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Introduction
Charophytes, often called stoneworts, are a group of autotrophic and macroscopic algae represented by more than 400 species assigned to the Characeae family within the Charophyta phylum (Schneider et al. 2015; Guiry & Guiry 2019). Its first members evolved nearly 420 million years ago (Graham & Wilcox 2000). Charophytes are a highly developed and diverse group of macroalgae (Kotta et al. 2004; Schneider et al. 2015). They are widely distributed in freshwater as well as brackish and marine habitats, from tropical to polar regions (Wood 1965). The occurrence of Characeae taxa is mostly limited to clear, freshwater lakes with low fertility (Dambska 1964; Krause 1969; 1981; 1997; Martin et al. 2003; Brzeska et al. 2015). Many stonewort species are characteristic of hard, oligo-mesotrophic water ecosystems (Krause 1981; 1997). Some species of the genus Chara are capable of developing in eutrophic or even hypertrophic habitats (Urbaniak & Gąbka 2014).
In some European countries, C. connivens is protected by law. In Poland, for example, C. connivens is a strictly protected species. The “Red list of plants and fungi in Poland” classifies this alga as extinct or most likely extinct (EX) species (Siemińska et al. 2006). In Great Britain, C. connivens is an endangered (EN) species (Stewart & Church 1992). In Ireland, the convergent stonewort is classified as a regionally extinct (RE) species. The conservation status of this species is inconsistent due to discoveries of new stands or confirmed recolonization of the historical ones. Initially, in 1993, the German government classified C. connivens as a regionally extinct species (Schmidt 1994), but following the identification of new stands, its status was changed to critically endangered (CE) in 2012 (Korsch et al. 2013). Finally, the latest update of the “Red list of stoneworts (Charophyceae) from Mecklenburg-Western Pomerania, Germany” presents C. connivens as an endangered (EN) species (Teppke et al. 2015). This alga is also a red-listed species in Sweden and, based on recent research, it should be likewise promptly classified as highly threatened in Finland (Appelgren et al. 2004). On the other hand, the Helsinki Commission (HELCOM) database from 2013 classified C. connivens as a “not native” species to the Baltic Sea area. Moreover, in some of the Baltic Sea countries (Denmark, Germany and Lithuania – verified in 2017), C. connivens is considered as an invasive species requiring special attention (DAISIE 2006; Drake 2009; NOBANIS 2015; Pagad et al. 2018). Unfortunately, the origin of the convergent stonewort in the Baltic Sea area is unclear and the Baltic Marine Biologists (BMB) working group “Non-indigenous estuarine and marine organisms” characterized it as cryptogenic (Torn & Martin 2003; Kautsky & Snoeijs 2004). According to some theories, this species arrived in the Baltic Sea as a consequence of shipping from the Mediterranean region and was introduced together with the ballast, e.g. wet sand (Luther 1979; Torn & Martin 2003; Tolstoy & Österlund 2003). This idea is supported by the fact that many of the identified Baltic Sea stands of C. connivens are convergent with the ballast sites of harbors. However, Appelgren et al. (2004) challenged this hypothesis based on coastal stands of C. connivens on the Åland Archipelago (Finland) outside the important shipping routes in the 19th or 20th century. Considering that the population of C. connivens in the Baltic Sea is quite stable at the regional scale, i.e. neither severely dispersed nor continually declining, the species is currently classified as the least concern (LC) species (HELCOM 2013).
The objectives of the current study were (1) to characterize the unknown and unique stand of C. connivens from Fuerteventura and (2) to determine the relationships between environmental gradients and distribution of this algae. This work presents revised data on the occurrence and ecology of the convergent stonewort.
Materials and methods
Research location
The study involved the Chara population from its stand located in the canyon El Barranco de las Peñitas on Fuerteventura (Spain), one of the largest (1657 km2) and oldest (20 million years) islands of the Canary Archipelago (Schmitz et al. 2018). This area is of volcanic origin and is currently highly eroded as well as the most arid region of Macaronesia. Temperatures are very stable (17–20°C) throughout the year, although air masses from neighboring Africa can suddenly increase the temperature (Schmitz et al. 2018). Precipitation is irregular and low, i.e. less than 200 mm per year, but winds and insolation (2800 hours per year) are very intense.
The research site was located in the western part of the island, between the cities of Mazquez and Vega de Rio Palmas (28°23’17.6”N; 14°06’13.2”W), at an elevation of 172 m a.s.l. (Fig. 1). The canyon is part of the rural park of Betancuria. The sampling site was located in a longitudinal, narrow rock crevice (25.31 × 3.56 m), partly filled with water (Fig. 2A), which constitutes a unique natural habitat on the island. A slight flow of water was observed. The bottom was rocky and sandy-organic.
Species identification
The identification of species was based on the examination of morphological features as presented by Cirujano et al. (2008), Bryant & Stewart (2011), and Urbaniak & Gąbka (2015). Moreover, the identification keys created by Dambska (1964), Moore (1986), Krause (1997), Pełechaty & Pukacz (2008) and John et al. (2011) were used. All specimens of Chara were classified to one species only – Chara connivens P. Salzmann ex A. Braun. The current nomenclature of algae, mosses and vascular plants was used according to AlgaeBase (Guiry & Guiry 2019) and the World Checklist of Selected Plant Families (WCSP 2019).
Field samples
Thalli and water sampling was conducted on 12 April 2019. Thalli specimens were collected directly from the central part of the Chara meadow. In total, two subsamples of thalli (2 × 5 g of fresh weight) were collected from this stand.
The samples of water (1 l) were collected from the central part of the stonewort meadow. Long-sleeve veterinary gloves were used in the process to prevent contamination. Samples of water were filtered through a coarse plastic sieve to separate vascular plants and filamentous macroalgae. Subsequently, the water samples were placed into two 0.5 l sterile plastic containers, preserved with 0.5 ml of chloroform and cooled in a mobile refrigerator. In the laboratory, before the chemical analysis, the samples were filtered through a microbiological filter with a pore size of 0.45 microns (Sartorius, Goettingen, Germany).
Physicochemical analysis
In the field, water temperature, pH, electrolytic conductivity, total dissolved solids (TDS), oxidation-reduction potential (ORP), salinity and oxygenation were measured using the Professional Plus Multi-Parameter Instrument (YSI, Yellow Springs, OH, USA). In addition, water depth was measured using a plastic staff gauge. Chemical analyses, i.e. ammonium nitrogen (N-NH3), nitrate nitrogen (N-NO3), total phosphate (P-PO4), sulfate (SO4) and total iron (Fe-total) concentrations, as well as water color, were performed in the laboratory using a HACH DR 2800 spectrophotometer (Fairborn, OH, USA) and applying standard hydrochemical methods (APHA 2002). Sodium chloride (NaCl) concentration was determined using the HACH Chloride Digital Titrator Kit (Fairborn, OH, USA). Water turbidity was measured using a EUTECH TN-100 turbidimeter (Thermo Scientific, Singapore). The applied equipment and standard analytical methods are presented in detail in Rybak & Gąbka (2018).
Microscopic observation
The morphology of the thalli was assessed directly after the collection of samples. A mobile MINI Magnum microscope (MINI, China) with a magnification of 5 to 20 and a magnifying glass were used in the field to quickly analyze the thalli.
Laboratory observations were performed using a Zeiss Stemi DV4 stereomicroscope (Zeiss, Germany) with a magnification of 8 to 32. Thalli were also analyzed under a light microscope (Zeiss Axioskop 2 MOT). Microphotographs of fresh samples (Fig. 3) were taken with the ProgRes Speed XT core 3 camera (Jenoptic, Germany).
Herbarium
Several individuals of Chara were selected for an herbarium. A herbarium sheet was prepared in accordance with the guidelines of Drobnik (2007), Kalmbach (2011) and Rybak (2015, 2018). Thalli samples were attached to the labelled sheet using Archer’s method. The herbarium sheet was digitized based on the Gilroy (2001) protocol (Fig. 4). A voucher specimen (in Merrill’s box) was deposited in the Natural History Collections at the Faculty of Biology [Poznań Algae Herbarium (acronym: POZA), voucher number: CH0009] at Adam Mickiewicz University in Poznań.
Results and discussion
Identification features
A detailed description of C. connivens presented by Dambska (1964), Wood (1965), Torn & Martin (2003), Moore (1986), Krause (1997), Cirujano et al. (2008), Pełechaty & Pukacz (2008), Bryant & Stewart (2011), Urbaniak & Gąbka (2014), Becker et al. (2016) was used. The plants are mostly small, up to 15 cm long, rarely longer (25–50 cm); slender thalli are delicate green and lustrous. An erect plant axis is usually 0.3–1.4 mm in diameter and not or slightly encrusted. C. connivens has 6–9 branchlets in a whorl. Fertile (male) plants often have branches that are strongly curved inwards (connivent) and sterile plants are similar. Internodes are shorter or as long as branches (in the upper part). Each branchlet has 6–10 segments, of which 6–8 are corticated; the last 1–3 segments can be without a cortex. The stem cortex is triplostichous and partly isostichous. Spine cells are lacking or rudimentary (papillous) if present. Stipulodes are also papillous, almost globular and occur in two rows. The plant is dioecious with gametangia at the lowest branchlet nodes. Bract cells (7–8) are very short, rudimentary, do not exceed the diameter of the branch. Bracteoles commonly papilliform or shorter than oogonium. Oogonia are 605–775 μm long and 330–410 μm wide. Oospores are long and ellipsoid-cylindrical in shape, usually dark brown or black, with a size of 485–595 × 205–325 μm. Antheridia (male gametangia) are solitary, clear orange-reddish, and up to 1100 μm in diameter.
Based on the description presented above, many morphological characteristics of Chara samples from Fuerteventura correspond to the C. connivens species, although only slightly encrusted male specimens were found (Figs 3 and 4). The fresh thalli were delicate, light green, up to 75 cm long, with axes from 0.4 to 1.1 mm in diameter. Antheridia were gaudy orange-reddish. Branches were strongly curved inward and ended with ecorticate terminal segments (Fig. 3).
C. connivens habitats
The C. connivens community on the Canary Islands grows mainly in hard freshwater, brackish water, small natural ponds and artificial irrigation tanks (del Arco Aguilar & Rodríguez Delgado 2018). To date, the Canary stands of C. connivens have been reported only from Lanzarote, a marine habitat (El Golfo) and Tenerife, marine (Las Cuevitas, and Adeje), riverine (El Barranco del Infierno) and artificial systems (Añocheza, Costa del Silencio) (Gil-Rodriguez et al. 1982; Raam & Gonzalez-Henriquez 1995) (Fig. 1, Table 1). The new stand of C. connivens on Fuerteventura Island was identified in a ravine-bed crevice of the canyon El Barranco de las Peñitas, 5.22 km from the ocean shoreline (Figs 1, 2). The sediment in this stand was rocky-organic. This type of sediment is not a necessary condition as in Germany C. connivens was found on sandy and organic substrates (Becker 2008; 2010), in Spain and Greece – on loamy soil (Espinar et al. 2002; Langangen 2010), and in the Baltic Sea region (data from Estonia and Sweden) – on sand, muddy sand, muddy clay or mixtures of sand with pebbles (Torn & Martin 2003).
Stands of Chara connivens on Canary Islands (Spain). The number assigned to a given stand is consistent with the numbering used in Figure 1
Island
Stand
Habitat
Date
Collector(s)
Herbarium, voucher number
References
Lanzarote
1. El Golfo
marine
07.1975
M.C. Gill Rodriguez
TFC Phyc., 2193
Rodriguez et al. 1981
Tenerife
2. Las Cuevitas
marine
12.1976
E. Beltrán
TFC Phyc., 2195
Rodriguez et al. 1981
Ibid.
marine
12.1978
W. Wildpret E. Beltrán C. Rodriguez
TFC Phyc., 2196
Rodriguez et al. 1981
3. Añocheza
artificial pool
04.1980
W. Wildpret E. Beltrán C. Rodriguez
TFC Phyc., 2197
Rodriguez et al. 1981
4. Adeje
marine
09.1982
J.C. van Raam
JVR & L., 8201
Raam & Gonzalez-Henriquez 1995
5. El Barranco del Infl erno
river
09.1982
J.C. van Raam
JVR & L., 8202
Raam & Gonzalez-Henriquez 1995
6. Costa del Silencio
artificial pool
09.1982
J.C. van Raam
JVR & L., 8203
Raam & Gonzalez-Henriquez 1995
7. Armeñime
artificial pool
09.1982
J.C. van Raam
JVR & L., 8204
Raam & Gonzalez-Henriquez 1995
Fuerteventura
8. El Barranco de las Peñitas
intermittent river
04.2019
A.S. Rybak A.M. Woyda-Ploszczyca
POZA, CH0009
(this paper)
Plant communities with C. connivens
The C. connivens population of El Barranco de las Peñitas presented in this work occurred together with other species, i.e. macroalgae and vascular plants. The C. connivens meadow was accompanied by Cladophora glomerata and Rhizoclonium sp. mats (Fig. 5). Previously, Barinova & Romanov (2015) also observed C. connivens coexisting with filamentous algae, but in freshwater artificial pools (Northern Israel, Ein Afeq region). In this stand, C. connivens thalli were covered with unbranched filaments of the green alga Oedogonium sp. Vascular plants were represented by Ruppia maritima, which was a co-dominant species in the plant community (Fig. 5). So far, C. connivens associations on the Canary Islands have only been reported from Tenerife (Gil-Rodriguez et al. 1982; Raam & Gonzalez-Henriquez 1995) and Lanzarote (Gil-Rodriguez et al. 1982), where the convergent stonewort co-occurred with R. maritima, Potamogeton pusillus, and Myriophyllum spicatum (del Arco Aguilar & Rodríguez Delgado 2018). Specifically, the Canary Island populations of C. connivens in brackish habitats (marine lagoons) observed by Raam & Gonzalez-Henriquez (1995) were accompanied only by Ruppia maritima, but in rivulet stands, this alga coexisted with the moss species Fontinalis antipyretica and the vascular plant Nasturtium officinale. Moreover, the aforementioned authors observed that C. connivens meadows in freshwater artificial ecosystems (dammed reservoirs and agricultural water basins) could grow in the company of P. pusillus, but the meadows consisted mainly of the convergent stonewort completely covering the water surface of habitats. Therefore, the plant communities with C. connivens from the Canaries are rather poor in terms of species composition.
In the Mediterranean Sea region, C. connivens forms its own association, which dominates a habitat and is called Ass.: Charetum conniventis Velayos, Carrasco & Cirujano 1989 (Corillion 1957). According to many phytosociologists, C. connivens is a characteristic species of the halophilic alliance All.: Charion canescentis Fukarek ex Krausch 1964 (Krause 1969; 1981; Schaminée et al. 1995; van Raam 1998). C. connivens is also an accompanying species for other communities, i.e. Ass.: Charetum hispidae and Charetum asperae (Schaminée et al. 1995; van Raam 1998). In other phytosociological works, the association Charetum conniventis is classified into a different alliance, namely All.: Charion fragilis Krausch 1964, which includes all groups of Chara communities from oligo-mesotrophic and calcium carbonate rich water (Becker et al. 2016; del Arco Aguilar & Rodríguez 2018).
In aquatic ecosystems of continental Europe, C. connivens occurs in various aquatic plant associations on habitats with different levels of salinity. In German inland lakes, C. connives occurred together with C. virgata, C. globularis and Nitella flexilis (Becker et al. 2016). The following vascular plant species can coexist with C. connivens, i.e. Elodea nuttallii, M. spicatum, Eleocharis acicularis, Elatine hexandra, and Potamogeton berchtoldii (Becker 2008; 2010; Becker et al. 2016). In marine brackish habitats (Fehmarn Island, Germany), C. connivens coexisted with other Chara species, i.e. C. aspera, C. canescens, C. vulgaris and C. globularis, while vascular plants were represented by Stuckenia pectinata, P. pusillus, Zannichellia palustris ssp. pedicellata and Ranunculus peltatus subsp. baudotii. This plant community also included the green alga Ulva intestinalis (Heinzel et al. 2010; Becker et al. 2016). On the other hand, in the coastal lake Cämmerer (Usedom Island, Germany), C. connivens grew together with C. aspera, C. canescens, C. tomentosa, C. papillosa, Najas marina ssp. intermedia, S. pectinate and M. spicatum (Becker et al. 2016). In the shallow peat lakes of the Netherlands, C. connivens grew together with C. aspera, C. globularis, C. hispida, C. aculeolata, C. contraria, Nitellopsis obtusa, Najas marina and the moss F. antipyretica (Simons et al. 1994; Becker et al. 2016). In the Baltic Sea, C. connivens species were usually recorded together with the algae C. aspera, C. globularis, C. tomentosa, C. contraria, N. obtusa and Tolypella nidifica as well as the vascular plants M. spicatum, S. pectinata, P. pusillus, and Z. palustris ssp. pedicellata, including nymphoides species and lemnids (Torn & Martin 2003; Brzeska et al. 2015; Becker et al. 2016). In addition, in the marine habitats of Finland and Estonia, C. connivens co-occurred with C. baltica and C. horrida (Appelgren et al. 2004; Torn et al. 2004; Becker et al. 2016). In the ephemeral lakes of Sardinia Island (Mediterranean Sea), C. connivens grew with C. aspera, Nitella opaca, Baldellia ranunculoides and R. peltatus ssp. baudotii (Becker et al. 2016). Furthermore, in salt marshes of southern Spain, C. connivens coexisted with C. canescens and Nitella hyalina (Espinar et al. 2002). Moreover, in freshwater lakes in Greece, C. connivens was observed only with C. globularis (Langangen 2010), but in France – with C. braunii and Nitella translucens (Corillion 1957; Becker et al. 2016).
Ecology of C. connivens
Unfortunately, the only literature concerning C. connivens on the Canary Islands (from the 1980s and 1990s) fails to mention physicochemical characteristics of water. The C. connivens population from Fuerteventura examined in this study grew in water with a neutral reaction and under good aerobic conditions (average oxygenation level – 83.4%, average concentration of oxygen – 7.4 mg l−1). A phytoplankton bloom did not occur. The water was colorless and its color was at the average level of 20 Pt-Co units. The turbidity level ranged from 0.6 to 1.06 NTU (Nephelometric Turbidity Unit). The water in the habitat was also characterized by a high concentration of mineral compounds, which was reflected in a high level of electrolytic conductivity (10.11 mS cm−1) and TDS (6.63 g l−1). The high concentration of sodium chloride (3206.5 mg l−1) was the most important feature of the C. connivens stand. Furthermore, the water also contained many sulfates (196 mg l−1), as well as nutrients, i.e., orthophosphates (2.73 mg l−1) and nitrate nitrogen (from 0.8 to 1.10 mg l−1). On the other hand, the total iron ion content was low (0.07 mg l−1; Table 2).
Physicochemical parameters of Chara connivens habitat. Number of repetitions: N = 6
Parameter
Units
Minimum
Mean
Maximum
Depth of water
m
0.8
1.6
1.6
Temperature
oC
19.3
19.3
19.3
pH
–
6.4
6.6
6.8
Electrolytic conductivity
mS cm−1
9.8
10.1
10.3
TDS
g l−1
6.6
6.6
6.6
ORP
mV
64.0
64.4
65.2
Water color
Pt-Co mg l−1
14.0
20.1
25.0
Turbidity
NTU
0.6
0.7
1.0
Oxygenation
%
80.0
83.4
86.4
Oxygen
mg l−1
7.0
7.4
7.8
N-NO3
0.8
0.8
1.1
N-NH3
0.1
0.1
0.2
Fe-total
0.06
0.07
0.07
PO4
2.7
2.7
2.7
SO4
168.0
196.0
216.0
NaCl
2821.5
3206.5
3382.5
Salinity
PSU
4.9
5.0
5.0
The available data describing the ecology of C. connivens are partly consistent with our results. Populations of this stonewort in north-western ponds of continental Spain grew in waters with turbidity ranging from 5 to 46 NTU, but the optimum turbidity value was 17 NTU (del Pozo et al. 2011). Moreover, the cover of C. connivens was significantly positively correlated with the level of orthophosphate. Such an observation was in line with findings that C. connivens prefers hypereutrophic waters (Lambert-Servien et al. 2006; Becker et al. 2016). However, mesotrophic to slightly eutrophic waters were also occupied by this alga (Felzines & Lambert 2012). Thus, C. connivens may tolerate relatively high concentrations of phosphates and ammonium (Simons et al. 1994). Nonetheless, it has been experimentally demonstrated that the growth of convergent stonewort thalli is significantly limited above 10 mg l−1 of ammonium.
Populations of C. connivens are often found on limestone or in water with a certain amount of limestone, thus neutral to alkaline habitats (van Raam 1998). Some evidence indicates that optimum pH for C. connivens is between 7.0 and 9.0, and the optimum calcium level ranges from 45.6 to 180 mg l−1 (Corillion 1957; Nat et al. 1994; Langangen 2010; Becker et al. 2016). Based on research performed in the Netherlands and Greece, this species tolerates a wide range of conductivity, i.e. from 0.35 to 3.07 mS cm−1 (Nat et al. 1994; Langangen 2010). Similarly, C. connivens can grow in ecosystems with varying chloride concentrations, ranging from 0 to 1070 mg l−1 (Simons & Nat 1996). According to van Raam (1998), C. connivens can settle in mesohaline habitats from 5.0 to 18.0 PSU (Practical Salinity Units). It was experimentally demonstrated that the optimal growth of this alga needs 124 mg l−1 of chloride and is significantly inhibited at approximately 2774 mg l−1 (Simons et al. 1994). In the Baltic Sea, populations of C. connivens occurred in habitats with salinity ranging from 0.8 to 9 PSU, which is in line with other parameters describing the degree of water mineralization for this stonewort (Blindow 2000; Torn & Martin 2003; Appelgren et al. 2004; Torn et al. 2004; Langangen 2007; Brzeska et al. 2015; Becker et al. 2016).
Undoubtedly, the distribution patterns and ecology of C. connivens from the Canary Islands require further detailed studies. It is difficult to monitor and protect inland macroalgae habitats on Fuerteventura, because its natural freshwater and brackish water ecosystems are rare, ephemeral and constantly exposed to anthropogenic pressure (eutrophication, tourist activity – see Figure 2A, and interference in the environment), as well as affected by extreme natural events (weather changes leading to the drying up of water bodies and rivers). We hope that our study will contribute to a better understanding of C. connivens ecology in the Atlantic Ocean region.