Pol. J. Environ. Stud. Vol. 32, No. 1 (2023), 833-841 ONLINE PUBLICATION DATE: 2022-12-05 DOI: 10.15244/pjoes/152725 Original Research Discovering the Rare Tolypella intricata (Trentepohl ex Roth) Leonh. 1863 (Charales, Charophyceae) in Poland Jacek Urbaniak1*, Rafał Krawczyk2 Department of Botany and Plant Ecology, Wrocław University of Environmental and Life Sciences, pl. Grunwadzki 24a, 50-363Wrocław, Poland 2 Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland 1 Received: 25 June 2022 Accepted: 10 August 2022 Abstract Tolypella intricata is one of the rarest charophytes from the genus Tolypella that can be found both in Poland and in Europe. Rarely found in the last 200 years, it was recently rediscovered in two localities in eastern Poland in the Lublin region. In this article, we present two new locations of T. intricata in Poland that make is a significant contribution to a closer understanding of the distribution of the biology and ecology of this species. The article provides basic information on the autecology of T. intricata together with the associated macrophyte vegetation. The morphology and ecology data are compared with the literature, along with a critical review of such information found in the literature. The results of molecular tests confirming the new position of T. intricata are also presented. Keywords: biogeography, charophyta, distribution, ecology, Tolypella intricata, Poland Introduction In general, genus Tolypella (A.Braun), belonging to the class Charophyceae (order Charales, family Characeae), is one of the rarest genera in the whole class. Currently, 21 species are accepted taxonomically in the genus under the Tolypella species name [1]. Of this genus, species Tolypella intricata (Trentepohl ex Roth) Leonh. 1863 seems to be one of the rarest charophytes species in Europe and worldwide [2]. It was first discovered and collected probably in the vicinity of Berlin and Leipzig (Eastern Germany). The available *e-mail: jacek.urbaniak@upwr.edu.pl information allows us to state that until the end of the 20th century, only a few sites where T. intricata was recorded were known. All currently known information on the distribution of T. intricata is provided in Table 1 and Fig. 1. So far, there are no more recent data on the prevalence of T. intricata in Europe. Certainly, T. intricata is an extremally rare species that is not easy to find, and this is one reason why no new localities of this specimen have been identified. This study aims to report the two new records of T. intricata in Eastern Poland located in the Lublin Region. The discovered populations of T. intricata are described in detail and compared with literature data. This new finding of T. intricata is discussed as a significant contribution to the knowledge on this species biogeography. 834 Urbaniak J., Krawczyk R. Materials and Methods All data presented in Table 1 on the known locations of T. intricata were collected from information obtained using queries of herbarium collections (C, GB, GLM, H, JE, KRA, L, LD, NY (C.V. Starr Virtual Herbarium), POZ, WRSL, WAW) and literature data. Acronyms of herbaria are given after NYBG Index Herbariorum [3] Algae scientific names are given after AlgaeBase [1] and IPNI [4] The abbreviated author names for plant names are given as in IPNI [4] following recommendations of ICN [5]. Below, we describe the two more recently found localities of T. intricata in Poland. The species was found by accident during many years of monitoring research in the area of over a dozen or so kilometers along the Vistula River. In further studies, no other localitites of T. intricata occurrence were found. In the case of the specimen collected near Witkowice, molecular DNA analyses were performed to confirm the discovery of T. intricata. The algae from the locality near Witkowice was grown in artificial conditions (greenhouses) in 2019 from soil samples (diaspors) collected in early spring 2018. We have also used determination keys [6, 7] to confirm both findings. Table 1. Summary of all information known so far on the occurrence of T. intricata in Europe Locality Herbarium code, References Belgium 1) East Flanders: Antwerp NY Denmark 1) Sydsjylland C, NY, Olsen [35] France 1) Centre-Val de Loire, Loir-et-Cher: Romorantin 2) Nouvelle-Aquitaine, Gironde: Bordeaux 3) Pays de la Loire, Sarthe: Le Chevain near Alençon, Sarthe Précigné Germany 1) NSG Bislicher Inseln 2) Brandenburg: Berlin, Schöneberg, Falkensee, Trebbin b. Berlin, Neuruppin vor Kränzlin 3) Baden-Württemberg: Karlsruhe (Brechtsee) 5) Saxen: Bienitz bei Leipzig 6) Niederlausitz: Golßen 6) Nordrhein-Westfalen: Krefeld NY, Corillion [36] NY NY NY U.Abts (private collection) NY NY, Weyer [29] NY NY NY Grece 1) Attica NY Hungary 1) Budapest-Rákosfalva NY Ireland 1) close to Dublin Moore [37] Italy 1) Gargano 2) Lombardia 3) Veneto 4) Emilia-Romagna WRSL Bazzichelli and Abdelahad [38] Bazzichelli and Abdelahad [38] Bazzichelli and Abdelahad [38] Netherland various localities Briunsma et al. [39] Poland 1) Warmia: Carlshof bei Altfelde 2) Wielkopolska: Valley of Warta River (Wielkopolska region) (C, GLM, H, LD, POZ, NY, W) Gąbka [40] Spain 1) Castilla - La Mancha: Cuenca, Torca del Agua NY Sweden 1) Gotland:Hangvar, Lerdala, Slite,Visby, 2) Øland 3) Skåne: Alnarp, Christianstad Lund, Trelleborg, Uppsala, NY [41] NY Serbia 1) Valjevac and Pačja 2) Zasavica [42] [43] Discovering the Rare Tolypella intricata... 835 Table 1. Continued. United Kindgom 1) England, Cambridgeshire: Harston, Welches Downs, The Wash below Mepal, Sutton 2) England, Yorkshire: Goole 3) England, Bedfordshire: Brammingham near Luton 4) England, Surrey: Egham 5) England: Shortwood Common near Staines 6) England: Gloucestershire NY, [37] NY NY NY NY NY [44] Fig. 1. Distribution of T. intricata in Europe - drawing based on Table 1 and contained therein references and data from Herbaria collection. Black dot - historical records<1945, white dot – record found near Karlsruhe, Germany at 2003 (NY), stars - recently found records (this paper). Phylogenetic Research The genomic DNA was isolated using the DNeasy Plant Mini Kit (Qiagen; Hilden Germany), according to the manufacturer’s protocol using Mixer Mill MM400 (Retsch; Haan, Germany) for plant tissue disruption. We have used psbC chloroplast DNA region that is used for phylogenetic studies in plants at all taxonomic levels that allowed confirmation of the newly sequenced cpDNA fragment from collected T. intricata, with present data available at NCBI (Gene Bank). The used region: psbC is often considered evolutionary conservative but employed in phylogeny and taxonomy, and some studies have found intraspecific variation in this biogeographically informative gene regions that has been contradicted [7]. PCR reaction mix included (in the total volume of 20 µl): 1U Taq recombinant polymerase (Thermo-Fisher Scientific), 10X Taq Buffer, 1 mM MgCl2, 0.5 µM of each primer, 0.4 mM dNTP and 1 µl DNA template. PCR cycle was performed with a Veriti Thermal Cycler (Life Technologies, Carlsbad, CA, USA) with the following parameters: 8 min at 95ºC, followed by 30 cycles of 45 s at 95ºC, 45 s at annealing temperature (51.2ºC) and 1min at 72ºC, followed by a final extension step of 10 min at 72ºC. Prior to sequencing, PCR products were purified using GeneMATRIX PCR/ DNA Clean Up Purification Kit (Eurx, Gdańsk, Poland). Sequencing, post-reaction purification and reading were done by Genomed (Warsaw, Poland) using an ABI 377XL Automated DNA Sequencer (Applied Biosystems, Carlsbad, CA, USA). All sequences are available in GenBank (accession numbers: SP114263-SP114265). All molecular analyses has been done at Department of Botany 836 Urbaniak J., Krawczyk R. and Plant Ecology Wrocław University of Environmental and Life Sciences). The obtained psbC sequences were aligned using DNA Baser Sequence Assembler v4 [8] and checked for nucleotide variation using Mega 11 software [9]. The cpDNA sequences were aligned using Muscle software implemented into Mega 11 [9, 10] before the phylogenetic analyses. We performed maximum likelihood (ML) to infer the phylogenetic relationships among selected individuals of Charophyte nucleotide collected from NCBI. Bootstraps analyses are based on 1000 replications of full heuristic searches. The results based on the new data and data collected from GeneBank are presented in Table 2. Results and Discussion Collection The new localities of T. intricata were located at: (1) a small river lake close to the Vistula river near the village Witkowice (Figs 2-3), near Stalowa Wola Table 2. Voucher details and GenBank psbC gene accession numbers of taxa used in this study. An-dash (-) indicates unavailable information; new sequences are written bold. Taxa Isolate, Voucher Locality psbC gene NCBI accession number Chara foliosa KGK0233, Proctor138 - MG880182.1 Chara foliosa KGK0333, NY02146579 - MG880181.1 Chara foliosa KGK0341, NY00739274 - MG880178.1 Chara globularis KGK1574, NY02282230 - KX430968.1 Chara tomentosa -,- - KJ395866.1 Lamprothamnium heraldii KGK0069, - - KJ395867.1 Lamprothamnium macropogon -,- - KJ395868.1 Lychnothamnus barbatus KGK4840, NY02020578 Lithuania MF166879.1 Lychnothamnus barbatus KGK3082, NY02146274 - MF166876.1 Nitella axiliaris KGK2327, NY00739255 - KX430974.1 Nitella flexilis KGK1769, NY02137614 - KX430978.1 Nitella hyalina KGK0190, NY02282228 - KX430979.1 Nitella hyalina KGK0059b, - - KJ395873.1 Nitella opaca F146, - - KJ395876.1 Nitellopsis obtusa KGK2013, NY02146765 - KX430980.1 Nitellopsis obtusa F131B, - USA KJ395878.1 Tolypella canadensis WP0270, NY02026386 USA KJ395828.1 Tolypella glomerata WP0168, NY01475172 USA KJ395848.1 Tolypella glomerata WP0273, NY02026388 Canada KJ395841.1 Tolypella glomerata WP0263, NY02026383 Canada KJ395840.1 Tolypella glomerata WP0285, NY01003601 USA KJ395831.1 Tolypella intricata WP0147, NY01475169 - KX430983.1 Tolypella intricata WP0040, NY01475199 USA KJ395844.1 Tolypella intricata WP0058, NY01474971 USA KJ395842.1 Tolypella intricata WP0073, NY01474970 USA KJ395829.1 Tolypella intricata JB1477, - this paper Poland SP114263.1 Tolypella intricata JB1478, - this paper Poland SP114264.1 Tolypella intricata JB1479, - this paper Poland SP114265.1 Tolypella prolifera WP0260, NY02026384 USA KJ395856.1 Tolypella prolifera WP0060, NY01003496 Canada KJ395843.1 Discovering the Rare Tolypella intricata... Fig. 2. Occurrence of T. intricata near Witkowice. Fig. 3. Small river lake close to Vistula river near village Witkowice where T.intricata was found. (Central Poland, Nadwiślańska Valley, Sandomierska Basin), 50°45′38″N, 21°50′46″E, 08-06-2009. T. intricata was collected using grapnels, then dried and stored in the collection of dry specimens in the Department of Botany and Plant Ecology Wrocław University of Environmental Sciences. (2) a temporary pond among arable fields near Kotlice village (Figs 4-5), near Hrubieszów (Kotlina Hrubieszowska, Wyżyna Wołyńska), 50°40′54″N, 23°35′28″E. T. intricata was grown in artificial conditions (greenhouses) in 2019 from soil samples (diaspors) collected in early spring 2018. Description of T. intricata Specimens from the Two New Localities Found in Poland Both newly found specimens of T. intricata were medium-size, 25-40 cm-high plants, branched with plant main axis 1.0-1.5 mm in diameter. Plants were greyish green and moderately to heavily encrusted (Witkowice, Figs 6-8). 837 Fig. 4. Occurrence of T. intricata near Kotlice. Fig. 5. Small temporary pond among arable fields near Kotlice village. The internodes were up to three times as long as the branches. We have observed 7-8 fertile branchlets in a whorl in very dense heads, divided once or twice with nodes bearing 3-6 lateral rays. Secondary rays with 1-2 nodes bore 5-7 celled tertiary rays. The end segment was 3-4 cells, with the last cell conical up to acute. About 7-8 sterile branchlets in a whorl were long, simple, and divided once or twice with one node bearing 2-3 lateral rays. The end cell was conical to acute. Lateral rays were elongated, composed of 4-6 cells with the end cell conical and acute. Dense heads consisted of two or more reduced fertile whorls on the upper shortened part of branch axes. All studied T. intricata specimens were monoecious plants with gametangia at fertile branchlet nodes at the base of fertile branchlets. Oogonia were numerous, aggregate, 2-4 at a node, 385-460 μm long, 280-465 μm wide. Oospores were dark brown 305-360 μm long and 220-360 μm wide. Antheridia were generally solitary and were about 310-360 μm in diameter (data are based on measurements of approximately 15-20 oospores and 5-7 antheridia). 838 Urbaniak J., Krawczyk R. Phylogenetic Analyses Fig. 6. General appearance with reduced fertile whorls of T. intricata. As similar as in case phylogenetic analysis presented prevoiusly [11, 12] genus Tolypella was monophyletic in performed analysis and both sections: sect. Tolypella to which belong T. glomerata, as well as sect. Rothia (T. intricata, T. prolifera, T. canadensis) were strongly supported in the ML bootstrap analysis (Fig. 9). Additionally, in a sect. Rothia all studied sequences that origin from two species T. intricata and T. prolifera formed two separate clades with strong bootstrap support. One of them contained seven individuals consisted of sequences that come from T. intricata (three newly sequenced and four other collected from NBCI), whereas second was formed by two specimens of T. prolifera. Single sequence of T. canadensis formed a sister clade to them, but weaker supported. Species from the genus Nitella (inside tribe Nitellae) were paraphyletic to Tolypella and as similar as in results obtained by [13, 14] was a sister clade to Chareae (Chara, Lamprothamnium, Lychnothamnus and Nitellopisis). In general, the newly collected in Poland sequences from T. intricata samples together with samples from NCBI was separated from T. prolifera. Both species formed monophyletic clades strongly supported by bootstrap analyses ≥99 (Fig. 9). Notices about the Occurrence of T. intricata Fig. 7. Branchlets fertile in a whorl in dense heads of T. intricata. Fig. 8. Fertile branchlets with oogonia and visible end segment with the last cell conical, acute of T. intricata. It seems that T. intricata in SE Poland can be found in two types of habitats. The first type near the town of Witkowice is small river lakes formed on the active floodplain of a large river, such as the Vistula or the Warta rivers. These are astatic ponds periodically drying out partially or completely. The substrate is dominated by a fine-grained mineral fraction. The aquatic vegetation in such reservoirs is poorly developed and not very rich in species; the species found are often pioneering species. The following species of vascular plants include Potamogeton trichoides Cham. & Schltdl., Ceratophyllum demersum L., Elodea canadensis Michx., Myriophyllum spicatum L., Potamogeton natans L. and Nuphar lutea (L.) and Chara globularis Thuil. Marsh vegetation is more well developed and built by patches of Glyceria maxima (Hartm.) Holmb., Phalaris arundinacea L., Bulboschoenus maritimus (L.), Palla Oenanthe aquatica (L.) Poir., and Rorippa amphibia (L.) Besser. Important components are also pioneering annual species growing at the edge of waterside habitats, such as Polygonum L. spp. and Bidens L. spp.; Agrostis stolonifera L., Ranunculus sceleratus L. and Alopecurus aequalis Sobol.; and pleuston species, such as Lemna minor L., Spirodela polyrhiza (L.) Schleid., and Salvinia natans (L.) All. The second type of habitat where T. intricata was found is a temporary pool with a diameter of 70-80 m that occasionally appears (in the years with abundant Discovering the Rare Tolypella intricata... 839 Fig. 9. Maximum Likelihood tree of the psbC gene analyses performed to confirm the discovery of T. intricata. Bootstrap analyses with values above 50 are included. The scale bar indicate 2% divergence. rainfall) in a water body in the agricultural landscape [15, 16]. Such a shallow water body developed in the Lublin region in karst depressions in arable fields. In this case, the substrate was clay soil covering shallow chalk rocks, and the formation of wetlands in this place was last observed in 2013 [17]. The vegetation found in this location was dominated by amphibious plants – dwarf ephemeral wetland species and low growing marsh species such as Alisma lanceolatum With., Alisma plantago-aquatica L., Alisma gramineum Lej., Isolepis supina (L.) R.Br., Limosella aquatica L., Lythrum hyssopifolia L., Elatine alsinastrum L., Potentilla supina L., Rorippa palustris (L.) Besser, Polygonum amphibium L., Polygonum lapathifolium L., Callitriche verna L., L. minor, Juncus bufonius L., Oenanthe auqatica (L.) Poir., Veronica beccabunga L., Plantago intermedia Gilib., Sparganium erectum L., and Typha angustifolia L. As mentioned earlier, T. intricata was not recorded here but grew out of the collected sediment in the laboratory together with Chara globularis Thuill., Chara vulgaris L.. Present Distribution of and Threats of T. intricata Currently, it is very difficult to unambiguously determine the distribution of T. intricata and the species conservation protection status in Europe [18-23]. Due to the sporadic records of the species and its periodical findings, almost all information concerning its distribution in concerns historical sites and only three 840 Urbaniak J., Krawczyk R. including both described in this paper were found in 21 century (Fig. 1). T. intricata, on the other hand, is much more common in the USA [13, 14, 24]. This is probably related to the biology of the species, which is found periodically in ephemeral sites and is often not recorded every year, as, for example, in the case of Kotlice. In most of the red lists of protected species, T. intricata has an endangered status [25-28] and is considered a very rare species [29]. It must also be remembered that the other species belonging to the genus Tolypella are also rare [11]: T. glomerata Leonh. 1863, T. canadensis T.Sawa 1973, T. hispanica Nordst. ex T.F. Allen 1888, T. normaniana (Nordst.) Nordst. 1868, T. salina Corill. 1960. Only T. nidifica (O.F.Müller) A.Braun 1857 is a locally quite common species found in the Baltic Sea. Fig. 1 and Table 1 show the places where T. intricata has been found and the two described sites from Poland. It is clear that there are not many locations. This is certainly influenced by the biology of the species and its sporadic finding and by the decrease in field research in recent years. It is often assumed that the distribution of plant species and the decline in biodiversity are influenced by the progressive anthropopression and destruction of habitats [19, 20, 30]. Furthermore, all known descriptions and locations of the occurrence of this species come from very specific sites, such as small water reservoirs, pits, canals and in-field reservoirs. They are, of course a subject of anthropopression, but the distribution of T. intricata is also influenced by a poor understanding of these specific habitats and the knowledge that this species can be found in such places [31-34]. This study reported the first record of T. intricata in Poland in over 150 years and also the most recent reliable record of the species in Europe. Results of our study enrich our knowledge distribution and the habitat characteristics and overall ecology of this rare Charophyte species. Our finding significantly contributes to the species biogeography, which is reviewed and discussed. Acknowledgments This research received support from the SYNTHESYS+ project http://www.synthesys.info/ which is financed by European Community Re-search Infrastructure Action under the H2020 Integrating Activities Programme, Project number 823827 (AT-TAF-TA3-1) and from the Leading Research Groups support project from the subsidy in-creased for the period 2020–2025 in the amount of 2% of the subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019 by Wrocław University of Environmental and Life Sciences” Conflict of Interest The authors declare no conflict of interest. References 1. GUIRY M.D., GUIRY G.M. AlgaeBase. World-wide electronic publication National University of Ireland Galway, 2021. Available online: http://www.algaebase.org (accessed on 18 September 2021). 2. URBANIAK J., GąBKA M. Polish Charophytes. An illustrated guide to identification. Publisher: Uniwersytet Przyrodniczy we Wrocławiu, Wrocław, Poland, 1, 2013. 3. 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