A review on the distribution and habitat features of Chara canescens in the Iberian Peninsula: sexual populations revisited

Asociación Ibérica de Limnología, Madrid. Spain. ISSN: 0213-8409 © A review on the distribution and habitat features of Chara canescens in the Iberian Peninsula: sexual populations revisited Adriana Arnal1, María. A. Rodrigo1 , Karl-Georg Bernhardt2 , Riccardo Guarino๎ , Angelo Troía๎Barbara Turnerํ, Johanna Weitzel๏, Hendrik Schubert๏ & Pablo García-Murillo๐ 1 Cavanilles Institute for Biodiversity and Evolutionary Biology, C/ Catedràtic José Beltrán 2, 46980-Paterna (Valencia). University of Valencia, Spain 2 Dep. Integrative Biology and Biodiversity Research, BOKU-University of Natural Resources and Life Sciences, Austria 3 Dep. Biological, Chemical and Pharmaceutical Sciences and Technologies, Univ. Palermo, Italy 4 Institute for Biosciences, Chair Aquatic Ecology, University of Rostock, Germany ๐ Dep. Plant Biology and Ecology, Faculty of Pharmacy, University of Sevilla, Spain *Corresponding author: [email protected] Recibido: 03/06/24 Aceptado: 11/07/24 ABSTRACT A review on the distribution and habitat features of Chara canescens in the Iberian Peninsula: sexual populations revisited. Chara canescens, mainly distributed in Europe and the African Mediterranean coast, is the only Charophyceae species capable of reproducing parthenogenetically, the asexual populations being common; however, the sexual ones are rare. In this study we compile all the literature and herbaria data concerning the presence of C. canescens in the Iberian Peninsula and the Balearic Islands over several decades, analysing the historical and spatial distribution of the species and the limnological characteristics of its habitats. A decline in the number of records in the literature and herbarium sheets since 2010 was detected. Most of the populations are concentrated in Castilla-La Mancha, Andalucía and Castilla-León. The species lives in brackish waterbodies, both in coastal areas and endorheic shallow small lakes, and the majority of these locations do not exhibit a good conservation status. The only three locations with sexual populations previously cited in the literature (Bodón Blanco –Valladolid- and Las Eras –Segovialakes and Lucio Largo – Doñana-) were revisited in June 2023. Sexual populations of C. canescens in the two former lakes occurred, but the lake in the Doñana National Park was completely dry. Two new sexual populations of the species were found in La Iglesia and Caballo Alba lakes 6HJRYLDLQFUHDVLQJWKHFLWDWLRQWR¿YHVH[XDOSRSXODWLRQV$GHVFULSWLRQRISRSXODWLRQFRYHUDJHPDOHIHPDOHUDWLRDQGLQGLYLGXDO (reproductive features) variables for the four C. canescens sexual populations are provided. The analysed limnological variables (water chemistry, other hydrophytes and marginal vegetation) showed that the lakes harbouring these populations exhibited signs of pollution, as well as altered and unstructured marginal vegetation. The genetic diversity of the species depends on the sexual populations, which are very rare in Europe, and are mainly concentrated in Spain. Therefore, the preservation of these inner brackish-water habitats LVHVVHQWLDOIRUWKHLPSOHPHQWDWLRQRIWUDQVQDWLRQDOH൵HFWLYHFRQVHUYDWLRQPHDVXUHVWRSURWHFWWKLVUHOHYDQWDQGSDUWLFXODUVSHFLHV KEY WORDS: Characeae, limnological characterisation, Iberian Peninsula, geographic parthenogenesis, sexual propagation, brackish aquatic ecosystems, aquatic macrophytes. RESUMEN Revisión de la distribución y de las características del hábitat de Chara canescens en la península Ibérica: revisitando las poblaciones sexuales. Limnetica, 44(1): 141-158 (2025). DOI: 10.23818/limn.44.10 Arnal et al. Limnetica, 44(1): 141-158 (2025) 142 INTRODUCTION Charophyta are a group of macroscopic green algae (Streptophyta infrakingdom, family Characeae) that play a fundamental role in the ecosystems in which they develop because they control and provide a multitude of ecological functions and ecosystem services (Coops & Doef, 1996, Meer- KR൵HWDODFWLQJIRUH[DPSOHDVHFRV\V- tem engineers. However, the level of knowledge about this group of algae is far from that of terrestrial vascular plants and has traditionally been scarcely studied in Portuguese and Spanish botanical surveys, with their records often being secondary and included in more general studies &LUXMDQRHWDO$PRQJWKHGL൶FXOWLHVLQ the study of these algae is the lack, in many cases, of specimens preserved in public accessible KHUEDULDWRFRQ¿UPWKHLULGHQWLW\DQGWRFRQWUDVW the information contained in bibliographical references. These circumstances, combined with a high phenotypic variability, complicate their taxonomy and cause some complex nomenclatural problems (Cirujano et al., 2007). Moreover, many species of charophytes are seriously threatened by the consequences of global change: enhanced eutrophication, an increase in temperatures, desiccation and other disturbanc- HVWKDWDUHD൵HFWLQJPDQ\ZDWHUERGLHVHVSHFLDOO\ in the Mediterranean region (Álvarez-Cobelas et al., 1991, Blindow, 1992, Jeppesen et al., 2011, Calero et al., 2015). Related to this, the studies assessing the distribution of the species from a historical perspective are very useful, and can help us to understand how the species, their ecological niches and the ecosystems that they inhabit have evolved over time, which may contribute WR GHYHORSLQJ H൵HFWLYH FRQVHUYDWLRQ PHDVXUHV (Chauvel et al., 2006, Nelson et al., 2013, Mosena et al., 2018, Büttner et al., 2022, Buldrini et al., 2023). The focus of the present study, is Chara canescens Desv. & Lois. (1810), a species mainly distributed in Europe and the African Mediterranean coast, which has its highest prevalence in the coastal areas near the Baltic Sea, with occasional localities in Australia, North America and Asia (Schubert et al., 2024). The Iberian populations are distributed throughout the peninsula, although they are scarcer in the western half (Cirujano et al., 2007, Schubert et al., 2024). Some populations were also recorded in the Balearic Islands in the past (Margalef, 1952, 1953; Comelles, 1984, 1986; Martínez-Taberner & Pericàs, 1988; La especie Chara canescens, principalmente distribuida en Europa y la costa africana mediterránea, es la única de la clase Charophyceae capaz de reproducirse partenogenéticamente, y posee poblaciones asexuales que son comunes pero las sexuales son raras. En este estudio reunimos datos de la literatura y de herbarios sobre la presencia de C. canescens en la península Ibérica y las Islas Baleares en las pasadas décadas, analizando su distribución histórica y espacial y las características limnológicas de sus hábitats. Se ha detectado un descenso del número de registros en la literatura y en herbarios desde 2010. La mayoría de las poblaciones se concentran en Castilla-La Mancha, Andalucía y Castilla-León. La especie vive en masas acuáticas salobres tanto en las zonas costeras como en lagunas endorreicas pequeñas y someras, y la mayoría de ellas no muestran un buen estado de conservación. Revisitamos las tres únicas localizaciones con poblaciones sexuales previamente citadas en la bibliografía (lagunas Bodón Blanco –Valladolid-, Las Eras –Segovia- y Lucio Largo – Doñana-) en junio de 2023. Encontramos poblaciones sexuales en las dos primeras lagunas, pero la del Parque Nacional de Doñana estaba completamente seca. Se descubrieron dos nuevas poblaciones sexuales para la especie en las lagunas La Iglesia y Caballo Alba (Segovia), aumentando así la cifra a cinco poblaciones sexuales. Se aporta la descripción de variables poblacionales (cobertura, proporción machos:hembras) e individuales (características reproductivas) para las FXDWUR SREODFLRQHV/DVYDULDEOHV OLPQROyJLFDVDQDOL]DGDVKLGURTXtPLFDRWURVKLGUy¿WRV\YHJHWDFLyQ PDUJLQDO LQGLFDQ que las lagunas con dichas poblaciones muestran signos de contaminación y de vegetación alterada y desestructurada. La diversidad genética de la especie depende de las poblaciones sexuales, las cuales son muy raras en Europa, y están concentradas principalmente en España. Por tanto, la preservación de estos hábitats salobres de interior es esencial para la implementación de medidas efectivas transnacionales para proteger a esta particular y relevante especie. PALABRAS CLAVE: &KDUDFHDHFDUDFWHUL]DFLyQOLPQROyJLFD3HQtQVXOD,EpULFDSDUWHQRJpQHVLVJHRJUi¿FDSURSDJDFLyQ VH[XDOHFRVLVWHPDVDFXiWLFRVVDOREUHVPDFUy¿WRVDFXiWLFRV This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) License. Chara canescens in the Iberian Peninsula Limnetica, 44(1): 141-158 (2025) 143 Martínez-Taberner et al., 1995). This species is unique among charophytes because it shows a combination of characteristics not found in any other species: on one hand, a combination of complete haplostic cortication and dioeciousness (Corillion, 1975) and, on the other, the occurrence of parthenogenetic reproduction, in which females produce oospores without fertilisation (Ernst, 1920). It has been described as an annual species, although overwintering individuals have been found in the Mediterranean region (Calero & Rodrigo, 2018). C. canescens inhabits brackish waters, often temporary, being unable to grow in freshwaters or marine conditions and it is also a species with a multitude of varieties and forms (Blindow & Schubert, 2004). Clearly, the most notorious characteristic of the species is its ability to reproduce parthenogenetically (Proctor, 1980). Parthenogenesis has been recorded in other organisms inhabiting temporary (Schmit et al., 2013) or brackish water bodies (Nascetti et al., 2003), and it has been well-documented in various species of zooplankton with similar habitats (Birky & Gilbert, 1971, De Meester, 1996, Serra et al., 2019). Parthenogenetic reproduction is not common in aquatic plants and does not occur in any other species of the genus Chara, nor in other charophytes. It has been suggested that this kind of reproduction may LQFUHDVH¿WQHVVHVSHFLDOO\LQPDUJLQDOVWUHVVIXO or extreme habitats with a high degree of subdivision, recolonization and local extinction (Haag & Ebert, 2004), being common in invasive species which are favoured by the fragmentation and reduction of the habitat caused by humans. Schaible and Schubert (2008) put forward some hypotheses for the success of this reproductive strategy in C. canescens, where parthenogenetic populations seem to have evolved from the sexual ones and to have surpassed them in distribution range. These hypotheses are: higher probability for asexuals to propagate in pioneer habitats, higher energetic costs of sexual reproduction, and GL൵HUHQFHVLQHFRSK\VLRORJLFDODFFOLPDWLRQDELO- ities between male and female organisms in general, or between sexually and asexually propagating populations. Currently, sexual populations are rare throughout the world, they are restricted to Europe, with the Mediterranean region standing RXWZKHUH¿YHVH[XDOSRSXODWLRQVZHUHUHFRUG- ed over several decades: two in Italy (Becker, 2019, Troia, 2020) and three populations in Spain (Comelles, 1986, García-Murillo et al., 1993). This reproductive strategy in C. canescens seems to follow the ecological pattern of “geographical parthenogenesis” (Vandel, 1928), where parthenogens occupy a wider geographic range while their close sexual relatives are restricted to a core DUHDSUHVHQWLQJGL൵HUHQWGLVWULEXWLRQZLWKSRWHQ- tial overlap (Hörandl, 2009), like the sympatric population of C. canescensZKLFKZDVFRQ¿UPHG in Austria by Schaible et al. (2011). The present study reviews bibliography and herbarium data concerning Chara canescens in order to establish the historical distribution and the main limnological features of the habitat of its parthenogenetic and sexual populations in the Iberian Peninsula and the Balearic Islands. Moreover, the sexual populations described in the literature have been revisited to gain information about population and individual characteristics of this species. The main aim of this work is to con- WULEXWHWRWKHHVWDEOLVKPHQWRIWKHVFLHQWL¿FDQG conceptual basis to protect the genetic diversity of C. canescensDQGWKHQHFHVVDU\JHQHÀRZEH- tween populations through the implementation of WUDQVQDWLRQDOH൵HFWLYHFRQVHUYDWLRQPHDVXUHV MATERIALS AND METHODS Revision of bibliography and herbarium data concerning Chara canescens and its habitat In order to list and catalogue the populations of C. canescens, the information contained in bibliographical references has been collected. Following the study by Cirujano et al. (2008) as a basis, the references citing C. canescens present in ResearchGate, Google Scholar and several other webpages have been reviewed. Moreover, additional bibliographical material (reports, books and thesis) has been also accessed. Two herbaria were reviewed: the Royal Botanical Garden of the Madrid Herbarium (MA), in Spain, and the Botanical Garden of the National History and Science Museum of the Lisboa Herbarium (LISU), in Portugal. In the MA-Algae, 27 sheets and 46 specimens preserved in con- Arnal et al. Limnetica, 44(1): 141-158 (2025) 144 servative solution were re-examined. Observations were made using a stereo-microscope. In the LISU, Dra. Ana Isabel Correia checked for the existence of C. canescens7KHLGHQWL¿FDWLRQ number of the material reviewed can be consulted in Table S1 of Supplementary Material (available at https://www.limnetica.net/en/limnetica). From the bibliography, the considered limnological features of the habitat were mainly water pH, conductivity, chloride and ammonium concentrations. Only the limnological data related to the occurrence of C. canescens in the same study are presented. The information about the current conservation status of the C. canescens locations has been obtained from the data gathered by Fundación Global Nature (2024) from the Spanish Inventory of Wetlands and the Autonomous communities’ reports. The sites of the occurrence of historical sexual populations The three sites where C. canescens sexual populations were cited in the bibliography were visited. Namely: Bodón Blanco Lake (Bocigas) in Valladolid, Las Eras Lake (Villagonzalo de Coca) in Segovia (Comelles, 1984, 1986) and Lucio Largo in Doñana National Park (Huelva) (García-Murillo et al., 1993) (Fig. 1). Las Eras and Bodón Blanco were sampled on 13th and 14th June 2023, respectively. Lucio Largo was visited on 21st May 2023, but the site was completely dry. In Doñana National Park, the rainfall for the September 2022-August 2023 hydrometeorological period was below-average for over more than a decade, this cycle being described as "very dry". Furthermore, the highest temperatures for the entire historical series were reached in those years in the area. The combination of high temperatures and low rainfall, as well as the increase in nearby groundwater abstractions for intensive agriculture and human supply have se- ULRXVO\D൵HFWHGWKH'RxDQDVZDWHUERGLHVV\V- tem, with a severe lack of water (De Felipe et al., 2023). Two other shallow endorheic lakes were visited during the sampling in Segovia on 14th June 2023, La Iglesia Lake (Villagonzalo de Coca) and Caballo Alba Lake (Villeguillo) where parthenogenetic populations were previously recorded by Comelles (1984). These lakes (together with Bodón Blanco and Las Eras lakes) are included in the Natura 2000 network (ZEC - ES4160062) called Lagunas de Coca y Olmedo Figure 1. Location and aerial view of the four sites studied in the complex “Lagunas de Coca y Olmedo” located in the inner Iberian Peninsula (provinces of Segovia and Valladolid) and Lucio Largo (Doñana National Park), in the SW Spanish littoral. The locations are also indicated with a white dot in the Spain map. Photographs taken from Google Earth. Localización y vista aérea de las cuatro localizaciones estudiadas en el complejo "Lagunas de Coca y Olmedo" situado en el interior de la Península Ibérica (provincias de Segovia y Valladolid) y El Lucio Largo (Parque Nacional de Doñana), en el litoral SW de España. La situación se indica en el mapa de España. Fotografías tomadas de Google Earth. Chara canescens in the Iberian Peninsula Limnetica, 44(1): 141-158 (2025) 145 and catalogued as “LIC” (site of communitarian importance for the Mediterranean biogeographic region). In this area, several wetlands between the provinces of Valladolid and Segovia are located. They are also important areas for the migration of birds. Nevertheless, these ecosystems KDYHEHHQD൵HFWHGE\KXPDQDFWLYLWLHVVXFKDV agriculture, especially cereal crops, the exploitation of pines for resin and pine nuts, the exploitation of the aquifer as well as other activities that KDYHPRGL¿HGWKHKDELWDWDQGD൵HFWHGWKHZDWHU available for aquatic macrophytes, such as charophytes (LIFE MedWetRivers, 2013). Limnological characterization of the Chara canescens sexual population sites Water temperature (ºC), conductivity (mS/cm), salinity (g/L), total dissolved solids (TDS) (g/L), pH and dissolved oxygen (mg/L and % saturation) were measured in situ by means of appropriate sensors in each lake. Water samples were taken by hand using a jar and kept in plastic bottles for further chemical analyses in the laboratory. Calcium carbonate, chloride, ammonium and silica concentrations (mg/L) were measured following A.P.H.A (2012). A study of the vegetation and plant communities surrounding the lakes was also carried out for the four locations. The communities were iden- WL¿HGXVLQJWKHSK\WRVRFLRORJLFDOPHWKRGRORJ\ of the Zurich-Montpellier school (Braun-Blan- quet, 1979), although it should be noted that the UHFRJQLWLRQ RI WKH GL൵HUHQW FRPPXQLWLHV ZDV GL൶FXOWGXHWRWKHSURIRXQGDOWHUDWLRQRIWKHOR- cal vegetation cover, which caused the absence of relevant species, as well as the coverage and frequency in which they were present. For this reason, some studies carried out in the area, such as the one by Ladero-Álvarez et al. (1984), were used as a reference. For vascular plants, the nomenclature of the Flora Ibérica (Castroviejo et al. (eds.), 1986-2021) was followed. Sampling and analysis of populations and individuals of Chara canescens In each small lake, the sampling of C. canescens LQGLYLGXDOVZDVGRQHLQWHQGL൵HUHQWVLWHVVHS- arated by two meters along a transect, from the shoreline to the deeper part of the waterbody. A round sampling unit with a surface area of 10 cm2 was used to sample each site of the transect. ,QWKH¿HOGWKHPD[LPXPFRYHUDJHRIWKHC. canescens meadow and the male:female ratio were determined visually (males are easily distinguishable by their large red antheridia). This UDWLRZDVFRQ¿UPHGODWHULQWKHODERUDWRU\IURP observation of the collected specimens. The fresh hand-collected material was transported to the laboratory in a portable cooler inside plastic bags. In the laboratory, the material was washed to remove the organic matter attached, and the individuals were measured from the apex to the beginning of the rhizoids using a ruler. Mean lengths were calculated for male and female individuals separately. The reproductive organs, oogonia and antheridia, were counted and expressed per fertile branchlet. An Olympus SZ61 (Shinjuku, Tokyo, Japan) stereomicroscope equipped with a camera (Model LC20-Olympus Soft Imaging Solutions GMBH) was used. The antheridia, oogonia and oospores were measured following the methodology established by Soulié-Märsche & García (2015). The length (longest polar axis, LPA) and width (largest equatorial diameter, LED) were measured for the female organs and the isopolarity index (ISI =LPA/LED×100) was calculated. The diameter for the male organs was also determined. The measurements were performed on photographs taken by the camera attached to the stereomicroscope using the LC micro measuring programme. Some C. canescens specimens collected that ZHUHQRWXVHGIRURWKHUVFLHQWL¿FSXUSRVHVZHUH deposited in the University of Valencia Herbarium “(VAL-Algae)” with the corresponding numbers: 3063, 3064, 3065, 3066, 3067, 3068, 3069, 3070, 3071. RESULTS AND DISCUSSION The historical distribution of Chara canescens Chara canescens Desv. & Loisel In Loisel., Not. Fl. France : 139 (1810) Arnal et al. Limnetica, 44(1): 141-158 (2025) 146 = Chara crinita Wallr., Annus Bot. 190 (1815) All the found citations of this species (under its GL൵HUHQWQDPHVLQWKH,EHULDQ3HQLQVXODDQGWKH Balearic Islands up to the present time are gathered in Table S1 (Supplementary material). In Portugal, the presence of the species was FRQ¿UPHG LQ WKH +HUEDULXP RI WKH %RWDQLFDO Garden of the National History and Science Museum of Lisboa (LISU), with only one record: LISU-P45314, from Costa da Trafaria LQ):HOZLWVFK,Q6SDLQWKH¿UVWUHFRUGV of C. canescens, date back to 1908 by E. Reyes- Prósper. Female individuals are represented in an herbarium sheet under the name of Chara crinita Wallr. var. toletana Prósper. from Quero (province of Toledo) and preserved in the cryptogamic collection of the Royal Botanical Garden of the Madrid Herbarium (MA-Algae). In 1910, Reyes-Prósper published the volume “The charophytes of Spain”, with detailed information and other records of C. crinita. Later, Caballero (1922,1929), carried out some other studies on Spanish charophytes; however, there is little literature recording C. canescens before 1950. Between 1950 and 1979, new records of this species appeared, but the locations from the SUHYLRXVSHULRGZHUHQRWUHFRQ¿UPHG)LJ$ and there is no herbarium material available. Most of the references for this species belong to the 1980-2010 period, when the maximum number of recorded locations was assessed, and some of WKHSUHYLRXVSRSXODWLRQVZHUHUHFRQ¿UPHG)LJ 2A). The increase in the number of C. canescens records after the 1980s is probably related to the increase in ecological studies, as well as social awareness regarding environmental matters, biodiversity, and conservation management (see Cirujano et al., 2007). Particularly relevant are the studies by Cirujano et al. (1980, 1982,1988, 1990, 1993, 2002, 2007, 2008, 2015), Comelles (1984,1986) and García-Murillo (1993, 1995). The number of herbarium specimens preserved during these years is also the highest recorded. Despite nearly two decades having passed, the number of records since 2010 has not yet surpassed the previous period (Fig. 2A). Regarding the spatial distribution of C. canescens in the Iberian Peninsula and the Balearic Islands, the maximum number of localities re- Figure 2. (A) Number of locations recorded in the literature with Chara canescens sorted by year periods including new records for each period and reconfirmed ones (B) Distribution of the number of locations with C. canescens within the Autonomous Communities of Spain and Provinces of each Community ordered by decreasing number of locations. The provincial codes used in the list are those used by Cirujano et al., (2008): see the list below. (C) Number of locations with C. canescens for each category of conservation status of the waterbody. (A) Número de localidades con presencia de Chara canescens registradas en la literatura en periodos de años incluyendo los nuevos registros para cada periodo y los reconfirmados (B) Distribución del número de localidades con presencia de C. canescens dentro de las Comunidades Autónomas de España y las provincias de cada Comunidad ordenadas de mayor a menor número de localidades. Los códigos provinciales usados son los establecidos por Cirujano et al., 2008: A=Alicante, Ab=Albacete, Al=Almería, Ca=Cádiz, CR=Ciudad Real, Co= Córdoba, Cu= Cuenca, Gu=Guadalajara, H=Huelva, Lo= Logroño, Ma=Málaga, Mll=Mallorca, Mn=Menorca, Mu=Murcia, Na=Navarra, P=Palencia, Sa=Salamanca, Sg=Segovia, Se=Sevilla, Te=Teruel, To=- Toledo, V=Valencia, Va=Valladolid, Vi=Álava, Za=Zamora, Z=Zaragoza. (C) Número de localidades con C. canescens para cada categoría de estado de conservación de los cuerpos de agua. Chara canescens in the Iberian Peninsula Limnetica, 44(1): 141-158 (2025) 147 corded with parthenogenetic populations of the species corresponds to the central and southern regions of the peninsula: Ciudad Real and Toledo provinces, belonging to the Castilla-La Mancha autonomous community, followed by the communities of Andalucía and Castilla León (Fig. 2B). Sexual populations of C. canescens also occurred in Castilla-León (Comelles, 1986) and Andalucía regions (García-Murillo et al., 1993) (Fig. 3). Despite most sites with populations of C. canescens are protected by the Natura 2000 network or other protection schemes, many of them have a bad conservation status (Fig. 2C). In Spain, WKHPDLQWKUHDWVWKDWD൵HFWWKHZHWODQGVDUHWKH transformation of the land, pollution and the climate-change consequences (MITECO, 2023). In addition, inner brackish water sites, which host C. canescensDUHHVSHFLDOO\VHQVLWLYHWRWKHDUWL¿FLDO freshwater inputs performed for other human purposes and economic interests. Some of the Spanish locations where C. canescens were recorded have been dried, drained, cultivated (Gobierno Vasco, 1994), with their original hydrographical QHWPRGL¿HGÈOYDUH]HWDODQGD൵HFWHG by sewage treatment plants and by grazing and slurry (Cirujano et al., 2015). The ignorance and lack of interest in aquatic vegetation causes the poor conservation status of habitats that host C. canescens, thus its presence (like other aquatic PDFURSK\WHVLVRQO\EULHÀ\PHQWLRQHGLQVWXGLHV focused on other ecological aspects or taxonomic groups (Cirujano et al., 2008). Limnological features of the habitat C. canescens is a representative species for waters of moderate salinity (Cirujano, 1990) being one of the few true brackish water species of charophytes, neither occurring in freshwater nor under fully marine conditions (Schubert et al., 2024). In the Iberian Peninsula, the habitat of C. canescens is characterized by neutral-basic pH values, above 7, reaching 9 in some lakes (Table 1). C. canescens populations inhabit waterbodies with a wide range of conductivity values, but most of them are between 4 and 20 mS/cm (Table 1). Chloride concentrations are often higher than 1000 mg/L, while ammonium concentrations are lower than 1.5 mg/L, despite this value is not often measured in the studies assessing C. canescens populations (Table 1). Sexual populations of Chara canescens in the revisited sites Previously recorded sexual populations in Bodón Figure 3. Historical distribution map of C. canescens populations in the Iberian Peninsula and Balearic Islands. The period of the cited location is indicated. Mapa de distribución histórica de las poblaciones de C. canescens en la Península Ibérica y las Islas Baleares. Se señala la época a la que corresponde la cita de la localización. Arnal et al. Limnetica, 44(1): 141-158 (2025) 148 Blanco and Las Eras endorheic shallow lakes &RPHOOHV  ZHUH UHFRQ¿UPHG GXULQJ WKH samplings in June 2023. For the same dates, male individuals were found in the populations of C. canescens occurring in Caballo Alba and La Iglesia lakes. These latter lakes have not been previously recorded as hosting sexual populations until the present study, but exclusively parthenogenetic populations (Comelles, 1984). Probably, the male individuals could have passed unnoticed in past surveys, since, in most cases, the male:female ratio is clearly biased to females (see below). The studied lake complex was previously highlighted by its uniqueness with respect to other lake complexes in Spain, or even in Europe, by authors such as Velasco et al. (2004) and Cabestrero (2018), because of its exceptionally high alkalinity, its saline hydrochemistry, the precipitation of rare minerals and high pH values. We corroborated the high water pHs in June 2023 (values ranging from 9 to 11) with the lowest in Bodón Blanco and the highest in Las Eras Lake (Table 2), These pH values are much higher than the ones measured for most of waterbodies where the parthenogenetic populations of C. canescens occur in Spain, which ranged from 7 to 8 (Cirujano, 1980, Aboal, 1985, Carretero, 1993, Gobierno Vasco, 1994, Cirujano et al., 2002, Cirujano et al., 2015, Calero & Rodrigo, 2018) (Table 1). Regarding water conductivity, its values ranged from 4 to 9 mS/cm, and the highest values were measured in Las Eras Lake (Table 2). These values are within the range measured in the waterbodies of parthenogenetic populations of the same species (Table 1). With respect to the values of other compounds, the greatest ammonium concentration in June 2023 was found in Bodón Blanco Lake, which might indicate a higher pollution in these waters than in the other ones from the same area, probably due to the wide area of agriculture crops surrounding the lake (Table 2). Compared to the concentrations in the parthenogenetic sites (Table 1), the values are similar, with the exception of some sites visited by Carretero Table 1. Limnological variables measured for different water bodies that host Chara canescens populations in the Iberian Peninsula. The reference for each data is indicated. Variables limnológicas medidas para diferentes masas de agua que albergan poblaciones de C. canescens en la Península Ibérica. Se indica la referencia de la que proviene cada dato. Name of the lake/area pH Cond. (mS/cm) Cl- (mg/L) NH๙+ (mg/L) Referemce Casa Bomba 20.2 García-Murillo et al. (1995) Casa Nueva 2 10.0 Cirujano (1990) Chica de Quero 4.7 700 Cirujano et al. (2002) Corral Rubio 9.8 9.4 1967 Cirujano (1990) ; Cirujano et al. (2002) Dehesilla 7.8 21.9 5160 Cirujano (1980) ; Cirujano et al. (2002) El Taray 3.8 430 Cirujano et al. (2002) Hoya Husilla 5.0 Cirujano (1990) Laguna Chica (Villafranca) 4.1 429.1 Cirujano et al. (2002) Laguna de Musco 7 4.3 297 0.1 Gobierno Vasco (1994) Las Eras/Bodón Blanco 1100-2500 Comelles (1986) Lucio de los Patos Reales 30 Espinar et al. (1997) Mallada “Canescens” 7.7 7.2 2110 0.3 Calero & Rodrigo (2018) Manjavacas 22.0-47.9 8329 -11 350 Cirujano et al. (2002) Nava Grande Malagón 14.8 Cirujano et al (1993) Navahornos 7.6-8.9 0.3-1.0 8.0-34.0 Cirujano et al. (2015) Murcia 7.4 10.5 2370.1 Aboal (1985) Salinas San Rafael 30.1 Cirujano et al. (1993) Puzol, Sagunto 7.4-8.1 5.0-9.3 1750-2320 0.1-1.1 Carretero (1993) Chara canescens in the Iberian Peninsula Limnetica, 44(1): 141-158 (2025) 149 (1993) in the Valencian Community, where they reached more than 1 mg/L. Despite the relative SUR[LPLW\RIWKHIRXUODNHVWKH\GL൵HUFRQVLGHUD- bly in silicate concentrations, with Bodón Blanco Lake standing out with the highest concentration and Caballo Alba Lake with the lowest value (Table 2). The vegetation within the water bodies and their surroundings was highly altered due to the impact of agricultural crops surrounding the shallow lakes and also, in some of them, due to the discharge of waste water into the basin. This ZDVUHÀHFWHGLQWKHREVHUYHGSODQWFRPPXQLWLHV which appeared quite disorganized, particularly the helophyte communities. Under these circum- VWDQFHV ¿QGLQJ WKH RUJDQL]DWLRQDO SDWWHUQV WKDW allow them to be assigned to phytosociological categories was a complicated task. In Bodón Blanco Lake, the submersed vegetation formed meadows of aquatic macrophytes with an uneven coverage; there were patches of around 1 m2 with total coverage; however, in other sites the coverage was only 20% (for the same surface area). The western, central, and the south-eastern shore areas were the most covered, without following a clear pattern. The predominant species was C. canescens (covered by a thin layer of epiphyton), although Zannichellia pedunculata Reichemb. (1830) was also abundant, particularly in the western and south-eastern areas, where it reached 100% coverage. In addition, some isolated individuals of Ruppia drepanensis Tineo ex Guss. (1845) were present, especially in the central area of the lake. Both Z. pedunculata and R. drepanensis VKRZHG ÀRZHUV DQG IUXLWV DW WKH WLPH RI sampling. This community can be assimilated into the phytosociological association Rupietum drepanensis Brullo &. Furnari 1976 (Fig. 4A). The water-margin vegetation was completely unstructured with only isolated and incomplete patches, remains of original plant communities that formed the vegetation belt around the lake (Table 3). In some cases, the crops that surrounded the lake extended almost to the edge. Only in the south-eastern part of the lake, a more developed portion of helophytic vegetation was found (10-15 m). The communities were organized according to the time passed since the area was ÀRRGHGDQGWKHVRLOPRLVWXUH)LJ$DQG7DEOH 3). Riella helicophylla (Bory & Mont.) Mont. (1852) and Tolypella salina Corillion. (1960) had also been cited from this lake (Cirujano et al., 1988; Comelles, 1984); however, they were not observed during the 2023 sampling. In Las Eras Lake, the vegetation structure was similar to the one described for Bodón Blanco Lake, although with a higher degree of alteration. The hydrophytic vegetation was less diverse than in the previous lake and consisted basically of C. canescens meadows. Additionally, Z. pedunculata and, more rarely, R. drepanensis were present. Table 2. Limnological variables measured for each lake studied in the Valladolid-Segovia area. Variables limnológicas medidas para cada laguna estudiada en el área Valladolid-Segovia. Bodón-Blanco Las Eras La Iglesia Caballo Blanco Water temperature (°C) 20.8 22.6 21.5 24.6 Conductivity (mS/cm) 4.2 8.9 5.9 8.5 Salinity (g/L) 2.7 5.0 3.8 5.6 TDS (g/L) 2.2 5.8 3.2 4.7 pH 9.6 10.7 10.1 10.3 Dissolved O๗ (mg/L) 4.7 14.0 12.6 13.0 O๗ saturation (%) 57.2 165.8 146.1 160.2 CaCO3(mg/L) 1680 1990 1940 810 Cl- (mg/L) 590 1440 1330 1670 NH๙ၰPJ/ 0.19 0.02 0.01 0.01 SiO๗ (mg/L) 1.62 0.41 0.17 0.06 Arnal et al. Limnetica, 44(1): 141-158 (2025) 156 dín Botánico De Madrid, 64(1), 87-102. DOI: 10.3989/ajbm.2007.v64.i1.57 Cirujano, S., Cambra, J., Sánchez-Castillo, P. M., Meco, A., & Flor Arnau, N. (2008). Flora LEpULFD $OJDV FRQWLQHQWDOHV &DUy¿WRV. Real Jardín Botánico, CSIC. Madrid. España Cirujano, S., Sánchez, S., Sánchez, R., Álvarez-Cobelas, M., Rubio, A., Soriano-Her- nando, Ó., Velasco, J.L., Laguna, C., Guerrero, N., Meco, A., & Martín, S. (2015). Caracterización limnológica de las Lagunas de Cantalejo (Segovia). Serie LimnoIberia nº 9. Grupo de Investigaciones del Agua. España. ISBN: 978-84-606-6234-1. &RPHOOHV01RYHVFLWDFLRQVGHFjUR¿WV a Espanya. Butlletí de la Institució catalana d'Història Natural, Sec. Bot., 5, 35-39. Comelles, M. (1986). Hallazgo de dos poblaciones sexuales de Chara canescens Desv. & Lois, en España. Anales del Jardín Botánico de Madrid, 42(2), 285-291. Coops, H., & Doef, R. W. (1996). Submerged vegetation development in two shallow, eutrophic lakes. Hydrobiologia, 340, 115-120. DOI : 10.1007/bf00012742 Corillion, R. (1975). Flore des Charophycées du Massif Armoiricain et des contraes voisines d'Europe occidentale, IV. C. N. R. S. Paris. France. De Felipe, M., Aragonés, D., & Díaz-Paniagua, C. (2023). Thirty-four years of Landsat moni- WRULQJUHYHDOORQJWHUPH൵HFWVRIJURXQGZDWHU abstractions on a World Heritage Site wetland. Science of the Total Environment, 880, 163329. DOI: 10.1016/j.scitotenv.2023.163329 'H 0HHVWHU /  /RFDO JHQHWLF GL൵HU- entiation and adaptation in freshwater zooplankton populations: patterns and processes. Ecoscience, 3(4), 385-399. DOI: 10.1080/11956860.1996.11682356 Ernst, A. (1920). Die Nachkommenschaft aus amphimiktisch und apogam entstandenen Sporen von Chara crinita. Induktive Abstammungs-und Vererbungslehre, 25, 185-197. Espinar, J. L., Cirujano, S., & García-Murillo, P. (1997). Contribución al conocimiento de los FDUy¿WRVGHO3DUTXH1DFLRQDOGH'RxDQDActa Botanica Malacitana, 22, 209-234 Fundación Global Nature (2024). Mapa interactivo de los humedales en España: El mapa más completo de las zonas húmedas de España. Accesed January 2024, <https://fundacionglobalnature.org> García-Murillo, P., Bernúes, M., & Montes, C. /RV PDFUy¿WRV DFXiWLFRV GHO3DUTXH Nacional de Doñana (SW España). Aspectos ÀRUtVWLFRV Actas VI Congreso Español de Limnología, 261-267. García-Murillo, P., Cirujano, S., & Minero, F. J. G. (1995). Contribución al conocimiento de ORVFDUy¿WRVGHO62GHOD3HQtQVXOD,EpULFD Studia Botanica, 13, 221-225. Gobierno Vasco. (1994). &DUDFWHUL]DFLyQ\7LSL¿- cación Ecológica de los Humedales de Interi- or de la Comunidad Autónoma del País Vasco. Dpto. de Urbanismo, Vivienda y Medio Ambiente del Gobierno Vasco. España. Haag, C. R., & Ebert, D. (2004). A new hypothesis to explain geographic parthenogenesis. Annales Zoologici Fennici, 41, 539-544. Hörandl, E. (2009). Geographical Parthenogenesis: Opportunities for Asexuality. In: Schön, I., Martens, K., Dijk, P. (eds.). Lost Sex: The evolutionary biology of parthenogenesis. (pp. 161-186). Springer, Dordrecht. DOI: 10.1007/978-90-481-2770-2_8 Jeppesen, E., Kronvang, B., Olesen, J. E., Au- GHW-6¡QGHUJDDUG0+R൵PDQQ&&« g]NDQD.&OLPDWHFKDQJHH൵HFWVRQ nitrogen loading from cultivated catchments in Europe: implications for nitrogen retention, ecological state of lakes and adaptation. Hydrobiology, 663, 1-21. DOI: 10.1007/s10750- 010-0547-6. Ladero-Álvarez, M., Navarro-Andrés, F., Valle-Gutiérrez, C. J., Marcos-Laso, B., Ruiz- Tellez, T., & Santos-Bobillo, M. T. (1984). Vegetación de los saladares castellano-leone- ses. Studia Botanica, 3, 17-62. LIFE MedWetRivers (2013). Plan básico de gestión y conservación del Espacio Protegido Red Natura 2000ZEC - ES4160062 - Lagunas de Coca y Olmedo. Margalef, R. (1952). Materiales para la hidrobiología de la isla de Menorca. Publicaciones del Instituto de Biología Aplicada, 11, 5-112. Margalef, R. (1953). Materiales para la hidrobiología de la isla de Mallorca. Publicaciones Chara canescens in the Iberian Peninsula Limnetica, 44(1): 141-158 (2025) 157 del Instituto de Biología Aplicada, 15, 5-111. Martínez-Abraín, A., Castejón-Silvo, I., & Roiloa, S. (2022). Foreseeing the future of Posidonia oceanica meadows by accounting for the past evolution of the Mediterranean Sea. ICES Journal of Marine Science, 79(10), 2597-2599. Martínez-Taberner, A., & Pericàs, J. J. (1988). 1RWHVÀRUtVWLTXHV/HV&KDUDFHDHGHO$OEXIHUD de Mallorca. Bolletí de la Societat d'Historia Natural de les Balears, 32, 145-150. Martínez-Taberner, A., Moya, G., Forteza, V., Rita, J., & Pericàs, J. (1995). La vegetació DTXjWLFD VXEPHUJLGD GH 6$OEXIHUD GH 0DO- lorca". In: Martínez-Taberner, A.G., Moya, V. (eds.). S'Albufera de Mallorca. 0RQRJUD¿HV de la Soc. Hist. Nat. Balears, 4. (pp. 97-111). Editorial Moll. Palma. España. ISBN: 84-273- 6506-3. 0HHUKR൵ 0 0D]]HR 1 0RVV %  Rodríguez-Gallego, L. (2003). The structur- LQJ UROH RI IUHHÀRDWLQJ YHUVXV VXEPHUJHG plants in a subtropical shallow lake. Aquatic Ecology, 37, 377-391. DOI: 10.1023/B:AECO.0000007041.57843.0b MITECO (2023). Plan Estratégico de Humedales a 2030. Ministerio para la Transición Ecológi- FD\HO5HWR'HPRJUi¿FR0DGULG(VSDxD Mosena, A., Steinlein, T., & Beyschlag, W. (2018). Reconstructing the historical spread of non-native plants in the North American West from herbarium specimens. Flora, 242, 45-52. '2,MÀRUD Nascetti, G., Bondanelli, P., Aldinucci, A., & Cimmaruta, R. (2003). Genetic structure of bisexual and parthenogenetic populations of Artemia from Italian brackish–hypersaline waters. Oceanologica Acta, 26(1), 93-100. DOI: 10.1016/S0399-1784(02)01233-1 Nelson, W. A., Dalen, J., & Neill, K. F. (2013). Insights from natural history collections: ana- O\]LQJWKH1HZ=HDODQGPDFURDOJDOÀRUDXV- ing herbarium data. PhytoKeys, 30, 1-21. DOI: 10.3897/phytokeys.30.5889 Proctor, V. W. (1980). Historical biogeography of Chara (Charophyta): an appraisal of the %UDXQ:RRG FODVVL¿FDWLRQ SOXV D IDOVL¿DEOH alternative for future consideration. Journal of Phycology, 16(2), 218-233. DOI: 10.1111/ j.1529-8817.1980.tb03023.x Reyes-Prósper, E. (1910). /DV FDUy¿WDV GH (V- paña. Madrid. España. Schaible, R., & Schubert, H. (2008). The occurrence of sexual Chara canescens populations (Charophyceae) is not related to ecophysiological potentials with respect to salinity and irradiance. European Journal of Phycology, 43(3), 309-316, DOI: 10.1080/09670260801930348 Schaible, R., Bergmann, I., & Schubert, H. (2011). Genetic structure of sympatric sexually and parthenogenetically reproducing population of Chara canescens (Charophyta). International Scholarly Research Notices, 2011, 501838. DOI: 10.5402/2011/501838 Schmit, O., Adolfsson, S., Vandekerkhove, J., 5XHGD - %RGH 6 1 6 5RVVHWWL * « Mesquita-Joanes, F. (2013). The distribution of sexual reproduction of the geographic parthenogen Eucypris virens (Crustacea: Ostracoda) matches environmental gradients in a temporary lake. Canadian journal of zoology, 91(9), 660-671. DOI: 10.1139/cjz-2012-0236 Schubert, H. & Blindow, I. (2004). Charophytes of the Baltic Sea. Gantner, Ruggell Liechtenstein. Schubert, H., Blindow, I., Nat, E., Korsch, H., Gregor, T., Denys, L., Stewart, N., Van de Weyer, K., Romanov, R., Casanova, M. T. (2024). Charophytes of Europe. Springer Nature. Germany. Serra, M., García-Roger, E. M., Ortells, R., & Carmona, M. J. (2019). Cyclically parthenogenetic rotifers and the theories of population and evolutionary ecology. Limnetica, 38(1), 67-93. DOI: 10.23818/limn.38.13 Soulié-Märsche, I., & García, A. (2015). Gyrogonites and oospores, complementary viewpoints to improve the study of the charophytes (Charales). Aquatic botany, 120, 7-17. DOI: 10.1016/j.aquabot.2014.06.003 Troia, A. (2020). Homage to Proserpina, or: why did the charophytes of the Pergusa lake vanish? In: La Mantia, T., Badalamenti, E., Carapezza, A., Lo Cascio, P., & Troia, A. (eds.). Life on islands. 1. Biodiversity in Sicily and surrounding islands. Studies dedicated to Bruno Massa. (pp. 47-51). Edizioni Danaus. Palermo. Italy. Arnal et al. Limnetica, 44(1): 141-158 (2025) 158 Vandel, A. (1928). La parthénogenèse géographique. Contribution à l’étude biologique et cytologique de la parthénogenèse naturelle. Bulletin biologique de la France et de la Belgique, 62, 164–281. Velasco, J., Soriano Hernando, Ó., Fernández, J., & Rubio, Y. (2004). Características físico-químicas de diferentes masas de agua: II Cuenca del Duero (España). Ecología, 18, 21- 34.