Colonisation and adaptation of

stygobiont species

PhD Thesis

By: Balázs Gergely

Supervisors: Herczeg Gábor, DSc Török János, DSc ELTE PhD school of Biology Head of school: Erdei Anna, DSc

Zootaxonomy, ecology, hydrobiology Programme Programme Leader: Török János, DSc

Budapest, 2019 Introduction

Caves as habitats can be an outstanding research topic for researchers from various fields of interest. From a taxonomical point of view the number of well-studied subterranean habitats is low, which results in a considerable difference between the estimated and the known number of species. In many cases the well-defined geographical and ecological isolation, the comparatively stable environment and the model systems containing few species offers opportunities for a wide range of research. Other than the obvious research potential, the investigation of the hardly or not reachable subterranean habitats and their fauna faces many difficulties (Schneider & Culver, 2004). Establishing a solid taxonomical and ecological background is well worth the effort, because with the simplified repetitively occurring systems provided by , we have the opportunity to investigate some of the basic questions of evolutionary biology (e.g. Jeffery, 2001). Speleobiological research in Hungary used to be leading edge with many forward-looking results and research directions (Dudich, 1932, 1959). Unfortunately, in the last few decades, the intensity of such Hungarian research has decreased remarkably. Hungary in terms of biology, is an outstandingly valuable region. This is due not only to the geographical location of the country, as it falls within the distribution range of numerous hypogean taxa, but also because the Hungarian karst areas are geographically separated from each other, and in several cases, there are also considerable distances between them. In comparison with the large continuous karst areas, these more or less separated areas provide better possibilities to detect any patterns of colonisation and adaptation. Caves are constantly emerging and vanishing throughout geological time. Although during their presence they can provide relatively balanced environments, their existence is temporary. The cave dwelling typically have surface- dwelling ancestors, which at some point have colonised the subterranean realm. The two major hypotheses explaining the driving forces behind the colonisation events are the Climatic Relict Hypothesis (CRH), and the Adaptive Shift Hypothesis (ASH) (Romero, 2009). According to the CRH the main driving force behind colonisation is climatic change resulting in transitional or permanently unfavourable environmental conditions on the surface. During such events, caves with their more balanced or stable environments, can act as refuges (Danielopol & Rouch, 2005; Peck & Finston, 1993). The ASH predicts that individuals colonise caves to exploit new food resources provided by the hypogean environment (Howarth, 1980, 1987). Cave animals show rather similar progressive and regressive adaptations commonly referred to as troglomorphisms (Christiansen, 1962). Troglobionts and eutroglophiles provide a unique opportunity to investigate the mechanisms of regressive evolution. One of the repeatedly occurring regressive changes is the reduction or loss of vision and associated structures. Knowledge about the underlying mechanisms can give us insights into general evolutionary processes (e.g. Protas et al., 2007). The success of a colonisation event is dependant upon multiple factors, one of the most important of which is undoubtedly the level of exaptation which describes the original capability of a given creature to survive and reproduce in the new environment. The (), even with its ever changing (Fišer et al., 2008) is the most suitable candidate in Hungary to investigate these general questions. This is not only because it contains the highest number of stygobiont species but also because it is a generally studied taxon, therefore offering a large amount of available ecological, behavioural and molecular data on other species to facilitate comparisons (Fišer, 2012). As the Niphargus genus entered the subterranean habitats circa 88 million years ago (McInerney et al., 2014) it is not the appropriate taxon to investigate the early stages of colonisation and adaptation. The waterlouse (Asellus aquaticus) being an epigean species which has repeatedly and successfully colonised caves, is a good model organism to examine those early stages.

Aims and scope

The first part of my work aimed to select which Niphargus species are members of the Hungarian fauna in comparison with the existing literature data. This first step was essential for all further studies. The objective of the second investigation was to clarify the phylogenetic background and close relatives of the Hungarian Niphargus species. The third investigation was intended to gain knowledge on the fauna of the Molnár János Cave and more specifically, on the hypogean and epigean populations of Niphargus hrabei and Asellus aquaticus inhabiting the cave and its surrounding surface habitats. The cave has a direct hydrological connection with the Malom Lake and the Danube. This natural setup represents good conditions in which to examine the processes of colonisation and adaptation. As the two species show different levels of exaptation, I assumed that despite the equal environmental conditions, different processes are shaping the emergence of the cave populations. The main questions of my dissertation were: 1. How many valid Niphargus species are part of the Hungarian fauna and which ones? What kind of new or known but never reported Niphargus species are to be expected in Hungary? 2. Which species are the close relatives of the Hungarian Niphargus species? What assumptions can we make regarding the geographic origin of the species and regarding the emergence of the Niphargus fauna of a certain habitat based on the phylogeny? 3. What are the macroscopic species of the Molnár János Cave and what is their origin? What kind of differences can be found in the genetic structure of the N. hrabei and A. aquaticus populations inhabiting the Molnár János Cave and the hydrologically connected Malom Lake and the Danube? What kind of isolation mechanisms can be detected on the species showing different levels of exaptation? What kind of genetic changes are at play behind the loss of vision of N. hrabei and A. aquaticus regarding the elements of the phototransduction cascade?

Material and methods

To clarify the valid Hungarian Niphargus species I narrowed down the list of possible species by evaluating the relevant literature data. Based on the reduced list I collected samples on the type localities wherever it was possible. The collected individuals underwent careful morphological investigation where both the preparation and the data collection were undertaken according to the modern Niphargus-taxonomic standard (Fišer et al. 2009). The morphological results were strengthened with molecular data when it was needed. The phylogenetic investigation of the Hungarian Niphargus species was based on specimens from type localities when it was possible, including three loci (28S, COI, H3). Trees were estimated using Maximum Likelihood and Bayesian inference methods with the use of sequences of 95 additional taxa available in different databases. The investigation of the fauna of the Molnár János Cave started with an intense sampling effort. As a result, five macroscopic invertebrates were found to be present in the cave, and on which we carried out investigations of different magnitudes. Two new Niphargus species were discovered, which are yet to be described. These two species were included in the phylogenetic and morphological research mentioned above, which I carried out. The Bythiospeum snail species found in the cave underwent genetic investigation based on the COI locus for species level determination. On the N. hrabei and A. aquaticus population we carried out population genetic and transcriptomic investigations. In the population genetic investigation, the Molnár János Cave, the Malom Lake and the Ráckeve-Soroksár Danube (RSD) populations were represented by 20 individuals per population and location (18 from N. hrabei cave population). For better comparison we also collected specimens from other parts of the country and used sequences from databanks. Four loci were selected and amplified with PCR method for both N. hrabei (16S, COI, ITS, NaK) and A. aquaticus (12S, 16S, COI, pseudoND2). Using the sequences we then built haplotype-networks, trees based on Maximum Likelihood and Bayesian inference methods and calculated Pairwise Genealogical Sorting Indices (pwgsi). The demographic history of the population was estimated using Extended Bayesian Skyline (EBS) analysis. The populations in the transcriptomic analysis were represented by two individuals per species and focal location, specifically collected for the purpose. Our primary aim with the investigation was to find out the differences and similarities between the expression of the phototransduction cascade elements in cave and surface populations of the two species showing different levels of exaptation. Therefore after extraction and sequencing, the de novo RNA sequences were filtered by Open Read Frame (ORF) detection and then narrowed by Phylogenetically-Informed Annotation (PIA) methods. Out of the cascade elements, the opsins were compared with reference opsin sequences. For further analysis, the opsin sequences were then used to build Maximum Likelihood phylogenetic tree and included the use of sequences from available datasets. Results

1. As a result of the study on Hungarian Niphargus species only eight species are considered valid and present in Hungary out of the 27 Niphargus species mentioned in the relevant literature. The rest of the species had to be excluded for various reasons. Species excluded due to international border changes: Niphargus dudichi HANKÓ, 1924; Niphargus baloghi DUDICH, 1940; Niphargus pater MÉHELŸ, 1941; Niphargus effosus DUDICH, 1943; Niphargus körösensis DUDICH, 1943; Niphargus stygocharis stygocharis DUDICH, 1943 Species excluded due to misidentifications and taxonomical changes: Niphargus stygius (SCHIÖDTE, 1847); Niphargus longicaudatus (A. COSTA, 1851), Niphargus puteanus (C. L. KOCH, 1836); Niphargus foreli HUMBERT, 1876; Niphargus leopoliensis JAWOROWSKY, 1893; Niphargus mediodanubialis DUDICH, 1941, Niphargus thermalis DUDICH, 1941 Out of the above listed species N. mediodanubialis, according to some authors, is a junior synonym of N.valachicus (S. Karaman, 1950). This assumption based on morphology is further strengthened by our genetic data. According to our morphological and genetic results N. thermalis is a junior synonym of N. hrabei. Niphargus species with uncertain taxonomic status (species inquirenda): Niphargus magyaricus MÉHELŸ, 1941; Niphargus matrensis MÉHELŸ, 1941; Niphargus Béldyi MÉHELŸ, 1941; Niphargus budensis MÉHELŸ, 1941; Niphargus ginsiensis MÉHELŸ, 1941; Niphargus parvus MÉHELŸ, 1941 Currently valid indigenous Niphargus species: Niphargus hrabei S. KARAMAN, 1932; Niphargus valachicus DOBREANU & MANOLACHE, 1933; Niphargus hungaricus MÉHELŸ 1937; Niphargus gebhardti SCHELLENBERG, 1934; Niphargus molnari MÉHELŸ 1927; Niphargus aggtelekiensis DUDICH, 1932; (WRZESNIOWSKY, 1890); Niphargus forroi G. KARAMAN, 1986. Thanks to our sampling effort, three new indigenous species were discovered and are yet to be described. Two inhabit the Molnár János Cave and one was found in the water-filled fissures of the Visegrád Mountains. The list of Niphargus species of Hungary can be further extended by the plausible Hungarian occurrence of Niphargus plurispinosus HUDEC & MOCK, 2014, which was described from the Slovakian side of the Zemplén Mountains.

2. Results of the phylogenetic analysis on the Hungarian Niphargus species: N. molnari and the two new species from Molnár János Cave share a common ancestor. The clade containing these three species also contains N. kieferi SCHELLENBERG, 1936 which is native to France and Germany and N. inopinatus SCHELLENBERG, 1932 which can be found from Germany to . According to our results, the closest relative of N. hrabei is N. plateaui CHEVREUX, 1901 with French distribution. This relationship is in agreement with previously published results (Copilaș- Ciocianu et al., 2018). The three other species belonging to this clade are from the Apennine Peninsula. N. gebhardti forming a well-defined clade together with species from the Crimean Peninsula (N. dimorphus BIRSTEIN, 1961, N. vadimi BIRSTEIN, 1961), Romania (N. bihorensis SCHELLENBERG, 1940) and Italy (N. ambulatory G. KARAMAN, 1975). The taxonomical rank of the Hungarian population of the N. aggtelekiensis as a species were strengthened by our results, while the Austrian populations identified on a morphological basis are not part of the species. The status of the populations of the Bükk Mountains morphologically identified as N. tatrensis could not be resolved by our results. According to our analysis, N. forroi which inhabits caves of the Bükk Mountains, is closely related to N. aggtelekiensis and N. tatrensis, yet showed a clear separation. The phylogenetic relations of N. valachicus and N. Hungaricus could not be resolved by our results. It is worth mentioning that our results contradict the previously assumed close relationship of N. valachicus and two Italian species, namely N. dolenianensis LORENZI, 1898 and N. krameri SCHELLENBERG, (Copilaș-Ciocianu et al., 2018).

3. Results of the investigations in Molnár János Cave: According to COI based genetic data the Bythiospeum snail species of the cave showed 99,6% similarity with B. acicula (HELD, 1838). This species is known from subterranean habitats of Southern Germany and it is the oldest described taxon of Clade I. defined by the latest summarizing molecular research on the genus (Richling et al. 2017). According to the haplotype network, tree and pwgsi analysis based on multiple loci, the hypogean population of N. hrabei showed no separation from the surface populations, while the hypogean population of A. aquaticus inhabiting the Molnár János Cave appeared to be genetically isolated from its surface counterparts. The lack of structure in the case of N. hrabei can be equally explained by the high level of exaptation or the recent cave colonization of the species. The demographic history modelling showed a sharp decline in population size with a bottleneck circa 10 k years ago, which was followed by a steady incline. This result is in accordance with previous assumptions suggesting the recent area expansion of the species (Copilaș-Ciocianu et al., 2018). The observed separation in A. aquaticus populations suggests that for this moderately adapted species, the cave environment acts as an isolation factor (isolation by environment, IBE (Wang & Bradburd, 2014)) playing an important role in the emergence of genetic differences. According to molecular clock based estimations, the divergence between the Malom Lake and the Molnár János Cave populations occurred approximately 60-140 k years ago. The calculated demographic history suggests a significant population decline circa 100-200 k years ago which could have been as a result of unfavourable surface conditions for the species. Considering the divergence time, it is plausible that the cave served as a climatic refugium emphasizing the role of the climatic relict hypothesis (CRH) in this colonisation event. Despite this differentiation, the A. aquaticus population in the Molnár János Cave still falls well within the species range for A. aquaticus in a phylogenetic context. The transcriptome investigation revealed no significant differences in the expression of the phototransduction cascade element between the sampled N. hrabei individuals from the one hypogean and two epigean populations. Despite the subterranean origin of its genus (McInerney et al., 2014), some elements of the cascade can be detected including a long wavelength sensitive (LWS) opsin. Because of the expression of the cascade elements, some sort of extraocular photoreception cannot be ruled out. In all three A. aquaticus, it was possible to detect the same key elements of the phototransduction cascade, including a short wavelength sensitive (SWS/UV) and a long wavelength sensitive (LWS) opsin. Therefore it can be assumed that even the troglomorphic A. aquaticus individuals are capable of light detection despite their reduced eyes. Comparing the A. aquaticus SWS/UV opsin with reference opsin sequences, we found lysin residue in the bovine position 90, which suggests a sensitivity shift into the UV direction. The LWS sequences for both species showed serine residue instead of alanine in bovine reference position 292, which difference can cause a spectral shift in both directions. Due to the quantitative nature of our study, the results do not give any insight into the regulation of the expression.

References

Christiansen K. (1962). Proposition pour la classification des animaux cavernicoles. Spelunca, 2, 75–78. Copilaș-Ciocianu, D., Fišer, C., Borza, P. & Petrusek, A. (2018). Is subterranean lifestyle reversible? Independent and recent large-scale dispersal into surface waters by two species of the groundwater amphipod genus Niphargus. Molecular Phylogenetics and Evolution, 119, 37–49. Culver, D.C. & Pipan, T. (2009). Biology of Caves and Other Subterranean Habitats. Oxford University Press, Oxford. 254 pp Danielopol, D.L. & Rouch, R. (2005). Invasion, active versus passive. In: D.C. Culver &W.B. White, eds. Encyclopedia of caves, pp. 305–310. Elsevier/Academic Press, Amsterdam, The Netherlands Dudich, E. (1932). Biologie der Aggteleker Tropfsteinhöhle ”Baradla“ in Ungarn. Spaläologischen. Monographien., 13, 246 pp. Dudich, E. (1959). A Barlangbiológia és problémái. (Biospeologica Hungarica VI). A Magyar Tudományos Akadémia Biológia Csoportjának Közleményei, 3(3–4), 323–57. Fišer, C., (2012). Niphargus: a model system for evolution and ecology. In: Culver, D. C. & White, W.B. (Eds.), Encyclopedia of Caves. Academic Press, New York, pp. 555–64. Fišer, C., Sket, B. & Trontelj, P. (2008). A phylogenetic perspective on 160 years of troubled taxonomy of Niphargus (Crustacea: Amphipoda). Zoologica Scripta, 37, 665–80. Fišer, C., Trontelj, P., Luštrik, R. & Sket, B. (2009). Towards a unified taxonomy of Niphargus (Crustacea: Amphipoda): a review of morphological variability. Zootaxa, 2061, 1–22. Howarth, F.G. (1980). The zoogeography of specialized cave animals: a bioclimatic model. Evolution, 34, 394–406. Howarth, F.G. (1987). The evolution of non-relictual tropical troglobites. International Journal of Speleology, 16, 1–16. Jeffery, W. R. (2001). Cavefish as a model system in evolutionary developmental biology. Developmental Biology, 231, 1–12. Karaman, S. (1950). Niphargus smederevanus N. sp. aus Nordserbien. Srpska Akademija Nauka 2, 1–9. McInerney, C. E., Maurice, L., Robertson, A. L., Knight, L. R. F. D., Arnscheidt, J., Venditti, C., Dooley, J. S. G., Mathers, T., Matthijs, S., Eriksson, K., Proudlove, G. S. & Hänfling, B. (2014). The ancient Britons: groundwater fauna survived extreme climate change over tens of millions of years across NW Europe. Molecular Ecology, 23, 1153–66. Peck, S.B., & Finston, T.L., (1993). Galapagos Islands troglobites: the questions of tropical troglobites, parapatric distributions with the eyed sister-species, and their origin by parapatric speciation: Memoires de Biospeleologie, 5(20), 19–37. Protas, M., Conrad, M., Gross, J. B., Tabin, C. & Borowsky, R. (2007). Regressive evolution in the Mexican cave tetra, Astyanax mexicanus. Current Biology, 17, 452–4. Richling, I., Makowsky, Y., Kuhn, J., Niederh€offer, H. J., & Boeters, H. D. (2017). A vanishing hotspot – The impact of molecular insights on the diversity of Central European Bythiospeum Bourguignat, 1882 (Mollusca: Gastropoda: Truncatelloidea). Organisms, Diversity and Evolution, 17(1), 67–85. Romero, A. (2009). Cave Biology: Life in Darkness. Cambridge University Press, Cambridge. 291 pp. Sket, B. (2008). Can we agree on an ecological classification of subterranean animals? Journal of Natural History, 42, 1549–63. Wang, I. & Bradburd, G. (2014). Isolation by Environment. Ecology Letters, 23, 5649–62. Publications that are parts of the thesis

Balázs, G. & Angyal, D. (2013). A magyarországi vakbolharákfajok (Amphipoda: Niphargus) kiértékelő irodalmi áttekintése. Állattani Közlemények, 98(1–2), 111–9. Balázs, G., Angyal D. & Kondorosy E. (2015). Niphargus (Crustacea: Amphipoda) species in Hungary: literature review, current taxonomy and the updated distribution of valid taxa. Zootaxa, 3974(3), 361– 76. Pérez-Moreno, J. L*., Balázs, G.*, Wilkins, B., Herczeg, G. & Bracken-Grissom, H.D. (2017). The role of isolation on contrasting phylogeographic patterns in two cave . BMC Evolutionary Biology, 17, 247. *divided first authorship Copilaș-Ciocianu, D., Fišer, C., Borza, P., Balázs, G., Angyal, D.& Petrusek, A. (2017). Low intraspecific genetic divergence and weak niche differentiation despite wide ranges and extensive sympatry in two epigean Niphargus species (Crustacea: Amphipoda). Zoological Journal of the Linnean Society, 181, 485–99. Pérez-Moreno, J. L., Balázs, G. & Bracken-Grissom, H. D. (2018). Transcriptomic and epigenetic insights into the evolution of vision loss in cave-dweling crustaceans. Integrative and Comparative Biology 58(3), 452-64.

Publications connected to the thesis

Angyal, D. & Balázs G. (2013). New data to the distribution of four aquatic troglobiont macroinvertebrate species in some caves of the Mecsek Mountains (SW Hungary). In: M. Filippi & Bosák P. (Eds.). Proceedings of the. 16th Congress on Speleology, Brno, Czech Republic, vol. 2, 426–9. Angyal, D. & Balázs, G. (2013). Distinguishing characters of Niphargus gebhardti Schellenberg, 1934 and Niphargus molnari Mehely, 1927 (Crustacea: Amphipoda): a clarification. Opuscula Zoologica, Budapest, 44(1), 3-8. Szederjesi T., Angyal D., Balázs, G. & Dányi L. (2014): Remarks on the earthworm genus Helodrilus Hoffmeister, 1845 with new epigean and subterranean records (Oligochaeta, Lumbricidae). Opuscula Zoologica 45(2): 181–188. Salamon, G., Dányi, L., Angyal, D., Balázs, G. & Forró L. (2014). A Baradla gerinctelen faunája. In: P. Gruber & Ľ. Gaál (Eds.): A Baradla-Domica-barlangrendszer. A Barlang, mely összeköt. Aggteleki Nemzeti Park Igazgatóság, Jósvafő, pp. 279–306. Angyal D., Balázs G., Zakšek V., Krízsik V. & Fišer C. (2015). Redescription of two subterranean amphipods Niphargus molnari Méhely, 1927 and Niphargus gebhardti Schellenberg, 1934 (Amphipoda, ) and their phylogenetic position. ZooKeys 509, 53–85. Angyal, D., Balázs, G., Krízsik, V., Herczeg, G. & Fehér, Z. (2018). Molecular and morphological divergence in a stygobiont gastropod lineage (Truncatelloidea, Moitessieriidae, Paladilhiopsis) within an isolated karstic area in the Mecsek Mountains (Hungary). Journal of Zoological Systematics and Evolutionary Research, 56, 493-504.

Oral presentations that are parts of the thesis

Balázs G. & Angyal D. (2013): A magyarországi Niphargus fajok kiértékelő áttekintése. X. Makroszkopikus Vízi Gerinctelenek Kutatási Konferencia, 2013. 04. 11-13., Szalafő, Hungary Balázs G. & Angyal D. (2013): Chase in history after the endemic Niphargus (Crustacea: Amphipoda) species of Hungary. 16th International Congress of Speleology, 2013. 07. 21-28. Brno, Czech Republic Mock A., Kováč L., Papáč V., Ľuptáčik P.,Parimuchová A., Forró L., Angyal D., Dányi L., Szél Gy., Balázs G., Košel V. & Fenďa V. (2015): Terrestrial and aquatic (Arthropoda) of the Domica-Baradla Cave System. 10th Scientific Conference Research and Protection of Caves of Slovak and Aggtelek Karst. 2015. 09. 22.-25. Rožňava, Slovakia, Aggtelek, Hungary Pérez-Moreno, J. L., Balázs, G., Bracken-Grissom, H. D. (2018). Transcriptomic and epigenetic insights into the evolution of vision loss in cave-dweling crustaceans. Annual Meeting of the Society-for-Integrative- and-Comparative-Biology (SICB). 2018. 01. 03-07. San Francisco (CA), USA

Other Publications

Noreikiene, K., Herczeg, G., Gonda, A., Balázs, G., Husby, A. & Merilä, J. (2015). Quantitative genetic analysis of brain size variation in sticklebacks: support for the mosaic model of evolution. Proceedings of the Royal Society B 282, 20151008. Balázs, G., Lewarne, B. & Herczeg, G. (2015). In situ underwater tagging of aquatic organisms: a test using the cave-dwelling olm, Proteus anguinus. Annales Zoologici Fennici, 52, 160-6. Herczeg, G., Gonda, A., Balázs, G., Noreikiene, K. & Merilä, J. (2015). Experimental evidence for sex- specific plasticity in adult brain. Frontiers in Zoology, 12, 38 ZItong, L., Baocheng, G., Jing, Y., Herczeg, G., Gonda, A., Balázs, G.,Takahito, S., Calboli, F. C. F. & Merilä, J. (2017). Deciphering the genomic architecture of the stickleback brain with a novel multi-locus gene-mapping approach. Molecular Ecology, 26, 1557–75. Balázs, G. & Lewarne, B. (2017). Observations on the olm Proteus anguinus population of the Vrelo Vruljak System (Eastern Herzegovina, Bosnia and Herzegovina). (EN) Opazovanja populacij močerila Proteus anguinus v sistemu izvira Vruljak (vzhodna Hercegovina, Bosna in Hercegovina).Natura Sloveniae, 19(1), 39-41. Angyal, D., Solís, E. C., Magaña, B., Balázs, G. & Simoes, N. (2018). Mayaweckelia troglomorpha, a new subterranean amphipod species from Yucatán state, México (Amphipoda, Hadziidae). Zookeys, 735, 1- 25. Dányi, L., Balázs, G. & Tuf, I. H. (2019). Taxonomic status and behavioural documentation of the troglobiont Lithobius matulici (Myriapoda, Chilopoda) from the Dinaric Alps: Are there semiaquatic centipedes in caves? Zookeys, 848, 1-20. Horváth, G., Garamszegi, L. Z., Bereczki, J., Urszán, T. J., Balázs, G. & Herczeg, G. (2019). Roll with the fear: environment and state dependence of pill bug (Armadillidium vulgare) personalities. Naturwissenschaften, 106, 3 p. 7.