The

Olm, Proteus anguinus in Croatia

Covjec ja ribica u Hrvatskoj
Final project report

The Olm, Proteus anguinus in Croatia Conservation research project plan

Croatian Institute for Biodiversity,

Croatian Herpetological Society HYLA
-Hungarian Natural History Museum
-Zagreb city ZOO
-University of Zagreb,
Veterinary Faculty
-FREATIK Society for Karst Research

Zagreb, 2013.

Content:

CHAPTER I. Distribution and conservation of Proteus

anguinus Laurenti, 1768 in Croatia

CHAPTER
Balkans

II.

Olm

as

cultural

CHAPTER
III.
Monitoring
populations in Croatia

herittage

of

Proteus

in

Western

anguinus

CHAPTER
IV.
Molecular
analysis
of
Croatian
populations
of
Proteus
anguinus
and
test
the
methodology for environmental DNA detection

CHAPTER V. Education on conservation of underground

habitats and raising public awerenes

CHAPTER I.

Distribution and conservation of Proteus anguinus

Laurenti, 1768 in Croatia

Duan Jeli, Katarina Koller, Ivona Buri

Croatian Institute for Biodiversity,
Croatian Herpetological Society HYLA,
I. Breznika 5a,
HR 10000 Zagreb, Croatia
jelic.dusan@gmail.com,
+385 98 608 099

Objective1:ImprovethegeneralknowledgeoftheOlmdistributionand

recordpossibleonsitethreats.

1.INTRODUCTION

1.1.Distribution
The Olm is the only specialized stygobiont vertebrata of Europe (Gottstein Matoec
et. all 2002). Stygobiont is an organism that is aquatic obligates in subterranean groundwaters
and cave streams (Muni i sur. 2000). Fossil proteids date from the Pleistocen of Germany,
and after the last glacial period inhabit underground waters of Dinaric region (Kovaevi
1984). The european blind cave salamander or an olm has been known for centuries as the
human fish because of it pale skin color. The species Proteus anguinus is limited to the
Dinaric Karst; it ranges from the surounding area of Trieste in Italy, Slovenia, Croatia and
Bosnia and Herzegovina. Although, the species are distributed to the southeastern border of
Bosnia and Herzegovina, but in neighbouring Montenegro has yet never been recorded (Jeli i
sur. 2012). The species has been introduced to the cave of the subterranean laboratory of the
CNRS France (Moulise), and in Germany (cave in the Harz province) (Ozimec i sur. 2009).
Taxonomic status among three distinct populations in Croatia are still uncertain: population
from Istria, which is considered as the most vulnerable, then the population from Gorski
Kotar and northern part of Lika (Gacko polje) and the population from Dalmatia (from the
Krka river throughout the south to the city of Dubrovnik) (Jeli i sur. 2012). What is
distinctive regarding the Olm distribution in Croatia is the existence of two gaps. The gap
between Slovenia population and those in Istria, and the gap among the population of
northwestern and southwestern Croatia. In the Lika region, as well as on Velebit mountain
and Zrmanja river the Olm has never been found. The exact reasons why there are no olmfindings are still unknown (Kleteki i sur. 1996).
Proteus anguinus is the only species in the Proteus genus. The most likely sister taxon to
Proteus is the genus Necturus from the New World. Within the genus Necturus, individual
species fall into three main lineages: N. lewisi, N. punctatus, and a group of three closely
related species consisting of N. alabamensis, N. beyeri i N. maculosus. The relationships
among these three species are uncertain. All species of Necturus, with the exception of N.
maculosus, are distributed along the coastal plain of the southeastern United States, from
southeastern Virginia to eastern Texas. Necturus maculosus is by far, the most widely
distributed species with a range extending from an apex in Louisiana to southeastern
Manitoba in the west and southestern Quebec in the east, essentially encompassing the entire
Mississippi River drainage system. The two coastal species, N. lewisi and N. punctatus are
separated from inland population of N. maculosus throughout the east by the Appalachian
Mountains (Grzimek 2003).

1.2.Threatsandconservation
Global category of threat: VU B2ab(ii,iii,v)
European category of threat: VU B2ab(ii,iii,v)
National category of threat: endangered species, EN B2ab(ii,iii,iv,v)

Conservation actions:

Strictly protected by Croatian Nature Protection Act (Official gazzet 70/05; 139/08;
57/11)
Annexes II and IV of the EU Habitats Directive, marked as priority species
Appendix II of the Bern Convention

Part of the range of the species is located within protected areas (National parks and Natural
parks). All subterranean objects are part of the Croatian Ecological Network. Olm is listed in
the National Ecological Network as a target species for the following types of
environmentally significant areas: Ogulinsko-plaansko area, Polje Jezero, Sinjsko polje,
Ombla, National park Krka, Rupeice spring, Rupeice sinkhole, Komareva, Crnaka pilja,
Rokina bezdana, Markarova pilja, Anti pilja, cave opposite to the lake Torak, cave
Miljacka II, Zagorska pe near Ogulina, sinkhole Crni Vir and Pincinova jama (Jeli i sur.
2012).

1.3.Systematicsofsalamanders
Phylum: CHORDATA
Class: AMPHIBIA
Order: CAUDATA
Family: PROTEIDAE
Genus: Necturus (North American genus)
Genus: Proteus (European genus)

The olm (Proteus anguinus Laurenti) belongs to the class Amphibia among 6 347 different
species (Kenneth Dodd 2010). Because many species have a diphasic life history, the class get
it's name from the Greek words amphi meaning two and bios meaning mode of life. The
name refers to the fact that amphibians have the ability to move, eat and breathe both on land
and beneath the water. Beneath the water level it is able to breath using gills, and it's lungs on
the land. The olm is permanently aquatic amphibia retaining the lungs and livivng in
subterranean lakes and streams througout it's lifetime. In Croatia, lives approximetely twenty
amphibian species, seven of which are salamanders (including the olm) and thirteen are
Anurans (Jeli i sur. 2012).
The salamander family Proteidae in its current conception consists of two extant genera of
permanently aguatic salamanders: Proteus and Necturus. The form that sets them apart from
most other species is neoteny. Neoteny is a well-known phenomenon in which adult individua
maintains larval morphologic features, a form in which it can breed (Trontelj i Goriki 2003).
This occurs because these animals do not go through the whole process of metamorphosis,
gaining the ability of reproduction in larvae state. The morphological features include three
pairs of large, bushy red gills, a relatively short, laterally compressed tail fin, and reduced
eyes. Some scientists therefore argued that the morphological characteristics shared by adult
individuals of Proteus and Necturus are the result of parallel evolution and thus not an
indicator of a common ancestory. Neoteny is thought to be developed as an adaptation to
permanent aquatic, subterranean habitats. Studies of morpfological similarities demonstrated
that both genera, Necturus and Proteus share the same diploid chromosome number (38),
which is not found in any other salamanders (Grimzek 2003). The distribution of proteids is
disjunct: Necturus is found only in the New World, whereas Proteus exclusively in the Old
World. All species of Necturus, with the exception of N. maculosus, are distributed along the
coastal plain of southeastern United States, from southeastern Virginia to eastern Texas. They
inhabit various types of surface waters. There are five currently recognized species of
Necturus. They are active during the night, but are by no means specialized as Proteus
(Goriki i Trontelj 2006).

Proteus anguinus species. Sket and Arntzen (1994) detected that within a species exist two
subspecies that are morphologically clearly recognizable. It is a white, troglomorphic
subspecies Proteus anguinus anguinus and dark-colored, nontroglomorphic Proteus anguinus
parkelj. Troglomorphy includes morphological, physical and behaviour changes as a result of
adaptation to constant darkness of cave systems (Aden 2005). Dark-colored individuals of
Proteus (Proteus anguinus parkelj) are found on two localities. One black specimen was
caught in the Dobliica spring, near rnomalj in southeastern Slovenia on October 18, 1986.
Another black specimen was sighted and photographed below the Jalevik spring, some 2,5
km north of Dobliica on April, 21, 1990. The Dobliica and Jalevik springs are situated in
close proximity to one another, and both discharge into the Dobliica river. No reliable data
about the current finding of the olm in the main Jelevik spring exists. The occurrence of
P.a.parkelj may in fact be restricted to the surface Karst of the Bela Krajina, while the deep
Karst of the sorrounding mountains is inhabited by white taxon (Sket i Arntzen 1994).
P.a.parkelj differs from P.a.anguinus in a dark pigmentation, fully developed eyes, a skull
with broader and shorter bones and fewer teeth, a voluminous jaw musculature, a
proportionally longer trunk with a higher number of vertebrae, shorter extremities, and a
shorter tail (Sket i Arntzen, 1994). The morphology of white subspecies includes pinkish
white skin, narrow head with tiny degenerated eyes as an adaptation to eternal darkness.
There are two hypothesis of the rudimentary state of the eyes. The first one is saying that the
degeneration of the eye is a secondary consequence of the adaptation to an underground
environment. The second one is that ocular atrophy is a kind of arrested development, linked
to a larval neotenic state (Durand 1976). The olm has small limbs with only three digits on the
forelimbs and two digits on the hand limbs. On the laterally compressed tail, distinctly shorter
than the rest of the body, a tail fin is visible (Margu 2008). P.a.parkelj achieves a total length
of approximately 199 mm 276 mm, while the P.a.anguinus has a total length of 147-299
mm. The black taxon has a different ecology. However, Proteus inhabits food poor karst
waters of deeper underground streams and lakes in limestone caves. In some places
individuals of Proteus actively come out of springs during dark nights because of better
feeding conditions in surface waters. On the bases of these evidently constant differences, the
black and white morph deserve an separate taxa attribute (Sket i Arntzen 1994).
Besides morphological differences, Sket and Arntzen (1994) have pointed out the genetic
differences between subspecies, and within some populations of the white subspecies, where
the subspecies were more similar to each other than populations Proteus anguinus anguinus.
However, their study failed to provide a complete overview of the relationship within
populations of species Proteus because of the small sample size and the limited number of
sampled populations. The new genetic research takes into account not just the morphology but
the geographic isolation of populations as well. According to the results, subspecies of
slovenian population are closer one to another than geographically isolated populations within
the white population. So, the dark taxon is part of SE Slovenian group and doesn't represent
an independent line, as would be expected from its distinctive morphology. Goriki (2006)
stated that supposedly there are five geographically isolated populations of Proteus anguinus
anguinus (Istria, SW Slovenia, SE Slovenija, Dalmatia and Gorski kotar) from which the
8

Istrian population genetically and morphologically differs the most. The Istrian population
appears to be the starting line, meaning it is different in a way it can be considered as a
separated species (Jeli i sur. 2012).

1.4.Biologyofspecies
Breathing. Olm breathes with gills and skin, but while in hypoxic conditions it breathes with
lungs. Proteus does not require high concentrations of oxygen, which corresponds to their low
metabolism level, which in turn is another adaptation to a subterranean lifestyle. While
conducting the study of resistance of the olms on the lackage of oxygen in the water, an
evident hyperemia (an increased amount of blood in the capillaries of an organ or a body part)
of gills and skin was noticed and animals often swam to the surface of the water in order to
catch the air. While observing the olm, the guestion of breathing air appears to still be active,
whereas in the literature various views can be found. Olms capability of "air swallowing" has
been known for a long time from a laboratory in Germany, while the French explorers denied
it. Briegleb (1962) noted it again and explained as " life necessary". Durand (1976)
considered that as an abnormal animal behaviour caused as a result of unsuitable condition
such as a high water temperature and lower oxygen concentration in it. The results of the
study Udisanje vazduha i problematika uloge plua kod proteusa suggest that frequent
inhalation of air is significant only in a very deoxygenated water. Thus, one can consider that
the lungs are an organ used for breathing when the concentration of oxygen in the water drops
below it's critical value (Sojar i sur. 1981).
Feeding. Olms are primarily predators; its natural food includes detritus and cave
invertebrates. They seem to feed on insect larvae, usually larvae of Trichoptera,
Ephemeroptera, Plecoptera and Diptera, mollusca (Belgrandiella) and freshwater amphipods
(Niphargus, Asellus, Synurella) (Bizjak-Mali i Bulog 2004). The groundwater ecosystems,
including cave and karstic aquifers, are characterized by limited food supplies during most of
the year. The reason lies in the fact that the groundwater ecosystems has the lack of
autotrophic production and sporadic, unpredictable, allochthonus input. Because of these
conditions, periods of prolonged starvation are common events in the life of subterranean
organisms, so olms are able to survive for a long period of time without food. In addition,
some authors reported that P. anguinus could survive food deprivation for exceptional period,
ranging between 18 and 96 months, withhout any signes of illness (Hervant i sur. 2001b). The
low and discontinuos food supplies along with darkness in subterranean environments
requires more time spent on food searching. Besides, the prey is invisible and often dead
(brought in by occasional floods). During th search for pray it compensates the absence of
eyes in several ways. The olm is capable of sensing very low concentrations of organic
compounds in the water. Using its scent Proteus evaluates the quality and quantity of the
pray. The sensory epithelia of the inner ear is very specifically differentiated, enabling the
olm to receive sound waves in the water, as well as vibrations from the ground. It is known

that the lateral line organs register low vibrations of the liquid environment (Durand 1976). In
addition, the Olm has the ability to register weak electronic fields (Hervant i sur. 2001b).
Reproduction. Breeding in Proteus appears to be aseasonal, reflecting the stability of their
subterranean habitat (Grzimek 2003). Relatively limited information is available on the
reproductive biology of Proteus anguinus, because of the fact that they live in total darkness
(Guillaume i sur. 1999). The olm can reproduce in two ways, what was discovered in a
research laboratory in Moulis in France (Kovaevi 1984). Proteus reportedly, has in some
degree a capability of viviparity, giving birth to a pair of well-developed youngs in low
temperatures and laying eggs in high temperatures. Although the adult individuals grupe in
places hidden under the rocks, in breeding season males determine their territory and defend
them from competing males. When the femail enters into that territory, courtship begins.
Courtship culminates in the male depositing a packet of sperm (spermatophore) which the
female catches and introduces it into its cloacal cavity. Spermatozoa are then stored in the
lumen of cloacal glands, the spermathecae, for six months or more. Thus, the fecundation
occurs when the eggs pass through the cloaca during the laying. When the mail leaves the
territory, the femail is searching for a place to lay her eggs. After two to three days the femail
lays eggs, which may number up to 100 or more per clutch. The eggs are usually attached
beneath some object, such as a rock or a log, and are guarded by the female. The eggs are
large (5-6 mm), full of yolk, and unpigmentated. The incubation period lasts two to six
months, depending on the species and the temperature (Grzimek 2003).
The olm is amphibia with the longest lifespan. It has a slow development, reaching adulthood
between 14 and 18 years of age and lives for more than 60 years (Hervant i sur. 2001b). The
Voituron et. all (2011) have suggested two possible explanations about animal's extreme
longevity. The first is the animal's exceptional laziness - it only eats about once a month, and
doesn't have to run away from predators, because it has none in its natural environment.
Because it doesn't extend much energy, its metabolic rate mostly stays at its baseline rates,
while most animals' metabolisms are often running much above their basal rate. Their second
theory is that the olm's mitochondria function differently from normal mitochondria- they are
able to process more ATP with less oxygen (Welsh 2010).
Habitat. Olm is an endemic stygobiont of the underground waters of Dinaric region
(Gottstein 2010). The main features of a subterranean life are lack of light, a day turning into
nights and a relative air humidity near full saturation value. The temperature of underground
waters tend to be more constant than in surface waters. The ideal temperature range for the
Olm is between 8C and 10C. Only the Rokina Bezdana in Lika region values 7 C, but
temperature here is variable and is reported to sometimes fall below 5C (Garai 1980a). In
some other localities, lower temperatures in caves probably occure during melting of snow or
after heavy cold rains in spring, when high quantities of cold surface water penetrate
underground. However, there is no locality in Slovenia where this animal is likely to live for a
longer period at temperatures below ispod 8 C (Sket 1997). The highest temperature mesured
in a proteus habitat has been 14 C in Istra (Raa 1980a).

10

Proteus populations are many, but its habitats are deep cracks and fissures in the limestone
caves, therefore it is not directly accessible to man and population estimation cannot easily be
performed. More often, the specimens can be found in marginal parts of its habitat, were the
animals were flushed out by heavy rains or were hunting for food (Grzimek 2003).

11

1.5. Review of previous research on the olm

Unusual creatures, which can be seen at karst springs when washed out of the cave after
heavy rainfall, have been known to local people long before they cought the attention of
scientists. Indeed there is a confirmation of this in carving on an ancient stone from Venezia
(10/11 century), which has been thought to represent Proteus. This carving used to be near the
church of San Nicol, Lido island, and now is in the Kunsthistorisches Museum in Wien
(Shaw 1999). Fairly realistic figure of olm is carved in a stone in Boljuni near Stock (Vega
1964). It stands for one of the most authentic monuments of Bosnian culture originated in
1477 (Picture 2). Carvings of the olm coming out of the pit represents forces of the
underground (Lui 2008).
Before Laurenti offered zoological description and naming, there was two descriptions of this
animal both written by people who had not seen it for themselves. The history of the name
Proteus goes back to Valvasor (1689) who mentioned the species, found in the intermittent
karst spring Lintvern near Vrhnika, in his treatise ''Die Ehre des Herzogtums Crain (Sket i
Arntzen, 1994). This information came from the postmaster who told him about ''supposed 20
cm long dragon (Shaw 1999).
However, Slovenians belived that there was a dragon living in one occasional spring. Dragon
would occasionally dry the spring up, but when he would spill out the water, his offsprings
would emerge alongside. None the less, it is unlikely that the human fish was found in the
Bela source near Vrhnika since it doesn't hydrogeographically and geologically match the
typical habitat, and it is different from all confirmed localities (Kakarigi 1993). The second
record is from 1751. Steinberg recorded that in time of high water rank, Primus Sicherle
caught five unknown fishes in the Unica river, 23 cm long, with snow-white skin and long
tails (Shaw 1999).
Joseph Nicolai Laurenti was the first to offer a formal draw and description of the olm in 1768
in his famous ''Specimen medicum (Picture 3 i Picture 4). Laurenti's description was short
but sufficient and has first ilustration of Proteus (which is today very famous). G.A. Scopoli
send him a dead specimens from the Stina spring, 40 km southeast of Ljubljana (Shaw
1999). The first specimen inside a cave were discovered in 1797 by Josef Jerinovi von
Lewengreif in the Pivka river at rna jama (Sket 1997). After a scientific writing Specimen
medicum followed a series of papers about human fish. Scopoli, in 1772, was the first to
mention that Proteus is an amphibian and Schreibers, in 1801, published a study about
anatomy of Proteus (Pretner 1968). Another more formal description gave George Shaw in
1802. It is less important than Laurenti's, not being the first, but it is much more detailed. Four
pages were devoted to this animal but its illustration was reverse and copy version of
Laurenti's picture from 1768 (Shaw 1999). MM. Cuvier and Rudolphi were examining the
internal structure of this animal and they discovered that the Proteus was not a larva, as
supposed, but a perfect animal (Configliachi i Rusconi 1821). John Russel in 1822 visited
Postojnska jama and wrote the first detailed description of the behaviour of Proteus. During
his visits to Postojna in 1828, Charles Babbage, who liked to collect various things, though
12

not connected with his own scientific area, took away some specimens. He described this
animal as ''a cresture living only in waters of dark caverns, with eyes, but cannot open the
eylids. These is probably the oldest description of Proteus in english (Juni 2005). Lionel
Smith Beale visited Postojnska jama on 24 August 1850 and probably then obtained his
Proteus, which he later continued to breed and record further development of individuals
outside their natural habitat. In 1861 Hugh Falconer visited Postojnska jama and wrote to
Charles Darwin about the Proteus anguinus he brought back with him. Falconer offer the
animal to Darwin who refused due he wasn't able to provide a good home for this animal and
suggested that London Zoo would be a better place for it (Shaw 1999).
Bosnia and Herzegovina. In the same time, in Bosnia and Herzegovina dr. Stjepan Bolkay
offered the first data about Proteus. He induced in 1895 that four animal was captured in
Studenci near Ljubuki. That is considered to be the oldest record of these species in Bosnia
and Herzegovina (Bolkay 1924, 1929). Another significant contribution about olm's habitat
for the area of Trebinje and Popovo polje gave Stevo ukovi, biology teacher from Trebinje
(ukovi 1967, 1983). He listed ten localities of Proteus for the area of Trebinje and its
surrounding. He also pointed out the danger of building a hydroelectric power plant on
Trebinjica river which floods Popovo polje. The HE was open shortly after, in 1979 and
Trebinjica river is no longer considered to be a gulf. Also Popovo polje is no longer being
flooded. On the territory of the municipality of Trebinje, ukovi ranked localities into two
groups. One group is located on the left side of Trebinjica river, and the second, much more
numerous, is in Popovo polje. In the eastern part of Trebinje, ukovi detected eight
localities, southwest of Trebinje two localities and northwest two more. In Popovo polje along
the left side of Trebinjica river, where no data was recorded in the past, ukovi found
seventeen localities. He believed that the number of localities in Popovo polje is even higher
and that Popovo polje is the richest part of ex Yugolavia in number of localities. He also
announced that Proteus population of Popovo polje is extremely endangered (ukovi
1983). Vojisalav Mikuli and Aleksandar Janii wrote about findings of olm near Mostar.
Mikuli mentioned that in the area of iroki Brijeg in 1935 some fisherman noted the olm at
the spring of Litica river (Litica river is also a gulf). In 1968 Janii caught the olm in
Radoblje cave, and later he breed and observed the olm (Janii 1970; Mikuli 1970).
Contribution to knowledge about the dispersion of olm in Bosnia and Herzegovina gave Boris
Sket, Slovenian biologist (1997). He noted 41 localities for Proteus in Bosnia and
Herzegovina. According to the last list made by Kotroan (2002), in Bosnia and Herzegovina
exists 57 localities. The new list contains localities described after 1997, with some of the
previously known localities that Sket didn't include into his previous list.
As the science developed, the nineties were characterized by molecular and genetic analysis.
The first description of Proteus anguinus parkelj was offered by Sket and Arntzen (1994).
They also pointed out the genetic differences between subspecies together with differences
inside population of Proteus anguinus anguinus. During the 2000, there was a lot of
phylogenetic analysis on Proteus populations, mostly made by Slovenian scientists. In 2006,
Goriki pela graduated on phylogenetic and morphological analysis of Proteus. She
indicated that there are five geographically isolated populations (Istra, SW Slovenija, SE
13

Slovenija, Dalmatia and Gorski kotar) and the Istrian population differs the most from other
populations in genetic and morphology due which should be considered as a separate
specimen. In years that followed, the olm caused the interest of many scientists and influential
people. It is interesting that ukovi, along with his coworkers (2008), wrote a study of
subterranean invertebrates in Montenegro and put the picture of Proteus with note that this
individual is from Niki spring near Montenegro. However, Proteus was never found on this
location and with no photograph evidence of this animal the Niki spring cannot be
considered as valid.

1.6.PublicationsoftheOlminCroatia
At the beginning of research of Proteus anguinus in Croatia, in early 19th century, foreign
scientists had a major impact on the direction of zoological research of Croatian region.
Approach was sistematicaly-faunistic, and the first explorers were mostly foreigners with
whom local naturalists were in touch. Besides foreigners, first collectors of fauna on these
area were wealthy citizens of Dalmatia, who were in contact with foreign, mainly Italian
biologists, with whom they exchanged the animal samples. In 1853, Bottieri wrote about
herpetofauna of some Dalmatian islands. ''Herpetologia europaea, by E. Schreibera (1875), is
of great importance since only Croatian herpetofauna was taken into account. The great
influence on knowledge about Croatian hepretofauna had Fr. Werner, scientist from Vienna,
who provided an article about knowing amphibians and reptiles of Istria and Dalmatia (Babi
1928).
Many authors wrote about the first specimen of Olm in Croatia found in 1840 at the Goruica
spring near Sinj (Fitzinger 1850). Seventy two years passed since the scientific description of
Olm (Laurenti 1768), until it was found at the croatian karst area (Kleteki i sur. 1996). Later
this findings had been described by many authors, like Paganetti-Hummler (1902) and
Brusina (1907), but Fitzinger (1850) was the first who wrote about this finding. He descriebed
the Olm as a new species Hypochthon carrarae. Fitzinger (1850) described seven new species
under the Proteus genus (Hypochton zoisii, H. schreibersii, H. freyer, H. haidingeri, H.
laurentii, H. xanthostictus, H. carrarae). Some of his typical localities are only a few
kilometres away from each other or are even in the same cave system what makes them
unlikely to be distinct subspecies (Sket 1997). Despite Fitzinger's taxonomy wasn't accepted,
Mertens and Wermuth (1960) mentioned twelve different names. Today we know that there is
one species and possibly two to three subspecies (P. a. anguinus i P. a. parkelj) (Gasc 1997).
There is an ongoing research by Croatian and Slovenian scientists who are trying to prove that
genus Proteus has two distinct species.
After finding the Olm at Goruica spring (near Sinj), data about new findings started to arrive
(other localities all around the karst region of Croatia) and several authors wrote about them.
Ottoman Tommasini (1875) wrote about the Olm from the Vrlika in Dalmatia. In 1880,
14

Mihovil Jurkovi gave Brusina a sample of Proteus. Sample was from Gacka river, near the
ruins of the Frankopan town. Since Brusina believed that this sample was not the same one as
in Slovenia and Dalmatia, he called it Proteus croaticus (Maor 1995). In 1883, Jurinac was
sent to explore the karst area and to collect some samples for Zagreb's National Zoological
Museum. Jurinac was encouraged by the museum's director himself. Director ordered him to
search Karlovac, Ogulin, Plitvika jezera and Kostajnicu and to collect amphibia, fisches,
mollusca, myriapods and crustaceans. Also, he was supposed to examine where exactly could
Proteus be found. After the research, he concluded that the human fish doesn't inhabit ''this
side of the Kapela (Jurinac 1886). The first finding of the Olm in Istria mentioned dr. C.
Marchesetti in 1885 during the penetration of water into the mine Krapan in Labin.
Kolombatovi mentioned in 1902 the Jadro river (near Solin) also as a finding of the Olm.
Until that, in Dalmatia were known only the Goruica spring (near Sinj) so he wasn't certain if
this sample of Proteus came to Jadro via underground streams. Clear overview about
amphibians in Croatia offered St. Karaman in 1921 in his ''Beitrge zur Herpetologie von
Jugoslavien. Ledi (1961) mentioned that in Mueva Hiica and estanovac were also found
specimens of Proteus. However, today we know this is not correct. Even Pretner didn't
mention it in his paper from 1963 where he gave the first distribution map of Proteus in
Croatia (Slika 5). On that map he noted 11 localities on which, according to the literature,
Proteus was found (three sites were in Lika). According the Pretner's map, there were known
37 localities of wich 35 were within area of Jugoslavia, and only 2 in Italia close to Bosnian
border. Abrami (1966) increased the number of localities of the areal in Italy.
Jela Pavleti recorded Ivina peina and Markarova pilja in 1964, when she was writing about
amphibians and reptiles of the National Zoological Museum in Zagreb. Gluevi (1969)
wrote about the Olm from Neretva river, and also mentioned villages Vid, Prud and Momii.
Further discoveries were Rokina bezdana near Jezerane in Lika in 1975, Pincinova peina
near Pore in 1976, and uderina jama near Dugo polje in Dalmatinska zagora in 1979
(Garai 1980).
By 1980 the several localities were known: Cetina spring, Markanova peina near Liko
village Stajnice, cave near Dabar, Gacka near Otoac, Goruica spring near Sinj, Vrlovka on
Kupi river near Kamanje, lower course of the Neretva river, Rokina bezdan in Lica, pit near
Pazina in Istra and uderina jama in Dugopolje what is all written by Gabri. Toni Raa
(1980b) gave a list of almost all previously known localities of the Olm in Croatia. Veernji
list (1985) published the findings of Olm in Zapolje. First finding of Olm in National park
Krka was the cave Miljacka II in 1989 and was noted by De Luca (1990). Tihomir Kovaevi
in his paper from 1992 reported a total of 25 certain localities of the Olm in Croatia. Kleteki,
Jali and Raa (1996) wrote about distribution of the Olm in Croatia and recorded 47
localities, 40 to be confirmed localities. Findings of the Olm in Zagorska pe near Mrenica
were published in Veernji list (17.1.1999.) on pages 26/27. These are the first findings for
Karlovac Country and also the northeasternmost evidence of Olm in Croatia (Ozimec 1999b).
According to Eduardo Kleteki, since 1999 there were approximately 50 confirmed localities
of Proteus anguinus in Croatia. Each of them are separate speleological objects for itselves. In
Slovenia 60 localities were recorded, but some of these cave systems often have more than
15

one entrence (Kozaranin i Boi 1999). The last review of Olm's distribution was given by
Sket (1997) and Durand (1998). Today we have approximately 300 localities of Proteus
anguinus (Laurenti, 1768) evaluated and listed, 150 in Slovenia, about 60 in Croatia and 30 in
Italia (Ozimec i Lui 2002).

16

2.MATERIALSANDMETHODS
2.1.Datacollectionandprocessing
2.1.1.Collectionofpublishedliteraturedata

Research has started with collecting literature and museum data from the museum collections
regarding Proteus in Croatia. For this purpose, we reviewed all published and unpublished
data in Croatian, Slovenian, German, English and Italian available in Natural History Museum
in Zagreb, and on the internet. A part of the research was the personal collection of Eduard
Kleteki. If the data were unable to gather in the above mentioned ways, we contacted the
authors directly. The whole data set, as well as evaluated criteria are given in Table 1.

2.1.2.Collectingunpublisheddata

The data about the presence of Proteus on new localities in Croatia were gathered during
fieldinvestigationsin 2012and2013.

Wecollecteddataabout:
existing literature localities wich were compared with experts oppinions (Branko
Jali, Eduardo Kleteki, Duan Jeli, Toni Raa, Vedran Jali, PetraKonradKova,
BorisSket,SilvioLegoviect.)
accidental Olm finding sites (individuals found in caves, individuals flushed during
highwater,ect.)
newlocalitiesdiscoveredbyactivespeleologists

During the Project, education training workshops were held in Ogulin, Pula and Sinj. The
workshops were focused on activating local people in contributing in the conservation of
Proteus.

17

2.2.Dataanalysis
2.2.1.Listoflocalities

CollecteddataforlocalitiesofProteuswereusedtomakeadigitalmapofdistributioninthe
computerprogramESRIArcMapInfo10.0.
Thedatawerepresentedintwomainways:
as a map with all known localities on the Croatian territory in the Gaus Kreger
coordinatesystem
asanUTMnetworkmap(UniversalTransverzeMercator)withbasicquadrant10x10
kmforlocalitieswithapproximatelocation

Followingthat,wemadeatablecontainingthe correctnamesofthelocalities,includingthe
districtandtowntowhichthementionedlocalitybelongs,andthefirstrecordoftheOlm,
includingthecertainityofthefinding.
- The certainty of the finding was devided into following groups:
a: certain confirmed finding (verified in recently published journals by
quolified authors or seen recently by reliable person)
b: certain, but unavailable finding (existing of historical data about finding of
the Olm published by oldish authors, but individuals were not seen afterwards the entrence into the object is unavailable)
c: potential finding (existing of historical data about finding of the Olm
published by oldish authors, but individuals were not seen afterwards due to the
inaccurate description of the locality in the original paper because the optimal
part of the object for Proteus residence is not visited)
d: most likely to be incorect finding (published data, but uncertan due to
geographical, hidrogeographical and/or environmental reasons, individuals
were not seen in recent time and the finding has not confirmed since
publishing)

Iftheobjectperseistypelocalityforaspecies,weuseditsfullnamefromtheliterature.
For other objects, I proposed a name using my own discretion and the proposal of
distinguishedspeleologists.
The section behind the table includes all the localities that I have been able to collect by
literature data review and by personal contacts with professionals within the field. The
findingsinsidethedistrictsweregroupedintofourcategoriesdependingonreliabilityofthe
finding, in the way that certain confirmed findings are separated from certain, but
unavailablefindings,potentialfindingsandmostlikelyto beincorectfindingsaccordingto

18

the above principles. The data in the table show the category of the locality, the most
importantfeaturesofthelocality,Olmfindingsandlocalityconditiones.

Dataaboutlocalityconditionesareusedto:
- defining the status of object according to hidrogeological function for further study:
a: cave
b: pit
c: spring
d: spring - cave
e: periodically spring
f: sinkhole
g: spring and sinkhole cave
h: wellspring
- develop a plane to eliminate causes of Olm and subterranean habitats voulnerability
- further educate local people about the need to take part in the species conservation
2.2.2.Literaturereferences

The collected literature was clasified into four groups:

a: scientific journals (journals that undergo some kind of reviews; the highest level of data
quality)
b: descriptive journals (journals that barely mention the Olm, and are written by
nonprofessional)
c: newpapers
d: book (monographs and tehnical books)

Within the scientific journals category we determined the country from which the
investigated Olm specimens came from. Moreover, the themes of scientific journals were
analyzed and divided into several categories: distribution, anatomy, behavior, biology,
conservation, genetics.
The analysis of the collected data was used to make a graphic presentation of the prevalence
of written scientific papers from 1959. untill today

19

3.RESULTS
3.1.Distributionresearch
The analysis of literature and museum data about the Olm findings in Croatia and by the
personal contacts with scientists and biospeleologists show that in Croatia here is 68 localities
where olm presence was recorded at least once. (Table 1). According to recent data, there are
37 (54%) confirmed localities, 10 (14%) certain, but unavailable, 14 (22%) potential and 7
(10%) which are most likely to be incorect. The atribute of confirmed locality gets the locality
on wich speleologists and speleodivers recorded the existance of the Olm in recent time. All
literature data which confirmed the existanc of the Olm, but in recent reasearch the Olm
presence wasn't recorded are certain, but unavailable localities or potential localities.
Proteus anguinus is recorded in three separate regions: : Istra, Gorski kotar and Dalmatia, and
in 8 districts: Istarian, Primorsko-goranska, Karlovaka, Liko-senjska, Zadarska, ibenskokninska, Splitsko-dalmatinska and Dubrovaka districts. In Istrian district have been recorded
11 localities, 4 of which are confirmed localities (Pincinova jama, Bregi sinkhole, Fontana
spring and Nimfej spring). 6 are certain, but unavailable localities and 1 is potential. In
Karlovaka district there is 9 localities and almost all are confirmed, even 7 of them (Klisura,
Zagorska pe, Bistrac spring, Rupeice spring and sinkhole, spring of Zagorska Mrenica,
Komareva pilja I and Komareva pilja II). Two localities are most likely to be incorect
(Vrlovka and Ozaljska pilja). For Primorsko-goranska district there are data about two
certain, but unavailable localities and one which are most likely to be incorect. From 9
recorded localities for Liko-senjska district 4 are confirmed (Rokina bezdana, Obajdin pilja,
Markarova pilja and Antia pilja or Ivina peina), 2 are potential and 3 are most likely to be
incorect. There is information about one potential locality for Zadar district. In ibenskokninska district exist 7 confirmed localities (Jama opposite Torak, Miljacka I, Miljacka II,
Miljacka III, pilja kod mlina, Miljacka V and Golubinka). The most localities were recorded
in Splitsko-dalmatinska district, even 19 of them, 9 of which are confirmed (Grab spring,
Stuba periodically spring, uderina jama, Krevac spring, Ponor Jasena, Marinovia Betina,
Vir spring, Matica river spring and Dropulia vrilo spring), 1 certain, but unavailable locality,
8 potential and 1 which are most likely to be incorect. In Dubrovako-neretvanska district
were noted 9 localities, 6 of which are confirmed (spring near village Bijeli Vir, spring near
Neretva, Gluci village, Momii village, Prud spring in Vid village and spring of river Norin),
1 certain, but unavailable localitie and two potential localities.
For better clarity, in following text are shown the basic features of each locality and related
information about the Olm findings. The data of basic features are accepted from literature.
The years and seeing of Proteus are resultats of personal comunication with professionals and
some are also used from literature. The features and location condition for which I could not
find any information I noted as unavailable data.
In last part of the results are given critical evaluation of literature data as well as related
diagram.
20

TABLE 1. Olm finding list (Proteus anguinus) in Croatia

S confirmed localities, SN certain, but unavailable localities, P potential localities, NK
most likely to be incorect localities; Capital letters indicate the authors who first mentioned
localitie as olm habitat; Collectors or observers are indicated by small letters in parentheses.
BD-BF: Biological Department of Biotechnological Faculty, Ljubljana. NHMW: Natural
History Museum Wien. SO PD Dubovac: Speleological department of Mountaineering
Society Dubovac.

FINDING

LOCALITY

DEFININGBY
HIDROGEOLOGICAL
FUNCTION

Istarriandistrict

Fontana,spring

StarskaVala,Tar

spring

2010.

(LegoviS.)

Pincinovajama
PonorBregi
(Graie,jama)

Tar,Pore,Baredine

pit

1976.

minj,sredinjaIstra

pit

3.07.2011.

RAA,1980a.
(Speleoloziupani,K.,
Grabar,L.SDPazin)

CaveinVodnjan
Effusioninan
artificialtunnelin
epipolje

Vodnjan,Istra

pit

SN

17.09.1895.

BOEGAN,1931.

Plomin

artificalhole

SN

1929.1931.

BOEGAN,1931.

Raa,boringhole
EffusioninaKrapan
colemine
Rakonek,boring
hole

Raa,Labin

artificalhole

SN

1948.i1950.

PRETNER,1962.

Raavillage,Labin
Raa(Rakovnikspring),
Labin

artificalhole

SN

1885.

artificalhole

SN

1986.

MARCHESETTI,1885.
Duringtheresearch
work

Pula

artificalhole

SN

PRETNER,1962.

Pula
Karolina Pula,below
theArena

Springcave

1894.,1895.

spring

2.11.2008.

BRIAN,1924.
(Kleteki,E.,Jali,V.,
Jali,B.)

Pula,waterworks
Ispringcaveunder
theVeliVrh

CERTAINTYOF
THEFINDING

DATAONFIRST
RECORDS

COLLECTOR,OBSERVER

Nimfej,spring
Primorsko
goranskadistrict
Cavernintunnel
Uka
Excavation(today
oilrafinery)in
Rijeka

Rijeka

artificalhole

SN

1978.

RAA,1980a.

Rijeka

artificalhole

SN

nakonI.
Svjetskograta

PRETNER,1962.

MuevaHiica

Skrad,Delnice

NK

LEDI,1961.

Karlovakadistrict

KomarevapiljaI

nearCrnovrelo,Drenica

pit

17./18.8.1999.

KomarevapiljaII

nearCrnovrelo,Drenica

pit

17./18.8.1999.

ZagorskaMrenica
spring,Ogulin

cave

2.01.1999.

Desmericevillage,Ogulin

spring

2.01.1999.

(Dowagne,A.M.)
GOTTSTEINMATOEC
etall.,2002.
(Dowagne,A.M.)
GOTTSTEINMATOEC
etall.,2002.
(JaliB.iLukai,D.)
GOTTSTEINMATOEC
etall.,2002.
(JaliB.)GOTTSTEIN
MATOECISUR.,2002.

Desmericevillage,Ogulin

26.08.2004.

1983.

(Jali,B.)
(Jali,B.)GOTTSTEIN
MATOECetall.,2002.

Zagorskape
ZagorskaMrenica,
spring
Bistrac,spring
Rupeica,spring
andsinkhole

Zagorjevillage,Ogulin

spring
Springandsinkhol
cave

Klisura

Perakovii,Ogulin

pit

20.08.2000.

(Jali,B.)

Vrlovkapilja

Kamanjevillage,Ozalj

cave

NK

1873?

LANGHOFFER,1912.

Ozaljskapilja
Likosenjska
district

Ozalj

cave

NK

HIRC,1905.

21

Rokinabezdana

Obajdinpilja

OpinaBrinje,near
Jezerane

pit

3.10.1975.

GARAI,1975.
(Barii,T.,Erhardt,R.,
Stipeti,Z.,SDUMS
"Velebit"Archive)
KLETEKIetall.1996.

cave/periodically
spring

1990.

cave

1962.

1983.(Antia
pilja)i
3.8.1961.(Ivina
peina)

PRETNER,1962.
(Antiapilja:
Cvitanovi,H.fromSO
PD"Dubovec"Karlovac;
info.Kleteki,E.)Ivina
peina:PRETNER,1963.

Markarovapilja

Jezerane,Ogulin
Stajnicevillage,Jezerane,
Lika

AntiapiljaorIvina
peina(Peinapod
Sitnikom)

Dabarirna
villages,Dabarskopolje,
Lika

Zapolje,hamlet

Dabar

23.3.1985.

CVITKOVI,1985.

Otoac

Otoac

7.7.1879.

JURINAC,1886.

Gacka
Gacka,nearruinsof
theoldcaste
Frankopan

Otoac

NK

BRUSINA,1880.

Otoac

NK

7.7.1879.

tirovaa

Velebit(centralVelebit)

NK

JURINAC,1888.
CONFIGLIACIand
RUSCONI,1819.

Zadarskadistrict
Effusionin
excavationfor
waterreservoirin
Bjelinavillage
ibenskokninska
district

BilianinearBenkovca

Artificialhole

(Raa,T.)KLETEKIet
all.,1996.

MiljackaI

N.P."Krka",ibenik

24.10.1998.

MiljackaII

N.P."Krka",ibenik

cave
Springandsinkhol
cave

22.8.1989.

MiljackaIII
piljakodmlina
(MiljackaIV)

N.P."Krka",ibenik

cave

24.10.1999.

N.P."Krka",ibenik

cave

30.6.1999.

MiljackaV
Caveoppositeto
thelakeTorak

N.P."Krka",ibenik
ikolariver,N.P."Krka",
ibenik

cave

24.10.1999.

pit

27.4.1998.

Viture,Koprnovillage,
Prgomet,ibenik

pit

1994.

spring

TVRTKOVIandVEEN,
2006.

Golubinka
Splitsko
dalmatinskadistrict

nalijevojobaliCetine,
seloOtok

cave

sinkhole

(Ludvig,.,Jali,B.,
Lukai,D.,Lackovi,D.)

(JaliB.andLukiO.)

(Jali,B.)GOTTSTEIN
MATOECetall.,2002.
(Jali,B.)
(Lackovi,D.)
GOTTSTEINMATOEC
etall.,2002.
(Jali,B.)GOTTSTEIN
MATOECetall.,2002.
(Raa,T.,izmi,F.,
Mimica,D.andJug
Dujakovi,J.)JURI,
1995.

Cetinaspring
Vrlika

Sinj

Goruicaspringand
stream

Sinj

spring

SN

1840.

Grab,spring

seloGrab,Trilj

spring

27.1.1979.

Periodicallyspring
(Stuba)

seloVedrinapokrajTrilja

spring

13.01.1997.

uderinajama

Dugopolje,Split

cave

6.12.1979.

WERNER,1897.
FITZINGER,1850.;
PAGANETTIHUMMLER,
1902.
(Vujevi,M.)RAA,
1980b.
(Raa,T.iRaa,B.)
GOTTSTEINMATOEC
etall.,2002.
(Raa,T.andGabriG.)
RAA,1980a.

Jadroriver
Wellsontheisland
olta
Surroundingsof
estanovac

Solin

spring

18.7.1900.

KOLOMBATOVI,1902.

Split

NK

WERNER1891.

Omi

LEDI,1961.

22

Surroundingsof
Gubavicawaterfal
Krevacspring

Cetinariver,Zadvorje
Grubinevillagesouthern
partofImotskopolje

Vrgorac

Vrgorac

Butina,spring

Vrgorac

spring

PonorJasena

sinkhole

2009.

Cave

1998.

VelikaBetina

Majii,Rava,Vrgorac
Kokoriivillagenear
Vrgorca
Kokoriivillagenear
Vrgorca

pit

Matica,riverspring

Vinavillage,Vrgorac

spring

1995.

Vir,spring
Dropuliavrilo
spring(Lukavac
spring)
Dubrovako
neretvanskadistrict
BijeliVirvillage
nearNeretvariver

Otriivillage,Vrgorac

spring

1994.1995.

Lukavacvillage,near
Vrgorca

spring

14.1.1999.

VillagenearNeretvariver
ThespringinNeretva
rivervallyontheborder
ofCroatiaand
Herzegovina
Glucivillagenear
Neretvariver
Momiivillagenear
Neretvariver,Metkovi
Vid,village,Neretvariver,
Metkovi
rijekaDubrovaka,
Dubrovnik

spring

1955?,1985?

spring

FITZINGER,1850.
(Arnaut,S.)KLETEKIet
all.,1996.

1968.

GLUEVI,1969.

1968.

spring

SN

25.8.1985.

GLUEVI,1969.
(Ciril,M.,slo.diver,
1986.)RAA,1980b.

spring

1987.

Doljane

Prudvillage,Metkovi
AreanearNeretva,
Metkovi

Stupahhle,
springcave

Stupavillage,Slano

1930.

MarinoviaBetina

Thespringin
Neretva
Gluci,village
Momii,village
Prud,spring
systemVilinacave
Omblaspring
Norinriverspring

spring

1981.,1988.

KLETEKIetall.,1996.

1927.

RAA,1980b.
(BDBF)KLETEKIetall.,
1996.

23

LEDI,1961.

(Raa,T.)
(Pervan,M.)GOTTSTEIN
MATOECetall.,2002.
(Prvan,Z.,info.Jali,
B.)
(Vukasovi,M.)
KLETEKIetall.,1996.
(oti,M.)KLETEKIet
all.,1996.
(alinovi,N.,inf.
Ajdukovi)GOTTSTEIN
MATOECetall.,2002.

(Ivankovi,A.)KLETEKI
etall.,1996.

GLUEVI,1969.
SPANDL,1926.

GRILLITSCHand
TIEDMANN,1994.
(NHMW)

3.2.Themapofdistribution

Slika 7. The map of olm findings with ceratinty of the finding in Croatia

3.4. Analysis of Proteus anguinus bibliography

The analysis of the literature data indicate that the major part of bibliography about the Olm
has been written between 1970-ies and today (Figure 50).
120

110 108

100
80

74

69

60
32

40

40

50

48

21
20
1

12

Slika 50. Number of the collected literature about Proteus anguinus specimens since
1659th till today

The first discussion (Die Ehre des Herzogtums Crain) where the Olm has been described in
the Karst spring near Vrhnika was written by Slovenian scientist Valvasor in 1689. Almost
100 years passed until the following group of studies has appeared. Laurenti (1768) was the
first who wrote after Valvasor, and gave zoological description of the specimen (Specimen
medicum). The following studies in the 18th and early 19th century have deal mainly with
anatomy, and autors were: Austrians (Schreibers), Germans (Oken, Rudolphi, Trevirano) and
Italians (Rusconi i Configliachi). The studies of the second half of the 19th century were
mostly dealt with the distribution of the Olm. After the first finding of Proteus at the Goruica
near Sinj, recorded by Fitzinger (1850), many authors started to discover and record the
findings. Scientists who followed Fitzinger were again Italians: Tommasini (1875) and
Marchesetti (1875, 1885), and two prominent Croatian scientists who gave their contribution
to the knowledge of Croatian fauna, Brusina (1880) and Jurinac (1886), as well as Germans:
Werner, Hamann and others. During this period, scientists have slowly started to write about
biology of the species, as well as behavior. The distribution studies have marked the begining
of the 20th century. Some of the most important authors were Kolombatovi (1902) who have
contributed to the knowledge of Dalmatian fauna, followed by Paganetti-Hummler, and again
Brusina, and Langhoffer (1912, 1915) who was the first who began to systematically explore
the Croatian cave fauna; Karaman, Brian, Babi, Hadi and Bolkay (1929) who were the first
that mentioned the findings for Bosnia and Herzegovina in Ein Beitrag zur geographischen

Verbreitung des Proteus anguinus Carrarae Fitzinger. Slovenian scientists DolivioDobrovolsky, in 1926 wrote an extend article about the skull anatomy of the Olm. At the
beginning of the 20th century the interest in the breeding of the Olm, which was enigma for a
long time, has incresed. Austrians and Germans: Nusbaum (1907), Kammerer (1912) and
Stieve (1918) who was a professor of anatomy at the Univerity of Berlin, also started to
explore the Proteus. In Slovenia, in 1924, by reorganizing the Company for cave exploring,
the systematic study of subterranean fauna has started. The most important names were
Hadi, Kenk, Kuer, Matjai, Pretner, Selikar.
More intensive research of the Olm started soon after the laboratory in Moulis was built in
1948. It is particulary interesting that the Franch in 1950 began to systematicaly solve a
problem of breeding in Proteus. So, in 1959, Vandel and Bouillon were the first who managed
to artificialy grow the Olm in the undrerground laboratory in Moulis, what they wrote in the
artical La reproduction du Prote. The first map of Olm distribution made by Slovenian
scientist Ergon Pretner (1963) and Italian scientists Giovanni Abrami (1966). The greatest
interest in the study of the Olm was recorded in the eighties and nineties. During these years,
the most researches were made by Franches (Vandel, Durand, Bouillon) and Slovenians
(Isteni, Bulog, Sket, Aljani), many of which wrote about anatomy, behaviour and breed in
the Olm. The prominent Croatian researches of that time were Raa, Garai, Gabri i
Kovaevi who all recorded the Olm findings in Croatia. Decade that followed also abound
with research. The exploring of anatomy was continued. Kleteki with co authors (1996), as
well as Sket (1997) made two very important distribution maps, which is also the last
compilation of Olm findings in Croatia. In the mid nineties, molecular-genetic researches
have been increasingly used in detecting of phylogeny, evolution, as well as in many other
areas of the Olm biology. Thus, an increasing number of Slovenian scientists started to
investigate the relationship within subspecies (Sket i Arntzen 1994), within the family
(Trontelj,Goriki, Murko-Buli,Sket 2002) and within the species (Goriki 2006, Goriki i
pela 2006, Goriki i Trontelj 2006).
This analysis shows that the most of the literature, associated with the Olm, are scientific
journals. Then follow books and descriptive journals, and more recently an increasing number
of the articles published in the newpapres (Figure 51).

26

5% 1%
8%

Scientificjournals
Books

17%

Descriptivejournals
Newpapers
Film
69%

Figure 51. Literature classification

27

4.DISCUSSION
4.1.Thefindings
This research about Proteus distribution shows that in Croatia there are 68 localities where the
Olm presence was recorded at least once. The findings are separated into four categories due
to reliability of the findings, following the next criteria. Attribute of confirmed finding gets
those localities for which there is available evidence published in the article by a qualified
author, as well as there is sample kept in some of the Museum, or the current conditions at the
locality allows future monitoring of the population. Those localities for which there is
available evidence published in the scientific journal, but individuals were not seen
afterwards, and the localities is unavailable are clasiffied as certain, but unavailable localities.
While the localities for which exist historical data about finding of the Olm published by
oldish authors, but individuals were not seen afterwards due to the inaccurate description of
the locality in the original paper because the optimal part of the object for Proteus residence is
not visited are potential localities. So confirmed, as well as certain, but unavailable and
potential localities are those for which there is at least one valid record of Proteus presence.
The validity of the findings includes seeing individuals by a professional, who is able to
recognize that this individuals are nothing else then species Proteus anguinus, and publication
of information in scientific journal. Evaluating the above facts this research confirmed a total
of 68 localities, 37 of which are confirmed localities, 10 are certain, but unavailable, while 14
of them are potential localities. Other 7 falls into category of most likely to be incorect
localities. In this category are those localities which are published data, but uncertan due to
geographical, hidrogeographical and/or environmental reasons, individuals were not seen in
recent time and the finding has not confirmed since publishing.
Istrian district recorded as many as 11 localities. Today, for just four of them we can say that
they are confirmed localities; two of them, Fontana spring in Tar near Pore and Bregi
sinkhole are the most recent Olm findings in Croatia. Problems with other seven is that this
localities are artificial holes incurred by digging canals (epi, penetration of water into the
tunnel) or by penetration of water during active mine operation (Raa, coal mine; Krapan
mine). Some of them are result of people need for reaching the water (Rakonek, wells for
water; Pula, works on plumbing). Most Istrian mines are closed now, and the parts in which
the Olm was found is unavailable and it is impossible to reach that parts furthermore. Another
example is the Cave in Vodnjan for which exist published data about Proteus finding, but
entrence is unavailable because of canalization that discharged into it, and the pit is buried.
Cave in Vodnjan is actually pit for its hydrogeological function. Slightliy different
explanation is related to the spring-cave under the Veli Vrh. In that part of Pula, respectively
the Veli Vrh are two to three wells which are relatively close one to another, but the exact
well from which the mentioned individua is unknown because of inaccurate description of the
wells in the original article. The well on Monte Grande (Pretner 1962) is described in all
older literature as the correct locality, but any of these three wells can be that well, and till
today in any of investigated wells is no confirmed the presence of Proteus.
28

In Primorsko-goranska district there are two certain, but unavailable localities, Cavern under
the tunnel through Uka mountain and excavation in Rijeka, and most likely to be incorect
locality Mueva Hiica near Skrad. Here is a similar situation as with described localities in
Istria. Therefore, excavation in Rijeka is rafinery today, and Proteus individua has been seen
during the digging for base of oil refinery. It is unable that this information can be check
anymore. Toni Raa (1980A) supposed that he saw a Proteus individua in Cavern under the
tunnel through Uka mountain in the end of seventies. He has never confirmed this
information by plausible finding. During the following research of these locality, Proteus was
never found. Mueva Hiica is cave near Skrad where the surface water were created by
directly filtration of rainwater from the surface and are not affiliated with the main
groundwater flow, so it is unlikely that this is Proteus habitat.
Two most likely to be incorect localities are confirmed for Karlovac district. That are Vrlovka
cave and Ozaljska cave, which both Langhoffer (1912) mentioned as Proteus findings.
Althought, Mr. ufflaj, who was a local aristocrat, recorded Vrlovka as Proteus finding
place, but Langhoffer himself doubted this finding, and Hirc (1893) tought that on these
locality the Olm was brought and blow out by groundwater. Till today not a single certain
founding exist, and the hydrological regime is completely changed from the days of the first
'finding', ie ground water are now in much greater depht and does not flow through accessible
part of the cave.
Clasiffying three localities in Liko-senjska district (Gacka, Gacka near the ruins of the old
castle Frankopan and Otoac) have been demanded. Five authors wrote about them, but all
five described localities relatively inexact. Brusina 1880 recorded that the teacher from
Otoac founds the individua of Proteus which he donated to Natural History Museum in
Zagreb. Six years later, Jurinac wrote about the finding of the Olm near Otoac on July, 7.
1879. After them, Langhoffer (1912) mentioned Gacka near the ruins of the old castle
Frankopan as recorded information about Proteus finding, with the same data - July, 7. 1879.
Karaman (1921) again described Gacka near Otoac, but Tvrtkovi (1993), Pretner (1968),
Raa (1980B), Kleteki et all. (1996), and Sket (1997) cited Brusina. The question is whether
the Olm can anymore be find in that part of the Gacka river, because that part of the flow is
completely changed. There is a newpaper artical in 'Veernji list' from 1985 about the seeng
of the Olm in Zapolje hamlet. Numerous investigation recently show no presence of this
species, so the locality has been checked jet. tirovaa is most likely to be incorect locality
because of hypothesis that assumed juvenil individua of the Olm allegedly was juvenil newt,
and there is no groundwater on these locality where the Olm could live. Confirmed locality
Antia cave or Ivina cave require more research to determine if these is one or two localities.
Is it really one object or two objects which are linked and makes one system. Some authors
reported different data of first finding for Antia cave and for the Ivina cave.
The well in Bliani near Benkovac in Zadar district, which could be the potential locality,
was mentioned for the first time by Kleteki et all. (1996). However, Toni Raa who receved
this information does not remeber where it came from. The locality have to be checked jet.

29

All localities in ibensko-kninska district are confirmed and there is data about Proteus
seeing in recent time for each locality.
A data about findings in Splitsko-dalmatinska district are quite confusing. Werner (1891)
mentioned Vrlika, but any subsequent information about this finding doesn't exist. Goruica
spring and sinkhole, which is the first finding of the Olm in Croatia, is certain, but unavailable
locality and have several synonyms in literature: Goruzizza (Raa 1980b), Gorizza
(Langhoffer 1912), Goruchizza (Raa 1980b), Goruica (Brusina 1908), Goruica (NN
1933; Kolombatovi 1902; Babi 1928), Goruica (NN 2007). It is impossible to dive into the
spring because the entrence has fall in and that represent a big problem. During the high
waters, there is possibility that indiviuals of the Olm are pull out, and it is necessary to
continue the research and examine the reliability of finding. Well on the island olta is most
likely to be incorect locality because individuals of Proteus can't live in such conditions
where sea water is the major influence. The Cetina spring could be potential locality due to
position of the finding as well as enough amount of water. In discussion with Branko Jali,
who occasionally dive in Cetina, I found that he has never seen the Olm on this locality so i
propose further research of the locality. Ledi (1961) is the only one who recorded the
surrounding of Gubavica waterfall as Proteus finding, but despite that fact, surrounding of
Gubavica waterfall is potential locality because the other localities along the Cetina river
suggests that it is adequvate place where Proteus could live. The same applies to the
surrounding of estanovac, which is also mentioned by Ledi (1961). I found literature data
about Proteus finding in spring of Jadro river. I noted it as potential locality because there is
no recent data about the presence of the Olm. Mr. Pervan gave information of Proteus seeing
in Velika Betina, but this informatio wasn't confirmed by professionals, so it is potential
locality which have to be checked jet. Vrgorac is potential locality because there is again the
same problem with inaccurente description of the locality in literature. There is some
indication that Vrgorac might actually be related to the Marinovia Betina, Vukuia Betina
and Velika Betina, three pits located relatively close one to another, all of which are in Karst
region at the Kokorii village near Vrgorac or that Vrgorac locality refers to some of the
springs Vir, Matica or Butina, which are located in the villages below Vrgorac.
In Dubrovako-neretvanska district were reported 6 confirmed localities, 1 certain, but
unavailable locality and 2 potential localities. In last category are Doljane and Stupa-hhle,
spring-cave near Slano. In Natural History Museum in Vienna is exemplar of Proteus from
the Stupa-hhle under the inventory number NHMW 19977/1, but till today the exact locality
has not been determined. System Vilina cave Ombla spring is certain, but unavailable
locality and data was given by Toni Raa who received information from Slovenian speleo
divers about Proteus seeing in lower siphon. After that, Petra Konrad-Kova has managed to
get into the siphon , which is quite inaccessible part of the spring, but has not seen a single
individua of Proteus. Lokalities such as the last mentioned, with inaccessible entrance for
people, in the further project 'Proteus Olm conservation project' will be subjected to
'environmental DNA' research based on the detection of genetic material of living organism
that can be collected from non-living environment (earth, air, water, fire).

30

I would like to mentio two more localities where still are not recorded the presence of the
Olm, but due to it's characteristics could be a potential findings in the future. One locality is
near Pazarite village located 13 km northwest of Gospi in Liko-senjska district in Velebit
Natural park. Still, we don't know exactly whether it is a Proteus finding because the only
information is from deliver who allegedly found it there. However, he sometimes gave fals
information about localities of the specimens that he found. Pursuant Branko Jali opinion I
suggest that these locality should be explored within the 'Proteus Olm conservation project'.
The second is Crni Vir sinkhole mentioned in 2006 by Tvrtkovi and Veen. According to
knowledge that in these sinkhole lives cave crustacean genera Troglocaris and Monolistra,
Crni Vir could be a potential locality of the Olm. Also, due to location of Crni Vir on the right
bank of the Neretva river, on these locality gravitated the grounwater of Popovo polje. Branko
Jali has not recorded a single specimens of the Olm for these locality, but because of
caracteristics it should be listed for the future researche as technique of eDNA since the dive
into Crni Vir is impossible because of narrow passage.

31

4.2.Literature
Literature data analyzing gave insight into the last three hundred years of the Olm researches.
It was necessary to see the status quo and prepare the base for further researches. Literature
data reviewing shown that the first articles were written by foreign authors. They were
primarly Austrians, Italians and Germans who were interested for the Olm because of its
unusual appearance. Soon after, samplers of Proteus were collected by travellers or naturalists
and has been used as a high status gift in 19th century. As well, 19th century was significantly
by the biospeleology beginnings which was initiated in the Dinara mountin region,
specifically in Postojna, Slovenia. Following journals about Proteus distribution were also
made by foreign scientists. Thus, Slovenian scientists have increasing influence in this area.
At the end of the 19th century, Dalmatian people were in contact with foreign scientists, with
whom they exchanged animal material and practical knowledge and as result arised different
travelouges usually about Dalmatian fauna. Accordance with the increasing number of
findings, more people came into the contact with this charismatic animal. A long period
passed before recognition of Proteus breeding due to the specific way of life in the
underground. Soon after the 1948th, when the underground station, a laboratory in Moulis
('Experimental Ecology Station of CNRS at Moulis') was constructed, the latest researches of
Proteus breeding were made. The goal of the laboratory is researching the physical and
biological aspects of the undreground cave systems, so the most researches of that time were
from laboratory in Moulis and were related to the Olm behaviour, reproduction and anatomy.
In general, up to the end of the 20th century, the primary articles were made by France and
Slovenian scientists who worked in superior underground laboratory in Moulis and Postojna.
The last two decades have been characterized by evolution and molecular genetics researches
which became a popular tehnique in scientific circles.
Scientific articles classification shows that the first scientific papers were books and scientific
journals, while in recent time there is increasing number of newpaper articles due to
increasing of sensibility and interest of the general public.

32

4.3.Threatsandconservationactionplanning
Human-induced threats to the speleological objects and associated fauna are varied and
numerous. The major threats on underground habitats are:

urban pollution such as garbage dumps in karstic springs and sinkholes (Cave in
Vodnjan, Markarova cave)
restriction of water levels
equipment and tourists excessive visitation of caves (uderina jama)
dams and small-scale hydroelectric power stations which cause a change in the stream
profile and reduces food intake into the underground (system Vilina cave Ombla
spring)
modification of water flow
destruction of caves and their associated networks of cracks by exploatation of
guarries, building of roads and highways and widening of highways, clandestine
excavations in the remoter parts of caves, or because of vandalism
pollution of the underground waters and dumping of organics acids in the underground
river (Rokina bezdana pollution of Stajniko polje as well as Jezerana leads to the
accumulation of waste water in the underground which the local people use as
drinking water)
illegal collection of this species for the pet trade (Bedek i sur. 2009, Ozimec 2006)

It is necessary to protect the speleological objects through water resource management and
regulations of tourists visiting the caves. Pincinova jama is given some form of statutory
protection because the cave hold especially high number of species (Proteus anguinus,
Niphargus heberer, Hadzia fragilis, Sphaeromides virei, Troglocaris schmidti and others), but
it's still in dangerous. Nimfer, the spring in the center of Pula is not currently threaten, and
this state should be maintaine and on the building should be make an education panel with
information about the Olm. Similar situation is with Krevac spring where should also be
make an education panel with the purpose to bring people together to realize conservation
benefits. In Istrian mines with Proteus presence, such as coal mine Raa, should be remove all
sources of pollution and remediate the surface water with the goal of protection the
underground fauna. On some localities is necessary to set up the tables to stop throwing
garbage in the cave and organize the educations and senzibilization of local people (RueicaZeleno jezero, Obajdin pilja, Golubinka). It is desirable to induce people in the consequences
of negative effects on underground systems, such as Zagorska Mrenica source which is by
underground streams associated with several localities that together supply the Ogulin region
with drinking water. Many caves needs further research (Klisura, cave opposite to the lake
Torak, Miljacka I, Miljacka II and Miljacka V) and should be raise founds for following
researches. Objects in the Krka National park, with a constant accumulation of water, stand
out for being particularly important as Olm habitats, as well as for many subterranean
crustaceans, snails and other cave visitors such as bats. Objects near the Krka river are in the
33

low protected area (Miljacka I, Miljacka II, Miljacka II, Miljacka IV, Miljacka IV and cave
opposite to the lake Torak), so they are not threaten for now. Miljacka III is located in the
canyon and are relatively inacessible to humans. Objects such as Markarova pilja, Antia
pilja and Ivina peina can be closed with a metal grate with lock doors to regulate human
activities and prevent waste dumping into the pit. Furthermore, it is necessary to continue
with education efforts to spread the world about such incredible creatures before it will be too
late to act.

34

5.CONCLUSIONS
In Croatia here are 68 localities, 37 of which are confirmed localities, 10 certain, but
unavailable, 14 potential and 7 which are most likely to be incorect. They are groupes in three
geographic entities: Istra, Gorski kotar and Dalmatia. The most localities, as many as 20, are
in the Splitsko-dalmatinska district. Followed by Istria district with 11 and Liko-senjska with
9 localities. The first specimen of Olm in Croatia was found in 1840 at the Goruica spring
near Sinj. Recent findings of the Olm are discavered in Istria, the Fontana spring and Bregi
sinkhole.
The first papers about Proteus were more related to distribution, while the later studies
include anatomy, behavior and biology of the species. The latest researches are dealing with
genetics and relationship within the order, species and subspecies.
The Olm is listed as Vulnerable because its limited area and small population size. The Olm is
in dangerous due to pollution of surface streams, degradation of underground systems,
economic development and industrial pollution.
There is still a little information about the aboundance of this species, so it is necessary to
provide guidance for future researches and monitoring of Proteus anguinus population in
Croatia, as well as protect habitat from negative human impacts.

35

CHAPTER II.

Olm as cultural herittage in Western Balkans

Duan Jeli, Katarina Koller, Ivona Buri

Croatian Institute for Biodiversity,
Croatian Herpetological Society HYLA,
I. Breznika 5a,
HR 10000 Zagreb, Croatia
jelic.dusan@gmail.com,
+385 98 608 099

36

INTRODUCTION
Unusual creatures, which can be seen at karst springs when washed out of the cave
after heavy rainfall, have been known to local people long before they cought the attention of
scientists. Indeed there is a confirmation of this in carving on an ancient stone from Venezia
(10/11 century), which has been thought to represent Proteus. This carving used to be near the
church of San Nicol, Lido island, and now is in the Kunsthistorisches Museum in Wien
(Shaw 1999).

Figure 1. Two supposed Proteus carved in a stone well-head of the 10th or 11th century, once near San Nicol
church at the Lido, Venezia4A, and now in the Kunsthistorisches Museum in Wien (Inv. No. 6825; Shaw, 1999)

Fairly realistic figure of olm is carved in a stone in Boljuni near Stock (Vega 1964). It
stands for one of the most authentic monuments of Bosnian culture originated in 1477
(Picture 2). Carvings of the olm coming out of the pit represents forces of the underground
(Lui 2008).

37

Figure 2. Olm carved on grave thombe from 1477 near Boljuni in Bosnia and Herzegovina (copy from Lui
2008)

Before Laurenti offered zoological description and naming, there was two descriptions of this
animal both written by people who had not seen it for themselves. The history of the name
Proteus goes back to Valvasor (1689) who mentioned the species, found in the intermittent
karst spring Lintvern near Vrhnika, in his treatise ''Die Ehre des Herzogtums Crain (Sket i
Arntzen, 1994). This information came from the postmaster who told him about ''supposed 20
cm long dragon (Shaw 1999). Then Steinberg recorded that:
In 1751, at a time of very great flow from the Malni springs near Planina, Primus
Sicherle [Primo Ziherl] caught five unknown fish in the Unica river, one span [c. 23
cm] in length, with snow-white skin and long tails. They each had four feet ... and they
cried and wailed as they were put from the net into the boat.
However, Slovenians belived that there was a dragon living in one occasional spring. Dragon
would occasionally dry the spring up, but when he would spill out the water, his offsprings
would emerge alongside. None the less, it is unlikely that the olm was found in the Bela
source near Vrhnika since it doesn't hydrogeographically and geologically match the typical
habitat, and it is different from all confirmed localities (Kakarigi 1993). The second record is
from 1751. Steinberg recorded that in time of high water rank, Primus Sicherle caught five
unknown fishes in the Unica river, 23 cm long, with snow-white skin and long tails (Shaw
1999).
Joseph Nicolai Laurenti was the first to offer a formal draw and description of the olm in
1768 in his famous ''Specimen medicum (Figure 3 and 4). Laurenti's description was short
but sufficient and has first ilustration of Proteus (which is today very famous). G.A. Scopoli
send him a dead specimens from the Stina spring, 40 km southeast of Ljubljana (Shaw
1999). The first specimen inside a cave were discovered in 1797 by Josef Jerinovi von
Lewengreif in the Pivka river at rna jama (Sket 1997). After a scientific writing Specimen
medicum followed a series of papers about the olm.

38

Figure 3. First discription of Proteus anguinus in J.N.Laurentia Specimen medicum

printed in Vienna in 1768

Figure 4. Drawing of olm in Laurenti`s work Specimen medicum

Scopoli, in 1772, was the first to mention that Proteus is an amphibian and Schreibers,
in 1801, published a study about anatomy of Proteus (Pretner 1968). Another more formal
description gave George Shaw in 1802. It is less important than Laurenti's, not being the first,
but it is much more detailed. Four pages were devoted to this animal but its illustration was
reverse and copy version of Laurenti's picture from 1768 (Shaw 1999). MM. Cuvier and
Rudolphi were examining the internal structure of this animal and they discovered that the
39

Proteus was not a larva, as supposed, but a perfect animal (Configliachi i Rusconi 1821). John
Russel in 1822 visited Postojnska jama and wrote the first detailed description of the
behaviour of Proteus. During his visits to Postojna in 1828, Charles Babbage, who liked to
collect various things, though not connected with his own scientific area, took away some
specimens. He described this animal as ''a cresture living only in waters of dark caverns, with
eyes, but cannot open the eylids. These is probably the oldest description of Proteus in
english (Juni 2005). Lionel Smith Beale visited Postojnska jama on 24 August 1850 and
probably then obtained his Proteus, which he later continued to breed and record further
development of individuals outside their natural habitat. In 1861 Hugh Falconer visited
Postojnska jama and wrote to Charles Darwin about the Proteus anguinus he brought back
with him. Falconer offer the animal to Darwin who refused due he wasn't able to provide a
good home for this animal and suggested that London Zoo would be a better place for it
(Shaw 1999).

Proteus as a gift
Especially in the early days when Proteus was a newly discovered as well as a very strange
animal, many specimens were sent away from Slovenia as gifts to interested scientists and
influential people. Scopoli, already mentioned as having supplied the specimen that Laurenti
described, sent preserved specimens to Carl Schreibers (1775-1852), Director of the
Naturhistorisches Museum in Wien, who passed some on to other similar institutions. Later,
live Proteus were sent there too. Baron Sigismund [iga] Zois (1747-1819), who himself
studied the animal, also sent specimens to Schreibers and elsewhere abroad, as did a 19th
century director of the Ljubljana Museum, Heinrich Freyer (1802-1866). It was Zois who
supplied Archduke Johann with the ones he kept in his aquarium in Wien.
Proteus has been used as a high status gift in more recent times too, as an animal specially
associated with Slovenia. Thus in the 1960s about five from Planinska jama were given by
President Tito to Emperor Hirohito of Japan, himself a biologist.

Proteus for sale

Proteus was offered for sale at least as early as 1816. It was in August of that year that Pietro
Configliachi and Mauro Rusconi had looked for them in rna jama and wrote in their Italian
monograph: ... the people of Adelsberg catch olm, which they call white fish, in rna jama
and they keep them alive in pots to sell later to travellers who come to Carniola and are
interested in such things, or else to take to the market at Trieste where they sell quite cheaply,
for two or three lire each. This market was renowned for the variety of fish etc. that it sold.
Sale of Proteus at Trieste was made known in English in 1820 when W. A. Cadell published
his account of visiting Postojnska jama in November 1817. Speaking of olm, he wrote The
country people sometimes bring them alive to Trieste, and sell them as objects of curiosity.
40

News of it was further spread when Configliachi and Rusconis statement was reprinted in the
several editions of the popular book The Caves of the Earth, first published in 1847.

Proteus for eating

Although Cadell had said that Proteus were sold in Trieste as objects of curiosity, their
availability in a fish market suggests that there at least they were sometimes sold as food. The
first documented occasion of Proteus eating was in 1834, as reported by Hohenwart a few
years later. In that year the people of Potiskavec in Dobrepolje (Dolenjska) were cleaning out
the cave where they obtained their drinking water (Potiskav{ka jama, from which the
villagers still get their water in times of drought15). Along with mud and stones they found
several Proteus which they put aside to return afterwards. They themselves did not plan to eat
the animals which were probably regarded as poisonous like some similar creatures; but a
group of gypsies fried them and ate them without any ill effect.

Even some guidance on how to transport olms was offered in a popular book, The
Subterranean World (Hartwig 1871; Figure 5), that was printed in many editions and in at
least three languages between 1863 and 1881:
The best method for transporting the Proteus is now perfectly understood, and living
specimens have been conveyed as far as Russia, Hungary, and Scotland. All that they
need is a frequent supply of fresh water, and a careful removal of all light. Their
food need cause no trouble, as the water contains all they require. It is recommended
to lay a piece of stalactite from their native grotto in the vase in which they are
transported. When resting or sleeping, they then coil themselves round the stone, as
if tenderly embracing it. In this manner they have already been kept above five years
out of their caverns. The guides to the Grotto of Adelsberg have always got a supply
on hand, and sell them for about two florins a-piece.

Figure 5: Proteus in The Subterranean World (Hartwig 1871) of 1871 which says that the animals can be bought
in Adelsberg (Postojna, Slovenia)

41

Historical facts on the sale of Proteus are interesting now but they will not have had any
influence on travellers and visitors at the time. Quite different is this statement in first edition
of Murrays Handbook for Travellers in Southern Germany (Murray 1890):
Specimens of the Proteus may generally be purchased at the inn at Adelsberg. The only
means of preserving it is by keeping it in water, which should be taken from a river, and
should be repeatedly changed, protecting it from the light, which is very hurtful to it,
and maintaining an equal temperature about it.
That travellers not only read but acted upon the Murrays statements is clear from their
repeated references to using river water and changing it frequently during their journey home.
As would be expected, the constant trade in live Proteus reduced their numbers but it is
unusual to find, in a century that was far from conservation-conscious, a statement like this in
the same book as early as 1863:
... as hundreds of specimens have since found their way to the cabinets of naturalists, to
be observed, dissected, or bottled up in spirits, their number has very much decreased,
and the time is perhaps not far distant when they will be entirely extirpated in the
grotto, where from time immemorial they had enjoyed an undisturbed security.

Figure 6. Watercolour of two olms in John Olivers manuscript (Oliver 1856)

42

Olm in Croatia (Hypochthon carrarae & Proteus croaticus)

At the beginning of research of Proteus anguinus in Croatia, in early 19th century,

foreign scientists had a major impact on the direction of zoological research of Croatian
region. Approach was sistematicaly-faunistic, and the first explorers were mostly foreigners
with whom local naturalists were in touch. Besides foreigners, first collectors of fauna on
these area were wealthy citizens of Dalmatia, who were in contact with foreign, mainly Italian
biologists, with whom they exchanged the animal samples. In 1853, Bottieri wrote about
herpetofauna of some Dalmatian islands. ''Herpetologia europaea, by E. Schreibera (1875), is
of great importance since only Croatian herpetofauna was taken into account. The great
influence on knowledge about Croatian hepretofauna had Fr. Werner, scientist from Vienna,
who provided an article about knowing amphibians and reptiles of Istria and Dalmatia (Babi
1928).
Many authors wrote about the first specimen of Olm in Croatia found in 1840 at the
Goruica spring near Sinj (Fitzinger 1850). Seventy two years passed since the scientific
description of Olm (Laurenti 1768), until it was found at the croatian karst area (elaborated in
Kleteki et al. 1996). Later this findings had been described by many authors, like PaganettiHummler (1902) and Brusina (1907), but Fitzinger (1850) was the first who wrote about this
finding. He descriebed the Olm as a new species Hypochthon carrarae. Fitzinger (1850)
described seven new species under the Proteus genus (Hypochton zoisii, H. schreibersii, H.
freyer, H. haidingeri, H. laurentii, H. xanthostictus, H. carrarae). Some of his typical
localities are only a few kilometres away from each other or are even in the same cave system
what makes them unlikely to be distinct subspecies (Sket 1997). Despite Fitzinger's taxonomy
wasn't accepted, Mertens and Wermuth (1960) mentioned twelve different names. Today we
know that there is one species and possibly two to three subspecies (P. a. anguinus and P. a.
parkelj) (Gasc 1997). There is an ongoing research by Croatian and Slovenian scientists who
are trying to prove that genus Proteus has two distinct species.

43

Figure 7. Olm prepared in wax in 1807 for the first exibition in Natural History Museum in Vienna (foto: Duan
Jeli with kind permition of NHMW)

After finding the Olm at Goruica spring (near Sinj), data about new findings started to arrive
(other localities all around the karst region of Croatia) and several authors wrote about them.
Ottoman Tommasini (1875) wrote about the Olm from the Vrlika in Dalmatia. In 1880,
Mihovil Jurkovi gave Brusina a sample of Proteus. Sample was from Gacka river, near the
ruins of the Frankopan town. Since Brusina believed that this sample was not the same one as
in Slovenia and Dalmatia, he called it Proteus croaticus (Maor 1995). In 1883, Jurinac was
sent to explore the karst area and to collect some samples for Zagreb's National Zoological
Museum. Jurinac was encouraged by the museum's director himself. Director ordered him to
search Karlovac, Ogulin, Plitvika jezera and Kostajnicu and to collect amphibia, fisches,
mollusca, myriapods and crustaceans. Also, he was supposed to examine where exactly could
Proteus be found. After the research, he concluded that the olm doesn't inhabit ''this side of
the Kapela (Jurinac 1886). The first finding of the Olm in Istria mentioned dr. C. Marchesetti
in 1885 during the penetration of water into the mine Krapan in Labin. Kolombatovi
mentioned in 1902 the Jadro river (near Solin) also as a finding of the Olm. Until that, in
Dalmatia were known only the Goruica spring (near Sinj) so he wasn't certain if this sample
of Proteus came to Jadro via underground streams. Clear overview about amphibians in
Croatia offered St. Karaman in 1921 in his ''Beitrge zur Herpetologie von Jugoslavien.
Ledi (1961) mentioned that in Mueva Hiica and estanovac were also found specimens of
Proteus. However, today we know this is not correct. Even Pretner didn't mention it in his
paper from 1963 where he gave the first distribution map of Proteus in Croatia (Slika 5). On
that map he noted 11 localities on which, according to the literature, Proteus was found (three
sites were in Lika). According the Pretner's map, there were known 37 localities of wich 35
were within area of Jugoslavia, and only 2 in Italia close to Bosnian border. Abrami (1966)
increased the number of localities of the areal in Italy.
Jela Pavleti recorded Ivina peina and Markarova pilja in 1964, when she was writing about
amphibians and reptiles of the National Zoological Museum in Zagreb. Gluevi (1969)
44

wrote about the Olm from Neretva river, and also mentioned villages Vid, Prud and Momii.
Further discoveries were Rokina bezdana near Jezerane in Lika in 1975, Pincinova peina
near Pore in 1976, and uderina jama near Dugo polje in Dalmatinska zagora in 1979
(Garai 1980).
By 1980 the several localities were known: Cetina spring, Markanova peina near Liko
village Stajnice, cave near Dabar, Gacka near Otoac, Goruica spring near Sinj, Vrlovka on
Kupi river near Kamanje, lower course of the Neretva river, Rokina bezdan in Lica, pit near
Pazina in Istra and uderina jama in Dugopolje what is all written by Gabri. Toni Raa
(1980b) gave a list of almost all previously known localities of the Olm in Croatia. Veernji
list (1985) published the findings of Olm in Zapolje. First finding of Olm in National park
Krka was the cave Miljacka II in 1989 and was noted by De Luca (1990). Tihomir Kovaevi
in his paper from 1992 reported a total of 25 certain localities of the Olm in Croatia. Kleteki,
Jali and Raa (1996) wrote about distribution of the Olm in Croatia and recorded 47
localities, 40 to be confirmed localities. Findings of the Olm in Zagorska pe near Mrenica
were published in Veernji list (17.1.1999.) on pages 26/27. These are the first findings for
Karlovac Country and also the northeasternmost evidence of Olm in Croatia (Ozimec 1999b).
According to Eduardo Kleteki, since 1999 there were approximately 50 confirmed localities
of Proteus anguinus in Croatia. Each of them are separate speleological objects for itselves. In
Slovenia 60 localities were recorded, but some of these cave systems often have more than
one entrence (Kozaranin i Boi 1999). The last review of Olm's distribution was given by
Sket (1997) and Durand (1998). Today we have approximately 300 localities of Proteus
anguinus (Laurenti, 1768) evaluated and listed, 150 in Slovenia, about 60 in Croatia and 30 in
Italia (Ozimec i Lui 2002).

Figure 8. Skeleton of Proteus anguinus from 1864 (foto: Duan Jeli with kind permition of NHMW)

Olm in Bosnia and Herzegovina

In the same time, in Bosnia and Herzegovina dr. Stjepan Bolkay offered the first data about
Proteus. He induced in 1895 that four animal was captured in Studenci near Ljubuki. That is
considered to be the oldest record of these species in Bosnia and Herzegovina (Bolkay 1924,
1929). Another significant contribution about olm's habitat for the area of Trebinje and
Popovo polje gave Stevo ukovi, biology teacher from Trebinje (ukovi 1967, 1983). He
listed ten localities of Proteus for the area of Trebinje and its surrounding. He also pointed out
the danger of building a hydroelectric power plant on Trebinjica river which floods Popovo
45

polje. The HE was open shortly after, in 1979 and Trebinjica river is no longer considered to
be a gulf. Also Popovo polje is no longer being flooded. On the territory of the municipality
of Trebinje, ukovi ranked localities into two groups. One group is located on the left side
of Trebinjica river, and the second, much more numerous, is in Popovo polje. In the eastern
part of Trebinje, ukovi detected eight localities, southwest of Trebinje two localities and
northwest two more. In Popovo polje along the left side of Trebinjica river, where no data
was recorded in the past, ukovi found seventeen localities. He believed that the number
of localities in Popovo polje is even higher and that Popovo polje is the richest part of ex
Yugolavia in number of localities. He also announced that Proteus population of Popovo
polje is extremely endangered (ukovi 1983). Vojisalav Mikuli and Aleksandar Janii
wrote about findings of olm near Mostar. Mikuli mentioned that in the area of iroki Brijeg
in 1935 some fisherman noted the olm at the spring of Litica river (Litica river is also a
gulf). In 1968 Janii caught the olm in Radoblje cave, and later he breed and observed the
olm (Janii 1970; Mikuli 1970). Contribution to knowledge about the dispersion of olm in
Bosnia and Herzegovina gave Boris Sket, Slovenian biologist (1997). He noted 41 localities
for Proteus in Bosnia and Herzegovina. According to the last list made by Kotroan (2002),
in Bosnia and Herzegovina exists 57 localities. The new list contains localities described
after 1997, with some of the previously known localities that Sket didn't include into his
previous list.
As the science developed, the nineties were characterized by molecular and genetic analysis.
The first description of Proteus anguinus parkelj was offered by Sket and Arntzen (1994).
They also pointed out the genetic differences between subspecies together with differences
inside population of Proteus anguinus anguinus. During the 2000, there was a lot of
phylogenetic analysis on Proteus populations, mostly made by Slovenian scientists. In 2006,
Goriki pela graduated on phylogenetic and morphological analysis of Proteus. She
indicated that there are five geographically isolated populations (Istra, SW Slovenija, SE
Slovenija, Dalmatia and Gorski kotar) and the Istrian population differs the most from other
populations in genetic and morphology due which should be considered as a separate
specimen. In years that followed, the olm caused the interest of many scientists and influential
people. It is interesting that ukovi, along with his coworkers (2008), wrote a study of
subterranean invertebrates in Montenegro and put the picture of Proteus with note that this
individual is from Niki spring near Montenegro. However, Proteus was never found on this
location and with no photograph evidence of this animal the Niki spring cannot be
considered as valid.

46

Modern conections
Even in modern times Proteus still manages to amaize people. It is still considered a symbol
of clear drinkable water and it would be hard to find a person that does not know this animal.
It is present in everyday life and it is used as symbol in various occasions.
There is a folk wisdom that is normaly said to people with pale skin colour:
You are as pale as the Proteus.[Blijed si kao ovjeja ribica.]

In many areas in Croatia and Bosnia and Herzegovina, when asked if the spring water is
drinkable, local people usually say that the water is even good enough for the Olm. Most of
them will also start to tell about their, or their friends encounter with the Olm in this or some
near spring. All this just to prove to you that the water is clear and good for drinking. In many
of this areas Olms were never found but the folk tales are being told around countries and are
often repeated even in areas where Olm is not present. All of these facts serve as a good
ground for explaining to the local people the need for conservation of Olms and their cave
habitats.

Figure 9. Postal stamp issued by Croatian Postal office in 2013 as a part of Croatian Fauna series. Note that
Proteus was incorectly drown with fully developed eyes and three fingers on both front and hind legs. Normally
adult Proteus do not have any eyes and three fingers on front and two on hind legs.

47

Figure 10. Proteus inspires Modern Art - Lady with a Proteus by Helena de Baross: inspired by da Vinci's
painting "Lady with an Ermine" (copyright Flickr)

Figure 11. Very rare 10 stotinov (tolar) coin with

Proteus anguinus from Slovenia (1993)

48

Figure 12. Postal stamps of Slovenia Cave Fauna 4v / Monolistra spinosissima, Aphaenopidius kamnikensis,
Proteus anguinus, Zospeum spelaeum. / Date of issue: 12 July 1993

Figure 13. a) Logo of Karst

Research Institute (Intitut za
raziskovanje krasa, Titov trg 2, SI6230 Postojna, Slovenia); b) logo of
Tular cave laboratory, Slovenia

49

Figure 14. Specificaly designed

chocolate with Proteus anguinus.
Sold in Lucifer Chocolate Shop in
Postojnska jama, Slovenia
(http://www.luciferchocolate.si/posute-cokoladesweetnes-of-slovenia)

Figure 14. Chocolate Souveniers with Proteus anguinus (made from 64% chocolate and white chocolate in
wooden box)

50

Literature:

Oliver, J. (1856): A description of the caverns of Adelsberg... together with ... supplementary
notes. Manuscript in library of Karst Research Institute, Postojna, 49+[56]pp. (p.[28] of the
Supplementary Notes and Proteus picture opp. p.29 of the main text) Oliver, J. 1856.
Opis Postojnske jame ... skupaj z ... dodatnimi opombami. Rokopis v knjinici Intituta za
raziskovanje krasa ZRC SAZU v Postojni, 49+[56]pp. (p.[28] Supplementary Notes and
Proteus picture opp. p.29 glavnega besedila)
Hartwig, G. (1871): The subterranean world. London, Longmans, Green. xix, 522pp. (p.167)
Murray, J. (1890): A handbook for travellers in South Germany and Austria. Part I. ...
London, J. Murray, xiii [1]-373, 567-626 (p.216)

51

CHAPTER III.

Monitoring of Proteus anguinus Laurenti, 1768

populations in Croatia

Objective1:ImprovethegeneralknowledgeoftheOlmdistributionandrecord
possibleonsitethreats.

Duan Jeli1, Petra Kova Konrad12, Vedran Jali2, Maja

Luka3, Ivan Cizelj4
1

Croatian Institute for Biodiversity,

Croatian Herpetological Society HYLA
2

FREATIK Society for karst research

Veterinary Faculty, University of Zagreb

4

Zagreb city ZOO

INTRODUCTION TO CAVE DIVING

Cave diving was used on this project for all inferred activities explained in Chapters I.,
III. and IV. Most of the cave diving was done to examine submerged cave systems in order to
confirm recent presence of olm (confirmed distribution - Chapter I.) and for setting up
monitoring programme (this Chapter). Monitoring of olm populations could only be done in
cave systems that are considerably easy to access and where it was possible to set up transect
lines. This was important for safetly reasons but also for ensuring statistical significance of
the results gathered (Figure 2.). Furthermore cave diving was done in interesting submerged
systems where Proteus anguinus was registered frequently in order to gather more data on
characteristics of their habitat. Behaviour of observed individuals was also carefully recorded,
photographed and filmed.
Specialized automatic data loggers were placed inside the four monitored cave
systems (Rupeica Swallow hole, Markarova cave, Miljacka II. cave and Picinova pit). These
data loggers are registering every 2 hours water temperature (C), water pressure (hPa) and
water depth change (m).Data loggers will be taken out after one year to download the data (~
April 2014).

Figure 1. Cave divers getting ready for diving

Figure 2. Scheme of the transect line

position in Rupeica spring and swallow
hole

1. Cave diving in submerged cave systems:

-Swallow hole Rupeica
-Spring of Rupeica
-Spring Krevac
- Spring-cave Miljacka II
-Spring-cave Miljacka V

2. Putting safety line and transect line in following cave systems:

-Swallow hole Rupeica

-Spring of Rupeica
-Spring Krevac
- Spring-cave Miljacka II
-Spring-cave Miljacka V
-Spring cave Markareva
-Spring of Zagorska Mrenica
-Cave Miljacka III

3. Diving in 15 submerged cave systems for gathering data on characteristics of the

habitat. The diving has been done in following submerged cave systems:
- Swallow hole Rupeica
-Spring of Rupeica
-Spring Krevac
- Spring-cave Miljacka II
-Spring-cave Miljacka V
-Spring cave Markareva
-Spring of Zagorska Mrenica
-Cave Miljacka III
-Spring of river Ombla
-Pincinova pit
-Pit Torak in canyon of river ikola
-mitovo lake
- Pit Rokina bezdana

- Spring Bistrac
-Cave system Peine-Veliko vrelo

4. Monitoring activities in 4 locations. The monitoring has been done in 6 instead of 4

locations in following submerged cave system:
- Swallow hole Rupeica: Monitoring on 19.1.2013., 12.5.2013.,6.7.2013. (Protocols in
Appendix)
- Spring of Rupeica: Monitoring on 19.1.2013., 12.5.2013.,6.7.2013. (Protocols in
Appendix)
- Spring Krevac: Monitoring on 3.9.2012.,18.11.2012., 29.3.2013., 30.6.2013., (Protocols in
Appendix)
- Spring-cave Miljacka II: Monitoring on 27.9.2012.,6.5.2013., 29.6.2013., 28.7.2013
(Protocols in Appendix)
- Spring-cave Miljacka V: Monitoring on 27.9.2012.,6.5.2013., 29.6.2013., 28.7.2013.,
(Protocols in Appendix)
- Markareva cave: Monitoring on 2.9.2012., 6.1.2013., 2.5.2013., (Protocols in Appendix)

5. Gathering photographic material on Proteus anguinus and other species in the

habitat. The underwater photography has been done in following submerged cave
systems:
- Swallow hole Rupeica
- Spring of Rupeica
- Spring Krevac
- Spring-cave Miljacka II
- Spring-cave Miljacka V

Methodology
Since cave diving is different from other recreational diving activities, many of the
techniques used are also much different. Because there is little to no visibility in caves and
cave divers must use their own source of light, guidelines must be placed to ensure divers can
find their way back to a cave's entrance. These are placed throughout the main tunnels of a
cave. The main line of a cave does not extend to the exit -- this prevents open-water divers or
untrained or uncertified people from viewing it as an invitation to enter the cave. Therefore, a
main guideline starts 10m inside the cave.
For activities in the project open circuit cave diving equipment has been used. This means that
divers used double thanks with valves and manifold, double first stages from the regulators
for cold waters, primary and secondary lamps, reels and spools.
Because of low temperature of the water and duration of the dive (90-120 min in 7-12C) the
divers used dry suits.
For monitoring of Proteus anguinus (since this is a new method) a special methodology has
been developed. The divers marked the 3mm synthetic line every 2 meters with numbers. The
numbers symbolised distance from the entrance. The line was attached to the bottom of the
cave channel in the middle of the cave channel (in ideal situations). The divers during the dive
separately count the number of Proteus anguinus and their distance from the transect line
(Figure 2).
The gathered information is written in note-books that are water resistant (Figure 3).
During cave-diving explorations of new submerged cave systems that are potential Proteus
anguinus habitats a safety line was used for orientation towards the exit.

The entire project diving was done by four project staff divers:

Vedran Jali, dive team

leader (FREATIK)

Branko Jali, senior advisor

(FREATIK)

Petra Kova Konrad, cave

diver (FREATIK)

Duan Jeli, project leader

(CHS-HYLA)

For monitoring of Proteus anguinus (since this is a new method) a special

methodology has been developed. The divers marked the 3mm synthetic line every 2 meters
with numbers. The numbers symbolised distance from the entrance. The line was attached to
the bottom of the cave channel in the middle of the cave channel (in ideal situations). The
divers during the dive separately count the number of Proteus anguinus and their distance
from the transect line (Figure 3).

The gathered information is written in note-books that are water resistant (Figure 4).
During cave-diving explorations of new submerged cave systems that are potential Proteus
anguinus habitats a safety line was used for orientation towards the exit.

Figure 3. Diver counting Proteus anguinus in Swallow hole Rupeica

During monitoring activities and cave diving explorations the following data about the habitat
has been collected:
1. Water temperature
2. Water level and force of the water current (flow): approximation made from corporations of
dry season and high water season (spring-winter)
3. Amount of sediment on the bottom of the cave channels and ceiling (approximation)

4. Geomorphology of cave channels ( size and shape of the cave channels)

5. Presence of other water fauna (type of species)
6. Presence of pollution (anthropogenic bulky waste in some of the monitored and explored
submerged cave systems)
7. Topographic mapping of cave channel (profile and layout of the cave channels-this gives
spatial data of the cave compared to environment.

Figure 4. Writing data in wet-notes during monitoring in Swallow hole Rupeica

For underwater photography 2 cameras have been used. Sea & Sea G1 camera with
underwater housing with underwater Y120 Sea&Sea flash and Canon 40 D camera with
Ikelite housing and DS 160 underwater flash.
Sea & Sea G1 camera has automatic focus and objective. For conditions of turbid
water instead of flash light the underwater light has been used. The light was 5600 Kelvin
temperature HID 35 W lamp. This had to be used when particles suspended in the water
reflected flash light and caused blurry photos.

With Canon 40D a 15mm fixed objective has been used. Also for the same reasons instead of
the flash light sometimes an underwater HID 21 lamp was used.
The underwater photography has been performed in Swallow hole Rupeica where in
the same dive two divers have been photographing. In spring Krevac only one diver did the
underwater photography since the other diver had to stay on the safety line because of sudden
reduction in visibility due to big amounts of sediment. In the Spring cave Miljacka II also
only one diver did underwater photography due to difficult transport of the equipment. The
sump is 250m from the entrance of the cave, and the cave is 200 m from the road so the whole
diving, caving and photo equipment has to be transported by only 2 divers 50 kg of equipment
per diver).

Incountered Difficulties
Since cave diving techniques combined with monitoring techniques was a new methodology
and due to extreme and difficult conditions in the caves some of the problems occurred.
The following difficulties have been recognized:
1. Transect line and safety line cannot be fixed to the bottom in the middle of cave channel.
This is due to big amounts of fine grained sediment and fixation of the line would cause
steering up of the silt. This would result in zero visibility conditions where monitoring or
other activities would be impossible to perform (Figure 5).
One of the solutions for the future is putting "anchors" on the sediment in form of steel bars or
weights. If this was done it would take several extra dives and a waiting period for the
suspended particles to settle. In some parts of cave systems where there is very little water
flow this could take days.
2. The silt is on the ceiling of the cave channel causing zero-visibility within seconds so
counting of Proteus anguinus and writing of the data into the wet-notes is very difficult. This
happens because with open circuit systems exhaled air in form of big bubbles hits the ceiling
and causes silt suspension. Possible solution is using rebreather systems for diving. This are

close circuit systems that do not out let exhale bubbles and thus do not cause silt from the
ceiling to get suspended in water (Figure 5).
3. Transportation of equipment in deep pits and narrow passages is very exhausting and this
problem can be solved by engagement of more cavers that will help transport equipment
along the vertical line using DED and cave rescue techniques (Figure 4).

Figure 4. Narrow passage in Markareva cave

Figure 5. Diving in low visibility

conditions

Conclusion
Directions of underground waters are still quite unexplored and this is why this
exploration is significant for future protection of Proteus anguinus habitat. During the
exploration pollution with bulky waist was noticed in several cave systems (Figure 6).
Construction of buildings, railroads and roads above or near cave passages may result in their
permanent pollution or collapse of channels and endangerment of Proteus anguinus and other
cave fauna. Exploration of cave passages and their mapping can insure adequate protection of
water and its unique underground fauna.
The new methodology for monitoring has been proved effective and will be used in
future projects. Results of this exploration will be represented to the local population
especially to the children in elementary and high-schools to achieve preventive protection of
underground waters and Proteus anguinus.

Figure 6. Waste near a Proteus anguinus habitat - Spring of Zagorska Mrenica

Figure 7. Olm photographed in Rupeica cave system during monitoring

FILMING OF THE OLM DOCUMENTARY

The goal of filming has been to capture Proteus anguinus in its natural habitat, in
various locations, some of the polluted. Also the goal was to film the habitat, its physical
parameters, and also monitoring activities done in a specific and extreme conditions as well as
other project activities.

Figure 8. Filming of start of the dive (descent)

Certain amount of filming has been done also on surface of Proteus anguinus habitats
to show a specific karst landscape.

Techniqual Equipment and Methodology

The filming has been done with camera Sony Z1. This camera provides HDV with the
1080i Standard and joining the existing DVCAM range, the HVR-Z1E offers affordable

migration path from Standard Definition whilst retaining the popular DVCAM benefits such
as ease of use and i.LINK (IEEE1394) connectivity. It does 60 frame, 50 frame, 30 frame, 25
frame and 24 frame standards, HD to SD down conversion,record in widescreen HDV and
output widescreen DVCAM and DV video.
For underwater filming for this camera Amphibico FXZ1 Phenom housing has been
used. For filming of habitat a 94 Super Wide Angle port has been used, Pal/NTSC Monitor, 2
x Sony NPF570 batteries for monitor, and for filming of Proteus anguinus and other species
and Micro lens has been used.
For underwater light an underwater video lamps have been used. Two different sets of
light have been used. For filming habitats, cave systems an HID Beer sub lamps have been
used. High-intensity discharge lamps (HID lamps) are a type of electrical gas-discharge lamp
which produces light by means of an electric arc between tungsten electrodes housed inside a
translucent or transparent fused quartz or fused alumina arc tube. HID lamps are used when
high levels of light over large areas are required, and when energy efficiency and/or light
intensity are desired.
For filming of Proteus anguinus and other fauna a halogen 50 W lamps have been
used. This lamps have been used because a halogen lamp produces a continuous spectrum of
light, from near ultraviolet to deep into the infrared. Since the lamp filament can operate at a
higher temperature than a non-halogen lamp, the spectrum is shifted toward blue, producing
light with a higher effective color temperature.

Filming locations
The filming has been done in several locations. The underwater filming has been done in
Pincinova pit, Swallow hole Rupeica,Markareva cave,Spring of Zagorska Mrenica.
1. Pincinova pit

Location

Pit in Istria, Village Nova vas

Type of habitat

Vertical pit with submerged bottom

Population size

40-70

Frames filmed

o Macro Proteus anguinus

o Habitat morphology

Minutes of filmed 45 minutes

material

Figure 8. Filming in the cave system of

Miljacka cave

2. Swallow hole Rupeica

Location

Ogulin-Plaki area, village Ivanci

Type of habitat

Submerged cave system

Population size

100-150

Frames filmed

Macro Proteus anguinus

Habitat morphology

Contamination with athropogenic waste

Monitoring activities

Sampling activities

Threats

Minutes of filmed 180 min

material

3. Markarova cave

Location

Stajnica karst polje, Village Stajnica, Jezerane

Type of habitat

Cave with vertical entrance and submerged channel

Population size

500-1000

Frames filmed

Macro Proteus anguinus

Habitat morphology

Vertical techniques used for caving

Monitoring activities

Minutes of filmed 120 min

material

4. Spring of Zagorska Mrenica

Location

Ogulin-Plaki area, Village Desmerice

Type of habitat

Spring

Population size

40-60

Frames filmed

Macro Proteus anguinus

Habitat morphology

Contamination with athropogenic waste

Monitoring activities

Cave diving tehniques

Threats-waste

Minutes of filmed 120 min

material

5. Miljacka 1

Location

Oklaj, Np Krka

Type of habitat

Submerged cave system

Population size

10-15

Minutes of filmed 45 min

material

Surface filming has been done in Ogulin-Plaki area.

LOCATION

Spring of Zagorska Mrenica

FRAMES FILMED

Duan Jeli preparation of

MIN

60

diving

PetraKova-Konrad

60

preparation of diving

Swallow hole Rupeica

60
Landscape

Sampling

60

Spring Bistrac

10

Simposium

60

Recomendations for future projects

For future projects it would be very attractive to film on more different locations like
cave Miljacka III that is a cave in sedra rock. This specific habitat conditions are very
interesting. Also filming in Rokina pit, that is 100 m deep pit with underground river and a
big population of Proteus anguinus will show very different environment.
Since Proteus anguinus feeds on different water fauna it would be useful to film this
water fauna. Some of them are Troglocaris sp., Monolistra sp., Alpioniscus sp., and others.
Other approach should be more focused on the threats. Like railroad construction in
Ogulin-Plaki area, road constructions, waste disposals near the Proteus anguinus habitats like
near Pincinova pit, Spring of Zagorska Mrenica and others.

In this moment 13 hours of raw material is made and captured, by the end of
editing probably there will be more then 20 hours of raw material.

PLEASE SEE EDITED 10 MINUTES OF SELECTED RAW MATERIAL

ATTACHED AS APENDIX I. TO THIS REPORT.

Objective3:EstablishcaptivebreedingandrecoveryCentre.

EX-SITU REHABILITATION AND CAPTIVE BREEDING CENTRE IN ZAGREB

ZOO

Keeping Olm's (Proteus anguinus) requiers

special room, because of special animal needs
(temperature, darkness, etc.). Room size of 3m x
3m x 2.8 m is therefore rebuilt. Before Olm
project, room was equiped with stainless steel sink
and electrical boiler for hot water, which are still
used. Becasue of low temperature requierement,
all walls and arch were isolated with 5 cm XPS
stiropor, which enables us to fully control room
temperature with low energy consumption.
Because of isolation factor, it was necessary to
Figure x. Cooling device for maintaining
exchange standard door with methal isolation door
room temperature on steady 6,0 - 10,0 C
filled with isolation foam. Room of 26 m is
cooled down by 3.7 kW cooling device that has fully automatic cooling and defrost system.
With souch equipment and isolation, we are able to control room temperature all year round,
and cool it down up to 6 C.

Figure x. Olm comming out of hiding

for feeding

Olm's are kept individually in 80 liter glass

aquariums, and till now, 18 aquariums are built
and prepared for animals. All aquariums are
standing on metal benches that are produced in a
way to fit into the room, and use maximum of
avaliable space. Every aquarium is aerated with
airstone connected to air pump. Surplus
aquariums are used for water preparation when
water changes is necessary. Water changes and
water distribution are done with aquarium pumps.
For desinfection of aquariums and working tools

F 10 SC desinfection solution is used.

Hiding places for animals are made of glass panels of different thickness, depending on
animal size. With souch hiding places, animals feel secure, and it is possible to monitor
animals quickly on daily basis.
Animals are fed with earthworms of suitable size and different Gammarus sp., which are bred
at zoo and readily accepted by animals.
Water quality is mesaured on weekly basis (oxygen level, conductivity, hardness, etc.) by zoo
staff.

MICROBILOGICAL TESTING (BACTERIOLOGY AND MICOLOGY)

In the period of November 2012 June 2013 we performed a microbiological testings

of 20 Proteus anguinus in total. Among them 12 animals were from Vedrine Sinj locality, two
from Fontana Istria locality, two from Markarova pilja Gorski kotar locality, two from
Rupecica Ogulin localiy, and two from Picinova jama Istria locality. All the animals from
Vedrine and Fontana localities were flushed animals with greater suspicion to harbor different
kinds of pathogens and oportunistic microorganisms.

The aim of the veterinary part of the project was to find out if it is possible to bring P.
anguinus (the olm) back to caves without harming the existing population if it will be
neccesary on some locations. Therefore it was of great importance to identify all the
microorganisms that may harm both olms found in the field and those living in the caves for
which we expected to be healthy. It was also of great importance to distinguish between
infectious pathogens and the opportunistic ones, that may appear depending on the
temperature of the environment. Since the olms who are out of caves are not kept at the
preferred temperature at which they usually live it was important to determine
microorganisms who are opportunistic, to distinguish between noncurable and curable
microorganisms and to find out the best way for prevention of infectious diseases found in
olms.
The aim of the project was also to establish the treatment of diseased animals based on
microorganisms found in each olm, and on sensitivity tests.
Finally, the proposal has the scientific aspect since the data about microorganisms harming
the olms is very scarce.
The following samples were taken from and tests were performed in each animal during the
first year:

Figure 9. Taking anal swabs from Proteus

1. Bacteriological and mycological cultures of skin, cloaca, oral cavity and the water from
the particular locality where olms were found.
Since the olms are aquatic animals it was also important to perfom microbiology tests
from the water to compare the differences between the microorganisms from the water
and from the animals.
Oral cavity, cloacal, and skin swabs, together with the water were plated on different kind
of agars, at aerobic conditions on two different temperatures: 22 and 8C. The aim of two
different temperatures was to distinguish between normal microbiota (at 8C) and the
opportunistic microorganisms at unnatural temperature (22C) on which they could be
found in the field. The plates were cheked for five days to determine the time when the
oportunistic bacteria develop. Further investigation of suspected colonies were performed
i.e. gram stainings, catalase, oxidase tests, and API commercial tests.

Figure 10. Some bacteriological and mycological cultures that were raised during testing

The following results were obtained:

VEDRINE 1
Oral cavity:
8C: Bacillus sp. (after 5 days)
22C: gram positive bacteria i.e. Bacillus sp. and Staphylococcus sp. (after two days)
Cloaca:
8C: Streptococcus sp. (after 5 days)
22C: Streptococcus sp. (after 5 days), Bacillus sp., E. coli (after more than 5 days)
Skin:
8C: negative
22C: Bacillus sp. (after more than 5 days)
Water from aquarium:
8C: negative
22C: Bacillus cereus, Bacillus sp.

VEDRINE 2:

Oral cavity:
8C: Bacillus sp. (after 5 days)
22C: Bacillus sp. (after two days)
Cloaca:
8C: negative
22C: Bacillus sp. (after more than 5 days)
Skin:
8C: negative
22C: Bacillus sp. (after more than 5 days)

VEDRINE 3 (male, flushed animal, bad general condition, wounds on the rostrum died
after few days):
Oral cavity:
8C: negative
22C: violacein producing bacteria (violacein producing gram negative bacteria further
identification neccesary to distinguish between Janchtinobacterium lividum and
Cromobacterium violaceum), P. fluorescens, Bacillus cereus (after 5 days)
Cloaca:
8C: negative
22C: Aeromonas hydrophila, Bacillus sp. (after 5 days)
Skin:
8C: negative
22C: Aeromonas hydrophila, Bacillus sp.
Intestines: negative

Liver: Micrococcus luteus

VEDRINE 4:
Oral cavity:
8C: negative
22C: Bacillus cereus, Aeromonas hydrophila, violacein producing bacteria (after 5
days), Penicillium sp. (after 6 days)
Cloaca:
8C: negative
22C: Enterobacter sp., Aeromonas hydrophila, violacein producing bacteria
Skin:
8C: negative
22C: Bacillus cereus, Aeromonas hydrophila, violacein producing bacteria, Penicillium
sp. (after 6 days)

VEDRINE 5:
Oral cavity:
8C: negative
22C: Aeromonas hydrophila, Cladosporium sp., Aspergillus sp. (after 6 days)
Cloaca:
8C: negative
22C: Aeromonas hydrophila, Rodotorula rubra
Skin:
8C: negative

22C: Aeromonas hydrophila, violacein producing bacteria, Cladosporium sp.,

Aspergillus fumigatus, Fusarium roseum (the growth of the fungi after 3 days)

VEDRINE 6:
Oral cavity:
8C: negative
22C: Aeromonas hydrophila, Enterobacter sp., violacein producing bacteria, Penicillium
sp. (after 6 days)
Cloaca:
8C: negative
22C: Aeromonas hydrophila, violacein producing bacteria, Penicillium sp. (after 6 days)
Skin:
8C: negative
22C: Aeromonas hydrophila, violacein producing bacteria

VEDRINE 7 (oral swab not taken due to the size of the animal):
Cloaca:
8C: negative
22C: Aeromonas hydrophila, E. coli (after more than 5 days)
Skin:
8C: negative
22C: Micrococcus roseus, E. coli, violacein producing bacteria, Aeromonas hydrophila
(after 6 days)

VEDRINE 8 (female, flushed animal, die after two weeks in captivity):

Liver: negative
Intestine: Bacillus cereus, Bacillus sp.

VEDRINE 9 (male, flushed animal, died after two weeks in captivity):

Oral cavity:
8C: negative
22C: Enterobacter sp., Bacillus sp.
Cloaca:
8C: negative
22C: Enterobacter sp.
Skin:
8C: negative
22C: negative
Liver:

VEDRINE 10 (female, flushed animal, die after two weeks in captivity):

Skin:
8C: Saprolegnia sp.
22C: Saprolegnia sp.
Liver: Bacillus sp., Bacillus cereus, violacein producing bacteria

VEDRINE 11 (male, flushed animal, died after two weeks in captivity):

Skin:
8C: Saprolegnia sp.
22C: Saprolegnia sp.
Liver: violacein producing bacteria
Intestine: Bacillus sp., violacein producing bacteria
VEDRINE 12 (female, flushed animal, died after two weeks in captivity):
Skin:
8C: Saprolegnia sp.
22C: Saprolegnia sp.
Intestine: Bacillus sp.

Water from the same site as the all animals except V1 and V2:
8C: negative
22C: Aeromonas hydrophila, violacein producing bacteria, Penicillius sp., Aspergillus
sp. (after 6 days)

NOTE: Six flushed animals from Vedrine locality died after 14 days in captivity.
Simptoms included cottony changes, loss of the gills and the toes, and dark red
pigmentation of distal parts of the body (tail, limbs) developed within 24 hours.The
animals were gasping at the surface due to the clogging of the skin pores and gills.
Despite of the Itraconazole 0,02% baths for 15 minutes in amphibian Ringer's solution 0,6
%, all the animals died within a few days. According to the findings, the mortality was
caused by stress due to unnatural conditions and secondary oportunistic microorganisms
development. Direct cause of the death was Saprolegnia sp. infection confirmed both
with standard microbiology test and patohistology of the skin.

Under standard conditions these fungi grow at 8oC in two to three days while much longer
time is required at 22 oC.

Collective samples of the dead animals:

Collective skin sample: Saprolegnia sp., Bacillus sp., Aspergillus fumigatus (after 3
days)
Collective sample of the leg changes: Saprolegnia sp., Bacillus sp.
Patohistology of the legs, gills and skin: Saprolegnia sp.

ISTRIA 1: (Only skin sample and feces from the aquaria were taken due to the size of the
animal)
Skin:
8C: negative
22C: negative
Feces:
8C: negative
22C: P. fluorescens, violacein producing bacteria, Bacillus sp., E. coli, Burkholderia sp.
(bacteria similar to those from the water from the same site with the exception of E. coli,
Staphylococcus sp. and Streptococcus sp.)

ISTRIA 2:

Oral cavity:
8C: E. coli ( after 2 days), Pseudomonas fluorescens (after 5 days)
22C: Bacillus sp. (after 2 days), E. coli, Bacillus sp., P. fluorescens (after 5 days)
Cloaca:
8C: E. coli, Bacillus sp. after days
22C: E. coli (after 24 hours), E. coli, Bacillus sp. (after 5 days), Aspergillus flavus (after
6 days)
Skin:
8C: negative
22C: E. coli, Bacillus sp., Aeromonas hydrophila (after 5 days)
Water from the same site:
8C: negative
22C: Staphylococcus sp., Streptococcus sp., Burkholderia sp., violacein producing
bcateria.
Water from aquaria:
8C: negative
22C: P. fluorescens, Bacillus sp.

MARKAROVA PILJA 1 (animal from the natural habitat):

Oral cavity:
8C: negative
22C: negative
Cloaca:
8C: negative

22C: negative
Skin:
8C: negative
22C: negative

MARKAROVA PILJA 2:
Oral cavity:
8C: negative
22C: P. fluorescens
Cloaca:
8C: negative
22C:
Skin:
8C: negative
22C: negative

Water from the same site as the animals:

8C: negative
22C: Bacillus cereus, Micrococcus luteus, violacein producing bacteria which grow on
22C, but not at 8C.

NOTE, Markarova pilja: After two days of incubation bacteria started to grow at 22C,
but not at 8C. This could mean that these opportunistic bacteria could potentially harm

the animals under the unnatural temperature i.e. after flushing from the underground, but
not in their natural habitat.

RUPEICA 1:
Oral cavity:
8C: negative
22C: Bacillus sp.
Cloaca:
8C: negative
22C: negative
Skin:
8C: negative
22C: Bacillus cereus, Bacillus sp.

RUPEICA 2:
Oral cavity:
8C: negative
22C: Bacillus sp.
Cloaca:
8C: negative
22C: Micrococcus luteus, P. fluorescens, Bacillus sp., Enterobacter sp.
Skin:
8C: negative

22C: Bacillus cereus, Bacillus sp., Micrococcus luteus, Micrococcus roseus Saprolegnia
sp.
NOTE, Rupeica 2: The animal from its natural habitat died after few weeks in captivity
with the same symptoms as the Vedrine animals. The direct cause was secondary
Saprolegnia sp. infection.

Water from the same site as the animals:

8 degree: negative
22C: Bacillus cereus, Bacillus sp., Micrococcus luteus, Aeromonas hydrophila, P.
fluorescens

PICINOVA JAMA 1 (Pore, Istria):

Oral cavity:
8C: negative
22C: Micrococcus luteus, P. fluorescens
Cloaca:
8C: negative
22C: Micrococcus luteus, P. fluorescens
Skin:
8C: negative
22C: Micrococcus luteus, P. fluorescens

PICINOVA JAMA 2 (Pore, Istria):

Oral cavity:

8C: negative
22C: Bacillus sp., P. fluorescens
Cloaca:
8C: negative
22C: P. fluorescens
Skin:
8C: negative
22C: Bacillus sp.

Water from the same site as the animals:

8C: negative
22C: Aeromonas hydrophila, Micrococcus luteus, P. fluorescens

From the above results it is visible that the olms have a saprofitic, soil microorganism on
their skin and in the gastrointestinal system. These are opportunistic bacteria that have a
better growth at higher temperatures, and slight or no growt at lower temperatures.
Therefore it is very important to monitor the temperature of the water in containers, and
also to collect the animals from the field as soon as possible, to avoid the growth of
opportunistic bacteria. On the other hand, Saprolegnia sp. fungus growth is much faster at
lower temperature and is dependent on immunocompetence of animals. Therefore, it is
very important to keep the animals under optimal conditions and with the minimum of
stress.

2. Parasitological tests
Fecal samples, intestine scrapings, livers, gall bladders and kidneys of dead animals were
tested for the parasitological findings.
Native smears were performed for the fecal samples and patohistology investigation of
the organs.
The aim of parasitological investigation was to find out what is normal parasitological
flora of healthy olms; this is currently not known. It is also important to detect the
parasites from thrown animals and note the differences (if any) between those animals
and healthy ones. Finally, the treatment could be established based on the findings if it
would be possible and necessary.

Figure 11. Organ check for parasitological investigation

Note: Due to their life in the water, it was difficult to obtain the correct animal's fecal
samples and to distinguish between their parasites and the free living parasites or ones
from their food. In all fecal samples ciliates and flagellates were found.

All intestine scrapings and livers of dead animals were parasitologically negative. The
organisms found could be normal intestinal flora of the olms.

The following results were obtained:

ISTRIA 1
Feces: Amoebas, flagellates, ciliates, nematode larvae, flagellate cysts - free living?
further identification necessary.

ISTRIA 2
Feces: Ciliates, flagellates (Colpoda sp.), amoebas

VEDRINE 1
Feces: Protozoans (Vorticella sp.), mites, Rotatoria sp., amoebas

VEDRINE 2
Feces: Rotatoria sp., ciliates, flagellates

VEDRINE 3
Kidneys: Myxospora sp. further identification needed

VEDRINE 8
Kidneys: Myxospora sp. further identification needed

MARKAROVA PILJA 1
Feces: negative

MARKAROVA PILJA 2
Feces: Trematode eggs

PICINOVA JAMA 1
Feces: negative

PICINOVA JAMA 2
Feces: negative

3. Rana virus identification

The aim od Rana virus detection was to find out weather the olms are receptive for this
very contagious disease, since this information is currently not available. It is also
important to determine all the amphibians carrying this virus and eradicate it to prevent its
spreading and to identify all infected olms so as not to bring them back to their natural
habitats.
Therefore all the olms were tested with real time PCR method from skin swabs.
All tested animals were negative for Rana virus.

4. Chlamydiacea sp. identification

Amphibians are also susceptible to infection with bacteria from Chlamydiaceae family
which is particulary important for the flushed animals who can get into contact with all
kinds of reservoirs like other amphibians, reptiles, birds etc...

Therefore Real-Time PCR method was used to detect the presence of bacteria from the
family Chlamydiaceae in the collective swabs of oral cavity and cloaca from each animal.
All tested animals were negative on Chlamydia sp.

5. Batrachochytrium dendrobatidis (BD) identification:

The aim of BD diagnostics was to find out weather the olms are receptive for this very
contagious disease, since this is currently not known. It is also important to determine the
BD in its very early phase, when it would be possible to give the supportive or preventive
therapy to affected animals. Real-Time PCR method was used to detect BD in swabs
taken from the ventrum of each animal.
All tested animals were negative for BD.

6. Ultrasound examination
The ultrasound examinations was performed in cooperation with Leibniz Institute for Zoo
and Wildlife Research. The aim was to detect overall healt status of all animals by
scanning the internal organs, and also sex determination which is of great importance for
future breeding program.
We measured the heart rate of each animal, to get the baseline information about normal
heart rate in olms since these data are not available at the moment. We also measured
gallbladder sizes of animals that were fasting for two weeks.

The sex determination was also performed in few animals to and the
ultrasonography was found to be useful tool for this purpose.

Figure 12. Ultrasound examination of animals to determine sex

Objective2:ConfirmingOlmintheinaccessiblecavesystemwiththe
environmentalDNAtechnique.

Figure 1. Sampling tissue for

population genetics study

CHAPTER IV.

MOLECULAR ANALYSIS OF CROATIAN POPULATIONS

OF PROTEUS ANGUINUS AND TEST THE METHODOLOGY
FOR ENVIRONMENTAL DNA DETECTION

Judit Vrs and Jlia Tnde Gl

Budapest
Hungarian Natural History Museum
Laboratory for Molecular Taxonomy, Collection of Herpetology

Budapest, 2013

53

FIRST SUBCHAPTER

Testing the methodology and confirming Olm in the inaccessible

cave system with the environmental DNA technique

1.INTRODUCTION
As partners of the olm conservation project, our task was to work out a method by which the
detection of the species becomes simpler. Our goal was to develop the eDNA method for
Proteus anguinus: (1) to test different sampling methods for cave water, (2) to develop
species-specific primers that target short sequences in the genom and (3) to optimise PCR
reactions, thermo profiles for the best result.

2. MATERIALS AND METHODS

2.1 SAMPLE COLLECTION
There are two basic methods for collecting water samples from the investigated areas:
(1) collecting 15 ml water (Ficetola et al. 2008) or
(2) collecting 2-10 litres of water and filtering it (Goldberg et al. 2011).
Since cave water has not been examined from this aspect before, we intended to test both
methods and see what quantity of water is needed to be collected for detecting Proteus DNA.
In the first phase we collected 15 ml water samples in June 2012 from aquariums where
Proteus anguinus specimens are kept in the Zoo of Zagreb, from Miljaka II cave (44.00035
N, 016.01723 E, Fig. 8) where a big population is known to live. After collection of the 15 ml
sample 1.5 ml of sodium acetate (3 M) and 33 ml of absolute ethanol was immediately added.
The samples were stored at -20C until procession.
In the second phase we took 10, 5 and 2 litre water samples in September 2012 from Rupeica
cave (45.18690 N, 015.22564 E) and filtered them through a 0.45 m cellulose nitrate filter
paper on the spot with a manual vacuum pump (Mityvac). The filters were preserved in 95%
ethanol in separate 2 ml tubes.

54

In the third phase we filtered 2 and 5 l samples from Miljaka II cave and Rupeica cave in
March 2013 when the water level was high, inaccessible for divers,
In the fourth phase we filtered 2 and 5 l samples and also collected 15 ml samples from
Rupeica cave and Picinova cave in June 2013. During these samplings we collected 15 ml
water from the bag where the captured animals were kept for about half an hour.

2.2 LABORATORY METHODS

2.2.1 DNA EXTRACTION
To get the precipitated DNA from the water samples (not filtered), we centrifuged the mixture
on maximum speed for 1 hour (SIGMA 2-16K centrifuge) and then discarded most of the
supernatant. We centrifuged the mixture again on 8000 g for 10 minutes and discarded the
remained supernatant. Afterwards, we took the samples into vacuum concentrator (Heto DNA
Mini, Thermo Fisher Scientific Inc., ID: 888872146) until all liquids evaporated. Then we
used QIAamp DNA Micro Protocol (Isolation of genomic DNA from small volumes of blood)
to isolate the DNA.

Figure 2. Sampling water from the Rupeica swolowhole

55

For DNA extraction from the filtered water samples, we used QIAamp DNA Micro Protocol
(Isolation of genomic DNA from dries blood spots). The filters were cut into halves and only
one of the halves was used. We cut these into small pieces (<3 mm) and then preformed the
extraction protocol.

2.2.2 PRIMERS, PCR PROTOCOLS, OPTIMISATION OF THE REACTIONS

We designed 35 primer pairs for Proteus anguinus targeting short sequences in the D-loop
region and in the cytochrom b gene and tested the first eight (Table 2).

Name of
the primer

PaF1

Primer
Direction

Region

Sequence (5'-3')

length
(bp)

forward

D-loop

CACCCAAGGCCAAAATTCTGAC

Melting

product

point

annealing

length

(Tm,

temperature

(bp)

C)

(C)

26

56,8
85

PaR1

reverse

D-loop

GCATAATATGTCKCCGCGTCT

21

PaF2

forward

D-loop

GACATATTATGCWTAATAGGAC

21

54,3
56,2
46,2

81
PaR2

reverse

D-loop

AATTGTGGATGMGAATTGT

25

PaF4

forward

D-loop

CAATATYCAGATCTACGGA

26

53
48,6
46,8

87
PaR4

reverse

D-loop

CWTAGATTCAAACCAGATGCC

20

PaF8

forward

D-loop

GTGGCATATAAATCTATGTC

19

53
51,3
45,6

70
PaR8

reverse

D-loop

TRTTATTCGTTTTCTAGAG

19

PaF9

forward

D-loop

AAATGAACYWTGCAAATCATC

20

53
42,5
48,5

98
PaR9

reverse

D-loop

GTTGGCTARAAAGTGTGATC

24

ProtcytF

forward

Cytochrome b

CAAATCGGMCARGCCGCCTC

20

60
50,2
61,6

82
ProtcytRI.

reverse

Cytochrome b

GTTTGTTCTCAABCMAKCCGGC

22

ProtcytFII.

forward

Cytochrome b

TCGGAGGRCAGCCRGTAGAA

20

na
58,4
60,3

57
ProtcytRII.

reverse

Cytochrome b

GGGCTGAGGCGGCYTGGCCG

20

ProtcytFIII.

forward

Cytochrome b

GCCGGMTKGVTTGAGAACAAAC

20

na
70,2
58,4

75
ProtcytRIII.

reverse

Cytochrome b

GTTTTGGTTTACAAGACCAA

Optimal

Total

20

64,7
48,7

Table 1. Tested mt-DNA primers for the environmental DNA experiment. The optimal annealing temperature
was not defined for ProtcytF-ProtcytRI. and ProtcytFII-ProtcytRII primer pairs because they did not amplify
clear fragments from Proteus anguinus DNA during optimisation.

56

The primers were designed based on the following sequence obtained from GenBank:
accession number GQ368659. We run 10 l reactions, consisting of Fermentas
DreamTaq compounds (Thermo Fisher Scientific Inc.):

1 l DreamTaq Buffer (10x),

0.2 l MgCl2 (2.5 M final concentration),
1 l dNTP mix (2mM),
0.05 l DreamTaq DNA Ploymerase (5 u/l),
1-1 l from each primer (5M),
0.4 l BSA (5 mg/ml),
3.35 or 4.35 l mQ water (depending on the quantity of the template),
2 l water template or 1 l DNA template.

First, we used DNA previously isolated from P. anguinus tissue (from Slovenia) to optimize
the annealing temperature for the primers with gradient PCR method. Additionally, we used
DNA templates from other amphibian species that occur at or near the olms habitat in
Croatia. These species were: Bombina variegata, Bufo bufo and Salamandra salamandra.
With gradient PCR, we determined the optimal annealing temperature for the primers (no
product from the other species) and the other steps (initial incubation, elongation, cycles, etc.)
were carried out based on Goldberg et al. 2011. The protocol consisted of:

initial incubation at 95C for 10 minutes,

55 cycles of 94C for 30 seconds, annealing temperatures according to the primers for
60 seconds and 72C for 60 seconds,

finishing at 72C for 60 seconds (MJ Dyad and MJ PTC-200 gradient PCR, Bio-Rad
Laboratories, Inc.).

Afterwards, we visualized the PCR products by gel-electrophoresis on 1.4% agarose gel. We

chose the two best primer pairs according to the gel photos, which were PaF4-PaR4 and
PaF8-PaR8. These did not amplify any product from the other amphibian species mentioned
above and the fragments from Proteus anguinus could be easily identified.

2.2.3 CLONING
PCR products made with the two best primer pairs mentioned above from Proteus anguinus
tissue, from the aquariums of the Zoo of Zagreb, from Miljacka cave (not filtered) and from
Rupeica spring (10 litres, filtered) were cleaned with Roche High Pure PCR Cleanup
Micro Kit (Roche Applied Sciences). Following this step, the cleaned PCR products were
cloned into pGEM T Easy vector system (Promega Corporation, Fig. 3).
57

This cloning step was needed because of the very low quantity and quality of DNA in the
samples that so far prevented us from succeeding in direct sequencing. The shortness of the
target sequences added to this problem.

Figure 3. Illustration of the pGEM - T Easy vector. Source: http://www.promega.com.

We used two different insert/vector proportions: 3 to 1 and 5 to 1. The reactions consisted of:

5 l Rapid Ligation Buffer (2x),

1 l pGEM T Easy vector, 1 l T4 Ligase,

different amounts of mQ water (0, 1, 2 l) and

different amounts of PCR products (1, 2, 3 l) depending on the DNA content

(Promega Corporation).

4 l of the ligation reaction was transformed into 25 l of JM 109 high efficiency competent
cells with heat shock and then 450 l of SOC medium were added to it. After 1.5 hours
incubation with shaking at 37C, 150 l of the transformation culture were spread out on the
surface of an LB agar plate. The ampicillin comprising LB medium selected the transformed
cells and the cells containing inserts were selected with the IPTG and X-gal system. The
plates were incubated overnight at 37C and the white colonies were picked for the colony
PCR. The 25 l reactions consisted of:

3 l template (supposedly transformed bacteria),

11.9 l mQ water,
2.5 l DreamTaq Buffer (10x),
0.5 MgCl2 (25 mM),
2 l dNTP mix (2 mM),
2.5-2.5 l T7 and SP6 primers for the plasmid (5 M) and
0.1 l DreamTaq DNA Ploymerase (5 u/l).

The PCR protocol consisted of:

initial denaturation step on 95C for 7 minutes,

58

39 cycles of 94C for 30 seconds, 50C for 45 seconds and 72C for 80 seconds
followed,

finishing with 72C for 5 minutes.

The positive colonies were visualized by agarose gel-electrophoresis. The size of the PCR
products comprising the insert was about 250 bp (Fig. 4). The PCR products were cleaned
with Roche High Pure PCR Cleanup Micro Kit (Roche Applied Sciences).

Figure 4. Colony PCR visualised by gel electrophoresis. Columns marked with * show no insertion, while
column marked with + show double insertion.

2.2.4 SEQUENCING
Sequencing PCR reactions were carried out with:

2 l BigDye Sequencing Buffer (5x),

2 l BigDye Terminator v3.1 Ready Reaction Mix (Life Technologies Corporation),

2 l of one primer,

3 l mQ water and

1 l diluted template.

All samples were sequenced for both directions. The thermal profil consisted of:

4 minutes of denaturation on 96C,

59

25 cycles of 96C for 30 seconds, 50C for 15 seconds, 60C for 4 minutes and

a finishing step for 7 minutes on 4C.

We cleaned the products with BigDye XTerminator Purification Kit (Life Technologies
Corporation). Sequencing was performed on The Applied Biosystems 3130 Genetic
Analyzer.
Sequences were cleaned and aligned with BioEdit v7.1.11 (sequence aligment editor by
Tom Hall, Ibis Biosciences), and results from water samples were compared to the sequence
of Proteus anguinus tissue DNA.

60

3. RESULTS
3.1 PRIMER DESIGN
We tested the first eight pairs of primers for the detection of Proteus anguinus DNA from
water samples. With gradient PCR we determined their optimal annealing temperatures
(Table 1). Two pairs were chosen from the primers that were proven to work best during PCR
reaction: PaF4 PaR4 and PaF8 PaR8. These amplified single, distinct fragments from the
olm that could be easily identified on agarose gel (Fig. 5 and 6).

Figure 5. Gel-electrophoresis picture of the products of the two selected primers. Left from the ladder: 1:
Salamandra salamandra, 2: Bufo bufo, 3: Bombina variegata, 4: Proteus anguinus (positive control), 5-6-7:
Miljacka water sample three repetitions, 8: Proteus anguinus (positive control), 9-10-11: Rupeica water sample
three repetitions.

61

Figure 6. Primers in the mitochondrial genome of P. anguinus. Upper row: primers PaF4 and PaR4 (see blue
anchors), lower row: primers PaF8 and PaR8 (see red flags).

3.2 WATER SAMPLES

We could amplify the correct fragments from isolated olm DNA, the aquarium sample (Zoo
of Zagreb) and from both the filtered (Rupeica collected in September 2012) and the not
filtered water samples (Miljacka-collected in June 2012).
The sequencing process and the aligment of the sequences ensured that the DNA we found in
the water samples were from Proteus anguinus (Fig. 7).

Figure 7. Alignment of the sequences obtained from water samples and from Proteus anguinus tissue to the
sequence from GenBank. The black lines show the position of the primers: from point 1 to 2: forward primer
PaF4, from point 3 to 4: reverse primer PaR4. The sequence parts outside the primers belong to the cloning
vector. Source: BioEdit Sequence Alignment Editor.

We failed to amplify Proteus d-loop fragments from the water samples collected in March
2013 and June 2013.
We succeeded to amplify target fragments from the water where the specimens were stored
during the sampling process in Rupecica and Picinova caves. These fragments were possible
to sequence without the cloning process, which shows that in case of sufficient DNA in the
sample cloning can be skipped from the detection process.

62

4. SUMMARY OF PROTEUS EDNA STUDIES

We successfully developed the laboratory protocol for environmental DNA detection

for Proteus anguinus, and provided primer sequences suitable for Proteus DNA
amplification

We tested two sampling methods for Proteus DNA detection from cave water. We
found that in case of significant population and low water level 15 ml water stored in
Sodium Acetate and 96% Ethanol can be suitable for detecting the species, but
filtering 5 or 10 liters of water gives more reliable results.

We found that the right timing for sampling is very important for the successful
detection of the species. Samples collected in September when water level was low
and water flow was slow could be successfully processed, while water samples
collected in March and with high water level was not possible to detect Proteus DNA
from.

Further development of the method is needed for more reliable detection of the
species.

5. REFERENCES
FICETOLA, G. F., MIAUD, C., POMPANON, F., TABERLET, P. 2008: Species detection using
environmental DNA from water samples. Biology Letters, 4. p. 423425.
GOLDBERG, C. S., PILLIOD, D. S., ARKLE, R. S., WAITS, L. P. 2011: Molecular detection of
vertebrates in stream water: a demonstration using Rocky Mountain tailed frogs and Idaho
giant salamanders. PloS One, 6. p. e22746.

63

64

SECOND SUBCHAPTER

MICROSATELLITE TESTING AND POPULATION GENETIC ANALYSES OF PROTEUS

POPULATIONS LIVING IN THREE DIFFERENT CAVE SYSTEMS

1.INTRODUCTION
Within this study our aim was to test several microsatellite primers developed for closely
related species to reveal the population genetic structure of Proteus anguinus. Instead of
testing 40 already developed primer pairs we decided to apply next generation sequencing
method (454 pyrosequencing) and develop Proteus-specific primers and test them on three
Proteus populations collected in three different cave systems in Croatia.

2. MATERIALS AND METHODS

We harvested DNA from a specimen sampled from Miljacka cave. The DNA extraction was
done using a Qiagen DNeasy Blood and Tissue kit and DNA (about 1.6 mg) was sent to
Macrogen (Seoul, Korea) for random 454 pyrosequencing and genomic library building. The
genomic data was screened for potential microsatellites using MSATCOMMANDER v1.0.8
(Faircloth 2008). We selected only di-, tri- and tetranucleotides with at least respectively 10, 6
and 6 repetitions. Sixty of them were selected and the primers designed with
MSATCOMMANDER were used for PCR amplifications. After testing the 60 fragments we
selected those which seemed to show polymorphism according to the gel electrophoresis. The
selected primers were then labelled with fluorescent dyes (FAM, VIC, PET, NED) and were
tested on 67 Proteus samples collected from three populations of three different cave systems
in Croatia (Rupecica, Picinova and Vedrine).
The test PCRs we run in 10 l reactions including:

1 l Fermentase Buffer (10x),

0.6 l MgCl2 (1.5 M final concentration),
0.5 l dNTP mix (2mM),
0.04 l Fermentase Taq DNA Ploymerase (5 u/l),
65

1-1 l from each primer (5M),

4.9 l mQ water (depending on the quantity of the template),
1 l template

The PCR protocol consisted of:

initial denaturation on 94C for 1:30

40 cycles of denaturation on 94C for 45 seconds, annealing on 60C for 60 seconds

and elongation on 72C for 2:30 minutes,

Final elongation on 72C for 5 minutes (MJ PTC-200 gradient PCR, Bio-Rad
Laboratories, Inc.).

Figure 8. Gel-electrophoresis picture of the PCR products of six fragments tested on 3 specimens from 3
populations (9 specimens altogether).

Fragment lengths were then analysed and visualized on The Applied Biosystems 3130
Genetic Analyzer.

66

67

3.RESULTS
3.1. PRIMER DEVELOPMENT AND FRAGMENT LENGTH ANALYSIS
The 454 pyrosequencing provided 82,915 reads and MSTAT-COMMANDER selected 178
microsatellite loci. Similar numbers of di- and trinucleotide microsatellites were detected,
whereas a very limited number (9) are tetranucleotides. The number of repetition varies
between 13.0 (minimum-maximum: 10-37) for dinucleotides, 7.0 for trinucleotides (6-11) and
6.9 for tetranucleotides (6-13). After selecting and testing 60 primer pairs 14 polymorphic
primer pairs were further selected for fluorescent labelling and testing on 3 populations of
Proteus anguinus (Table 2.).

Primer
Pang4
Pang8
Pang10
Pang15
Pang16
Pang20
Pang22
Pang23
Pang24
Pang29
Pang31
Pang32
Pang33
Pang41

PRIMER_F
GCCTAGAGGCGACATGCTG
AGACCCAAAGAGACGGTGC
GACACCATGCGACTGGAAC
AAGTGCACCGTGGTCTGG
AAGTGCACCGTGGTCTGG
GCACACATGATCCCAGTGC
TCCCTCCCAACAAGAAGCC
GCCGGAGCCAATGTAACTG
CATTGTCTGTGCTGGAGCC
CATTGTCTGTGCTGGAGCC
TGTTCCTGTGCAGGTCTCC
GGCTTCAAGTGCTTCTGGG
ACAGGTCACTCTGCACTGG
TCTGCCTTCTCGTTGGGAG

PRIMER_R
CTTGTTCCTTGCCTGCCAC
GGGAAGAGTCACCGAGTGC
CTTTGGGTCAGCTTCGTGC
TGTGCTCCAAAGGGACAGG
TGTGCTCCAAAGGGACAGG
GCAGCCAATTGACCCAGC
GGAGTGACAGAACTCCTGGG
CTTGCACTGACCGGTGTTC
CACACACCGCCTTCAGTTG
CACACACCGCCTTCAGTTG
AGAAGGAGCAAATCAGGGC
CACAGTCTTGTGACGTGGC
TTGCCAGCACCATAACAGG
TCTCTGATCCCACCAGTGC

Length
185
215
232
253
254
291
305
306
307
324
332
337
339
369

Repeat
(ATT)^6
(AGC)^6
(AGC)^6
(AC)^16
(AGAT)^6
(GCT)^6
(ATTT)^6
(AC)^10
(GAT)^6
(CTT)^6
(ATCT)^6
(AAT)^6
(CAGT)^6
(AGG)^6

Type
Trinucleotide
Trinucleotide
Trinucleotide
Dinucleotide
Tetranucleotide
Trinucleotide
Tetranucleotide
Dinucleotide
Trinucleotide
Trinucleotide
Tetranucleotide
Trinucleotide
Tetranucleotide
Trinucleotide

dye
NED
FAM
FAM
FAM
VIC
VIC
VIC
PET
PET
PET
NED
NED
NED
FAM

Table 2. Selected 14 polymorph microsatellite loci for Proteus anguinus.

We then run fragment length analyses on 67 Proteus anguinus specimens from 3 populations
(Picinova, Rupecica, Vedrine).

Figure 9. Fragment length analysis of locus Prota4 for a specimen from Picinova cave.

68

The microsatellite loci were generally characterized by low number of alleles and high
heterozygosity (Table 3.).
Locus
name

Number of
alleles

Size range of
amplification
product

Ho

He

Annealing
temperature

Prota4
Prota8
Prota10
Prota20
Prota22
Prota31
Prota48
Prota52

2
7
5
8
6
6
5
13

185-198
200-222
222-236
258-295
298-308
288-335
393-449
402-445

0,988
0,473
0,069
0,570
0,381
0,651
0,535
0,623

0,500
0,472
0,136
0,415
0,310
0,375
0,319
0,489

60 C
60 C
60 C
60 C
60 C
60 C
60 C
60 C

Table 3. Characterization of eight polymorphic microsatellite loci for Proteus anguinus. N=67 individuals
analysed.

3.2. GENETIC CHARACTERIZATION OF THREE PROTEUS ANGUINUS POPULATIONS

Based on analyses of the eight polymorphic microsatellite loci we tried to characterize the
population genetic structure of three Proteus anguinus populations from three different cave
systems. The three populations were:
33 specimens from Picinova cave
8 specimens from Vedrine
26 specimens from Rupecica cave

Figure 10. DNA sampling for population genetics study. All animals were collected in sterile plastic bag and
released imediately after the sampling on the same place in the cave (marked on capture). Population was
monitored twice afterwards to be sure there is no mortalities.

69

The analyses of 8 microsatellite loci indicated that in general populations show low allele
number, low heterozygosity, and high percentage of polymorphyc loci (Table 4.). Number of
private alleles compared to the low number of alleles was high.

0,600

5,000
4,000
3,000
2,000
1,000
0,000

0,400
0,200
0,000
Pop1

Pop2

Heterozygosity

Mean

AllelicPatternsacrossPopulations

Pop3

Na
NaFreq.>=5%
Ne
I
No.PrivateAlleles
No.LCommAlleles(<=25%)

Populations

Figure 11. Allelic patterns across three populations of Proteus anguinus. Pop1=Picinova, Pop2=Vedrine,
Pop3=Rupecica.

Population
Picinova
Mean
SE
Vedrine
Mean
SE
Rupecica
Mean
SE

Nr. of
alleles
3,625
0,498
2,125
0,295
2,625
0,532

Nr. of
effective
alleles
2,068
0,317
1,841
0,203
1,645
0,293

% of
Observed
Expected
polymorphic
heterozygosity heterozygosity loci
100%
0,557
0,445
0,135
0,074
75%
0,679
0,391
0,157
0,087
87,5%
0,373
0,294
0,139
0,084

Table 4. Microsatellite data of three populations based on eight microsatellite loci.

When conductiong principal coordinates analysis (PCoA) to visualize similarity/dissilarity of

data, the individuals belonging to the same population grouped together, showing the
differences in genetic structure of microsatellites. The first axes explained the 44.97% of the
variance, while the second and third axes explained the 20.13% and 11.74% of the variance
(Fig.12.).

70

Coord.2

PrincipalCoordinates

Rup20
Rup22
Rup19Rup23
Rup8Rup25
Rup18
Rup7
Rup10
Rup6
Rup5
Rup9
Rup26
Rup2Rup21
Rup16
Rup12
Rup1
Rup4
Rup3
Rup24
Rup13
Rup14
Rup15
Rup11
Rup17

Pic25

Pic8
Pic10
Pic14
Pic6
Pic11
Pic20Pic9
Pic15
Pic12
Pic23
Pic27
Pic17
Pic3 Pic28
Pic21
Pic31
Pic18
Pic19
Pic30
Pic4Pic16 Pic26
Pic22 Pic7
Pic1
Pic13
Istra1 Pic29 Pic5
Istra2Pic2
Pic24

Ved4
Ved2
Ved5
Ved6
Ved1
Ved12
Ved3
Ved22
Coord.1

Figure 12. Principal coordinates analysis of the three Proteus anguinus populations using eight microsatellite
loci.

Figure 13. For reaching some of the localities, we had to use speleological gear to reach deeper parts of the cave.
Sampled animals were held separated for health reasons.

71

4. SUMMARY OF MICROSATELLITE DEVELOPMENT AND ANALYSES

We successfully developed primers for analyses of polymorphic microsatellite loci

suitable for future population genetic studies of Proteus anguinus

We applied the developed Proteus-specific microsatellite loci to reveal the basic

population genetic characteristics of Proteus anguinus based on 67 specimens

We tested the method on three Proteus populations from three cave systems and gave
characterization of population genetic measures for the three populations

Further development of the method test of further primers (loci) is needed in order to
find more polymorphic microsatellite loci for future conservation genetic studies of
the species.

5. REFERENCES
FAIRCLOTH, B. C. 2008: Msatcommander: detection of microsatellite repeat arrays and
automated, locus-specific primer design. Molecular Ecology Resources 8. p. 92-94.

72

CHAPTER V.

Education on conservation of underground habitats

and raising public awareness

Objective4:Getlocalpeopleinvolvedintheconservationofunderground
habitatsasoneoftheprimeconservationactions.

Duan Jeli, Ivona Buri, Ana tih, Ivana Sui

Croatian Institute for Biodiversity,
Croatian Herpetological Society HYLA

EDUCATION AND PROMOTION

Our objective during this project was to give special attention to local, regional and
global education on karst underground and olm as umbrella species. In this manner our
activities were divided on local, regional and global, based on their expected reach.
During the project we discovered that we are mainly working only on horizontal
distribution of material (through our activities) and identified that as a problem for reaching
wider number of people. To resolve this we developed VERTICAL scheme of distributing
material and news about the project. Main idea is to make all of our presentation material
(ppt and pdf presentations) available online and ask our volunteers to give lectures where ever
they can, and we will send them educational material in quantity needed for each case. Our
aim was to get our volunteers to try to recruit new volunteers in their lectures and in this way
to build up a pyramid of volunteers and promoters.
One publication that makes us particularly proud is an educative poster prepared by
one of our youngest volunteers Luka Prelogar (6th grade of elementary school) who
prepared educative lecture and a poster about Olm conservation and presented it to his
class. Poster also went to the school science competition. CHS-HYLA provided the
PowerPoint presentation, material and literature. This is the best example of vertical
distribution of materials and knowladge.

Local activities
During August 2012 we presented the PROTEUS project official web page which can be
visited at www.proteus.hibr.hr. It is currently only available in Croatian language, but we are
preparing the English version as well that will be published by the end of 2013. Web page is
the place where all of the project activities are being presented and news are being put up
regularly. Web page is being interconnected with CHS-HYLA and Croatian Institute for
Biodiversity official web pages and Facebook accounts. In this way all the news are
distributed more efficiently.

Figure 1. PROTEUS project official web page www.proteus.hibr.hr

During August 2012 PROTEUS project visual identity and official logo were
finished. Project has one main logo and several variations. Project leaflets have also been
prepared and printed in order to start with the material distribution through project activities.
Due to the general economic crisis the prices went down and we managed to print 30.000
bookmarks and 30.000 leaflets (funded by ZSL & MAVA together; Fig. 2 & 3). This was

about 20% more than originally planned. Leaflets were distributed through all of the local,
regional and global activities.

Figure 2. Educational leaflet about Olm and our PROTEUS project

Figure 3. Bookmark about Olm and our PROTEUS project (front and back side)

Project team was asked to give a talk on 9th Conference on conservation of Karst
adopted animals, held in Otoac (June 13th 2012), on the subject of research and
conservation of the Olm and cave adopted endemic fish in Karst region of Dinaric Alps.
Conference was organised by Croatian Centre for breeding of endemic fish and crayfish from

Otoac (Fig. 4). Duan Jeli presented project activities and distributed CHS-HYLA and
PROTEUS project gift packet with promotional material (bookmarks, leaflets, pencils etc.).

Figure 4. Croatian Centre for breeding of endemic fish and crayfish in Otoac

Promotional lectures were held on the large speleological convention in Rakovica

on 10th and 11th November 2012. This is the largest gathering of speleological societys in
region and this offered great opportunity to promote PROTEUS project. Round table has been
made with the representatives of speleological societies from Croatia and some from Bosnia
and Herzegovina and they were introduced to the project objectives. These people were asked
to spread the word about the project and invited to sign in for the Olm conservation news mail
where they will get all the updates on how to help. Many people approached the project
team with useful data on sightings of olm in different cave systems.
During September 2012 (9th 10th) Duan Jeli was invited to give a lecture on J.J.
Strossmayer University in Osijek to the biology students. Lecture was a part of Nature
conservation course held by prof. dr. Oleg Antoli. We hope this will help to get the
university staff and students involved in the project activities. Osijek is situated in Pannonia
plan flatlands of Croatia and people here have very poor knowledge on Dinaric karst
phenomena. Focus of the lecture was to raise awareness on karst and its hidden underground.

On 3rd and 4th of September we organized the first international Olm workshop in
the city of Ogulin. On this workshop we gathered experts from Croatia and whole region to
discuss about Olm research and conservation. Even though this animal is only found in small
part of Italy, Slovenia, Croatia and Bosnia and Herzegovina, there are also many foreign
scientists and experts working on this subject (UK, Hungary, France etc.).

Figure 5. First international workshop on olm conservation in Croatia and region

Early in the morning and after the opening greetings, an introduction of main goals and
activities of PROTEUS project was given by the ZSL fellow and project manager Duan
Jeli. After that we heard very interesting lecture on the known distribution and condition of
populations in Croatia by Eduard Kleteki from Croatian Natural History Museum in Zagreb,
followed by Judit Vrs from Hungarian Natural History Museum in Budapest. Dr. Vrs
told us about current research on population genetics of Croatian populations of the Olm, as
part of our PROTEUS project. The secrets of cave diving and possibilities of research with
that method on PROTEUS project was presented to us by expert diver Petra Kova Konrad
from State Institute for Nature Protection (Croatia). Prof. Boris Sket from Biotechnical
Faculty in Ljubljana told us about the olm differentiation through the whole distribution area
and indicated even the possibilities that there are several undescribed species currently
considered as Proteus anguinus. About olm current status and known distribution in Bosnia

and Herzegovina we heard from Emina unje from the International University of Sarajevo,
and Gergely Balzs from NGO Caudata Hungarian Cave Research (Hungary) told us about
several years of research in Bosnia and Herzegovina (Trebinje area).
Story about the whole Olm project and the conference was published in National Park
Krka magazine called Buk, several daily newspapers, local and national radio
stations.
During October 2012 PROTEUS team organized two educational lectures to promote
olm conservation and PROTEUS project itself. First lecture was held in Zagreb for general
public and it was organized in cooperation with one of the largest mountaineering
societys PD Matica, Zagreb on 5th of October 2012. Mountaineers are great nature lovers
and they showed this by arriving to the lecture in unexpectedly large number (>50). This
opportunity was used to distribute promotional material to the visitors, but also additional
material was provided for all that wanted to further distribute them and become our volunteer.
The presentation that was shown there is available online and they could have taken it and
presented it to other communities of people (mountaineering societys, schools, etc.). In this
they would be provided with material to distribute on that occasion.
On 2nd of April 2013 we had an project second local workshop in city library in
Sinj. Workshop attracted around 35 participants from Splitsko-dalmatinska County, mainly
speleologist and students. Participants were mostly interested in local research being done in
their area and possibilities of cooperation with PROTEUS project. Sinj is very important in
our project because of locality in Vedrine, where the olms are being flushed out annually and
in next stage of the project we are planning to do a reconstruction of the site to enable the
olms to return back into the system. For this we need good cooperation with local decision
makers and community.
On 23rd of April diving team was invited to make an 10 min interview and
reportage for the Croatian National Television HRT which was broadcasted during the
first week of May 2013. The subject of the report was on usage of new and innovative
technologies in NATURA2000 monitoring. Monitoring program developed for olm by the
PROTEUS project team was selected as the most extreme and presented as partly science
fiction that it is possible to detect presence of the animals that you can not see. This is done by
our eDNA method for distance sampling of olm presence. Cave diving is also for the first
time, for olm, used for NATURA2000 monitoring.

Figure 6. Croatian National Television filming PROTEUS project reportage

On 8th and 9th of April Duan Jeli and Petra Kova Konrad attended a TAIEX
office Workshop on Preparation for programme LIFE+ of European commission.
Workshop was held by consultants from Poland (Peter Jany), Slovakia (Andrzej Muter) and
Chech Republic (Markta Konen). LIFE+ is one of the EC financial instruments for the
environment for years 2007-2013 worth 2,143 billion . Duan and Petra were later
responsible for application of LIFE+ project for conservation of Proteus anguinus in Croatia.
Project proposal was submitted to the European Commission in June 2013 and results are
expected in April 2014. Whole project value is set to around 750.000,00 .

On 9th of April 2013 PROTEUS personnel had a meeting in Zagreb with mr. Boris
Erg, the head of IUCN South and Eastern Europe office in Belgrade. Meeting was held
because of IUCN interest to publish a story about PROTEUS project and olm
conservation story on their official web site. Another subject was further cooperation and
possibly partnership in future LIFE+ project application. Both sides agreed to strengthen the
cooperation and proceed with LIFE+ project application. As a result of this cooperation IUCN

published 4 stories about CHS-HYLA activities through their COUNTRY FOCUS ON

CROATIA in September 2013. One of the stories is about the PROTEUS project.

On 26 and 27th of July PROTEUS project had its second international workshop
on Proteus conservation issues. This year we hosted visitors from five countries: Croatia,
Slovenia, Bosnia and Herzegovina, Romania and Hungary. Aim of the workshop was to
present current work on this species in region and to start developing mutual cooperation
among projects. During the workshop project leader Duuan Jeli presented the monitoring
results of PROTEUS project, Julia Thunde Ghal presented the molecular part that was done in
the Hungarian Natural History Museum (Budapest) and Ivan Cizelj (Zagreb ZOO) presented
the part on captive held animals and veterinary research done on Veterinary Faculty of
University of Zagreb. Visiting lecturers were given from Gregor Aljani (Fig. 7) from Tular
Laboratory (Slovenia) where Olms are captive breed for several generations now and Silvio
Legovi from Baredine Grotta (show cave in Istria) where Olm is presented to the wide
public. At the end, Duan Jeli presented a PROTEUS project plan for next 5 years
(2014-2018) and presented new project partners: Public Institution Krka National Park
(Croatia), Baredine Grotta show cave, Centre for mapping flora and fauna (Slovenia),
Ecological Society of Montenegro and Herpetological Society of Bosnia and Herzegovina
(Figure 8).

Figure 7. Lecture by Gregor Aljani (Slovenia) on second international workshop

Figure 8. Project leader Duan Jeli presenting new PROTEUS project 2014-2018

Regional education

Two lectures of regional character were organized and held in Kraljevo and
Kragujevac (Serbia) on 8th and 9th September 2012 for high school and university students
(first day) and general public (second day). This lecture was organized in cooperation with
nature conservation NGO BALKAN from Kraljevo.

On 18th of April 2013 Duan Jeli held another international lecture in Debrecen
University (Hungary) for PROTEUS project regional promotion. Lecture was held for
University personnel and students of 4th and 5th year of Biodiversity conservation course.
Around 60 people intended the lecture. We thank dr. sc. Mihaly Foldvari for the organisation
of this event and logistics provided during stay.

Working on our project we found that there is a great lack of knowledge in working
with GIS throughout our partners in PROTEUS project. This is why we organized on 26th and
27th of February 2013 a two day workshop on Quantum and ESRI GIS use in nature
conservation. Altogether 23 people attended. Four person from Sarajevo and one from Banja
Luka (Bosnia and Herzegovina), two from Begrade (Serbia) and 16 students and our
colleagues from NGOs from Croatia. All the participants were introduced to working in GIS
environment and provided with wide range of GIS mapps that are currently freely available.
They were also introduced to how to produce their own maps and how to manage their field
data.

Croatian Herpetological Society also participated on MAVA foundation conference

in Tirana (Albania) from 4-6th of March where further plans for PROTEUS project were
made and new possible partnerships were established. It was decided to go forward to the
second phase of the project during 2013/2014 and to add more partners from Bosnia and
Herzegovina and Montenegro. After the MAVA conference from 7-9th of March IUCN held
a conference of members for South and East Europe. CHS is a member of IUCN since
2008 and during the conference IUCN members were also introduced to the PROTEUS
project. During this meeting CHS-HYLA represented the attitudes of PROTEUS project and
tried to push through certain useful ideas into the IUCN strategy for South and East Europe.

Figure 9. MAVA foundation conference in Tirana (Albania) from 4-6th of March (photo by
curtsy of MAVA)

Global education

During June 2012 project team had participated in the filming of BBC documentary
called Attenborough Ark. Prime target was filming the olm and its underground water
habitats. We did 8 days of filming and it was not easy diving twice a day into the cold water
for underground/underwater shots. Premiere of the documentary was organised on BBC1 in
November 2012. Documentary is talking about 10 animals in the whole World that Sir. David
Attenborough (Figure 10 & 11) would select to place on his imaginary Ark, in order to save
them from extinction. One of the selected 10 animals is our olm. His imaginary Ark is
actually representing nature conservation projects that are being done to conserve these 10
animals, and in this manner PROTEUS project is presented directly in the documentary.
Local newspapers also took a moment to write about the project and the BBC filming. As a
project leader, Duan Jeli gave several interviews about the olm conservation.

Figure 10. Filming of BBC documentary Attenborough Ark with Sir. David Attenborough

Figure 11. Vedran Jali (cameraman) and Petra Kova Konrad during the filming in Miljacka II.

In addition during the cooperation of the project team with Canadian Educational
office Scholastic Canada the example of our Olm prime research locality the
Rupeica cave was taken as an example of 10 most extreme animal habitats in the
World. These books are sold through educational system in Canada and USA.

Figure 12. Proteus anguinus in The 10 most extreme Animal Habitats journal

From 19-22nd of March Duan Jeli participated on Student Conference on

Conservation Science in Cambridge and presented a talk "The olm as an umbrella species
for karst conservation in Croatia". The talk raised a lot of positive comments and new
innovative ideas how to improve the work.

One of the PROTEUS project sponsors The Mohamed bin Zayed Species
Conservation Fund published a 3 minute video about our PROTEUS project and diving
activities. Story focuses on importance of underground water resources in Dinaric karst.

Video can be seen on MBZ official website http://www.speciesconservation.org/ (under

MEDIA: http://www.youtube.com/watch?feature=player_embedded&v=-WTl1Irg-4k).

On 8-11 May 2013 project team participated on "International Conference on

Diseases of Zoo and Wild animals" and presented a poster titled The olm (Proteus
anguinus) in Croatia conservation research project (Lukac, M., Horvatek Tomic, D.,
Cizelj, I., Jelic, D. & Prukner Radovcic, E.) extended abstract was published during July
2013 in conference abstract book. Contribution is dealing with comprehensive study of
physiological microflora and pathogens that may jeopardize Proteus existence (in wild and
captive conditions). We tested skin, pharyngeal and cloaca swabs for microflora and
parasitological and microbiological analyses of fecal samples were also performed. For
comparison we also tested water samples from the habitat and aquarim. This was a mutual
contribution of project partners: Faculty of Veterinary Medicine, University of Zagreb,
Zagreb Zoo & Croatian Herpetological Society HYLA).