Convergent evolution underground - two new troglomorphic collembolan species of the genus Schaefferia Absolon, 1900 (Collembola, Poduromorpha, Hypogastruridae) from Iran and Montenegro

Convergent evolution underground - two new troglomorphic collembolan species of the genus Schaefferia Absolon, 1900 (Collembola, Poduromorpha, Hypogastruridae) from Iran and Montenegro Ľubomír Kováč1, Marko Karaman2, Mahmood Mehrafrooz Mayvan1 1Department of Zoology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Šrobárova 2, 041 54 Košice, Slovakia 2Natural History Museum of Montenegro, Trg vojvode Bećir bega Osmanafića 16, Podgorica, Montenegro Corresponding author: Mahmood Mehrafrooz Mayvan ([email protected]) Academic editor: Louis Deharveng|Received 28 April 2025|Accepted 21 October 2025|Published 5 November 2025 https://zoobank.org/F2343D01-6810-4530-8D62-CDBB6BEC97DF Citation: Kováč Ľ, Karaman M, Mehrafrooz Mayvan M (2025) Convergent evolution underground - two new troglomorphic collembolan species of the genus Schaefferia Absolon, 1900 (Collembola, Poduromorpha, Hypogastruridae) from Iran and Montenegro. Subterranean Biology 54: 1–22. https://doi.org/10.3897/ subtbiol.54.157228 Abstract Two new subterranean springtail species of the genus Schaefferia Absolon, 1900 are described: S. greensladeae sp. nov. from Danial Cave in the Alborz Mountains, Iran, and S. montenegrina sp. nov. from Babatuša Cave in Dinaric Karst, Montenegro. Both species are characterized by the absence of eyes and postantennal organ (PAO) having more than 5 lobes in the contrary to other eyeless congeners that have less than 5 lobes in PAO. Morphological traits of both species indicate high level of association with the subterranean environment. The phylogenetic relationships and morphological adaptations of two new species to subterranean life are discussed. Schaefferia greensladeae sp. nov. and S. montenegrina sp. nov. serve as a good example of convergent evolution that is characteristic of invertebrates highly adapted to deep subterranean realm. An updated key to the world species of the genus and a table summarizing diagnostic characters of the world species of Schaefferia are provided. Keywords Cave springtails, convergent evolution, phylogeny, Schaefferia, taxonomy Subterranean Biology 54: 1–22 (2025) doi: 10.3897/subtbiol.22.157228 https://subtbiol.pensoft.net Copyright Ľubomír Kováč et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Subterranean Biology Published by The International Society for Subterranean Biology A peer-reviewed open-access journal Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 2 Introduction The genus Schaefferia Absolon, 1900 (Hypogastruridae) was first established for S. emucronata, a species described from a cave in the Czech Republic. The genus is characterized by the following features: body pigment reduced, eyes from 0+0 to 7+7 (mostly less than 5+5), furca reduced or absent - if present, then dens usually with 3 or 4 setae (sometimes 5 or 6), anal spines long (more or less equal to claw in length), Ant IV with a simple apical bulb and 5 to 12 sensilla, body covered with well-differentiated setae, Ti I–III without knobbed tenent hairs, claws with or without inner tooth, empodium well-developed with basal lamella. Moreover, the genus belongs to the Ceratophysella denticulata line, i.e. its representatives are characterized by a specific setal pattern (Thibaud et al. 2004). Schaefferia emucronata Absolon, 1900 is the nominal species of the genus, described from Kateřinská jeskyně Cave in the Moravian Karst, Czech Republic. Later, S. willemi (Bonet, 1930) was described from the Rochefort and Han caves in Belgium and two species from caves in Germany: S. decemoculata (Stach, 1939) from Kellerhöhle, Westfalia and S. quadrioculata (Stach, 1939) from Diebshöhle and Heimkehle caves in Harz Mountains. The first species that was found to inhabit surface environment was S. pouadensis Delamare Deboutteville, 1945 from France. Schaefferia guerrerense (Bonet, 1945) was the first species reported outside Europe, discovered in Mexico in the popular Cacahuamilpa show cave. The genus has Holarctic distribution (Thibaud 1972) with 35 species described up to date (Jordana et al. 2020), involving both edaphic and subterranean species. The representatives of the genus often inhabit caves in low altitudes, while humus, soil and mosses in mountains, being frequent in relatively cold microclimate with air temperature around 10 °C and relatively high humidity (Thibaud 1972). A recent study, carried out by Jordana et al. (2020) in Sierra de Gaudarrama Mountains, Spain, yielded a species associated with colluvial “milieu souterrain superficiel” (MSS - mesovoid shallow substratum) documenting variety of subterranean habitats that the congeners of this genus occupy. In this paper, we provide comprehensive descriptions of two new eyeless Schaefferia species that were discovered during the recent cave explorations in Iran and Montenegro, with their morphological traits showing high level of association with the subterranean environment. A table summarizing diagnostic characters of eyeless Schaefferia congeners is included together with an updated key to the world species of the genus. The phylogenetic relationships and morphological adaptations of two new species to subterranean life are discussed. Two new species of eyeless cavernicolous Schaefferia 3 Material and methods Cave descriptions Danial Cave (IRAN) Danial Cave is the second largest river cave in Iran, which is located in the Alborz Mountain range in the Mazandaran karst area, on the southern edge of the Caspian Sea near the Salman Shahr of Abbasabad city (36°39'35.2"N, 51°10'53.2"E) at altitude of 204 m above sea level (Fig. 1), its name derived from the close village. Alternative name of this cave is Gerde Kooh. The cave is situated in the Hyrcanian Forest type on the slope of the mountain with the main entrance 5 m long and 6 m high (Fig. 2). Its length is 2,158 m, with a subterranean river flowing along the entire cave (Yusefinia 2017; Ghelich Khani et al. 2022; Barjadze et al. 2024; Mehrafrooz Mayvan et al. 2025). It has constant air temperature (around 12 °C) and relative humidity (RH) is over 73%, measured with a thermo-hygrometer TFA 30.5015 DTH in April 2024. Babatuša Cave (MONTENEGRO) This cave is situated near the village of Trnovo, Virpazar, (42°17'56"N, 19°01'58"E) at altitude of 381 m above sea level (Fig. 7). The entrance is a horizontal fissure about 10 m wide and about 2 m high (Fig. 8) (Vujačić et al. 2024). The cave is relatively short (a little over 290 m), with the great main chamber rich in flowstones and small sinter pools (Djurovic and Djurovic 2021). Its galleries are filled with clay deposits up to 10 m thick (Vujačić et al. 2024). Methods For observation in an optical microscope with phase contrast, specimens were firstly boiled softly in evaporation glass dish with 95% ethyl alcohol on electric cooking plate for approximately 1 minute to remove fat from the body. For clearing, the specimens were transported to concave glass dish with 10% water solution of KOH for ca. 1 minute and then transported to a dish containing Chlorophenol until the specimens were fully transparent. At the end, specimens were mounted on Swann medium on permanent slides. After slide was sufficiently dried out, the cover glass was ringed with Canada balsam to seal the mounting medium and prevent its desiccation. The specimens were observed in Leica DM 2500 light microscope equipped with phase and DIC contrasts, and a drawing arm. Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 4 Figure 1. A map of Iran, red area - Mazandaran province; location of the Danial Cave, locus typicus of Schaefferia greensladeae sp. nov., is marked with a red asterisk on an enlarged map of the province (upper) B Hyrcanian Forest near the cave in summer (Photo: M. Mehrafrooz). Abbreviations used. a—anterior row of seta(e); Ant—antennal segment(s); Asp—anal spines; Cl—claw; m—median row of seta(e); Mc—macroseta(e); ms—microsensillum; os—subapical organite (sub-apical sensory peg); p—posterior row of seta(e); PAO—postantennal organ; s—sensory seta; Ti—tibiotarsus; VT—ventral tube; CoPJSU—Collembola collection of the Department of Zoology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University, Košice, Slovakia; ZMFUM—Zoology Museum of Ferdowsi University of Mashhad, Mashhad, Iran. Results Species description Schaefferia greensladeae Mehrafrooz Mayvan & Kováč, sp. nov. https://zoobank.org/424A45B7-3B5F-41C2-992B-788B3B16DC29 Figs 3–6, Table 1 Diagnosis. Eyes absent, PAO large with 7 subequal lobes formed around central circle. Ant IV with 7–8 sensilla and simple apical vesicle. Claw enlarged, with an internal tooth and pair of lateral teeth. Retinaculum with 3–4 teeth, furca welldeveloped, with 5 dental setae. Two new species of eyeless cavernicolous Schaefferia 5 Type locality. Iran, Mazandaran province, Salman Shahr County, Abbasabad city, Danial village, Danial Cave, Coordinates: 36°39'35.2"N, 51°10'53.2"E, cave entrance 204 m a.s.l. Type material. Holotype: • Female on slide [DaSc101IR], dark zone, collected from surface of water puddle, 15.x.2023, temperature = 11.9 °C, RH 73.0%, leg. M. Mehrafrooz. Paratypes: • 2 specimens on slides [DaSc102IR and DaSc103IR], dark zone, collected from surface of water puddle, 15.x.2023, temperature = 12.0 °C, RH 73.4%, leg. M. Mehrafrooz. Holotype deposited in CoPJSU; two paratypes deposited in ZMFUM. Description. Body appearance. Length 1.9 mm on average (n = 3, max. length 2 mm). Habitus typical of Ceratophysella Börner in Brohmer, 1932 without eyes. Colour alive white without traces of pigment. Cuticular granulation rather uniform, slightly coarser on Abd V–VI, macroand mesosetae differentiated. Antenna. It is nearly as long as head, with apical bulb (Fig. 3A–D). Length of Ant I, II, III, IV in holotype as 0.07, 0.06, 0.07, 0.1 mm, respectively. Ant I with 7 setae; Ant II with 6 dorsal and 6 ventral setae in a single row and 1 short smooth seta near its base (dorsal setae clearly coarser and thicker); Ant III and IV clearly Figure 2. A ground plan of the Danial Cave (adopted from Ghelich Khani et al. 2024) B cave decorations in Rizan chamber (Photo: M. Mehrafrooz) C main entrance of the cave (Photo: M. Mehrafrooz). Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 6 separated, Ant III sensory organ with 2 central short and curved rods flanked by 2 long guard sensilla (Fig. 3B), a small ventrolateral microsensillum in a ventral pit, and 7 dorsal and 8 ventral ordinary setae; Ant IV with a simple apical bulb (Fig. 3C), 7–8 cylindrical blunt sensilla (a, b, c, d, e, f, g and h) (after Yosii 1962), subapical ms near base of sensillum a, apical organite os placed near sensillum b and 20 setae around sensilla. Head. Eyes absent. PAO near base of Ant I well developed, large, about 33 µm in diameter, with 7 subequal lobes formed around central circle (Fig. 3F). Labrum typical of the genus with 2+2 prelabral and 5, 5, 4 labral setae. Labium with all normal papillae (A–E) and 6 proximal setae. Left mandible with 4 teeth and right mandible with 5 teeth. Abdomen. Ventral tube with 4+4 apical setae. Retinaculum with 3–4 teeth (asymmetry observed). Furca well-developed (Fig. 5B), length of manubrium, dens, Figure 3. Schaefferia greensladeae sp. nov. A antennal segments I–IV B Ant III sensory organ C tip of antenna D Ant IV blunt sensillum E dorsal side of head, setal pattern F postantennal organ with the nearest setae and granulation of cuticle G shape of macroseta. Scale bars: 0.025 mm (A); 0.01 mm (B, C); 0.025 mm (E); 0.02 mm (F–G). Two new species of eyeless cavernicolous Schaefferia 7 Figure 4. Schaefferia greensladeae sp. nov. A–C setal pattern of legs A leg I B leg II C leg III D–F foot complex of legs D leg I E leg II F leg III. Scale bars: 0.025 mm (A–C); 0.01 mm (D–F). mucro in holotype as 0.11, 0.06, 0.01 mm, respectively; manubrium with 12 setae; dens with 5 setae (basal seta longer); mucro about 1/4 of dens length, with lateral lamella, mucro and dens taken together are as long as inner edge of claw III. Anal spines long (86 µm), on papillae (Fig. 6C). Ratio anal spines (including papilla): claw III = 1.6; ratio anal spines: dens = 1.9. Legs. Legs I–III with normal setae, tenent hair acuminate. Setal pattern as in Fig. 4A–C. Leg I–III with 3, 8, 8 setae on coxae, 7, 7, 7 setae on trochanters, 14, 13, 12 setae on femora and 19, 19, 18 setae on tibiotarsi, respectively. Claw slender, 78 µm long in holotype, internal edge with strong medial tooth and lateral edge with pair of Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 8 Figure 5. Schaefferia greensladeae sp. nov. A dorsal side of thorax, setal pattern B furca, setal pattern. Scale bars: 0.05 mm (A); 0.01 mm (B). Figure 6. Schaefferia greensladeae sp. nov. A Dorsal side of Abd I–III, setal pattern B dorsal side of Abd IV–VI, setal pattern C detail of anal spine, lateral view. Scale bars: 0.05 mm (A, B); 0.01 mm (C). Two new species of eyeless cavernicolous Schaefferia 9 basal teeth; pretarsus with 1 lobe and 2 setae (1 external and 1 internal) reaching base of lateral teeth (Fig. 4D–F). Ratio of claw I length: width = 3.2. Setal pattern of tergites. Dorsal setal pattern generally of type II with welldifferentiated setae. Setae on head and body slightly dentated (Fig. 3G). Head. Setal pattern as in Fig. 3E. Seta a0 present; d1–d5 (d2 and d4 as Mc); sd1–sd5; v1–v2 (v2 as Mc); c1–c5; p1–p3, p5 (p3 and p5 as Mc, p4 absent); oc1–oc3 (oc1 as Mc). Thorax. Setal pattern as in Fig. 5A. Th I with 3+3 setae as m1, m3 and m4. Th II–III with a1–a6 (a3 as Mc); m1, m4’–m6 (m6 as s), lateral microsensillum present on Th II in front of m6 (s); p1–p4 (p4 as s), p5–p6 (p2, p5 and p6 as Mc). Abdomen. Setal pattern as in Fig. 6A, B. Abd I–III with 6+6 setae between p5 (s) in rows ‘a’ and ‘m’: a1, a2, a4, m2, m3 and m4 (p2, p4, p6 and p7 as Mc). Abd IV with 7+7 setae between p5 (s) in rows ‘a’ and ‘m’: a1, a2, a4, a5, m1, m3 and m5 (a4, a5, p2, p4 and p6 as Mc). Abd V with 2+2 setae between p3 (s) on row ‘a’: a1 and a3 (a5, p1 and p5 as Mc). Etymology. The species is named in the honour of prof. Dr. Penelope Greenslade, an outstanding Collembola taxonomist and ecologist. Taxonomic remarks. Based on absence of eyes, S. greensladeae sp. nov. is similar to five representatives of the genus: S. coeca Cassagnau, 1959, S. guerrerense (Bonet, 1945), S. oaxacana Palacios-Vargas & Thibaud, 1985, S. profundissima Jordana & Baquero, 2012 and S. subcoeca Deharveng & Thibaud, 1980. The new species is characterized by 7 lobes in PAO, while there are 4 (6) lobes in PAO in S. coeca and S. subcoeca, 4–5 lobes in S. oaxacana and 4 lobes in S. guerrerense and S. profundissima. Moreover, the new species is separated from S. coeca in number of sensilla on Ant IV: 8–12 in S. coeca and 7–8 in S. greensladeae. The both species differ also in number of dental setae: 3–5 setae in S. coeca and 5 setae in S. greensladeae. Mucro is absent in S. guerrerense and S. subcoeca, but it is present in the new species. Schaefferia greensladeae sp. nov. has 5 dental setae, while S. oaxacana has 4 dental setae. Also, S. greensladeae sp. nov. in terms of the setal pattern of the dorsal body is close to S. oaxacana and S. profundissima (see Table 1). Schaefferia greensladeae sp. nov. differs from S. profundissima by the presence of seta a1 on Abd IV, and from S. oaxacana in having seta m4 on Abd IV, whereas S. oaxacana has m3. The new species also represents the first record of this genus in the country (Mehrafrooz Mayvan et al. 2023). Schaefferia montenegrina Kováč & Mehrafrooz Mayvan, sp. nov. https://zoobank.org/13F15544-55EE-4D2A-AF61-80B6939D2D10 Figs 9–12, Table 1 Diagnosis. Eyes absent, PAO small, forming a rosette of 5–6 regular oval vesicles. Ant IV with 7–8 sensilla and simple apical vesicle. Th I with 2+2 setae, Abd V without seta p2. Claw without internal or lateral teeth. Retinaculum with 3–4 teeth, furca welldeveloped, with 5 dental setae. Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 16 Remarks to phylogeny of the genus Thibaud (1972) outlined Schaefferia as a valid genus, representatives of which show morphological regressions in pigmentation, number of eyes and furcal complex. He regarded these features as manifestations of adaptation to the eudaphic or cave lifestyle, having evolutionary significance, and the genus as a phyletic line of the species group Ceratophysella denticulata, formed as a result of postglacial colonization of the underground environment (deep soil, caves). The genus Schaefferia, similarly as genera Typhlogastrura Bonet, 1930 and Bonetogastrura Thibaud, 1974, is apparently derived from Ceratophysella groups of species, which have successfully invaded caves and became more or less troglomorphic (Christiansen and Wang 2006). In the contrary to Schaefferia, the both genera are characterized by the maintenance of a fully developed furca. After Christiansen (1985), reduction of furca in Schaefferia may represent an edaphic phenomenon. It is important to note that all three genera have common Holarctic distribution. However, any assumptions to phyletic relationships within the genus Schaefferia, or presumably related genera, remain to be clarified by a thorough phylogenetic study combining morphological traits with mitochondrial and nuclear DNA data. It is indeed not an easy task when considering demanding field collections of these collembolans with usually rare and dispersed distribution in subterranean spaces. Remarks on morphological adaptations to subterranean environment In the previous section, we briefly discussed troglomorphic and edaphomorphic adaptations in Schaefferia representatives. Thus, blidness itself doesn’t necessarily imply association with cave life, well documented in this genus by three edaphic species, all from France: S. ariegica Cassagnau, 1959, S. coeca and S. subcoeca. Regarding troglobiotic and guanobiotic forms, altogether six species have been reported in this genus exclusively from caves (Lukić 2019): S. guerrerense and S. oaxacana from Mexico, S. profundissima from the Caucasus, S. kitakamiana Yosii, 1991 from Japan, S. baschkirica Kniss, 1985 from the Urals, Russia, and S. hubbardi Thibaud, 1995 from USA. Schaefferia montenegrina sp. nov. has clearly elongated claw and missing internal teeth, the combination of both characters considered troglomorphic adaptations (Christiansen 2012). It has been noticed that some guanobiotic species of the family Hypogastruridae exhibit this character (Deharveng and Bedos 2018) as an adaptation to easier movement on fresh bat guano (Lukić 2019), such as in Acherontides eleonorae Palacios-Vargas and GnaspiniNetto, 1992 from Brazil. Nevertheless, there are three blind species of Schaefferia with elongated unguis: two species described in this paper and S. profundissima from KruberaVoronya, a deep cave system in the Caucasus Mountains. Elongation of unguis (claw) in these species is an obvious troglomorphic character that evolved independently in three geographically distant karst complexes (Dinaric Karst, Alborz Mountains and Caucasus Mountains) as an example of convergent evolution, a well-known phenomenon in several invertebrate groups inhabiting deep subterranean realm. Two new species of eyeless cavernicolous Schaefferia 17 Key to world species of genus Schaefferia The following key was prepared based on the diagnostic characters of worldwide Schaefferia species (adapted from Jordana et al. 2020). 1 Mucro absent ..............................................................................................2 – Mucro reduced or well-developed ...............................................................9 2 With 7+7 eyes; dens with 6 setae; Abd IV without seta a1 ............................ .......................................................S. oculea Babenko, 1999 (Russia, soil) – With less than 7+7 eyes; dens with less than 6 setae; Abd IV with seta a1 ...3 3 With 5+5 eyes ....S. canigouensis Deharveng & Thibaud, 1980 (France, soil) – With less than 5+5 eyes...............................................................................4 4 With 3+3 eyes .............................................................................................5 – With less than 3+3 eyes...............................................................................6 5 Dens with 4 setae; Abd II–III with 6+6 setae in aand m-row together (m3 present) ................... S. emucronata Absolon, 1900 (Czech Republic, cave) – Dens with 3 setae; Abd II–III with 5+5 setae in aand m-row together (m3 absent) ........................................S. lindbergi Gama, 1963 (Portugal, cave) 6 With 2+2 eyes .............................................................................................7 – With 0+0 eyes .............................................................................................8 7 Dens with 4 setae; Ant IV with 8–9 sensilla; Abd II–III without setae in mrow; Abd IV without seta a4 ......................................................................... ........................................S. quadrioculata (Stach, 1939) (Germany, cave) – Dens with 3 setae; Ant IV with 7 sensilla; Abd II–III with 2 setae in m-row (m2 and m4); Abd IV with seta a4................................................................ .................................................S. ariegica Cassagnau, 1959 (France, soil) 8 Dens with 4–5 setae; Abd IV with seta a4; Abd IV with setae a1, a2, a4, a5 . .............................................. S. guerrerense (Bonet, 1945) (Mexico, cave) – Dens with 3 setae; Abd IV without seta a4; Abd IV with setae a1, a3, a5 ...... ............................ S. subcoeca Deharveng & Thibaud, 1980 (France, soil) 9 Mucro reduced .........................................................................................10 – Mucro well-developed ...............................................................................15 10 With 5+5 eyes ...........................................................................................11 – With less than 5+5 eyes.............................................................................12 11 Dens with 5 setae; Abd II–III with 2+2 setae in m-row (m2, m4) ................. ............................. S. maxima Deharveng & Thibaud, 1980 (France, soil) – Dens with 3–4 setae; Abd II–III with 3+3 setae in m-row (m2, m3, m4) ...... .........................................S. decemoculata (Stach, 1939) (Germany, cave) 12 With 4+4 eyes .............................S. willemi (Bonet, 1930) (Belgium, cave) – With less than 4+4 eyes.............................................................................13 13 With 3+3 eyes ...........................................................................................14 – With 0–1 eye .................................S. coeca Cassagnau, 1959 (France, soil) Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 18 14 Dens with 3 setae; PAO with 4 vesicles; Abd II–III without setae in m-row; Abd V without seta a2 .................................................................................. ..................... S. pouadensis Delamare Deboutteville, 1945 (France, cave) – Dens with 6 setae (sometime 5); PAO with 6–8 vesicles; Abd II–III with 2+2 setae in m-row (m2, m4); Abd V with seta a2 ............................................... .......................................S. scossirolii Dallai & Sabatini, 1981 (Italy, soil) 15 With 6+6 eyes ...........................................................................................16 – With less than 6+6 eyes.............................................................................24 16 Ant IV with 7 sensilla ...............................................................................17 – Ant IV with 6 sensilla ...............................................................................23 17 Dens with 3–4 setae ............................... S. cheoha Wray, 1963 (USA, cave) – Dens with more than 4 setae .....................................................................18 18 Abd V with seta a2 ........ S. fjellbergi Jordana & Baquero, 2020 (USA, soil) – Abd V without seta a2 ..............................................................................19 19 Abd IV with 5+5 setae in a-row (a1–a5) ....................................................... .................................. S. vandalica (Jordana & Arbea, 1990) (Spain, soil) – Abd IV with less than 5+5 setae in a-row ..................................................20 20 Abd IV with 3+3 setae in m-row ...............................................................21 – Abd IV with 2+2 setae in m-row ...............................................................22 21 Abd II–III with setae m2, m4, m5; dens with 5 setae; ratio Asp: dens as 4.5; ratio dens: mucro as 4 ........... S. babenkoi Jordana & Baquero, 2020 (Russia, soil) – Abd II–III with setae m2, m3, m4; dens with 5–6 setae; ratio Asp: dens as 2.3; ratio dens: mucro as 2.7 ..................................................................... ..................................... S. duodecimocellata Bonet, 1945 (Mexico, soil) 22 Dens with 5 setae; Abd IV with setae a1, a3, a5 in a-row; Abd IV with setae m1, m4 in m-row; ratio Asp: dens as 1; ratio dens: mucro as 2.3 .................. ................................................S. elegans (Cassagnau, 1959) (France, soil) – Dens with 6 setae; Abd IV with setae a1, a2, a4, a5 in a-row; Abd IV with setae m1, m5 in m-row; ratio Asp: dens as 2.2; ratio dens: mucro as 2.5 ....... ..................................... S. sendrai Jordana & Baquero, 2020 (Spain, soil) 23 Dens with 4 setae; tenaculum with 3+3 teeth; Abd IV with 2+2 setae in mrow (m1, m3) ...........S. deharvergi (Jordana & Arbea, 1990) (Spain, soil) – Dens with 5 setae; tenaculum with 4+4 teeth; Abd IV with 1+1 seta in m-row (m1) ..................................S. jarae (Jordana & Arbea, 1990) (Spain, soil) 24 With 5+5 eyes ...........................................................................................25 – With less than 5+5 eyes.............................................................................29 25 Ant IV with 5 sensilla; dens with 3 setae ....................................................... ............................................S. bidentata (Cassagnau, 1954) (France, soil) – Ant IV with more than 5 sensilla; dens with more than 3 setae .................26 Two new species of eyeless cavernicolous Schaefferia 19 26 PAO with 5 vesicles; Abd V without seta p1 ................................................. S. raulmugnizi Palacios-Vargas & Castaño-Meneses, 2008 (Mexico, soil) – PAO with 4 vesicles; Abd V with setae p1 .................................................27 27 Dens with 4–5 setae; Abd IV without seta a1 ................................................ ...................................................S. bermani Babenko, 2019 (Russia, soil) – Dens with 5–6 setae; Abd IV with seta a1 .................................................28 28 Labial papilla C absent; Abd V without seta a2’ ............................................ ............................................. S. czernovi (Martynova, 1978) (Russia, soil) – Labial papilla C present; Abd V with seta a2’ ................................................ ........................................... S. quinqueoculata (Yosii, 1956) (Japan, cave) 29 With 4+4 eyes ...........................................................................................30 – With less than 4+4 eyes.............................................................................31 30 PAO with 4 vesicles; Ant IV with 6–7 sensilla; Abd IV with 4+4 setae in mrow (m1, m3, m4, m5) .......S. fukugakuchiana (Yosii, 1956) (Japan, cave) – PAO with 4–6 (7–8) vesicles; Ant IV with 7–8 sensilla; Abd IV with 1+1 seta in m-row (m4) ............................S. baschkirica Kniss, 1985 (Russia, cave) 31 With 3+3 eyes ...........................................................................................32 – With less than 3+3 eyes.............................................................................33 32 Ant IV with 6–7 sensilla; dens with 5 setae; Abd II–III with 3+3 setae in mrow (m2, m3, m5) ........................S. hubbardi Thibaud, 1995 (USA, cave) – Ant IV with 9 (7–8) sensilla; dens with 4 setae; Abd II–III without setae in m-row .................................S. sexoculata (Gisin, 1947) (Switzerland, soil) 33 With 2+2 eyes; Ant IV with 6 sensilla ........................................................... ...................................................S. kitakamiana Yosii, 1991 (Japan, cave) – With less than 2+2 eyes; Ant IV with more than 6 sensilla ........................34 34 Ant IV with trilobed apical vesicle; dens with 4 setae; Ti I–III with 18, 18, 17 setae, respectively .......................................................................................... ................. S. oaxacana Palacios-Vargas & Thibaud, 1985 (Mexico, cave) – Ant IV with simple apical vesicle; dens with 5 setae; Ti I–III with 19, 19, 18 setae, respectively ......................................................................................35 35 PAO with 4 vesicles; dorsal head without seta a0; Abd IV without seta a1 in a-row ............S. profundissima Jordana & Baquero, 2012 (Georgia, cave) – PAO with more than 4 vesicles; dorsal head with seta a0; Abd IV with seta a1 in a-row ....................................................................................................36 36 PAO with 7 vesicles; Th I with 3+3 setae (m1, m3, m4); Abd V with seta p2; Cl with a strong inner tooth and pair of lateral teeth..................................... ............................................................S. greensladeae sp. nov. (Iran, cave) – PAO with 5–6 vesicles; Th I with 2+2 setae (m1, m4); Abd V without seta p2; Cl without inner or lateral teeth .....S. montenegrina sp. nov. (Montenegro, cave) Ľubomír Kováč et al. / Subterranean Biology 54: 1–22 (2025) 20 Acknowledgements The study was supported from the Slovak Research and Development Agency, project APVV-21-0379, and from the Slovak Scientific Grant Agency, project VEGA 1/0438/22. The third author was supported from the postdoctoral program of the Faculty of Science, Pavol Jozef Šafárik University in Košice, Slovakia. We would like to thank Mohammad Ali Jari (head of the climbing board of Abbas Abad county, Iran), Dr. Saeid Esmaeilpour and Sahand Sarvari Nouri for assistance during the expeditions to Danial Cave. Also, we are grateful to Kavoshgaran Caving Club in Mashhad for to help increase knowledge and protect Iranian caves. References Absolon K (1900) Vorläufige Mittheilung über einige neue Collembolen aus den Höhlen. 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