Two new species of Fridericia (Annelida: Enchytraeidae) from Hungarian caves Dózsa-Farkas, Klára Nagy, Hajnalka Felföldi, Tamás European Journal of Taxonomy 2019 2019-09-17 553 1 18 869W4 Dózsa-Farkas & Nagy & Felföldi, 2019 Dózsa-Farkas & Nagy & Felföldi 2019 [618,872,1600,1627] Clitellata Enchytraeidae Fridericia Animalia Enchytraeida 2 3 Annelida species baradlana sp. nov.   urn:lsid:zoobank.org:act: 692C37DF-0429-44CA-B989-495C256D2D43  Figs 1–3    Diagnosis The new species can be recognized by the following combination of characters: (1) medium sized ( 9– 15 mm in vivo); segments 42–61; (2) chaetae maximum 4–5 per bundle; (3) clitellum weakly developed, girdle shaped; (4) body wall 25–35 μm thick, cuticle thin ( <1 μm); (5) oesophageal appendages long with many branches at the end; (6) all pharyngeal glands united dorsally; (7) five pairs of preclitellar nephridia; (8) coelomo-mucocytes scarce, a/c-type, lenticytes numerous and large; (9) dorsal vessel from XIX–XXI; (10) chylus cells in XII–XIV, occupying 2 segments; (11) bursal slit T-shaped; (12) seminal vesicle in XI, not brown; (13) subneural glands in XIII–XIV; (14) sperm funnels approximately as long as half the body diameter, collar as wide as funnel body, spermatozoa 250–320 μm long, heads 110–150 μm in vivo; (15) spermatheca with two long, arm-like diverticula variously bent or bifurcate in proximal part; ectal ducts of variable length with two ectal glands and ampullae fused proximally with common opening into oesophagus.    Etymology Named after the typelocality, Baradla cave.    Material examined    Holotype HUNGARY• Baradla cave (Aggtelek Karst), entrance of Styx branch, from bits of decaying wood (these are remains of an old wooden bridge); 48°28′55.0″ N, 20°30′0.85″ E;  6 Dec. 2014; L. Dányileg.; (F. 30. slide No. 2624, adult, stained, whole mounted specimen); ELTE.    Paratypes HUNGARY• 2 specimens; same data as for the holotype (P. 128.1–128.2 slide No. 2598, 2623, adult, stained, whole mounted specimens)• all other paratypesfrom same type locality;  2 Oct. 2015; D. Angyal, G. Balázs& L. Dányileg.; P. 128.3–128.5 slide No. 2086, 2643, 2644, three adult, stained, P. 128.6 slide No.2664 only the forepart (with 26 segments) of an adult, stained, whole mounted specimens P.128.7 No. 2638 not stained, whole mounted specimens; P.128.8. slide No. 2639 (body end used for molecular analysis, DNA ID number 911b) not stained, whole mounted specimens; ELTE.  Additional material   HUNGARY• 2 subadultspecimens; same type locality; (one of them used for molecular analysis, DNA ID number 911); only in vivo. One adult specimen was studied in vivo, stained and whole-mounted. 4 subadultspecimens and 2 specimensfrom typelocality, only in vivo.    Description MEASUREMENTS. Medium-sized, whitish worms. Holotype 9.7 mmlong, 370 μm wide at VIII and 380 μm at the clitellum when fixed, 53 segments. Body length of the paratypes 9–15 mm, width 300–390 μm at VIII and 360–400 μm at the clitellum in vivo; fixed specimens: length 6.6–11.3 mm, width 310–420 μm at VIII and 310–420 μm at the clitellum, segments 42–61. CHAETAE. Chaetal formula: 2,3,4–4,3,2: 2,3,4,(5)–4,(5),3,2. Inner chaetae being slightly shorter and thinner than outer: 38–40 × 5 μm and 30–35 × 2,5 μm (in preclitellar bundles). Behind the clitellum from ca. XXVII-XXXV only two chaetae per lateral bundle, posteriorly length about 45–55 × 5 μm. Often detached chaetae in coelom ( Fig. 2E). HEAD PORE. At 0/I longitudinal slit. DORSAL PORES. From VII. Three or four rows of hyaline epidermal gland cells per segment. CLITELLUM. In XII–1/2XIII, weakly developed, girdle-shaped, hyalocytes and granulocytes arranged in rows. Body wall about 25–35 μm and cuticle <1 μm thick ( Fig. 2F), in forepart slightly stronger than at the body end.   Table 1(opposite page). List of  Fridericiaspecimens used for molecular taxonomic analyses with collection data and GenBank accession numbers. Sequences determined in this study appear in bold. Holotypes and paratypes of new species are indicated with H and P in parentheses.     Species  Specimen  Locality  Habitat  Reference  GenBank accession numbers   ID  ITS  CO1  H3    Fridericia baradlana sp. nov. 911 Hungary, Aggtelek, Baradla cave bits of decaying wood this paper   MK560154   MK580963   MK562741    Fridericia baradlana sp. nov. 911b (P) Hungary, Aggtelek, Baradla cave bits of decaying wood this paper  –  –   MK562742    Fridericia spelaeophila sp. nov. 1283 (H) Hungary, Bükk Mts. Kis-kőháti shaft rotten wood debris this paper   MK560155   MK580964   MK562739    Fridericia spelaeophila sp. nov. 1284 (P) Hungary, Bükk Mts. Kis-kőháti shaft rotten wood debris this paper   MK560156   MK580965   MK562740    Fridericia dura 907 Hungary, Őrség National Park on the edge of alder swamp  Dózsa-Farkas & Felföldi 2018, Nagy et al. 2018  MF547696  –  KX985894    Fridericia floriformis 1088 South Korea, Jeju Island clayey soil, meadow  Dózsa-Farkas et al. 2019  MH128733  MH124586  MH124597    Fridericia floriformis 1136 South Korea, Jeju Island clayey soil, meadow  Dózsa-Farkas et al. 2019  MH128729  MH124589  MH124598    Fridericia galba 1103 Hungary, Kőszeg Mts meadow  Nagy et al. 2018  MF547697  MF547667  MF547688    Fridericia galba 1123 Hungary, between Szolnok and Szajol riverine poplar-ash wood-lands  Nagy et al. 2018  MF547698  MF547668  MF547693    Fridericia glandifera 883 Hungary, near Pilisszentlélek beech wood  Dózsa-Farkas & Felföldi 2018, Nagy et al. 2018  –  MF547671  KX985889    Fridericia hegemon 776 Hungary, Mezőföld meadow  Nagy et al. 2018  –  –  MF547681    Fridericia heliota CE324 Russia, Krasnoyarsk lab culture  Erseus et al. 2010  –  GU902064  –    Fridericia miraflores CE801 Sweden, Alingsås at a lake  Erseus et al. 2010  –  GU902074  –    Fridericia ratzeli 844 Hungary, Őrség National Park hay meadow  Dózsa-Farkas & Felföldi 2018  KX985875  KX985884  KX985895    Fridericia ratzeli CE782 Sweden, Vårgårda at a lake  Erseus et al. 2010  –  GU902070  –    Fridericia raxiensis 1096 Hungary, Kőszeg Mts alder carr at creekside  Nagy et al. 2018  –  MF547673  MF547685    Fridericia raxiensis 1097 Hungary, Kőszeg Mts alder carr at creekside  Nagy et al. 2018  MF547695  MF547674  MF547686    Fridericia regularis 782 Hungary, Mezőföld meadow  Nagy et al. 2018  MF547703  –  MF547682    Fridericia sohlenii 928 Hungary, Őrség   Sphagnummire this paper   MK560157  –   MK562743    Fridericia sohlenii 1045 Hungary, Velem creekside under  Salixtree this paper   MK560158   MK580966   MK562744    Fridericia ventrochaetosa 1102 Hungary, Kőszeg Mts montane hay meadow  Nagy et al. 2018  MF547699  MF547675  MF547687    Fridericia ventrochaetosa 1114 Hungary, Kőszeg Mts montane hay meadow  Nagy et al. 2018  MF547700  MF547676  MF547690    Fridericia waldenstroemi CE897 Sweden, Strömstad at a stream  Erseus et al. 2010  –  GU902076  –    Hemifridericia parva(out-group) 511a Hungary, Kiskunság open sand steppe oak woodlands  Dózsa-Farkas & Felföldi 2015  KM591939  KM591923  KM591931 BRAIN. Egg-shaped, about 160–175 μm long, about 2 times as long as wide in vivo; 130–155 μm long and 1.5–2 times as long as wide when fixed ( Fig. 2A).   Fig. 1. A–D.  Fridericia baradlana sp. nov. A–B. Spermathecae. C. Oesophageal appendage. D. Coelomocytes. E–F.  Fridericia spelaeophila sp. nov. E. Coelomocytes. F. Spermathecae. Scale bars: 50 μm.   Fig. 2.Micrograph of  Fridericia baradlana sp. nov. A. Brain. B–D. Coelomocytes (C. several large lenticytes). E. Detached chaetae in coelom. F. Thick body wall, thin cuticle. G. Pharyngeal glands (marked with arrows). H. Subneural glands in XIII–XIV (marked with arrows). I–K. Sperm funnels (funnel’s body marked with black arrows, sperm head marked with white arrows). (B–C, F, I–K in vivo, A, F, H fixed, stained, D, G fixed, not stained. Scale bars: 50 μm, in D 20 μm. B, E from holotype.)   Fig. 3.Micrograph of  Fridericia baradlana sp. nov. A.Sperm funnels (marked with arrows). B–C.Male copulatory organs (marked with arrows). D.Bursal slit (marked with arrow). E–H, K–L.Spermathecae. I–J.Spermathecal ectal glands (marked with arrows). (A, C, E–F in vivo, B, J, K–L fixed, stained, D, G, H–I fixed, not stained). Scale bars: 50 μm, in H, J–L 20 μm, J–L from holotype. OESOPHAGEAL APPENDAGES. Long with many branches at the end in V ( Fig. 1C). All pharyngeal glands united dorsally, those in 5/6 and 6/7 with ventral lobes, the third pairs largest ( Fig. 2G). CHLORAGOCYTES. From V, 20–23 μm long preclitellarly when fixed. Dorsal vessel from XIX–XXI, blood colourless. MIDGUT PARS TUMIDA. Not visible. NEPHRIDIA. Five pairs of preclitellar nephridia from 6/7 to 10/11, length ratio anteseptale: postseptale 1: 1.3–1.6, medial origin of the efferent duct. COELOMO- MUCOCYTES. Scarce, a/c-type of Möller (1971), elliptic ( Figs 1D, 2B, D), length 30–40 μm in vivo. LENTICYTES. In large numbers, large 10–15 μm long ( Figs 1D, 2C). Chylus cells in XII–XIV, occupying 2 segments. SEMINAL VESICLE. In XI, not brown. Sperm funnels cylindrical ( Figs 2I–K, 3A), about 150–230 μm long and about 1.5–2.3 times as long as wide in vivo. FUNNEL. Length in fixed specimens 95–150 μm, funnel body 1.2–2 times as long as wide but sometimes wider than long; collar as wide as funnel body. SPERMATOZOA. Length: 250–320 μm, heads 110–150 μm in vivo( Fig. 2J–K); when fixed 140–170 μm and heads 80–95 μm. Diameter of sperm ducts 7–9 μm in vivo(9–10 μm, when fixed). MALE COPULATORY ORGANS. 115–140 μm long, 70–80 μm wide and 50–59 μm high in vivo( Fig. 3C); 70– 130 μm long, 50–70 μm wide and 40–60 μm high when fixed ( Fig. 3B). Bursal slits T-shaped ( Fig. 3D). SUBNEURAL GLANDS. In XIII–XIV ( Fig. 2H). SPERMATHECAE ( Figs 1A–B, 3E–H). Two ectal glands of variable length (25–50 μm long) ( Figs 3I–J); ectal ducts contractile, thus the length variable, about 150–250 μm long and 16–17 μm wide, canal 2 μm wide in vivo(85–185 μm long, 16–17 μm wide when fixed), not widened entally, projecting into ampulla, ental bulbs about 30–40 μm wide when fixed. AMPULLAE. With two long, arm-like diverticula, in their proximal parts dividing into an upper and a lower branch and variously bent (not easily visible, since the branches are overlapping with each other). Proximal part of ampullae fused, with common opening into oesophagus dorsally.    Distribution and habitat Only known from the typelocality: Baradla cave, Aggtelek Karst, Hungary.    Differential diagnosis There are only four valid species of  Fridericiawith two elongate spermathecal diverticula and with the proximal parts of the two spermathecal ampullae fused, forming one common opening into the oesophagus:  F. waldenstroemiRota & Healy, 1999,  F. montafonensisSchmelz, 1998,  F. profundicolaDózsa-Farkas, 1991, and  F. longeauritaBoros & Dózsa-Farkas, 2015. The new species differs from all these species (leaving other characters out of consideration) by the form of diverticula, which divide into an upper and a lower finger-like branch from their base at the ampulla. 5023595302 2014-12-06 ELTE L. Danyi Hungary 48.481945 Baradla cave (Aggtelek Karst), entrance of Styx branch 1 20.500237 Styx 3 4 1 holotype 5023595304 2014-12-06 ELTE L. Danyi Hungary 48.481945 HUNGARY 1 20.500237 Styx 3 4 2 paratype 2425015702 2015-10-02 ELTE D. Angyal & G. Balazs & L. Danyi Hungary 3 4 3 3 paratype 2425015705 Hungary HUNGARY 3 4 2 2