NATURA SLOVENIAE 22(2): 69-77 Prejeto / Received: 4. 12. 2020 
SHORT COMMUNICATION Sprejeto / Accepted: 21. 12. 2020 
Biotehniška fakulteta Univerze v Ljubljani in Nacionalni inštitut za biologijo, Ljubljana, 2020 
 
 
 
New data on the distribution of six morphologically 
cryptic species of Niphargus stygius species complex 
(Amphipoda: Niphargidae) 
 
 
 
Janko ŠET
1
, Špela BORKO
2
 
 
 
1
Jovan Hadži Institute of Biology ZRC SAZU, Zagorica 20, SI-1292 Ig, Slovenia; E-mail: janko.set@zrc-sazu.si 
2
SubBio Lab, Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000 Ljubljana, 
Slovenia; E-mail: spela.borko@bf.uni-lj.si 
 
 
 
Abstract. Subterranean amphipods, morphologically resembling Niphargus stygius species complex, were 
molecularly analysed. We isolated genomic DNA and amplified the subunit I of mitochondrial cytochrome oxidase 
gene (COI) for 94 specimens from 37 localities. We report on six new localities for N. chagankae, one for N. cvajcki, 
eight for N. gottscheeanensis, two for N. kenki, one for N. malagorae and two for N. zagrebensis. New data extend 
previously known distribution ranges of N. gottscheeanensis and fill the missing gap between the remote N. kenki 
occurrences. We report on new co-occurrence data for two species pairs, i) N. chagankae and N. likanus, and ii) 
N. gottscheeanensis and N. podpecanus. 
 
Key words: cryptic species, Niphargus stygius, molecular taxonomy, distribution, COI, co-occurrence 
 
 
Izvleček. Novi podatki o razširjenosti šestih morfološko kriptičnih vrst kompleksa Niphargus 
stygius (Amphipoda: Niphargidae) – Raziskovali smo razširjenost kriptičnih vrst v kompleksu N. stygius. 
Izolirali smo DNA in pomnoževali podenoto I mitohondrijskega gena za citokrom oksidazo (COI) 94 osebkov iz 37 
lokalitet. Navajamo šest novih nahajališč vrste N. chagankae, eno za N. cvajcki, osem za N. gottscheeanensis, dve 
za N. kenki, eno za N. malagorae in dve za N. zagrebensis. Izboljšali smo znanje o razširjenosti N. gottscheeanensis 
in opisali novo lokaliteto N. kenki sredi obširnega območja med obema do sedaj znanima skupinama nahajališč. 
Opisali smo prvo sopojavljanje N. chagankae in N. likanus in novo lokacijo, kjer se sopojavljata vrsti  
N. gottscheeanensis in N. podpecanus. 
 
Ključne besede: kriptične vrste, Niphargus stygius, molekularna taksonomija, razširjenost, COI, sopojavljanje 
 
 
 
Introduction 
 
 
The use of molecular methods has shown that morphology alone poorly reflects species 
composition of some taxa (Witt et al. 2006, Adams et al. 2014, Katouzian et al. 2016). This was 
thoroughly utilized to distinguish morphologically cryptic species which resemble each other to 
the point of being impossible to identify without usage of molecular approach (Bickford et al. 

70 Janko ŠET & Špela BORKO: New data on the distribution of six morphologically cryptic ... / SHORT COMMUNICATION 
NATURA SLOVENIAE 22(2): 69-77 
2007). Cryptic species often represent unique evolutionary lineages (Trontelj et al. 2009, Delić 
et al. 2017a) with much narrower distribution than nominal species (Trontelj et al. 2009, Eme 
et al. 2018). Therefore, they play an important role in understanding patterns of local or regional 
biodiversity and often appeal for additional conservational efforts (Delić et al. 2017a). 
 
Cryptic species are common in the subterranean realm (Trontelj et al. 2009, Niemiller et al. 
2012, Parimuchová et al. 2020). Fragmentation of subterranean environments, coupled with low 
dispersal abilities of subterranean fauna, increases the probability of allopatric speciation 
(Trontelj et al. 2009, Garcia-Machado et al. 2011). In addition, common properties of 
subterranean habitats, i.e. darkness and lack of food sources, enable acquisition of convergent 
morphologies through the processes of directional selection (Trontelj et al. 2009, Juan et al. 
2010). Both mentioned processes, allopatric speciation and convergent evolution, lead to a 
cryptic speciation within subterranean habitats.  
 
Genus Niphargus, the largest genus of freshwater Amphipoda in the world (Väinölä et al. 
2008), comprises more than 430 described species (Fišer 2019). All representatives, including 
the three known fossil species, show morphological adaptations to subterranean habitats – 
troglomorphies, i.e. reduction of eye, body pigments, elongation of antennae and appendages 
(Fišer 2019). Many morphologically cryptic complexes of Niphargus species have been 
recognized (e. g. Lefébure et al. 2006, 2007, Fišer & Zagmajster 2009, Trontelj et al. 2009, Delić 
et al. 2017a, Eme et al. 2018), including Niphargus stygius sensu lato, consisting of 16 cryptic 
species (Delić et al. 2017a). 
 
The N. stygius s.l. species are distributed in Slovenia, North-Western Croatia, and Northern 
Italy (Delić et al. 2017a). Individual species distribution is often poorly understood, six of them 
are known only from their type locality (Delić et al. 2017a). Although boundaries of species’ 
ranges are not thoroughly explored, it has been established that ranges of several species from 
this complex overlap and that specimens can even co-occur. To improve our knowledge on 
factors limiting geographical distributions, as well as ecology and evolutionary history of these 
species, sequencing of all available individuals from every locality is of high importance. 
Therefore, we analysed additional samples of N. stygius s.l. and herein report on the obtained 
results. 
 
 
 
Material and methods 
 
 
We analysed the specimens from Niphargus stygius s.l. (as defined in Delić et al. 2017a) 
from the Zoological Collection of the Department of Biology, Biotechnical Faculty, University of 
Ljubljana, Slovenia, collected from 2002 to 2014. The samples were preserved in 96% or 70% 
ethanol. Samples were morphologically classified before the study. We performed DNA isolation 
and amplification of subunit I of mitochondrial cytochrome oxidase gene (COI) of 94 specimens 
from 37 localities. 
 
 

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NATURA SLOVENIAE 22(2): 69-77 
We isolated DNA from one of the pereopods, using GenEluteTM Mammalian Genomic DNA 
Miniprep Kit (Sigma-Aldrich). We amplified COI fragment, using LCO 1490 and HCO 2198 
primers (Folmer et al. 1994) and PCR protocol as described in Švara et al. (2015). PCR products 
were purified using Exonuclease I and Fast AP Thermosensitive Alkaline Phosphatase (Thermo 
Fisher Scientific Inc., USA). Purified PCR products were sequenced by Macrogen Europe 
(Amsterdam, Netherlands) with same amplification primers. We assembled and edited the 
resulting chromatograms in Geneious 6.0.5. The acquired sequences were then aligned and 
compared with the ones available in publicly available database GenBank using »blastn« 
(Boratyn et al. 2013) to assign the analyzed specimens to formally described species. A general 
threshold for determining the species was 98% similarity or, in case that sequence contained 
ambiguities, as close as possible to it. To reconstruct the species’ distributions, we combined 
our species distribution data with those of Delić et al. (2017a). 
 
 
 
Results and discussion 
 
 
We obtained COI barcodes for 62 specimens (out of 94 in total) from 30 of the 37 original 
localities. On three localities we found only specimens that did not belong to N. stygius s.l.  
(N. dobati and N. spoeckeri). We acquired new presence data for six Niphargus stygius s.l. 
species from 20 localities: six new localities for N. chagankae, one for N. cvajcki, eight for  
N. gottscheeanensis, two for N. kenki, one for N. malagorae and two for N. zagrebensis. 
Sequences were submitted to GenBank under accession numbers MW370352–MW370416. 
 
A new record from Cerkniško polje significantly extended the known distribution range of  
N. gottscheeanensis westward for more than 30 km. Additionally, we identified a new population 
of N. kenki. According to Delić et al. (2017a), this species has a disjunct range with known 
localities from Central Slovenia (Ljubljana, Škofja Loka) and Eastern Slovenia (Kozjansko). The 
newly discovered locality, Maroltova jama near Letuš, fills the gap between the two regions. 
 
A new locality with co-occurring N. podpecanus and N. gottscheeanensis and a locality with 
the first-time reported co-occurrence of N. chagankae and N. likanus were recorded.  
N. gottscheeanensis co-occurs according to current records with N.  chagankae (one locality), 
with N. podpecanus (four localities) and with N. novomestanus (one locality). Besides with  
N. gottscheeanensis (one locality mentioned above), N. chagankae co-occurs according to 
current records also with N.  likanus (one locality). Syntopy between cryptic species, based on 
current knowledge, is rare (Delić et al. 2017a). However, the assembled data suggest that co-
occurrences and syntopies between species of N. stygius species complex could be generally 
overlooked. The data from seven remaining localities correlate with Delić et al. (2017a). All 
results are summarized in Tab. 1 and Fig. 1. 
  

72 Janko ŠET & Špela BORKO: New data on the distribution of six morphologically cryptic ... / SHORT COMMUNICATION 
NATURA SLOVENIAE 22(2): 69-77 
Table 1. Analized samples of Niphargus stygius s.l. in SE Slovenia. Locality, coordinates (WGS84), number and species of 
individuals from samples are given, along with their legator and date of sampling. We were unable to classify any 
specimen from seven localities. Species marked with * are not N. stygius s.l. species. On localities marked with 
+
 we are 
reporting on the presence of N. stygius s.l. species for the first time. On some localities from Delić et al. (2017a), we 
are reporting on the presence of species that have previously not been reported from there. They are marked with **. 
Tabela 1. Analizirani vzorci za Niphargus stygius s.l. v JV Sloveniji. Za vsak vzorec podajamo lokaliteto, koordinate 
(WGS84), število in vrste obravnavanih osebkov, datume vzorčenj ter legatorje. Iz vzorcev sedmih lokalitet nam ni 
uspelo določiti nobenega osebka. Vrst, označenih z *, ne uvrščamo v N. stygius s.l. Na lokalitetah, označenih s 
+
, smo 
prvič potrdili pojavljanje vrst N. stygius s.l. Na nekaterih že znanih lokalitetah N. stygius s.l. (Delić et al. 2017a) smo 
dodatno potrdili vrste, ki tam še niso bile zabeležene. Označene so z **. 
Locality (Cave ID) 
Lat. (N) 
Long. (E) Species 
Number of 
classified/ 
all 
specimens Date Legator 
Izvir pri črpališču, 
Slovenska vas; 
Kočevje; SLO 
45.66765 14.83022 
N. gottscheeanensis** 3/8 
22.2.2007 
B. Sket, 
G. Bračko N. podpecanus 3/8 
Jama Bedara (ID NA), 
Tihočaj; Žumberak; 
HRV 
45.74657 15.54649 / 0/2 
4.11.2005 S. Minihofer 
18.12.2005 M. Pavlek 
Andrejčkov štibelc 
(1263), Zagozdec; 
Stari trg; SLO
+
 
45.52798 15.05823 N. chagankae 2/2 26.1.2002 P. Presetnik 
Brezno presenečenj 
(4500), Krašica 
planina; Dobrovlje; 
SLO 
46.26638 14.92125 / 0/2 12.5.2012 R. Kvas 
Izvir Črnega potoka, 
Črni potok; Kočevje; 
SLO
+
 
45.59128 14.89939 N. gottscheeanensis 6/6 7.1.2007 
B. Sket, 
M. Zagmajster 
Izvir Ribjek, Osilnica; 
SLO
+
 
45.53333 14.73162 N. gottscheeanensis 1/1 28.4.2007 
B. Sket, 
J. Jugovic 
Tounjčica (ID NA), 
Tounj; HRV 
45.24882 15.32263 / 0/2 27.1.2008 M. Pavlek 
Potok Zege, Dolga 
vas, pri pokopališču; 
Kočevje; SLO
+
 
45.62472 14.87689 N. gottscheeanensis 1/1 9.2.2002 R. Verovnik 
Jama Džud (3341), 
Lahinja; Belčji vrh; 
Črnomelj; SLO
+
 
45.50240 15.20062 N. zagrebensis 1/1 6.1.2007 
V. Zakšek, 
B. Šarac 
Kekčevo brezno 
(4672), Dolenja vas; 
SLO
+
 
45.68130 14.77650 N. malagorae 2/2 21.12.2013 
T. Delić, 
M. Zagmajster, 
N. Sivec 
Izvir v kamnolomu 
Vražji kamen, Miklarji; 
Črnomelj; SLO
+
 
45.55997 15.08765 N. chagankae 2/2 24.6.2013 
T. Delić, 
J. Tramte, 
D. Šinigoj 
Jama Trbušnjak 
(HR02378), 
Grahovljani; Pakrac; 
HRV 
45.49513 17.25975 / 0/3 10.6.2007 M. Faller 
Jama pod Zimzelom 
(ID NA), Potok 
Musulinski; Ogulin; 
HRV
+
 
45.28196 15.11917 N. chagankae 1/2 
25.6.2008 B. Jalžić 
14.12.2008 K. Gašpić 

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Locality (Cave ID) 
Lat. (N) 
Long. (E) Species 
Number of 
classified/ 
all 
specimens Date Legator 
Jama v kamenolomu 
Tounj (HR00692), 
Tounj; HRV
+
 
45.24800 15.31985 N. chagankae 2/2 7.10.2007 M. Pavlek 
Jama Drobovnik (ID 
NA), Kunčani; 
Radatoviči; Žumberak; 
HRV 
45.72509 15.32496 / 0/2 20.4.2007 M. Pavlek 
Izvir 200m S od 
Zajčjega Polja; Črni 
potok; Kočevje; SLO
+
 
45.59635 14.89227 N. gottscheeanensis 2/2 7.1.2007 
B. Sket, 
M. Zagmajster 
Mala jama nad 
Trebnjem (394), 
Trebnje; SLO
+
 
45.89980 15.00770 N. cvajcki 2/2 28.7.2009 M. Zagmajster 
Jama pod gradom 
Luknja (575), Prečna; 
Novo mesto; SLO 
45.81841 15.09978 N. novomestanus 3/3 
3.9.2006 
V. Zakšek, 
B. Šarac 
29.11.2006 B. Sket 
Viršnica (571), Velika 
Račna; Grosuplje; 
SLO
+
 
45.90392 14.70549 N. gottscheeanensis 1/1 17.7.2010 
M. Zagmajster, 
L. Mrzelj, 
P. Dovč 
Studenec Pahle, Črni 
potok; Kočevje; SLO
+
 
45.58795 14.89512 N. gottscheeanensis 3/3 7.1.2007 
B. Sket, 
M. Zagmajster 
Zelške jame (576), 
Zelše; Unec; SLO 
45.79066 14.30349 N. spoeckeri* 4/4 
8.8.2003 
P. Trontelj, 
M. Zagmajster 
6.7.2011 
T. Delić, 
J. Matičič 
Pod Malim naravnim 
mostom, Rakov 
Škocjan; Rakek; SLO 
45.79097 14.30554 / 0/2 23.3.2003 B. Sket 
Izvir pri Kočevskih 
poljanah, Kočevske 
poljane; Dolenjske 
Toplice; SLO
+
 
45.72466 15.05483 N. zagrebensis 2/2 27.2.2007 
B. Sket, 
G. Bračko 
Dekmanca-Lastnič, 
studenec ob cesti 
levo, Bistrica ob Sotli; 
SLO
+
 
46.07968 15.63265 N. kenki 2/4 
6.8.2002 B. Sket 
8.8.2002 unknown 
Sustav Matešička-
Popovača (HR00957), 
Matešiči; Slunj; HRV 
45.10808 15.61389 / 0/2 11.4.2010 R. Baković 
Svinjska jama (534), 
odtočni sifon, 
Cerknica; SLO 
45.77179 14.32378 
N. dobati* 1/2 
2.12.2006 
M. Zagmajster, 
J. Jugovic 
N. spoeckeri* 1/2 
Jama v Bobnaricah 
(11036), Otok; 
Cerknica; SLO
+
 
45.75211 14.35560 N. gottscheeanensis 1/1 19.8.2011 
J. Matičič, 
M. Matičič 
Izvir pri Otovcu, 
Otovec; Črnomelj; 
SLO
+
 
45.59228 15.16412 N. zagrebensis 1/3 30.6.2011 G. Bračko 

74 Janko ŠET & Špela BORKO: New data on the distribution of six morphologically cryptic ... / SHORT COMMUNICATION 
NATURA SLOVENIAE 22(2): 69-77 
Locality (Cave ID) 
Lat. (N) 
Long. (E) Species 
Number of 
classified/ 
all 
specimens Date Legator 
Maroltova jama 
(4895), Letuš; SLO
+
 
46.30750 15.02148 N. kenki 2/2 8.2.2012 R. Kvas 
Jama Lesina (1811), 
Lipa; Vinica; SLO
+
 
45.47002 15.20277 N. chagankae 2/2 3.9.2014 C. Fišer 
Planinska jama (748), 
Planina; Postojna; 
SLO 
45.81989 14.24566 N. spoeckeri* 3/8 
16.9.2002 NA 
14.9.2004 R. Verovnik 
24.11.2009 
J. Jugovic, 
A. Moškrič 
2.6.2011 V. Zakšek 
Đulin ponor-
Medvedica 
(HR00728), Ogulin; 
HRV 
45.26704 15.22399 N. chagankae** 1/3 
25.2.2012 
R. Baković, 
S. Minihofer 
25.2.2012 
R. Baković, 
T. Mihoci, 
S. Minihofer 
Podpeška jama (17), 
Podpeč; Videm; SLO 
45.83926 14.68632 N. gottscheeanensis 1/1 6.11.2010 M. Zagmajster 
Jama Gadina-Žopenca 
(235), Loka; 
Črnomelj; SLO 
45.56461 15.18202 N. zagrebensis 1/1 6.1.2007 
V. Zakšek 
B. Šarac 
Velika jama nad 
Trebnjem (104), 
Trebnje; SLO 
45.89989 15.00723 N. cvajcki 4/4 12.7.2011 S. Polak 
Lukova jama pri 
Zdihovem (91), 
Suhor; Kočevje; SLO 
45.52580 14.89410 N. brachytelson 2/2 20.11.2003 B. Sket 
Jama v Kamnolomu 
(2950), Vinica; 
Črnomelj; SLO 
45.45443 15.24431 N. chagankae 1/2 5.4.2007 S. Polak 
 
 
Further targeted and wider sampling (filtering epikarst water, e.g., drips from crevices in the 
ceiling; traps, etc.) is needed for obtaining a better insight into the species’ habitat preferences 
and distribution, a solid baseline for establishing conservational efforts. 
 
  

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NATURA SLOVENIAE 22(2): 69-77 
 
Figure 1. Distribution of Niphargus stygius s.l. species, based on the analyzed samples. Distributions of N. stygius s.l. 
species without any new localities found are not included (N. brachytelson, N. goricae, N. iskae, N. kapelanus, N. karamani, 
N. kordunensis, N. likanus, N. novomestanus, N. podpecanus, and N. stygius). Circles indicate new localities from our 
samples, crosses are older localities from Delić et. al. (2017b). Arrows point to the two new localities of syntopy of two 
species from N. stygius s.l. complex; co-occurrence of N. podpecanus and N. gottscheeanensis (grey circle) and co-
occurrence of N. chagankae and N. likanus (circle with black upper half and white lower half). 
Slika 1. Razširjenost vrst postranic Niphargus stygius s.l. ki smo jih določili v naših vzorcih. Razširjenosti vrst N. stygius 
s.l., za katere v raziskavi nismo opisali nobenega novega nahajališča, na sliki nismo prikazali (N. brachytelson, N. goricae, 
N. iskae, N. kapelanus, N. karamani, N. kordunensis, N. likanus, N. novomestanus, N. podpecanus in N. stygius). Krožci 
označujejo nove lokalitete iz naših vzorcev, križci pa lokalitete iz Delić et al. (2017b). S puščicama sta označeni novo znani 
lokaciji sopojavljanja dveh vrst tega morfološko kriptičnega kompleksa; N. podpecanus in N. gottscheeanensis (siv krožec) 
ter N. chagankae in N. likanus (krožec, katerega zgornja polovica je črna, spodnja pa bela). 
 
 
 
Acknowledgements 
 
 
The study was part of J. Š.’ master thesis, done at SubBio Lab, Department of Biology, Biotechnical Faculty, University 
of Ljubljana. We are grateful to Teo Delić and Cene Fišer for all the help kindly offered to us. We thank all legators, including 
members of the Croatian Biospeleological Society (HBSD) for Croatian samples. The study was funded by the Slovenian 
Research Agency (Programs P1-0184 and P1-0236, Project J1-2464). Š. B. was supported by the Slovenian Research Agency 
through PhD grant (contract KB139 382597). 

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NATURA SLOVENIAE 22(2): 69-77 
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