molecular characterization by barcodes as well as the description of morphological characters.
The genus Acerentomon Silvestri, 1907 presently comprises 38 species [1], [4], [5]), all of which have a West Palaearctic distribution (Europe and northern Africa). Most species are recorded solely from their type localities. Thirteen species of the genus Acerentomon have been reported from Austria ( [6]). In soil samples from the Leopoldsberg (near Vienna) we found an undescribed species of Acerentomon together with A. italicum Nosek, 1969, both belonging to the "doderoi" group sensu Nosek (1973) [7]. The present paper contains a description of the new species from Austria, along with its barcoding sequence, and an identification key for all Acerentomon species of the doderoi group.

Ethics Statement
The species used in this study are neither CITES-species nor endangered species according to regional Red Lists. Our sampling permission was RU5-BE-939/001-2013.

Nomenclature Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: 1F2DDC05-5E40-4127-AB12-FC089F40EEAE. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Molecular Approach
To obtain COI and 28S (region D2-D3) sequences, genomic DNA was extracted from complete animals applying a non-destructive extraction method (NDE) [13]. Protocols for DNA extraction, amplification, and sequencing of 41 published individuals from the genus Acerentomon are given in [2]. In the present study, DNA of 27 individuals was extracted by means of an NDE-method with the Blood & Tissue Kit (Qiagen). After DNA extraction the cuticle was transferred to 100% EtOH, before wholemounts were prepared. Thermocycler profiles differed for the amplification of the COI and 28S rDNA fragment: initial denaturation of 30 sec, 30 cycles of 1 min at 94°C, 1 min at 46°C (COI) / 48°C (28S rDNA) and 1 min (COI) / 1.5 min (28S rDNA) at 68°C and a final extension step for 5 min at 68°C.
Each PCR reaction consisted of 2 μl DNA, 5 μl PCR buffer (5x containing 18 mM MgCl 2 ; BioLabs OneTaq), 0.7 μl dNTP (10 mM each; BioLabs Desoxynucleotide Solution Mix), 0.7 μl each primer (10 μM, VBC Biotech), 0.1 μl Polymerase (BioLabs OneTaq), 10.8 μl ddH 2 O. A second PCR-repeat, applying the same conditions, was used to increase the yield of DNA. PCR products were purified using GeneJET PCR Purification Kit (Thermo Scientific) and eluted in 20 μl with ddH 2 O. Sequencing reaction was performed at LGC Genomics, Germany. Different primer pairs were needed to sucessfully amplify the COI and 28S rDNA fragments (Table 1). In this study we successfully sequenced 13 sequences of the COI, and 27 sequences of the 28S rDNA. All sequences from the study of [2] are deposited at the Barcode of Life Data Systems (BOLD) under the project name PROTAT. New sequences are deposited at BOLD under the project name PROTA ( Table 2). The entire dataset of the manuscript can be downloaded at: http://www.boldsystems.org/index.php/MAS_Management_OpenDataSet?datasetcode=DS-2016ADOD Alignment and NJ tree based on K2P distances were performed using MUSCLE, as implemented in Mega v. 6.0 [14]. COI and 28S rDNA were analyzed separately. The reliability of trees was assessed with 1000 bootstrap replicates.

Systematics of the Genus Acerentomon
Protura of the genus Acerentomon Silvestri, 1907 are characterized by the presence of three pairs of anterior setae on the mesonotum and four pairs of anterior setae on the metanotum, well developed maxillary palps with an apical tuft of setae and basal sensilla, a striate band with distinct striae, a claviform foretarsal sensillum t1, a leaf-like sensillum t3, a very long seta δ4 which contrasts with the length of other δ-setae, and the absence of sensillum b'. The second and third abdominal legs usually carry two setae differing in length (apical seta half the length of subapical seta). The maxillary gland possesses a small calyx and a short distal part. The genus has been subdivided into four groups, which differ in the presence or absence of seta x on tergite VII, and seta P1a on sternite VIII ( [7], [10]). However, a cladistic analysis performed on 36 Acerentomon species using 24 morphological characters supports a split into only two main groups: "doderoi" and "aceris" [15]. Of these the "doderoi" group is characterized by the presence of seta x on tergite VII, and comprises a total of 21 species. Two Acerentomon spp. described after the study can be assigned to the aceris group.
The new species described in this paper belongs to the "doderoi" group of Acerentomon species in the sense of [15] and is characterized by the presence of seta x on tergite VII and a pair of posterior setae P1a on sternite VIII, presence of seta Pc on tergite VII and sternite VII, absence of seta P3a on tergite VII, long mesonotal and metanotal setae P1a that are longer than P1, long foretarsal sensillum c, short and slender sensillum b, and broadened sensillum a. The new species is most similar to A. gallicum Ionescu, 1933 in chaetotaxy and measurements. A detailed description is given below.
Accessory setae on tergite I differing in length: seta P1a slightly shorter than P1, seta P2a one third length of P2, equal in length to P3, P4 and P5 setae (Fig 2B). Accessory setae on tergites II-VII setiform, nearly equal in length, about half the length of the principal setae (Fig 2C).  Tergite VII with seta Pc and seta x (Fig 2D). Tergite I with 1+1 psm pores only (Fig 2B). Tergites II -VII with three pairs of pores (psm, psl and al) (Fig 2C and 2M). Tergites I-VII anteriorly with two parallel cuticular lines (Fig 2C and 2D). Pleural structures not developed on tergites I-V; on tergite VI 20-22 teeth present anterior to pore al, on tergite VII some distinct teeth near to pore al (Fig 2J and 2K).
Abdominal appendages with 4, 2, 2 setae. Apical seta of abdominal legs II and III less than half the length of subapical seta, 26 and 17 μm, respectively (Fig 2H and 2I). Accessory setae on sternites setiform, shorter than principal setae, seta Pc on sternite VII shorter than P1a setae (Fig 2M). Sternite I without pores, sternites II-V with single spm pore posterior to Ac (Fig 2H  and 2I). Sternite VI and VII with two pairs of spsm and spsl pores and with a group of three sternal anteromedial pores (sam) anterior to Ac and above cuticular lines (Fig 2M). Sternites II-III anteriorly with a cuticular line (Fig 2H and 2I), sternites IV-VII with two parallel cuticular lines (Fig 2M).
Abdominal segment VIII with distinct striate band and with a regular row of small, scattered denticles anteriorly (Fig 2D and 2M). Comb VIII composed of 14-16 slender, teeth of varying lengths (Fig 1H). Pore psm with several surrounding teeth, other pores absent ( Fig  2D). Laterotergites VIII with row of granules in anterior part and with 8-10 small teeth on posterior margin (Fig 2L). Pore psm with 1-2 accompanying teeth. Setae 1a present on sternite VIII. Pores absent (Fig 2M).
Hind margin of tergites and sternites IX-XI smooth. Dorsal lobe of segment XII with simple median pore, hind margin smooth. Ventral lobe of segment XII with about 10 teeth on hind margin ( Fig 2E) and with 1+1 sternal anterolateral pores.
Type material and deposition: Holotype female (slide no. 77.1) from sample collected in litter and soil, steep southwest slope with Quercus pubescens over platy marl, Leopoldsberg, Remarks: Acerentomon christiani sp. nov. is similar to the group of species characterized by a short foretarsal sensillum b, the apex of which does not reach the base of γ3, and a broadened sensillum a (A. gallicum, A. tenuisetosum, A. brevisetosum, A. italicum, A. fageticola and A. nemorale). Furthermore, A. christiani differs from all Acerentomon spp. of the"doderoi" group in possessing a very long foretarsal sensillum c, which is longer than sensillum a and three times longer than b. In the remaining species sensillum c is shorter than a and about 1.5 times longer than sensillum b. The chaetotaxic pattern of the new species is most similar to A. gallicum, A. tenuisetosum and A. brevisetosum. However, Acerentomon italicum, A. fageticola and A. nemorale differ in seta Pc being absent from tergite VII, in the shape of the comb with lower number of teeth and in the length of the foretarsi (see the identification key). Acerentomon christiani sp. nov. is closest to A. gallicum in foretarsus and body lengths, and in the shape of laterotergal lines (lines on tergites II-V smooth, line of tergite VII with about 15 teeth and line on tergite VII with one or two strong teeth). These two species clearly differ in the length and position of sensillum e on the foretarsus (in the new species e is shorter and located at half the length between the bases of d and f, in A. gallicum this sensillum is long and very close to the base of sensillum d), in the shape of the comb (which possesses 12-14 long teeth in A. gallicum), and in the relative length ratio of mesonotal setae P1 and P1a (in A. christiani sp. nov. the seta P1a is longer than seta P1, whereas in A. gallicum P1a is shorter). The position of the foretarsal sensillum e of the new species is similar to A. tenuisetosum, but the new species differs in possessing longer foretarsus and rostral setae and in the shape of the comb (see identification key below). Acerentomon christiani sp. nov. is closest to A. brevisetosum in the relative length ratio of mesonotal setae (P1a longer than P1), but differs in the length of the foretarsus and dorsal setae, in the shape of the anterolateral lines on tergites VI and VII, and in the shape of the comb (A. brevisetosum is characterized by shorter foretarsal length, very short dorsal setae of approximately 20 μm, smooth anterolateral lines on tergites VI and VII and possession of about 13 teeth on the comb).
Molecular description. The DNA barcode of Acerentomon christiani sp. nov. is clearly delimited from all other Acerentomon species sequenced so far. The new species clusters with either Acerentomon maius (Fig 3, COI), or Acerentomon dispar (Fig 3, 28S).

Discussion
The monophyly of the Acerentomon "doderoi" group is supported by the presence of supplementary seta x on tergite VII. Two other characters were briefly discussed by [15] as additional support: a distinctly protruded rostrum (LR 2.8 to 4.5) and the presence of 6 setae on tergite XI. Within the doderoi group only A. novaki has a short rostrum (LR 14). The presence of seta P1a on sternite VIII mentioned by [7] and [10] as an important character for distinguishing their doderoi group, was not confirmed by [15], who added species with only four setae on sternite VIII (A. franzi and A. noseki) into the group. Within the group only A. franzi is mentioned as having just four setae on tergite XI in the original description of [16]. However, in two male specimens from Vienna tergite XI has 6 setae. Two species (A. skuhravyi and A. granulatum) form a small subgroup, characterized by 6 setae on sternite IX and by an anterior position of setae P1a on sternite VIII ( [17], [18]). All other chaetotaxic characters are uniform among all species of the group, with the exception of the chaetotaxy of segment VII. The presence or absence of seta Pc on tergite VII and sternite VII, and the absence of seta A1 and presence of setae P3a on tergite VII, are of limited phylogenetic value, since [9] noted interspecific variability in these characters. The number of anterior setae on sternites I and III varies from 5 to 7 in different species, but this variation can be intraspecific, as observed both in the new species and mentioned by [9]. Species of the Acerentomon doderoi group clearly differ in length of sensillum b, shape of sensilla a and b, position of sensillum e on the foretarsus, shape of comb and in foretarsal length. The new species differs from all others by a very long foretarsal sensillum c, which reaches the base of the sensillum t3 and is about three times longer than sensillum b (see identification key).
Both the tree based on the COI barcoding fragment and the tree based on the 28S rDNA fragment are fully congruent with morphological systematics. Since A. christiani sp. nov. is known only from a single locality little can be said with respect to its intraspecific variation. Distances are large among populations of Acerentomon dispar, sampled from four different locations in Austria (Fig 3, COI), a result congruent with [2]. However, intraspecific distances in the same species are nearly lacking in 28S rDNA sequences (Fig 3, 28S). A more conclusive contribution of molecular data to relationships among Acerentomon species awaits a denser sampling at both population-and species-level.
Identification Key for All Described Species of the Acerentomon "doderoi" Group