UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Špela BORKO ADAPTIVE RADIATION OF Niphargus (CRUSTACEA: AMPHIPODA) AND ITS CONTRIBUTION TO THE DIVERSITY OF DINARIC SUBTERRANEAN FAUNA DOCTORAL DISSERTATION Ljubljana, 2022 UNIVERSITY OF LJUBLJANA BIOTECHNICAL FACULTY Špela BORKO ADAPTIVE RADIATION OF Niphargus (CRUSTACEA: AMPHIPODA) AND ITS CONTRIBUTION TO THE DIVERSITY OF DINARIC SUBTERRANEAN FAUNA DOCTORAL DISSERTATION ADAPTIVNA RADIACIJA SLEPIH POSTRANIC (CRUSTACEA: AMPHIPODA: Niphargus) IN NJEN PRISPEVEK K PESTROSTI DINARSKE PODZEMNE FAVNE DOKTORSKA DISERTACIJA Ljubljana, 2022 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. 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 24th September 2019 it has been confirmed that the candidate meets the requirements for pursuing a PhD in the interdisciplinary doctoral programme in Biosciences, Scientific Field Biology. Doc. Dr. Cene Fišer is appointed as supervisor. Doctoral dissertation was conducted at the Department of Biology, Biotechnical Faculty, University of Ljubljana. Commission for assessment and defence: President: Prof. Dr. Peter TRONTELJ University of Ljubljana, Biotechnical Faculty, Department of Biology Member: Prof. Dr. Florian ALTERMATT University of Zurich, Department of Evolutionary Biology and Environmental Studies Member: Doc. Dr. Maja ZAGMAJSTER University of Ljubljana, Biotechnical Faculty, Department of Biology Date of the defence: 7th June 2022 Špela BORKO II Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KEY WORDS DOCUMENTATION DN Dd DC UDC 575.8:551.44:595.371(043.3) CX evolutionary radiations, subterranean, Niphargus, the Dinarides, phylogenetics, biodiversity AU BORKO, Špela AA FIŠER, Cene (supervisor) PP SI-1000, Ljubljana, Jamnikarjeva 101 PB University of Ljubljana, Biotechnical Faculty, Interdisciplinary Doctoral Programme in Biosciences, Scientific Field Biology PY 2022 TI ADAPTIVE RADIATION OF Niphargus (CRUSTACEA: AMPHIPODA) AND ITS CONTRIBUTION TO THE DIVERSITY OF DINARIC SUBTERRANEAN FAUNA DT Doctoral dissertation NO VIII, 116 pages, 5 appendices, 130 references LA en AL en/sl AB Speciation and dispersal are among key processes that drive biodiversity. Adaptive radiation (AR), a monophyletic species proliferation accompanied by ecological diversification, may be generating much of Earth’s biodiversity, but it has never been tested in subterranean environment. We reconstructed deep evolutionary history of amphipods. Next, we comprehensively analysed the speciation and ecomorphological diversification of the subterranean genus Niphargus, on continental scale, within a subterranean hotspot, and within one community. Amphipods evolved in several diversification pulses that correspond to ecological opportunities. Niphargus followed the course of AR that unfolded in the time of emerging mountain ranges. Several clades independently radiated, adaptively and nonadaptively. The clades adapted to distinct sets of adaptive optima with limited convergence. The speciation and dispersal contributed differently within the Dinarides. The diversity of the south-eastern Dinarides emerged mostly through local diversification of several clades. They acted as a donor area. The north-western hotspot arose through a combination of dispersal and local diversification. Although the general patterns support the AR hypothesis, explicit tests for ecological speciation are lacking. A community in Melissotrypa Cave is a potential case of ecological speciation of Niphargus. AR is thus a universal phenomenon, present also in subterranean environment, but more research is needed to understand the contribution of different processes to biodiversity, especially on microevolutionary level. III Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 KLJUČNA DOKUMENTACIJSKA INFORMACIJA ŠD Dd DK UDK 575.8:551.44:595.371(043.3) KG Evolucijske radiacije, Niphargus, podzemlje, Dinaridi, filogenije, biodiverziteta AV BORKO, Špela, mag. ekol. biod. SA FIŠER, Cene (mentor) KZ SI-1000, Ljubljana, Jamnikarjeva 101 ZA Univerza v Ljubljani, Biotehniška fakulteta, Interdisciplinarni doktorski študijski program Bioznanosti, znanstveno področje Biologija LI 2022 IN ADAPTIVNA RADIACIJA SLEPIH POSTRANIC (CRUSTACEA: AMPHIPODA: Niphargus) IN NJEN PRISPEVEK K PESTROSTI DINARSKE PODZEMNE FAVNE TD Doktorska disertacija OP VIII, 116 strani, 5 prilog, 130 virov IJ en JI en/sl AI Speciacija in disperzija sta ključna procesa, ki poganjata biotsko pestrost. Adaptivna radiacija (AR), hitro vznikanje vrst iz skupnega prednika, pospremljeno z ekološko diverzifikacijo, bi lahko bila ključen proces nastanka biotske pestrosti na Zemlji, a še ni bila testirana v podzemlju. Najprej smo rekonstruirali globoko evolucijsko zgodovino postranic. Nato smo celovito analizirali vzorce speciacije in ekomorfološke diverzifikacije slepih postranic iz rodu Niphargus, na kontinentalnem nivoju, znotraj vroče točke podzemne biotske pestrosti in znotraj ene združbe. Postranice so se razvile v več diverzifikacijskih pulzih, ki sovpadajo z ekološkimi priložnostmi. Slepe postranice so diverzificirale v procesu AR, na območju dvigajočih se kraških gorstev. Več kladov je neodvisno radiiralo, adaptivno in neadaptivno. Čeprav deloma konvergirajo, so se prilagodili na različne adaptivne optimume. Prispevek speciacije in disperzije se vzdolž Dinaridov spreminja. Biotska pestrost jugovzhodnih Dinaridov je večinoma posledica lokalne diverzifikacije več kladov in deluje kot prispevno območje vrst. Severozahodna vroča točka pa je posledica kompleksne kombinacije disperzije in lokalne diverzifikacije. Čeprav splošni vzorci pritrjujejo hipotezi AR, pa ekološka speciacija še ni bila primerno testirana. Združba slepih postranic v jami Melissotrypa bi lahko predstavljala primer ekološke speciacije. AR je tako univerzalni fenomen, prisoten tudi v podzemlju. Za razumevanje prispevka različnih procesov, še posebej na mikroevolucijskem nivoju, pa bodo potrebne dodatne raziskave. IV Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. 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 ANNEXES ........................................................................................................ VIII ABBREVIATIONS AND SYMBOLS ............................................................................... IX 1 INTRODUCTION .................................................................................................... 1 1.1 EVOLUTIONARY RADIATIONS ....................................................................... 2 1.1.1 Adaptive radiations ............................................................................................... 2 1.1.1.1 Ecological opportunity ............................................................................................ 4 1.1.1.2 Independent replicated radiations ........................................................................... 4 1.1.2 Nonadaptive radiations ........................................................................................ 5 1.2 ORIGINS OF BIODIVERSITY IN THE SUBTERRANEAN ENVIRONMENT 6 1.3 STUDY MODEL .................................................................................................... 7 1.4 AIMS AND HYPOTHESES .................................................................................. 8 2 SCIENTIFIC WORKS ............................................................................................ 9 2.1 THE LATE BLOOMING AMPHIPODS: GLOBAL CHANGE PROMOTED POST-JURASSIC ECOLOGICAL RADIATION DESPITE PALAEOZOIC ORIGIN ...... 9 2.2 A SUBTERRANEAN ADAPTIVE RADIATION OF AMPHIPODS IN EUROPE .............................................................................................................................. 22 2.3 AMPHIPODS IN A GREEK CAVE WITH SULPHIDIC AND NON- SULPHIDIC WATER: PHYLOGENETICALLY CLUSTERED AND ECOLOGICALLY DIVERGENT ...................................................................................................................... 35 2.4 A HOTSPOT OF GROUNDWATER AMPHIPOD DIVERSITY ON A CROSSROAD OF EVOLUTIONARY RADIATIONS ..................................................... 52 2.5 HOW DID SUBTERRANEAN AMPHIPODS CROSS THE ADRIATIC SEA? PHYLOGENETIC EVIDENCE FOR DISPERSAL–VICARIANCE INTERPLAY MEDIATED BY MARINE REGRESSION–TRANSGRESSION CYCLES .................... 66 3 DISCUSSION AND CONCLUSIONS .................................................................. 80 V Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3.1 DISCUSSION ....................................................................................................... 80 3.1.1 Adaptive radiations in the subterranean environment ................................... 81 3.1.2 The genus Niphargus: adaptive radiation within the amphipod radiation ... 82 3.1.3 Distinguishing between ecological and nonecological speciation ................... 85 3.1.4 Processes that have shaped a subterranean biodiversity hotspot ................... 86 3.2 CONCLUSIONS .................................................................................................. 90 4 SUMMARY ............................................................................................................. 91 4.1 SUMMARY .......................................................................................................... 91 4.2 POVZETEK .......................................................................................................... 95 5 REFERENCES ..................................................................................................... 107 ACKNOWLEDGEMENTS ANNEXES VI Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 TABLE OF CONTENTS OF SCIENTIFIC WORKS Copilaş-Ciocianu D., Borko Š., Fišer C. 2020. The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin. Molecular Phylogenetics and Evolution, 143: 106664, https://doi.org/10.1016/j.ympev.2019.106664: 12 p. Borko Š., Trontelj P., Seehausen O., Moškrič A., Fišer C. 2021. A subterranean adaptive radiation of amphipods in Europe. Nature Communications, 12: 3688, https://doi.org/10.1038/s41467-021-24023-w: 12 p. Borko Š., Collette M., Brad T., Zakšek V., Flot J.-F., Vaxevanopoulos M., Sarbu S. M., Fišer C. 2019. Amphipods in a Greek cave with sulphidic and non-sulphidic water: phylogenetically clustered and ecologically divergent. Systematics and Biodiversity, 17, 6: 558-572 Borko Š., Altermatt F., Zagmajster M., Fišer C. 2022. A hotspot of groundwater amphipod diversity on a crossroad of evolutionary radiations. Diversity and Distributions, 00: 1–13 Delić T., Stoch F., Borko Š., Flot J.-F., Fišer C. 2020. How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for dispersal–vicariance interplay mediated by marine regression–transgression cycles. Journal of Biogeography, 47: 1875– 1887 VII Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 LIST OF ANNEXES ANEX A: Consent from publishers for the re-publication of article entitled The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin in the print and electronic versions of the doctoral dissertation ANEX B: Consent from publishers for the re-publication of article entitled Amphipods in a Greek cave with sulphidic and non-sulphidic water: phylogenetically clustered and ecologically divergent in the print and electronic versions of the doctoral dissertation ANEX C: Consent from publishers for the re-publication of article entitled How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for dispersal– vicariance interplay mediated by marine regression–transgression cycles in the print and electronic versions of the doctoral dissertation VIII Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ABBREVIATIONS AND SYMBOLS MOTU: Molecular Operational Taxonomic Unit IX Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 1 INTRODUCTION Biodiversity can be measured in different ways: as number of species (species diversity), phenotypic or ecological disparity (phenotypic diversity), or phylogenetic relatedness (phylogenetic diversity) (Devictor et al., 2010). Understanding the origins of the various facies of biodiversity is a primary goal of evolutionary biology and macroecology. The current biodiversity patterns emerged through an interplay of speciation, dispersal, and extinction (Davies et al., 2007). Speciation, the evolution of two or more descendant species from a common ancestor, is the ultimate mechanism that generates biodiversity. Speciation can be driven by geographic distance. When a geographic barrier arises between populations, reproductive isolation will eventually follow regardless of the type or strength of selection (allopatric speciation). However, selection accelerates speciation. If speciation is driven by selection, it can be ecological or nonecological. In the first case, reproductive isolation evolves as a side product of the adaptation to different ecological niches. In the second case, reproductive isolation develops through the fixation of different advantageous mutations in separate populations experiencing similar selection pressures (Rundell and Price, 2009; Schluter, 2009). Phenotypic diversification commonly goes hand in hand with the speciation, either in response to local selection or simply due to random evolution of phenotype. If phenotypic diversification is due to exploitation of different ecological niches (ecological speciation), morphological diversification will relate to ecological diversity (Michaud et al., 2018; Schluter, 2000). In these occasions, phenotypic diversification may be causally linked to speciation. However, the link between form and function is not always clear and is sometimes difficult to establish (Guillerme et al., 2020). The second process that influences the biodiversity is dispersal. Dispersal is a process of movement by which individuals shift their geographic position. It has implications on gene flow, population dynamics, resource competition, and species distribution. Despite its importance, it is often poorly understood (Duarte and Mali, 2018). Disentangling how speciation and dispersal contributed to biodiversity patterns is a challenging task. Combining different metrics of biodiversity, such as species richness and phylogenetic diversity, can elucidate the relative contributions of the two respective processes to overall biodiversity patterns (Davies et al., 2007; Fritz and Rahbek, 2012; Li and Yue, 2020). Phylogenetic diversity is a measure of diversity that incorporates the information on shared ancestry of species (Faith, 1992). It can unravel the biogeographic history and interactions among species in an assemblage, especially if combined with other diversity measures. For example, species-rich areas with low phylogenetic diversity may emerge through rapid recent speciation or high temporal turnover of lineages and rare dispersal events. In contrast, 1 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 species-rich areas with high phylogenetic diversity may emerge through long periods of steady speciation or high dispersal. Individual dispersal events may have a small impact on species richness, but elevate phylogenetic diversity. High phylogenetic diversity can also be a result of extinction, a process of disappearance of a species (Davies et al., 2007). However, the extinction rates of clades without fossil evidence, such as soft tissue invertebrates, are difficult to reconstruct from phylogenies alone. Biodiversity hotspots form through high diversification, low extinction, high immigration, or a combination of these processes (Wiens and Donoghue, 2004). It has been proposed that most of the extant diversity in many major clades across the globe emerged through sudden bursts of speciation, often accompanied by extensive ecological diversification – evolutionary radiations (Stroud and Losos, 2016). We tested this view in an ecologically simple, permanently dark, and nutrient-deprived subterranean realm. 1.1 EVOLUTIONARY RADIATIONS The tempo and mode of speciation vary among clades, in space and in time. Speciation can be slow or fast, gradual or abrupt. Evolutionary radiations, rapid proliferations of species within a single lineage, have been recognised as important drivers of biodiversity. Evolutionary radiations may or may not be accompanied by relevant niche differentiation (Rundell and Price, 2009). Adaptive radiations are species proliferations accompanied by ecological divergence among descendants, i.e. differentiation of relevant ecological niches (Czekanski-Moir and Rundell, 2019; Rundell and Price, 2009; Schluter, 2000), while nonadaptive radiations are not accompanied by relevant niche differentiation and descendant species do not exhibit ecological divergence (Gittenberger, 1991). Clades may contain elements of both adaptive and nonadaptive diversification, i.e. ecologically differentiated sympatric lineages, as well as ecologically similar allopatric or parapatric lineages. Moreover, ecological differentiation is sometimes difficult to define. Consequently it can be difficult to draw a line between these two phenomena (Rundell and Price, 2009). 1.1.1 Adaptive radiations Adaptive radiation is a monophyletic species proliferation accompanied by ecological diversification (Rundell and Price, 2009; Schluter, 2000). Adaptive radiation causally integrates species and phenotypic components of biodiversity (Losos and Mahler, 2010; Stroud and Losos, 2016). The adaptive radiation model uses eco-evolutionary dynamics operating on a local scale such as local adaptation and interspecific interactions, to explain macroevolutionary processes and global biodiversity patterns, including speciation, clade diversification, and latitudinal gradients of species richness (Schluter, 2000). According to the strict definition of adaptive radiation, ecology-based divergent selection among populations leads to barriers to gene flow and consequent ecological speciation. Strong 2 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 divergent natural and sexual selection in different environments results in rapid ecological divergence and hence rapid speciation. However, sometimes it is hard to reconstruct the sequence of processes. It is possible that reproductive isolation evolves as a result of nonecological processes and ecological differentiation happens later. If these species later enter into sympatry the resulting pattern would resemble the ecological speciation (Rundell and Price, 2009). The classic definition of adaptive radiation incorporates four criteria: common ancestry of species, phenotype-environment correlation, trait utility, and rapid speciation (Schluter, 2000). Common ancestry can be inferred from phylogenies. A clade that has undergone adaptive radiation does not need to be monophyletic, i.e. it does not need to include all the descendants of the ancestral lineage. In fact, many classic examples of adaptive radiation, such as anoles exhibit old species that dispersed into environments that did not promote diversification. Phenotype-environment correlation is necessary for ecological specialization. This means that differences in phenotype are associated with features of the environment, such as the use of different resources. Trait utility is the advantage of a trait value in its environment. To prove trait utility in the strict sense, i.e. that a trait value improves performance in a given environment or task, experimental evidence is needed, which is often difficult to achieve (Gillespie et al., 2020). Finally, an initial rapid diversification followed by a slowdown in net diversification through time is described by an early-burst model (Harmon et al., 2010). Rapid speciation often begins after a period of steady pace of evolution, triggered by newly emerged ecological opportunity. The adaptive radiation process depends on intrinsic and extrinsic factors. Internal factors of a clade include sexual selection, hybridization and developmental plasticity. An external prerequisite is an ecological opportunity, “a wealth of unexploited ecological resources” (Schluter, 2000). The relative importance and interaction of intrinsic and extrinsic factors are poorly understood. It is also not clear whether these factors also have predictive power or if they can only be used for post-hoc explanation of the adaptive radiation (Losos and Mahler, 2010; Stroud and Losos, 2016). Adaptive radiations have primarily been studied in discrete, replicated habitats, such as on islands or in lakes, where the study design can be relatively easily framed in space and time (Grant and Grant, 2008; Losos, 2009; Seehausen, 2006). In this type of settings the dispersal from source areas is limited, the onset of diversification coincides with initial colonisation, and the ecological niches for the incoming species can be better defined and quantified (Losos, 2009; Seehausen, 2006). Famous examples are anole lizards in the Greater Antilles islands, Darwin’s finches in Galapagos, or cichlids in the African Great Lakes (Grant and Grant, 2008; Losos, 2009; Seehausen, 2006). Less attention has been paid to continental adaptive radiations (Blom et al., 2016), or radiations of invertebrates. Habitat-specific diversification and allopatric speciation have been shown as drivers of radiation of shrimps in Sulawesi's ancient lake (Von Rintelen et al., 2010). Competition and heterogeneity of 3 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 resources were recognised as ecological opportunities in the adaptive radiation of Galápagos land snails (Parent and Crespi, 2009). Similar sets of ecomorphs arose through both dispersal and diversification in Hawaiian spiders (Gillespie, 2004). Studies of radiation of amphipods in Lake Baikal were mainly descriptive (Macdonald et al., 2005; Naumenko et al., 2017; Sherbakov, 1999). No study explicitly asked whether adaptive radiation could unfold in the subterranean environment. Although ecologically harsh, subterranean habitats are both ecologically heterogeneous and geographically fragmented (Culver and Pipan, 2019) and as such a candidate environment for it (Naciri and Linder, 2020). 1.1.1.1 Ecological opportunity Adaptive radiation requires empty or underutilised adaptive zone, a multitude of ecological resources that are free of competitors (Schluter, 2000). Ecological opportunities can arise from different key events. The first is the colonization of a new area. The Hawaiian archipelago was colonised by several groups of animals that radiated within new area: birds, insects and arachnids (Gillespie, 2004; Lovette et al., 2002). The aquatic equivalent of islands are ancient lakes with multitude of cichlid radiations (Seehausen, 2015). Next ecological opportunity is the emergence of new resources. The uplift of new mountain ranges like the Andes or the Miocene appearance of grasslands in North America were such events (Stroud and Losos, 2016). The extinction of the ecologically dominant group also opens an ecological opportunity within ancestral range of taxon. Mass extinction events were usually followed by rapid radiations of surviving species, such as the rise of birds and mammals after the Cretaceous-Paleogene extinction (Hull, 2015). Finally, a taxon can evolve a feature that enables new interaction with the environment without specific change of the environment: a key innovation. The origin of wings in birds and bats opened a way to the aerial realm. New study even suggests that anoli adaptive radiation was driven by a key innovation rather than colonisation of islands, specifically the adhesive toe pads that facilitated the exploitation of arboreal niches (Burress and Muñoz, 2022). The diversification of one group of organisms can also in turn create ecological opportunity for another unrelated group of species, a phenomenon that is described by the “diversity begets diversity” model. In this case, new species increase the number of available ecological niches (Losos and Mahler, 2010). 1.1.1.2 Independent replicated radiations Large adaptive radiations within discrete ecosystems often comprise of several parallel radiations that result in similar suites of ecomorphs. Ecomorphs are morphologically and behaviourally similar species that occupy similar microhabitats but are not necessarily phylogenetically closely related (Mahler et al., 2013). Such cases are above mentioned anole lizards on islands (Mahler et al., 2013) and cichlid fishes in lakes (Elmer et al., 2014) or South American rivers (Burress et al., 2018), where clades repeatedly and independently evolved into a similar sets of habitat specialists that cluster in functional morphological 4 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 space. These parallel radiations are not necessarily replicates. The anole radiation comprises of convergent phenotypes and also of species with unique phenotypes that have evolved a morphology and ecology not found on other islands. It has been shown, that anole lizards exhibit significant pairwise phenotypic similarity between species on different islands, even when the full ecomorphological diversity of the lineages is taken into account (Mahler et al., 2013). The repeated occurrence of not closely related ecomorphs has also been reported from subterranean environments, within amphipod genus Niphargus (Trontelj et al., 2012). 1.1.2 Nonadaptive radiations A counterpoint to adaptive radiation, nonadaptive radiation is a lineage diversification with minimal ecological differentiation, resulting in related and ecologically similar species. Most often, the speciation in nonadaptive radiation would be allopatric or parapatric, where gene flow reduces due to geographic isolation (Rundell and Price, 2009), at times fortified by intensified genetic drift in smaller populations (Nürk et al., 2020). The process is affected by the similarity of environments of the diverging populations. It may also be possible for ecologically similar species to coexist in sympatry. One such case is sexual selection leading to reproductive isolation (Rundell and Price, 2009). The main difference between adaptive and nonadaptive radiation is that species proliferation from common ancestor is due to nonecological speciation. However, the definition of nonadaptive radiation is even vaguer than that of adaptive radiation and published cases of nonadaptive radiation often do not exhibit exceptionally fast proliferation of species, but are nevertheless referred to as such. Nonadaptive radiations often involve organisms with limited dispersal abilities or those living in highly subdivided environments with similar habitats and conditions on either side of barriers (Czekanski-Moir and Rundell, 2019). However, at larger spatial scales, organisms with intermediate dispersal abilities may diversify fastest, because they can colonize new areas but cannot maintain gene flow (Agnarsson et al., 2014). One optional mechanism underlying nonadaptive radiation could be also phylogenetic niche conservatism, the tendency of species to retain ancestral ecological characteristics. In spatially and temporally fluctuating environment, this mechanism would promote vicariant isolation and speciation (Kozak et al., 2006). The first case of a highly diversified clade, where diversification cannot be associated with its environment, was recognised already in 1872 in land snails in Hawaii (Gulick, 1872). Sexual selection has been identified as a driver of nonadaptive radiation in several genera of damselflies, either as nonadaptive divergence in colouration, behaviour, or genital structures differentiation (Wellenreuther and Sánchez-Guillén, 2016), and also as an early driver of radiation of electric fish (Arnegard et al., 2010). A high degree of speciation but low phenotypic specialization was found in sigmodontine rodents (Maestri et al., 2017) and cold-climate lizards (Reaney et al., 2018). Evidence of cryptic species of amphipods in Lake Baikal suggests a possible combination of adaptive and nonadaptive events (Schön and 5 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Martens, 2004). A similar case reveals in subterranean realm, where highly diversified clades on one hand, and eco-morphologically similar or even cryptic clades of Niphargus on the other (Delić et al., 2017a; Delić et al., 2017b; Trontelj et al., 2012) hint that a combination of nonadaptive and adaptive processes may be at work in this subterranean hyper-speciose clade. 1.2 ORIGINS OF BIODIVERSITY IN THE SUBTERRANEAN ENVIRONMENT The subterranean environment is harsh, compared to the surface. Due to the complete absence of light and consequently photosynthetic production, subterranean ecosystems are among the most resource-limited environments on Earth. Only a few species have successfully colonized them. The subterranean environment has long been perceived as an evolutionary dead end, whose inhabitants are doomed to extinction before they can diversify further (Barr and Holsinger, 1985; Culver and Pipan, 2019; Poulson and White, 1969). However, the dead-end hypothesis has soon been challenged by some authors (Stoch, 1995), and studies in past two decades have shown that evolutionary processes continue after the colonisation of the subterranean environment, including speciation within the subterranean environment (see below). Yet, the hypothesis of entirely subterranean adaptive radiation has never been tested. A considerable number of subterranean species derived from subterranean ancestor, apparently in both, nonecological and ecological speciation. Some species retained similar morphology due to similar environmental conditions, and some of them are morphologically indistinguishable (morphologically cryptic) (Esposito et al., 2015; Faille et al., 2013; Fišer et al., 2015). It has been shown that species diversity of European groundwater isopods and amphipods is probably two to three times greater than expected due to cryptic species (Eme et al., 2018). Most cryptic species probably emerged in allopatry, by geographic fragmentation. Sometimes they later came into sympatry, examples can be found among amphipod genus Niphargus or cave shrimps (Fišer et al., 2018; Zakšek et al., 2009). On the other hand, a number of species have further diversified morphologically and ecologically, apparently in a process of specialization to different subterranean habitats and trophic niches in both, terrestrial and aquatic environment. Within the group of Australian groundwater dytiscid beetles multiple sympatric pairs or triplets of sister species evolved, that exhibit high morphological disparity (Leijs et al., 2012; Vergnon et al., 2013). Another case come from subterranean spiders. Two sister species from the genus Troglohyphantes, ecomorphologically adapted to different subterranean habitats, apparently evolved through spatial niche partitioning (Mammola et al., 2018). Several sympatric sister species of the subterranean Leiodidae beetles were hypothesised to evolve adaptively (Njunjić et al., 2018). Adaptation to different habitats has been shown in the genus Niphargus (Delić et al., 2016; Trontelj et al., 2012). There are other examples of subterranean diversification, such as in freshwater crayfishes (Stern et al., 2017), anchialine cave shrimps (Jurado-Rivera, Pons, et 6 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 al., 2017) or Carabidae beetles (Faille et al., 2013). The amount of evidence, albeit indirect, suggests that evolutionary processes in subterranean environment closely resemble those on the surface and that the phenomenon of adaptive radiation cannot be ruled out. Although mostly viewed as ecologically simple, stable and extreme compared to surface counterpart, subterranean environment is also diverse and variable. The emerging alternative view on subterranean environment is that it should at least theoretically promote diversification and adaptive radiation. It offers various habitats, both terrestrial and aquatic, with spaces of different sizes, with different flow velocity and variously distant from the surface. It is also highly fragmented (Culver and Pipan, 2019). Subterranean environment thus provides a complex landscape for lineages to ecologically differentiate and also barriers for gene flow that promote allopatric speciation (Naciri and Linder, 2020). Karst massifs represent discrete natural replicates and offer an interesting model system analogous to lakes and islands. To date, no comprehensive analysis of the speciation-diversification of the exclusively subterranean group has been made. The aim of this PhD programme is to fill this gap. 1.3 STUDY MODEL Our study model is the amphipod genus Niphargus, the largest genus of freshwater amphipods in the world (Väinölä et al., 2008). It is distributed through the Western Palearctic, from Ireland to Iran, with the highest diversity in south-eastern Europe, area of the modern South-Eastern Alps, the Dinarides, and the Carpathians. With more than 420 described species, and a high number of undescribed species, this mega-diverse genus importantly contributes to biodiversity patterns in the aquatic subterranean habitats of the Western Palearctic (Fišer, 2019; Horton et al., 2021; Zagmajster et al., 2014). The genus presumably evolved from a subterranean ancestor, and the diversification of this monophylum apparently took place entirely in the subterranean environment (Fišer et al., 2008b). Nowadays, members of the genus are found in virtually all subterranean aquatic habitats from the surface to great depths. Their morphology is related to habitat properties, suggesting that species adapted to subterranean habitats (Delić et al., 2016; Trontelj et al., 2012). Niphargus thus comprises three key elements defining adaptive radiation: monophyly, massive speciation, and ecological disparity, making it an excellent candidate to test the model of subterranean adaptive radiation. Second, Niphargus species constitute a major part of the European groundwater fauna and importantly contribute to the biodiversity patterns in the region. As such, it is also a suitable model system for studying groundwater biodiversity patterns of the global subterranean hotspot, the Dinarides. The Dinarides are a 166,000 km2 large mountain range in south-eastern Europe. The carbonate part of the Dinarides is the Dinaric karst. This 650 km long and up to 150 km wide area with more than 2,000 m of elevation span and more than 20,000 known caves, is the paramount karst region in Europe (Hajna, 2019). The area is a global 7 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 biodiversity hotspot for surface and subterranean ecosystems (Myers et al., 2000; Sket, 2012; Zagmajster et al., 2014). More than ¼ of all described Niphargus species have been found here, and several large monophyletic clades occur only in this region (Fišer et al., 2008b). 1.4 AIMS AND HYPOTHESES The aim of this dissertation was to comprehensively analyse the speciation and ecomorphological diversification of the genus Niphargus across different spatio-temporal scales, and to evaluate its contribution to the origin of a subterranean biodiversity hotspot. We followed five specific aims. First, we aimed to reconstruct phylogenetic relationships and major diversification patterns of order Amphipoda and solve Niphargus position within the order. Second, we quantified the tempo and mode of diversification patterns of Niphargus and explored if they corroborate with adaptive radiation model expectations. We explored whether shifts in speciation and diversification spatially and temporally concur with geologic and paleogeographic events that might have grounded the ecological opportunity. We expected that the evolutionary history of the genus consist of multiple independent adaptive and nonadaptive radiation events in the areas of the emerging mountain ranges in south-eastern Europe. Third, we in analysed in detail a possible case of ecological speciation within Niphargus on a level of a single community, that can be considered as an adaptive radiation in a narrow sense. We analysed a monophylum of three species occupying three distinct habitats within the Melissotrypa Cave in Greece, forming a possible case of ecological speciation. Fourth, we explored in what extent speciation, either through adaptive or nonadaptive radiations, and dispersal from neighbouring regions contributed to the overall biodiversity of Niphargus in Dinarides, a global hotspot of a subterranean fauna. Given the exceptionally high diversity and morphological disparity in the Dinaric Karst, a combination of processes seems to be involved. Finally, we evaluated the importance of Dinarides for the development of subterranean fauna in Southern Europe. We questioned whether the Dinaric region, where intense speciation took place, acted as a donor of species for the neighbouring areas, especially Apennine Peninsula. 8 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2 SCIENTIFIC WORKS 2.1 THE LATE BLOOMING AMPHIPODS: GLOBAL CHANGE PROMOTED POST-JURASSIC ECOLOGICAL RADIATION DESPITE PALAEOZOIC ORIGIN Copilaş-Ciocianu D., Borko Š., Fišer C. 2020. The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin. Molecular Phylogenetics and Evolution, 143: 106664, https://doi.org/10.1016/j.ympev.2019.106664: 12 p. The ecological radiation of amphipods is striking among crustaceans. Despite high diversity, global distribution and key roles in all aquatic environments, little is known about their ecological transitions, evolutionary timescale and phylogenetic relationships. It has previously been proposed that the amphipod ecological diversification began in the Late Palaeozoic. By contrast, due to their affinity for cold/oxygenated water and absence of pre-Cenozoic fossils, we hypothesized that the ecological divergence of amphipods arose throughout the cool Late Mesozoic/Cenozoic. We tested our hypothesis by inferring a large-scale, time-calibrated, multilocus phylogeny, and reconstructed evolutionary patterns for major ecological traits. Although our results reveal a Late Palaeozoic amphipod origin, diversification and ecological divergence ensued only in the Late Mesozoic, overcoming a protracted stasis in marine littoral habitats. Multiple independent post-Jurassic radiations took place in deep-sea, freshwater, terrestrial, pelagic and symbiotic environments, usually postdating deep-sea faunal extinctions, and corresponding with significant climatic cooling, tectonic reconfiguration, continental flooding, and increased oceanic oxygenation. We conclude that the profound Late Mesozoic global changes triggered a tipping point in amphipod evolution by unlocking ecological opportunities that promoted radiation into many new niches. Our study also provides a solid, time-calibrated, evolutionary framework to accelerate research on this overlooked, yet globally important taxon. 9 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 10 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 11 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 12 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 13 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 14 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 15 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 16 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 17 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 18 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 19 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 20 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 21 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.2 A SUBTERRANEAN ADAPTIVE RADIATION OF AMPHIPODS IN EUROPE Borko Š., Trontelj P., Seehausen O., Moškrič A., Fišer C. 2021. A subterranean adaptive radiation of amphipods in Europe. Nature Communications 12: 3688, https://doi.org/10.1038/s41467-021-24023-w: 12p. Adaptive radiations are bursts of evolutionary species diversification that have contributed to much of the species diversity on Earth. An exception is modern Europe, where descendants of ancient adaptive radiations went extinct, and extant adaptive radiations are small, recent and narrowly confined. However, not all legacy of old radiations has been lost. Subterranean environments, which are dark and food-deprived, yet buffered from climate change, have preserved ancient lineages. Here we provide evidence of an entirely subterranean adaptive radiation of the amphipod genus Niphargus, counting hundreds of species. Our modelling of lineage diversification and evolution of morphological and ecological traits using a time-calibrated multilocus phylogeny suggests a major adaptive radiation, comprised of multiple subordinate adaptive radiations. Their spatio-temporal origin coincides with the uplift of carbonate massifs in South-Eastern Europe 15 million years ago. Emerging subterranean environments likely provided unoccupied, predator-free space, constituting ecological opportunity, a key trigger of adaptive radiation. This discovery sheds new light on the biodiversity of Europe. This article is licensed under a Creative Commons Attribution 4.0 International License. 22 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 23 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 24 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 25 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 26 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 27 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 28 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 29 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 30 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 31 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 32 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 33 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 34 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.3 AMPHIPODS IN A GREEK CAVE WITH SULPHIDIC AND NON-SULPHIDIC WATER: PHYLOGENETICALLY CLUSTERED AND ECOLOGICALLY DIVERGENT Borko Š., Collette M., Brad T., Zakšek V., Flot J.-F., Vaxevanopoulos M., Sarbu S. M., Fišer C. 2019. Amphipods in a Greek cave with sulphidic and non-sulphidic water: phylogenetically clustered and ecologically divergent. Systematics and Biodiversity, 17, 6: 558–572 We characterized taxonomically, ecologically, and phylogenetically the amphipod community of Melissotrypa Cave (Central Greece), which comprises both freshwater and sulphidic lakes. We found four amphipod species: Niphargus jovanovici, Niphargus lindbergi, Niphargus gammariformis sp. nov. and an unknown species of Bogidiella. The three Niphargus species form a well-supported monophylum but differ in their ecology and morphology: N. jovanovici is a small and slender species inhabiting small freshwater voids, N. lindbergi is a large and stout species living in freshwater lakes, whereas N. gammariformis sp. nov. is a small and stout species found predominantly in a sulphidic lake. Available evidence suggests that diversification may have happened in a geographically restricted area and was driven by ecological differentiation. Niphargus gammariformis sp. nov. shows morphological convergences in diagnostic traits with two species hitherto classified into the genus Pontoniphargus. As molecular phylogenies show Pontoniphargus nested within Niphargus, we synonymize here Pontoniphargus with Niphargus. The species originally named Pontoniphargus ruffoi needed to be renamed into Niphargus pontoruffoi. 35 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 36 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 37 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 38 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 39 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 40 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 41 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 42 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 43 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 44 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 45 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 46 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 47 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 48 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 49 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 50 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 51 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.4 A HOTSPOT OF GROUNDWATER AMPHIPOD DIVERSITY ON A CROSSROAD OF EVOLUTIONARY RADIATIONS Borko Š., Altermatt F., Zagmajster M., Fišer C. 2022. A hotspot of groundwater amphipod diversity on a crossroad of evolutionary radiations. Diversity and Distributions, 00: 1–13 Groundwater harbours an exceptional fauna and provides invaluable ecosystem services, yet is among the least explored and consequently least protected ecosystems. Successful protection of its biodiversity depends on complete species’ inventories, knowledge of species’ spatial distribution, and quantification of biodiversity patterns, as well as disentanglement of the processes that shaped biodiversity patterns. We studied the hyper-speciose amphipod genus Niphargus as a model system within a global subterranean biodiversity hotspot, the Western Balkans. We linked the biodiversity patterns with possible underlying processes and discuss the needs to include information on different origins of biodiversity into conservation approaches. We analysed biodiversity patterns of Niphargus using two biodiversity metrics, species richness and phylogenetic diversity, on a grid-based approach. To account for high cryptic diversity, we replaced nominal species with taxonomic units identified in unilocus delimitations (MOTUs). We built a time-calibrated multilocus phylogeny of 512 Niphargus MOTUs from within and outside the study area, and calculated Faith’s phylogenetic diversity, standardized effect sizes of phylogenetic diversity, and residual of phylogenetic diversity regressed onto species richness. Within the study area, we recognized 245 MOTUs, belonging to different Niphargus clades. Species richness is highest in a north-western hotspot, although some species-rich cells were detected also in the southeast. High phylogenetic diversity coincides with high species richness in the northwest, while in the southeast it is lower than expected. We have shown that species richness does not predictably correlate with phylogenetic diversity. This difference suggests that different processes have led to the formation of species-rich areas in the Western Balkans: through a combination of dispersal and speciation in the northwest, and local radiation in the southeast, respectively. This calls for caution in conservation strategies relying solely on number of species and may change the view on conservation priorities within this region. This article is licensed under a Creative Commons Attribution 4.0 International License. 52 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 53 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 54 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 55 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 56 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 57 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 58 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 59 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 60 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 61 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 62 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 63 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 64 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 65 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 2.5 HOW DID SUBTERRANEAN AMPHIPODS CROSS THE ADRIATIC SEA? PHYLOGENETIC EVIDENCE FOR DISPERSAL–VICARIANCE INTERPLAY MEDIATED BY MARINE REGRESSION–TRANSGRESSION CYCLES Delić T., Stoch F., Borko Š., Flot J.-F., Fišer C. 2020. How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for dispersal–vicariance interplay mediated by marine regression–transgression cycles. Journal of Biogeography, 47: 1875–1887 Freshwater subterranean amphipods with low dispersal abilities are known from both sides of the impermeable barrier, the Adriatic Sea. We tested the hypothesis that historical marine regression–transgression cycles shaped the distribution patterns of subterranean amphipods through repeated cycles of dispersal and vicariance against the hypothesis that subterranean amphipods colonized both sides of the Adriatic Sea independently. Our study model was genus Niphargus, a clade of freshwater subterranean amphipods (Crustacea: Amphipoda). The taxonomic structure of the studied clade was revised using unilocus species delimitation methods. The timeframe of cladogenetic events was inferred using a multi-locus time-calibrated phylogeny and compared to the main regression–transgression events in the Miocene and Pleistocene. The geographical origin of the studied clade, species range expansions and contractions, as well as vicariance events were assessed through modelling of historical biogeography. Subterranean amphipods of the genus Niphargus, found on both sides of the Adriatic Sea, form a monophylum. The reconstructions of ancestral ranges suggest that the clade emerged in the Balkan Peninsula, dispersed three times independently to the Apennine Peninsula and once back to the Balkans. Adriatic Islands were colonized multiple times, predominantly from the Balkan Peninsula. The dispersal–vicariance events correspond to historical regression–transgression cycles in Miocene and Pleistocene. Marine regression–transgression cycles apparently shaped the distribution patterns of subterranean amphipods while the alternative hypothesis received no support. The actual distribution of subterranean faunas apparently reflects old biogeographical events. 66 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 67 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 68 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 69 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 70 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 71 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 72 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 73 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 74 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 75 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 76 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 77 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 78 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 79 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3 DISCUSSION AND CONCLUSIONS 3.1 DISCUSSION We are living in a time of global biodiversity crisis, where biodiversity loss is occurring at speed and magnitude that is unprecedented in human history. It is therefore of utmost importance to understand the origins and drivers of the various facets of biodiversity, not only for the sake of knowledge, but also to be able to successfully protect them (Barnosky et al., 2011; Myers et al., 2000). Speciation, dispersal, and extinction are key processes that shape biodiversity patterns. Adaptive radiation has been recognised as a major generator of the extant diversity (Stroud and Losos, 2016). We tested this view in subterranean realm – invariant, if compared to variability of surface habitats, yet inhabited by species that exhibit a variety of forms and functions (Culver and Pipan, 2019). In this dissertation, we explore diversification and diversity patterns of the groundwater amphipod genus Niphargus on continental scales, in a global subterranean hotspot, the Dinarides, and within a cave community. The diversification patterns of Niphargus indeed are consistent with those expected under the adaptive radiation hypothesis. Sudden independent bursts of speciation and ecomorphological diversification within multiple Niphargus clades unfolded in the area of emerging karst massifs (Borko et al., 2021). Nevertheless, Niphargus is only one of many groups within the mega radiation of amphipods, where several diversification bursts resulted in more than 10,000 extant species (Copilaş-Ciocianu et al., 2020). On one hand, we will question the generality of adaptive radiations in extreme environments, and on the other hand, we will place the radiation of Niphargus in the context of a broader amphipod radiation. Next, we question ecological speciation, a key process of adaptive radiation, within a Niphargus community (Borko et al., 2019). We discuss the challenging and sometimes elusive distinction between adaptive and nonadaptive radiations, especially when analysing individual speciation events. Finally, we focus on the origins of biodiversity in subterranean biodiversity hotspot, the Dinarides. We compare different biodiversity patterns across the region and discuss the imprints and implications of the evolutionary processes behind them (Borko et al., 2022). We evaluate the contribution of speciation and dispersal to the formation of Dinaric subterranean hotspot. Several adaptive radiations have unfolded in this region. However, even though dispersal is less likely in a fragmented subterranean environment where species generally have small ranges, massive dispersal events did take place (Delić et al., 2020). While the south-eastern part of the region acted as a donor of species, the situation in the north-western part is more complex. 80 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3.1.1 Adaptive radiations in the subterranean environment Adaptive radiations are a universal phenomenon and an important global generator of biodiversity (Schluter, 2000). Simpson (1953) even claimed that adaptive radiations could explain the entire diversity of life on Earth. Although the importance of adaptive radiations is widely recognised, case studies remain limited and the processes and mechanisms behind them are poorly understood (Gillespie et al., 2020). Despite growing numbers of studies of adaptive radiations in recent years, the generality of the adaptive radiation hypothesis has never been tested in an extreme environment. The recovered patterns of speciation and ecological and morphological diversification of the genus Niphargus were consistent with the adaptive radiation theory (Borko et al., 2021). We have shown for the first time that adaptive radiation can unfold in subterranean environment, traditionally considered as ecologically harsh and highly resource-limited environment (Culver and Pipan, 2014). The genus Niphargus underwent massive subterranean speciation and ecomorphological diversification composed of nested patterns of adaptive and nonadaptive radiations (Borko et al., 2021). Although we did not specifically question ecological versus nonecological speciation, it is plausible to expect that Niphargus radiation contains both elements. Our results support the hypothesis of the generality of adaptive radiations (Schluter, 2000), which can apparently unfold even in ecologically simple and resource-limited environments such as the subterranean one, and contrary to the evolutionary dead-end hypothesis (Culver and Pipan, 2019). However, Niphargus is only one of many subterranean taxa and for now remains a rare exception of adaptive radiation in subterranean environment, rather than the rule. We suggest that further studies of subterranean radiations in various clades are needed to better understand the origins of subterranean biodiversity. Such potential candidate groups would be aquatic or terrestrial clades with known ecomorphological diversification, such as isopods of genus Monolistra (Prevorčnik et al., 2010) or beetles from the family Leiodidae (Njunjić et al., 2018). Subterranean adaptive radiation seems even less likely if we consider a degree of morphologically cryptic species. Two to three times greater molecular species diversity than expected by nominal species in crustaceans (Eme et al., 2018), and even seven time greater in collembolans (Lukić et al., 2020) suggest massive allopatric speciation within similar habitats. On the other hand, it is possible that ecological differentiation exists within seemingly similar species but has not been recognised as such; the differences could manifest in physiology (Delić et al., 2017a) or life histories (Cieslak et al., 2014). Diversification patterns of the genus Niphargus suggest a complex combination of adaptive radiations, dispersal, and nonadaptive radiations (Borko et al., 2021). Further studies are needed to assess the contribution of colonisation, dispersal, and speciation within the subterranean realm. 81 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Not only that subterranean environment can sustain adaptive radiations, it can also protect radiated clades from climatic perturbations at the surface. The extensive adaptive radiations in Europe were mostly wiped out by tectonic changes or climatic perturbations, and most modern European fauna arose through immigration (Hewitt, 2000; Neubauer et al., 2015). In contrast, we have shown that Niphargus originated in north-western Europe, dispersed and radiated in the south and east, and survived major extinction events (Borko et al., 2021). Our results suggest that more radiations may be expected and preserved in environments that are insulated from the effects of climate fluctuations but have been so far overlooked due to their apparent simplicity, such as the subterranean realm and deep soil (Von Saltzwedel et al., 2016). 3.1.2 The genus Niphargus: adaptive radiation within the amphipod radiation With more than 10,000 species, amphipods are among the most ecologically diverse and speciose crustaceans inhabiting all aquatic and semiaquatic habitats worldwide (Horton et al., 2021). Analysis of the entire order revealed a major eco-evolutionary radiation that occurred in pulses, with multiple convergent shifts in ecology (Copilaş-Ciocianu et al., 2020). Amphipods diversified in several pulses that followed the periods of delayed speciation. Although their ancestors originated already in the Permian, the first diversification was delayed until the Jurassic-Cretaceous, followed by a second pulse of speciation and ecological radiation during the Cretaceous-Paleogene (Copilaş-Ciocianu et al., 2020). The pattern of delayed burst of speciation was also observed in Niphargus (Borko et al., 2021). These pulses can be associated with environmental conditions and possible key events that triggered diversification, i.e., an ecological opportunity: a newly available abundance of unexploited ecological resources (Schluter, 2000). Although it is impossible to explicitly test the hypothesis of which key event triggered adaptive radiation (Stroud and Losos, 2016), the temporal concurrence of the onset of radiation and the event may indicate possible ecological opportunity. Amphipods are generally cold-adapted animals with low tolerance to hypoxia. We hypothesise that the first ecological opportunity for amphipods emerged after the Permo-Triassic mass extinction event, and when oxygenation of marine environments was restored (Copilaş-Ciocianu et al., 2020). From the theory of ecological opportunity, we can assume that the extinction emptied the space of predators and paleoclimatic changes made it hospitable again (Schluter, 2000). The second pulse of diversification may have been driven by vicariance after the breakup of Pangea. The last major spike of speciation and ecological diversification in the deep history of amphipods occurred during a period of high sea level, with a variety of shallow marine habitats and highly oxygenated environments. Multiple transitions into freshwaters or deep sea occurred (Copilaş-Ciocianu et al., 2020). Although plausible, our hypotheses are mostly speculative in nature. Deep-time reconstructions are 82 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 accompanied with multitude of uncertainties, especially when it comes to poorly fossilised groups such as amphipods. Without extensive fossil data and lacking a good share of molecular data from extant groups, detailed reconstructions are impossible. Within amphipods, many nested radiations likely occurred. Transition to freshwater was recognised as ecological opportunity that promoted diversification in amphipod genus Gammaraus (Hou et al., 2011). Multiple colonisations and convergent adaptations were shown for deep sea lysianassoid amphipods (Corrigan et al., 2014). Occurrence of gelatinous plankton and adaptation to symbiotic lifestyle in hyperiid amphipods resulted in one mega-radiation and several smaller radiations (Copilaş-Ciocianu et al., 2020). Multiple radiations unfolded also in ancient lakes of temperate climates. The amphipods in Lake Baikal appear to be among the largest adaptive radiations of extant amphipods, although their studies were mainly descriptive and adaptive radiation was never explicitly tested (Macdonald et al., 2005; Naumenko et al., 2017; Sherbakov, 1999). A recent study suggests that the Pontocaspian amphipods, with more than 80 species that have convergently evolved into at least four ecomorphs, may meet adaptive radiation criteria (Copilaș-Ciocianu and Sidorov, 2022). Less studied are smaller potential radiations in Lake Titicaca, Lake Ohrid, and lake Fuxian (Copilaș-Ciocianu and Sidorov, 2022; Jaume et al., 2021). Niphargus can thus be viewed as a case of nested radiation, albeit the largest one among freshwater amphipods, generating 20–25% of all freshwater and 5 % of all amphipods in the world (Horton et al., 2021; Väinölä et al., 2008). Interestingly, Niphargus radiation unfolded in a very different environment than ancient lakes, although some parallels can be drawn, such as diversity of habitats and geographic size of the area in which these radiations unfolded. Niphargus ancestors were shallow-water marine amphipods (Copilaş-Ciocianu et al., 2020) in western Europe in the early Eocene (Borko et al., 2021), with slow pace of speciation for the first 20 to 30 million years. During this time, the genus dispersed through interstitial, probably in coastal or brackish waters. We hypothesise that the genus accumulated genetic variation during this time that supported subsequent rapid diversification (Seehausen, 2015). The timing of the diversification burst in Niphargus coincides with the uplift of karst massifs in south-eastern Europe, subsequent karstification and favourable hydrogeological conditions (e.g. Miocene Dinaric lakes (de Leeuw et al., 2012)). The South-Eastern Alps, Dinarides, and Carpathians uplifted in south-eastern Europe as islands in the Parathetys. Exposure of the carbonate rocks to atmospheric processes triggered karstification and consequently the formation of new, diverse subterranean habitats (Culver and Pipan, 2019). The vast new freshwater environments, initially free of predators and competitors, likely constituted an ecological opportunity for the Niphargus ancestors. Early habitat diversification from interstitial to cave lakes and streams and to unsaturated fissure system and shallow subterranean begun, followed by within-habitat diversification in several clades in the area. We developed a plausible explanation for the ecological opportunity, however the intrinsic factors of Niphargus that promote diversification were not explored yet (but see Fišer et al., 2008a). The next interesting question would be to investigate the evolvability 83 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and possible drivers, such as hybridization and phenotypic and behavioural plasticity, of Niphargus and comparably among subclades (Stroud and Losos, 2016). Although the molecular and morphological data of Niphargus are well resolved, they are still limited. We acknowledge that we were not able to directly evaluate the contribution of extinctions to the recovered diversification patterns. However, we assume that extinctions were negligible and randomly distributed under assumption that subterranean environment acts as a refugium from major catastrophic disturbances on the surface (Borko et al., 2021). Second, many areas are still under sampled and many species have not yet been discovered. It is possible that other emerging karst areas have supported similar events, but we have not detected them – recent discoveries hint that the Zagros or Caucausus mountain ranges may be possible candidate areas (Esmaeili-Rineh et al., 2015; Rendoš et al., 2021). In exceptional settings, large adaptive radiations can be the sum of independent radiations occurring in closely related lineages. As noted above, Niphargus is likely just one of many amphipod radiations. Furthermore, this pattern of nested radiations is present also within Niphargus. The genus consists of several large monophyletic and geographically well-defined clades. All six major clades displayed an early-burst speciation, but without increased habitat diversification, that unfolded earlier in the evolutionary history of the genus. Four clades exhibited significantly high morphological disparification, namely the West Balkan, North Dinaric, Pontic, and Pannonian clades. The South Dinaric clade was not significant, but the high number of morphotypes and newly obtained data suggests adaptive radiation. Finally, the youngest Apennine clade has only few morphotypes. We hypothesise that this clade could be a predominantly nonadaptive radiation that onset after the final formation of the Apennine Peninsula 10 million years ago (Popov et al., 2004), with morphologically similar species living predominantly in one habitat type. We hypothesise that the diversification may have been driven by allopatric speciation in a highly fragmented karst environment. Since one lineage from the Apennine clade dispersed also into the Dinarides, this is consistent with nonadaptive radiation hypothesis of intermediate dispersers (Agnarsson et al., 2014). However, due to the lack of morphological and distributional data, we have not been able to reliably estimate the course of diversification of this clade (Borko et al., 2021). We found that these clade-level radiations show evidence of between and within clade convergent evolution. Although showing some degree of convergence, the radiations overall adapted to distinct sets of adaptive optima. We hypothesise that the early habitat diversification detected in the tree-wide analysis may have constrained further clade-level morphological diversification, which mostly unfolded within one or a few habitat types, such as cave lakes (West Balkan and South Dinaric clade), cave streams (North Dinaric), or interstitial groundwater (Pontic and Pannonian clade). We may expect that diversification analysis of other traits, such as gnathopods that are associated with trophic ecology, would reveal additional level of diversification within habitats (Premate et al., 2021). These within-84 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and between-habitat diversifications prompted high levels of sympatry, resulting in uniquely species-rich communities, counting up to nine Niphargus species per site (Fišer et al., 2019; Trontelj et al., 2012). Alternative constraining force could be a competition among Niphargus lineages. It is possible that radiations have evolved within limited habitat types because others were already occupied. One such example is competing cichlid genera in Lake Mweru that do not overlap in morphospace (Meier et al., 2019). We have shown that parallel and partially convergent adaptive radiations have unfolded within several Niphargus clades. Available information from the under sampled clades in Greece and Iran, or a lineage in the Iberian Peninsula morphologically described as separate genus Haploginglymus suggests that there may be more radiation events (Borko et al., 2019; Esmaeili-Rineh et al., 2015; Jurado-Rivera, Álvarez, et al., 2017). 3.1.3 Distinguishing between ecological and nonecological speciation Although overall macroevolutionary patterns of the group imply adaptive nature of radiation, the distinction between adaptive and nonadaptive processes becomes difficult on the level of individual speciation events. Not only that many clades contain elements of adaptive and nonadaptive radiations, ecological and nonecological speciation cannot be separated from each other in many cases (Rundell and Price, 2009). For example, species can first speciate in allopatry, then diverge ecologically, and only later come into sympatry (Zakšek et al., 2019). The resulting pattern of ecomorphologically distinct species living in sympatry would imply ecological speciation. At the level of population genetics, one must thoroughly analyse the evolution of lineages on a case-by-case basis to gain insight into the underlying processes. Studied individual speciation events show complex scenarios. For example, within a West Balkan clade one lineage evolved in a series of habitat fragmentations and parapatric ecological divergence (Delić et al., 2017a), while another lineage exhibited allopatric speciation due to dispersal, ecomorphological differentiation in allopatry, and only later post-speciation dispersal that resulted in sympatry (Zakšek et al., 2019). We studied a Niphargus community in a Greek cave that at a first glance fits the adaptive radiation hypothesis. We have shown that three closely related and morphologically highly dissimilar sympatric species occupy different aquatic habitats within a cave – small pores in the lake bottom, phreatic waters of freshwater lake and phreatic waters of a sulphidic lake. Phylogenetic clustering and ecological divergence indicate ecological speciation of the species triplet within one cave. Moreover, morphological adaptation to sulphidic water is a showcase of convergent evolution, that evolved at least four times in Niphargus (Borko et al., 2019). However, similarly as in studies on dytiscid beetles (Vergnon et al., 2013) we did not specifically test whether reproductive isolation evolved due to ecological differentiation and thus cannot rule out an alternative hypothesis. In the alternative scenario, a speciation 85 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and ecomorphological differentiation happened in allopatry, followed by secondary contact. This view is supported by the fact that two of the studied species have a wide distribution outside the studied cave, although the occurrences were mostly not confirmed by molecular methods. To answer this question, one would first need a reliable distribution and molecular data to infer a detailed phylogeography and reconstruct past dispersal. Inadequate sampling is a common problem in subterranean biology (Culver and Pipan, 2019). Second issue is a well-resolved molecular phylogeny, a key tool for successful disentangling of evolutionary history. Relationships in times of abrupt speciation often remain unclear, as is the case in Niphargus. While the overall pattern inferred from the available data is unlikely to be challenged, higher resolution of individual splitting events would overcome the gap between macro- and microevolutionary processes in Niphargus. The use of next-generation sequencing technologies, such as ddRADseq (Parker et al., 2022), is therefore a plausible next step in Niphargus studies. 3.1.4 Processes that have shaped a subterranean biodiversity hotspot The Dinarides are a global subterranean biodiversity hotspot, a region with the highest diversity of groundwater amphipods and subterranean fauna in general (Zagmajster et al., 2014). Most of the species found here are endemic to the region. 123 nominal species of Niphargus have been recorded in the Dinarides (Horton et al., 2021). We were able to obtain molecular data for 79% of them. However, the exceptionally high number of new, previously unrecognised Molecular Operational Taxonomic Units (MOTUs) suggests that the number of species is still underestimated and in reality 2 to 2.6 times higher than acknowledged (Borko et al., 2022). A somewhat surprising result of the massive sequencing was 148 new MOTUs that do not correspond to any of nominal species and were not recognised in previous studies. Despite the lack of reliable ecological and morphometric data for most of this vast, previously hidden diversity, we were able to disentangle main processes that have shaped the Niphargus’ diversity in the region. The species found in this area belong to several clades. The central and south-eastern Dinarides are mainly occupied by the West Balkan, South Dinaric, North Dinaric and Apennine clade. The Aegean clade is only marginally present in southeast and a clade of broadly distributed shallow subterranean species is present in the eastern alluvial planes ( N. sphagnicolus lineage), but they do not spatially overlap with Dinaric clades. In the north-western Dinarides, two of the Dinaric clades occur, the West Balkan and the North Dinaric clade. The first is distributed only within the inner boundary of the Dinarides, while the second penetrates into the South-Eastern Alps. Additionally, also species of the Pannonian and Pontic clades are present in this region. Another four lineages with five or more species occur in the north-western Dinarides: shallow subterranean or interstitial species from lineages N. sphagnicolus, N. julius – N. kenki and N. spinulifemur, and predominantly stream 86 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 species from lineage N. stygius s. str. Lastly, 18 species from various non-Dinaric clades or with unclear phylogenetic position occur here (Borko et al., 2022). The distribution and phylogenetic relations of species along the Dinarides suggest that speciation and dispersal contributed differently in different parts of the region, with predominant local diversification within the south-eastern Dinarides and a combination of dispersal and local diversification in the north-western Dinarides and surrounding regions. However, more work will be needed to reliably evaluate the contribution of adaptive and nonadaptive speciation events within the region, mostly due to lack of ecomorphological data for newly discovered MOTUs. The second insight is that Dinaric lineages importantly contributed to the niphargid fauna of neighbouring regions. In the south-eastern Dinarides local fauna derives predominantly from three Dinaric clades, whereas other clades are lacking and occupy only boundary regions. The ancestors of the West Balkan, South Dinaric and North Dinaric clades have diversified entirely within the region (Borko et al., 2021), to a high number of 143 MOTUs, which is probably the largest Niphargus radiation within areas of similar size. The overall diversification patterns of three clades are consistent with the adaptive radiation hypothesis (Borko et al. 2021), but more data is needed be able to reliably reconstruct the diversification course of each clade. Three Dinaric clades were an important source of niphargid fauna of the Apennine Peninsula. Dispersal from the Dinarides to the Apennine Peninsula took place along land bridges that emerged during marine regressions: during the connection between the Dinaric Karst and Apulia 10–15 million years ago, during the Messinian salinity crisis, and most recent during the Pleistocene glaciations (Delić et al., 2020). Conversely, only one, the ancestral lineage of N. hebereri, dispersed back into the central Dinarides during the Messinian salinity crisis and diversified in the coastal area (Delić et al., 2020). No other immigrant lineage is present in the central Dinarides, despite the several dispersal routes to Dinarides. One possible reason is a priority effect – an impact that a first arrival species can have on the development of a community. The Apulia is younger than the Dinarides and was colonised from the Dinarides. Therefore, Apulia is unlikely to be a donor of older lineages. Even if younger lineages dispersed into Dinarides, the competition with ecologically similar species prevented their speciation. The phylogeographical history of the Apennine clade is less clear. The Apennine clade is nested within major Niphargus radiation, but its relation to other Dinaric clades is not resolved. The basal split of the clade divides it into smaller south Dinaric subclade and larger Apennine subclade. Given the paleogeographic data it seems plausible that ancestors of Apennine subclade migrated into Apennine Peninsula and massively diversified there, now occupying the entire peninsula. If the ancestor was indeed from the Dinarides, the question arise why south Dinaric subclade is much less speciose and narrowly distributed, compared 87 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 to other equally old Dinaric clades. More molecular and distributional data is needed to answer these questions. Dispersal from the Dinarides also occurred on the other side of the mountain ridge, into the Pannonian alluvial plains and toward the Carpathian foothills, as in the case of N. mirocensis from the West Balkan clade (Petkovič et al., 2015). We expect that comprehensive sampling could reveal more such cases of dispersal from the Dinarides towards the east, and possibly also in completely understudied south (Albania). To sum up, the south-eastern Dinarides acted as a donor region from where species dispersed and occupied surrounding regions that later emerged from the sea. The existing Dinaric clades, which occupied all subterranean aquatic habitats, probably blocked the immigration of foreign lineages into this area. Consequently, the south-eastern Dinarides have relatively low phylogenetic diversity, despite their high species richness. The evolutionary history of Niphargus was different in the north-western Dinarides, especially at the junction with the South-Eastern Alps and the Pannonian lowlands. Here, on the intersection of karst areas and alluvial plains, the Niphargus species richness peaks and representatives of most of Niphargus phylogenetic lineages occur. Unrelated clades that diverged from each other already in the Eocene spatially overlap (Borko et al., 2022). The area was occasionally submerged (Kováč et al., 2018). Aquatic connections between the Adriatic basin and the Paratethys Sea probably opened migration routes, resulting in congregation of species from several phylogenetically and geographically independent lineages. The established sympatries lead to high phylogenetic diversity and species richness, the highest in the region and overall. This north-western hotspot is thus the result of a combination of dispersal from outside the Dinarides and diversification within the region. The north-western Dinarides acted as recipient area for various unrelated Niphargus lineages, that consequently diversified here, mostly within one habitat type. Such case are lineages from Pannonian and Pontic clades, that migrated in the north-western Dinarides. While Pannonian species only marginally penetrate into the Dinarides and are more common in the border regions of the South-Eastern Alps and the Pannonian lowlands, a subclade of 10 species from the Pontic clade has apparently diversified in the north-western Dinarides, in a specific habitat (fissure system) (Borko et al., 2022). We hypothesise that they prevented diversification of species from Dinaric clades in this particular habitat, where only few species occur (a case of competition driven constrain of adaptive radiation (Meier et al., 2019)). This would be an interesting study of colliding adaptive radiations and among clade competition. As in the south-east, we noted some dispersal from the north-western Dinarides into neighbouring regions. A N. rhenorhodanensis lineage of six species in western Italy according to latest phylogenetic reconstructions belongs to the West Balkan clade. While 88 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 500 km away from closest relatives, they occur within relatively small area with radius around 50 km (Borko et al., 2022). More data is needed to confirm the lineage’s phylogenetic position and to get insight into its phylogeography. Given the contrasting differences between the patterns of species richness and phylogenetic diversity of Niphargus in the Dinarides and in general small distributional ranges (Bregović et al., 2019; Trontelj et al., 2009), speciation can be considered a universal generator of species richness patterns along the entire Dinarides (Borko et al., 2022). Nevertheless, dispersal played an important role in the formation of this hotspot. The dispersal acted mostly in the north-west, while in south-east the Dinarides were the donor area for surrounding regions (Borko et al., 2022). We acknowledge that the aforementioned inability to evaluate extinctions presents a potential source of misinterpretation of phylogenetic diversity patterns. However, the most recent driver of extinctions in Europe were Pleistocene glaciations, and the Balkan Peninsula was recognised as a southern refugium (Hewitt, 2000). Additionally, Alpine glaciers in the northwest of the study area prompted Niphargus speciation (Delić et al., 2022). Therefore, we consider dispersal as a more likely cause of high phylogenetic diversity in northwest. 89 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 3.2 CONCLUSIONS We have comprehensively analysed the patterns of speciation and ecomorphological diversification of the genus Niphargus at the continental scale, within the subterranean hotspot, and within a community. This is the first in-depth analysis of Niphargus diversification and the first study of adaptive radiation in the subterranean realm. The conclusions from our work can be summarised as follows: 1. Amphipods evolved in several diversification pulses that correspond to various ecological opportunities. A pattern of nested radiations is present within amphipods, Niphargus being one of them. 2. Tempo and mode of Niphargus speciation and ecomorphological diversification are consistent with the adaptive radiation hypothesis. Thus, adaptive radiation is indeed a universal phenomenon that also occurs in subterranean environment. 3. Several independent radiations unfolded within Niphargus. At least five Niphargus clades adaptively radiated, and one clade shows evidence of nonadaptive radiation. Although showing some degree of convergence, the clades adapted to distinct sets of adaptive optima. 4. The timing of increased diversification corroborate with the uplift of karst massifs in South-Eastern Europe and the subsequent formation of new subterranean habitats, representing a plausible ecological opportunity. 5. Although general patterns support the adaptive radiation hypothesis, explicit tests for ecological speciation are lacking. A community in a Greek cave is a possible case of ecological speciation of Niphargus, but more detailed analyses are needed. 6. The diversity of Niphargus within the Dinarides is 2 to 2.6 times higher than the number of nominal species indicates. Speciation and dispersal contributed differently in different parts of the Dinarides. 7. The diversity of the south-eastern Dinarides emerged mostly through local speciation. Southeastern Dinarides are a donor area, with repeated dispersal from the region, while dispersal in the region is limited. 8. In the north-western Dinarides, the high diversity is due to a combination of dispersal and local diversification. 9. More studies are needed for understanding the contribution of adaptive and nonadaptive speciation events within the Dinarides. 90 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4 SUMMARY 4.1 SUMMARY Understanding the origins of the various facies of biodiversity is a primary goal of evolutionary biology and macroecology. The current patterns of biodiversity have emerged through an interplay of speciation, dispersal, and extinction. Biodiversity hotspots form through high diversification, low extinction, high immigration, or a combination of these processes. It has been proposed that most of the extant diversity in many major clades around the globe emerged through adaptive radiations: a sudden burst of speciation accompanied by extensive ecological diversification. In adaptive radiation, ecology-based divergent selection among populations leads to cessation of gene flow and, consequently, ecological speciation. The classic definition of adaptive radiation includes four criteria: common ancestry of species, phenotype-environment correlation, trait utility, and rapid speciation. An external prerequisite for adaptive radiation to unfold is an ecological opportunity, “a wealth of unexploited ecological resources,” such as the extinction of a dominant group, the emergence of new resources, or the colonisation of a new area. Large adaptive radiations within discrete, replicated habitats, such as on islands or in lakes, often comprise of multiple parallel radiations that result in similar suites of ecomorphs. Famous examples of adaptive radiation include anole lizards in the Greater Antilles, Darwin’s finches in Galapagos, or cichlids in African Great Lakes. Less attention has been paid to continental adaptive radiations, or radiations of invertebrates. A counterpoint to adaptive radiation, nonadaptive radiation is a lineage diversification with minimal ecological differentiation that results in related and ecologically similar species. Most often, the speciation would be allopatric or parapatric, where gene flow reduces due to geographic isolation, sometimes fortified by intensified genetic drift in smaller populations. The process is affected by the similarity of environments of the diverging populations. Nonadaptive radiations often involve organisms with limited dispersal abilities or those living in highly subdivided environments with similar habitats and conditions on either side of barriers. Due to the complete absence of light and thus photosynthetic production, subterranean ecosystems are among the most resource-limited environments on Earth. The subterranean fauna has long been considered an evolutionary dead end. Studies over the past two decades have shown that evolutionary processes continue after the colonisation of the subterranean environment, including speciation within it. A considerable number of subterranean species derived from subterranean ancestors. Some species retained similar morphology due to similar environmental conditions, and many species have further diversified morphologically and ecologically. In subterranean amphipod genus Niphargus, a highly diversified clades on one hand, and eco-morphologically similar or even cryptic clades on 91 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 the other hand, suggest that this clade evolved through a combination of nonadaptive and adaptive processes. To date, no comprehensive analysis of speciation and ecomorphological diversification of the exclusively subterranean group has been made. The aim of this dissertation was to comprehensively analyse the speciation and ecomorphological diversification of the genus Niphargus at the continental scale, within subterranean biodiversity hotspot, and within a community. The work was divided into five chapters. First, we analysed the diversification patterns of the order Amphipoda and provided the evidence for monophyly of Niphargus. Second, we explored whether the tempo and mode of Niphargus diversification patterns corroborate with adaptive radiation hypothesis. We tested whether shifts in speciation and diversification are spatially and temporally consistent with geological and paleogeographic events that may have grounded the ecological opportunity. Third, with aim to analyse a possible case of ecological speciation within Niphargus, we studied a community within a Melissotrypa Cave in Greece. Fourth, we aimed to investigate the extent to which speciation and dispersal from neighbouring regions contributed to the overall biodiversity of Niphargus in the Dinarides. Finally, we evaluated the Dinarides as a donor region of species, especially for the neighbouring Apennine Peninsula. Amphipods are among the most speciose crustacean orders. Despite their global distribution and key role in aquatic environments, little is known about their phylogenetic relationships and diversification patterns. In the first chapter, we inferred a large-scale, time-calibrated multilocus phylogeny and reconstructed evolutionary patterns for major ecological traits. We showed that amphipods originate in the Late Palaeozoic, but their speciation and ecological diversification exploded only in the Late Mesozoic, after a protracted stasis in marine littoral habitats. Multiple independent post-Jurassic radiations occurred in deep-sea, freshwater, terrestrial, pelagic, and symbiotic environments. We identified deep-sea faunal extinctions, significant climatic cooling, tectonic reconfiguration, continental flooding, and increased oceanic oxygenation as potential ecological opportunities. Niphargus is a monophyletic lineage, sister to another subterranean clade comprised of Pseudoniphargus and Microniphargus. Multiple potential adaptive radiations are nested within amphipods. One of these is the subterranean genus Niphargus, which accounts for 20-25 % of all amphipod species. In the second chapter, we inferred calibrated multilocus phylogeny of Niphargus and modelled the lineage diversification and the evolution of morphological and ecological traits. We have shown that the diversification of the genus Niphargus is a case of adaptive radiation, consisting of multiple subordinate adaptive radiations. Their spatio-temporal origin coincides with the uplift of carbonate massifs in south-eastern Europe 15 million years ago. The emerging subterranean environments likely provided an unoccupied, predator-free space that represented an ecological opportunity, a key trigger for adaptive radiation. Although dark and food-deprived, subterranean environment supported adaptive radiation 92 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 and preserved it through major climatic perturbances on the surface. Our results support the hypothesis of the generality of adaptive radiations that can indeed unfold even in ecologically simple and resource-limited environments such as the subterranean. However, Niphargus is only one of many subterranean taxa and for now remains a rare exception of adaptive radiation in the subterranean realm. We suggest that further studies of subterranean radiations in various clades are needed to better understand the origins of subterranean biodiversity. Although the macroevolutionary patterns suggest adaptive radiation in Niphargus, distinguishing between ecological and nonecological speciation can be challenging. In the third chapter, we characterized the amphipod community of Melissotrypa Cave (Greece) taxonomically, ecologically, and phylogenetically. We found three Niphargus species and one Bogidiella sp. The three Niphargus species form a well-supported monophylum, but differ in their ecology and morphology: N. jovanovici is a small and slender species inhabiting small freshwater voids, N. lindbergi is a large and stout species living in freshwater lakes, whereas N. gammariformis sp. nov. is a small and stout species found predominantly in a sulphidic lake. The first two are broadly distributed, although their occurrences are mostly not supported by molecular data. Third species is a single-site that shares convergent morphology with unrelated species from different sulphidic caves. Available evidence suggests that diversification was likely driven by ecological differentiation. However, more data is needed to rule out the alternative hypothesis that species evolved in allopatry and only later came into secondary contact. To answer this question, one would need reliable distributional and molecular data to infer detailed phylogeography and reconstruct past dispersal. In the fourth chapter we focused on processes that shaped a subterranean biodiversity hotspot. We studied the biodiversity patterns of Niphargus in the Dinarides, using two biodiversity metrics, species richness and phylogenetic diversity, with a grid-based approach. To account for high cryptic diversity, we replaced nominal species with Molecular Operational Taxonomic Units (MOTUs) identified in unilocus delimitations. We built a time-calibrated multilocus phylogeny of 512 Niphargus MOTUs from within and outside the study area, and calculated Faith’s phylogenetic diversity, standardized effect sizes of phylogenetic diversity, and residual of phylogenetic diversity regressed on species richness. Within the study area, we recognized 245 MOTUs belonging to different Niphargus clades. Species richness is highest in a north-western hotspot, although some species-rich cells were also detected in the southeast. High phylogenetic diversity coincides with high species richness in the northwest, while in the southeast it is lower than expected. This difference and the detailed analysis of clades distribution suggest that the north-west and the south-east hotspots emerged through different processes: by a combination of dispersal and speciation in the northwest, and by local radiation in the southeast, respectively. The south-eastern Dinarides can therefore be considered as a donor area of species for the surrounding regions, while the situation in the north-west, at the junction with the South-Eastern Alps and the 93 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Pannonian lowlands, is more complex, with many immigrant species from various non-Dinaric clades, some of which diversified within specific habitats. In the last chapter, we tested whether the distribution patterns support the hypothesis of repeated dispersal and vicariance between the Dinarides and the Apennine Peninsula caused by marine regression–transgression cycles. We collected data for most of Niphargus lineages, focusing on species living on both sides of the Adriatic Sea. We applied unilocus delimitations and calculated a time-calibrated multilocus phylogeny, which we used for modelling dispersal, vicariance, extinction, cladogenesis and ancestral ranges. We compared the time frame of events with the main regression–transgression events in the Miocene and Pleistocene. A clade containing South Dinaric and West Balkan clades is distributed on both sides of the Adriatic Sea. The reconstructions of the ancestral ranges suggest that the clade originated in the Dinarides, dispersed three times independently to the Apennine Peninsula and once back to the Dinarides. Adriatic Islands were colonized multiple times, mostly from the Dinarides. The dispersal–vicariance events correspond to historical regression– transgression cycles in the Miocene and Pleistocene, namely a land bridge between the Dinarides and the newly emerged Apulia, a regression during the Messinian salinity crisis, and most recent during the Pleistocene glaciations. The Dinarides indeed acted as a donor region for surrounding land masses that later emerged from the sea, while dispersal back into the area was limited, probably due to the priority effect. We comprehensively analysed the patterns of speciation and ecomorphological diversification of the genus Niphargus at the continental scale, within the subterranean hotspot, and within a community. This is the first in-depth analysis of amphipod and Niphargus diversification and the first study of adaptive radiation in the subterranean realm overall. We were unable to distinguish between adaptive and nonadaptive radiations in the Dinarides due to unexpectedly high number of new MOTUs without reliable ecological and morphological data. Nevertheless, we provided a solid phylogenetic and methodological framework that will enable to further explore processes that shaped the evolutionary history of Niphargus, as well as other subterranean groups. 94 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 4.2 POVZETEK Biotsko pestrost lahko merimo na več načinov: kot vrstno, fenotipsko ali filogenetsko pestrost (Devictor in sod., 2010). Razumevanje izvora biotske pestrosti je prvotni cilj evolucijske biologije in makroekologije. Današnji vzorci biotske pestrosti so nastali skozi preplet speciacije, disperzije in izumrtja (Davies in sod., 2007). Glavni mehanizem nastanka biodiverzitete je speciacija, t. j. evolucija novih vrst iz skupnega prednika. Lahko je ekološka ali neekološka. Pri ekološki speciaciji se reproduktivna izolacija razvije s prilagoditvijo na različna okolja ali okoljske niše. Pri neekološki speciaciji pa se razvije skozi fiksacijo različnih koristnih mutacij v populacijah pod podobnim selekcijskim pritiskom (Rundell in Price, 2009; Schluter, 2009). Speciacijo običajno spremlja fenotipska diverzifikacija, ki je lahko naključna, ali vzročno povezana s speciacijo. Pri ekološki speciaciji je fenotipska diverzifikacija povezana z izkoriščanjem različnih ekoloških niš. Morfološka pestrost v tem primeru sledi ekološki pestrosti (Michaud in sod., 2018; Schluter, 2000). Disperzija je proces premikanja s katerim vrste spreminjajo svoje areale. Vpliva na pretok genov, populacijsko dinamiko, kompeticijo in razširjenost vrste. Čeprav je pomembna, jo dostikrat slabo razumemo (Duarte in Mali, 2018). Razumevanje disperzije olajša filogenetska pestrost, mera pestrosti, ki vključuje sorodstvene odnose med vrstami (Faith, 1992). Vrstno pestra območja z nizko filogenetsko pestrostjo lahko nastanejo zaradi hitre nedavne speciacije in majhne disperzije. Nasprotno pa visoka disperzija ali dolga obdobja enakomerne speciacije vodijo v visoko vrstno in filogenetsko pestrost. Izumiranje povečuje filogenetsko pestrost, a ga je dostikrat težko rekonstruirati (Davies in sod., 2007). Vroče točke biotske pestrosti nastanejo zaradi povečane speciacije, majhne stopnje izumiranja, obsežnega priseljevanja ali kombinacijo naštetega (Wiens in Donoghue, 2004). Večina današnje biotske pestrosti naj bi nastala v povečanih pulzih speciacije, t. i. evolucijskih radiacijah (Stroud in Losos, 2016). To tezo smo testirali v nepričakovanem okolju: ekološko preprostem in s hranili osiromašenem podzemlju. Speciacija pri različnih skupinah poteka različno hitro. Lahko je počasna ali hitra, zvezna ali nenadna. Tempo speciacije se lahko skozi čas spreminja. Hitro vznikanje novih vrst iz skupnega prednika imenujemo evolucijska radiacija. Če pri tem pride do diferenciacije ekoloških niš, govorimo o adaptivni radiaciji. Pri neadaptivni radiaciji do te diferenciacije ne pride. Kladi lahko vsebujejo elemente obeh procesov, adaptivne in neadaptivne diferenciacije. Razmejitev je nejasna, ekološko diferenciacijo je tudi pogosto težko opredeliti (Czekanski-Moir in Rundell, 2019; Gittenberger, 1991; Rundell in Price, 2009; Schluter, 2009). Adaptivna radiacija je hitro vznikanje novih ekološko diferenciranih vrst iz skupnega prednika (Rundell in Price, 2009; Schluter, 2000). Klasična definicija adaptivne radiacije 95 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 vključuje štiri pogoje: skupnega prednika, povezavo med fenotipom in okoljem, funkcionalnost znaka in hitro speciacijo (Schluter, 2009). Model z ekoevolucijskimi procesi, ki delujejo na lokalni ravni (lokalna adaptacija, medvrstne interakcije), pojasnjuje makroevolucijske procese in globalne vzorce biotske pestrosti. Ekološko pogojena divergentna selekcija med populacijami vodi v zmanjšan genski pretok in posledično ekološko speciacijo. Hitrost ekološke divergence in speciacije je odvisna od moči divergentne selekcije. Včasih je težko določiti sosledje procesov, ko, na primer, pride do neekološke speciacije v alopatriji, ki ji sledi ekološka divergenca in sekundaren kontakt med novonastalima vrstama (Losos in Mahler, 2010; Schluter, 2009; Stroud in Losos, 2016). Adaptivna radiacija je odvisna od notranjih in zunanjih dejavnikov. Notranji dejavniki klada so na primer spolna selekcija, hibridizacija in razvojna plastičnost. Zunanji dejavnik, oziroma predpogoj za zagon adaptivne radiacije pa je ekološka priložnost, t. j. neizkoriščen ekološki vir. Primeri ekoloških priložnosti so kolonizacija novega območja, pojav novega vira, izumrtje ekološko dominantne skupine, ali evolucija t. i. ključne inovacije (ang. key innovation), ki omogoča novo interakcijo z okoljem (Losos in Mahler, 2010; Schluter, 2009; Stroud in Losos, 2016). Prve študije adaptivnih radiacij so se osredotočale na diskretne, ponavljajoče se habitate, kjer je primere relativno enostavno prostorsko in časovno vrednotiti. Klasični primeri adaptivne radiacije so ostrižniki v velikih afriških jezerih, ali kuščarji anoli in Darwinovi ščinkavci na otokih Velikih Antilov oziroma na Galapagosu (Grant in Grant, 2008; Losos, 2009; Seehausen, 2006). V takšnem okolju je omejeno priseljevanje in začetek diverzifikacije sovpada s poselitvijo. Prav tako je lažje definirati ekološke niše (Losos, 2009; Seehausen, 2006). Manj pozornosti je bilo namenjeno kontinentalnim radiacijam, ali radiacijam nevretenčarjev, med katerimi najdemo študije sulaveških jezerskih kozic, galapaških polžev ali havajskih pajkov (Blom in sod., 2016; Gillespie, 2004; Gillespie in sod., 2020; Von Rintelen in sod., 2010). Velike adaptivne radiacije, ki so se odvile v diskretnih in ponavljajočih se ekosistemih pogosto sestojijo iz več vzporednih neodvisnih radiacij, skozi katere se oblikujejo podobni seti ekomorfov. Ekomorfi so ekološko in vedenjsko podobne vrste, ki živijo v podobnih habitatih, a niso nujno sorodne (Mahler in sod., 2013). Primeri vzporednih neodvisnih radiacij so že omenjeni anoli in afriški ostrižniki, pri katerih so se v več kladih neodvisno razvili podobni habitatni specialisti, ki so si morfološko podobni (Elmer in sod., 2014; Mahler in sod., 2013). Te vzporedne radiacije niso nujno identične replike. Radiacija anolov na primer sestoji iz fenotipsko konvergentnih vrst, pa tudi unikatnih vrst, s samosvojimi ekomorfološkimi značilnostmi (Mahler in sod., 2013). Kadar hitro vznikanje vrst ne poteka sočasno z ekomorfološko diferenciacijo, govorimo o neadaptivni radiaciji. Speciacija poteka v ekološko podobnih okoljih. Večinoma je takšna speciacija alopatrična ali parapatrična, torej do omejenega genskega pretoka pride zaradi 96 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 geografske izolacije (Rundell in Price, 2009). Učinek se še poveča v majhnih populacijah, kjer je prisoten genetski drs (Nürk in sod., 2020). Nastajajoče vrste so lahko tudi simparične, na primer v primeru spolne selekcije (Rundell in Price, 2009). Neadaptivna radiacija je bolj pogosta pri organizmih z omejeno sposobnostjo razširjanja, ali pa v močno razčlenjenih okoljih, kjer so v posamičnih enotah podobni okoljski pogoji (Czekanski-Moir in Rundell, 2019). Na večji prostorski skali verjetno najhitreje speciirajo organizmi s srednjimi sposobnostmi razširjanja, saj še vedno lahko kolonizirajo nova območja, a nato ne zmorejo vzdrževati genskega pretoka (Agnarsson in sod., 2014). Drugi možni mehanizem neadaptivne radiacije je konservativizem filogenetskih niš, to je tendenca vrst, da ohranjajo predniške ekološke lastnosti. V nestabilnih okoljih bi tak mehanizem vodil v vikariantno izolacijo (Kozak in sod., 2006). Na splošno so definicije neadaptivnih radiacij še bolj ohlapne in raznolike od definicij adaptivnih radiacij, verjetno tudi zaradi težavnosti opredelitve (odsotnosti) ekološke diferenciacije. Neadaptivna radiacija je bila leta 1872 opredeljena na primeru Havajskih kopenskih polžev (Gulick, 1872). Spolno selekcijo kot gonilo radiacije so potrdili pri več rodovih kačjih pastirjev in pri električnih ribah (Arnegard in sod., 2010; Wellenreuther in Sánchez-Guillén, 2016). Kriptična speciacija postranic v Bajkalskem jezeru, v kombinaciji z že omenjeno visoko fenotipsko pestrostjo nekaterih skupin, pa nakazuje na kombinacijo adaptivnih in neadaptivnih dogodkov (Schön in Martens, 2004). Podoben primer najdemo v podzemlju, kjer znotraj rodu Niphargus najdemo tako kriptične klade, kot močno morfološko raznolike klade (Delić in sod., 2017a; Delić in sod., 2017b; Trontelj in sod., 2012). Do danes še nobena študija ni testirala adaptivne radiacije v podzemlju. Čeprav je ekološko zahtevno, je to okolje hkrati geografsko zelo razdrobljeno in ekološko pestro, torej dopušča razvoj adaptivnih in neadaptivnih radiacij. Naravnih virov hrane v podzemlju je malo, v veliki večini so zunanjega izvora. Le malo vrst je uspešno koloniziralo podzemlje, zato je dolgo veljalo, da so podzemne vrste evolucijska slepa veja, obsojene na izumrtje še preden uspejo diverzificirati (Barr in Holsinger, 1985; Culver in Pipan, 2019; Poulson in White, 1969). A študije zadnjih desetletij kažejo, da se evolucijski procesi nadaljujejo tudi v podzemlju. Mnogo vrst se je razvilo iz podzemeljskega prednika, prek neekološke in ekološke speciacije. Nekatere vrste so ohranile podobno morfologijo, lahko so celo morfološko nerazpoznavne (Esposito in sod., 2015; Faille in sod., 2013; Fišer in sod., 2015). Druge so ekomorfološko diverzificirale, v procesu specializacije na različne podzemne habitate in trofične niše. Tak primer so ditiscidni hrošči iz avstralskih vodonosnikov, kjer so se večkratno razvili dvojčki ali trojčki sestrskih, a morfološko zelo različnih vrst, domnevno prilagojenih različnim ekološkim nišam (Leijs in sod., 2012; Vergnon in sod., 2013). Do podobnega sklepa so prišli v študijah jamskih pajkov (Mammola in sod., 2018) in slepih postranic Niphargus (Delić in sod., 2016; Trontelj in sod., 2012). 97 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Objavljeni podatki nakazujejo, da evolucijski procesi v podzemlju potekajo podobno kot na površju. Še več, podzemlje ustreza zgoraj naštetim lastnostim okolij, ki promovirajo radiacije. Nudi kompleksno strukturo raznolikih in zelo razdrobljenih habitatov, v katerih bi se linije lahko ekološko diferencirale in prostorsko ločevale. A celovita makro analiza speciacije in diverzifikacije v podzemlju še ni bila narejena. Namen pričujoče naloge je zapolniti to vrzel. Kot modelni organizem za analizo adaptivne radiacije v podzemlju smo izbrali rod slepih postranic, Niphargus, največji rod sladkovodnih postranic na svetu (Väinölä in sod., 2008). Z več kot 420 opisanimi in številnimi še neopisanimi vrstami ima ta izjemno pestra skupina pomembno vlogo v vodnih podzemnih habitatih zahodne Palearktike in pomembno prispeva k njihovi biotski pestrosti (Fišer, 2019; Horton in sod., 2021; Zagmajster in sod., 2014). Slepe postranice so se najverjetneje razvile in podzemnega prednika in v celoti speciirale in ekomorfološko diverzificirale v podzemlju (Fišer in sod., 2008b). Dandanes jih najdemo v praktično vseh podzemnih vodnih habitatih, vse od površja do izjemnih globin. Morfološke značilnosti vrst lahko povežemo z lastnostmi habitatov v katerih živijo, kar kaže na morebitno adaptacijo znotraj podzemlja (Delić in sod., 2016; Trontelj in sod., 2012). Niphargus združuje vse ključne elemente adaptivnih radiacij: izvor iz skupnega prednika, veliko število vrst in ekomorfološko diferenciacijo. Kot tak je odličen model za testiranje hipoteze podzemne adaptivne radiacije. Namen disertacije je bil celovito analizirati vzorce speciacije in ekomorfološke diverzifikacije rodu Niphargus na kontinentalnem nivoju, znotraj vroče točke biotske pestrosti in znotraj ene združbe. Disertacijo smo razdelili v pet sklopov. V prvem sklopu smo preučili filogenetsko zgodovino redu postranic in pokazali, da obdobja povečane diverzifikacije sovpadajo s potencialnimi ekološkimi priložnostmi (Copilaş-Ciocianu in sod., 2020). V drugem sklopu smo ovrednotili tempo in hitrost diverzifikacije rodu Niphargus in preverili, ali ustrezajo pričakovanjem modela adaptivne radiacije, ter če tudi te dogodke lahko pojasnimo z ekološko priložnostjo. Hipotetično ekološko priložnost bi lahko predstavljal dvig karbonatnih gorstev v jugovzhodni Evropi, ki je ustvaril množico prostih, še nenaseljenih podzemnih habitatov. Testirali smo dve hipotezi: evolucijsko zgodovino rodu sestavljajo adaptivni in neadaptivni dogodki; in da bodo večkratne neodvisne adaptivne radiacije na območjih dvigajočih se gorstev ugnezdene znotraj filogenije rodu (Borko in sod., 2021). V tretjem sklopu smo preučili potencialni primer ekološke speciacije znotraj rodu: združbo jame Melissotrypa, v kateri živijo tri vrste slepih postranic, ki zasedajo različne habitate (Borko in sod., 2019). V četrtem sklopu smo preučevali pestrost slepih postranic znotraj vroče točke podzemeljske biotske pestrosti – Dinaridov. Vrstno bogate regije lahko nastanejo z lokalno speciacijo ali z doseljevanjem iz okoliških regij (Rosindell in Phillimore, 2011). Namen tega sklopa je bil analizirati doprinos speciacije, bodisi adaptivne ali neadaptivne, in disperzije k pestrosti slepih postranic na Dinaridih. Izredno visoka pestrost in morfološka raznolikost vrst, ki živijo na Dinarskem krasu kaže na kombinacijo procesov in raznolik prispevek procesov znotraj regije (Borko in sod., 2022). 98 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 V petem sklopu smo analizirali disperzijo med Dinaridi in Apeninskim polotokom (Delić in sod., 2020). Postranice so z več kot 10.000 vrstami, ki živijo v praktično vseh vodnih habitatih, med najbolj pestrimi redovi rakov (Arfianti in sod., 2018; Horton in sod., 2021). Kljub njihovi številčnosti, globalni razširjenosti in pomembni vlogi v vodnih ekosistemih, filogenetski odnosi med njimi do zdaj niso bili razrešeni. Temeljili so predvsem na morfologiji, hipoteze o časovnem poteku diverzifikacije pa so si močno nasprotovale (Lowry in Myers, 2017). Zato smo izračunali obsežno časovno kalibrirano molekularno filogenetsko drevo in rekonstruirali potek speciacije in ekološke diverzifikacije. Zbrali smo molekularne podatke rodov postranic na način, da bi zajeli čim več filogenetske in ekološke pestrosti skupine. Kalibrirano filogenetsko drevo smo izračunali s štirimi informativnimi genskimi markerji: mitohondrijsko citokrom oksidazo (COI), delom gena 28S rRNK, delom 18S rRNK in histonom (H3). Filogenetske odnose smo izračunali z različnimi metodami: Bayesovim pristopom (Aberer in sod., 2014; Drummond in sod., 2012), metodo največjega verjetja (Stamatakis, 2014; Trifinopoulos in sod., 2016), in metodo največje varčnosti. Za kalibracijo smo uporabili pet kalibracijskih točk, ki temeljijo na fosilnih najdbah (Copilaş-Ciocianu in sod., 2019). Iz literature smo zbrali ekološke podatke o habitatu (morski/sladkovodni/kopenski, bentoški/pelaški, načinu življenja (simbiotski/prostoživeči), globini (5 diskretnih kategorij) in temperaturni preferenci (hladnoljubni/toploljubni) za vsak rod. Speciacijo skozi čas smo izračunali z metodo drsečega okna (Meredith in sod., 2011). Rekonstruirali smo predniška stanja za vseh pet ekoloških znakov z metodo največjega verjetja in Bayesovim pristopom (Revell, 2012) in rekonstruirali hitrost ekološke diverzifikacije. Rekonstruirane filogenije so bile dobro podprte in so v veliki meri sovpadale. Čeprav skupina obstaja že od Perma, je do največje diverzifikacije prišlo kasneje in sicer v več pulzih: sredi jure, na prehodu v kredo, sredi krede in na prehodu v paleogen. Z izjemo prehoda na kopno so se vse spremembe habitata ponovile večkrat neodvisno in relativno pozno v evolucijski zgodovini redu (Copilaş-Ciocianu in sod., 2020). Postranice so v glavnem hladnoljubne živali z nizko toleranco na hipoksijo. Prva ekološka priložnost bi lahko sledila permsko-triasnemu množičnemu izumrtju, po tem ko so oceani zopet postali oksigenirani. Iz teorije adaptivne radiacije lahko sklepamo, da je izumrtje izpraznilo prostor, ki je po paleoklimatskih spremembah postal prosto dostopno okolje za postranice. Drugi izbruh speciacije je najverjetneje sledil razpadu Pangeje, speciacija bi v bila tem primeru vikariantna. Zadnji veliki izbruh speciacije in ekološke diverzifikacije pa se je zgodil v pozni kredi, ko je zaradi povišane gladni morja nastala obilica plitvomorskih habitatov in so imela morja visok delež kisika. Čeprav smiselne, pa so našteto zgolj hipoteze, ki jih z dostopnimi podatki ni moč zanesljivo testirati. Rekonstrukcije tako oddaljenih 99 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 dogodkov so zahtevne, še posebej za skupine z malo fosili, kot so postranice (Copilaş- Ciocianu in sod., 2020). Postranice so najverjetneje doživele več ugnezdenih radiacij, še posebej se to kaže v starodavnih velikih jezerih, na primer v Bajkalskem jezeru (Naumenko in sod., 2017). Rod Niphargus je po številu vrst sodeč največja takšna ugnezdena radiacija, ki predstavlja kar 20 do 25 % vseh sladkovodnih postranic in 5 % vsega redu (Horton in sod., 2021). Zanimivo je, da se je ta izjemna radiacija zgodila v na prvi pogled precej drugačnem okolju kot so velika jezera: v podzemlju. Za analizo vzorcev diverzifikacije rodu Niphargus smo zbrali vse dostopne molekularne, morfološke in ekološke podatke za opisane in še neopisane vrste slepih postranic. Da bi zajeli vso kriptično pestrost, smo kot osnovno enoto pestrosti namesto vrste uporabili MOTU (ang. Molecular Operative Taksonomic Unit, v prevodu molekularna operativna taksonomska enota). Za namene delimitacije smo upoštevali obstoječe večlokusne študije, če le-ta ni obstajala pa smo ubrali konservativni enolokusni pristop. Končni nabor podatkov je obsegal 377 MOTUjev. MOTUje smo razdelili v šest kategorij, glede na habitat v katerem se pojavljajo: sistem razpok v nezasičeni coni jam, intersticij, podzemna jezera, podzemni potoki, plitvo podzemlje in podzemna voda s specifičnimi kemijskimi lastnostmi. Podatke smo zbrali iz literature, terenskih zapiskov in relacijske baze SubBioDB. Morfološki podatki so vključevali 11 telesnih mer, ki odražajo lastnosti habitata, v katerem živi vrsta. Podatki so bili zbrani z merjenjem osebkov in iz literature. Kalibrirano filogenetsko drevo smo izračunali na podlagi sedmih informativnih genskih markerjev: COI, dveh segmentov 28S rRNK, H3, dela 18S rRNK, del sekvence gena za fosfoenolpiruvat karboksinazo (PEPCK), gena za glutamil-prolil-tRNK sintetazo (EPRS), gen za beljakovino toplotnega šoka 70 (HSP70) in gena za arginin kinazo (ArgKin). Kalibrirano filogenijo smo izračunali z Bayesovim pristopom (Bouckaert in sod., 2014). Uporabili smo štiri notranje kalibracijske točke: 1) fosil slepe postranice, pri katerem se prvič pojavi trnast krempelj (Jazdzewski in Kupryjanowicz, 2010); 2) zadnjo zalitje povezave med Evrazijo in severno Ameriko kot zgornjo mejo starosti rodu (Brikiatis, 2016); 3) starost otoka Kreta, na katerem živijo monofiletske vrste z najbližjim sorodnikom na celinski Grčiji (Allegrucci in sod., 2011); 4) starost vodne povezave med Paratetido in Mediteranom, ki je ločila dinarske in bližnjevzhodne klade (Popov in sod., 2004). V naslednjem koraku smo rekonstruirali potek speciacije, poseljevanja habitatov in morfološke diverzifikacije ter jih primerjali z modeli naključne diverzifikacije (ang. null model), kot bi jih pričakovali pri nevtralni evoluciji (Harmon in sod., 2003; Revell, 2012). Nato smo z metodo največjega verjetja testirali kateri model evolucije najbolje opiše naše podatke (Clavel in sod., 2015). Del analiz smo ponovili na šestih monofiletskih kladih z več kot 25 vrstami. Testirali smo konvergenco fenotipov različnih linij, da bi ugotovili, ali so se 100 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 določeni fenotipi večkrat neodvisno razvili (Ingram in Mahler, 2013). Rekonstruirali smo predniške areale in analizirali potek poselitve (Pagel in sod., 2004). Kalibrirana filogenija rodu Niphargus obsega vsaj dvakrat več vrst kot druga največja analizirana podzemeljska skupina (Morvan in sod., 2013). Pokazali smo, da rod slepih postranic izvira iz srednjega miocena (56 do 39 milijonov let nazaj). Analiza kopičenja filogenetskih linij skozi čas je pokazala pospešitev celokupne speciacije pred 15 milijoni let, z upočasnjevanjem zadnjih pet milijonov let. To je v skladu z predvidevanji adaptivno-radiacijskega modela, tako imenovano dinamiko zgodnjega izbruha (ang. early burst). Primerjava alternativnih modelov speciacije je pokazala, da proces speciacije rodu slepih postranic najbolje opiše model s spremembo hitrosti speciacije okoli 15 milijonov let nazaj. Sledi mu model s spremembo hitrosti speciacije in spremembo nosilnosti okolja (Borko in sod., 2021). Rekonstruirali smo predniške habitate in pokazali, da so predniki danes živečih vrst prve 20 do 30 milijonov let živeli in se razširjali v intersticijskih obalnih ali aluvialnih habitatih. Nato so večkrat kolonizirali raznolike nove podzemne habitate. Pričetek kolonizacije novih habitatov sovpada s povišano speciacijo (Borko in sod., 2021). Funkcionalna morfologija slepih postranic odraža pogoje, v katerih vrsta živi, na primer velikost podzemnih prostorov, hitrost toka, način premikanja ali prehranjevalne navade (Trontelj in sod., 2012). Povezava med morfologijo in habitati ni enoznačna, zato je smiselno uporabljati obe metriki komplementarno. Analizirali smo evolucijsko dinamiko morfološke raznolikosti skozi čas. Morfološka raznolikost se je prvič skokovito povišala približno 35 milijonov let nazaj. Sledilo je 15 milijonov let divezifikacije, kakršno bi pričakovali pri nevtralnem modelu evolucije. Pred približno 15 milijoni let je raznolikost spet poskočila, kar nakazuje na neodvisno diverzifikacijo morfologije znotraj več kladov. Faza visoke fenotipske diverzifikacije ustreza fazama visoke speciacije in ekološke diverzifikacije. Pri vseh 11 telesnih merah se je stopnja diverzifikacije sistematično višala skozi čas. Primerjava multivariatnih modelov evolucije morfologije je pokazala, da morfološko diverzifikacijo rodu najbolje opiše model s preskokom iz Brownovega gibanja v model zgodnjega izbruha okoli 15 milijonov let nazaj (Borko in sod., 2021). V izjemnih pogojih so velike adaptivne radiacije lahko seštevek neodvisnih vzporednih radiacij znotraj klada. Na šestih geografsko opredeljenih monofiletskih kladih smo ponovili analize diverzifikacije. Speciacija znotraj kladov ustreza modelu zgodnjega izbruha, ki se je pričel pred 15 do pet milijoni let nazaj. Modeli spreminjanja ekološke pestrosti niso odstopali od pričakovanih. Analiza morfološke disparitete je pokazala adaptivno-radiacijski vzorec pri pontskem, panonskem, zahodnobalkanskem in severnodinarskem kladu, pri južnodinarskem kladu sprememba ni bila statistično značilna. Apeninski klad sestavljajo večinoma morfološko podobne vrste, za katere nismo imeli dovolj morfoloških podatkov, sklepamo pa, da bi lahko bil to primer neadaptivne radiacije. Preverili smo tudi, ali so se v neodvisnih 101 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 vzporednih radiacijah razvili podobni morfotipi. Na podobno evolucijo morfotipov smo sklepali s pomočjo modeliranja t.i. adaptivnih vrhov morfoloških lastnosti. Adaptivni vrh v tem primeru predstavlja določeno vrednost morfološki lastnosti (ali skupka lastnosti); ena lastnost ima v različnih okoljih lahko različne vrednosti, ki na drevesu vznikenjo večrat neodvisno. Najverjetnejši modeli so predvideli 11 do 12 konvergentnih adaptivnih vrhov, od teh sta se dva razvila večkrat znotraj klada, devet pa se jih je razvilo v več kladih. Tri do štiri adaptivni vrhovi so se pojavili le enkrat. Čeprav kladi izkazujejo določen nivo konvergence so imel posamezne radiacije določene skupine adaptivnih vrhov (Borko in sod., 2021). Rekonstrukcija predniških območij je pokazala, da slepe postranice izvirajo iz današnje zahodne Evrope. Preko intersticija so se razširjale proti jugovzhodni Evropi. Časovni in prostorski potek diverzifikacije analiziranih kladov sovpada z dvigom in zakrasevanjem kraških masivov jugovzhodnih Alp, Karpatov in Dinaridov, zato sklepamo, da novi habitati predstavljajo verjetno ekološko priložnost. Ob dvigu kraških masivov iz morja se je začelo zakrasevanje, to je formacija jam in množice drugih podzemnih habitatov. Proces je dosegel vrhunec v srednjem miocenu. Kraški masivi so takrat delovali kot otoki v Paratetidi, občasno povezani med seboj. Obsežna nova sladkovodna podzemna okolja so nudila popolnoma nov in še nenaseljen ekološki prostor, in ekološko priložnost za slepe postranice, ki so že bile prilagojene na podzemno okolje (Borko in sod., 2021). Čeprav makroevolucijski vzorci kažejo na adaptivno radiacijo rodu Niphargus, je razlikovanje med ekološko in neekološko speciacijo na nivoju posamičnih speciacijskih dogodkov zapleteno. Kladi lahko vsebujejo elemente obeh procesov, včasih pa vzorci ustrezajo obema procesoma in jih je nemogoče razložiti (Rundell in Price, 2009). Taksonomsko, filogenetsko in ekološko smo ovrednotili združbo slepih postranic v grški jami Melissotrypa, ki bi se lahko razvila prek ekološke speciacije. V jami živijo štiri vrste postranic, tri iz rodu Niphargus in ena iz rodu Bogidiella. Tri vrste slepih postranic živijo v različnih habitatih: v majhnih medzrnskih prostorih na dnu jezera, v freatski vodi sladkovodnega jezera in v freatski vodi jezera bogatega z žveplovodikom. Filogenetsko drevo 103 vrst slepih postranic smo izračunali s štirimi informativnimi genskimi markerji: COI, dvema odsekoma 28S rRNK in H3. Filogenetske odnose smo izračunali z Bayesovim pristopom (Ronquist in sod., 2012) in metodo največjega verjetja (Trifinopoulos in sod., 2016). Izvedli smo klastrsko analizo funkcionalnih morfoloških znakov za vrste z znano ekologijo. V Melissotrypi smo našli vrste N. lindbergi S. Karaman, 1956, N. jovanovici, S. Karaman 1931, in še neopisano vrsto iz žveplenega jezera, ki smo jo opisali kot N. gammariformis sp. nov. Tri vrste so si ozko sorodne in tvorijo svojo evolucijsko linijo z nejasno pozicijo znotraj rodu. Vrste se med seboj razlikujejo v habitatu in morfologiji. Klastrska analiza je pokazala, da so morfološko podobne nesorodnim vrstam, ki živijo v podobnih habitatih (Borko in sod., 2019). 102 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Morfološke prilagoditve na s strupenim žveplovodikom bogato okolje so se pri slepih postranicah neodvisno razvile vsaj štirikrat. Ekomorfološka raznolikost in filogenetsko združevanje nakazujeta ekološko speciacijo znotraj jame. A bi morali za nedvoumno potrditev ekološke speciacije pokazati, da se je reproduktivna izolacija zares razvila zaradi ekološke diferenciacije, za kar nimamo dovolj podatkov. Alternativna možnost bi bila, da so se vrste razvile v alopatriji, se prilagodile na različna okolja, nato pa prišle v sekundarni kontakt (Rundell in Price, 2009). Četudi je N. gammariformis znan samo iz študirane jame, pa sta drugi dve vrsti gelede na razpoložljive podatke razširjeni širše. Za razrešitev vprašanja bi potrebovali natančne podatke o razširjenosti in tudi dobro podprto molekularno filogenetsko drevo na nivoju populacij (Borko in sod., 2019). V četrtem sklopu disertacije smo se osredotočili na vprašanje biotske pestrosti in procesov v prostoru. Analizirali smo filogenetsko in vrstno pestrost slepih postranic v območju vroče točke podzemeljske biotske pestrosti, Dinaridov (Borko in sod., 2022). Pregledali smo celotno zbirko slepih postranic Skupine za speleobiologijo, BF, UL. Za namene pokritja vseh obstoječih lokalitet znotraj Dinaridov in čim več morfološke variabilnosti smo odbrali preko 1000 osebkov za izolacijo DNK in pomnoževanje COI gena. Uspešno pomnožene sekvence smo analizirali skupaj z že obstoječimi COI sekvencami. Uporabili smo pet enolokusnih delimitacijskih metod (Puillandre in sod., 2012, 2021; Zhang in sod., 2013), jih primerjali z delimitacijami iz obstoječih večlokusnih študij in izbrali konsenzno določitev vsakega osebka. Zaradi visokega deleža novih, prej nepoznanih MOTUjev, za katere še ne obstajajo zanesljivi ekološki in morfološki podatki, nismo mogli rekonstruirati evolucije morfologije in poseljevanja dinarskih kladov, zato smo ubrali drugačen pristop. Ponovili smo računanje kalibrirane filogenije z enakimi markerji in metodološkim pristopom kot v drugem sklopu (Borko in sod., 2021). Nato smo na analizirano območje položili mrežo kvadratov velikosti 20 km x 20 km in znotraj vsakega kvadrata računali vrstno in filogenetsko pestrost (Faith, 1992). Filogenetsko pestrost smo standardizirali glede na število vrst v celici (Kembel in sod., 2010). Nato smo primerjali vzorce obeh metrik v prostoru. Glede na rezultate enolokusnih delimitacij na Dinaridih živi med 212 in 474 MOTUjev. Po preverjanju kakovosti in navzkrižnem preverjanju z že objavljenimi večlokusnimi študijami smo oblikovali končen nabor podatkov, ki je obsegal 598 lokalitet in 245 MOTUjev, med katerimi je 79% vseh nominalnih vrst na Dinaridih. Presenetljivo odkritje je bilo kar 148 novih MOTUjev, ki ne ustrezajo nominalnim vrstam in še niso bili obravnavani v preteklih študijah. Filogenetski odnosi so bili v večji meri podobni prvotnim rekonstrukcijam, a se je število MOTUjev znotraj dinarskih kladov (južnodinarski, severnodinarski in vzhodnobalkanski klad) močno povečalo (Borko in sod., 2022). Vrstno najbolj bogate celice so na severozahodu Dinaridov. Vzorec filogenetske pestrosti le deloma sledi vzorcu vrstne pestrosti. Večina celic je imela nižjo filogenetsko pestrost kot bi 103 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 pričakovali, če bi bile živali razvrščene v prostoru naključno. Celoten jugovzhodni del Dinaridov ima izjemno nizko filogenetsko pestrost. Severozahodni Dinaridi imajo razmeroma višjo filogenetsko pestrost. Dodatno vpogled v vzorce vrstne in filogenetske pestrosti na Dinaridih dobimo, če analiziramo razporeditev kladov v prostoru (Borko in sod., 2022). Osrednje in jugovzhodne Dinaride poseljujejo predvsem dinarski kladi, to so zahodnobalkanski, južnodinarski in severnodinarski klad. Za njih smo pokazali, da so v celoti diverzificirali na območju Dinaridov. Egejski klad seže v Dinarski prostor le bežno, na skrajnem jugovzhodu. Na vzhodnih aluvialnih ravnicah pa so prisotni predstavniki široko razširjenih plitvo podzemeljskih vrst iz linije N. sphagnicolus. Vsi trije kladi so se iz Dinaridov tudi preseljevali, predvsem na Apeninski polotok, v obdobjih povezav med obema kopninama. Nasprotno pa je le ena linija prišla v Dinaride in se razširila po obmorskem delu (glej sklop 5). Kljub obstoju povezav z drugimi kraškimi masivi nobena druga linija ni uspešno naselila jugovzhodnih Dinaridov. Filogenetska pestrost na jugovzhodu je nizka in vzorec diverzifikacije vseh treh dinarskih kladov nakazuje, da je adaptivna radiacija dominirala nad disperzijo. Drugače je v severozahodnih Dinaridih, še posebej na stičišču z jugovzhodnimi Alpami in Panonsko nižino. V območju, kjer se kraška območja stikajo z aluvialnimi ravnicami, sta tako vrstna kot filogenetska pestrost visoki, tu živijo predstavniki večine linij rodu slepih postranic. Dva dinarska klada segata do severozahoda. Zahodnobalkanski klad je razširjen le znotraj meja dinaridov, medtem ko je severnodinarski prisoten tudi v jugovzhodnih Alpah. Prisotne so vrste iz pontskega in panonskega klada. Panonske vrste le bežno segajo v ožje območje Dinaridov in so bolj prisotne v jugovzhodnih Alpah in Panonski nižini. Iz panonskega klada je v severozahodnih Dinaridih prisotna linija desetih MOTUjev, ki je najverjetneje diverzificirala v tem prostoru, znotraj specifičnega habitata (sistem razpok). Nadalje v severozahodnih Dinaridih najdemo še štiri nesorodne klade z vsaj petimi MOTUji na klad, ter 18 MOTUjev brez najbližjih sorodnikov znotraj tega prostora. Ta kompleksna struktura nesorodnih vrst, ki so se priselile v regijo in sorodnih vrst, ki so diverzificirale tu, se odraža v visoki biotski pestrosti (Borko in sod., 2022). Glede na razlike v vzorcih vrstne in filogenetske pestrosti in na splošno majhne areale razširjenosti vrst slepih postranic lahko zaključimo, da je lokalna diverzifikacija najverjetneje glavno gonilo vrstne pestrosti v Dinaridih, disperzija pa je bila prisotna predvsem v severozahodnem delu regije (Borko in sod., 2022). Potencialna pomanjkljivost študije je nezmožnost oceniti delež izumiranja, ki lahko pomembno prispeva k visoki filogenetski pestrosti. A iz več razlogov sklepamo, da izumiranje ni imelo vloge pri nastanku današnjih vzorcev filogenetske pestrosti: podzemlje je v času katastrofičnih dogodkov predstavljalo pribežališče vrstam, ki na površju drugače ne bi preživele. V Evropi so bile poledenitve glavni vzrok izumiranja, Dinaridi pa so prepoznani kot južni refugij. Še več, 104 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 pokazano je bilo da so alpski ledeniki na severozahodu spodbudili speciacijo slepih postranic in ne obratno (Delić in sod., 2022). Z analizo vzorcev pestrosti smo pridobili pomemben vpogled v možne procese, ki so se odvijali v regiji. A zaradi višjega števila novih MOTUjev, kot smo jih pričakovali, in posledično pomanjkanja zanesljivih ekomorfoloških podatkov, nismo mogli oceniti doprinosa neadaptivnih in adaptivnih radiacij. Za podrobnejšo in predvsem zanesljivo analizo procesov na lokalni ravni bo potrebno še veliko terenskega in laboratorijskega dela (Borko in sod., 2022). V zadnjem sklopu smo analizirali disperzijo rodu Niphargus med Dinaridi in širšim območje, konkretno Apeninskim polotokom (Delić in sod., 2020). Obdobja regresije in transgresije morja bi lahko povzročili izmenjavo disperzije in vikariance. Zbrali smo podatke za 494 osebkov slepih postranic, s poudarkom na vrstah ki živijo ob Jadranskem morju. Z enolokusnimi delimitacijami s COI markerjem (Puillandre in sod., 2012; Zhang in sod., 2013) smo prepoznali 169 MOTUjev, za katere smo izračunali filogenijo. Filogenijo smo izračunali s pomočjo sekvenc štirih markerjev: H3, COI in dvema fragmentoma 28S, z metodo največjega verjetja (Minh in sod., 2020) in z Bayesovim pristopom (Ronquist in sod., 2012). Kalibrirano filogenijo smo izračunali z Bayesovim pristopom (Bouckaert in sod., 2014) in enakimi kalibracijskimi točkami kot v sklopih 2 in 4. Nato smo na t. i. transjadranskem kladu, ki vključuje sestrska zahodnobalkanski in južnodinarski klad, z metodo zveznih markovskih procesov modelirali disperzijo, izumiranje, vikarianco, kladogenezo in predniške areale (Matzke, 2013). Znotraj analiziranega klada so vgnezdeni trije dobro podprti podkladi z vrstami, prisotnimi na obeh straneh Jadranskega morja. Prav tako so nekatere sestrske vrste prisotne na Dinarskem krasu in na Jadranskih otokih. Dinaridi so bili, tako kot v študiji sklopa 2, prepoznani kot izvorno območje klada. Cepitve med vrstami na obeh straneh morja so se zgodile v treh časovnih oknih. Prva serija cepitev sega med 10 do 20 milijonov let nazaj, druga 4,4 do 8,9 milijonov let nazaj, tretja pa se je odvila v zadnjih dveh milijonih let. Vsi modeli so pokazali, da je bila disperzija mnogo večja od izumiranja. Najbolj podprt je bil model skokovite disperzije (ang. jump dispersal) (Delić in sod., 2020). Prva serija cepitev sovpada s kopenskim mostom med Dinaridi in novo nastalo kopnino Apulija (Mazza in Rustioni, 2008). Potopitev mostu, ki je sledila, je razdelila populacije, kar je vodilo v obdobje, ko so potekle prve vikariantske speciacije. Sledila je regresija Jadranskega morja med mesinijsko krizo slanosti (Garcia-Castellanos in Villaseñor, 2011), ko je predniška linija N. hebereri najverjetneje migrirala nazaj iz Apeninskega polotoka na Jadranske otoke in obalo Dinaridov. Zadnje cepitve sovpadajo s pleistocensko poledenitvijo (Correggiaii in sod., 1996). Vsaj šest linij je takrat iz Dinaridov koloniziralo Jadranske otoke (Delić in sod., 2020). 105 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Znotraj zahodnobalkanskega in južnodinarskega klada smo pokazali na večkratno disperzijo iz Dinaridov na Apeninski polotok. Disperzija iz Dinaridov se je najverjetneje zgodila še večkrat. Na vznožju karpatov je razširjena vrsta N. mirocensis z najbljižimi sorodniki na Dinaridih. Linija šestih vrst iz kompleksa N. rhenorhodanensis v zahodni Italiji je dispergirala iz severozahodnih Dinaridov. Dinaride zato lahko obravnavamo kot donorsko območje slepih postranic, ki so iz Dinaridov poseljevale kasneje vzdignjene kopnine. Nasprotno je bila disperzija v osrednje in jugovzhodne Dinaride od zunaj močno omejena, pokazali smo le en prehod nazaj iz Apeninskega polotoka. Najverjetneje so že prisotne dinarske vrste preprečevale uspešno priseljevanje. V okviru doktorske disertacije smo celovito analizirali vzorce speciacije in ekomorfološke diverzifikacije znotraj rodu Niphargus na kontinentalnem nivoju, znotraj vroče točke podzemne biotske pestrosti in znotraj jamske združbe. Pokazali smo, da diverzifikacija slepih postranic ustreza adaptivno-radiacijskemu modelu, ter da se je znotraj rodu odvilo več neodvisnih radiacij na območjih dvigajočih se kraških gorstev. Pomembno smo razširili podatkovni nabor na območju Dinaridov in razkrili skrito pestrost slepih postranic znotraj vroče točke podzemeljske biotske pestrosti. Pokazali smo, da imajo slepe postranice znotraj Dinaridov heterogeno filogenetsko strukturo v prostoru, kar je posledica različnega doprinosa speciacije in disperzije k nastanku vrstne pestrosti tega območja. To je prva tovrstna študija adaptivne radiacije v podzemlju in kot taka predstavlja pomemben doprinos k razumevanju evolucije v podzemlju. Ne le, da odkriva potencial za nadaljnje raziskovanje radiacij v podzemlju, pomembno prispeva tudi k prepoznavanju Evrope kot kontinenta z visoko biotsko pestrostjo v okoljih, kjer je na prvi pogled ne bi pričakovali. 106 Borko Š. 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The limits of cryptic diversity in groundwater: Phylogeography of the cave shrimp Troglocaris anophthalmus (Crustacea: Decapoda: Atyidae). Molecular Ecology, 18, 5: 931–946 Zhang J., Kapli P., Pavlidis P., Stamatakis A. 2013. A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29, 22: 2869–2876 116 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ACKNOWLEDGEMENTS First, I would like to thank my supervisor and mentor, Doc. Dr. Cene Fišer, for all your support, patience, and guidance. Without your vast knowledge of Niphargus and your inexhaustible flow of ideas, we would all be lost. I am also grateful for your support of my expeditions into deep caves, the escapes I need to appreciate the beauties of the surface. I would like to thank the commission for assessment and defence: Prof. Dr. Peter Trontelj, Prof. Dr. Florian Altermatt and Doc. Dr. Maja Zagmajster for your contribution, which significantly improved the dissertation and the studies contained therein. I am thankful to Prof. Dr. Florian Altermatt and the team at Eawag (Swiss Federal Institute of Aquatic Science and Technology) for your hospitality during my study visit, during which I gained invaluable experience. The members of the Altermatt Lab welcomed me and shared their insights, knowledge and ideas with me, which helped me develop my work and broaden my horizons. Florian, you are an excellent scientist and environmentalist and I am honoured to have the opportunity to learn from you. A big thank you goes to the members of SubBio Lab for all the nice moments. It is a pleasure to work with you. Finally, I would like to thank my partner Matic Di Batista. You are my rock and the most honest critic. It is a great joy and privilege to travel this life with you. Thank you for believing in me and remembering me that I can. Funding This work was supported by the Slovenian Research Agency – ARRS (“Young Researcher” PhD grant, project J1-2464 and program P1-0184). Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ANNEXES ANEX A Consent from publishers for the re-publication of article entitled The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin in the print and electronic versions of the doctoral dissertation Copilaş-Ciocianu D., Borko Š., Fišer C. 2020. The late blooming amphipods: Global change promoted post-Jurassic ecological radiation despite Palaeozoic origin. Molecular Phylogenetics and Evolution, 143: 106664, https://doi.org/10.1016/j.ympev.2019.106664: 12 p. Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ANEX B Consent from publishers for the re-publication of article entitled Amphipods in a Greek cave with sulphidic and non-sulphidic water: phylogenetically clustered and ecologically divergent in the print and electronic versions of the doctoral dissertation Borko Š., Collette M., Brad T., Zakšek V., Flot J.-F., Vaxevanopoulos M., Sarbu S. M., Fišer C. 2019. Amphipods in a Greek cave with sulphidic and non-sulphidic water: phylogenetically clustered and ecologically divergent. Systematics and Biodiversity, 17, 6: 558-572 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 ANEX C Consent from publishers for the re-publication of article entitled How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for dispersal–vicariance interplay mediated by marine regression–transgression cycles in the print and electronic versions of the doctoral dissertation Delić T., Stoch F., Borko Š., Flot J.-F., Fišer C. 2020. How did subterranean amphipods cross the Adriatic Sea? Phylogenetic evidence for dispersal–vicariance interplay mediated by marine regression–transgression cycles. Journal of Biogeography 47: 1875–1887 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022 Borko Š. Adaptive radiation of Niphargus … and its contribution to the Dinaric subterranean fauna. Doct. Dissertation. Ljubljana, Univ. of Ljubljana, Biotechnical Faculty, 2022