UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Seyed Mohammad Javad MALEK-HOSSEINI DISCOVERY, DIVERSITY, AND THE ORIGINS OF TROGLOBIOTIC FAUNAS IN IRAN DOCTORAL DISSERTATION Ljubljana, 2022 UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Seyed Mohammad Javad MALEK-HOSSEINI DISCOVERY, DIVERSITY, AND THE ORIGINS OF TROGLOBIOTIC FAUNAS IN IRAN DOCTORAL DISSERTATION ODKRIVANJE, PESTROST IN IZVOR TROGLOBIOTSKIH FAVN V IRANU DOKTORSKA DISERTACIJA Ljubljana, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Based on the Statute of the University of Ljubljana and the decision of the Biotechnical Faculty senate, as well as the decision of the Commission for Doctoral Studies of the University of Ljubljana adopted on October 22, 2018, it has been confirmed that the candidate meets the requirements for pursuing a PhD in the interdisciplinary doctoral programme in Biosciences, Scientific Field Biology. Assoc. Prof. Dr. Matjaž Kuntner is appointed as supervisor. Doctoral dissertation was conducted at the Jovan Hadži Institute of Biology, Scientific Research Centre of the Slovenian Academy of Sciences and Arts and Organisms and Ecosystems Research Department, National Institut of Biology (NIB), Ljubljana, Slovenia. Commission for assessment and defense: President: Prof. Dr. Peter TRONTELJ University of Ljubljana, Biotechnical Faculty, Department of Biology Member: Prof. Dr. Cene FIŠER University of Ljubljana, Biotechnical Faculty, Department of Biology Member: Prof. Dr. Tanja PIPAN Slovenian Academy of Sciences and Arts, Karst Research Institute Date of defense: Seyed Mohammad Javad Malek-Hosseini II Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KEY WORDS DOCUMENTATION DN Dd DC UDC 551.44(235.15)(043.3) CX biodiversity, Zagros, Iran, Carabidae, hotspot, origin of troglobionts AU MALEK-HOSSEINI, Seyed Mohammad Javad, MSc. biol. AA KUNTNER, Matjaž (supervisor) PB University of Ljubljana, Biotechnical Faculty, Interdisciplinary Doctoral Programme in Biosciences, Scientific Field Biology PY 2022 TI DISCOVERY, DIVERSITY, AND THE ORIGINS OF TROGLOBIOTIC FAUNAS IN IRAN DT Doctoral dissertation NO XI, 186 p., 4 ann., 12 fig, 4 tab, 148 ref. LA en AL sl/en AB We investigated the troglobiont biodiversity of Iran, with a focus on Zagros karstic areas. This study followed three main goals. First, a discovery component involved faunistic investigations into the troglobiont organisms in Zagros Mountains of Iran, as well as taxonomic descriptions of new taxa. Three new troglobiont species were described: a carabid beetle, an asellid isopod and a truncatelloid gastropod. Additionally, we report on the discovery of six species under description (Oligochaeta, Decapoda, Isopoda) and over 10 species that are awaiting description. DNA extraction and PCR of selected markers from most of these taxa have been done, enabling species delimitation and phylogenetic analyses. We recorded new habitats for previously described Iranian cave fishes. Our second aim was to examine patterns of troglobiont biodiversity and endemism in the Zagros Mountains of Iran. We published a checklist of Iranian subterranean arthropods in 2017. In an additional chapter, we listed all Iranian troglobiont animals (previously published and our new discoveries, including yet undescribed species). The species richness and distribution patterns of Iranian troglobiont diversity were presented, including one currently recognized biodiversity hotspot. As the third goal, we aimed to analyze Iranian troglobiont animals phylogenetically to understand their relationships and to identify their origin and estimate a timeframe for their colonization into subterranean habitats. The new troglobiont species and some of sampled surface relatives were used for DNA isolation and molecular analysis. Related sequences were mined from GenBank. Given the poor knowledge of Iranian subterranean fauna, this speleobiological dissertation enriched this knowledge by reporting new findings and describing new species. The discovery component of this thesis enabled us to establish Iran as an important part of the world map of subterranean biodiversity. We now understand more about the origins of Iranian troglobionts and the time of their adaptation to subterranean environment. This fauna is likely to show high endemism and is important from the conservation perspective. III Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KLJUČNA DOKUMENTACIJSKA INFORMACIJA ŠD Dd DK UDK 551.44(235.15)(043.3) KG biodiverziteta, Zagros, Iran, Carabidae, vroča točka, izvor troglobiontov AV MALEK-HOSSEINI, Seyed Mohammad Javad, MSc in Zoology (Animal Biosystematics) SA KUNTNER, Matjaž (mentor) KZ SI-1000 Ljubljana, Jamnikarjeva 101 ZA Univerza v Ljubljani, Biotehniška fakulteta, Interdisciplinarni doktorski študij Bioznanosti, znanstveno področje Biologija LI 2022 IN Odkrivanje, pestrost in izvor troglobiotskih favn v Iranu TD Doktorska disertacija OP XI, 186 str., 8 sl., 4 pril., 148 vir. IJ en JI sl/en AI Raziskali smo biotsko raznovrstnost troglobiontov v Iranu s poudarkom na kraških območjih gorovja Zagros. Disertacija je sledila trem glavnim ciljem. Prvi cilj, komponenta odkrivanja, je vključevala favnistične raziskave troglobiontskih organizmov v gorovju Zagros v Iranu ter taksonomske opise novih taksonov. Opisane so bile tri nove troglobiontske vrste: karabidni hrošč, asellidni izopod in truncatelloidni gastropod. Poleg tega poročamo o odkritju šestih vrst v postopku opisovanja (Oligochaeta, Decapoda, Isopoda) in več kot 10 vrst, ki čakajo na opis. Iz večine teh taksonov sta bili opravljeni ekstrakcija DNK in PCR izbranih markerjev, kar omogoča razmejitev vrst in filogenetske analize. Za prej opisane iranske jamske ribe smo zabeležili nove habitate. Naš drugi cilj je bil preučiti vzorce biotske raznovrstnosti in endemizma troglobiontov v gorovju Zagros v Iranu. Leta 2017 smo objavili vrstni seznam iranskih podzemeljskih členonožcev. V dodatnem poglavju smo navedli vse iranske troglobionte (prej objavljene in naša nova odkritja, vključno s še neopisanimi vrstami). Predstavili smo vrstno bogastvo in vzorce razširjenosti iranske troglobiontske pestrosti, vključno z eno trenutno priznano vročo točko biotske raznovrstnosti. Tretji cilj je bil filogenetska analiza iranskih troglobiontov, da bi razumeli njihove medsebojne odnose ter ugotovili njihov izvor in ocenili časovni okvir njihove kolonizacije podzemnih habitatov. Nove vrste troglobiontov in nekatere vzorčene površinske sorodnike smo uporabili za izolacijo DNK in molekularno analizo. Relevantna zaporedja DNK so bila dodatno pridobljena iz baze GenBank. Glede na slabo poznavanje iranske podzemeljske favne je ta biospeleološka disertacija obogatila to znanje s poročanjem o novih najdbah in opisom novih vrst. Del disertacije posvečen odkrivanju nam je omogočil uveljavitev Irana kot pomembnega dela svetovnega zemljevida podzemeljske biotske raznovrstnosti. Zdaj bolje razumemo izvor iranskih troglobiontov in čas njihovega prilagajanja podzemnemu okolju. Ta favna verjetno izkazuje visok endemizem in je pomembna z vidika ohranjanja narave. IV Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 TABLE OF CONTENTS KEY WORDS DOCUMENTATION ...................................................................... III KLJUČNA DOKUMENTACIJSKA INFORMACIJA ........................................... IV TABLE OF CONTENTS .......................................................................................... V TABLE OF CONTENTS OF SCIENTIFIC WORKS .......................................... VII LIST OF FIGURES .............................................................................................. VIII LIST OF TABLES ................................................................................................... IX LIST OF ANNEXES ................................................................................................. X ABBREVIATIONS AND SYMBOLS ..................................................................... XI 1 INTRODUCTION ................................................................................................... 1 1.1 SUBTERRANEAN ENVIRONMENT ................................................................... 1 1.1.1 A brief history of Speleology and Speleobiology ............................................. 1 1.1.2 Characteristics of the subterranean environment ........................................... 1 1.1.3 Energy resources in caves ................................................................................. 2 1.1.4 Cave animals ..................................................................................................... 3 1.1.5 Troglomorphic traits ........................................................................................ 4 1.1.6 Global biodiversity of troglobiotic animals ..................................................... 4 1.1.7 Hotspots of troglobiotic biodiversity ................................................................ 5 1.1.8 Origin of troglobionts ....................................................................................... 7 1.2 SPECIFICATION OF THE RESEARCH PROBLEM ............................................ 7 1.3 REVIEW OF LITERATURE ............................................................................... 12 1.4 CONSERVATION ............................................................................................... 15 1.5 THESIS AIMS AND OUTLINE .......................................................................... 16 V Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2 SCIENTIFIC WORKS ......................................................................................... 19 2.1 PUBLISHED SCIENTIFIC WORKS ................................................................... 19 2.1.1 First insights into the origin of Iranian cave beetle diversity with description of two new species of the genus Duvalius (Carabidae) ........................................... 19 2.1.2 Description of a new genus and species as the first gastropod species from caves in Iran. ............................................................................................................ 37 2.1.3 A new obligate groundwater species of Asellus (Isopoda: Asellidae) from Iran ........................................................................................................................... 53 2.1.4 A checklist of subterranean arthropods of Iran ............................................ 82 2.2 REMAINING LINKING SCIENTIFIC WORK ................................................. 111 2.2.1 Overview of troglobiont biodiversity in Iran ............................................... 111 3 DISCUSSION AND CONCLUSIONS ................................................................ 150 3.1 DISCUSSION .................................................................................................... 150 3.1.1 Goal 1: Discovery of troglobiotic fauna in Iran (Zagros karsts) ................ 150 3.1.2 Goal 2: Iranian troglobiotic fauna species richness and hotspot detection 151 3.1.3 Goal 3: Phylogeny and evolutionary history of Iranian troglobionts ......... 154 3.1.4 Conservation ................................................................................................. 155 3.2 CONCLUSIONS ................................................................................................ 156 4 SUMMARY ......................................................................................................... 160 4.1 SUMMARY ....................................................................................................... 160 4.2 POVZETEK ....................................................................................................... 166 5 REFERENCES .................................................................................................... 175 ACKNOWLEDGEMENTS ANNEX VI Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 TABLE OF CONTENTS OF SCIENTIFIC WORKS Malek-Hosseini MJ., Muilwijk J., Gregorič, M., Kuntner M., Čandek, K. 2021. First insights into the origin of Iranian cave beetle diversity with description of two new species of the genus Duvalius (Carabidae). Journal of Zoological Systematics and Evolutionary Research, 59: 1453-1469. Fatemi Y., Malek-Hosseini M. J., Falniowski A., Hofman S., Kuntner M., Grego J. 2019. Description of a new genus and species as the first gastropod species from caves in Iran. Journal of Cave and Karst Studies, v. 81, no. 4, p. 233-243. Malek-Hosseini M. J., Jugovic J., Fatemi Y, Kuntner M, Kostanjšek R, Douady C. J., Malard F. 2022. A new obligate groundwater species of Asellus (Isopoda: Asellidae) from Iran. Subterranean Biology, 42: 97-124 Malek-Hosseini M. J., Zamani A. 2017. A checklist of subterranean arthropods of Iran. Subterranean Biology, 21: 19-46. VII Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF FIGURES Figure 1: Map of terrestrial ecoregions in Iran .................................................................... 10 Figure 2: Carbonate location map of Iran ............................................................................ 10 Figure 3: Some of Iranian obligate subterranean dwelling animals .................................... 14 Figure 4: Opening of Chah-Kabootari cave ...................................................................... 133 Figure 5: Population of Gara tashanensis in Chah-Kabootari cave .................................. 134 Figure 6: Eidinemacheilus cf. smithi fromTuveh spring ................................................... 134 Figure 7: Tuveh Spring, habitat locality of Iranian cave fish ............................................ 135 Figure 8: Some of Iranian troglobiont Crustacea .............................................................. 136 Figure 9: Map of Iran, showing the distribution of troglobiont animals. .......................... 139 Figure 10: Distribution of troglobiont species number in Iran at grid cell size 100 × 100 km. ........................................................................................................................................... 144 Figure 11: Map of Iran showing species richness per localities (caves, wells, springs) ... 145 Figure 12: Map of Iran showing species richness per localities (caves, wells, springs) ... 146 VIII Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF TABLES Table 1: age of some karst formations in Iran ..................................................................... 11 Table 2 (S1): Our original sequences and additional sequences from GenBank were used in the phylogenetic analysis. .................................................................................................... 49 Table 3: All Iranian troglobint animals ............................................................................. 137 Table 4: Locality details and species number of Iranian troglobiont animals ................... 141 IX Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF ANNEXES ANNEX A: Permission from publisher for the publication of article Malek-Hosseini et al. (2021) in the printed and electronic versions of the doctoral dissertation ANNEX B: Permission from publisher for the publication of article Fatemi et al. (2019) in the printed and electronic versions of the doctoral dissertation ANNEX C: Permission from publisher for the publication of article Malek-Hosseini et al. (2022) in the printed and electronic versions of the doctoral dissertation ANNEX D: Permission from publisher for the publication of article Malek-Hosseini and Zamani (2017) in the printed and electronic versions of the doctoral dissertation X Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ABBREVIATIONS AND SYMBOLS Mya million years ago COI cytochrome c oxidase subunit I PCR polymerase chain reaction H3 Histone H3 UTM universal transverse mercator XI Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1 INTRODUCTION 1.1 SUBTERRANEAN ENVIRONMENT 1.1.1 A brief history of Speleology and Speleobiology Subterranean environments, mostly caves, have been fascinating subjects for many scientists in several fields such as biology, geology, and archaeology. Speleology is a cross-disciplinary field that combines the knowledge of chemistry, biology, geology, physics, meteorology, and cartography to develop portraits of caves as complex, evolving systems. Speleology has a recent origin as a field of science. Cave study traces back to 1680s when John Beaumont studied caves in the Mendip Hills of England and presented his conclusions to the Royal Society in 1676. During that decade, Janez Vajkard Valvasor from Slovenia, wrote about caves and karst hidrology, also presenting to the Royal Society. Although, several sporadic speleological studies had been carried out by 1900, Édouard-Alfred Martel (1859–1938), the 'father of modern speleology', who through his extensive and well-publicised cave explorations introduced in France the concept of speleology as a distinct area of study. In 1895 Martel founded the Société de Spéléologie, the first organization devoted to cave science in the world. The French entomologist René Jeannel and the Romanian biologist Emil Gheorghe Racoviță studied more than 1400 caves in southern Europe and North Africa and published descriptions of the caves and their fauna. Emil Racoviță" Institute of Speleology, the first one in the world dealing with cave research, was founded by Racoviță in Cluj, Romania. The first obligate cave-dwelling animal to be studied scientifically and described was the cave salamander Proteus anguinus Laurenti, 1768 from Slovenian caves. However, first time, it was described from springs and wrongly as living in Lake Cerknica. The first troglobiont invertebrate was the cave beetle Leptodirus hohenwarti Schmidt, 1832 from Slovenia (Camacho, 1992; White and Culver, 2012). During recent 100 years, lots of studies have been carried out on subterranean biology in different parts of the world and many books and articles have been published. Also, several journals are specially focused on publications of different disciplines of speleobiology. 1.1.2 Characteristics of the subterranean environment Subterranean environment represents an interesting and unique ecosystem. Several kinds of subterranean environments exist that harbor troglobiont animals such as caves, karstic 1 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 springs, wells, interstitial waters, and lava tubes. Regarding the biodiversity and evolutionary points of view, caves are the most important subterranean environment. Several kinds of caves exist but our main interest in this study is natural solution caves in limestone. A series of general features (such as stability, predictability, being simple and isolated, nutrient poor) have been emphasized as the main characteristics of the subterranean environments (Camacho, 1992; Culver and Pipan, 2009) which are both terrestrial and aquatic. Light is the origin of most life, and primary production is the source of biodiversity; nevertheless, life also exists in darkness. Based on the amount of light and the interaction of the subterranean environments with the surface, three main zones are considered for caves: a) entrance (where the light reaches), b) twilight zone (transition zone), c) dark zone (deep zone or total darkness) (Vandel, 1965; Camacho, 1992). The most obvious characteristic of caves (except at the entrance zone) is darkness. Subterranean environments are characterized not only by continuous darkness but also by a reduced variability in the number of specific abiotic conditions such as moisture, temperature, and water chemistry, in some cases low oxygen, as well as by isolation and restriction in space. Additionally, hypogean systems are relatively energy-limited (restricted food supply) compared to photosynthetically based epigean systems (Vandel, 1965; White and Culver, 2012). 1.1.3 Energy resources in caves Due to the lack of sunlight, there is no photosynthesis in subterranean environments. So, the green plants are not the primary producer. The utilization of the energy of chemical bonds and chemoautotrophy by bacteria are the basic source of energy. Some truly chemoautotrophic-based cave ecosystems sch as Frasassi in Italy and Movile in Romania are the two best studied examples to date. Fatemi et al. (2019) mentioned Tashan cave in Iran likely represents a chemoautotrophically based cave ecosystem. This cave is a subterranean habitat with sulfide-rich waters. The sulfide caves host interesting ecosystems with unusually rich and diverse invertebrate communities (Brad et al., 2021) such as crustaceans (Peterson et al., 2013; Por, 2014; Brad et al., 2015), insects (Tobler et al., 2013) gastropods (Falniowski and Sarbu, 2015; Fatemi et al., 2019) and occasionally fishes (Reisch et al., 2010; Roach et al., 2011; Mousavi-Sabet at al., 2016). External energy sources enter subterranean habitats in a variety of ways. Flowing water, especially streams entering caves, carries with it not only dissolved organic material, but also particulate organic material. Percolating water provides input of dissolved organic matter. Wind and gravity bring nutrients into caves when organic material comes into an entrance. Examples include falling leaves as well as the body of dead animals. Guano is one of the most important food sources in many caves. (Culver and Pipan, 2009; Fatemi et al., 2019). Culver et al. (2003) hypothesized a connection between surface productivity and the amount of food available in 2 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 caves: in nearly all caves, there is no significant chemoautotrophy so that all available food results from the surface productivity. 1.1.4 Cave animals Subterranean organisms can be classified into three main groups, according to their affinity to life in this habitat (ecologically, in relation to their association with subterranean/cave environments): a) Troglobiont: any of the organisms found in caves (subterranean habitats) that display the typical, convergent phenotypes (morphological, physiological, and behavioral) such as loss of eyes and pigmentation, and elongation of appendages. However, the degree of troglomorphy could be different in different troglobiont animals. Sket (2008) Mentioned that we have to accept that the degree of troglomorphy does not necessarily correspond to the strength of the ‘‘troglobiosis‘‘. Troglobionts are obligatory cave species (troglos: cave and bios: life). The term stygobiont is used for the aquatic obligate troglobionts (Sket, 2008; Culver and Pipan, 2009). b) Troglophile: an organism that can complete its life cycle in caves but may also do so outside of caves. This group could be essentially epigean species able to maintain a permanent subterranean population (which may become troglobiont). Sket, 2008 considered two unnecessary categories for troglophiles: a) eutroglophile is an essentially epigean species, but able to maintain a permanent subterranean population; b) subtroglophile is inclined perpetually or temporarily to inhabit a subterranean habitat but is bound to the surface for some biological functions (e.g., feeding) (Sket, 2008; Romero, 2009). c) Trogloxene: species only occurring sporadically in a hypogean habitat and unable to establish a subterranean population. An organism that habitually enters caves but must return periodically to the outside for certain of its living requirements, usually food. Bats and cave birds are good examples. This term was introduced by Racovitza (1907) to replace accidental or ‘occasional guest’ (Camacho, 1992; Culver and White, 2005; Sket, 2008; Culver and Pipan, 2009; Romero, 2009). 3 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1.1.5 Troglomorphic traits These adaptations are the response of animal to survive in the lightless, food-poor cave environment. There are some regressive and convergent characters such as varying degrees of eye loss and depigmentation. Some predictive adaptations are increase in tactile and chemical sensitivities (elongation of appendages and increase in numbers of sensory hairs, setae and other receptors), and increase in metabolic economy (reduction of the routine metabolic rate, aggressive and territory behavior and fright reactions). Some adaptations in terrestrial troglobionts are to the moisture. Development of paedomorphic forms is another predictive adaptation in obligate cave-dwellings. There are some adaptations regarding the life cycles such as longer duration of phases of life cycle. They show some developments of certain reproductive strategies such as low fecundity, increase in egg size and decrease in the number of eggs (Camacho, 1992; Christiansen, 1992). Many abovementioned adaptations could be toward saving energy. The ability of saving more fat in body has been improved in these animals. They have an increase in food-finding ability. Although an increase in food-finding ability in cave animals often seems to go along with an increase in food-searching activity, changed motion patterns result in a reduction of energy expenditure, sometimes to exaggerated extension. For example, in the most cave-adapted species of the amblyopsid fish in North America, over 90% of the total energy savings by adaptations are based on the reduced activity (Poulson, 1985; Camacho, 1992; Culver and white, 2005; Culver and Pipan, 2009). 1.1.6 Global biodiversity of troglobiotic animals Although relatively poorly studied in comparison with the epigean ecosystems, subterranean ecosystems show a rather high biodiversity at the global scale. However, subterranean biodiversity is much lower than epigean biodiversity, but it is not as poor as it seems to be. Extensive biological studies have been carried out in the main karst areas around the world, namely in the eastern United States of America and in the region from Pyrenees to Slovenia and the Dinaric Karst (Culver et al., 2000; Culver and Pipan, 2009). Globally, over 7000 aquatic subterranean dwellers have been catalogued (Botosaneanu, 1986), but unfortunately, such data are not available for the terrestrial subterranean fauna. It is likely that subterranean habitats harbor even larger numbers of terrestrial species, e.g., Coleoptera alone includes more than 1927 underground-limited species (Juberthie, 1998; Gibert and Deharveng, 2002). Culver and Holsinger (1992) proposed a potential biodiversity of about 50,000 to 100,000 obligate terrestrial subterranean species on the basis of nested regional estimates. Sket et al. (2004) reported more than 975 terrestrial and 650 aquatic obligate subterranean species for the Balkan Peninsula. More than 1,138 obligate cave-dwelling 4 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 species and subspecies from 112 families and 239 genera have been described in the United States (Hobbs, 2012). The subterranean aquatic fauna in most karst areas in Europe has been fairly well studied, with around 1800 stygobiont species known to date (Stoch and Galassi, 2010). Two groups of Vertebrata have troglobiont representatives: salamanders (Amphibia Gray, 1825; Caudata Scopoli, 1777; Urodela Duméril, 1806) (13 species) and fishes (Actinopterygii Klein, 1885) (more than 280 species) (White and Culver, 2012). There are troglobiotic forms of many groups of invertebrates such as Porifera, different groups of worms such as Platyhelminthes (flatworms), Nematoda (roundworms), Annelida (segmented worms), Mollusca (mollusks) especially Gastropoda, many groups of Arthropoda such as Crustacea, Arachnida, Myriapoda (millipedes and centipedes) and Insecta (insects) (Camacho, 1992; Romero, 2009). 1.1.7 Hotspots of troglobiotic biodiversity The syntagm “biodiversity hotspot”, one of the most important global tools for nature conservation, has been used in different ways, but with the same goal: identifying areas around the world that have high biological diversity (Myers, 1988; Reid, 1998; Hughes et al. 2002; Brooks et al., 2006). Biodiversity hotspots were defined by Myers et al. (2000) as the richest places in terms of biodiversity, with high numbers of species found nowhere else and that have already lost 70% of their original vegetation. Hotspots were most commonly referred to areas with high biodiversity that also are with high productivity (Briscoe et al., 2016). Culver and Sket (2000) used the term hotspots of subterranean biodiversity to define subterranean habitats with an arbitrary cutoff of twenty or more obligate subterranean species. Understanding patterns of subterranean biodiversity requires an understanding of regional patterns. In general, the number of species found in any one cave or subsurface site is small relative to the number of species in the region. Culver and Pipan (2009) have set the value to 25 stygobionts or 25 troglobionts per site. Whether an area is a hotspot or not is thus arbitrarily defined, but can have value within a comparative framework. A particularly vexing problem in the analysis of species numbers in subterranean sites is the nearly universal problem of undescribed species. The difficulty is that not all records reported as new species turn out to be new species (Culver et al., 2012, 2021). 5 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Culver and Sket 2000, published a paper listing 20 caves and karst wells with 20 or more known species. In 2021, there was a Special Issue of the journal of Diversity: “Hotspots of Subterranean Biodiversity” expanded and enriched previous hotspot lists (Gibert and Deharveng, 2002; Culver and Pipan, 2009, 2013; Deharveng and Bedos, 2019). These hotspots may not capture all regions of high subterranean species richness; all these hotspot caves are worthy of study and protection in their own right, and any protection strategy should include these exceptional sites as well as regional areas of species richness (Culver et al., 2021). It is worthy reiterating that, compared to surface habitats, the underground diversity is low (Sket, 1999a) and that most diversity in subterranean habitats is expressed regionally rather than locally. The fragmented nature of the cave habitat and restricted opportunities for dispersal keep local diversity much lower than regional diversity (Culver and Sket, 2000). To improve our knowledge of the diversity of a region, it is essential to have large inventories of well-identified species and georeferenced localities for them in order to conduct standardized geospatial analyses that can be compared among regions and at different spatial scales (Culver and Pipan, 2013; Zagmajster et al., 2018). In a study by Trajano et al., 2016, they determined some spots of high diversity of troglobionts in Brazil. They described five Brazilian areas and a cave with their unique troglobiotic faunas, which were considered as spots of high diversity of troglobionts based on the following criteria: (1) high number of troglobionts, when compared to other studied cave areas, (2) presence of higher taxa (families, order) so far not recorded in subterranean habitats elsewhere in Brazil, (3) presence of highly troglomorphic taxa, and (4) presence of phylogenetic relicts (taxa which have no close living relatives, and distributional relicts, whose relatives survived somewhere else sensu Holsinger, 1988) (Trajano et al., 2016). Their criteria seem to be logical, especially for areas that have been poorly studied in regards of troglobiotic faunas. These criteria with modifications and rationalization, considering the alpha and beta species divesrsity, phylogenetic and functional diversity and habitat diverstity in Iran, could be considered for Iran, as well as the entire Middle-East. Prior to this study, the cave with the highest diversity in Iran was Loven cave in Zagros that harbors three species of Iranian cave fishes (Vatandoust et al., 2019). There has been no hotspot of troglobiotic biodiversity or spots of high diversity of troglobionts detected in Iran. Based on some studies on Iranian caves conducted on some regions the presence of some hotspots is expected. However, some parts of Iran are completely arid and as deserts do not support any subterranean hotspots. 6 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1.1.8 Origin of troglobionts From evolutionary and biogeographic points of view and for the study of speciation and adaptation, subterranean environment and its fauna is a natural laboratory. Reduced aboveground dispersal of cave organisms produces phylogenetic patterns of area distribution that largely match the geological history of mountain ranges and cave habitats. Most current hypotheses assume that subterranean lineages arose recently from surface dwelling, dispersive close relatives, but for terrestrial organisms there is scant phylogenetic evidence to support this view. Understanding the origin of subterranean fauna and adaptations have been an interesting topic of numerous evolutionary studies (Culver, 1982; Culver and Pipan, 2009; Ribera et al., 2010). There are two general hypotheses regarding the origin of the subterranean fauna: the climatic relict and the habitat shift hypotheses. The climatic relict hypothesis (Peck and Foinston, 1993) claims that during special climatic conditions, the populations of epigean species are forced into a subterranean environment, so it acts as a refuge for them. These populations become isolated from their surface relatives, and then develop the morphological and physiological adaptations to this new habitat (Peck and Finston, 1993). The adaptive shift hypothesis (Howart, 1980) posits that the opportunity to exploit new resources leads to the colonization of the subterranean environment by epigean populations. These populations are not forced below ground by changing conditions, and there may be limited gene flow between the two environments for some time (Howarth 1980; 1987). Both scenarios assume that subterranean lineages have originated from closely related epigean relatives, with recurrent colonization of the subterranean medium (Ribera et al., 2010). 1.2 SPECIFICATION OF THE RESEARCH PROBLEM Comprising a land area of 1,648,195 km2, Iran is the second-largest country in the Middle East and the 17th-largest in the world. Iran is geologically a part of the Alpine-Himalayan orogenic belt. It lies between latitudes 24° and 40° N, and longitudes 44° and 64° E. Five major structural zones, different in structural history and tectonic style, can be distinguished in Iran (Stöcklin, 1968): (a) The Zagros Range, (b) The Sanandaj-Sirjan Range, (c) Central Iran, (d) East and South-East Iran, and (e) The Alborz and Kopet-Dagh Ranges. The Zagros and Alborz Mountains are the two main mountain chains in western and northern Iran, which 7 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 comprise nearly a third of the Iranian land area (Figure 1) (Raeisi et al., 2012; Malek-Hosseini and Zamani, 2017). More than 10% of the earth's surface is composed of karst. Karstic carbonate formations cover about 11 % of Iran's land area (Figure 2). The total area of the karstified carbonate rocks in Iran is about 185.000 km2, with 55,2 % in the Zagros, 24,3 % in Central Iran, 15,2 % in Alborz, 4,7 % in East and South Iran, and less than 0,5 % in the Sanandaj-Sirjan Range (Raeisi, 2004; Raeisi et al., 2012). Most of the outcrops of carbonate rocks are of the Cretaceous and Tertiary Age. Alavi (2004) recently revised the Zagros stratigraphic column to consist of four age-groups of rocks from Cambrian to Post-Miocene. The age of some main karstic formations in Iran is listed in Table 1. Orogeny and then speleogenesis occurred after the mentioned times in Table 1 and referred to Paleocene- Eocene- Oligocene and after that (Cucchi and Zini, 2003; Popov et al., 2004; Agard et al., 2011; Raeisi et al., 2012). As Iran has arid areas, deserts, low rainfall in many parts of the country, the importance of karstic areas as a resource for groundwater is significant. Groundwater represents about 30% of global freshwater resources (Shiklomanov, 2000). This part of the aquatic ecosystems hosts numerous species that cannot be found in surface freshwaters (Culver et al., 2009; Trontelj et al., 2009; Esmaeili-Rineh et al., 2018). Most of the cave studies in Iran concentrate merely on visiting, photography, and/or mapping the explored caves. Sometimes the complete information about a cave has not been published or it cannot easily be found. The speleological committee of Iran has started to collect cave data, but this has not yet been published. Marefat (1994) reported 258 caves, but the report did not cover all the caves in Iran, and had only partial information about location, altitude, survey, and length of some of the caves; neither geological settings nor hydrologic features were reported. There are many highland karst aquifers without any cave systems yet known, while big springs emerge at their bases of erosion. Several levels of caves are expected in the karst mountains of Iran, as a result of the rapid rates of uplifting and local valley incision. There are two main reasons for the small number of known caves. First, most of the karst areas are high mountains with steep slopes, so that many cave entrances have been filled in by talus or transported sediments or have been blocked by entrance breakdown in the high-risk earthquake regions of Iran. Second, many springs with high discharges are of the vauclusian type, with no explorable dry cave systems. Conduit systems of these springs, which have developed at higher levels in the past, cannot presently be seen on the surface. The deepest cave in Iran is Ghar Parau, 751 m deep (Raeisi and Kowsar, 1997; Gunn, 2003). Also, some other deep caves have been discovered in Iran. Vast amount of faunistic data from Iran has been collected during the last decade but its subterranean fauna remains inadequately known and most reported species from the caves 8 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 are in fact trogloxenes (Malek-Hosseini and Zamani, 2017; Tahami et al., 2017; Esmaeili-Rineh, 2018; Fatemi et al., 2019). Nevertheless, 46 obligate subterranean organisms have been reported from Iran, of which as many as 31 have been described since 2013. 43 species of these troglobionts are restricted to Iran (Löffler, 1959; 1961; Pesce, 1979, 1980 1981; Pesce and Maggi, 1982; Malek-Hosseini and Zamani, 2017; Mamaghani-Shishvan and Esmaeili-Rineh, 2019; Bargrizaneh et al., 2021). The only known troglobiont vertebrates are four fish species. Troglobiont invertebrates include a spider, a diplopod, a beetle and 38 crustaceans. More than 20 troglobionts are waiting to be reported or described. The most species-rich group in subterranean environment is Amphipoda with the genus Niphargus Schiødte, 1849. All Iranian troglobionts were described from the central and northern parts of Zagros and northern slopes of Alborz (Mousavi-Sabet et al., 2016; Malek-Hosseini and Zamani, 2017). This small knowledge of Iranian caves fauna is due to a lack of comprehensive studies of all taxa. So, according to Malek-Hosseini and Zamani (2017), the seemingly low species richness of Iranian troglofauna is not realistic. The number of described caves of Iran is more than 2000 (Raeisi et al., 2012), with many more to be added. There are potential subterranean hotspots in the karst regions of western Caucasus in Georgia and possibly the Tien Shan Mountains in Kyrgyzstan. Although biologically poorly-studied, based on their positive relationship to the productivity maps and the presence of major cave regions they seem to harbor potential subterranean hotspots (Gvozdetski et al., 1994, Klimchouk 2004a, b). These regions are the closest to Iran which somehow have been studied in term of caves fauna. There are a lot of similarities between them and the north and western parts of Iran. Based on the studies conducted on these regions as well as some sporadic studies on Iranian caves in the mentioned areas, we could consider that there are some hotspots there (Malek-Hosseini and Zamani, 2017). From east of Kyrgyzstan to the eastern coast of the Asian continent, the climate is probably too dry to support any subterranean hotspots (Culver et al., 2006). 9 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 1: Map of terrestrial ecoregions in Iran (Map derived from WWF (Olson et al. 2001) and Safaei-Mahroo et al. (2015). Figure 2: Carbonate location map of Iran (according to Raeisi and Kowsar 1997). 10 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Table 1: Age of some karst formations in Iran (Raeisi et al., 2012) Age Zone Formation Surmeh Early to Late Jurassic (20.3-145.4 Mya) Fahliyan Lower Cretaceous (145-100.5 Mya) Bistoon Upper Triassic / Upper Cretaceous (237-66.72 Mya) Sarvak Upper Cretaceous (100.5-66.72 Mya) Zagros Ilam Upper Cretaceous (100.5-66.72 Mya) Jahrum Tertiary (65-2.58 Mya) Asmari Tertiary (65-2.58 Mya) Guri Tertiary (65-2.58 Mya) Jamal Paleozoic (541-251.9 Mya) Central Iran Orbitolina Cretaceous (145-66 Mya) Lar Upper Jurassic (163.5-145.4 Mya) Alborz Tizkuh Lower Cretaceous (145-100.5 Mya) Kopet Mozduran Upper Jurassic (163.5-145.4 Mya) Dagh Tirgan Lower Cretaceous (145-100.5 Mya) The origin of Iranian troglobionts is mostly unknown. However, some taxonomical studies on Iranian cave fishes and the genus Niphargus from amphiopds just mentioned some time period for their divergence, but they did not focus on their origin and time of the colonisation of subterranean environments by these troglobiont animals (Esmaeili-Rineh et al., 2015b; Hashemzadeh Segherloo et al., 2018). Speleobiological studies are required to address many questions. What was the time of cave colonization by these organisms? While such questions have been addressed in most of other known karst biodiversity hotspots such as Balkan Peninsula, such knowledge is currently not available in the Middle-East, the Caucasus and in Central Asia. In order to address this problem, the first step is to survey these troglofaunas. Although faunistic studies in Iran have produced preliminary data, these are largely unavailable for numerous neighboring countries, e.g., Iraq, Afghanistan, Tajikistan, and Armenia. This means that there is a gap of knowledge that extends from the Dinaric karst in the west, through Turkey into Iran, and further east to oriental faunas. Most diverse groups 11 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 of troglobionts in subterranean environments of Middle East are aquatic crustaceans and fish. Some scattered research has been done on biogeography, phylogeography and molecular phylogeny of Middle Eastern subterranean organisms (Bargrizaneh et al., 2021; Kirchner et al., 2021). Iranian subterranean biology must be included into the story. The question is what is the origin of this fauna? Is this the same for all parts of Iranian subterranean environments or different for each structural zone? Speleobiological studies should address this problem for Zagros karst. With taking a look at lithological-paleogeographic maps of Paratethys, we will face with these questions: where did the Iranian troglofauna originate from? From which successors of Tethys or Paratethys? 1.3 REVIEW OF LITERATURE At present, information on the taxonomic composition of the Iranian troglobioict fauna is far from complete. The discovery goes slowly but it has shown that the numbers of discovered organisms is increasing. Most of studies have been faunistic. Some sporadic studies have been carried out on the phylogeny of these troglobiont animals. First study on Iranian troglofauna was done on Iranian cave fish. Bruun and Kaiser (1944) described a cave fish from a subterranean habitat (Loven cave) accidentally in the place where the railway was on constructions. This species had been collected in 1937. They named the new genus and species as Iranocypris typhlops ( Bruun & Kaiser, 1944). Recent studies showed that this species belongs to the genus Garra (Farashi et al., 2014) . Results of a study by Mousavi‐Sabet and Eagderi, 2016 showed that Garra fishes in Loven cave belong to two different species: Garra typhlops (Bruun & Kaiser, 1944) and they also described Garra lorestanensis Mousavi‐Sabet & Eagderi, 2016 (Greenwood, 1976). In 1950, Anthony Smith, who led a group of Oxford University students, visited Iran in search of a reputedly blind white cave fish (Smith, 1953). Their quest failed. Twenty-six years later, Anthony Smith returned to Iran and, using information supplied by Bruun and Kaiser in their paper, visited (not without considerable difficulty) the type locality of Garra typhlops. There, exactly as described by Bruun and Kaiser, he found a small group of fishes swimming in the well-like outlet of a subterranean water-body (Greenwood, 1976). This species was described by Greenwood, 1976 as Noemacheilus smithi. When Nalbant and 12 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Bianco (1998) reviewed the loaches of Iran, they transferred N. smithi to Paracobitis without giving reasons. Hashemzadeh Segherloo et al., 2016 studied this species molecularly as well. They named Eidinemacheilus, as a new generic name for Noemacheilus smithi Greenwood, 1976. Another species of this genus is living in Iraqi Kurdistan: Eidinemacheilus proudlovei Freyhof, Abdullah, Ararat, Ibrahim & Geiger, 2016 (Freyhof et al., 2016). Mahjoorazad and Coad (2009) and Vatandoust et al., 2019 reported G. typhlops and G. lorestanensis from two different localities that first one is 130 km far from Loven cave and the second one is 30 km to the south of Loven cave. These reports may reveal the potential presence of a large freshwater aquifer in the Zagros Mountains. Mousavi‐Sabet et al., 2016 described Garra tashanensis Mousavi-Sabet, Vatandoust, Fatemi & Eagderi, 2016 from Tashan Cave in southeastern part of Iran in Zagros Mointains. Karaman (1998), Esmaeili-Rineh and Sari (2010, 2013), Esmaeili-Rineh et al. 2015a, 2016, 2017, Mamaghani-Shishvan and Esmaeili-Rineh, 2019, Zamanpoore et al., 2019, Bargrizaneh et al., 2021 published several new species of the genus Niphargus in Iran. They have done some molecular analysis on genus Niphargus of Middle-East. According to Esmaeili-Rineh et al., 2015 b, the origin of one of the identified clades from Iran and Lebanon corresponds to marine transgression between the Black Sea and Mediterranean approximately 12 Mya. Three species of troglobiont Isopoda have been discovered from Zagros karstic areas in Iran. Microcharon raffaellae Pesce, 1979 was reported from a well in Shahr-e Kord city (Pesce, 1979). Gakal cave in south-western Iran in Zagros harbors Protracheoniscus gakalicus Kashani, Malek-Hosseini & Sadeghi, 2013, a member of Oniscidea that shows high level of troglomprphism (Kashani et al., 2013). Stenasellus tashanicus Khalaji-Pirbalouty, Fatemi, Malek-Hosseini & Kuntner, 2018 from Tashan cave was the first representative of Stenasellids from the country (Khalaji-Pirbalouty et al., 2018). Trilacuna qarzi Malek-Hosseini & Grismado, 2015 as the first troglobiont spider from Iran, was discovered from Gakal Cave (Malek Hosseini et al., 2015a, b). Chiraziulus troglopersicus Reboleira, Malek-Hosseini, Sadeghi & Enghoff, 2015 as the first troglobiont species of Diplopoda from Iran was reported from Neyneh cave in south-western part of the country in Zagros (Reboleira et al., 2015). 13 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 About 12 species of Copepoda have been reported from subterranean waters of different parts of Iran as troglobionts during 1937-1981 by several authors. Unfortunately, the precise localities for these species have not been determined in literature (Löffler, 1959; 1961; Pesce, 1980; 1981; Pesce and Maggi, 1982). Erpobdella borisi Cichocka, Bielecki, Kur, Pikuła, Kilikowska & Biernacka, 2015 from Erpobdellidae family of Hirudinida was discovered from Sahoolan Cave in north-western Iran as the first troglobiont Hirudinea from the country (Cichocka et al., 2015). Foranotum perforatum Nabozhenko and Sadeghi, 2017 (Coleoptera: Tenebrionidae) as the first troglobiont beetle from Iran was reported from from a cave in Southern Zagros Mountains (Nabozhenko and Sadeghi, 2017). Figure 3: Some of Iranian obligate subterranean dwelling animals: a: Garra lorestanensis; b: Garra typhlops; c: Eidinemacheilus smithi; d: Garra tashanensis; e: Trilacuna qarzi; f: Chiraziulus troglopersicus; g: Protrachaeoniscus gakalicus (photos: Kashani et al., 2013; Malek-Hosseini et al., 2015a; Reboleira et al., 2015; Esmaeili et al., 2016; Mousavi‐Sabet et al., 2016) 14 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1.4 CONSERVATION The identification and protection of priority areas are common goals in managing and conserving biodiversity (Margules et al., 2002). However, our knowledge of subterranean biodiversity is inconsistent and often deficient in many areas. With few exceptions, cave ecosystems and habitats are poorly sampled when compared with surface ecosystems. Caves have an important role in the cycle of nature. Caves are valuable in many perspectives such as ecological (environmental), scientific, cultural heritage, tourism and economic (Romero, 2009). Considering the fact that the life cycles of animals in subterranean environments and especially caves depend on all elements of these environments from abiotic and inanimate parts such as the habitat itself and to all types of cave animals (troglobiont, troglophile, trogloxene), and regarding the vulnerability and high sensitivity of these habitats, they must be protected by applying especial management principles. Conserving an ecosystem implies the protection of habitats and species. Causes of deterioration in subterranean environments are mostly related to human activities. Organic pollutions, problem of heavy metals, petroleum products, excessive visiting by cavers and tourists even scientists, destructions and diggings or vandalism are kinds of these causes. These negative factors that have human origin, threaten the animal population in subterranean environments. Cave species are extremely limited in numbers an also many of them have extremely limited climatic or other environmental requirements (Camacho, 1992). The entry of surface contaminants by such as pesticides and chemical fertilizers, also water extraction from underground aquifers threatens the life in subterranean habitats. Scientific depredation is one of the main causes of some decline even extinction of troglobiont species. Numerous samplings of Iranian cave fishes by different research groups for unnecessary studies have been done as for a single study 44 specimens were collected (Abbasi and Gharezi, 2008; Sargeran et al., 2008). Collecting cave fishes from the same locality and for several times per year and even using invasive methods such as electroshock by researchers is scientific depredation. Reboleira et al., 2015 mentioned some destruction in Neyneh cave in Iran due to the activities of treasure hunters. Fatemi et al., 2019 mentioned that due to the negligence of authorities, cavers, locals, and even the research groups, a lot of habitat destruction has occurred in the recently-discovered Tashan cave that harbors some troglobiont species such as fish, gastropod and isopod. Troglobionts are unique and seem to be restricted to a single cave or karstic system. Many cave species are extremely limited in number. Subterranean environments and specially 15 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 caves, karstic springs and Qanats1 are the most important habitats for troglofauna in Iran. Despite the shortage of water, drought, excessive water extraction from underground, many pollutions from surface to the groundwaters, the Karstology and Speleobiology and thus, conservation of these resources, have been neglected in Iran. Only Iranian cave fishes from Loven cave have the status of Vulnerable (VU) in IUCN list (Coad 1996; Coad et al., 2009; IUCN Red List of Threatened Species, 2018; World Conservation Monitoring Centre, 2018 a, b). In general, biodiversity in Iran is under serious threat; modern infrastructures such as road systems cutting across natural or semi-natural habitat have damaging effects on biodiversity via wildlife-vehicle collision (Farashi and Shariati, 2017). 1.5 THESIS AIMS AND OUTLINE This dissertation follows three main goals to improve the knowledge about Iranian troglobiont animals in several aspects such as faunistic and biodiversity, evolutionary and the origin. First, a discovery component involves faunistic investigations into the troglobiont organisms in Zagros Mountains of Iran, as well as taxonomic descriptions of new taxa. We aim to substantially increase the taxonomic and faunistic knowledge about Iranian subterranean fauna. The putative presence of representatives of all major animal lineages in subterranean environments of Iran that are known as troglobionts from other karst areas is tested. The second aim is to establish a georeferenced database of Iranian subterranean fauna and conduct an analysis of troglobiotic diversity patterns. The presence of one, or several troglobiotic diversity hotspots in Iran is tested. 1 A qanat or kariz, is a system for transporting water from an aquifer or water well to the surface, through an underground aqueduct. 16 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 The third aim is to propose a temporal and phylogenetic frame for the origin of the subterranean fauna of the Zagros Mountains. Considering the age of some karstic areas in Iran and also some preliminary studies on Iranian troglobiont animals, the colonization of subterranean environments by these animals and also some divergence in surface relatives could have occoured in Late Miocene and Pliocene or even as recent as Pleistocene. In the first part of the thesis, we present the description of a troglobiont carabid beetle genus Duvalius Delarouzée, 1859 and one of its surface-dwelling congeners as a step towards the first aim of this study. This chapter also includes a part of the third aim. A species level phylogeny of Duvalius and closely related genera, and a dated phylogeny that enables understanding of the timing of cave colonization by this troglobiont beetle are presented. The description of a new obligate cave-dwelling genus and species of gastropods is presented in this thesis. Using our original sequences and sequences from GenBank, we reconstruct the phylogenetic trees of the related taxa with our new genus. We present the description of a new subterranean-dwelling species of the genus Asellus from Iran. Both morphological and molecular studies confirm that this is a new species. Species phylogenetic relationships using original and datamined mitochondrial and nuclear rDNA, and estimation of molecular divergences with other Asellus species, suggest that A. ismailsezarii sp. nov. is sister to a larger clade that also contains the European A. aquaticus species complex. This indicates that this species does not belong to the European A. aquaticus species complex. In this thesis, we present the checklist of subterranean arthropods of Iran as a published article. In an addition to this article, we represent an overview of troglobiotic biodiversity in Iran. In this part, we represent all Iranian troglobiotic animals (previously published and our new discoveries, including yet undescribed species). The species richness and distribution patterns of Iranian troglobiotic diversity are presented, including the currently recognized hotspot. Given the poor knowledge of Iranian subterranean fauna, this speleobiological dissertation adds critical knowledge by reporting new findings and describing new species. The discovery component of this thesis enables us to establish Iran as an important part of the world map of subterranean biodiversity by discovery of some new species that previously 17 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 had not been reported from the country even to the order or family level. This dissertation attempts to detect the origins of Iranian troglobionts and the time of their adaptation to subterranean environment. This fauna shows high endemism. This small discovered species and what will be discovered is very important from conservation perspective. Such studies about cave fauna should lead to decisions about conservation approaches. 18 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2 SCIENTIFIC WORKS 2.1 PUBLISHED SCIENTIFIC WORKS 2.1.1 First insights into the origin of Iranian cave beetle diversity with description of two new species of the genus Duvalius (Carabidae) Malek-Hosseini M. J., Muilwijk J., Gregorič, M., Kuntner M., Čandek, K. 2021. First insights into the origin of Iranian cave beetle diversity with description of two new species of the genus Duvalius (Carabidae). Journal of Zoological Systematics and Evolutionary Research, 59, 7:1453-1469, doi: 10.1111/jzs.12537 Naslov: Prvi vpogled v izvor raznolikosti iranskih jamskih hroščev z opisom dveh novih vrst rodu Duvalius (Carabidae) Izvleček: Podzemna okolja Irana so zelo slabo raziskana. V tem prispevku želimo izboljšati znanje o iranski jamski biotski raznovrstnosti, pri čemer sledimo trem ciljem: (i) raziskati iranske jame za troglobiontske hrošče; (ii) razumeti filogenetske odnose znotraj rodu Duvalius Delarouzée, 1859 in oceniti časovno obdobje kolonizacije iranskih jam; in (iii) komentirati trenutno poznavanje iranske troglobiontske favne za lažje prihodnje raziskave. S terenskim delom in morfološkim pregledom smo opisali dve novi vrsti rodu Duvalius iz jam v gorovju Zagros v Iranu: troglobiotski Duvalius nezelensis sp. nov. in netroglobiotski Duvalius achaemenius sp. nov. Za filogenetske analize smo pridobili izvirna zaporedja dveh mitohondrijskih (COI, 16S) in dveh jedrnih (18S, 28S) genov za tri vrste rodu Duvalius iz gorovja Zagros in jih združili z objavljenimi molekularnimi podatki drugih vrst in rodov hroščev. Z uporabo Bayesovega sklepanja in največje verjetnosti smo rekonstruirali filogenijo rodu Duvalius in tesno sorodnih rodov, nato pa s programom BEAST izračunali čas razhajanja glavnih linij. Naše filogenije so dobro podprle klad "Zagros", katerega ločitev od drugih sorodnikov je ocenjena na 9,7 milijona let. V kladu Zagros je razcep D. nezelensis in njegove sestrske skupine ocenjen na 7,8 milijona let, medtem ko je razcep D. achaemenius in D. kileri ocenjen na nedavnih 0,78 milijona let. V študiji smo prispevali nekaj razrešitve pri razumevanju vrstnega bogastva jamskih hroščev v Iranu in časa njihove podzemne kolonizacije. Vendar naše filogenije potrjujejo taksonomske težave, saj je več rodov ugnezdenih globoko v “rodu” Duvalius. Ključne besede: Duvalius, molekularna filogenija, Trechini, troglobiont, Zagros 19 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 20 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 21 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 22 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 23 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 24 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 25 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 26 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 27 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 28 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 29 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 30 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 31 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 32 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 33 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 34 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 35 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 36 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.2 Description of a new genus and species as the first gastropod species from caves in Iran. Fatemi Y., Malek-Hosseini M. J., Falniowski A., Hofman S., Kuntner M., Grego J. 2019. Description of a new genus and species as the first gastropod species from caves in Iran. Journal of Cave and Karst Studies, v. 81, no. 4, p. 233-243, https://caves.org/pub/journal/PDF/v81/81_4_233.pdf Naslov: Opis novega rodu in prve vrste polžev iz jam v Iranu Izvleček: Poročamo o novem stigobiontskem trunkateloidnem polžu iz sulfidnih jezerc v jami Tašan v gorovju Zagros v jugozahodnem Iranu. Habitat bogat z vodikovim sulfidom je podoben doslej znanim habitatom polžev iz s sulfidi bogatih jam v Romuniji, Italiji in Grčiji. Novo opisani rod Trogloiranica n. gen. z novo opisano vrsto T. tashanica n. sp. predstavljata prve prave stigobiontske polže, najdene v Iranu. Na podlagi filogenije, pridobljene iz mitohondrijskega (citokrom oksidaza podenota I) in jedrnega (histon 3) genoma ter anatomskih znakov, je T. tashanica uvrščena v družino Moitessieriidae Bourguignat, 1863. Novi rod lahko predstavlja evolucijski relikt iz obdobja po miocenu, preden sta se razcepili družini Moitessieriidae in Cochliopidae Tryon, 1866. 37 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 38 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 39 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 40 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 41 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 42 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 43 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 44 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 45 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 46 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 47 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 48 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Table 2 (S1): Our original sequences and additional sequences from GenBank were used in the phylogenetic analysis. Species name COI H3 Agrafia wiktori JF906762 MG543158 Alzoniella finalina AF367650 Anagastina zetavalis EF070616 Avenionia brevis berenguieri AF367638 Belgrandiella cf. kusceri KT218511 MG551366 Belgrandiella cf. kuesteri MG551327 Bithynia tentaculata AF367643 Bythinella austriaca JQ639858 Bythinella micherdzinskii JQ639854/- Bythiospeum acicula KU341350 Bythiospeum acutum HM107120 Bythiospeum alzense KU341354 Bythiospeum francomontanum HM107128/- Bythiospeum cf. gebhardti KP296922 Bythiospeum hungaricum KP296923 Bythiospeum husmanni HM107134 Bythiospeum pellucidum HM107124 Bythiospeum quenstedti HM107113 Bythiospeum saxigenum HM107125 Bythiospeum suevicum HM107118 Bythiospeum sp. AF367634/- Bythiospeum sp . Achenwald KU341341 Bythiospeum sp. Hinterfallbach KU341347 Continuation 49 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 2 (S1) Bythiospeum sp. Rheinfelden KU341360 Bythiospeum sp . Schwangau KU341338 Dalmatinella fluviatilis KC344541 Daphniola louisi KM887915 Ecrobia maritima KJ406200 MG551322 Emmericia expansilabris KC810060 Fissuria boui AF367654 Graziana alpestris AF367641 Grossuana codreanui EF061919 Hauffenia michleri - KY087878 Heleobia dalmatica JQ973022 Heleobia dobrogica EU938131 Heleobia foxianensis JQ973023 Heleobia maltzani KM213723 Heleobia stagnorum JQ973024 Heleobops carrikeri AF213347 Horatia klecakiana KJ159128 Hydrobia acuta AF278808 Iglica cf. gracilis MH720985-MH720989 MH721003- MH721004 Iglica cf. hauffeni MH720995 Iglica cf. forumjuliana MH721006 Iglica hellenica KT825581 MH721007 Islamia zermanica KU662362 MG551320 Lithoglyphus prasinus JX073651 Littoridinops monroensis AF367644 Continuation 50 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 2 (S1) Littorina littorea KF644330 Marstoniopsis insubrica AF322408 Moitessieria cf . puteana AF367635 MH721012 Montenegrospeum bogici () KM875510 MG880218 Onobops jacksoni AF367645 Paladilhiopsis cf. absoloni MH721021 Paladilhiopsis blihensis MH721015 Paladilhiopsis bosniaca MH721020 Paladilhiopsis bosnica MH721021 Paladilhiopsis grobbeni MH720990-MH720991 MH721013 Paladilhiopsis maroskoi MH721017 Paladilhiopsis turrita MH720992 MH721015 Paladilhiopsis gittenbergeri MH720993 MH721025 Peringia ulvae AF118302 Pontobelgrandiella sp . KU497024 MG551321 Pseudamnicola sp. KT710579 Pyrgophorus platyrachis AF367632 Radomaniola curta KC011814 Sadleriana fluminensis KF193067 Salenthydrobia ferrerii AF449213 Sardopaladilhia plagigeyerica HQ623176 Semisalsa dalmatica AF367631 Spurwinkia salsa AF367633 Tanousia zrmanjae KU041812 Trogloiranica tashanica MK906040-MK906039 MK906042-MK906041 Continuation 51 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 2 (S1) Truncatella pulchella AF253085 Truncatella scalaris JX970621 52 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.3 A new obligate groundwater species of Asellus (Isopoda: Asellidae) from Iran Malek-Hosseini M. J., Jugovic J., Fatemi Y, Kuntner M, Kostanjšek R, Douady C. J., Malard F. 2022. A new obligate groundwater species of Asellus (Isopoda: Asellidae) from Iran. Subterranean Biology, 42: 97-124, doi: 10.3897/subtbiol.42.79447 Naslov: Nova obligatna podzemeljska vrsta iz rodu Asellus (Isopoda, Asellidae) iz Irana Izvleček: Z le 43 opisanimi stigobionti in le dvema vrstama enakonožnih rakov je obligatna podzemeljska favna Irana, obsežne države z več kot 10 % površine apnenca, nezadostno poznana. Tu poročamo o odkritju vrste Asellus ismailsezarii sp. nov. iz gorovja Zagros, prvega brezokega in depigmentiranega asellidnega enakonožca iz Irana. Nova vrsta je morfološko podobna vrsti Asellus monticola Birstein, 1932, vendar je brez oči in popolnoma depigmentirana, ima rahlo ukrivljen pereopod IV in nima sete na proksimalnih robovih eksopodita pleopodov IV in V. Filogenetska razmerja med vrstami z uporabo izvirne in podatkovno rudarjene mitohondrijske DNK in jedrne rDNK ter ocena molekularnih razlik z drugimi vrstami rodu Asellus kažejo, da je A. ismailsezarii sp. nov. sestrska vrsta večjega klada, ki vsebuje tudi evropski kompleks vrst A. aquaticus. Površinske populacije vrst iz rodu Asellus so ob različnih priložnostih in v več krajih v Evropi in Aziji kolonizirale podtalnico, pri čemer so nastale vrste in podvrste, ki so razvile troglomorfizme, kot sta depigmentacija in izguba oči. Od 37 uradno opisanih vrst in podvrst rodu Asellus jih je 15 iz podzemne vode, vključno z A. ismailsezarii sp. nov. Predvidevamo, da bo v Aziji odkritih še veliko vrst rodu Asellus, ki obligatno poseljujejo podzemne vode. Ključne besede Azija, Crustacea, podsemeljske vode, molekularna filogenija, taksonomija, troglomorfija 53 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 54 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 55 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 56 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 57 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 58 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 59 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 60 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 61 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 62 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 63 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 64 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 65 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 66 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 67 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 68 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 69 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 70 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 71 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 72 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 73 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 74 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 75 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 76 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 77 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 78 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 79 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 80 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 81 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.1.4 A checklist of subterranean arthropods of Iran Malek-Hosseini M. J., Zamani A. 2017. A checklist of subterranean arthropods of Iran. Subterranean Biology, 21: 19–46, doi: 10.3897/subtbiol.21.10573 Naslov: Vrstni seznam podzemnih členonožcev Irana Izvleček: Razumevanje podzemeljske biotske raznovrstnosti je pomembno, vendar so obsežna območja sveta še vedno slabo raziskana. V tem prispevku predstavljamo prvi korak k popisu podzemeljskih členonožcev Irana. Po pregledu in analizi dostopne literature ter pregledu vzorcev, ki smo jih zbrali v iranskih jamah, smo našteli 89 jamskih vrst (iz 42 jam in 5 kraških izvirov), ki pripadajo štirim poddeblom členonožcev: Chelicerata (1 razred, 4 redovi, 36 vrst), Crustacea (2 razreda, 3 redovi, 15 vrst), Hexapoda (2 razreda, 5 redov, 34 vrst) in Myriapoda (2 razreda, 3 redovi, 4 vrste). Ključne besede Jame, favna, Srednji vzhod, Zagros, troglofavna 82 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 83 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 84 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 85 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 86 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 87 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 88 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 89 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 90 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 91 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 92 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 93 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 94 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 95 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 96 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 97 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 98 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 99 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 100 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 101 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 102 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 103 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 104 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 105 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 106 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 107 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 108 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 109 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 110 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.2 REMAINING LINKING SCIENTIFIC WORK 2.2.1 Overview of troglobiont biodiversity in Iran This part of dissertation represents updated information to the checklist we published in 2017. In that checklist 2017 (Malek-Hosseini and Zamani, 2017), 16 troglobiont species were listed for Iran. However, already at the time of this publication, some new troglobionts were being described from Iran. The list also missed 12 species of copepods from older literature (mostly in French and German languages) and one oligochaete. Including these missing sources, 29 species had been reported as troglobionts from Iran in 2017. Here, we represent all Iranian troglobiont animals (previously published and our new discoveries, including yet undescribed species). The species richness and distribution patterns of Iranian troglobiotic diversity are presented, including the currently recognized hotspots. So, the aims of this chapter are: - to provide the updated checklist of troglobiont species of Iran - to map the distribution of troglobionts in Iran - to detect any hotspots of troglobiont biodiversity in Iran 2.2.1.1 Introduction Iran is 1.648 million km2 large country (the second-largest country in the Middle East after Saudi Arabia), situated in western Asia. Most of the land lies in an arid environmental zone. The area coverage of different types of climates in Iran is 35.5% hyper-arid, 29.2% arid, 20.1% semi-arid, 5% Mediterranean and 10% wet (of the cold mountainous type). Thus, more than 82% of Iran’s territory is located in the arid and semi-arid zone of the world. The average rainfall in Iran is about 250 mm, which is less than 1/3 of the average rainfall in the world (860 mm) (Amiri and Eslamian, 2010). In addition, this sparse precipitation is also unfavorable with respect to time and location. Another important climatic element is extreme temperature changes that at some places range from -20 to +50°C. Severe drought is also recognized as a feature of Iran’s climate (Amiri and Eslamian, 2010). The rainfall is different from more than 1800 mm in northern parts of Iran on the Caspian seashore and in some parts of the Zagros about 800 mm, but average is about 400 mm in the sloping region of Alborz and Zagros mountains, and in arid parts and central Iran less than 100 mm (Raziei et al., 2005; Soltani et al., 2012). During this decade, the country has suffered severe desiccation 111 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and this lack of rainfall has resulted in extensive losses of water resources, vegetation, and productivity. It is one of the world's most mountainous countries, its landscape dominated by rugged mountain ranges that separate various basins or plateaux from one another. The Zagros and Alborz Mountains are two main mountain chains in western and northern Iran, which comprise nearly a third of the Iranian surface. Geologically, Iran is a part of the Alpine-Himalayan orogenic belt, divided into five major structural zones: a) Zagros Range, b) Sanandaj- Sirjan Range, c) Central Iran, d) East and South-East Iran and e) Alborz and Kopet- Dagh Ranges (Raeisi et al. 2012). The proportion of carbonate karst surface in Iran land area is more than 11% (Raeisi et al. 2012), with more than 2000 discovered caves up to now (Raeisi et al. 2012), and many new expected. This value of carbonate karst surface increases to about 23% in the south-central region of Iran (Raeisi and Kowsar, 1997). During the last decade, several faunistic studies have been carried out in Iran, resulting in new records of already known species, as well as the descriptions of many new ones. However, despite the vast amount of collected faunistic data, the Iranian subterranean fauna remains inadequately known, and most reported species from the caves are in fact troglophiles and trogloxenes (Malek-Hosseini et al., 2015a, b; Malek-Hosseini and Zamani, 2017; Malek-Hosseini et al., 2021). During this decade, more discoveries have been carried out in underground environments of Iran and mostly in Zagros Mountains. Another new species of cave fishes, new habitats and new troglobiont arthropods have been discovered in Iran. 23 species of the genus Niphargus Schiødte, 1849 from amphipods have been recorded from Iran from 28 localities. So, the genus Niphargus is with highest species richness of troglobionts in Iran (Mousavi‐Sabet et al., 2016; Bargrizaneh et al., 2021). Other troglobionts include a spider, a millipede, a terrestrial oniscid isopod, few species of copepods and a coleopteran beetle. Most of these troglobionts have been described during last 15 years (Kashani et al., 2013; Malek-Hosseini et al., 2015a; Reboleira et al., 2015; Malek-Hosseini et al., 2021). Endemism and species richness are highly relevant to the prioritization of conservation efforts in which Iranian subterranean environments have remained relatively neglected. The discovery component and preparation of a checklist of Iranian troglobionts with the map of distribution, will enable us to establish Iran as an important part of the world map of subterranean biodiversity. This will therefore facilitate conservation decisions. 112 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.2.1.2 Material and methods We used all published literature in any language and supplemented it with our own data. Some new taxa were described recently, but there are many more new species that were identified via DNA analysis and await morphological descriptions. Records of yet undescribed species are referenced in the separate section of this list. In this chapter, we provide the updated checklist of Iranian troglobiont animals based on the available literature, our collection and published and unpublished material. We mentioned the species that have been reported as troglobiont in literature. For our own observations of collected species in this study, we used the term of troglobiont that is determined in the introduction of this thesis. Up to date, more than 100 caves, wells and springs have been surveyed for subterranean animals (arthropods, mollusks, oligochaetes and fishes). The caves that have been reported as habitats for bats only, are not included. However, many of these caves out of these 100 habitats, harbor bats. Most of reported animals are indeed troglophiles and trogloxenes (Malek-Hosseini and Zamani., 2017; Tahami et al., 2017). Prior to this study, 46 species had been reported as troglobiont from Iran. Some taxa that were reported for the first time from Iran as troglobiont, were discovered by a group that the author of this thesis (Mohammad Javad Malek-Hosseini) was involved in (e.g. Malek-Hosseini et al., 2015a; Khalaji-Pirbalouti et al., 2019; Zamanpoore et al., 2020), and so, most of discoveries are recent (2010-2021) (e.g. Hekmatara et al., 2013; Mousavi-Sabet at al., 2016; Esmaeili-Rineh, et al., 2018; Bargrizaneh et al., 2021). When mapping species richness and sampled localities distribution, the problem of finding suitable scale for analyses needs to be addressed because different scales shows that the pattern depends on the scale size we use (Stoms, 1994). If the basic sample cells are too large, the regional patterns and details are lost, but if the cells are too small, the data are over-scattered (Fortin and Dale, 2005). Regarding this fact that not many subterranean habitats have been studied in Iran and thus considering the vastness of the country and number of known caves and karstic springs from Iran, the density of caves and springs in each area, we decided to consider 100 × 100 km scale for analyses. We generated a troglobiont species-richness map by calculating the number of species present in each 100 × 100-km UTM grid cell. 113 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Our research area covers the whole Iran but as showed in the maps, most species have been reported from west and north of the country. Considering the low number of species, we mentioned all troglobiont species in this study. Localities were identified according to the descriptions in the literature sources, and we also used our own data. For some old literatures without the exact locality, we could not determine exact position, so we put the coordinates to the central parts of the closest town or village; this was done for five localities, harboring 14 species. In the first part of the analysis, we prepared maps with pointed localities by numbers. The details are shown in table 2. Number of species are determined for each subterranean habitat. Richness per locality is shown on map by the size of circles and points. We used QGIS to prepare the distribution maps of species. 2.2.1.3 Updated checklist of troglobiont species of Iran From all 49 identified and described troglobiont species of Iran (five terrestrial, 44 aquatic), 43 species are arthropods, and of these, 39 species are crustaceans, and 23 belong to the genus Niphargus (Niphargidae). In this study, we discovered 26 new species of troglobiont animals. We have described three of them. We described also a troglophile species of beetles, sister to the new discovered troglobiont one (Malek-Hosseini et al., 2021; Fatemi et al., 2019). In this thesis, we only presented the three published articles including the descriptions of two of our new troglobiont species ( Duvalius nezelensis Muilwijk and Malek-Hosseini, 2021 of carabid beetles; Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego 2019 of gastropods and Asellus ismailsezarii Malek-Hosseini, Jugovic, Fatemi & Douady, 2022 from Isopoda) (Malek-Hosseini et al., 2022) and a troglophile Duvalius achaemenius Muilwijk and Malek-Hosseini, 2021. The description of another species of obligate cave-dwelling species of crustacea (a Caridina atyid shrimp) is under preparation. List of species 114 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Phylum Chordata Class Actinopterygii Klein, 1885 Order Cypriniformes Bleeker, 1859 Family Cyprinidae Linnaeus, 1758 Garra lorestanensis Mousavi-Sabet & Eagderi, 2016 Synonym: Iranocypris typhlops, Bruun, E. W. Kaiser, 1944 Records: Tang-e-Haft- Darreh Bagh - Sirom Village, Pol Dokhtar City, Lorestan Province, Loven Cave, N33°4'39" E48°35'33" (Mousavi-Sabet et al., 2016), Figure 9, locality 20. Records: Tuveh Village, Andimeshk City, Khuzestan Province, Tuveh Spring, N32°48'48.8" E48°43'7", (Vatandoust et al., 2019), Figure 9, locality 19. Records: Talkhab Hendmini Village, Darrehshah City, Ilam Province, Drainage, the Simarreh River basin, intake tunnel for a dam, N33°16'56" E47°12'16", (Mahjoorazad and Coad, 2009), Figure 9, locality 21. Garra typhlops (Bruun & Kaiser, 1944) Synonym: Iranocypris typhlops, Bruun & Kaiser, 1944 Records: Tang-e-Haft- Darreh Bagh - Sirom Village, Pol Dokhtar City, Lorestan Province, Loven Cave, N33°4'39" E48°35'33" (Bruun & Kaiser, 1944; Mousavi-Sabet et al., 2016), Figure 9, locality 20. Records: Tuveh Village, Andimeshk City, Khuzestan Province, Tuveh Spring, N32°48'48.8" E48°43'7", (Vatandoust et al., 2019), Figure 9, locality 19. Garra tashanensis Mousavi-Sabet, Vatandoust, Fatemi & Eagderi, 2016 115 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", (Mousavi-Sabet et al., 2016), Figure 9, locality 13. Family Nemacheilidae Regan, 1911 Eidinemacheilus smithi (Greenwood, 1976) Synonym: Paracobitis smithi (Greenwood, 1976) Synonym: Noemacheilus smithi Greenwood, 1976 Records: Records: Tang-e-Haft- Darreh Bagh - Sirom Village, Pol Dokhtar City, Lorestan Province, Loven Cave, N33°4'39" E48°35'33" (Greenwood, 1976; Hashemzadeh Segherloo et al., 2016), Figure 9, locality 20. Phylum Annelida Lamarck, 1809 Class Clitellata Michaelsen, 1919 Subclass Oligochaeta Grube, 1850 Subclass Hirudinea Lamarck, 1818 Family Erpobdellidae Blanchard, 1894 Erpobdella borisi Cichocka, Bielecki, 2015 Records: Sahoolan-Eisa Kand Village, Mahabad City, West Azerbaijan Province, Sahoolan Cave N36°39'16.9" E45°57'10.8", (Cichocka et al., 2015), Figure 9, locality 37. Class Gastropoda Cuvier, 1795 Subclass Caenogastropoda Cox, 1960 116 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Order Littorinomorpha Golikov & Starobogatov, 1975 Moitessieriidae Bourguignat, 1863 *2Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego 2019 * Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29" (Fatemi et al., 2019), Figure 9, locality 13. Phylum Arthropoda von Siebold, 1848 Subphylum Chelicerata Heymons, 1901 Class Arachnida Lamarck, 1801 Order Araneae Clerck, 1757 Family Oonopidae Simon, 1890 Trilacuna qarzi Malek Hosseini & Grismado, 2015 Records: Basht City, Kohgiluyeh and Boyer-Ahmad Province, Gakal Cave, N30°18' E51°9' (Malek-Hosseini et al., 2015a, b), Figure 9, locality 5. Subphylum Myriapoda Latreille, 1802 Class Diplopoda De Blainville in Gervais, 1844 Order Spirostreptida Brandt, 1833 2 The asterisk symbol (*) in the beginning and end of the species names means that this species has been discovered and described in part of this thesis 117 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Family Cambalidae Hoffman, 1979 Chiraziulus troglopersicus Reboleira, Malek Hosseini, Sadeghi & Enghoff, 2015 Records: Khaeez Vilage, Kohgiluyeh and Boyer-Ahmad Province, Neyneh Cave, N30°40'18" E50°21'71", (Reboleira et al., 2015), Figure 9, locality 12. Subphylum Crustacea Brünnich, 1772 Class Malacostraca Latreille, 1802 Order Amphipoda Latreille, 1816 Family Niphargidae Bousfield, 1977 Niphargus alisadri Esmaeili-Rineh & Sari, 2013 Records: Hamedan Province, Alisadr Cave, N35°16' E48°17' (Esmaeili-Rineh and Sari, 2013), Figure 9, locality 32. Niphargus bisitunicus Esmaeili-Rineh, Sari & Fišer, 2015 Records: Kermanshah Province, Sarab-e-Bisitun, N34°25' E47°28', (Esmaeili-Rineh et al., 2015), Figure 9, locality 28. Niphargus borisi Esmaeili-Rineh, Sari & Fišer, 2015 Records: Choram City, Kohgiluyeh and Boyer-Ahmad Province, Belqis Spring, N30°45' E50°44', (Esmaeili-Rineh et al., 2015), Figure 9, locality 10. Niphargus daniali Esmaeili-Rineh & Sari, 2013 118 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Lireh Sar Village, Salmanshahr City - Abbasabad district, Mazandaran Province, Danial Cave, N36°41' E50°53', (Esmaeili-Rineh and Sari, 2013), Figure 9, locality 40. Niphargus darvishi Esmaeili-Rineh, Sari & Fišer, 2015 Records: Kohrang-Chelgerd Village, Kohrang City, Chaharmahal and Bakhtiari Province, Dimeh Spring, N32°30' E50°13', (Esmaeili- Rineh et al., 2015), Figure 9, locality 17. Niphargus fiseri Mamaghani-Shishvan & Esmaeili-Rineh, 2019 Records: Badin Abad Village, Piranshahr City, West Azerbaijan Province, Badin Abad Spring, N36°34'55" E45°10'34" (Mamaghani-Shishvan and Esmaeili-Rineh, 2019), Figure 9, locality 36. Niphargus hakani Esmaeili-Rineh, Mirghaffari & Sharifi, 2017 Records: Razan City, Hamedan Province, Kheder- Goli Spring, N35°27' E49°07', (Esmaeili-Rineh et al., 2017a), Figure 9, locality 33. Niphargus hosseiniei Esmaeili-Rineh, Sari, Fišer & Bargrizaneh, 2017 Records: Sahneh City, Kermanshah Province, Nojivaran Spring, N34°29' E47°29', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 29. Records: Bazargan City, West Azerbaijan Province, Brolan Spring, N39°47' E44°35', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 45. Records: Ravansar City, Kermanshah Province, Ghori-Ghale Cave, N34°53' E46°34', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 31. Niphargus ilamensis Esmaeili-Rineh, Sari, Fišer & Bargrizaneh, 2017 119 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Darreh Shahr City, Ilam Province, Sarab-e-Moord , N33°14' E46°54', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 23. Records: Darreh Shahr City, Ilam Province, Sarab-e-Kanipahn, N33°20' E46°46', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 24. Niphargus keeleri Zamanpoore & Bakhshi, 2019 (Figure 8b) Records: Roodrish Village, Dishmook County, Kohgiloyeh va Boyer-Ahmad Province, Keeler Cave, N31°18' E50°35' (Zamanpoore et al., 2019), Figure 9, locality 11. Niphargus kermanshahi Esmaeili-Rineh, Heidari, Fišer & Akmali, 2016 Records: Sahneh City, Kermanshah Province, Kangarshah Spring, N34°36'53.7" E47°39'44.2", (Esmaeili- Rineh et al., 2016), Figure 9, locality 30. Niphargus khayyami Hekmatara, Zakšek, Heidari Baladehi & Fišer, 2013 Records: Ravansar City, Kermanshah Province, Ghoori-Ghaleh Cave, N34°53' E46°34', (Hekmatara et al., 2013), Figure 9, locality 31. Niphargus khwarizmi Hekmatara, Zakšek, Heidari Baladehi & Fišer, 2013 Records: Kharvana village, Tabriz City, East Azarbayjan Province, Cheshmeh Kahriz (Qanat), N38°41' E46°10', (Hekmatara et al., 2013), Figure 9, locality 44. Niphargus kurdistanensis Mamaghani-Shishvan, Esmaeili-Rineh & Fišer, 2017 Records: Baneh City, Kurdistan Province, Shoei Cave, N36°0'18" E45°53'45”, (Mamaghani-Shishvan et al., 2017), Figure 9, locality 35. Records: Marivan City, Kurdistan Province, Darvish-Olya Cave, N35°37'14" E46°37'59" (Mamaghani-Shishvan et al., 2017), Figure 9, locality 34. 120 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Niphargus lorestanensis Esmaeili-Rineh, 2018 Records: Aligudarz City, Lorestan Province, Shol-Abad Spring, N33°09′ E49°5′ (Esmaeili-Rineh, 2018), Figure 9, locality 22. Niphargus nasrullahi Bargrizaneh, Esmaeili-Rineh & Fišer, 2021 Records: Kakan Village, Yasuj City, Kohgiloyeh va Boyer-Ahmad Province, Nahr Spring , N30°40' E51°43' (Bargrizaneh et al., 2021), Figure 9 locality 6. Niphargus persicus Esmaeili-Rineh, Sari, Fišer & Bargrizaneh, 2017 Records: Tir-e-Bagh Village, Marvdasht City, Fars Province, Tir-e-Bagh Spring, N30°14' E52°40' (Esmaeili- Rineh et al., 2017b), Figure 9, locality 4. Niphargus sari Esmaeili-Rineh, Niyakan & Akmali, 2018 Records: Arkavaz Village, Chardavol City Ilam Province, Jo-Khanem Spring, N33°19' E46°40' (Esmaeili-Rineh, et al., 2018), Figure 9, locality 25. Niphargus sharifi Esmaeili-Rineh, Sari & Fišer, 2015 Records: Khoram-Abad City, Lorestan Province, Sarab-e-Robat, N33°35' E48°18', (Esmaeili-Rineh et al., 2015), Figure 9, locality 26. Niphargus sohrevardensis Esmaeili-Rineh, Sari, Fišer &Bargrizaneh, 2017 Records: Zanjan City, Zanjan Province, Sohrevard Spring, N36°40' E48°29', (Esmaeili-Rineh et al., 2017b), Figure 9, locality 39. 121 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Khoram-Abad City, Lorestan Province, Razbashi Spring, N33°38' E48°35' (Esmaeili- Rineh et al., 2017b), Figure 9, locality 27. Niphargus urmiensis Mamaghani-Shishvan & Esmaeili-Rineh, 2019 Records: Oshnavieh City, West Azerbaijan Province, Randole Spring, N37°3'25" E45°2'48" (Mamaghani-Shishvan & Esmaeili-Rineh, 2019), Figure 9, locality 38. Niphargus valachicus Dobreanu & Manolache, 1933 Records: Ghaem Shahr, Mazandaran Province, unidentified cave (Karaman, 1998), Approximate point: Figure 9, locality 43. Niphargus yasujenis Bargrizaneh, Esmaeili-Rineh & Fišer, 2021 Biareh Village Sisakht City, Kohgiloyeh va Boyer-Ahmad Province, Biareh Spring, N30°51' E51°27 '(Bargrizaneh et al., 2021), Figure 9, locality 8. Order Isopoda Latreille, 1817 Suborder Oniscidea Latreille, 1802 Protracheoniscus gakalicus Kashani, Malek Hosseini & Sadeghi, 2013 (Figure 8c) Records: Basht City, Kohgiluyeh and Boyer-Ahmad Province, Gakal Cave, N30°18' E51°9' (Kashani et al., 2013), Figure 9, locality 5. Suborder Asellota Latreille, 1802 Family Lepidocharontidae Galassi & Bruce, 2016 Microcharon raffaellae Pesce, 1979 122 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Shahr-e-Kord City, Chaharmahal va Bakhtiari Province, undetermined well in literature (Pesce, 1979), Approximate point: Figure 9, locality 16. Family Stenasellidae Dudich, 1924 Stenasellus tashanicus Khalaji-Pirbalouty, Fatemi, Malek-Hosseini, Kuntner, 2018 (Figure 8f) Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29" (Khalaji-Pirbalouty et al., 2018), Figure 9, locality 13. Family Asellidae Rafinesque, 1815 *Asellus ismailsezarii Malek-Hosseini, Jugovic, Fatemi & Douady, 2022* (Figure 8a) Records: Tuveh village, Andimeshk, Khuzestan Province, Ganow spring, N32°48'31'' E48°43'32'', (Malek-Hosseini et al., 2022), Figure 9, locality 18. Class Maxillopoda Dahl, 1956 Order Cyclopoida Burmeister, 1834 Family Cyclopidae Rafinesque, 1815 Megacyclops dusssarti orientalis Pesce, Pace & Maggi, 1984 Synonymy: Acanthocyclops dussarti orientalis Pesce, Pace & Maggi, 1984 Records: Cialestore Village, Shahr-E-Kord City, Chaharmahal va Bakhtiari Province, undetermined point in literature. (Pesce et al., 1984), Approximate point: Figure 9, locality 16. Diacyclops zschokkei (Graeter, 1910) Synonymy: Cyclops zschokkei Graeter, 1910; Diacyclops languidoides zschokkei (Juberthie, 2001). 123 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Kelarabad City, Mazandaran Province, undetermined point in literature. (Pesce et al., 1984), Approximate point: Figure 9, locality 41. Records: Cialestore Village, Shahr-E-Kord City, Chaharmahal va Bakhtiari Province, undetermined point in literature. (Pesce et al., 1984), Approximate point: Figure 9, locality 16. Diacyclops iranicus Pesce & Maggi, 1982 Records: Kelarabad City, Mazandaran Province, Approximate point: Figure 9, locality 41. Records: Shahr-e-Kord City, Chaharmahal va Bakhtiari Province, undetermined point in literature (Pesce and Maggi, 1982), Approximate point: Figure 9, locality 16. Haplocyclops neuter Kiefer, 1955 Synonymy: Bryocyclops neuter (Kiefer, 1955) Records: Shahr-E-Kord City, Chaharmahal va Bakhtiari Province, undetermined point in literature (Kiefer, 1955), Approximate point: Figure 9, locality 16. Order Harpacticoida G. O. Sars, 1903 Family Ameiridae Boeck, 1865 Parapseudoleptomesochra iranica (Löffler, 1959) Synonymy: Nitocrella iranica Löffler, 1959 Records: Neyriz City, Fars Province, Qualeh Kirmiz, bord du lac Niriz, undetermined point in literature (Löffler, 1959), Approximate point: Figure 9, locality 3. Nitocrella kirgizica Borutsky, 1972 124 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Neyriz City, Fars Province, Qualeh Kirmiz, bord du lac Niriz, undetermined point in literature, (Borutzky, 1972), Approximate point: Figure 9, locality 3. Nitocrella mara Löffler, 1959 Records: Neyriz City, Fars Province, Qualeh Kirmiz, bord du lac Niriz, undetermined point in literature, (Löffler, 1959), Approximate point: Figure 9, locality 3. Nitocrella paceae Pesce, 1980 Records: Shahr-e-Kord, Chaharmahal va Bakhtiari Province (Pesce, 1980), Approximate point: Figure 9, locality 16. Records: Farokhshahr, Chaharmahal va Bakhtiari Province, (Pesce, 1980), Approximate point: Figure 9, locality 15. Nitocrella petkovskii Pesce, 1980 Records: Shahr-e-Kord, Chaharmahal va Bakhtiari Province (Pesce, 1980), Approximate point: Figure 9, locality 16. Records: Farokhshahr, Chaharmahal va Bakhtiari Province, (Pesce, 1980), Approximate point: Figure 9, locality 15. Nitocrella stentinai Sterba, 1973 Records: Saidabad, no more data, (Sterba, 1973; Juberthie, 2001) Nitokra fallaciosa fallaciosa Klie, 1937 125 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Shahr-e-Kord, Chaharmahal va Bakhtiari Province (Juberthie, 1994; 1998; 2001), Approximate point: Figure 9, locality 16. Family Leptastacidae Paraleptastacus spinicaudus triseta Noodt, 1954 Synonymy: Paraleptastacus spinicauda trisetosa Serban, Lang, 1957 Record: Mahmoodabad City, Mazandaran Province, (Juberthie,1994; 1998; 2001), Approximate point: Figure 9, locality 42. Class Insecta Linnaeus, 1758 Order Coleoptera Linnaeus, 1758 Family Tenebrionidae Latreille, 1802 Foranotum perforatum Nabozhenko & Sadeghi, 2017 Record: Ashna village, Khonj City, Fars Province, Khan Cave, N27°44'41" E53°20'15" (Nabozhenko and Sadeghi, 2017), Figure 9, locality 1. Family Carabidae Latreille, 1802 *Duvalius nezelensis Muilwijk & Malek-Hosseini , 2021* Records: Jookhooneh Village, Sarparyab Town, Kohgiloyeh va Boyer-Ahmad Province, Nezel Cave, N30°47'28.6" E50°56'53.8" (Malek-Hosseini et al., 2021), Figure 9, locality 9. 2.2.1.4 Unpublished or undescribed new troglobiont species 126 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Rather than discovered (described and undescribed) species in this study, there is another new species of the genus Asellus from an Iranian caves in literature (Beko, 2016) that has not been described yet. Some animals have been identified to the family even order level. For three species of Oligochaeta, we may be dealing with new family for science. Regarding the molecular studies, it shows that this group is not included in any of other families. For morphology the same, but we need fresh and well-preserved specimens. Despite this fact that in many cases, undescribed species are problematic and counting them as new species are doubtful, but in this study, the collected specimens from different groups of animals that we considered them as new species, first: are geographically far from other congeners; second: some have been discovered from the country for the first time even in order level; third: as we mentioned, initial DNA analysis showed that they are indeed new species and even far from the congeners from other parts of the world and also placed in distinct clade; and forth: the initial morphological check, dissection and comparisons, revealed that they are new species and different from described taxa. List Phylum Annelida Lamarck, 1809 Class Clitellata Michaelsen, 1919 Subclass Oligochaeta Grube, 1850 Order Opisthopora Michaelsen, 1930 Lumbricidae Rafinesque-Schmaltz, 1815 Helodrilus sp. Records: Dangezloo village, Padena County, Isfahan Province, Dangezloo Cave, N30°51' E51°38', Figure 9, locality 7. 127 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Order Haplotaxida Brinkhurst, 1971 Oligochaeta sp. Not identified Records: Torang Village, Baft City, Kerman Province, Torang Cave, N28°45' E56°49', Figure 9, locality 2. Oligochaeta sp. Not identified Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Oligochaeta sp. Not identified Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10’1”, Figure 9, locality 14. Order Cyclopoida Burmeister, 1834 Family Cyclopidae Rafinesque, 1815 Mesocyclops sp. Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Tropocyclops sp. A (Figure 8h) Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. 128 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Tropocyclops sp. B Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Tropocyclops sp. C Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Records: Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10’1”, Figure 9, locality 14. Order Harpacticoida G. O. Sars, 1903 Family Ameiridae Boeck, 1865 Nitocrella sp. A (Figure 8g) Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29, Figure 9, locality 13. Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10’1”, Figure 9, locality 14. Order Thermosbaenacea Monod, 1927 Family Monodellidae Taramelli, 1954 Tethysbaena sp. (Figure 8e) Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29, Figure 9, locality 13. 129 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Order Decapoda Latreille, 1802 Family Atyidae De Haan, 1849 Caridina sp. (Under description) (Figure 8d) Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29, Figure 9, locality 13. Order Amphipoda Latreille, 1816 Family Melitidae Bousfield, 1973 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10'1'', Figure 9, locality 14. Family: Stegocephalidae Dana, 1852 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Family Niphargidae 3Bousfield, 1977 Records: Torang Village, Baft City, Kerman Province, Torang Cave, N28°45' E56°49', Figure 9, locality 2. Order Isopoda Latreille, 1817 Suborder Asellota Latreille, 1802 3 The specimens are in the collection of Shiraz University, Iran. 130 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Family Stenasellidae Dudich, 1924 Stenasellus sp. Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10'1", Figure 9, locality 14. Asellidae Rafinesque, 1815 4 Asellus sp. Records: Sahoolan-Eisa Kand Village, Mahabad City, West Azerbaijan Province, Sahoolan Cave, N36°39'16.9" E45°57'10.8", (Bevk, 2016), Figure 9, locality 36. Order Araneae Clerck, 1757 Family Oonopidae Simon, 1890 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Araneae sp. Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Chah-Kabootari Cave, N30°52'10" E50°10'1", Figure 9, locality 14. Subclass Acari Leach, 1817 Mesostigmata G. Canestrini, 1891 4 This species is not from our collection. It is from Bevk, 2016. 131 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13 Class Diplopoda De Blainville in Gervais, 1844 Order Polydesmida Leach, 1815 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29", Figure 9, locality 13. Class Insecta Linnaeus, 1758 Order Zygentoma Börner, 1904 Family Nicoletiidae Escherich, 1905 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29, Figure 9, locality 13. Subclass Collembola Lubbock, 1871 Records: Sarjoosher Village, Tashan district, Behbahan County, Khuzestan Province, Tashan Cave, N30°51'54" E50°10'29, Figure 9, locality 13. Class Gastropoda Cuvier, 1795 Subclass Caenogastropoda Cox, 1960 Order Littorinimorpha Golikov & Starobogatov, 1975 Hydrobiidae Stimpson, 1865 132 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Records: Tuveh Village, Andimeshk City, Khuzestan Province, Tuveh Spring, N32°48'48.8" E48°43'7", (Vatandoust et al., 2019), Figure 9, locality 19. 2.2.1.5 New records of cave fish During field works, we discovered some new localities for described Iranian cave fishes. Chah-Kabootari cave (Figures 4-5), a limestone cave with sulfur water, a kilometer to west of the Tashan cave harbors Garra tashanensis. Several pools were discovered in Tashan cave as well. Although, the difference in morphology, DNA analysis determined that the isolated populations of Tashan cave belong to a single species. Tashan cave and Chah-Kabootari populations belong to the single species of G. tashanensis. Figure 4: Entrance of Chah-Kabootari cave (Photo: Malek-Hosseini M. J., 2018). 133 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 5: Population of Gara tashanensis in Chah-Kabootari cave (Photo: Malek-Hosseini M. J., 2018). Tuveh spring, is another locality was discovered as habitat for Eidinemacheilus smithi (Figure 6). Tuveh spring (Figure 7) that was reported in 2019 as a new locality for the Iranian cave barbs Garra lorestanensis and G. typhlops, harbors the cave loach fish as well. Although Vatandoust et al., 2019 mentioned that E. smithi potentially could exist in this habitat, they did not collect this species there. These three species were reported previously from Loven cave, about 30 km to the north of Tuveh Spring. Figure 6: Eidinemacheilus cf. smithi fromTuveh spring (Photo: Fatemi Y., 2018). 134 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 7: Tuveh Spring, habitat locality of Iranian cave fish (Photos: Malek-Hosseini M. J., and Fatemi Y., 2018). 135 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 8: Some of Iranian troglobiont Crustacea a) Asellus ismailsezari. b) Niphargus keeleri c) Protrachaeoniscus gakalicus; d) Caridina sp.; e) Tethysbaena sp.; f) Stenasellus tashanicus; g) Nitocrella sp.; h) Tropocyclops sp. (Photos: Malek-Hosseini M. J., and Fatemi Y., 2015-2019). 136 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Table 3: All Iranian troglobiont animals Group of animals Number Genus- Family Terrestrial Aquatic Described, Described Undescribed of Genera known and Species from published literature recently in this study Amphipoda 26 4 3 0 26 23 0 3 Copepoda 17 9 3 0 17 12 0 5 Decapoda 1 1 1 0 1 0 0 1 Isopoda 6 4 4 1 5 3 1 2 Thermosbaenacea 1 1 1 0 1 0 0 1 Fishes 4 2 2 0 4 4 0 0 Oligochaeta 5 5 3 0 5 1 0 4 Araneae 3 3 2 3 0 1 0 2 Diplopoda 2 2 2 2 0 1 0 1 Coleoptera 2 2 2 2 0 1 1 0 Gastropoda 2 2 2 0 2 0 1 1 Continuation 137 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 3 Zygentoma 1 1 1 1 0 0 0 1 Collembola 1 1 1 1 0 0 0 1 Acarina 1 1 1 0 1 0 0 1 Total 72 38 28 10 62 46 3 23 2.2.1.6 Biodiversity patterns Despite only modest research on subterranean fauna in Iran in comparison with some well-studied parts of the world such as Balkan Peninsula, (Sket, 2008), the number of species at some sites or areas are increasing with more studies. Thus, even if preliminary, preparing the overview of current knowledge is informative both from the taxonomic as well as the conservation point of view. Recent discoveries have enabled the updated distributions for all troglobiont species from Iran. 45 localities are shown harboring 72 obligate subterranean-dwelling species from Iran (caves, wells, and karstic springs). 36 localities are located in Zagros karstic areas. As discussed in this thesis, this is due to more substantial research in these areas. The localities of Iranian troglobiont animals are shown in Figure 9. Details are found in Table 4. 138 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 9: Map of Iran, showing the distribution of troglobiont animals. For the localities details see Table 2. Species names are found in the checklists. 2.2.1.7 Overview of species richness per cave and richness of taxa In the published sources, prior to this study, Loven cave with three species of cave fishes was the richest cave in number of troglobionts (Vatandoust et al., 2019). Some caves and springs with two troglobionts are listed as well (Malek-Hosseini et al., 2015a; Esmaeili-Rineh et al., 2017b). In our study, we identified two caves (Table 4) as having over five troglobionts. There is Tashan-Chah-Kabootari cave system with 21 troglobiont species (Figures 8, 9). Three species have been described, and more are under description. 18 species have been analyzed molecularly, which revealed they are new species for science. These two caves that are connected by their groundwater, have been visited several times. In any new visits, we 139 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 discovered new paths, pools, streams, and kinds of microhabitats. So, with more surveys, we collected more species. This means that more fieldwork is needed to understand this cave system. Some species are found in both caves. According to our DNA and morphological analyses, two species including a fish and a copepod are living in both caves. Crustacea with 11 representatives, is the richest group in this cave system. There are some springs and wells in Tashan area around Tashan and Chah-Kabootari caves just in four to six square kilometers that all have sulfur water as same as these two caves. Obviously, they have formed a big aquifer. These caves and aquifer potentially harbor more terrestrial and aquatic troglobiont species Tuveh spring with four species is the second richest habitat after Tashan-Chah-Kabootari caves system. Tuveh spring is part of a big aquifer including Loven cave as well. Loven-Tuveh aquifer harbors three species of Iranian cave fishes that have been found in both sites. A new species of troglobiotic gastropods have been collected from Tuveh spring. Also, we discovered the new species Asellus ismailsezarii from Ganow spring about 1 km to the east of Tuveh spring from the same aquifer. The most diverse group of troglobionts in subterranean environments of Iran is Crustacea with 51 species, of which 39 species are described and 12 have not been officially described yet (Table 3). Amphipoda with 26 species is the most diverse group and within Amphipoda, the genus Niphargus Schiødte, 1849 including 23 species from 28 localities shows the highest species richness of troglobionts in Iran. Niphargus is the largest freshwater amphipod genus in the world, with species living in subterranean waters of the Western Palearctic (Fišer et al. 2008). Copepoda with 17 species is the second most diverse group in Iranian groundwaters. The family Ameiridae with seven described species from three genera is species richest Copepoda in groundwaters of Iran, with one more species not described yet (Table 3). Cave fishes with four representatives from two families and two genera from five localities are the next diverse group of troglobionts in Iran after copepods. 140 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Distribution of richness of troglobiont animals is shown in Figures 10, 11, 12. As showed in figure 10, cells with highest species numbers are situated in western Iran and especially Southwestern in the border of Khuzestan and Kohgiluyeh and Boyerahmad Provinces. These two cells include 27 troglobiont species. Together with the eastern cell, they have 30 troglobiont species, which is considered as the richest part of the country in regard of troglobiotic fauna. Table 4: Locality details and species number of Iranian troglobiont animals (localities with asterisk symbol are not the exact points) No Locality name Number Terrestrial Aquatic Described Undescribed Locality . of number on species the map 1 Tashan Cave 17 4 13 3 14 13 2 Chah-Kabootari 1 6 1 6 14 Cave 7 3 Tuveh Spring 4 _ 4 3 1 19 4 Loven Cave 3 _ 3 3 _ 20 5 Gakal Cave 2 2 0 2 0 5 6 Torang Cave 2 _ 2 0 2 2 7 Ghoori-Ghaleh 2 _ 2 2 _ 30 Cave 8 Sahoolan Cave 2 _ 2 1 1 36 9 Alisadr Cave 1 _ 1 1 _ 31 system 10 Khan Cave 1 1 0 1 _ 1 Continuation 141 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 4 11 Tir-e-Bagh Spring 1 _ 1 1 _ 4 12 Nahr Spring 1 _ 1 1 _ 6 13 Dangezloo Cave 1 _ 1 0 _ 7 14 Biareh Spring 1 _ 1 1 _ 8 15 Nezel Cave 1 1 0 1 _ 9 16 Cheshmeh-Belqeis 0 1 1 _ 10 (Spring) 1 17 Keeler Cave 1 0 1 1 _ 11 18 Neyneh Cave 1 1 0 1 _ 12 19 Dimeh Spring 1 _ 0 1 _ 17 20 Ganow Spring 1 _ 1 0 _ 18 21 Simarreh River _ 1 1 _ 21 basin 1 22 Shol-Abad Spring 1 _ 1 1 _ 22 23 Sarab-e-Moord 1 _ 1 1 _ 23 24 Sarab-e-Kanipahn 1 _ 1 1 _ 24 25 Jo-Khanem Spring 1 _ 1 1 _ 25 26 Sarab-e-Robat 1 _ 1 1 _ 26 Continuation 142 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 4 27 Razbashi Spring 1 _ 1 1 _ 27 28 Sarab-e- Bisitun 1 _ 1 1 _ 28 29 Nojivaran Spring 1 _ 1 1 _ 29 30 Kangarshah Spring 1 _ 1 1 _ 30 31 Kheder- Goli 1 _ 1 1 _ 33 Spring 32 Darvish-Olya Cave 1 _ 1 1 _ 34 33 Shoei Cave 1 _ 1 1 _ 35 34 Badin Abad Spring 1 _ 1 1 _ 36 35 Randole Spring 1 _ 1 1 _ 38 36 Sohrevard Spring 1 _ 1 1 _ 39 37 Danial Cave 1 _ 1 1 _ 40 38 Cheshmeh Kahriz - 1 _ 1 1 _ 44 Qanat 39 Brolan Spring 1 _ 1 1 _ 45 40 ⁕5Qualeh Kirmiz, _ 3 3 _ 3 bord du lac Niriz 3 Continuation 5 ⁕ Symbol: These localities with this symbol are not the exact point 143 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Continuation of Table 4 41 ⁕Farokhshahr 2 _ 2 2 _ 15 42 ⁕Shahr-E-Kord _ 6 6 _ 16 City 6 43 ⁕Kelarabad City 2 _ 2 2 _ 41 44 ⁕Mahmoodabad 1 _ 1 1 _ 42 45 ⁕Ghaem Shahr 1 _ 1 1 _ 43 Figure 10: Distribution of troglobiont species number in Iran at grid cell size 100 × 100 km. 144 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 11: Map of Iran showing species richness per localities (caves, wells, springs). 145 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Figure 12: Map of Iran showing species richness per localities (caves, wells, springs). 2.2.1.8 Endemism The degree of endemism is very high in Iranian troglobionts. However, we should consider this fact that that not many caves, wells and karstic springs in Iran have been faunistically studied, and not many troglobiont species have been reported from the country. Most of the known species are restricted to a single cave, a spring or an aquifer. In general, subterranean communities are with high endemism but low local diversity relative to regional diversity (Gibert and Deharveng, 2002). Regarding grid cell map, 93% of Iranian troglobiont animals are restricted to a single cell and only 7% including five species have been reported for more than an area as a single grid cell. So, this is congruent with the worldwide characteristics (from Gibert and Deharveng, 146 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2002) of subterranean faunas. We have to consider that low sampling effirts could artifactually concluded in short-range endemism. Garra lorestanensis has been reported from three localities: 19, 20 and 21. Niphargus sohrevardensis has been reported from two far localities: 27 and 39. Niphargus valachicus Dobreanu & Manolache, 1933 was reported by Karaman (1998) from undetermined site from northern Iran. It has wide distribution from Europe to Iran. This species was reported first time from Romania (Dobreanu and Manolache, 1933). However, the molecular data is not available for this species from Iran and potentially, it could be a separate species. Iran represents the eastern boundary of Niphargus range. Two species of copepods, Diacyclops zschokkei and D. iranicus have been reported from two far localities 16 and 41. This degree of endemism implies the importance of priorities for the protection of the habitats of these troglobiont species in Iran. 2.2.1.9 Conclusion In this chapter, we compiled faunal lists of troglobiont animals of Iran based on scattered published literature together with published and unpublished data from our own collections. As showed in the maps (Figures 9, 10, 11, 12), most of Iranian troglobionts have been discovered in west and northern parts of the country from Alborz and Zagros. These parts of the country are mostly mountainous with limestone karsts that harbor many caves and karstic springs. The other reason for being more studied is that some research groups in the universities in these providences are studying troglofauna of these areas. Our original field works focused on Zagros. From all 72 species (both described and undescribed), more than 60 have been collected from Zagros karstic areas. Zagros, as host many karstic areas and thus many caves and springs, as well variety of vegetations, in some parts with 800 mm rainfall (Raziei et al., 2005; Soltani et al., 2012), many rivers, also rich biodiversity, it harbors most of Iranian troglobiont animals. However, this region has been better studied. The number of troglobionts is rising in Iran due to ongoing researches. However, the relatively low species richness of Iranian caves is not realistic, and it is due to a lack of 147 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 comprehensive studies of all taxa. Eastern parts of the country have been neglected in the aspect of subterranean biology. Despite this fact that most part of Eastern-Iran is located in very arid zones, there are some caves and karstic spring that would be very interesting. Northeast of the country is also mountainous with many caves that potentially could harbor troglobiont animals. As for many other taxa for Zagros, the putative presence of troglobiont representatives of many taxa is predicted Central Zagros and especially west of Kohgiluyeh and Boyer-Ahmad and East of Khuzestan Provinces harbor about 30 troglobiont species and many more to be discovered. Speleobiological studies are needed in Iran. Compared to some karstic areas such as Dinaric or Balkan Peninsula, the number of studies and discovered troglobionts are low. Most diverse groups are aquatic crustaceans and fish (Mousavi‐Sabet et al., 2016; Bargrizaneh et al., 2021). Considering the vastness of Iran and big karstic areas harboring more than 2000 caves and thousands of karstic springs, more field work studies are needed to understand the subterranean environments of the country. For the above reasons, it is too preliminary to discuss the troglobiotic fauna of Iran within a global context. With over 2000 records of specialized subterranean species from both Europe and the United States, we could say that these parts of the world have been the most studied parts in regards of subterranean fauna. Several datasets on the georeferenced occurrence of these obligate subterranean species have been presented till now (Deharveng et al., 2009; Zagmajster et al., 2014; Bregović and Zagmajster, 2016; Christman, et al., 2016; Pipan et al., 2020; Culver et al., 2021). Until 1999, more than 45000 caves were known from the USA and many more have been added up to date (Culver et al., 2009). More than 2000 caves and approximately 10000 karstic springs and wells are known for Iran. Of course, not all caves and also karstic springs and other groundwaters need to be sampled to obtain a good estimate of species richness for a region, but a rather large number do (Pipan et al., 2020). Since 2010 and including this study, about 56 troglobiont species have been discovered (including non-described ones) from Iran that is 77 % of discovered troglobiont species from Iran. 33 species have been described since 2010. Nonetheless, this dissertation is a start of a more methodical study of the cave biotas in Iran and wider. We developed a georeferenced database of troglobionts (cave-obligate species) in Iran to examine spatial patterns of species richness and endemism, including >88 records 148 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 for 48 described and 24 under-description or undescribed species. Although, we have discovered more troglobiont taxa from Iran, due to obvious time constraints of a single work, all initial results could not be included in this thesis. 149 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3 DISCUSSION AND CONCLUSIONS 3.1 DISCUSSION 3.1.1 Goal 1: Discovery of troglobiotic fauna in Iran (Zagros karsts) Several taxa that prior to this study had no troglobiont representatives in Iran, are now included in the troglobiotic list of fauna of the country. Although we discovered several new troglobiont species from subterranean environments of Iran that represent a significant number for the troglobiotic fauna of the country, this still is a relatively small amount of data in comparison with the epigean fauna of the country or the subterranean fauna of some well-studied parts of the world such as Balkan Peninsula. This seemingly low species richness of obligate subterranean animals in Iran is likely an artefact stemming from lack of focused research on this fauna. Most caves, wells and karstic springs of the country that might potentially harbor troglobiont species are not easy to reach and there are not many particular surveys on Iranian troglobiont organisms. Moreover, given the vastness of the country and extension of the karstic areas from north to south, an extensive study would require tremendous effort. Some cave-adapted groups of animals such as fishes (4 species), Crustacea (amphipods and isopods, copepods, more than 30 species), diplopods, and insects have been reported from Iran (Malek-Hosseini and Zamani, 2017; Fatemi et al., 2019; Bargrizaneh et al., 2021). We suggest the putative presence of several groups of animals in hypogean environments of Iran, but to test this presumption, much more intensive field work is needed. Although Coleoptera and especially carabids have many representatives in subterranean environments, no troglobiont carabid beetle has been discovered from Iran prior to this study and the only known troglobiont coleopteran from Iran was the tenebrionid Foranotum perforatum (Nabozhenko and Sadeghi, 2017). In this study, we investigated the subterranean beetle fauna of Iranian caves of the Zagros Mts. We diagnosed and described two newly discovered species of Duvalius Delarouzée, 1859 from two caves in Zagros. The troglobiont Duvalius nezelensis Muilwijk and Malek-Hosseini, 2021 that occurs in the Nezel cave, while the troglophile Duvalius achaemenius Muilwijk and Malek-Hosseini, 2021 occurs in the small Suq cave (Malek-Hosseini et al., 2021). Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner & Grego, 2019 was the first obligate cave-dwelling snail from Iran. We reported the discovery of the 150 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 sixth sulfidic stygobiont gastropod species of the world in the Tashan Cave of Iran with the general ecological importance of the habitat. Asellus ismaeilsezarii Malek-Hosseini, Jugovic, Fatemi & Douady, 2022, is a new obligate groundwater asellid isopod from Ganow spring close to Tuveh spring and 30 km to the Loven Cave, both localities harboring Iranian cave fishes. Ganow spring is a part of big aquifer that harbors three species of cave fishes and a new troglobiont species of gastropods. We described this Asellus species based on morphology and molecular data. Several troglobiont species that have been discovered in this study are under-description. A troglobiont species of atyid shrimps: Caridina sp. from the sulfidic water of Tashan cave as the first obligate cave-dwelling species of shrimps from Iran that is also first troglobiont one from Atyidae from sulfidic hypogean waters in the world, has been described and awaiting publication. This family has several troglobiont represantatives worldwide. Another troglobiont isopod is under description. Stenasellus sp. From Chah-Kabootari that genetically is sister to Stenasellus tashanicus from Tashan cave. Molecular analyses of copepods from Tashan-Chah-Kabootari cave system showed that we have five different species from three genera of two families. Two new species of Amphipoda have been collected in Tashan and Chah-Kabootari caves. They are waiting to be described. A new species from Thermosbaenacea that is the first representative of this order from Iran was collected in Tashan cave. Molecular and morphological analyses both showed it belongs to the genus Thetysbaena Wagner, 1994. We discovered four new species of Oligochaeta. A new troglobiont species of the genus Helodrilus (family Lumbricidae) from water of Dangezloo cave, and two interesting species from sulfidic waters of Tashan and Chah-Kabootari caves. These oligochaetes should be sampled again for more appropriate specimens for morphology. DNA analyses have been done and very interesting results have been obtained. 3.1.2 Goal 2: Iranian troglobiotic fauna species richness and hotspot detection Iran covers 164.8 million km2 of area and about 11% of the country’s land are karstic carbonate formations. The country located in western Asia at the interface between three zoogeographic realms, Palaearctic, Oriental (Indo-Malayan) and Afrotropical (Ethiopian). 151 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Iran is situated at the conjunction of three climatic zones: the Mediterranean, the arid West Asian, and the temperate humid/semi-humid Caspian zone (Raeisi et al., 2012; Amiri and Eslamian, 2010). Variety of climate, vegetation, rainfall, temperature and then very different productivity exist in Iran from south to the north and from east to west. Although many parts of the country, especially central parts, are desert, a significant difference could be seen in western and northern parts of the country. The Zagros region exemplifies the continental variant of the Mediterranean climate pattern, while the northern slopes of Alborz are distinctly humid. So, these areas have a high level of productivity. Determining the global patterns of subterranean species richness has remained elusive (Pipan et al., 2020). There are several poorly studied subterranean faunas in the world especially in Central Asia and Middle Eastern countries such as Iran. To understand the subterranean fauna of Iran, considering the karstic areas of the country, at least 600 sites from all around the country that potentially could harbor troglobiont animals need to be studied. As up to date, more than 100 caves, wells and springs have been surveyed for subterranean animals (arthropods, mollusks, oligochaetes and fishes) and only 49 troglobiont species have been reported from them, and considering this fact that the subterranean environments of huge areas of the country have not been studied yet, we suggest 600 sites to obtain a realistic estimation of the troglofaunal of the country. For Iran, to determine spots of high subterranean biodiversity, we should consider some criteria that used by Trajano et al. (2016) for Brazil with consideration to the difference of climate in Iran with Brazil. First, we should consider the number of troglobionts, when compared to other studied cave areas in the region. Presence of higher taxa (families, order) so far not recorded in subterranean habitats elsewhere in Iran even in the region must be considered. Regarding the country and the region, with a comparative point of view, any site with more than 15 troglobiont species could be considered as hotspot of troglobiont diversity for this region. Undescribed species are problematic in the analysis of species numbers in subterranean sites because sometimes some of them are not new species. According to our analyses (morphological and molecular), this has not been true regarding Iranian troglobiont diversity as all discovered species are new for science. There is a remarkable concentration of subterranean sites in the Dinaric karst of Slovenia, Croatia, and Bosnia-Herzegovina, which is also the richest known region of subterranean biodiversity in the world (Sket, 1999b). This concentration and connection of subterranean sites in Iran may be just referred for Loven-Tuveh aquifer and for the sulfidic water system that we detected in Tashan and Chah-Kabootari caves. This system harbors a unique 152 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ecosystem and represents a specific hypogean habitat with several cave ponds containing groundwater rich in hydrogen sulfide. The first studied cave containing a sulfide ecosystem was Movile Cave in Romania (Sarbu and Popa, 1992). According to the local people and our observations, there are more wells and springs with sulfur water in that area close to Tashan. With more field works, we could understand the fauna of this big unique system. As we are preparing the species level testing to corroborate the species status of several taxa, we saw some different species from the same genera in Tashan and Chah-Kabootari caves that are sisters genetically, and some groups of animals that only exist in one of these caves and even in a single isolated pool, we could say that with detection of more places in these two caves, new taxa would be discovered that are restricted to a microhabitat. Despite numerous samplings, covering the different microhabitats (chambers, passages, guano, stream, ponds, lakes), large sections of Tashan cave remain unexplored, from both a topological (e.g., permanently flooded sections, dangerous streams down to the pools with loose, unstable, and falling walls) and biological perspectives. The inability to sample the entirety of the karst system implies an underestimation of its species richness. During several visits and samplings from Tashan-Chah-Kabootari system, we collected new taxa that we were not able to collect them in previous field works. So, here, we considered Tashan-Chah-Kabootari cave system with 21 obligate cave-dwelling species as a hotspot for troglobiont diversity in Iran. Regarding the topography of this cave system with sulfidic water, new paths in these caves, and unused techniques of sampling in both aquatic and terrestrial parts of these caves, undiscovered species are expected. The number of Iranian troglobiont species is likely to increase in the future since new records are occurring regularly. The most diverse group of troglobionts in Iran was Crustacea. This is congruent with global pattern of subterranean diversity. Crustaceans constitute a major animal group in groundwater (Culver and Pipan, 2013) with around 3400 species known worldwide, 1200 of which are known to occur in Europe (Camacho and Puch, 2021; Stoch and Galassi, 2010). This is more than 65% of the total groundwater species currently known from Europe (Iannella et al. 2020; Iepure et al., 2021). The new, developing Iranian database on subterranean biodiversity can be build on a clearer premises than other databases in the world. All other databases needed to clean masses of data and find the coordinates for localities, here the database can be constructed in a modern way from the scratch.. This enables not only more exact mapping, but also spatial modeling, including spatial autocorrelation (Zagmajster et al., 2008), conditional autoregression (Christman and Culver, 2001), and kriging (Christman and Zagmajster, 2012). 153 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3.1.3 Goal 3: Phylogeny and evolutionary history of Iranian troglobionts With this study, the discovery of several new taxa adds to phylogenetic resolution of several groups such as Trechinae beetles, Isopods, and Gastropoda family Moitessieriidae. Also, some time frames for their origins and colonization of subterranean environments were estimated. The phylogenetic relationships were reconstructed in big frames of taxa from Palearctic and Africa. For some groups, even species from Oriental were included to see a better results. Here we discuss about the abovementioned groups separately and then compare the estimated time of colonization of Iranian subterranean environments by different terrestrial and aquatic taxa. Due to incomplete sampling of some taxa and as there is no close relatives in surface (epigean relatives), for some Iranian troglobiont taxa, our analyses cannot unequivocally pinpoint the immediate phylogenetic relative these troglobiont taxa. So due to that, providing a chronogram (datation) was totally irrelevant at the moment. The problem is not to make a chronogram but first to get enough sequences of different genes from more taxa within the ‘’Iranian pattern” clades and second to have a reliable calibration point for time estimation (datation) within those clades. For some taxa, at the moment, we have neither the first nor the second. The monophyly of some taxa of Iranian troglobionts have been shown in our analysis. Duvalius species group of beetles from the Zagros Mts. made the “Zagros clade”. The split of the Zagros clade to have occurred between 7.3 and 12.9 Mya (mean 9.7 Mya) in Late Miocene. Two diversifications of the Zagros clade that estimated to have occurred between 5.4 and 10.6 Mya (mean 7.8 Mya) in Late Miocene for the split of D. nezelensis from its sister group, and more recently in Middle Pleistocene between 0.4 and 1.3 Ma (mean 0.78 Mya) for the split of D. achaemenius and D. kileri. The colonisation of subterranean environments by D. nezelensis could have occurred in any time from 10.6 Mya. Molecular analysis of newly discovered gastropod from Tashan cave confirmed that it belonged to the family Moitessieriidae. This new genus and species, Trogloiranica tashanica may represent an evolutionary relict from the post Miocene before the split of the families Moitessieriidae and Cochliopidae. The separation of this species from its surface relatives was estimated on 3 Mya. The molecular analyses we did on the first troglobiont Asellus from Iran that was discovered and described in this study, showed that this species did not belong to the A. aquaticus complex. Reconstructed trees showed that A. ismailsezarii is sister to a larger clade that also contains the European A. aquaticus species complex. In this case, lack of data from Iran and 154 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 neighboring countries did not allow us to understand the phylogenetic relationship of this Iranian species and also estimate a timeframe for its divergence from its congeners. A. monticola have been reported from the Caspian Sea region. This region could be considered as a contact zone between species of the European A. aquaticus complex and Asian species, including A. ismailsezarii and A. monticola. Regarding the similarities in morphological characters of A. ismailsezarii and A. monticola, we expect that these two species are phylogenetically close as well. 3.1.4 Conservation Iran harbors many caves with historical values. Several caves with troglobiont species are located in historical sites. Unfortunately, there is no protection of valuable and unique subterranean habitats of the country. Some caves such as Tashan and Chah-Kabootari should be in priority for special protection. Up to date, much of destruction has been caused by cavers, treasure hunters and local people in these caves. Scientific depredation is one of the main reasons for Iranian cave fishes to be in danger. Considering available literature and our studies, most of Iranian troglobionts are restricted to a single cave and any changes in the environment, could end up in extinction. Although subterranean fishes of Iran have been reported from more than one locality, all threats and risk of extinction still exist. The importance of preparation of the databases on subterranean biodiversity of Iran is obvious. So, the management and strategies for protection of Iranian subterranean environments could be organized better. Identification of troglobiont diversity hotspots should results in protection of these unique habitats. Tashan-Chah-Kabootari system, Loven-Tuveh aquifer and Gakal cave are the most important habitats in terms of subterranean fauna in Iran. Unfortunately, there is no protection of these habitats. Tashan-Chah-Kabootari system likely represents another chemoautotrophically based cave ecosystem harboring 21 obligate cave dwelling species that some of them in order level, are recorded from the country for the first time. Although, most of species are waiting to be described morphologically, molecular studies on these animals from this system showed that they are new species and also some of them as distinct clades sister to the rest of congeners. From evolutionary view, the estimated time for this undescribed species showed that study of this cave system is very important and will reveal worthy stories about the origins of Iranian troglobiotic faunas. This cave system has not been completely studied yet and much evidence may be lost and destroyed as there is no protection at all. Regarding the threats for this cave system, especially scientific depredation and uncontrolled visits, the priority should be in direction of protection of this valuable and unique hotspot of troglobiont biodiversity. Regarding our visits and also some geologist and archaeologist visits of Tashan Cave, lot of valuable historical clues and objects from ancient times have been completely manipulated and 155 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 plundered by treasure hunters during last 7 years. Their destructive activities affect cave life of animals as well. Four fish species are known as the only true subterranean vertebrates from Iran: Garra typhlops (VU on IUCN), G. lorestanensis, G. tashanensis and Eidinemacheilus smithi (VU) (Cypriniformes). Three species have been described from Loven cave and Tuveh spring and G. tashanensis is known only from Tashan cave and Chah-Kabootari cave, Khuzestan Province. First threat for Iranian cave fish is scientific depredation. Fish are sampled by several research teams. Just in a study by well-known professors in 2008, 44 specimens were killed. Although Loven cave fish are as national natural heritage of Lorestan province, they are considered as ornamental fish and are easily caught. Loven cave is close to road and railroad. Dam and road construction activities are underway. Soil texture at the cave opening is sandy and alluvial. Wall rocks are sloping and loose. Flooding has been problematic and could clogged the entrance with soil. Extreme earthquakes are potentially risky and fallen rocks could completely fill the opening pool of Loven Cave. In addition to scientific depredation and treasure hunters, water extraction is a big threat for Tashan Cave. Water well is very close to this cave and directly extract water from the cave. The rainfall in this area has decreased significantly. This cave is in middle of three villages. Pesticides and human wastes directly flow into the system. So, without exaggeration and overstatement, these Iranian cave fishes are headed towards extinction. All effort should be made to avoid this. Considering all habitats and species as national heritages, education to research groups and local communities, installing appropriate doors, impose heavy fines and control all visits must be applied. Also fencing loose walls at the opening of Loven Cave is necessary. 3.2 CONCLUSIONS In this thesis, we followed three main goals related to the Iranian troglobiotic fauna. We studied several caves and karstic springs in Zagros Mountains. We tried to discover more localities that harbor troglobiotic animals. Considering the promises in the dissertation proposal, we tried to describe some new species and new habitats for Iranian troglobiotic 156 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 animals. However, there remain several new species to be described. Regarding our original data and available data on GenBank, we tried to reconstruct phylogeny of Iranian troglobionts from different groups of animals. We focused on a dated phylogeny to estimate a timeframe for their colonization to the subterranean environment. Then, considering the available faunistic data and our discoveries, we did a species richness analysis and looked for any hotspot for troglobiotic diversity in Iran. We can draw the following conclusions, coming from our work: 1. The presence of several taxa in obligate cave-dwelling forms is confirmed by this study. For the discovery and description chapter, we described three new troglobiont species including: Coleoptera: Carabidae: Duvalius nezelensis Muilwijk and Malek-Hosseini, 2021 (Malek-Hosseini et al., 2021). Gastropoda: Hydrobiidae: Trogloiranica tashanica Fatemi, Malek-Hosseini, Falniowski, Hofman, Kuntner and Grego, 2019 (Fatemi et al., 2019). Isopoda: Asellid isopods Asellus ismailsezarii Malek-Hosseini, Jugovic, Fatemi and Douady, 2022 (Malek-Hosseini et al., 2022) Several new species are under-description such as a new troglobiont atyid shrimp from the genus Caridina Milne-Edwards, 1837. Although diverse, Iranian's subterranean fauna remains poorly studied and many additional species await discovery and description. We identified several under-sampled regions and outlined conservation and management priorities to improve our knowledge and aid in protection of the subterranean biodiversity in Iran and specially Zagros karstic areas. We envisage that the exploration of Iranian troglobiont animals has just begun and that many more species still await formal description. Although the available data clearly demonstrate that the subterranean biodiversity in the Zagros karstic areas of Iran is relatively high (at least in some parts), especially compared with that of other caves in the world and when you 157 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 consider the climate, amount of rainfall and productivity (Deharveng and Bedos, 2000; Pipan et al., 2020). 2. 72 obligate cave-dwelling species (49 described and 23 undescribed and under-description) have been discovered from Iran. Out of this, more than 60 species have been collected from Zagros karstic areas. Zagros, as hosts many karstic areas and thus many caves, wells and springs and the karstic parts of the Zagros have been more understudy. 3. Clearly, the currently reported diversity of Iranian troglofauna is a vast underestimation and it remains poorly known. The low species richness of Iranian subterranean environments is not realistic, and it is due to a lack of comprehensive studies of all taxa. 4. Central Zagros and especially west of Kohgiluyeh and Boyer-Ahmad and East of Khuzestan Provinces harbor about 30 troglobiont species and many more to be discovered. Tashan-Chah-Kabootari cave system harbors 21 troglobiont species (five terrestrial and 16 aquatic). Considering the discoveries of new paths in this cave system, more troglobiont species are expected with more field work and sampling. 5. Phylogenetic relationships of Iranian troglobiont animals with their epigean congeners were studied and in some cases monophyly was renonstructed. Lack of complete samplings from many taxa made the understanding of these relationships difficult. This problem is global for some taxa. 6. Different timeframes were estimated for the colonisations of the subterranean environments of Iran by different taxa. In addition to finding the closely relatives of Iranian troglobiotic animals, phylogenetic analyses were done to estimate a timeframe for their colonisation to the subterranean environment. For some taxa, it was not, and it is not easy to find their close relatives. In some cases, the order is first record from the country. Also, no surface-dwelling species have been collected from the same genera. Three million-year was estimated for the separation of the obligate cave-dwelling gastropod: Trogloiranica tashanica from its surface relatives. 158 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Time frames estimated for the colonisation of subterranean environments by animals are affected by the rates of evolution used in analyses. The colonisations of subterranean environments of Iran by different groups of animals could have occurred in different times and as independent populations colonising. Our study adds to phylogenetic resolution of Trechinae by adding three Duvalius species from Iran. The split of the troglobiont carabid beetle D. nezelensis from its sister group occurred between 5.4 and 10.6 Ma (mean 7.8 Mya) in Late Miocene. So, the colonisation of the subterranean environment by D. nezelensis must have occurred at any recent time since the Late Miocene (from 10.6 Mya). This implies that D. nezelensis developed its troglomorphic features between the Late Miocene and today. estimated split overlaps with the potential time frame of the Nezel cave formation. 7. Most of the habitats for Iranian troglobiont animals, especially Tashan-Chah-kabootari cave system and Loven-Tuveh aquifer are not protected. The evidence suggests protection of both is the main conservation priority. 159 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4 SUMMARY 4.1 SUMMARY One of the interesting environments for biological studies such as taxonomy, evolution, ethology, etc., are subterranean habitats, both aquatic and terrestrial. The main feature of such environments is continuous darkness. Some abiotic conditions such as moisture, temperature, water chemistry and oxygen are almost constant during the year. Additionally, hypogean systems are relatively energy-limited compared to photosynthetically based epigean systems. These habitats harbor three main categories of animals based on degree of their dependence on subterranean environments. The focus of this study was on troglobionts, a term that refers to organisms found only in caves and that display cave-specific phenotypes (morphological, physiological, and behavioral) such as loss of eyes and pigmentation, and elongation of appendages. Troglobionts are obligatory cave-dwelling species. Although relatively poorly studied in comparison with the epigean ecosystems, subterranean ecosystems show a rather high biodiversity at the global scale. However, in comparison with epigean environments, this subterranean biodiversity is low, but not as low as it seems to be. Over 7000 aquatic subterranean dwellers have been globally catalogued but such data are not available for the terrestrial faunas. About 50,000 to 100,000 obligate terrestrial subterranean species are estimated to exist worldwide. The syntagm “biodiversity hotspot” is one of the most important global tools for nature conservation. Several hotspots of subterranean biodiversity have been detected mostly in Europe and United States of America, and an additional few are known from Australia and Asia. This is according to the literature that used an arbitrary cutoff of 20-25 obligate subterranean species for a site to be considered as a hotspot for troglobiotic biodiversity. This cutoff depends on comparative value to others. Prior to this study, the cave with the highest diversity in Iran was Loven cave in Zagros that harbors three species of Iranian cave fishes. There has been no hotspot of troglobiont biodiversity or spots of high diversity of troglobionts detected in Iran. Iran with 1,648,195 km2 of land area, is the second-largest country in the Middle East and the 17th-largest in the world. This country is geologically a part of the Alpine-Himalayan orogenic belt. 160 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 The total area of the karstified carbonate rocks in Iran is about 185.000 km2, with 55,2 % in the Zagros, 24,3 % in Central Iran, 15,2 % in Alborz, 4,7 % in East and South Iran. More than 2000 caves, 10000 springs and wells have been reported from Iran and mostly from Zagros and Alborz. The subterranean fauna of Iran remains inadequately known not only faunistically, but also from the evolutionary view, especially when considering species origins. In this study, we followed three aims. The first aim was discovery of troglobiont organisms in Zagros Mountains of Iran, and taxonomic descriptions of new taxa. Three new troglobiont species were described: a carabid beetle, an asellid isopod and a truncatelloid gastropod. Six species (Oligochaeta, Decapoda, Isopoda) are under description and more than 10 species are awaiting description. We also recorded new habitats for previously described Iranian cave fishes. The second aim was to prepare a checklist of all Iranian troglobionts to examine patterns of troglobiont biodiversity and endemism in the Zagros Mountains of Iran. In this part of the thesis, we detected and discussed the first hotspot for troglobiont biodiversity in Iran. The third aim was to study the phylogenetic relationships among selected troglobiont groups, and investigate the origins of this troglofauna. DNA extraction and PCR for several markers was performed for numerous new troglobiont species we discovered as well as for their relevant surface relatives, and we have published a subset of these original data in scientific publications. DNA isolations and PCRs were done at Evolutionary Zoology Laboratory, Biological Institute Jovan Hadzi, ZRC SAZU, Slovenia. Related sequences were mined from GenBank for meaningful reconstructions of phylogenetic relationships of studied taxa, for tests of their species status, and for estimations of the timeframe of their colonization of subterranean environments. In a scientific paper within this dissertation, we described two new species of cave beetles of the genus Duvalius from caves of Zagros Mts. in Iran. Duvalius nezelensis was the first troglobiont carabid beetle from the country. The main troglomorphic features of D. nezelensis were absence of eyes and long appendages. We also described D. achaemenius as a troglophile species. Although Coleoptera and especially carabids have many representatives in subterranean environments, no troglobiont carabid beetle had been discovered from Iran prior to this study and the only known troglobiont coleopteran from Iran was Foranotum perforatum from Tenebrionidae. Now, a total of six Duvalius species are known from Iran. 161 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 For phylogenetic analyses, we obtained original sequences of two mitochondrial (COI, 16S) and two nuclear (18S, 28S) genes for three Duvalius species from the Zagros Mts. and combined them with published molecular datasets using other Duvalius species. We applied Bayesian inference and maximum likelihood for reconstruction of a species level phylogeny of Duvalius and closely related genera. To explore divergence times of the major lineages of Duvalius, we used BEAST. Our phylogenies recovered a well-supported “Zagros clade” with the three examined Iranian species. About 9.7 Mya (Late Miocene) was estimated for the isolation time of this clade from other congeners. Within the Zagros clade, the split of D. nezelensis and its sister group was estimated at 7.8 Mya (Late Miocene). We concluded that the colonization of subterranean habitats by D. nezelensis could have occurred at any time since 7.8 Mya. In the second publication, we described a new gastropod species from the sulfidic ponds of Tashan Cave in the Zagros Mts. of Southwest Iran. Trogloiranica tashanica represented the first troglobiont/stygobiont gastropods found in Iran and sixth stygobiont gastropod species of the world from sulfide rich water. We used two markers (COI and H3) to infer the phylogeny of this species and relatives, and studied their anatomical evidence for the placement of T. tashanica in the family Moitessieriidae. The phylogeny inferred with COI suggested a close relationship with the Moitessieriidae but did not confirm the monophyly of the family. The phylogeny inferred with nuclear H3 clearly demonstrated that T. tashanica belongs to the Moitessieriidae. Likewise, the concatenated analysis of COI and H3 supported this relationship. We commented on the unique hydrogen sulfide habitat of the newly discovered gastropod. Tashan Cave harbors a unique ecosystem. The recent discovery of a blind fish, Garra tashanensis, and the isopod Stenasellus tashanicus, have been followed by other discoveries including the new gastropod genus and species reported in this study. Hydrogen sulfide is toxic for most organisms and only five of the around twenty so far known sulfide cave ecosystem sites worldwide, host stygobiont gastropod species. Another troglobiont species we described in this study and published the finding within a paper was Asellus ismailsezarii from Asellidae family, order Isopoda, from Ganow spring close to Tuveh Village, Andimeshk City, Khuzestan Province, Zagros Mts, Western Iran. Asellus ismailsezarii was the first obligate groundwater Asellus reported from the country. This species showed troglomorphies such as depigmentation and loss of eyes. We provided morphological and molecular evidence that supported the species status of A. ismailsezarii. COI and 28S genes of A. ismailsezarii were analyzed with available sequences of the genus 162 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Asellus on GenBank. 11 MOTUs within the Asellus pattern were found, including one MOTU corresponding to A. ismailsezarii. The resulting tree of the nuclear 28S rDNA sequences was congruent with the COI phylogeny. These analyses showed that this species was not nested within the European A. aquaticus clade. This new species of Asellus from Ganow spring showed that this aquifer, which includes important cave of Loven, Tuveh and Ganow springs, harbors more troglobiont animals. Previously reported Iranian cave fishes Garra lorestanensis, G. typhlops and Eidinemacheilus smithi from this aquifer from three sites, as well as some other unpublished gastropods confirm this hypothesis. With the new discovery, up to date, two Asellus species have been reported from Iran. Asellus monticola was reported from a spring in the extreme Northwest of Iran. With the new discovery, the number of obligate subterranean-dwelling isopod species from Iran raised up to four (three aquatics and one terrestrial). We suggested the putative presence of many more obligate groundwater Isopoda and especially Asellus taxa in Iran. The main problem with the study of phylogenies and finding the origin of Iranian troglobionts was the lack of data for close relatives of studied taxa in this dissertation. There was not enough data available on GenBank. Also, as some genera were new for the country and science, there were no epigean/hypogean close relatives for those taxa, neither genetically nor geographically. Regarding our results and the available literature, most of colonization of Iranian subterranean habitats by animals, have occurred during Middle Miocene to Early Pleistocene (15 Mya – 2 Mya). However, few data were available in this regard and most of Iranian troglobionts had not been tested yet for understanding their origins. Several groups of troglobionts in different karstic areas must be examined. Also, the estimated times could be compared in both aquatic and terrestrial troglobionts. To examine the species distribution patterns of troglobiont diversity in Iran, we used available literature and our own data. Complementing the checklist of subterranean arthropods of Iran which we published in 2017, we represented all Iranian troglobiont animals (previously published and our new discoveries, including yet undescribed species) in this dissertation to update the checklist. One of the main problems for diversity analyses is whether or not to include yet undescribed species that had not been taxonomically evaluated. In our case, we believe this not to be the problem, because we examined most of the sampled taxa morphologically and molecularly, and, considering the geographical distribution of the taxa in question, we eliminated reasonable doubt as to their taxonomic status. 163 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 From all 49 identified and described troglobiont species from Iran (six terrestrial, 43 aquatic), 42 species were arthropods, and out of these, 39 species were crustaceans, and 23 species of them belonged to the genus Niphargus (Niphargidae). Terrestrial troglobionts included two species of beetles, a spider, a diplopod, and an isopod. In this study, we discovered 26 new species of troglobiont animals. To date, we described three of them. During field works, we discovered some new localities for previously described Iranian cave fishes. Chah-Kabootari cave, a limestone cave with sulfur water, a kilometer to west of the Tashan cave harbors Garra tashanensis. Tuveh spring is another locality, which was discovered to harbor Eidinemacheilus smithi. In total 45 localities we listed 72 obligate subterranean-dwelling species from Iran (caves, wells, and karstic springs). Most (36) of localities were in Zagros Mts, potentially indivating samping bias. The most diverse group of troglobionts in subterranean environments of Iran was Crustacea with 51 species, of which 12 await taxonomic description. With 26 species Amphipoda was the most diverse group. Within Amphipoda, the genus Niphargus counted 23 species (28 localities), making it the genus with the highest number of troglobionts in Iran. The second most-diverse group in Iranian groundwaters was Copepoda with 17 species. Cavefishes with four representatives from two families and two genera from five localities were the next most diverse groups of Iranian troglobionts. We prepared maps to show the distribution and richness of troglobiont species per site. We showed the number of species present in each 100 × 100-km UTM grid cell prepared using QGIS. Cells with the highest species numbers were situated in western Iran and especially Southwest. There were two cells, which included 27 troglobiont species. Together with the eastern cell, they showed to have 30 troglobiont species, which was considered as the richest part of the country regarding troglobiont diversity. The richest cave system that we detected consisted of Tashan and Chah-Kabootari caves that harbor 21 troglobiont species, of which five species were terrestrial, and 16 species were aquatic. In addition to these cave systems, other springs and wells in that area within six square kilometers all have sulfidic water. Obviously, they have formed a big aquifer. These caves and aquifer could harbor more terrestrial and aquatic troglobiont species. To understand this, more fieldwork, and sampling are needed. More springs with sulfidic water in that area have been observed by locals, which mostly are seasonal, flowing during winter and spring, and they harbor cave-fish as reported by local people as “red-pink fish”. Potentially, Tashan district is an important part for Iranian subterranean biodiversity as its 164 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 sulfidic aquifer makes it unique. This cave system was the first hotspot for troglobiotic biodiversity in Iran that needs high priority for protection. Considering the checklist we prepared, Tashan-Chah Kabootari cave system, Loven-Tuveh aquifer and Gakal cave, as important habitats for troglobionts, must be protected. Subterranean habitats are neglected parts of Iranian environments in protection point of view. The degree of endemism in Iranian troglobiont animals was high and most of species were restricted to one cave, spring, or aquifer. This implies the importance of priorities for the protection of the habitats of these troglobiont species in Iran. Caves and springs have been to a large extent neglected from the conservation point of view. According to our observations and some reports from locals, cavers and scientific publication, lots of destruction has occurred in these habitats. On the top of it, scientific depredation endangeres Iranian cave-fishes. Excessive samplings using rather invasive sampling as electroshock can be reconstructed from the publications. Although, most of the Iran land lies in an arid environmental zone, and considering the low rainfall in many parts of the country, there are many karstic areas in western and northern Iran. So, the currently reported Iranian subterranean biodiversity, even after this thesis, is still an underestimation. Speleobiology is in its first stage in Iran and regarding the discoveries of the recent decade, forthcoming decades will reveal more facts about this biodiversity and its evolutionary history. Given the poor knowledge of Iranian subterranean fauna and its evolutionary history, this biospeleological dissertation enriched our knowledge by reporting new findings, describing new species, reporting on troglobiont biodiversity patterns, hotspot recognition, performing phylogenetic analyses to test species validity, as well as to understand the relationships and the origin of examined taxa. Our discoveries will therefore facilitate conservation decisions. 165 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4.2 POVZETEK Eno najzanimivejših okolij za biološke študije, kot so taksonomija, evolucija, etologija itd., so podzemni habitati, tako vodni kot kopenski. Glavna značilnost takšnih okolij je stalna tema. Nekateri abiotski pogoji, kot so vlaga, temperatura, kemija vode in kisik, so skorajda konstantni vse leto. Poleg tega so hipogeni sistemi v primerjavi s fotosintetičnimi epigeni relativno energetsko omejeni. V teh habitatih živijo tri glavne kategorije živali glede na vrsto uporabe podzemnih okolij. V tej študiji smo se osredotočili na troglobionte, izraz, ki se nanaša na organizme, ki jih najdemo samo v jamah in ki kažejo konvergentne fenotipe (morfološke, fiziološke in vedenjske), kot so izguba oči in pigmentacije ter podaljšanje okončin. Troglobionti so torej vrste, ki obligatno živijo v jamah (Vandel, 1965; Camacho, 1992; Culver in White, 2005; Sket, 2008; Culver in Pipan, 2009; Romero, 2009; White in Culver, 2012). Čeprav so podzemni ekosistemi v primerjavi z epigeičnimi ekosistemi razmeroma slabo raziskani, kažejo precej visoko biotsko raznovrstnost na svetovni ravni. Na svetu je bilo popisanih več kot 7000 vodnih podzemeljskih vrst živali (Botosaneanu, 1986), za kopenske favne pa takšnih podatkov ni na voljo. Ocenjuje se, da na svetu obstaja približno 50.000 do 100.000 obligatnih kopenskih podzemeljskih vrst (Culver and Holsinger, 1992; Culver in sod., 2000; Culver and Pipan, 2009). Sintagma "vroča točka biotske raznovrstnosti" je eno najpomembnejših globalnih orodij za ohranjanje narave (Myers, 1988; Reid, 1998; Hughes in sod., 2002; Brooks in sod., 2006). Več vročih točk podzemeljske biotske raznovrstnosti je bilo odkritih predvsem v Evropi in Združenih državah Amerike, nekaj pa jih je znanih tudi iz Avstralije in Azije (Culver in Sket, 2000; Culver in sod., 2021). To je v skladu z literaturo, ki je uporabila arbitrarno mejo 20-25 obligatnih podzemeljskih vrst, da se območje šteje za vročo točko troglobiontske biotske raznovrstnosti (Culver in Sket, 2000; Souza Silva in Ferreira, 2016; Pipan in sod., 2020). Ta meja je odvisna od primerjalne vrednosti z drugimi (Culver in sod., 2021). Pred to študijo je bila jama z največjo raznolikostjo v Iranu jama Loven v Zagrosu, v kateri živijo tri vrste iranskih jamskih ribic (Vatandoust in sod., 2019). V Iranu doslej ni bila znana vroča točka troglobiontske biotske raznovrstnosti. Iran je z 1 648 195 km2 površine druga največja država na Bližnjem vzhodu in 17. največja na svetu. Večina ozemlja leži na sušnem okoljskem območju. Različne vrste podnebja v Iranu pokrivajo 35,5 % površine, 29,2 % je sušnega, 20,1 % polsušnega, 5 % sredozemskega in 10 % vlažnega podnebja (hladnega gorskega tipa). Tako se več kot 82 % iranskega 166 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ozemlja nahaja na sušnem in polsušnem območju sveta. Povprečna količina padavin v Iranu znaša približno 250 mm, kar je manj kot tretjina povprečne količine padavin na svetu (860 mm) (Amiri in Eslamian, 2010). Padavine se razlikujejo od več kot 1800 mm v severnih delih Irana na obali Kaspijskega morja in v nekaterih delih gorovja Zagros do približno 800 mm, vendar je povprečje približno 400 mm na nagnjenem območju gorovja Alborz in Zagros, v sušnih delih in osrednjem Iranu pa manj kot 100 mm (Raziei et al., 2005; Soltani et al., 2012). Iran je geološko del alpsko-himalajskega orogenega pasu. Skupna površina kraških karbonatnih kamnin v Iranu je približno 185.000 km2, od tega 55,2 % v Zagrosu, 24,3 % v osrednjem Iranu, 15,2 % v Alborzu ter 4,7 % v vzhodnem in južnem Iranu. Zagros in Alborz sta dve glavni gorski verigi v zahodnem in severnem Iranu, ki obsegata skoraj tretjino iranske površine. V Iranu je bilo popisanih več kot 2000 jam, 10000 izvirov in vodnjakov, večinoma v Zagrosu in Alborzu (Raeisi, 2004; Raeisi in sod., 2012). Podzemna favna Irana je še vedno nezadostno poznana, ne le s favnističnega, temveč tudi z evolucijskega vidika, zlasti pri obravnavi izvora vrst. V tej disertaciji smo sledili trem ciljem. Prvi cilj je bilo odkrivanje troglobiontskih organizmov v gorovju Zagros v Iranu, skupaj s taksonomskimi opisi novih taksonov. Opisane so bile tri nove troglobiontske vrste: karabidni hrošč, asellidni izopod in truncatelloidni gastropod. Šest vrst (Oligochaeta, Decapoda, Isopoda) je opisanih, več kot 10 vrst pa čaka na opis. Zabeležili smo tudi nove habitate že opisanih iranskih jamskih rib. Drugi cilj disertacije je bil pripraviti seznam vrst vseh iranskih troglobiontov, da bi preučili vzorce biotske raznovrstnosti in endemizma troglobiontov v gorovju Zagros v Iranu. V tem delu disertacije smo odkrili in obravnavali prvo vročo točko biotske raznovrstnosti troglobiontov v Iranu. Tretji cilj disertacije je bil preučiti filogenetske odnose med izbranimi skupinami troglobiontov in raziskati izvor te troglofavne. Za številne nove vrste troglobiontov, ki smo jih odkrili, ter za njihove ustrezne površinske sorodnike smo izvedli ekstrakcijo DNK in PCR za več markerjev, del teh izvirnih podatkov pa smo objavili v znanstvenih publikacijah. DNA izolacije ter reakcije PCR so bile narejene v Laboratoriju za Evolucijsko Zoologijo na Biološkem inštitutu Jovana Hadžija, ZRC SAZU. Sorodna zaporedja smo pridobili iz baze GenBank za smiselne rekonstrukcije filogenetskih odnosov proučevanih taksonov, za preizkuse njihovega vrstnega statusa in za ocene časovnega okvira njihove kolonizacije podzemnih okolij. V znanstvenem članku v okviru te disertacije smo opisali dve novi vrsti jamskih hroščev iz rodu Duvalius iz jam v gorovju Zagros v Iranu. Duvalius nezelensis je bil prvi troglobiontski 167 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 karabidni hrošč iz te države. Glavne troglomorfne značilnosti vrste D. nezelensis so bile odsotnost oči in podaljšane okončine. Opisali smo tudi vrsto D. achaemenius kot troglofilno vrsto. Čeprav imajo Coleoptera in zlasti karabidi številne predstavnike v podzemnih okoljih, pred to študijo iz Irana ni bil odkrit noben troglobiontski karabid, edini znani troglobiontski hrošč iz Irana pa je bil Foranotum perforatum iz družine Tenebrionidae. Zdaj je iz Irana znanih šest vrst rodu Duvalius. Za filogenetske analize smo pridobili izvirna zaporedja dveh mitohondrijskih (COI, 16S) in dveh jedrnih (18S, 28S) genov za tri vrste Duvalius iz gorovja Zagros in jih združili z objavljenimi zbirkami molekularnih podatkov o drugih vrstah Duvalius. Za rekonstrukcijo filogenije rodu Duvalius in tesno sorodnih rodov smo uporabili Bayesovo sklepanje in največjo verjetnost. Za raziskovanje divergenčnih časov glavnih linij rodu Duvalius smo uporabili BEAST. Naše filogenije so pokazale dobro podprt klad "Zagros" s tremi preučevanimi iranskimi vrstami. Za čas izolacije tega rodu od drugih sorodnikov smo ocenili približno 9,7 milijonov let (pozni miocen). Znotraj klada Zagros je bila ločitev D. nezelensis in njene sestrske skupine ocenjena na 7,8 milijonov let (pozni miocen). Sklepali smo, da bi se lahko kolonizacija podzemeljskih habitatov z D. nezelensis zgodila kadar koli po 7,8 milijoni let. Ta znanstveni prispevek je bil objavljen z naslovom »Prvi vpogled v izvor raznolikosti iranskih jamskih hroščev z opisom dveh novih vrst rodu Duvalius (Carabidae)« leta 2021 v reviji Journal of Zoological Systematics and Evolutionary Research. Izvleček: Podzemna okolja Irana so zelo slabo raziskana. V tem prispevku želimo izboljšati znanje o iranski jamski biotski raznovrstnosti, pri čemer sledimo trem ciljem: (i) raziskati iranske jame za troglobiontske hrošče; (ii) razumeti filogenetske odnose znotraj rodu Duvalius Delarouzée, 1859 in oceniti časovno obdobje kolonizacije iranskih jam; in (iii) komentirati trenutno poznavanje iranske troglobiontske favne za lažje prihodnje raziskave. S terenskim delom in morfološkim pregledom smo opisali dve novi vrsti rodu Duvalius iz jam v gorovju Zagros v Iranu: troglobiotski Duvalius nezelensis sp. nov. in netroglobiotski Duvalius achaemenius sp. nov. Za filogenetske analize smo pridobili izvirna zaporedja dveh mitohondrijskih (COI, 16S) in dveh jedrnih (18S, 28S) genov za tri vrste rodu Duvalius iz gorovja Zagros in jih združili z objavljenimi molekularnimi podatki drugih vrst in rodov hroščev. Z uporabo Bayesovega sklepanja in največje verjetnosti smo rekonstruirali filogenijo rodu Duvalius in tesno sorodnih rodov, nato pa s programom BEAST izračunali čas razhajanja glavnih linij. Naše filogenije so dobro podprle klad "Zagros", katerega ločitev od drugih sorodnikov je ocenjena na 9,7 milijona let. V kladu Zagros je razcep D. nezelensis in njegove sestrske skupine ocenjen na 7,8 milijona let, medtem ko je razcep D. achaemenius in D. kileri ocenjen na nedavnih 0,78 milijona let. V študiji smo prispevali nekaj razrešitve pri razumevanju vrstnega bogastva jamskih hroščev v Iranu in časa njihove podzemne kolonizacije. Vendar 168 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 naše filogenije potrjujejo taksonomske težave, saj je več rodov ugnezdenih globoko v “rodu” Duvalius. V drugi objavi smo opisali novo vrsto polža iz sulfidnih ribnikov v jami Tašan v gorovju Zagros v jugozahodnem Iranu. Trogloiranica tashanica je prvi troglobiontni/stigobiontni gastropod, najden v Iranu, in šesta vrsta sulfidnega stygobiontnega gastropoda na svetu. Z dvema markerjema (COI in H3) smo sklepali o filogeniji te vrste in sorodnikov ter preučili njihove anatomske dokaze za uvrstitev T. tashanica v družino Moitessieriidae. Filogenija, izpeljana s COI, je nakazovala tesno sorodstvo z družino Moitessieriidae, vendar ni potrdila monofilije družine. Filogenija, izpeljana z jedrnim H3, je jasno pokazala, da T. tashanica spada v družino Moitessieriidae. Podobno je to potrdila tudi združena analiza COI in H3. Razpravljali smo o edinstvenem habitatu novo odkritega polža, ki je poln vodikovega sulfida. V jami Tašan se nahaja edinstven ekosistem. Nedavnemu odkritju slepe ribe Garra tashanensis in izopoda Stenasellus tashanicus so sledila druga odkritja, vključno z novim rodom in vrsto gastropoda, o katerih poroča ta študija. Vodikov sulfid je strupen za večino organizmov in le pet od približno dvajsetih doslej znanih nahajališč sulfidnih jamskih ekosistemov po vsem svetu gosti stigobiontske vrste gastropodov. Ta znanstveni prispevek je bil objavljen z naslovom »Opis novega rodu in prve vrste polžev iz jam v Iranu« leta 2019 v reviji Journal of Cave and Karst Studies. Izvleček: Poročamo o novem stigobiontskem truncatelloidnem gastropodu iz sulfidnih ribnikov v jami Tašan v gorovju Zagros v jugozahodnem Iranu. Habitat vodikovega sulfida je podoben doslej znanim habitatom polžev iz s sulfidi bogatih jam v Romuniji, Italiji in Grčiji. Novo opisani rod Trogloiranica n. gen. z novo opisano vrsto T. tashanica n. sp predstavljata prve prave stigobiontske polže, najdene v Iranu. Na podlagi filogenije, pridobljene iz mitohondrijskega (citokrom oksidaza podenota I) in jedrnega (histon 3) genoma ter anatomskih znakov, je T. tashanica uvrščena v družino Moitessieriidae Bourguignat, 1863. Novi rod lahko predstavlja evolucijski relikt iz obdobja po miocenu, preden sta se razcepili družini Moitessieriidae in Cochliopidae Tryon, 1866. Druga vrsta troglobiontov, ki smo jo opisali v disertaciji in objavili v članku, je Asellus ismailsezarii iz družine Asellidae, red Isopoda, iz izvira Ganow blizu vasi Tuveh, mesto Andimeshk, provinca Khuzestan, Zagros Mts, zahodni Iran. To je bil prvi oblikatni stigobiontski Asellus iz Irana. Družina Asellidae je ena redkih družin metazojev, ki vsebuje veliko število površinskih in podzemnih vodnih vrst. Do danes družina vsebuje 23 rodov ter 428 vrst in podvrst. Od teh 428 vrst in podvrst je 279 vrst brez oči in depigmentiranih vrst, ki so značilne za podzemne organizme, 18 vrst je večinoma omejenih na podzemne vode, 169 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 pogosto imajo zmanjšane oči in pigmentacijo, 131 vrst pa je takih, ki imajo oči in pigment ter poseljujejo površinske vode. Družina je splošno razširjena na severni polobli z vrstami v severnem delu Severne Amerike, Evropi, severni Afriki in Aziji. Pri vrsti Asellus ismailsezarii so bile ugotovljene troglomorfije, kot sta depigmentacija in izguba oči. Navedli smo morfološke in molekularne dokaze, ki potrjujejo status vrste A. ismailsezarii. Gena COI in 28S vrste A. ismailsezarii smo analizirali z zaporedji vrst rodu Asellus, ki so na voljo v GenBank. Najdenih je bilo 11 MOTU znotraj rodu Asellus, vključno z enim MOTU, ki ustreza vrsti A. ismailsezarii. Nastalo drevo jedrnih zaporedij 28S rDNK se je ujemalo s filogenijo COI. Te analize so pokazale, da ta vrsta ni v evropskem kladu A. aquaticus. Ta nova vrsta rodu iz izvira Ganow je pokazala, da ta vodonosnik, ki vključuje pomembne jame Loven, Tuveh in izvire Ganow, gosti več troglobiontskih živali. Prej opisane iranske jamske ribe Garra lorestanensis, G. typhlops in Eidinemacheilus smithi iz tega vodonosnika s treh najdišč ter nekateri drugi neobjavljeni polži potrjujejo to hipotezo. Z novim odkritjem sta bili doslej iz Irana najdeni dve vrsti rodu Asellus. Asellus monticola je bil najden na izviru na skrajnem severozahodu Irana. Z novim odkritjem se je število obligatnih podzemeljsko živečih izopodov iz Irana povečalo na štiri vrste (tri vodne in ena kopenska). Predlagali smo domnevno prisotnost še več obligatnih podzemeljskih izopodov in zlasti taksonov rodu Asellus v Iranu. Naše ugotovitve skupaj s prejšnjimi poročili o asellidih iz Irana in sosednjih držav (Turkmenistan, Gruzija, Armenija) kažejo, da je območje Kaspijskega morja kontaktno območje med vrstami evropskega kompleksa A. aquaticus, vključno z A. aquaticus Linnaeus, 1758 in A. aquaticus messerianus ter azijskih vrst, vključno z A. monticola in A. ismailsezarii. Ta znanstveni prispevek je bil objavljen z naslovom »Nova obligatna podzemeljska vrsta iz rodu Asellus (Isopoda, Asellidae) iz Irana« leta 2022 v reviji Subterranean Biology. Izvleček: Z le 43 opisanimi stigobionti in le dvema vrstama izopodov je obligatna podzemeljska favna Irana, obsežne države z več kot 10 % površine apnenca, nezadostno poznana. Tu poročamo o odkritju vrste Asellus ismailsezarii sp. nov. iz gorovja Zagros, prvega brezokega in depigmentiranega asellidnega izopoda iz Irana. Nova vrsta je morfološko podobna vrsti Asellus monticola Birstein, 1932, vendar je brez oči in popolnoma depigmentirana, ima rahlo ukrivljen pereopod IV in nima sete na proksimalnih robovih eksopodita pleopodov IV in V. Filogenetska razmerja med vrstami z uporabo izvirne in podatkovno rudarjene mitohondrijske DNK in jedrne rDNK ter ocena molekularnih razlik z drugimi vrstami rodu Asellus kažejo, da je A. ismailsezarii sp. nov. sestrska vrsta večjega klada, ki vsebuje tudi evropski kompleks vrst A. aquaticus. Površinske populacije vrst iz rodu Asellus so ob različnih priložnostih in v več krajih v Evropi in Aziji kolonizirale podtalnico, pri čemer so nastale vrste in podvrste, ki so razvile troglomorfizme, kot sta depigmentacija in izguba oči. Od 37 uradno opisanih vrst in podvrst rodu Asellus jih je 15 iz 170 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 podzemne vode, vključno z A. ismailsezarii sp. nov. Predvidevamo, da bo v Aziji odkritih še veliko vrst rodu Asellus, ki obligatno poseljujejo podzemne vode. Glavna težava pri preučevanju filogenij in iskanju izvora iranskih troglobiontov je bilo pomanjkanje bližnjih sorodnikov za preučevane taksone v tej disertaciji. V GenBank ni bilo na voljo dovolj podatkov. Ker so bili nekateri rodovi novi za državo in znanost, za te taksone ni bilo bližnjih sorodnikov iz epigejskega/hipogejskega območja, niti genetsko niti geografsko. Glede na naše rezultate in dostopno literaturo se je večina kolonizacij iranskih podzemnih habitatov z živalmi zgodila v srednjem miocenu do zgodnjega pleistocena (15 mio - 2 mio). Vendar je bilo v zvezi s tem na voljo malo podatkov, saj večina iranskih troglobiontov še ni bila testirana, da bi bolje razumeli njihov izvor. V prihodnosti bo potrebno preučiti še več skupin troglobiontov na različnih kraških območjih. Prav tako bi lahko primerjali ocenjene čase pri vodnih in kopenskih troglobiontih. Za preučitev vzorcev razporeditve vrstne pestrosti troglobiontov v Iranu smo uporabili razpoložljivo literaturo in lastne podatke. Kot dopolnitev vrstnega seznama podzemeljskih členonožcev Irana, ki smo ga objavili leta 2017, smo v tej disertaciji predstavili vse iranske troglobionte (prej objavljene in naša nova odkritja, vključno s še neopisanimi vrstami) in tako posodobili seznam. V letu 2017 smo objavili seznam podzemeljskih členonožcev Irana z naslovom »Vrstni seznam podzemnih členonožcev Irana« v reviji Subterranean Biology. Izvleček: Razumevanje podzemeljske biotske raznovrstnosti je pomembno, vendar so obsežna območja sveta še vedno slabo raziskana. V tem prispevku predstavljamo prvi korak k popisu podzemeljskih členonožcev Irana. Po pregledu in analizi dostopne literature ter pregledu vzorcev, ki smo jih zbrali v iranskih jamah, smo našteli 89 jamskih vrst (iz 42 jam in 5 kraških izvirov), ki pripadajo štirim subfilumom členonožcev: Chelicerata (1 razred, 4 redovi, 36 vrst), Crustacea (2 razreda, 3 redovi, 15 vrst), Hexapoda (2 razreda, 5 redov, 34 vrst) in Myriapoda (2 razreda, 3 redovi, 4 vrste). Eden glavnih problemov pri analizah raznolikosti je, ali vključiti še neopisane vrste, ki še niso bile temeljito taksonomsko preverjene. Menimo, da v našem primeru to ne predstavlja težave, saj smo večino vzorčenih taksonov pregledali morfološko in molekularno ter, glede na geografsko razširjenost obravnavanih taksonov, odpravili dvom o njihovem taksonomskem statusu. Od vseh 49 ugotovljenih in opisanih vrst troglobiontov iz Irana (šest kopenskih, 43 vodnih) je bilo 42 vrst členonožcev, od teh je bilo 39 vrst rakov, 23 vrst pa jih je pripadalo rodu Niphargus (Niphargidae). Med kopenskimi troglobionti sta bili dve vrsti hroščev, pajek, diplopod in izopod. V tej študiji smo odkrili 26 novih vrst troglobiontov. Do danes nam je 171 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 uspelo opisati in objaviti tri od njih. Med terenskim delom smo odkrili nekaj novih nahajališč že prej opisanih iranskih jamskih rib. V jami Chah-Kabootari, apnenčasti jami z žvepleno vodo, kilometer zahodno od jame Tashan, živi Garra tashanensis. Izvir Tuveh je še eno najdišče, kjer je bil odkrit Eidinemacheilus smithi. Glede na literaturo in naše podatke je bilo ugotovljeno, da je na 45 nahajališčih v Iranu (jame, vodnjaki in kraški izviri) prisotnih 72 vrst obligatnih podzemljskih vrst. Kar 36 nahajališč je bilo na kraških območjih Zagrosa. Kot je bilo obravnavano v tej disertaciji, je to posledica večjega števila raziskav na teh območjih. Vendar pa je gorovje Zagros večinoma kraško in vsebuje največ jam in izvirov v državi. Tako majhno število raziskanih nahajališč podzemeljske biotske raznovrstnosti ni primerljivo z ZDA in Balkanskim polotokom ter njihovimi odkritimi troglobiontskimi vrstami. Do leta 1999 je bilo v ZDA znanih več kot 45.000 jam, do danes pa so jih dodali še veliko več (Culver et al., 2009). Sket in sodelavci (2004) so za Balkanski polotok navedli več kot 975 kopenskih in 650 vodnih obligatnih podzemeljskih vrst. V Združenih državah Amerike je bilo opisanih več kot 1138 obligatnih jamskih vrst in podvrst iz 112 družin in 239 rodov (Hobbs, 2012). Podzemna vodna favna na večini kraških območij v Evropi je dokaj dobro raziskana, saj je doslej znanih okoli 1800 vrst stigobiontov (Stoch in Galassi, 2010). V Iranu je znanih več kot 2000 jam ter približno 10000 kraških izvirov in vodnjakov. Čeprav ni treba vzorčiti vseh jam in tudi kraških izvirov ter drugih podzemnih voda, da bi dobili dobro oceno vrstnega bogastva za določeno regijo, pa bi jih bilo potrebno povzorčiti znatno število (Pipan in sod., 2020). Najbolj raznolika skupina troglobiontov v podzemnih okoljih Irana so bili raki (Crustacea) z 51 vrstami, od katerih je bilo 39 vrst opisanih, 12 pa jih še ni bilo uradno opisanih. Najbolj raznolika skupina je Amphipoda s 26 vrstami, v okviru Amphipoda pa je največje vrstno bogastvo troglobiontov v Iranu pokazal rod Niphargus, ki vključuje 23 vrst iz 28 lokalitet. Druga najbolj raznolika skupina v iranskih podzemnih vodah je Copepoda s 17 vrstami. Jamske ribe s štirimi predstavniki iz dveh družin in dveh rodov iz petih lokalitet so bile na naslednjem mestu raznolikih skupin troglobiontov v Iranu. Pripravili smo zemljevide, ki prikazujejo razširjenost in bogastvo troglobiontskih vrst na posameznem območju. Prikazali smo število vrst, prisotnih v vsaki 100 × 100-kilometrski celici mreže UTM, ki smo jo pripravili s programom QGIS. Celice z največjim številom vrst so se nahajale v zahodnem Iranu in zlasti na jugozahodu. V dveh celicah je bilo 27 vrst troglobiontov. Skupaj z vzhodno celico sta imeli 30 troglobiontskih vrst, kar je veljalo za najbogatejši del države glede raznolikosti troglobiontov. 172 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Najbogatejši jamski sistem, ki smo ga odkrili, sta sestavljali jami Tashan in Chah-Kabootari, v katerih se je nahajalo 21 vrst troglobiontov, od katerih je bilo pet vrst kopenskih, 16 vrst pa vodnih. Poleg teh jamskih sistemov imajo sulfidno vodo tudi drugi izviri in vodnjaki na tem območju v območju 6 kvadratnih kilometrov. Očitno je, da so ustvarili velik vodonosnik. V teh jamah in vodonosniku bi lahko živelo še več kopenskih in vodnih vrst troglobiontov. Da bi to razumeli, je potrebno več terenskega dela in vzorčenja. Domačini so na tem območju opazili več izvirov z žvepleno vodo, ki so večinoma sezonskega značaja, tečejo pozimi in spomladi, v njih pa se zadržujejo jamske ribe, ki jih domačini imenujejo "rdeče-rožnate ribe". Okrožje Tashan je potencialno pomemben del iranske podzemne biotske raznovrstnosti, saj je zaradi sulfidnega vodonosnika edinstveno. V sulfidnih jamah so zanimivi ekosistemi z nenavadno bogatimi in raznolikimi nevretenčarji, med katerimi prevladujejo raki in žuželke, in občasnimi ribami. Ekosisteme sulfidnih jam so bili preučevali Sarbu s sodelavci (1996). Prva raziskana jama s sulfidnim ekosistemom je bila jama Movile v Romuniji, sledile so jama Frasassi in Grotto Azzurra v Italiji, jama Melissotrypa v Grčiji ter jama Ayalon in izvir Tabgha v Izraelu, zdaj pa še študija vodonosnika Tashan-Chah Kabootari v Iranu. V teh zgoraj omenjenih sulfidnih jamah živijo zanimive troglobiontske živali in njihove favne bi lahko primerjali, da bi ugotovili morebitne podobnosti. V prihodnjih študijah želimo preveriti, ali so sulfidne jame Tashan-Chah Kabootari resnično kemoavotrofne ali ne. Tudi z evolucijskega vidika je še vedno vprašanje, kdaj so jamski sistem Tashan-Chah Kabootari naselili organizmi in ali je bila ta kolonizacija enkraten dogodek ali zaporedje neodvisnih dogodkov. Jamski sistem Tashan in Chah-Kabootari je tako prva vroča točka troglobiontske biotske raznovrstnosti v Iranu, ki jo je treba prednostno zaščititi. Glede na seznam vrst, ki smo ga pripravili, je treba zaščititi jamski sistem Tashan-Chah Kabootari, vodonosnik Loven-Tuveh in jamo Gakal kot pomembne habitate troglobiontov. Podzemni habitati so z vidika varstva zanemarjeni deli iranskega okolja. Stopnja endemizma pri iranskih troglobiontih je bila visoka in večina vrst je bila omejena na eno jamo, izvir ali vodonosnik. To pomeni, da je treba prednostno varovati habitate teh troglobiontskih vrst v Iranu. Jame in izviri, ki so pomemben del iranskega okolja, so bili z vidika ohranjanja v veliki meri zanemarjeni. Po naših opažanjih in nekaterih poročilih domačinov, jamarjev in znanstvenih publikacij, je v teh habitatih prišlo do številnih uničenj. Žal je ena glavnih groženj za iranske jamske ribe znanstvena plenitev. Domačini so poročali o številnih vzorčenjih ihtiologov, celo o invazivnih metodah, kot je elektrošok, kar smo zlahka razbrali iz njihovih publikacij. 173 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Čeprav večina iranskega ozemlja leži na sušnem okoljskem območju in glede na majhno količino padavin v številnih delih države, je v zahodnem in severnem Iranu veliko kraških območij. Zato je trenutno poznana podzemna biotska raznovrstnost v Iranu tudi po tej disertaciji še vedno podcenjena. Speleobiologija je v Iranu v prvi fazi in glede na odkritja zadnjega desetletja bodo prihodnja desetletja razkrila več dejstev o tej biotski raznovrstnosti in njeni evolucijski zgodovini. Glede na slabo poznavanje iranske podzemeljske favne in njene evolucijske zgodovine je ta biospeleološka disertacija obogatila naše znanje s poročanjem o novih najdbah, opisom novih vrst, poročanjem o vzorcih biotske raznovrstnosti troglobiontov, prepoznavanjem vročih točk, izvedbo filogenetskih analiz za preverjanje veljavnosti vrst ter za razumevanje odnosov in izvora preučevanih taksonov. Naša odkritja bodo tako olajšala odločitve o ohranjanju. 174 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 5 REFERENCES Abbasi M., Gharezi, A. 2008. 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Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Zagmajster M., Culver, D. C., Sket B. 2008 . Species richness patterns of obligate subterranean beetles in a global biodiversity hotspot - effect of scale and sampling intensity. Diversity and Distributions, 14: 95-105 Zagmajster M., Eme D., Fišer C., Galassi D., Marmonier P., Stoch F., Cornu J., Malard F. 2014. Geographic variation in range size and beta diversity of groundwater crustaceans: Insights from habitats with low thermal seasonality. Global Ecology and Biogeography, 23: 1135-1145 Zagmajster M., Malard F., Eme D., Culver D. C. 2018. Subterranean biodiversity patterns from global to regional scales. In: Cave Ecology, Ecological Studies. Moldovan O. T., Kovác L., Halse S. (eds.). Cham, Springer: 195-228 Zamanpoore M., Bakhshi Y., Sadeghi S., Malek-Hosseini, M. J. 2019. Niphargus keeleri n. sp., a new cave Amphipod (Arthropoda: Crustacea) from the Zagros Mountains, Iran. Journal of Natural History, 53, 43-44: 2621-26 187 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ACKNOWLEDGEMENTS I would first like to thank my parents for all supports during my Ph.D. study. Thank you, my loves, for being patient during my long stay in Slovenia. It was hard for me as well, not to see you for more than 1283 days (till now). Special and deep thanks to Dr. Matjaž Kuntner, my supervisor, a great friend and knowledgeable leader of EZ LAB group, who guided me through this journey. I highly appreciate his supports during my study in Slovenia. Regarding the vastness of my dissertation topic, I had to be in touch with several experts of different taxa of troglobiont animals which Matjaž was patient and supportive to being in corporation with this big groups from all around the world. The job has not been finished yet! Matjaž supported me as he could, and it was my great motivation to pursue and finish my study. I would like to thank the members of my doctoral commission dr. Cene Fišer, dr. Tanja Pipan, and prof. Peter Trontelj for all of their guidance during writing of the theme of dissertation, writing articles and improving the dissertation scientifically. I thank all of my friends at Jovan Hadži Institute of Biology ZRC SAZU, especially dr. Klemen Čandek, dr. Matjaž Gregoric and Tjaša Lokovšek for all of their support regarding my thesis. I learned a lot from them about laboratory work and working with phylogenetic software. Thank you for all of helps during writing the articles and doing the analyses. I wish to thank dr. Maja Zagmajster, dr. Teo Delić and Kuang-Ping Yu for all their helps and constructive comments for writing the thesis. I also thank my brother, Ali, my cousin, Vahid, my dear friends Yaser Fatemi, Shahab Azizi, Saadat Rahideh, Barooninezhad family in Sarjosher Village, and all other friends from caving groups for all their assistance during field works in Iran. I highly appreciate all guidance and helps of. Jure Jogovic, Jan Muilwijk, Florian Malard, Serban Sarbu, Jozef Grego, Rok Kostanjšek and Jean-François Flot regarding scientific publication of this dissertation. Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Funding This work was supported by the Biotechnical Faculty of the University of Ljubljana for assistance in doctoral research. Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Annex A Permission from publisher for the publication of article Malek-Hosseini et al. (2021) in the printed and electronic versions of the doctoral dissertation Malek-Hosseini M. J., Muilwijk J., Gregorič M., Kuntner M., Čandek K. 2021. First insights into the origin of Iranian cave beetle diversity with description of two new species of the genus Duvalius (Carabidae). Journal of Zoological Systematics and Evolutionary Research 59, 7: 1453–1469 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Annex B Statement on publisher permissions for the inclusion of own published articles in the printed and electronic versions of the doctoral thesis The following paper was published by The National Speleological Society. The Journal of Cave and Karst Studies is a Free Access journal. “If you are the author of a published article in this journal, you have the right to reuse the full text of your published article as part of your thesis or dissertation. In this situation, you do not need to request permission from The National Speleological Society for this use.” Fatemi Y, Malek-Hosseini M. J., Falniowski A., Hofman S., Kuntner M., Grego J. 2019. Description of a new genus and species as the first gastropod species from caves in Iran. Journal of Cave and Karst Studies 81: 233–243 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Annex C Permission from publisher for the publication of article Malek-Hosseini et al., (2022) in the printed and electronic versions of the doctoral dissertation The following paper was published by a PENSOFT journal, where “If you are the author of a published article in this journal, you have the right to reuse the full text of your published article as part of your thesis or dissertation. In this situation, you do not need to request permission from PENSOFT for this use.” Malek-Hosseini M. J., Jugovic J., Fatemi Y, Kuntner M, Kostanjšek R, Douady C. J., Malard F. 2022. A new obligate groundwater species of Asellus (Isopoda: Asellidae) from Iran. Subterranean Biology, 42: 97-124 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Annex D Permission from publisher for the publication of article Malek-Hosseini and Zamani (2017) in the printed and electronic versions of the doctoral dissertation The following paper was published by a PENSOFT journal, where “If you are the author of a published article in this journal, you have the right to reuse the full text of your published article as part of your thesis or dissertation. In this situation, you do not need to request permission from PENSOFT for this use.” Here, we also have the permission letter from the editor in chief of the journal. Malek-Hosseini M. J, Zamani A. 2017. A checklist of subterranean arthropods of Iran. Subterranean Biology 21: 19–46 Malek-Hosseini S. M. J. Discovery, diversity, and the origins of troglobiotic faunas in Iran. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022