49 Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology Abstract This is the first record of the liverwort Riccia atromarginata for the Western Balkans. It was found on conglomerate terraces in the city of Podgorica, Montenegro, at the confluence of the rivers Ribnica and Morača. The material from Montenegro as well as living samples from Tenerife, Gran Canaria and Cyprus served as a basis for a detailed description of the main characteristics of the species, on the basis of which Riccia atromarginata can be reliably identified. However, for some characteristics there is a clear variability. Iz vleček V prispevku poročamo o prvem nahajališču jetrenjaka Riccia atromarginata na zahodnem delu Balkanskega polotoka. Nahajališče se nahaja na konglomeratnih terasah sotočja rek Ribnice in Morače v Podgorici (Črna gora). Za natančen opis glavnih značilnosti vrste smo uporabili primerke iz Črne gore ter z otokov Tenerife, Gran Canaria in Ciper Za nekatere značilnosti vrste smo ugotovili jasno variabilnost. Key words: bryophytes, liverworts, thallus, spores, comparative analysis, Podgorica, Montenegro. Ključne besede: mahovi, jetrenjaki, steljka, spore, primerjalna analiza, Podgorica, Črna gora. Received: 23. 7. 2020 Revision received: 23. 9. 2020 Accepted: 24. 9. 2020 1 Natural History Museum of Montenegro, T rg Vojvode Bećir-bega Osmanagića 16, 81000 Podgorica, Montenegro. E-mail: sneza.dragicevic72@gmail.com 2 Studienzentrum Naturkunde, Universalmuseum Graz, Weinzöttlstraße 16, 8045 Graz, Austria. E-mail: martina.poeltl@museum-joanneum.at 3 Intitute of Biology, Department of Plant Science, University of Graz, Holteigasse 6, 8010 Graz, Austria. E-mail: edith.stabentheiner@uni-graz.at; christian.berg@uni-graz.at Snežana Dragićević1  , Martina Pöltl2  , Edith Stabentheiner3  & Christian Berg3  DOI: 10.2478/hacq-2020-0020 20/1 • 2021, 49–56 20/1 • 2021, 49–56 50 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology Introduction The thallous liverwort Riccia atromarginata Levier has a Holarctic distribution. It grows from the Mediterranean to the Arab peninsula, central Asia, and in North and Central America (Jovet-Ast 1986, Schuster 1992, Bischler 2004). According to Bischler (2004), in the Mediterrane- an, the species is distributed across Portugal, Spain, Alge- ria, Tunisia, Libya, Egypt, Israel, the Canary and Balearic Islands, Morocco, France, Italy, Sardinia, Sicily, Greece, and Crete. R. atromarginata was also reported in Turkey (Özenoğlu et al. 2019). The recently published checklist of European Bryophytes (Hodgetts & Lockhart 2020) lists additional records from Madeira and Cyprus in accord- ance with Sérgio et al. (2006) and Frahm et al. (2009). Dierßen (2001) described the range of the liverwort R. atromarginata by vegetation zones – occurring in tropical and Mediterranean regions with evergreen hardwood and coniferous forests, as well as meridional steppes and semi- deserts in the continental parts. It grows on dry, moder- ately acidic to basic, compact soils with low to medium nutrient content, fully exposed to light and often wind. Habitats range from dune sand with some trampling in- fluence, to deep or thin open soil between woody plants, stones and rocks. Study area Podgorica, the capital city of Montenegro, is located in the south-eastern part of the country. The largest part of Podgorica-Skodra valley, as well as the northwestern, northern and eastern parts of the surrounding mountain slopes (Kučke and Piperske mountains) administratively belong to Podgorica, as do the hills of the Lješanska na- hija (Radojičić 1996; Radojičić 2002). The hydrological network consists of the Morača River and its tributaries: Zeta, Cijevna, Ribnica, Matica, and Sitnica, as well as the Mareza springs (Ičević 2003). Geologically, the area is characterized by Quaternary sediments, represented by glaciofluvial, alluvial, terrace and lake sediments. (Đokić & Petrović 1974). The calcareous alluvial depos- its of gravel and sand turned into the conglomerate on which a shallow and skeletal layer of brown eutric soil was formed (Fuštić 1997; Fuštić & Đuretić 2000). The area of Podgorica belongs to the thermophytic deciduous for- est vegetation zone, characterized mainly by the associa- tion Rusco-Carpinetum orientalis (Blečić & Lakušić 1976). Only small fragments of natural vegetation are present (on the city’s hills: Gorica, Malo Brdo, Dajbabska Gora and Kakaricka Gora, (Stešević & Jovanović 2008), since it is replaced by secondary vegetation comprised of grass- lands, agricultural fields, urban areas, etc. According to the Köppen climate classification (Köppen 1936), Pod- gorica and the surroundings belong to the climate sub- type Csa – Mediterranean climate characterized by hot, dry and clear summers, and mild and rainy winters; while summer is the driest period of year, maximum rainfall oc- curs in November, and is lowest in July. The climate is different from the real Mediterranean climate because of its slightly colder winters (Burić et al. 2014). According to Burić et al. (2012), the mean annual precipitation in Pod- gorica is 1654 mm, occurring mainly during the winter. There are 121 rainy days and 129 summer days with mean temperature above 24 °C. The mean annual temperature is 15.3 °C, the mean highest temperature is 26.4 °C in summer and 7.6 °C in winter. Snowfall is very rare. The area Skaline, were the Ribnica River flows into the Morača River, is a frequently visited historic place in the city centre of Podgorica. During the Holocene, the rivers incised deep erosion valleys into the fluviatile conglomer- ate. The cliffs are steep, and often vertical. Methods Our work was based on descriptions of the main morpho- logical characters of the R. atromarginata from Podgorica and our living samples from Tenerife, Gran Canaria and Cyprus, and its comparison with available data from the literature: the shape and width of the thallus, margins, sides, groove, ventral scales, cilia, and spores. To do so, we measured a thalli, and made cross sections of our ma- terial, and inspected and measured spores under a light microscope. We tested various agents commonly used in microscopy for bleaching Riccia spores because of its very dark spores which not allow a clear view of all spore characteristics (for example the wall, which is impor- tant for determination). Bleaching with chloral hydrate only sometimes worked. Otherwise, we used sodium hypochlorite, which leads to completely hyaline spores within a couple of minutes (Berg & Pöltl 2020b). Results Riccia atromarginata, a new species for the bryophyte flora of Montenegro Riccia atromarginata Levier was found in Skaline, at the confluence where the river Ribnica flows into the river Morača, in Podgorica (Figure 1). Its habitat is in shallow soil on conglomerate terrace along the rivers, with south- ern exposure (N 42°26'22.49" E 19°15'30.06", 22 m a.s.l., leg. S. Dragićević & Ch. Berg, 27.02.2020, GJO 101207, Riccia project-ID ABry_517). 20/1 • 2021, 49–56 51 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology Figure 1: Skaline, at the confluence of the Ribnica and Morača rivers, the location where Riccia atromarginata was discovered. Slika 1: Skaline, sotočje reke Ribnice z reko Moračo, nahajališče vrste Riccia atromarginata. Figure 2: Habitat of liverwort Riccia atromarginata on Skaline in Podgorica. Slika 2: Habitat vrste Riccia atromarginata na območju Skaline v Podgorici. 20/1 • 2021, 49–56 52 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology These terraces are often covered by city buildings or parks, or are used as pastures (sometimes only sporadi- cally). Nearby terraces with deeper soil layers are covered by more mesophilous grasslands, while in areas with a thin soil layer, which are more suitable for liverworts, dry grass- land dominates (Figure 2). The conglomerate terraces of the river Morača in Podgorica provide an extraordinary habitat for thallose liverworts, where besides R. atromar- ginata, Reboulia hemisphaerica, Mannia androgyna, Ox- ymitra paleacea, Riccia nigrella, R. ciliata, R. papillosa, R. michelii and some not yet identified Riccia species occur. Inside this habitat type, R. atromarginata prefers patches of open soil at or near the upper cliff edge, where grassland vegetation is even sparser due to erosion processes. Morphology of the species Descriptions of Riccia atromarginata were given in the protologue of Levier (1889) and later Müller (1906– 1911), Jovet-Ast (1986), and Schuster (1992). For the description presented here, we use primary data from our “Riccia morphology and sequencing project”, which in- clude collections of R. atromarginata from Tenerife (GJO 101208), Gran Canaria (GJO 100874), Montenegro (GJO 101207) and Cyprus (GJO 101206, GJO 101209). Riccia atromarginata belongs to the larger Riccia spe- cies of the subgenus Euriccia, with thallus segment width often exceeding 1 mm. In our samples, thalli width is (0.4–)0.8–1.7(–1.8) mm. The thallus segments are ovate Figure 3: Riccia atromarginata A Thalli from Gran Canaria with numerous cilia even on ventral thallus side; B cross section of thallus from Montenegro with epidermal cells of different shape and size; C, D cross section of ultimate thallus segment with dorsal cilia, collection from Tenerife. Scale bar 200 µm. Slika 3: Riccia atromarginata A steljka s Kanarskih otokov s številnimi cilijami tudi na ventralnem delu; B prerez steljke iz Črne gore s celicami epidermide različnih oblik in velikosti; C, D prerez sprednjega dela steljka z dorzalnimi cilijami; zbirka s Tenerifa. Merilo 200 µm. 20/1 • 2021, 49–56 53 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology to oblong with rounded apices growing in loose batches or small groups, but not in rosettes, sometimes as undi- vided segments, often with one or two furcations, rarely with more. The upper side is vivid green in young parts, and dark bluish green in older ones. Margins and sides are conspicuous deep purple, visible from dorsal view. The groove is sharp and deep on the thallus apex but opens just behind the top becoming wide and flat on the termi- nal thallus segments, while older thallus parts are mostly flat or even convex on the dorsal surface. Ventral half scales at the thallus apex are small, deep purple to hyaline and disappear towards thallus centre. The marginal thal- lus edge is sharp to obtuse. On this edge, sometimes even on the upper surface and the sides, conic, obtuse to acute, hyaline cilia (in some determination keys called “papil- lae”) are formed with a length up to 270 µm (Figure 3A, C and D). At the ultimate segment, short conical cilia nearly always exist at the marginal edges. They are bristle- like, and stand dense together in two or more rows form- ing a fringe (Figure 3C). The epidermal cells are normally globose or pyriform with high variability (Figure 3B). The appearance and length of cilia varies greatly within the distribution range, and even within one population, where thalli with many cilia can occur right next to nearly glabrous ones. All epidermal cells including enlarged ones erode away with age. In cross section, the youngest thal- lus-segments are (1)2–3 times wider than high. In contrast to other descriptions in the literature and our living samples from the Canary Islands and Cyprus, the thalli from Podgorica do not show cilia on the dorsal surface, and the marginal cilia are comparable short (up to 50 µm). The marginal edges are rather sharp in cross section (Figure 4). Figure 4: Riccia atromarginata from Podgorica, A–D habitus view and E–G cross section of ultimate thallus segment. Slika 4: Riccia atromarginata iz Podgorice, A–D habitus, E–G prečni prerez sprednjega dela steljke. 20/1 • 2021, 49–56 54 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology The species is considered as dioecious by Jovet-Ast (1986). Levier (1889) wrote “planta ut videtur dioica” means “looks like dioicous”. Schuster (1992) considered the species as monoicous with the remark “sometimes ap- pearing dioicous!”. This feature is not easy to verify. In our living collection, R. atromarginata was sporulate in all our samples, while dioicous species like Riccia cilliifera- complex or R. michelii remain without capsules over more than two years of cultivation. all around the spore. Proximal triradiate ridges are only weakly indicated, but a weak edge is visible giving an idea of proximal triangles (Figure 5B, 5D). In all Riccia-liter- ature, spore size is considered as a stable and diagnostic feature. Within all analysed R. atromarginata-samples, the spores from those of Podgorica are comparably small with 64 µm minimum, 90 µm mean, and 116 µm maximum size; 93% of all spores are 80 µm or larger (54 spores measured). Our samplings from Tenerife, Montenegro and Cyprus differ significantly in spore size, with a ma- jority of spores larger than 80 µm and a mean diameter around 100 µm (Figure 6). We measured 140 spores so far from three individuals of three different population from Tenerife, Montenegro and Cyprus, with minimum size of 64 µm, lower quartile 91 µm, median 101, mean 106 µm, upper quartile 120 µm and maximum 159 µm. Figure 5: Spores of Riccia atromarginata from Podgorica, A distal view light microscope, B proximal view light microscope, C distal view scanning electron microscope, D proximal view scanning electron microscope. Scale bar 100 µm. Slika 5: Spore vrste Riccia atromarginata iz Podgorice. A distalno (svetlobni mikroskop), B proksimalno (svetlobni mikroskop), C distalno (elektronski mikroskop), D proksimalno (elektronski mikroskop). Merilo 100 µm. The spores are rather characteristic, with a somewhat apolar appearance, i.e., spores look similar from every side view, without a pronounced proximal and distal face (Figure 5). Spores are more globose than round triangular like in typical polar spores (f.e. R. glauca or R. bifurca, see Berg & Pöltl 2020a). The marginal wing is nearly lacking or small, and under a light microscope, a light margin can appear due to the loose exospore (Figure 5B), but the wing is never typical smooth, and never without ornamentation. The distal face of the spores shows 10–20 areoles in diameter, with nearly the same sized areoles Figure 6: Distribution of spore diameter (in µm) of Riccia atromar- ginata based on 140 spores from three individuals from Tenerife, Montenegro and Cyprus. Slika 6: Premer spor vrste Riccia atromarginata na osnovi meritev 140 primerkov s Tenerifa, Cipra in Črne gore. The histogram of this measurements (Figure 6) shows a striking bimodal distribution. This could indicate (a) a high variability of spore size between populations or be- tween capsules, or (b) the genetic influence of R. trabu- tiana (or possibly another cryptic species) with much smaller spores, or (c) a continuum of characteristics be- tween both taxa. Mature spores are dark brown to black, and are difficult to observed under a light microscope. 20/1 • 2021, 49–56 55 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology Discussion Even in light of the taxonomic uncertainties, we consider our sample to be Riccia atromarginata, because under the current state of knowledge, the sum of characteristics of thallus and spores points clearly in the direction of this species. Schuster (1992) discussed the high global vari- ability of the species, but concluded that the common characteristics were so striking that it nevertheless count- ed as a unique species. However, uncertainty always re- mains on a global scale, especially because there are simi- lar species outside the known area of R. atromarginata, like the Australian R. inflexa Taylor, whose identity has not yet been finally clarified (Cargill et al. 2016). The example that we present here shows that many problems are still unsolved in this genus. In our “Ric- cia morphology and sequencing project” (Berg & Pöltl 2020a, 2020b) we try to detangle the European taxa using morphological and molecular data, and with the help of living collections. Many species, like Riccia sub- bifurca or R. ligula, are unclear, and many species pairs (e.g. R. ciliata/intumescens, R. ciliifera/gougetiana, R. com- mutata/warnstorfii, R. beyrichiana/michelii) need deeper analysis of their delimitation. However, R. atromarginata seems to be one of the more easily identifiable taxa; only the delimitation to R. trabutiana could be problematic. This taxon is similar with dark red margins and spores of similar structure, but smaller in all parts including spore size, and never seen with papillae or ciliae, which are characteristic for R. atromarginata. This new record is important as it helps us in our aim to resolve morphological differences between European Ric- cia taxa, but also fills the distribution gap between Italy and Greece. Acknowledgements The authors are grateful to reviewers who made contri- butions to improve the quality of the manuscript and to Adam Clark (University of Graz), who revised the Eng- lish. Also, thanks to Boštjan Surina from Natural history museum Rijeka/University of Primorska, Koper who con- tibuted with useful suggestions and translated text into the Slovenian. This research has been partially funded by the Ministry of Science of Montenegro. Snežana Dragićević  https://orcid.org/0000-0002-2788-5211 Martina Pöltl https://orcid.org/0000-0002-3001-058X Edith Stabentheiner  https://orcid.org/0000-0001-5228-8390 Christian Berg  https://orcid.org/0000-0002-0587-3316 References Berg, C. & Pöltl, M. 2020a: Spore development during capsule maturation of three Riccia species. Herzogia 33: 179–187. DOI: https://doi.org/10.13158/heia.33.1.2020.179 Berg, C. & Pöltl, M. 2020b: Comparison of different bleaching agents for bryophyte spores – the genus Riccia as an example. Herzogiella 7: 56–62. Bischler, H. 2004: Liverworts of the Mediterranean. Bryophytorum Bibliotheca 61: 252. Blečić, V. & Lakušić, R. 1976: Prodromus biljnih zajednica Crne Gore. Glasnik Republičkog zavoda za zaštitu prirode i Prirodnjačkog muzeja, Titograd, 9: 57–98. Burić, M., Micev, B. & Mitrović, L. 2012: Atlas klime Crne Gore. CANU, Leksikografski centar, Knjiga 2, Podgorica, 132 pp. Burić D., Ducić V. & Mihajlović J. 2014: The climate of Montenegro: modificators and types – Part T wo. Bulletin of the Serbian Geographical Society 94: 73–90. Cargill, D. C., Neal, W. C., Sharma, I. & Gueidan, C. 2016: A preliminary molecular phylogeny of the genus Riccia L. (Ricciaceae) in Australia. Australian Systematic Botany 29, 197–217. DOI: https://doi.org/10.1071/SB16018 Dierßen, K. 2001: Distribution, ecological amplitude and phytosociological characterisation of European bryophytes. Bryophytorum Bibliotheca 56: 1–289. Đokić, V . & Perović, Z. 1974: Geologija i tektonika terena sliva Morače uzvodno od ušća Zete. Geološki glasnik. Zavod za geološka istraživanja Crne Gore, Titograd, Knjiga 7: 5–35. Frahm, J., Lüth, M., & van Melick, H. 2009: Die Moose Zyperns. Archive for Bryology 46: 1–8. Fuštić, B. 1997: Zemljišta sliva Skadarskog jezera. Prirodne vrijednosti i zaštita Skadarskog jezera. Naučni skupovi, CANU, Knjiga 44: 187–195. Fuštić, B. & Đuretić, G. 2000: Zemljišta Crne Gore. Univerzitet Crne Gore, Biotehnički institut, Podgorica, 626 pp. Hodgetts, N. & Lockhart, N. 2020: Checklist and country status of European bryophytes – update 2020. Irish Wildlife Manuals, No. 123. National Parks and Wildlife Service, Department of Culture, Heritage and the Gaeltacht, Ireland, 87 pp. Köppen, W. 1936: Das geographische System der Klimate. In: Köppen, W. & Geiger, R. (eds.): Handbuch der Klimatologie. Gebrüder Borntraeger, Berlin, pp. 1–44. Jovet-Ast, S. 1986: Les Riccia de la région méditerranéenne. Cryptogamie, Bryol. et Lichenol 7: 283–431. Levier, E. 1889: Riccia atromarginata Levier spec. nov. Nuovo Giornale Botanico Italiano 21: 291–292. Müller, K. 1906–1911: Die Lebermoose Deutschlands, Österreichs und der Schweiz mit Berücksichtigung der übrigen Länder Europas. I. Abteilung. In: Dr. Rabenhorst‘s Kryptogamen-Flora Deutschlands, Österreichs und der Schweiz, Band 6, Verlag von Eduard Kummer, Leipzig, pp. 1–870. 20/1 • 2021, 49–56 56 Snežana Dragićević, Martina Pöltl, Edith Stabentheiner & Christian Berg Riccia atromarginata (Ricciaceae, Marchantiophyta) new to the Western Balkans, with notes on its morphology Özenoğlu, H., Kirmaci, M. & Kiremit, F . 2019: Contributions to the genus Riccia L. (Ricciaceae) in T urkey. T urkish Journal of Botany 43: 253–261. DOI: doi:10.3906/bot-1801-25 Radojičić, R. 1996: Geografija Crne Gore – prirodna osnova. Unireks Nikšić, Nikšić, 252 pp. Radojičić, R. 2002: Geografija Crne Gore – društvena osnova i regije. Dukljanska akademija nauka i umjetnosti, Podgorica, 416 pp. Schuster, R.M. 1992: Riccia [Micheli] L. In: Schuster R. M. (ed.): The Hepaticae and Anthocerotae of North America East of the hundredth Meridian. Field Museum of Natural History Chicago, Vol. 6: 421–709. Sérgio, C., Sim-Sim, M. & Carvalho, M. 2006: Annotated catalogue of Madeiran bryophytes. Boletim do Museu municipal do Funchal (Historia natural), Suppl. 10, 163 pp. Stešević, D. & Jovanović, S. 2008: Flora of the city of Podgorica, Montenegro (taxonomic analysis). Archives of Biological Sciences 60 (2): 245–253.