Corresponding author: Dorottya Angyal (
Academic editor: C. O.Coleman
A detailed redescription of two endemic, cave-dwelling niphargid species of the Hungarian Mecsek Mts.,
Angyal D, Balázs G, Zakšek V, Krízsik V, Fišer C (2015) Redescription of two subterranean amphipods
Fragmented mountain areas in East-Central Europe had been suggested to be centres of endemisms that evolved through a complex geological history including Eocene marine regression-transgression cycles and Pleistocene glacial cycles (
Location of the Mecsek Mts. and the nearby isolated mountain ranges within Europe.
The holotypes of both species are either in an unknown place or had been destroyed. Although we identified the distinguishing characters of
Samples for the redescription were collected in the Abaligeti Cave (
Distribution of the two species within the Abaligeti Cave.
Cleared and stained exoskeletons of 10 (
DNA extraction was performed using QIAamp DNA Microcit® (Qiagen) or Sigma Aldrich GenElute Mammalian Genomic DNA Miniprep Kit® following the manufacturer’s instructions. Only a few pereopods were used for DNA isolation of each animal. The following primer pairs were used for PCR amplifications of COI, 28S rDNA fragment and histone (H3). For COI: LCO 1490 – HCO 2198 (
In order to recover phylogenetic relationships of
7 females and 3 males from the stream of the Western 2. collateral of the Abaligeti Cave (Cadastre number: 4120-1, Hungarian Cave Cadastre), collected in 23 March 2013 (leg. D. Angyal and A. Illés), dissected and mounted on slides; additional 4 specimens not dissected. Slides were deposited in the Collection of Crustaceans of the Hungarian Natural History Museum with the following codes: N.MOL-02, N.MOL-03, N.MOL-04, N.MOL-06, N.MOL-07, N.MOL-08, N.MOL-09, N.MOL-10, N.MOL-11, N.MOL-12. Diagnostic voucher number of specimen used for molecular studies: NB555 (
COI Gen Bank Accession Number:
Small to medium-sized niphargid; epimeral plate III postero-ventral corner sharply inclined. Telson with 3–4 apical spines, 1–3 lateral spines, 0–2 lateral plumose setae, 0–2 spines in cleft, dorsal surface with 1–3 spines in mediobasal position. Maxilla I outer lobe with 7 spines, 1.-3. pluri-toothed, 4.-7. variable (uni-, bi-, pluri-toothed). Gnathopod I and gnathopod II dactyli with single seta on outer margin. Gills II-VI ovoid, approximately same size as pereopod VI coxa, posterior margin slightly concave. Pleopods I-III with 2 retinacles on each. Uropod I lenght of endopodite: length of exopodite ratio as 1.00: (1.00–1.20) on males and 1.00: (1.15–1.18) on females. Uropod III sexually dimorphic, exopodite rod-shaped, distal article of exopodite on males 83–115% of proximal article length and 18–73% on females.
Body and telson. Small to medium-sized species, females are 6.4 mm to 9.0 mm, males are 7.8 mm to 10.6 mm. Head length up to 13% of body length; rostrum absent. Pereonites I–VI without setae; pereonite V, VI, VII with 1 postero-ventral seta each. Pleonites I–III with 3–6 setae along dorso-posterior margin (Fig.
Telson length: width as 1.0: 0.6–0.8; cleft 71–87% of length; lobes apically rounded. Telson spines (per lobe): 3–4 apical spines; lateral margins with 1–3 spine, 0–2 plumose setae; 0–2 in cleft spines, dorsal surface with 1–3 basal spines in mediobasal position (Figs
Antennae and mouthparts. Antenna I 35–48% of body length. Flagellum with up to 19 articles; each article with 1 long aesthetasc. Peduncle article 1: 2: 3 proportions 1.0: 0,78 (0.72–0.88): 0,4 (0.36–0.46). Proximal article of peduncle dorso-distally slightly produced. Accessory flagellum biarticulated; distal article shorter than one-half of the proximal article. Lengths of antennae I: II as 1.0: 0.50. Flagellum of antenna II with 6–8 articles. Lengths of peduncle articles 4: 5 as 1.0: (0.84–0.95); flagellum 54–70% of peduncle length (articles 4 + 5) (Fig.
Inner lobes of labium longer than half of outer lobes (Fig.
Left mandible: incisor with 5 teeth, lacinia mobilis with 4 teeth; between lacinia and molar 6–9 thick, serrated setae, long seta at base of molar absent (Fig.
Right mandible: incisor processus with 4–5 teeth, lacinia mobilis with several small denticles (more then 12), between lacinia and molar 6–7 thick, serrated setae, long seta at base of molar present. Proportions of mandibular palp articles 2: 3 (distal) as 1.0: 1,20 (1.17–1.32). Proximal palp article without setae; second article with 9–11 seta in 5–6 groups; distal article with 1 group of 3–5 ’A setae’; 3 groups of ’B setae’; 16–24 ’D setae’; 3–5 ’E setae’ (Fig.
Maxilla I distal palp article with 2–3 apical and subapical setae. Outer lobe of maxilla I with 7 spines, 1–3 spines are always pluri-toothed with 3–6 lateral tooth while 4–6 spines are uni-, or bitoothed. Inner lobe with 1–2 setae (Fig.
Maxilla II inner lobe slightly smaller than outer lobe; both of them setose apically and subapically, number of setae is approximately 13–23 per lobe (Fig.
Maxilliped palp article 2 with 11–17 rows of setae along inner margin; distal article with dorsal seta and group of small setae at base of nail. Maxilliped outer lobe with 6–12 flattened, thick setae and 3–8 serrated setae; inner lobe with 2–3 flattened, thick setae apically and 5–9 serrated setae (Fig.
Coxal plates. Coxal plate I width: depth as 1.00: 1.03 (0.89–1.16), of flattened rhomboid shape, antero-ventral corner subrounded; anterior and ventral margin of coxa I with 3–6 setae (Fig.
Gnathopods. Basis width is 38 (33–45)% of basis length. Gnathopod I ischium with 4–8 posterodistal setae in 1 row. Carpus length 62 (57–75)% of basis length and 87 (80–100)% of propodus length. Anterior margin of carpus only with distal group of setae; carpus posteriorly with transverse rows of setae proximally and a row of lateral setae, posterior enlargment small. Propodus subquadrate, palm convex. Along posterior margin 6–8 rows of denticulated setae. Anterior margin with 10–17 setae in 2–3 groups, antero-distal group with 6–12 setae. Group of 2–4 facial setae below (proximal of) palmar spine; 2–4 single surface setae present. Palmar corner with palmar spine, single supporting spine on inner surface, and 3 (rarely 4) denticulated, thick spiniform setae on outer side. Nail length 36 (34–37)% of total dactylus length; along anterior margin single seta; along inner margin 4–5 setae (Fig.
Gnathopod II basis width: length as 1.0: 0.26 (0.21–0.29). Ischium with 2–6 postero-distal setae. Carpus length 56 (50–61)% of basis length and 86 (71–94)% of propodus lenght. Anterior margin of carpus only with distal row of setae; carpus posteriorly with transverse rows of setae proximally, a row of lateral setae; postero-proximal bulge small, positioned proximally. Propodus medium-sized (sum of length, diagonal and palm length measures up to 19 (15–21)% of body length) and larger than propodus of gnathopod I (1.0: 0.57 (0.65–0.85)). Propodus rectangular, palm convex. Posterior margin convex with 6–9 rows of denticulated setae. Anterior margin with 10–20 setae in 3–5 groups; antero-distal group with 7–9 setae. 1 group of 2–3 facial setae below (distal of) palmar spine; 1–4 individual surface setae present. Palmar corner with strong palmar spine, single supporting spine on inner surface, and 1 denticulated, thick spiniform seta on outer side. Nail length 31 (22–36)% of total dactylus length. Along anterior margin single seta; along inner margin 4–6 short setae (Figs
Pereopods III-IV. Proportions of pereopods III: IV as 1: 0.95 (0.93–0.97). Dactylus IV 45 (39–51)% of propodus IV; nail length 47 (39–52)% of total dactylus length. Dactyli III–IV with one dorsal plumose seta, one spine-like seta at the base of the nail, and tiny seta near the spine-like seta (sometimes not visible or absent). Additional spiniform setae on posterior margin are absent (Fig.
Pereopods V-VII. Proportions of pereopods V: VI: VII as 1.00: 1.4 (1.37–1.54): 1.5 (1.42–1.61). Pereopod VII length 47 (42–52)% of body length. Basis V-VII narrow with convex posterior margins. Basis V width is 70 (60–78)% of length, basis VI is 67 (59–76)% of length and basis VII is 66 (56–76)% of length. Basis V with small posterodistal lobe, posterior margin with 8-13 setae, anterior margin with 6-8 groups of setae. Dactylus V with one dorsal plumose seta, one spine-like seta at the base of the nail, and tiny seta near the spine-like seta (sometimes not visible or absent). Additional spiniform setae on posterior margin are absent (Fig.
Basis VII posterior margin with 6–13 setae, anterior margin with 6–11 groups of setae. Total number of basis setae is 15–21. Dactylus VII length 26 (24–29)% of propodus VII length; nail length 26 (16–33)% of total dactylus length. Dactylus VII with one spine-like seta at the base of the nail. Additional spiniform setae on posterior margin are absent (Fig.
Pleopods. Pleopods I-III with 2-hooked retinacles. Pleopod II rami of 16–20 articles each (Figs
Uropods. Uropod I basipodite with 6 dorso-lateral and 6 dorsomedial spinifom setae. Length ratio endopodite: exopodite as 1.00: 0.89 (0.83–1.0); rami slightly curved. Endopodite total setae number 2–4 in 2–3 groups, apically 5 spinifom setae. Exopodite with 2–7 spines; apically 5 spinifom setae (Fig.
Uropod II endopodite: exopodite length as 1.00: 0.81 (0.77–0.9) (Fig.
Uropod III up to 38–46% (males) and 12–42% (females) of body length. Basipodite with no lateral seta and 3–6 apical spiniform and thin setae. Endopodite 58–61% (males) and 48–70% (females) of basipodite length, endopodite apically with 1–2 thin-flexible and spiniform setae; laterally 0–1 seta. Exopodite of uropod III rod-shaped, distal article of exopodite 83–115% (males) and 18–73% (females) of proximal exopodite article length. Proximal article with 4–5 groups of plumose, thin-flexible and spiniform setae along inner margin and 4 groups of thin-flexible and spiniform setae along outer margin. Distal article with 3–6 apical setae; lateral setae only in males (Fig.
7 females and 4 males from a permanent pool in the main passage near ’Karthago romjai’ hall of the Abaligeti Cave (Cadastre number: 4120-1, Hungarian Cave Cadastre), collected on 23 March 2013 (leg. D. Angyal & A. Illés), dissected and mounted on slides; additional 4 specimens not dissected. Slides were deposited in the Collection of Crustaceans of the Hungarian Natural History Museum with the following codes: N.GEB-02, N.GEB-03, N.GEB-04, N.GEB-05, N.GEB-08, N.GEB-10, N.GEB-14, N.GEB-15, N.GEB-17, N:GEB-18, N.GEB-20. Diagnostic voucher numbers of specimens used for molecular studies: NB 550 (
COI Gen Bank Accession Numbers:
Small-sized niphargid; epimeral plate III postero-ventral corner subrounded. Telson with 3–6 apical spines, 0–2 lateral spines, 0–1 lateral plumose setae, 0–1 spines in cleft and 0–1 dorsal surface spines. Maxilla I outer lobe with 7 spines, pluri-, uni-, bi-toothed spines alternating. Gnathopod I and gnathopod II dactyli with single seta on outer margin. Gills II-VI ovoid. Pleopods I-III with 3, rarely 4 retinacles on each. Uropod I lenght of endopodite: length of exopodite ratio as 1.00: (1.09–1.11) on males and 1.00: (1.03–1.17) on females. Uropod II sexually dimorphic, exopodite rod-shaped, distal article of exopodite on males 95–155% of proximal article length and 52–72% on females.
Body and telson. Small-sized niphargid species, females 4.9–5.9 mm, males 5.9–7.0 mm. Head length up to 9% of body length; rostrum absent. Pereonites I-VI without setae; pereonite V, VI, VII with 1 postero-ventral seta each. Pleonites I-III with 1–2 setae along dorso-posterior margin. Epimeral plate II posterior and ventral margins convex, ventro-postero-distal corner rounded. Along ventral margin 1–3 spiniform setae; along posterior margin 3–4 thin setae. Epimeral plate III ventral and posterior margins convex, ventro-postero-distal corner rounded; along ventral margin 2–3 spiniform setae; along posterior margin 4 thin setae. Urosomite I postero-dorso-laterally with 1 seta; urosomite II postero-dorso-laterally with 1 spiniform seta; urosomite III without setae. Near insertion of uropod I 1 spiniform seta (Figs
Telson length: width as 1.0: 0.88; cleft 74 (70–79)% of length; lobes apically widely rounded. Telson spines (per lobe): 2–4 apical spines, 33.5 (28–39)% of telson length; lateral margins with 0–2 spine and 0–1 plumose setae; 0–1 in cleft spines, 0 or 1 dorsal surface spines, 1 basal spine (Figs
Antennae and mouthparts. Antenna I 37 (34–41)% of body length. Flagellum with up to 13–16 articles; each article with 1 long aesthetasc (Fig.
Inner lobes of labium longer than half of outer lobes (Fig.
Left mandible: incisor with 5 teeth, lacinia mobilis with 4 teeth; between lacinia and molar 5–7 thick, serrated setae, long seta at base of molar absent (Fig.
Right mandible: incisor processus with 4 teeth, lacinia mobilis with 5–6 denticles, between lacinia and molar 6–8 thick, serrated setae, 1 long seta at base of molar present. Proportions of mandibular palp articles 2: 3 (middle: distal) as 1.0: 1.1 (1.00–1.21). Proximal palp article without setae; second article with 4–6 seta in 3–4 groups; distal article with 1 group of 3–4 ’A setae’; 2–4 of ’B setae’ (single or in groups); 9–13 ’D setae’ and 3–5 ’E setae’ (Fig.
Maxilla I distal palp article with 3–6 apical and subapical setae. Outer lobe of maxilla I with 7 spines, pluri-, uni-, bi-toothed spines alternating. Inner lobe with 1 seta (Fig.
Maxilla II inner lobe slightly smaller than outer lobe; both of them setose apically and subapically, number of setae is approximately 6–11 on inner lobe and 8–12 on outer lobe (Fig.
Maxilliped palp article 2 with 8–11 rows of setae along inner margin; distal article with dorsal seta and group of small setae at base of nail. Maxilliped outer lobe with 6–8 flattened, thick setae and 3–5 serrated setae; inner lobe with 2–3 flattened, thick setae apically and 2–4 serrated setae (Fig.
Coxal plates. Coxal plate I width: depth as 1.00: 0.76 (0.6–0.9) of flattened rhomboid shape, antero-ventral corner subrounded; anterior and ventral margin of coxa I with 4–6 setae (Fig.
Gnathopods. Gnathopod I basis width 42 (38–47)% of basis length. Ischium with 3–4 posterodistal setae in 1 row. Carpus length 61 (52–82)% of basis length and 98 (87–110)% of propodus length. Anterior margin of carpus only with distal group of setae; carpus posteriorly with transverse rows of setae proximally and a row of lateral setae, posterior enlargment small. Propodus subquadrate, palm and posterior margin convex. Along posterior margin 3–4 rows of denticulated setae. Anterior margin with 6–11 setae in 2–3 groups, antero-distal group with 4–8 setae. Group of 2–3 facial setae below (proximal of) palmar spine; 1–4 surface setae in 1–2 groups present. Palmar corner with palmar spine, single supporting spine on inner surface, and 2–3 denticulated, thick spiniform setae on outer side. Nail length 33 (30–39)% of total dactylus length; along anterior margin single seta; along inner margin 3–4 setae (Fig.
Gnathopod II basis width: length as 1.0: 0.34 (0.27–0.45). Ischium with 3–4 postero-distal setae in 1 row. Carpus length 59 (48–69)% of basis length and 106 (96–111)% of propodus length. Anterior margin of carpus only with distal row of setae; carpus posteriorly with transverse rows of setae, proximally a row of lateral setae; postero-proximal bulge small and positioned proximally. Propodus small to medium-sized (sum of length, diagonal and palm length measures up to 12–15% of body length) and larger than propodus of gnathopod I (1.0: 0.87 (0.78–0.96)). Propodus rectangular, palm convex. Posterior margin straight or convex with 4–5 rows of denticulated setae. Anterior margin with 3–9 setae in 1–2 groups; antero-distal group with 4–8 setae. Group of 2–4 facial setae below (proximal of) palmar spine; 2–3 surface setae in 1–2 groups present. Palmar corner with strong palmar spine, single supporting spine on inner surface, and 2–3 denticulated, thick spiniform setae on outer side. Nail length 34 (29–42)% of total dactylus length. Along anterior margin single seta; along inner margin 3 short setae (Fig.
Pereopods III–IV. Proportions of pereopods III: IV as 1: 0.96 (0.89–1). Dactylus IV 51 (46–57)% of propodus IV lenght; nail length 53 (44–61)% of total dactylus length. Dactyli III-IV with dorsal plumose seta (sometimes not visible or absent), one spine-like seta at the base of the nail, and tiny seta near the spine-like seta (sometimes not visible or absent). Additional spiniform setae on posterior margin are absent (Fig.
Pereopods V–VII. Proportions of pereopods V: VI: VII as 1.00: 1.3 (1.27–1.49): 1.5 (1.46–1.58). Pereopod VII length 42–45% of body length. Basis V-VII with convex posterior margins. Basis V width is 71 (66–80)% of length, basis VI is 68 (64–73)% of length, and basis VII is 66 (63–69)% of length. Basis V with small posterodistal lobe, posterior margin with 4–6 setae, anterior margin with 4–9 setae in 3+1 groups (Fig.
Pleopods. Pleopods I-III with 3, rarely 4 hooked retinacles. Pleopod II rami of 11–13 articles each (Figs
Uropods. Uropod I basipododite with 4–5 dorso-lateral and 1–3 dorsomedial spiniform setae including spiniform setae in distal position. Length ratio endopodite: exopodite as 1.00: 0.91 (0.87–0.97); rami slightly curved. Endopodite with 1–2 setae, apically 5 spinifom setae. Exopodite with 1–4 setae or spines in 1–2 groups; apically 5 spinifom setae (Figs
Uropod II endopodite: exopodite length as 1.00: 0.84 (0.77–0.95) (Figs
Uropod III 38 (37–39)% (males) and 26 (24–30)% (females) of body length. Basipodite with 0–1 lateral setae and 5–6 apical spiniform and thin setae. Endopodite 41 (39–44)% (males) and 48 (41–54)% (females) of basipodite length; endopodite apically with 0–2 thin-flexible and spiniform setae; laterally with 0–1 seta. Exopodite of uropod III rod-shaped, distal article of exopodite 100 (95–105)% (males) or 60 (52–78)% (females) of proximal article length. Proximal article with 3–4 groups of plumose, thin-flexible and spiniform setae along inner margin and 2–4 groups of thin-flexible and spiniform setae along outer margin. Distal article without lateral seta (males) or with 3 setae in 1 group (females); apically 4–7 setae (Figs
High morphological similarity to the focal pair of species reveal another four species phylogenetically related to
Phylogenetic relationship of
The following species are compared with
Comparison of the main diagnostic characters of
Species | No. apical telson spines | No. lateral telson spines | Pleopod I. no. hooks in retinacle | Pleopod II. no. hooks in retinacle | Pleopod III. no. hooks in retinacle | Uropod I endopodite/exopodite length | Gnathopod dactylus anterior margin seta no. | Shape of gills II-IV | Epimeral plates postero-ventral corner shape | Source of data |
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3–4 | 1–3 | 2 | 2 | 2 | endopodite slightly longer | single | ovoid | sharply inclined | own slides | |
3–6 | 0–2 | 3 (rarely 4) | 3 (rarely 4) | 3 (rarely 4) | endopodite slightly longer | single | ovoid | subrounded | own slides | |
4–5 | 1–2 | 4–5 | 4–5 | 4–5 | exopodite slightly longer | single | ? | subrounded | ||
3+1 | 2 | 3–4 | 3–4 | 3–4 | nearly equal | single | narrow | subrounded |
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? | 3 | ? | ? | ? | ? | ? | ? | sharply inclined |
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3–5 | 1–2 | 4–7 | 3–5 | 4–5 | equal | single | narrow | subrounded |
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5–7 | 1 pair, plumose | 4–6 | 4–6 | 4–6 | exopodite slightly longer | single | long and recurved | I., II. subrounded, III. angular |
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5–7 | 2–5 | 4–6 | 3–5 | 3–6 | exopodite slightly longer | more than 1 | ? | subrounded |
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2 | 2 | 2 | 2 | 2 | endopodite longer | single | narrow | subrounded |
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3–5 | 1–2 | ? | ? | ? | endopodite 2x longer | more than 1 | ? | subrounded | ||
3–4 | 0–3 | 2 | 2 | 2 | nearly equal | more than 1 | large, irregularly ovoid | III. sharply inclined |
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Phylogenetic relationships within the genus
Bayesian phylogenetic tree of 104 amphipod taxa (including
Among the studied two species,
Distribution of
Small pool formed by dripping water, one of the occupied microhabitats of
Due to its protected geographical situation, since the Tertiary, the area of Mecsek may have played a refugial role during the alternating warmer and colder eras, preserving old lineages of Crustaceans. They presumably ensconced into subterranean aquatic habitats from searing creaks of the Paratethys Sea, that encompassed the islands of the Mecsek. Then, by degress, they had been adapted to the subterranean conditions in both physiological and morphological features (
The distribution range of the two endemic species is small, the most distant caves are seven kilometers far. These caves belong to three different catchment areas (Fig.
Hidrologically connected caves are in quadrats.
Some highly endemic, troglobiont invertebrate taxa are known from the Southern Hungarian Mecsek Mts. Two of them, the blind amphipod
Professional trips of DA and GB to the Biotechnical Faculty of University of Ljubljana were funded by the Balassi Institute, Campus Hungary Short Therm Study Program (grants B2/1R/1411 and B2/1R/10676). VZ and CF were partially funded by Slovenian Research Agency Program P1-0184. We are grateful to Teo Delić (University of Ljubljana) and Mária Tuschek (Molecular Taxonomic Laboratory, Hungarian Natural History Museum) for their help in molecular taxonomic work. Dr. László Forró, Katalin Kovács and Dr. László Dányi (Department of Zoology, HNHM) are also acknowledged for providing literature and infrastructure for our studies. Dr. Krisztina Buczkó (Department of Botany, HNHM) is gratefully acknowledged for her help in scanning electron microscopy. Our thanks are due to speleologist colleagues from the Pro Natura Karst and Cave Research Society, namely Andrea Illés, Zoltán Tegzes, Artúr Nyírő and Béla Nacsa for their help during the sample collections. We are grateful to Dr. Jenő Kontschán (Plant Protection Institute, Hungarian Academy of Sciences) for his advices regarding the early phase of the manuscript. Gabriella Koltai is acknowledged for the linguistic checking of the manuscript. We also thank Dr. John Holsinger, Dr. Ronald Vonk and Dr. Oliver Coleman for their helpful and constructive commentaries that remarkably improved the early version of the manuscript.
Protocols and thermo profiles used in molecular studies
Primers, molecular protocols
List of taxa and sequence data used in phylogenetic analysis
Sequence data
Original descriptions of
Descriptions