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Five million years in the darkness: A new troglomorphic species of Cryptops Leach, 1814 (Chilopoda, Scolopendromorpha) from Movile Cave, Romania
expand article infoVarpu Vahtera, Pavel Stoev§, Nesrine Akkari|
‡ University of Turku, Turku, Finland
§ National Museum of Natural History, Sofia, Bulgaria
| Naturhistorisches Museum Wien, Vienna, Austria
Open Access

Abstract

A new species of Cryptops Leach, 1814, C. speleorex sp. nov., is described from Movile Cave, Dobrogea, Romania. The cave is remarkable for its unique ecosystem entirely dependent on methane- and sulfur-oxidising bacteria. Until now, the cave was thought to be inhabited by the epigean species C. anomalans, which is widespread in Europe. Despite its resemblance to C. anomalans, the new species is well-defined morphologically and molecularly based on two mitochondrial (cytochrome c oxidase subunit I COI and 16S rDNA) and one nuclear (28S rDNA) markers. Cryptops speleorex sp. nov. shows a number of troglomorphic traits such as a generally large body and elongated appendages and spiracles, higher number of coxal pores and saw teeth on the tibia of the ultimate leg. With this record, the number of endemic species known from the Movile Cave reaches 35, which ranks it as one of the most species-rich caves in the world.

Keywords

Biospeleology, Cryptops speleorex sp. nov., Dobrogea, molecular phylogenetics, new species, troglomorphism

Introduction

Located in the southeastern part of Romania not far from the Black Sea Coast, Movile Cave is the first known subterranean chemosynthesis-based ecosystem (Sarbu et al. 2019). Being completely isolated from the outside environment for 5.5 million years, the cave is remarkable for its unique ecosystem entirely dependent on methane- and sulfur-oxidising bacteria, which release nutrients through chemosynthesis for fungi and other cave animals along the food chain. This subterranean ecosystem is also notable for being rich in hydrogen sulfide, methane (1–2%), ammonia and CO2 (1.5–3.5%) whereas it is poor in O2 (7–16%). Relative humidity in the cave is 100% and there is no detectable air movement. The cave was first discovered in 1986 and since then, only a handful of people have visited it (Sarbu et al. 2019).

Despite its harsh living conditions, Movile Cave ecosystem is known to harbor a diverse and unique fauna. The cave hosts 51 invertebrate species, of which 34 species are endemic (Sarbu et al. 2019). Among these species, some present a number of unique adaptations to a troglobitic life in caves, such as the troglobiont water scorpion Nepa anophthalma Decu, Gruia, Keffer & Sarbu, 1994 (Hexapoda, Hemiptera, Nepidae); the nesticid and liocranid spiders Kryptonesticus georgescuae Nae, Serban & Weiss, 2018 (Araneae: Nesticidae) and Agraecina cristiani (Georgescu, 1989) (Araneae, Liocranidae); the cave leech Haemopis caeca Manoleli, Klemm & Sarbu, 1998 (Annelida, Hirudinea, Haemopidae) and the isopod Armadillidium tabacarui Gruia, Iavorschi & Sarbu, 1994 (Crustacea, Isopoda, Armadillidiidae) (Sarbu et al. 2019).

Five species of myriapods are hitherto discovered from the innermost parts of Movile viz. Archiboreoiulus serbansarbui Giurginca, Vănoaica, Šustr, & Tajovský, 2020 (Diplopoda), Symphylella Silvestri, 1902 sp. (Symphyla), Geophilus alpinus Meinert, 1870 and Clinopodes carinthiacus (Latzel, 1880) (Geophilomorpha) and a troglobitic population of Cryptops anomalans Newport, 1844 (Negrea 1993; Sarbu et al. 2019). It is worth mentioning that the latter taxon has been only studied morphologically (Negrea 1993, 2004).

Recently, we had the occasion to study freshly collected specimens of an undetermined species of the genus Cryptops Leach, 1814 from Movile Cave. Using both, morphological and molecular evidence, the cave specimens were compared with those of C. anomalans living on the surface, outside the cave. A phylogenetic analysis of 29 Cryptops specimens from different parts of Europe, including two from inside Movile Cave, based on two mitochondrial (cytochrome c oxidase subunit I COI and 16S rDNA) and one nuclear (28S rDNA) markers was performed. Morphological and molecular analyses confirmed that the cave specimens from Movile correspond to a new species, Cryptops speleorex sp. nov., that we describe herein. Additionally, we provide an annotated list and a key to the troglobitic Cryptops species in the world.

Material and methods

All Cryptops specimens from Movile Cave were hand-collected by the biospeleologists Serban Sarbu and A. Hillebrand and preserved in 70% or 96% ethanol. Microphotographs were obtained with a Nikon DS-Ri-2 camera mounted on a Nikon SMZ25 stereomicroscope using NIS-Elements Microscope Imaging Software with an Extended Depth of Focus (EDF) patch. Images were edited in Photoshop CS6 and assembled in InDesign CS6. Material is shared between the ISER – Emil Racoviță Institute of Speleology, Bucharest, Romania; IZB – University of Belgrade – Institute of Zoology, Faculty of Biology, Belgrade, Serbia; NHMW – Naturhistorisches Museum Wien, Austria; NMNHS – National Museum of Natural History, Sofia, Bulgaria and the ZMUT – University of Turku – Zoological Museum, Finland. In addition to the type material of the new species we have morphologically studied material of C. anomalans from Serbia and Romania.

Morphological terminology follows Bonato et al. (2010).

Abbreviations: T – tergite, S – sternite.

Molecular methods

Altogether 29 specimens from both inside and outside the Movile Cave were included in the phylogenetic analysis. Of these, 14 were sequenced in this study. Total DNA was extracted from the legs using NucleoSpinTissue kit (Macherey-Nagel) according to the standard protocol for human or animal and cultured cells. Samples were incubated overnight. One nuclear (28S rRNA) and two mitochondrial (cytochrome c oxidase subunit I, COI, and 16S rRNA) fragments were chosen for amplification since they have proven informative between closely related taxa (Vahtera et al. 2012, 2013). 28S rRNA fragment was amplified with the primers 28Sa/28Sb (Whiting et al. 1997), COI fragment with the primers LCO1490/HCO2198 (Folmer et al. 1994) and 16S rRNA with the primers 16Sa/16Sb (Xiong and Kocher 1991; Edgecombe et al. 2002). All primers had a universal tail (T7Promoter/T3) attached to them.

Polymerase chain reaction (PCR) amplifications were performed with MyTaqTM HS Red Mix. PCR was performed in a total volume of 23 μL containing 7.5 μL of MQ, 12.5 μL of MyTaq HS Red Mix, 2×, 0.5 μL of each primer (10 μM) and 2 μL of DNA template. PCR started with initial denaturation at 95 °C for 1 min and was followed by denaturation at 95 °C for 15 s. Annealing temperature for 28S rRNA and COI was 49 °C and 43 °C for 16S rRNA. Annealing lasted for 15 s and was followed by extension at 72 °C for 10 s. The last three steps were repeated 35 times. A negative control was included. PCR products were run in electrophoresis on 1% Agarose gel using Midori Green Advanced DNA Stain (Nippon Genetics). Samples were purified with an A’SAP PCR clean-up kit (ArcticZymes). Sequencing was performed by Macrogen Europe. The resulting chromatograms were visualized and assembled using the software Sequencher 5 (Gene codes corporation, USA). All new sequences are deposited in GenBank (See Table 1 for accession numbers).

Table 1.

Specimens used in the molecular phylogeny and their GenBank accession numbers (specimens sequenced in this study in bold). Institutional abbreviations: ISER–Emil Racoviță Institute of Speleology, Bucharest, Romania; IZBU–University of Belgrade–Faculty of Biology, Institute of Zoology, Belgrade, Serbia; MCZ–Museum of Comparative Zoology, Harvard University; ZFMK–Museum Koenig, Bonn; ZSM–Bavarian State Collection of Zoology, Munich; ZMUT–Zoological Museum, University of Turku, Finland.

Species Lab code Voucher ID number Voucher Country COI 16S 28S
Cryptops speleorex sp. nov. K3 http://mus.utu.fi/ZMUT.MYR-TYPE001 ZMUT Romania MW240507 MW243978 MW243648
C. speleorex sp. nov. K4 ISER Romania MW240508 MW243977 MW243649
C. anomalans 1a IZB Serbia MW240504 MW243967 MW243651
C. anomalans 1b IZB Serbia MW240505 MW243968 MW243652
C. anomalans 2 IZB Serbia MW240511 MW243642
C. anomalans 3 IZB Serbia MW240515 MW243970 MW243643
C. anomalans 4 IZB Serbia MW240503 MW243979 MW243654
C. anomalans 7 IZB Serbia MW240506 MW243969 MW243653
C. anomalans 8 IZB Serbia MW240512 MW243971 MW243644
C. anomalans 9 IZB Serbia MW240514 MW243973 MW243645
C. anomalans 12 IZB Serbia MW240516 MW243974 MW243646
C. anomalans 13 IZB Serbia MW240513 MW243972 MW243647
C. anomalans 54a ISER Romania MW240510 MW243975 MW243650
C. anomalans 57a ISER Romania MW240509 MW243976 MW243641
C. anomalans ZFMK-MYR 1048 ZFMK Germany KM491639
C. anomalans ZFMK-MYR 1047 ZFMK Germany KM491699
C. anomalans ZFMK-MYR 1379 ZFMK Germany KM491703
C. anomalans ZFMK-MYR 4072 ZFMK Germany KM491706
C. anomalans ZSM-ART-JSP130812-004 ZSM Germany KU497151
C. anomalans ZSM-ART-JSP110624-001 ZSM Germany KU497158
C. anomalans ZSM-ART-JSP141105-017 ZSM Germany KU497159
C. anomalans IZ-131458 MCZ UK KF676499 KF676457 KF676353
Cryptops sp. ZFMK-MYR-1185 ZFMK Austria KM491620
Cryptops sp. ZFMK-MYR 3662 ZFMK Germany KU342042
Cryptops sp. ZSM-ART-JSP150118-047 ZSM Slovenia KU497143
Cryptops sp. ZSM-ART-JSP110425-008 ZSM Croatia KU497153
C. croaticus ZFMK-MYR 3320 ZFMK Austria KU342049
C. hortensis IZ-130582 MCZ UK JX422662 JX422684 JX422582
C. parisi IZ-130592 MCZ UK KF676502 KF676460 KF676356
Scolopendra cingulata IZ-131446 MCZ Spain HM453310 HM453220 AF000782

Phylogenetic analyses

Most specimens included in the analysis had all three markers successfully sequenced. To obtain more geographic variation in the dataset, 15 Cryptops specimens (mostly from Wesener et al. 2016) from GenBank (Table 1) were additionally included in the phylogenetic analysis. Of these, 12 had only COI available. Multiple sequence alignments were performed in MAFFT7 online service (Katoh et al. 2019; Kuraku et al. 2013). Sequences were trimmed in Mesquite v 3.10 (Maddison and Maddison 2019) after which the three separate data sets were concatenated with SequenceMatrix (Vaidya et al. 2011) for the phylogenetic analyses. The final molecular matrix including all three data sets (COI, 16S, 28S) consisted of 1561 characters and 29 taxa (excluding outgroup).

Phylogenetic analysis was conducted using both parsimony and maximum likelihood as optimality criteria. Parsimony analysis was done with TNT v. 1.5 (Goloboff and Catalano 2016) treating gaps as missing data. The search strategy consisted of 100 replications, and of 10 rounds of both ratchet and tree drifting followed by tree fusing (Goloboff 1999). Command xmult was executed until 50 independent hits of the shortest tree were found. A strict consensus of the most-parsimonious trees was produced. The command ‘blength’ was used to report the branch lengths of the resulting trees. Jackknife (Farris et al. 1996) resampling method with 1000 replicates and with a probability of a character removal being 0.36 was applied to estimate nodal support. Maximum likelihood analysis of the combined data was conducted RAxML v. 8 (Stamatakis 2014) in the CIPRES portal (Miller et al. 2010). The three genes were separated into different partitions. Unique general time-reversible (GTR) model of sequence evolution (RAxML implements only GTR-based models of nucleotide substitutions) with corrections for a discrete gamma distribution (GTR+ Γ) was used. Nodal support values were estimated using the rapid bootstrap algorithm with 1000 replicates together with GTR-CAT model (Stamatakis et al. 2008). The mitochondrial genes (16S+COI) and the nuclear ribosomal 28S were additionally analysed separately using the same search strategy as was used for the combined data.

Uncorrected p-distances of aligned COI, 16S and 18S data were calculated with MEGA v. 7.0.21 (Kumar et al. 2016).

Results

Order Scolopendromorpha Pocock, 1895

Family Cryptopidae Kohlrausch, 1881

Genus Cryptops Leach, 1814

Cryptops (Cryptops) anomalans Newport, 1844

Material examined

Romania: SE Romania: Lalomiţa County, Călugărească Forest, 18.II.2016, leg. and det. S. Baba, 1 subad. ex. (ISER); Lalomiţa County, Călugărească Forest, oak forest, 28.II.2019, leg. and det. S. Baba, 2 ex. (ISER) (lab code 54a); Lalomiţa County, Călugărească Forest, rotten wood, 13.III.2016, leg. and det. S. Baba, 1 ex. (ISER); Bucharest, Herăstrău Park, under stones, 10.X.2019, leg. and det. S. Baba, 1 ex. (ISER) (lab code 57a); Mangalia, Obanul Mare, Cave Drilling, -3 m, 10.VIII.1999, det. St. and A. Negrea, 1 ex. (ISER); Mangalia, Obanul Mare, Cave Drilling, -8 m, 27.V.2000, det. St. and A. Negrea, 1 ex. (ISER); Mangalia, Obanul Mare, Cave Drilling, -8 m, 28.VI.2000, det. St. and A. Negrea, 1 ex. (ISER); Mangalia, Obanul Mare, Cave Drilling, -12 m, 27.V.2000, det. St. and A. Negrea, 1 ex. (ISER). Serbia: Valley of the Izbice River, v. Izbice, near Novi Pazar, SW Serbia (43°07.333'N, 20°34.354'E; elevation about 700 m a.s.l.): 5♂, 5♀, collected in 2012 (May-October), leg. D. Stojanović (lab code 1) (IZB); Prolom Banja Spa, near Kuršumlija, southern Serbia (43°02.449'N, 21°23.448'E; elevation about 620 m a.s.l.): 3♀, collected 30.04.2016., leg. D. Stojanović (lab code 2) (IZB); village Kacabać, near Bojnik, Leskovac, southern Serbia (43°03.415'N, 21°46.368'E; elevation about 200 m a.s.l.): 2♂, 1♀, collected 01.05.2016., leg. D. Stojanović (lab code 3) (IZB); Pećina Rasnica 1 Cave, village Rasnica, near Pirot, SE Serbia: 1♂, 1♀, collected 18.07.2018., leg. D. Antić (lab code 4) (IZB); Novopazarska Banja Spa, near Novi Pazar, SW Serbia (43°09.269'N, 20°33.132'E; elevation about 650 m a.s.l.): 3♂, 4♀, collected 30.05.2012., leg. D. Stojanović (IZB); Spomen Park, Leskovac, southern Serbia (42°59.051'N, 21°56.349'E; elevation about 200 m a.s.l.): 1♀, collected 28.07.2012., leg. D. Stojanović (IZB); pine forest near the Đurđevi Stupovi Monastery, Novi Pazar, SW Serbia (43°09.183'N, 20°30.049'E): 1♀, collected 15.05.2015., leg. D. Stojanović (lab code 7) (IZB); village Dobanovci, near Surčin, Belgrade, Serbia (44°49.197'N, 20°13.334'E): 1♀, collected 03.11.2013., leg. D. Stojanović (lab code 8) (IZB); Bojčinska šuma forest, village Progar-Jakovo, near Surčin, Belgrade, Serbia (44°43.528'N, 20°09.245'E): 3♀, 1♂, collected 09.06.2013., leg. D. Stojanović, K. Bjelanović (lab code 9) (IZB); Vrla River, Mt. Vlasina, near “Rosa” water factory, v. Topli Do, near Surdulica, SE Serbia (42°38.213'N, 22°17.565'E; elevation about 1070 m a.s.l.): 1♂, collected 07.07.2011, leg. D. Stojanović (IZB); Višnjička Banja, Belgrade, Serbia (44°49.073'N, 20°32.337'E; elevation about 350 m a.s.l.): 1♂, collected 09.06.2006, leg. Ž. Pavković (IZB); Spomen Park, Leskovac, southern Serbia (42°59.051'N, 21°56.349'E; elevation about 200 m a.s.l.): 3♀, 1♂, collected 14.04.2012., leg. D. Stojanović (lab code 12) (IZB). Bulgaria: Pirin Mts, between Sandanski and Lilyanovo, 12.8.1988, litter, mainly Platanus, P. Beron leg. 1 ex. (NMNHS) (Figs 2B, 3B, 4B,D, 5B).

Cryptops (Cryptops) speleorexsp. nov.

Figs 1A, B, 2A, 3A, 4A, C, 5A, 6A–C

Previous records

Cryptops anomalans: Negrea, 1993: p. 87 and all subsequent records (Negrea 1994, 1997, 2004; Negrea and Minelli 1994; Sarbu et al. 2019).

Figure 1. 

Cryptops speleorex sp. nov. A holotype, habitus, dorsal view B paratype (ZMUT), posteriormost segments and ultimate legs, dorsal view.

Material examined

Holotype : Romania: Constanța County, Mangalia, Movile Cave (Peștera Movile), Lake Hall, June, 2014, leg. S. Sarbu, 1 ex. (NMNHS, Myriapoda Collection Id: 10 812); Paratypes: same locality and collector leg. S. Serban, 1 ex. (NHMW10177); same locality, 22.XI.2017, leg. A. Hillebrand, 1 ad. ex., identified as C. anomalans by Stefan Baba (ISER); 1 ad. ex., same locality, date and collector, identified as C. anomalans by Stefan Baba (http://mus.utu.fi/ZMUT.MYR-TYPE001).

Diagnosis

A species morphologically similar to Cryptops anomalans, but differing from it by the much elongated antennae and legs, generally less setose forcipules and body, coxopleures with more than 300 coxal pores (vs. less than 100 in anomalans), ultimate leg with 13–17 saw teeth on tibia (usually 7–10, occasionally 12 in anomalans), and larger and elongated spiracles (see Table 2). Genetically, Cryptops speleorex sp. nov. differs from the C. anomalans specimens from Romania and Serbia by 9.2–12.2% in COI and 6.6–8.7% in 16S rDNA.

Table 2.

Differences in morphological characters between Cryptops anomalans and C. speleorex sp. nov.

Morphological character Cryptops anomalans Cryptops speleorex sp. nov.
Body size (mm) 25–50 >46–52
Antennae length Until posterior end of T3 Until mid of T5
Antennal article 7 L/W (mm) 0.5 × 0.25 1.0 × 0.5
Antennae: spines on basal articles Present, numerous Lacking or just a few
Ultimate leg length 7.65 mm 13.25 mm
Ultimate leg pretarsus (mm) 0.25 1
Ultimate leg saw teeth on tibia and tarsus 1 Tibia: 7–12 (usually 7–10); Tarsus: 3–5 Tibia: 13–17; Tarsus: 5–6
Legs Short, compact, pretarsus short Strongly elongated, pretarsus long
Spiracles Ovoid, small to medium sized (Fig. 4D) Strongly elongated, large (Fig. 4C)
Forcipular trochanteroprefemur With spines medially (4–6) Without spines, just stout setae
Coxopleural pore field Approx. 2/3 of coxapleura; composed of less than 100 pores (86–90) Approx. 4/5 of coxopleura; composed of more than 310 pores (317–320)

Description (holotype)

Length (anterior margin of head plate to posterior margin of telson) approx. 52 mm (46 mm in an adult paratype) (Figs 1A, B). Head plate (Fig. 2A) 3.2 mm long, 3.4 mm broad; antenna approx. 10 mm long. Body yellow-brownish (Fig. 1A); antennae and legs pale yellow; posterior edge of head and tergites with irregular light brownish band, darker in the middle (Figs 1B, 2A); forcipular tarsungulum and leg claws dark brown. Head plate overlaps approx. 1/3 of tergite 1; head plate slightly broader than long (3.2 mm × 3.4 mm), posterior corners strongly rounded, sides convex outwards, anterior apex slightly indented at the base of antennae, bisected by longitudinal median furrow; paramedian sutures diverging anteriorly on head plate; head punctate, sparsely covered with fine setae.

Figure 2. 

Cryptops spp., head and anteriormost segments A Cryptops speleorex sp. nov., holotype, dorsal view B Cryptops anomalans, Pirin Mts (Bulgaria), dorsolateral view (slightly apical).

Antenna relatively long, extending to the middle of tergite 5 when folded backward (Figs 1A, 2A); composed of 17 articles; article length formula: 17<1<2=16<3=4=13=14<5=6=11=12<7–10; basal two articles relatively stout, in general articles increase in length to a maximum at articles 7–10, then gradually shortening; article 17 is more than half length of article 16 (approx. 60%); articles 5–10 much longer than wide, length up to 3 times the width. All surfaces of antennal articles with scattered long setae, densest on articles 1–3; short, fine setae abundant on all articles except for articles 1 and 2, as well as basal part of 3.

Clypeus with 2 setae; prelabral setae in one row of 21–22; 4 short setae between clypeus and prelabral row, irregularly or more evenly scattered. Labral mid piece with a short, but well-developed tooth; side pieces rounded (Fig. 3A).

Figure 3. 

Cryptops spp., forcipular coxosternum, ventral view A Cryptops speleorex sp. nov., holotype B Cryptops anomalans, Pirin Mts (Bulgaria).

Forcipular segment anterior margin of coxosternite convex on each side, with a weak median diastema, fringed by 2 marginal setae on each side. Surface of coxosternite (Fig. 3A) covered with scarce short setae, 10–15 in total; trochanteroprefemur stout, median margin slightly expanded proximally, with 4 setae; femur and tibia very short; tarsungulum long, curved, almost equal in length to trochanteroprefemur’s height.

Maxilla 2 with a well-developed pretarsus; dorsal brush white, dense, situated on the distalmost part of article 3 of telopodite. Proximal side of first maxillary telopodite covered by 10–15 setae (Fig. 3A).

Tergites Tergite 1 with a complete anterior transverse suture and cruciform sutures (Figs 1A, 2A). Oblique sutures present on tergites 2–8; complete paramedian sutures on tergites 2–20; lateral crescentic sulci visible on tergites 6–20; all tergites nearly devoid of setae, occasionally individual scattered short setae. Tergite 21 longer than wide, posterior margin subtriangular, with rounded apex; shallow median depression along posterior half of tergite (Fig. 1B).

Sternites 1–2 and 19–21 without transverse and median sutures; S 3–18 with median longitudinal and curved transverse sutures, more prominent from sternite 5 onward (Fig. 4A). All sternites covered by minute setae. Endosternite: subtrapezoidal, lateral margins very slightly convex, posterior margin slightly concave in the middle; surface with several (6–10) moderately long and sparse setae.

Figure 4. 

Cryptops spp., sternites 8–9 and spiracles A, B sternites 8–9 A Cryptops speleorex sp. nov., holotype, arrow indicating the endosternite B Cryptops anomalans, Pirin Mts (Bulgaria) C, D spiracles C Cryptops speleorex sp. nov., holotype D Cryptops anomalans, Pirin Mts (Bulgaria).

Spiracles strongly elongated on T3, reducing in size towards the posterior end of the body; slit-like (Fig. 4C).

Coxopleural pore field elliptical, covering 4/5 of surface, with more than 310 coxal pores (317–320), extending nearly to posterior margin of coxopleuron (Fig. 5A). Approx. 15–20 sparse spiniform setae emerging between pores and from the dorsal and posterior margins of coxopleuron.

Figure 5. 

Cryptops spp., Coxopleural pore field A Cryptops speleorex sp. nov., holotype B Cryptops anomalans, Pirin Mts (Bulgaria).

Legs generally long; leg 10: prefemur 1.47 mm long, femur 1.59 mm, tibia 1.76 mm, tarsus 2.35 mm, pretarsus 0.7 mm. All tarsi single (Fig. 6A). Walking legs (Fig. 6A, B) smooth, generally poor in setae; spiniform setae sparsely present on the surface of prefemur, and occasionally also on the femur; all pretarsi long, with an anterior and posterior accessory spines of different size, the larger being 2/3rd of pretarsus; accessory spines absent on leg 21; 20 leg: prefemur, femur and tibia slightly swollen; femur and tibia being slightly concave at midlength; a specific field of dense, minute setae present on the ventral, lateral and mesal sides of prefemur, femur and part of tibia.

Figure 6. 

Cryptops speleorex sp. nov., legs. A holotype, walking leg B paratype (ZMUT), walking leg, close-up of apical claw C holotype, ultimate legs, lateral view D, E paratype (NHMW), distal articles of ultimate teeth showing saw teeth.

Ultimate leg (Fig. 6C): prefemur 3.61 mm long, femur 3.05 mm, tibia 1.94 mm, tarsus 1: 1.28 mm, tarsus 2: 2.22, pretarsus 0.56 mm.; numerous robust spiniform setae on the ventral, mesal and less so on lateral and dorsal sides of prefemur; spiniform setae present also on the ventral and mesal sides of femur; tibia, tarsus 1 and tarsus 2 covered by tiny dense setae on all sides; 13–14 saw teeth on tibia (17 in an adult paratype) and 5–6 on tarsus 1.

Etymology

The species epithet is a noun in apposition, meaning "king of the cave", referring to the species top position in the food chain of the Movile ecosystem.

Distribution

The species is hitherto known only from the aphotic zone of the Cave Movile in the southern part of Romanian Dobrogea.

Ecological remarks

Cryptops speleorex sp. nov. is the largest invertebrate species in Movile Cave. It has been observed feeding on terrestrial isopods (Trachelipus troglobius Tabacaru & Boghean, 1989, Armadillidium tabacarui Gruia, Iavorschi & Sarbu, 1994), smaller beetles, Diplura or spiders (Sarbu et al. 2019).

Phylogenetic analyses

Parsimony analysis resulted in a single most-parsimonious (MP) tree of length 1586 steps (Fig. 7). Two C. speleorex sp. nov. specimens collected from Movile Cave (samples K3 and K4) group within C. anomalans as a separate clade supported by jackknife resampling value (hereafter JF) of 99. The phylogeny shows the Movile Cave clade being evolutionary most closely related to the clade (JF = 75) including C. anomalans samples from southern Serbia and Belgrade area (JF = 100) and Romania and SW Serbia (JF = 84). This Serbian/Romanian clade forms a sister group with the clade (JF = 95) containing a single C. anomalans specimen (lab code 4) from southeast Serbia (collected from a cave) and identical sequences of C. anomalans from London, UK and different parts of Germany (JF = 100). All specimens above form a clade with strong support (JF = 92). Outside this clade are Cryptops sp. from Austria and an unsupported clade containing Cryptops spp. from Croatia and Slovenia together with C. hortensis (Donovan, 1810). Basal to these are resolved C. parisi Brolemann, 1920 and C. croaticus Verhoeff, 1931 (JF = 82) followed by Cryptops sp. from Germany.

Figure 7. 

The single most parsimonious tree of length 1586 steps with jackknife resampling values > 50% shown on the nodes. Branch lengths represent the number of optimized character-state changes.

Regarding the placement of C. speleorex sp. nov. and the relationships among the C. anomalans specimens, the likelihood analysis (Fig. 8) resulted in a mostly congruent tree topology with the parsimony tree, the only difference being that in the parsimony analysis C. speleorex sp. nov. is resolved basal to the Serbian/Romanian clade whereas in the likelihood tree it is resolved within it. The C. speleorex sp. nov. specimens form a clade supported by bootstrap value (hereafter BS) of 100. Cryptops speleorex sp. nov. groups together with the C. anomalans specimens from Serbia (excluding a single Serbian C. anomalans specimen, lab code 4) and Romania. All the specimens above form a sister clade to a group including C. anomalans specimens from Serbia (lab code 4), Germany and the UK. As in the parsimony analysis, the additional Cryptops species (other than C. anomalans) were resolved as basal to C. anomalans. Their internal grouping varies from that in the parsimony tree, which is not surprising due to the lack of nodal support in the basal-most nodes.

Figure 8. 

Likelihood tree with bootstrap values > 50% shown for each node.

When analyzed separately (only likelihood, tree not shown), the mitochondrial COI and 16S resolved C. speleorex sp. nov. as a distinct clade (BS = 100) within C. anomalans specimens, the tree topology regarding C. speleorex sp. nov./C. anomalans being identical to that of the parsimony tree. Not surprisingly, the level of variation in the nuclear 28S was low and the likelihood analysis based on it could not resolve the relationships among the C. anomalans/C.speleorex sp. nov. specimens (tree not shown).

Pairwise distances

Pairwise distances between the samples by each marker are shown in Tables 35. The differences between C. speleorex sp. nov. and the closest clade (Fig. 7) comprising of C. anomalans specimens from Romania and Serbia are 9.2–12.2% (COI) and 6.6–8.7% (16S rDNA). Nuclear 28S rDNA was conservative and showed almost no variation (0–0.3%) between these specimens. The difference between the new species and the rest of the C. anomalans specimens (Serbia (lab code 4), Germany and UK) is 13.8–15.5% (COI). In respect to 16S the differences were 10.7–12.5% and 9.9–11.2% between the new species and the Serbian (lab code 4) and C. anomalans from London, UK, respectively. Intraspecific difference between the two C. speleorex sp. nov. specimens is 8.5% in COI and 6.6% in 16S.

Table 3.

Estimates of evolutionary divergence between sequences. COI: The number of base differences per site from between sequences are shown. The analysis involved 30 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding.

All positions containing gaps and missing data were eliminated. There were a total of 556 positions in the final dataset.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1 Scolopendra cingulata (IZ-131446)
2 Cryptops anomalans UK (IZ-131458) 0.248
3 C. anomalans Germany (KM491639) 0.248 0.000
4 C. anomalans Germany (KM491699) 0.248 0.000 0.000
5 C. anomalans Germany (KM491703) 0.248 0.000 0.000 0.000
6 C. anomalans Germany (KM491706) 0.248 0.000 0.000 0.000 0.000
7 C. anomalans Germany (KU497151) 0.248 0.000 0.000 0.000 0.000 0.000
8 C. anomalans Germany (KU497158) 0.248 0.000 0.000 0.000 0.000 0.000 0.000
9 C. anomalans Germany (KU497159) 0.248 0.000 0.000 0.000 0.000 0.000 0.000 0.000
10 C. anomalans SE Serbia cave (4) 0.237 0.110 0.110 0.110 0.110 0.110 0.110 0.110 0.110
11 C. anomalans SW Serbia (1a) 0.223 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.144
12 C. anomalans SW Serbia (1b) 0.223 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.144 0.000
13 C. anomalans SW Serbia (7) 0.225 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.149 0.005 0.005
14 Cryptops speleorex sp. nov. Movile cave, Romania (K3) 0.246 0.155 0.155 0.155 0.155 0.155 0.155 0.155 0.155 0.153 0.121 0.121 0.126
15 Cryptops speleorex sp. nov. Movile cave, Romania (K4) 0.225 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.142 0.095 0.095 0.101 0.085
16 C. anomalans Bucharest Romania (57a) 0.212 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.138 0.135 0.074 0.074 0.079 0.112 0.103
17 C. anomalans SE Romania (54a) 0.225 0.146 0.146 0.146 0.146 0.146 0.146 0.146 0.146 0.142 0.092 0.092 0.097 0.122 0.117 0.040
18 C. anomalans southern Serbia (2) 0.243 0.129 0.129 0.129 0.129 0.129 0.129 0.129 0.129 0.138 0.103 0.103 0.108 0.104 0.097 0.088 0.099
19 C. anomalans Belgrade, Serbia (8) 0.239 0.133 0.133 0.133 0.133 0.133 0.133 0.133 0.133 0.140 0.097 0.097 0.103 0.110 0.094 0.086 0.094 0.023
20 C. anomalans Belgrade, Serbia (13) 0.239 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.138 0.095 0.095 0.101 0.108 0.092 0.085 0.092 0.022 0.002
21 C. anomalans Belgrade, Serbia (9) 0.239 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.138 0.095 0.095 0.101 0.108 0.092 0.085 0.092 0.022 0.002 0.000
22 C. anomalans southern Serbia (3) 0.239 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.131 0.138 0.104 0.104 0.110 0.112 0.092 0.099 0.106 0.032 0.027 0.025 0.025
23 C. anomalans southern Serbia (12) 0.241 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.137 0.135 0.094 0.094 0.099 0.106 0.090 0.090 0.101 0.040 0.029 0.027 0.027 0.043
24 C. hortensis UK (IZ-130582) 0.243 0.203 0.203 0.203 0.203 0.203 0.203 0.203 0.203 0.182 0.174 0.174 0.178 0.173 0.189 0.180 0.187 0.198 0.200 0.198 0.198 0.191 0.191
25 Cryptops sp. Austria (KM491620) 0.228 0.178 0.178 0.178 0.178 0.178 0.178 0.178 0.178 0.167 0.182 0.182 0.185 0.174 0.167 0.176 0.183 0.180 0.173 0.173 0.173 0.185 0.182 0.169
26 Cryptops sp. Croatia (KU497153) 0.230 0.182 0.182 0.182 0.182 0.182 0.182 0.182 0.182 0.180 0.200 0.200 0.203 0.201 0.191 0.203 0.209 0.198 0.196 0.194 0.194 0.194 0.192 0.156 0.156
27 C. parisi UK (IZ-130592) 0.221 0.192 0.192 0.192 0.192 0.192 0.192 0.192 0.192 0.173 0.176 0.176 0.182 0.191 0.182 0.171 0.180 0.171 0.169 0.167 0.167 0.167 0.171 0.196 0.192 0.185
28 C. croaticus (KU342049) 0.239 0.201 0.201 0.201 0.201 0.201 0.201 0.201 0.201 0.185 0.173 0.173 0.176 0.192 0.178 0.174 0.180 0.194 0.194 0.192 0.192 0.196 0.196 0.169 0.192 0.192 0.137
29 Cryptops sp. Slovenia (KU497143) 0.255 0.192 0.192 0.192 0.192 0.192 0.192 0.192 0.192 0.185 0.189 0.189 0.192 0.201 0.203 0.189 0.203 0.201 0.205 0.207 0.207 0.201 0.196 0.173 0.207 0.180 0.187 0.165
30 Cryptops sp. Germany (KU342042) 0.223 0.201 0.201 0.201 0.201 0.201 0.201 0.201 0.201 0.187 0.192 0.192 0.196 0.210 0.194 0.178 0.192 0.198 0.194 0.194 0.194 0.205 0.198 0.210 0.185 0.187 0.196 0.203 0.216
Table 4.

Estimates of evolutionary divergence between sequences. 16S: The number of base differences per site from between sequences are shown. The analysis involved 17 nucleotide sequences. All positions containing gaps and missing data were eliminated.

There were a total of 392 positions in the final dataset.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 Scolopendra cingulata (IZ-131446)
2 Cryptops anomalans UK (IZ-131458) 0.390
3 C. anomalans SW Serbia (1a) 0.372 0.092
4 C. anomalans SW Serbia (1b) 0.372 0.092 0.000
5 C. anomalans SW Serbia (7) 0.372 0.094 0.003 0.003
6 C. anomalans southern Serbia (3) 0.372 0.082 0.036 0.036 0.038
7 C. anomalans Belgrade, Serbia (8) 0.367 0.092 0.041 0.041 0.041 0.018
8 C. anomalans Belgrade, Serbia (13) 0.372 0.089 0.043 0.043 0.043 0.020 0.008
9 C. anomalans Belgrade, Serbia (9) 0.372 0.087 0.041 0.041 0.041 0.010 0.008 0.010
10 C. anomalans southern Serbia (12) 0.365 0.084 0.033 0.033 0.036 0.018 0.031 0.033 0.023
11 C. anomalans SE Romania (54a) 0.372 0.092 0.059 0.059 0.059 0.048 0.054 0.051 0.048 0.051
12 C. anomalans Bucharest Romania (57a) 0.372 0.099 0.066 0.066 0.066 0.054 0.059 0.056 0.054 0.059 0.013
13 Cryptops speleorex sp. nov. Movile cave, Romania (K4) 0.365 0.099 0.082 0.082 0.082 0.066 0.074 0.071 0.069 0.071 0.066 0.069
14 Cryptops speleorex sp. nov. Movile cave, Romania (K3) 0.383 0.112 0.087 0.087 0.084 0.071 0.077 0.074 0.069 0.082 0.079 0.082 0.066
15 C. anomalans SE Serbia cave (4) 0.385 0.084 0.094 0.094 0.097 0.077 0.087 0.084 0.082 0.079 0.097 0.107 0.107 0.125
16 C. parisi UK (IZ-130592) 0.355 0.217 0.209 0.209 0.212 0.214 0.227 0.224 0.222 0.219 0.224 0.227 0.232 0.235 0.232
17 C. hortensis UK (IZ-130582) 0.360 0.230 0.217 0.217 0.219 0.222 0.235 0.230 0.232 0.224 0.235 0.245 0.230 0.219 0.230 0.260
Table 5.

Estimates of evolutionary divergence between sequences. 28S: The number of base differences per site from between sequences are shown. The analysis involved 18 nucleotide sequences.

All positions containing gaps and missing data were eliminated. There were a total of 316 positions in the final dataset.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 Scolopendra cingulata (IZ-131446)
2 Cryptops anomalans UK (IZ-131458) 0.187
3 C. anomalans Bucharest Romania (57a) 0.187 0.000
4 C. anomalans southern Serbia (2) 0.190 0.003 0.003
5 C. anomalans southern Serbia (3) 0.190 0.003 0.003 0.000
6 C. anomalans Belgrade, Serbia (8) 0.190 0.003 0.003 0.000 0.000
7 C. anomalans Belgrade, Serbia (9) 0.190 0.003 0.003 0.000 0.000 0.000
8 C. anomalans southern Serbia (12) 0.190 0.003 0.003 0.000 0.000 0.000 0.000
9 C. anomalans Belgrade, Serbia (13) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000
10 Cryptops speleorex sp. nov. Movile cave, Romania (K3) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000
11 Cryptops speleorex sp. nov. Movile cave, Romania (K4) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000
12 C. anomalans SE Romania (54a) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
13 C. anomalans SW Serbia (1a) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
14 C. anomalans SW Serbia (1b) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
15 C. anomalans SW Serbia (7) 0.190 0.003 0.003 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
16 C. anomalans SE Serbia cave (4) 0.184 0.006 0.006 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009 0.009
17 C. parisi UK (IZ-130592) 0.196 0.054 0.054 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057 0.057
18 C. hortensis UK (IZ-130582) 0.190 0.063 0.063 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.066 0.070 0.079

Key for identification of cave-specialized (troglomorphic/troglophilic) Cryptops

1 Forcipular coxosternal margin with blunt, rounded or slightly flattened, hyaline lobes; tarsungulum very short C. (Paracryptops) indicus
Forcipular coxosternal margin without hyaline lobes; tarsungulum moderate or long 3
3 Trigonal sutures present on the posterior part of sternites. Tarsus of most legs bipartite Cryptops (Trigonocryptops) 1
Sternal trigonal sutures absent. Tarsus of most legs a single article Cryptops (Cryptops)
5 Ultimate legs with saw teeth present from prefemur to tarsus 2, saw teeth formula: 28-30-14-17-17 C. spelaeoraptor Ázara & Ferreira, 2014
Ultimate legs with saw teeth present on tibia and tarsus 1 only 7
7 T1 with transverse suture only 9
T1 with transfer and other sutures 11
9 Head without paramedian sutures; length: 19 mm, antennae short, 3+3 saw teeth on tibia and tarsus of ultimate legs C. beroni
Head with incomplete paramedian sutures on the posterior half and the anteriormost quarter of the cephalic plate; length: 28–29 mm; antennae long, 4+9 saw teeth on tibia and tarsus 1 of ultimate leg C. illyricus
11 T1 with inverted Y-shaped sutures C. legagus Edgecombe, Akkari, Netherlands, Du Preez, 2020
T1 with transverse and/or paramedian sutures 13
13 T1 with transverse suture and two paramedian sutures; prefemur and femur of ultimate legs with dorsodistal spinous process; small species, ca 15 mm, cave in India C. kempi
T1 with transverse suture and U-shaped or cruciform suture; prefemur and femur of ultimate legs without dorsodistal spinous process; caves in Europe 15
15 T1 with transverse and cruciform sutures; head with 2 complete paramedian sutures, large species Cryptops speleorex sp. nov.
T1 with transverse suture and characteristic U-shaped suture attached to it; head with incomplete paramedian sutures 17
17 Labrum tridentate 19
Labrum unidentate 21
19 Antennae short, head plate with incomplete anterior and posterior paramedian sutures; saw teeth on tibia and tarsus in combination 13+6 C. dianae
Antennae long, head plate with posterior paramedian sutures only C. umbricus umbricus
21 Head with two incomplete posterior paramedian sutures only; anterior margin of forcipular coxosternite strongly convex and covered by spiniform setae, cave in France C. umbricus lewisi
Head with two incomplete short posterior paramedian sutures only; anterior margin of forcipular coxosternite slightly rounded and barely protuberant; spiniform setae missing, cave on Tenerife C. vulcanicus

Discussion

Scolopendromorphs are strictly terrestrial and most species are found in forest leaf litter, decomposed wood, under bark of dead trees, in the soil, under stones or in caves in the temperate and tropical areas of the world. Few species are well adapted to eremic environments (Minelli and Golovatch 2013), occasionally in atypical habitats such as forest canopy (Lewis 1982; Phillips et al. 2020) or tropical rivers (Siriwut et al. 2016). Although less common than lithobiomorphs, scolopendromorphs may occur in caves, where they are represented with some highly adapted species, mainly from the family Cryptopidae. Other families are only marginally recorded in caves: Scolopocryptopidae (genera Thalkethops Crabill, 1960 and Newportia Gervais, 1847 with several species from American caves, including several troglobites), Plutoniumidae (genera Plutonium Cavanna, 1881 and Theatops Newport, 1844) in European caves and Scolopendridae (genus Otostigmus Porat, 1876; O. cooperi Chamberlin, 1942 inhabits Chilibrilo caves in Panama (Chamberlin 1942); Otostigmus troglodytes Ribaut, 1914 found in a cave near Tanga, Tanzania (Ribaut 1914)). The genus Cryptops is by far the most frequent in the caves worldwide with some 18–20 species found in caves in South Europe (Spain, France, Italy, Greece), Canary Islands, Cuba, Brazil, Australia and Africa. Troglomorphic species are known from the nominate subgenus, and the subgenera Trigonocryptops and Paracryptops (see Table 6).

Table 6.

An annotated list of the troglobitic/troglophilic Cryptops species in the world.

Species Distribution Category References
Cryptops (Cryptops) beroni Matic & Stavropoulos, 1988 Greece: Crete, Acrotiri, Cave Katholiko Troglobite? Matic and Stavropoulos (1988)
Cryptops (Trigonocryptops) camoowealensis Edgecombe, 2006 Australia: Queensland, Camooweal area, Five O’Clock Cave Troglobite Edgecombe (2006)
Cryptops (Trigonocryptops) cavernicolus Negrea & Fundora Martinez, 1977 Cuba Troglobite Matic et al. (1977)
Cryptops (Cryptops) dianae Matic & Stavropoulos, 1990 Greece: Thassos Island, cave Dracotrypa unknown Matic and Stavropoulos (1990)
Cryptops (Trigonocryptops) hephaestus Ázara & Ferreira, 2013 Brazil: known from three iron ore caves of the “Quadrilátero Ferrífero” (Iron Quadrangle) in Minas Gerais in Mariana and Itabirito municipalities Troglophile Ázara and Ferreira (2013), Chagas-Jr and Bichuette (2018)
Cryptops (Cryptops) illyricus Verhoeff, 1933 Caves only?; Slovenia and Croatia Verhoeff 1933
Cryptops (Trigonocryptops) iporangensis Ázara & Ferreira, 2013 Brazil: known from four caves (Ressurgência das Areias de Água Quente, Gruta Monjolinho, Caverna Alambari de Baixo, Caverna Santana) in Iporanga, São Paulo Troglobite Ázara and Ferreira (2013), Chagas-Jr and Bichuette (2018)
Cryptops (Paracryptops) indicus (Silvestri, 1924) India: Assam, Garo Hills, Siju Cave Troglophile (Silvestri 1924)
Cryptops (Cryptops) kempi Silvestri, 1924 India: Assam, Garo Hills, Siju Cave Troglophile (Silvestri 1924)
Cryptops (Cryptops) legagus Edgecombe, Akkari, Netherlands, Du Preez, 2020 Botswana: Diviner’s Cave (Koanaka Hills) and Dimapo Cave (Gcwihaba Hills) Epigean/Troglophile? Edgecombe et al. (2020)
Cryptops (Trigonocryptops) longicornis (Ribaut, 1915) Caves in Spain Troglobite Ribaut (1915)
Cryptops (Cryptops) speleorex sp. nov. Romania: Mangalia, Movile Cave Troglobite This paper (see also Negrea 1993)
Cryptops (Trigonocryptops) roeplainsensis Edgecombe, 2005 Australia: known from three caves (Nurina Cave 6N-46, Burnabbie Cave, cave 6N-1327), Roe Plains Troglobite Edgecombe (2005)
Cryptops (Cryptops) spelaeoraptor Ázara & Ferreira, 2014 Brazil: Bahia, Campo Formoso, only known from the type locality, Toca do Gonçalo Cave Ázara and Ferreira (2014), Chagas-Jr and Bichuette (2018).
Cryptops (Trigonocryptops) troglobius Matic, Negrea & Fundora Martinez, 1977 Cuba Troglobite Matic et al. (1977)
Cryptops (Cryptops) umbricus umbricus Verhoeff, 1931 Caves in France and Italy but also found outside caves Troglophile Verhoeff (1931), Matic (1960), Iorio and Minelli (2005), Iorio and Geoffroy (2007, 2008), Iorio (2010)
Syn. Cryptops jeanneli Matic, 1960
Cryptops umbricus ischianus Verhoeff, 1942
Cryptops (Cryptops) umbricus lewisi Iorio, 2010 France: Alpes-Maritimes, Gourdon, Aven du Fourchu Cave Troglobite Iorio (2010)
Cryptops (Cryptops) vulcanicus Zapparoli, 1990 Spain: Tenerife Island, Cueva Felipe Reventón Troglobite Zapparoli (1990)

Several morphological characters traditionally used in centipedes taxonomy could be subject to intraspecific variation related to postembryonic development, animal life stage and ecology (Akkari et al. 2017). This might render species identification problematic in some cases and generates taxonomic errors. This is also true for such a highly variable and widely distributed species as C. anomalans. In fact, nine species and subspecies were hitherto synonymised with this species (see Krapelin 1903; Verhoeff 1931; Crabill 1962; Zapparoli 2002). Three subspecies are still listed as valid for it (Chilobase 2.0). Now the identity of these taxa and the presence of any possible cryptic species within C. anomalans could only be revealed via an integrative study combining morphological and molecular markers. Whereas clear molecular differences are here indicated by the different markers and the high interspecific distance between C. anomalans and the newly described species C. speleorex sp. nov., the morphological comparison was not as straightforward since both species show several similarities, including an overlapping in size. While several of the differences observed between both species (Table 2) could be understood as a clear indication of troglomorphism in C. speleorex sp. nov. such as the elongation of appendages, a few other characters including the number of saw teeth on tibia and tarsus 1 of the ultimate legs, number of coxal pores and the shape of spiracles were diagnostic to separate both species.

Intraspecific distance between the two sequenced Cryptops speleorex sp. nov. specimens is relatively high in comparison to the detected interspecific variation (Tables 35) raising a question whether these two specimens could actually be interpreted as two separate species. However, this variation is only shown in the two mitochondrial markers – there are no morphological differences (or any difference in their nuclear 28S marker) between the C. speleorex sp. nov. specimens. As Morgan-Richards et al. (2017) well explains, cryptic speciation should never be used as a null hypothesis in the absence of phenotypic or nuclear data supporting it. Instead, “the origin of the divergent mtDNA haplogroups might result from complex biogeographical scenarios or they might simply represent normal, stochastic processes of mutation and extinction of a non-recombining locus within a large population”.

Taxonomic and evolutionary implications of C. speleorex sp. nov

The type locality of C. anomalans is unknown and therefore it is impossible to conclude which part (if any) of the studied population is the actual C. anomalans described by Newport (1844). Before this study, only a handful of C. anomalans specimens from a limited geographic range had been sequenced (Spelda et al. 2011; Vahtera et al. 2013; Wesener et al. 2016). We acknowledge that describing C. speleorex sp. nov. as a new species leaves C. anomalans paraphyletic and that monophyly is violated by this taxonomic act. However, we view this as an inevitable consequence of speciation with a particular evolutionary implication, i.e., that C. speleorex sp. nov. evolved within what is currently known as C. anomalans. It is worth noting that the closest evolutionary relatives of C. speleorex sp. nov. appear to be the C. anomalans specimens from Serbia (excluding the sample number 4) and Romania (Figs 8, 9). This means that they are most closely related to each other than either of them is to the rest of the studied C. anomalans populations. The current situation with C. anomalans should not be seen as a failed taxonomy but as a natural consequence when new data from a widespread species is obtained.

Figure 9. 

Map of Europe showing geographic distribution of Cryptops specimens analyzed herein. Asterisk – C. speleorex sp. nov. Dot – other Cryptops spp. used in the study (see Table 1 for details).

Acknowledgements

We are especially grateful to Serban M. Sarbu (Adjunct Faculty, California State University Chico) for calling our attention to this interesting material and for committing samples from Movile Cave for study. Stefan Baba (“Emil Racoviță” Institute of Speleology & Faculty of Biology, University of Bucharest, Romania), Dragan Antić and Dalibor Stojanović (both from University of Belgrade – Faculty of Biology) committed further specimens of C. speleorex and C. anomalans from Serbia and Romania for study. The study was partially funded by project #KP-06-H21/1-17.12.2018 of the National Science Fund, Ministry of Education and Science of the Republic of Bulgaria to PS and by Helsinki Entomological Society to VV. We thank G.D. Edgecombe, C. Martínez-Muñoz, A. Schileyko and Ivan H. Tuf for their constructive comments that greatly benefited the manuscript.

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1 Here belong: C. camoowealensis, C. cavernicolus, C. hephaestus, C. iporangensis, C. longicornis, C. roeplainsensis, C. troglobius