234
Fottea, Olomouc, 16(2): 234–243, 2016
DOI: 10.5507/fot.2016.014
Multifaceted characterization of a Lemanea luviatilis population (Batrachospermales, Rhodophyta) from a glacial stream in the south–eastern Alps
Abdullah A. Saber1*, Marco Cantonati2, Morgan L. ViS3, Andrea aneSi4 & Graziano Guella4,5
1
Botany Department, Faculty of Science, Ain Shams University, Abbassia Square–11566, Cairo, Egypt;* Corresponding author e–mail: abdullah_elattar@sci.asu.edu.eg, tel.: +20 111 28 99 55 7, fax: +2 0226857769
2
Museo delle Scienze – MUSE, Limnology and Phycology Section, Corso del Lavoro e della Scienza 3, I–38123
Trento, Italy. email: marco.cantonati@muse.it. Tel: +39 320 92 24 755
3
Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA; e–mail: vis–
chia@ohio.edu, tel.: +1 740–593–1134
4
Department of Physics, Bioorganic Chemistry Lab, University of Trento, Via Sommarive 14, 38123 Povo,
Trento, Italy; e–mail: graziano.guella@unitn.it, andrea.anesi@unitn.it
5
CNR, Institute of Biophysics, Trento, Via alla Cascata 56/C, 38123 Povo, Trento, Italy
Abstract: The aim of this study was a combined and multifaceted characterization (morphological, molecular,
lipid, pigment, and ecological data) of a Lemanea (freshwater red alga) population from the south–eastern Alps,
exploring its adaptive strategies to the montane habitat, (turbulent, very–cold glacial stream with extremely
low–conductivity). Although the thalli were small (only up to 1 cm), the morphology was within the current
circumscription of Lemanea luviatilis. The molecular data placed this population within a clade of specimens
identiied as L. luviatilis and L. fucina. This L. luviatilis population was determined to possess lipid classes,
especially phosphatidylcholine and monogalactosyldiacylglycerol with high unsaturation index (UI) and long
acyl chains, which are typical adaptations for maintaining adequate membrane luidity and consequently all the
metabolic processes associated to the plasma membrane. The carotenoids proile revealed that, besides α /β–
carotene, there are signiicant amounts of zeaxanthin and lutein. This study further demonstrated that red algae
are a rich source of important food web w–3 fatty acids and may play an important role in the diets of grazers.
L. luviatilis is reported from one of the highest elevations (2,170 m a.s.l.) known for the genus Lemanea and
this species. This study conirms the presence of L. luviatilis in a cold, unpolluted, turbulent stream and this
type of stream may be its preferred habitat.
Key words: Lemanea luviatilis, freshwater, red alga, glacial stream, pigment analysis, lipidomics, adaptive
mechanisms
IntroductIon
Within the freshwater order Batrachospermales, there
are four genera, Lemanea, Paralemanea, Psilosiphon
and Petrohua, with a unique pseudoparenchymatous
tubular gametophytic thallus construction (EntwiSle
et al. 2009). This thallus morphology appears to have
evolved three times in this order with Lemanea and Paralemanea being sister taxa and both Psilosiphon and
Petrohua being distantly related in other clades of the
Batrachospermales phylogeny (ViS et al. 2007). The
genus Lemanea is easily distinguished from these other
three pseudoparenchymatous tubular genera by the following suite of characters: a central axis lacking internal cortical filaments, T– /or L–shaped ray cells closely abutting the outer cortex, and spermatangia present
The International Workshop on Benthic Algae Taxonomy Proceedings, Part II
Editors: Dr. Marco Cantonati & Prof. Aloisie Poulíčková
in discrete patches on the nodes (ViS & Sheath 1992).
Lemanea appears to be widely distributed in boreal
and temperate regions of North America (e.g., ViS
& Sheath 1992), and in fast–flowing streams in Europe (e.g., eloranta & KwandranS 2007; Kučera et
al. 2008; eloranta et al. 2011, 2016). It has been frequently reported from India, but appears to be localized
to Manipur state (GaneSan et al. 2015). In addition, this
genus has been infrequently reported from China (Xie
et al. 2004). L. fluviatilis has been frequently reported
from Europe, North America and India (ViS & Sheath
1992; eloranta et al. 2011; GaneSan et al. 2015 and
the references therein).
Lemanea is known from a wide range of stream
habitats from lowlands to mountains in both North
America and Europe (ViS & Sheath 1992; Kučera
Saber et al.: Multifaceted characterization of a Lemanea luviatilis
et al. 2008). However, this genus appears to be more
prevalent in cooler, faster flowing streams (Sheath
& HambrooK 1990; eloranta & KwandranS 2007;
Kučera et al. 2008; eloranta et al. 2016). There is
scatter data in the literature regarding stream chemistry
including nutrients (Sheath & HambrooK 1990 and the
references therein; ViS & Sheath 1992; Carmona et al.
2011. In Austria, it is considered indicative of high–altitude streams with low nutrient content (PiPP & rott
1994; rott et al. 1999). Likewise, Lemanea species
have been incorporated as an indicator of low nutrient streams in other European countries (e.g., rott &
SChneider 2014).
Previous research on Lemanea has covered a
variety of applied and basic topics. There have been a
few applied studies of Lemanea in India ranging from
use as an herbal remedy, diabetes treatment and biofuel production (GaneSan et al. 2015 table 2 and the
references therein). Much of the basic Lemanea research has focused on the systematics, biogeography
and potential use in biomonitoring (HardinG & Whitton 1981; ViS & Sheath 1992; eloranta et al. 2011).
There have been a few studies of the ecophysiology of
this genus primarily relating temperature and current
velocity to chlorophyll a, dry mass and carbon assimilation as measures of vigor and growth (Thirb & BenSon–EVanS 1982, 1984, 1985). To our knowledge, there
have been no studies regarding the lipid content of this
taxon and how this may relate to thermal niche. However, the effects of temperature on fatty acids composition patterns have been addressed by some studies on
micro– (e.g., FuSChino et al. 2011; Flaim et al. 2012;
Leblond et al. 2015) and macroalgae (e.g., BeCKer et
al. 2010).
The primary goal of the present study was to
characterize a high–mountain population of Lemanea from a glacial stream in the south–eastern Alps
by means of a multifaceted approach (genetics, morphological, membrane lipid and pigment analyses,
and ecology), and relating these to potential adaptive
mechanisms of this taxon to its glacial–stream habitat.
MaterIals and Methods
Study site. The Lemanea specimens were sampled at an elevation of 2170 m a.s.l., in the south–eastern Alps (Chiese
stream at Levade, in the upper part of the Fumo Valley in
the Adamello batholith, 46° 07′ 23″ N, 10° 33′ 54″ E), in a
glacier–fed, turbid and turbulent, well–oxygenated and cold
(5.86 °C, August 28th 2014 noon) stream. This stream lows
over holocrystalline bedrock in the Adamello–Brenta Nature
Park (Figs 1a–c).
Hydrochemical characterization. Water sampling was
conducted using polyethylene bottles, which had previously
been cleaned with ultrapure water and superpure nitric acid
(1%). Water temperature, pH, and speciic conductivity were
assessed in the ield using a HYDROLAB H20 multiprobe
235
datasonde. Detailed hydrochemical characteristics of the
stream including major ions, nutrients, trace elements and
metals were investigated following standard procedures and
methods (APHA 2000). Metals were analyzed by means of
ICP–OES (Optima 5300 Perkin Elmer Corp.). The ions, Na+,
Ca2+, Mg2+, Cl– and SO42– were measured using ionic chromatography (ICS 1500 Dionex Corp.). The nutrients (N–NO3–,
N–NH4+, TN, TP and SRP) were by molecular absorption
spectrometry and silicates as SiO2 by the molybdosilicate
method (APHA 2000; Cantonati et al. 2011).
Sampled algal materials. A stream reach about 20 m in
length was inspected with an aquascope to identify the most
suitable sampling points and to gain some information on the
distribution of the species. Specimens included in this study
were collected from rocks using forceps and placed in 100–
ml sterile clean polyethylene (PET) bottles for transport.
The specimens were transported on ice to the laboratory for
further studies. In the lab, the specimens were cleaned using distilled water to remove epiphytes and debris. This step
was veriied by microscopic examination. Subsequently, the
specimens were divided into four portions. The irst portion
was dried in silica desiccant for DNA extraction, the second
portion was ixed in 4% (v/v) formaldehyde solution for
morphological identiication, the third portion that was well–
cleaned was immediately used for lipidomics and bioorganic
screening, and the fourth portion was stored as a voucher
specimen in the Museo delle Scienze–MUSE, Trento, Italy,
and the Phycology Unit NO. 341 in the Botany Department,
Faculty of Science, Ain Shams University, Cairo, Egypt.
For morphological identiication and to visualize chromoplast autoluorescence, specimens were examined using both
Zeiss Axioskop 2 microscope (Zeiss, Jena, Germany) in Italy
and BEL® photonics biological microscope (Italy) in Egypt.
Morphometric diagnostic features were measured and photographed using Axiocam and Canon Powershot G12 digital
cameras. A total of 20 measurements were made for the taxonomically important morphometric features: thallus height,
diameter without and with the spermatangial papillae, and
the length of internodes. Specimens were morphologically
identiied using the relevant literature: ViS & Sheath (1992),
Sheath & Sherwood (2011) and eloranta et al. (2011). Photomicrographs were arranged into plates using Adobe Photoshop (version CS 4, Adobe Systems Inc.).
Molecular data generation. For DNA extraction, thalli
were ground in liquid N2 and extracted using Nucleospin
Plant Genomic DNA kit (Macherey–Nagel) according to the
manufacturer’s protocol. The rbcL gene was PCR ampliied
using the primer set (F160 & rbcLR; ViS et al. 1998) and
Extaq Polymerase system (Clontech Laboratories Inc.) in the
thermocycler conditions described in Keil et al. (2015). PCR
product was puriied using UltraClean PCR Clean–up DNA
Puriication Kit (Mo Bio Laboratories). The PCR product
was sequenced using an ABI 3100 Genetic Analyzer (Applied Biosystems); the ampliication primers and two internal
primers (F650, R897.lem) were used to completely sequence
the sense and anti–sense strand. Sequences were compiled in
Sequencer 5.2.4 (GeneCodes Corp.). In order to explore the
phylogenetic afinities of the new sequence, all rbcL sequences available in GenBank for Lemanea (34) and Paralemanea
(14) were obtained (GenBank accessed 25 March 2016). Two
sequences were excluded AF029153 due to sequence quality and DQ523257 as it appeared to be a Batrachospermum
and not a Lemanea sequence. Additional sequences of Ba-
236
Fottea, Olomouc, 16(2): 234–243, 2016
DOI: 10.5507/fot.2016.014
Fig. 1. Lemanea luviatilis sampling site: (a) Chiese Stream at Levade in the Fumo Valley (Adamello–Brenta Nature Park, south–eastern Alps);
(b) large boulders overlown by turbulent, swift, and cold water; (c) L. luviatilis tufts on a boulder in the stream viewed using an aquascope.
trachospermum gelatinosum (L) DC, Sirodotia delicatula
Skuja, S. huillensis (Welwitsch ex West & G.S.West) Skuja,
S. suecica Kylin and Tuomeya americana (Kützing) Papenfuss were utilized as outgroup sequences as these taxa have
been shown to be most closely related to Lemanea and Paralemanea in previous studies of the Batrachospermales (ViS
et al. 1998; entwiSle et al. 2009). If sequences were longer
than 1282 bp, they were trimmed to that length before further
analyses since most sequences were 1282 or shorter. The new
sequence from this study was aligned with the previously
published GenBank sequences using Muscle (edGar 2004)
as implemented in Geneious Pro version 8.1.5 (Biomatters,
Ltd., New Zealand, KearSe et al. 2012). The phylogenetic
placement of the specimen was explored using Bayesian Inference (BI) Analysis in Mr.Bayes v.3.2.6 (ronquiSt et al.
2012) and Maximum Likelihood (ML) Analysis in PhyML
(Guindon & GaSCuel 2003) as implemented in Geneious Pro
version 9.1.2. For both analyses, the general time reversible
model was implemented with gamma for the BI and both
gamma and invariant sites estimated by the program for ML.
The BI analysis was run for 1,100,000 generations with a
burn–in of 100,000. The ML analysis was run for 1000 generations using a parsimony–inferred starting tree with 1000
bootstrap replicates using a random starting tree. The new
rbcL sequence was submitted to GenBank (KU343187).
Lipid and pigment analyses. Total lipids were extracted by
a slightly modiied Folch method (FolCh et al. 1957). Briely,
cell clusters were collected into 15 ml glass tubes, re–suspended in 10 ml of chloroform/methanol 2:1 (v/v), sonicated
for 15 min in an ultrasonic bath (Sonorex Super, Bandelin
electronics, Berlin, Germany), and centrifuged at 3000× g for
10 min at room temperature to separate the organic phase
(bottom layer). For each cell pellet, the extraction procedure
was repeated three times. All the organic phases were collected, iltered by using glass ilters under vacuum, and reduced
to dryness on a rotary evaporation (Büchi Labortechnik AG,
Flawil, Switzerland) to obtain crude lipid extracts. Extracts
were re–suspended in 300 μl of methanol/chloroform 9:1
(v/v).
Crude lipid extracts were subjected to Reverse Phase
Liquid Chromatography–Electrospray Ionization–Ion Trap–
Mass Spectrometry analyses (RPLC–ESI–IT–MS). Under
this chromatographic setup, lipid molecular species were
separated primarily according to the hydrophobicity of their
acyl chains. Details are reported in Guella et al. (2003). Hydrophilic Interaction LIquid Chromatography (HILIC) was
also used to establish the membrane lipid class composition
based on the different polarity of lipids head groups (aneSi
& Guella 2015).
Each lipid molecular species was quantiied with respect to the total area of all lipid species belonging to the
same class (e.g., relative quantiication of MGDGx was performed with respect to total area of MGDG). The unsaturation index (UI) and the average chain length (ACL) were
calculated for each lipid class, using the formulas
UI class y = Σ (relative area lipidx * double bond number of
lipidx) and
ACL class y = Σ (relative area lipidx * acyl chain length of
lipidx)
where lipidx represents each single molecular species belonging to the y lipid class, respectively.
Pigments (chlorophylls and carotenoids) were simultaneously analyzed in the same chromatographic conditions through
a Photo–Diode–Array detector (PDA) operating at 470 nm
(carotenoids) and 660 nm (chlorophylls). Chromatographic
peaks were identiied by comparing retention times and online spectra (UV–Vis and positive and negative–ion ESI–MS
spectra) against known standards (FraSSanito et al. 2005).
Saber et al.: Multifaceted characterization of a Lemanea luviatilis
results
Ecological features
The Lemanea population was thriving on the downstream edge of large boulders overlown by swift currents (>1 m.s–1). The thalli were in tufts or lawns on
the boulders. In the stream reach examined the species
colonized larger boulders in strong currents.
Stream chemistry including the primary ions,
nutrients, trace elements, and metals’ concentrations
are listed in Table 1. Despite the extremely low conductivity (9 µS.cm–1), the water was only very–slightly
acidic (pH 6.2). Nutrient concentrations were very low,
especially phosphorus (S.R.P. 3 µg.l–1). Trace elements
and metals were measured in parts per trillion. The
only elements having notable concentrations were Al,
Fe, U, Ti, Zn due to the lithological characteristics of
the bedrock.
Morphological Characterization
The gametophyte thalli were small (up to 1 cm long),
olive green, primarily unbranched and in growing in
dense tufts (Figs 2a,b). The base of the thalli are stalked and the spermatangia in rusty–brown discrete
patches (Figs 2c,d). The thalli are pseudoparenchymatous, tubular, 150–500 µm in diameter without the
spermatangial papillae and 200–600 µm in diameter
with the spermatangial zones, with the surface covered
in numerous hair cells (Figs 2e–g). Internodes 0.25–
0.35 mm long. In addition, the carposporophytes can
be seen to project inwardly into the hollow centre (Figs
2f,g). The outer thallus is composed of small cells with
several, parietal, disc–shaped chromoplast that can be
easily seen in autoluorescence (Figs 2d,h,i). In cross–
section, it is evident that much of the inner thallus is
hollow with no cortical ilaments illing the center and
ray cells abutting the larger cells of the inside of the
tube (Figs 2 f,g,j,k).
Both the qualitative and quantitative characteristics of the population were assessed and the most recent key to European Lemanea species consulted (eloranta et al. 2011). The diagnostic characteristics in the
key showed the specimens to be Lemanea luviatilis.
However, the feature thallus height (6–30 cm long) in
the description was longer than the population study.
Given that thallus length may vary considerably and all
other characteristics were within the ranges provided,
the population was assigned to Lemanea luviatilis on
the basis of morphology.
Molecular data analysis
An examination of sequence similarity showed that the
sequence from this population was most closely related
(99.1%, similar or 11 bp different) to a group of GenBank sequences (AY575163, AY575164, AY575167,
AY575170). Both the BI and ML analyses produced
trees with similar topologies such that only the BI tree
with both the posterior probabilities and ML bootstrap
237
values is shown (Fig. 3). The sequence from this study
was sister to a clade containing sequences labelled as
Lemanea luviatilis (9), L. fucina (5) and Lemanea sp.
(1) and its inclusion with the clade was well support
(1.0/89). Like the sequence from the current study, all
within this clade were collected from various European locations. L. borealis was shown to be sister to this
clade but the support was low (0.73/–). In the current
analysis, L. luviatilis, L. fucina and L. borealis were all
shown to be paraphyletic.
Pigments and lipids
The carotenoids proile of this alga contains signiicant
amounts ofα /β–carotene, zeaxanthin and lutein – (Fig.
4a). Among chlorophylls (Fig. 4b), chlorophyll a is
dominant; the absence of chlorophyllide–a (loss of
phytyl chain, not detected) and the very low amount of
phaeophytin–a (loss of the magnesium ion, about 1%)
Table 1. Physical and chemical characteristics of Chiese stream inhabited by Lemanea luviatilis.
Variable
Chiese Stream at Levade
Temperature (°C)
5.9
–1
Conductivity (µS.cm )
pH
9
6.2
N–NO (µg.l )
–
3
–1
136
+
4
–1
9
N–NH (µg.l )
–1
TN (µg.l )
219
–1
TP (µg.l )
6
S.R.P. (µg.l )
–1
3
SiO2 (mg.l )
2.4
Na+ (mg.l–1)
0.70
Ca2+ (mg.l–1)
1.1
–1
2+
–1
Mg (mg.l )
0.13
SO4 (mg.l )
0.6
2–
–1
–
–1
Cl (mg.l )
0.2
–1
58.08
–1
Ba (µg.l )
1.26
–1
Rb (µg.l )
1.90
–1
0.18
–1
Al (µg.l )
Cu (µg.l )
Fe (µg.l )
51.79
Mn (µg.l–1)
1.56
Pb (µg.l–1)
0.06
–1
0.22
–1
11.82
–1
2.84
U (µg.l )
Ti (µg.l )
Sr (µg.l )
–1
Zn (µg.l )
–1
Mo (µg.l )
11.74
0.39
238
determined in all the LC–UV chromatographic runs indicated both optimal storage conditions of the alga and
mild extraction conditions.
Our LC–MS methodology also allowed identiication
of the membrane lipids contained in the raw extract
of Lemanea luviatilis (Table 2; Fig. 4c) relying on
retention time on different chromatographic stationary phases (RP18 and HILIC), on full scan and tandem
MS spectra obtained by positive and negative ESI
ion–modes ionization. Among the structural components of chromoplast, we proiled 20 monogalactosyl
diacylglycerols (MGDG), 22 digalactosyl diacylglycerols (DGDG) and 10 sulfoquinovosyl diacylglycerols
(SQDG) with other cell membrane lipids were in the
Fottea, Olomouc, 16(2): 234–243, 2016
DOI: 10.5507/fot.2016.014
classes 3 diacylglyceryl N,N,N–trimethylhomoserine
(DGTS) and 26 phospholipids belonging to the class
of phosphaditylcholine (PC). Among galactolipids,
MGDGs and DGDG bearing polyunsaturated and long
fatty acyl chain (in particular EPA, the w–3 eicosapentaenoyl chain, 20:5) were the most abundant in this
class (about 67% and 52%, respectively). Curiously,
concerning plasma membrane lipids, this PUFA chain
is very abundant (about 85%) in PC lipid species and
not present at all in any detectable DGTS species.
This hint is also in agreement with the averaged
unsaturation index (UI) of each lipid class. The PUFA–
richest (PC and MGDG) are also the most unsaturated
lipid classes (UI = 7.6 and 6.8 respectively), followed by DGDG (4.1); the PUFA–poorest (SQDG and
DGTS) are the less unsaturated lipid classes (UI = 3.3
and 2.8 respectively).
As expected, a similar trend is found for the
average chain length, with PC and MGDG having
the highest ACL values (38.5 and 37.9 respectively),
DGDG intermediate (ACL = 35.9) whilst SGDG and
DGTS have the lowest values (34.1 and 34.0).
dIscussIon
Most of the morphological characteristics examined
would suggest that this Lemanea population belongs
to the species Lemanea luviatilis. However, the thalli
collected in this study were distinctly short. The small
stature of this population has been observed in other
collections at the same site in different years (Cantonati personal observation). The small size may be due to
the glacial mountain habitat as Cantonati et al. (2001)
collected similarly small thalli from the typical high
discharge/high turbidity glacial Niscli Stream (Adamello) at an elevation of 2372 m a.s.l. (46°06'50.25"
N, 10°37'31.30" E). The smaller size of L. luviatilis in
this study might be an adaptive phenotypic mechanism to avoid the high current velocity and low–related
Fig. 2. Morphological characteristics for the population of Lemanea luviatilis: (a) overall habit showing small–sized olive–green
unbranched thalli in dense clumps; (b) basal disc from which unbranched, pseudoparenchymatous thalli arise; (c) lower part of the
thallus showing a short stalk (arrowhead); (d) magniied node showing spermatangia in rusty–brown patches (arrowhead); (e) close–up
view of the thallus surface showing plentiful hair cells (arrowhead).
(f) cross–section of thallus showing the developing carposporophyte
with ilaments (arrowhead) projecting into the center of the thallus;
(g) autoluorescent plastids in the outer cortical cells, and carposporophyte ilaments; (h) several, parietal, disc–shaped chromoplasts;
(i) autoluorescent chromoplasts; (j) light green–stained transverse
section showing a thin layer of small outer cortical cells, larger inner cortical cells with an empty central region; (k) magniication of
the transverse section characterized by the small outer cortical cells
and the larger inner cortical ones with ray cells (arrowhead) running
parallel to the cortex. Scale bars 5 mm (a), 20 µm (e, h–i, k), 200 µm
(b–d), 100 µm (f–g, j).
Saber et al.: Multifaceted characterization of a Lemanea luviatilis
Fig. 3. Phylogenetic tree based on rbcL sequence data showing the
relationships of Lemanea luviatilis (Italy) and previously published
sequences from GenBank using Bayesian Inference analysis in
Mr.Bayes v.3.2. Support values are shown as BI pp/ML bootstrap.
Branches without values had BI pp <0.7 and ML <60%.
stress in the montane habitat in the south–eastern Alps.
EVeritt & BurKholder (1991), ViS et al. (1991), eloranta & KwandranS (1996) and FilKin & ViS (2004) in
their studies of Lemanea and Paralemanea observed
these taxa at high current velocity (>1 m.s–1), but with
greater sized thalli. Perhaps, the smaller size is not related to current velocity, but the montane habitat (characterized, e.g., by scarce nutrient, in particular phosphorus availability). It likely seems that further studies
are needed to assess how widespread the small sized
thalli of L. luviatilis are in high mountain populations
of the Alps, and the possible adaptive signiicance of
this feature.
The molecular data placed the Lemanea population from the current study as sister in a clade with
sequences labelled as Lemanea luviatilis, L. fucina
and Lemanea sp. Unfortunately, there is no associated
morphological data with the specimens from GenBank.
Therefore, it is assumed that they were identiied using
the current circumscriptions for L. luviatilis and L.
fucina. These two taxa appear to be morphologically
distinguished with L. fucina having many thalli bran-
239
ched per population in ViS & Sheath (1992) and the
stalk being more tapered as well as thalli not growing
as compact tufts (eloranta et al. 2011; see Table S1).
These morphological characters seem like they may be
open to interpretation and could result in the species
epithets being utilized differently. As well, there is the
distinct possibility that these characteristics used for
morphological identiication may not be phylogenetically informative. Nevertheless, when future systematic studies are conducted, current Lemanea population will have both morphological and molecular data
documented so that it can be easily included and may
provide data from a more extreme montane habitat.
From the ecological standpoint, this study conirmed that L. luviatilis prefers unpolluted, fast–lowing, oligotrophic, mountain streams with cold water.
Accordingly, lederer & SouKuPoVá (2002) recorded
this species in rocky mountain streams and rivers in
Central Europe. ViS & Sheath (1992) suggested that
cold waters with an average temperature of 13 ºC are
the typical environments for members of the Lemaneaceae. They recorded populations in streams with
water temperatures between 7 and 24 ºC. Kučera et al.
(2008) showed that L. luviatilis is a widespread species in Czech Republic, usually growing on boulders
and cobbles in rifles, on weirs or in waterfalls which
are partly–shaded or well–illuminated. They also conirmed that L. luviatilis is not restricted to mountain
streams, but occurs across an altitudinal gradient ranging from 305 to 888 m a.s.l. Accordingly, eloranta
& KwandranS (1996) described this species from a
wide range of low velocity including strong currents
(0.2–1.9 m.s–1). GutowSKi et al. (2004), eloranta &
KwandranS (2007), and CeSChin et al. (2012) reported
L. luviatilis from oligotrophic streams. ViS & Sheath
(1992) reported that L. luviatilis in North America
occurs over a wide pH range (5.0–8.6). CeSChin et al.
(2012) determined this species in Italian watercourses with pH regimes ranging from neutral to alkaline
(7.4–8.6).
One of the most notable features of our study
site is the high elevation (2170 m a.s.l.). Cantonati et
al. (2001) sampled L. luviatilis from a stream in the
same mountain range at an elevation of 2372 m a.s.l.
These reports might be some of the highest altitudes for
Lemanea luviatilis occurrence (compare e.g. Sheath &
ViS 2015, who reported elevations up to 1200 m a.s.l.).
Recent studies of KwandranS & eloranta
(2010) and eloranta et al. (2011) on freshwater
red algae biodiversity in Italy lamented a scarcity of
knowledge about the biogeographical patterns of this
group in some Central European countries including
Italy. Our record might contribute to a more detailed
knowledge on the distribution of this species, particularly in regards to elevation and pH. Recently, combined molecular /morphological studies have provided
new insights into Lemanea/Paralemanea biodiversity
in so far poorly studied geographic areas (GaneSan et
240
Fottea, Olomouc, 16(2): 234–243, 2016
DOI: 10.5507/fot.2016.014
Fig. 4. Chromatograms of carotenoids, chlorophylls, and lipids for Lemanea luviatilis: (a) RP8 chromatogram as detected by PDA at l 470 nm
(carotenoids); (b) RP8 chromatogram as detected by PDA at l 665 nm (chlorophylls) (c) HILIC chromatogram as detected by full scan ESI(+)
mass spectrometer (lipid classes). Intra–class lipid distribution was obtained in reversed–phase chromatographic conditions.
Saber et al.: Multifaceted characterization of a Lemanea luviatilis
Table 2. Results of the membrane lipidomics analysis of Lemanea
luviatilis detailed per lipid class.
Items
Lipid classes
MGDG
DGDG
SQDG
PC
DGTS
species
20
22
10
26
3
UI
6.76
4.11
3.28
7.58
2.83
ACL
37.93
35.94
34.06
38.48
34
Total number of lipid molecular species
81
MGDG: monogalactosyl diacylglycerols; DGDG: digalactosyl diacylglycerols; SQDG: sulfoquinovosyl diacylglycerols; PC: phosphaditylcholine; DGTS: diacylglyceryl N,N,N–trimethylhomoserine;
UI: the unsaturation index of each lipid class; ACL: average chain
length for each lipid class.
al. 2015).
The carotenoids proile of this alga is in fair agreement
with that expected for a macrophytic type Rhodophyta
(Schubert & García–Mendoza 2008). Moreover, this
inventory of pigments, especially zeaxanthin and lutein, might have light–harvesting and protective functions in this harsh mountain habitat (TaKaiChi 2011).
Temperature and nutrient availability (speciically P) are the most inluential environmental drivers
affecting algal cell membrane luidity and permeability by the presence of special algal membrane–lipids’
classes, as an adaptive mechanism. DGTS are primitive lipids that are mainly determined in lower plants,
algae, dinolagellates, and mosses (Kumari et al. 2013;
RieKhoF et al. 2014). They are structurally similar to
PC, but lack the phosphate group. In general, DGTS
lipids can completely replace PC lipids in the plasma
membranes, especially in organisms living in harsh
environments, like those with low or very low phosphorus availability. However, the average UI and ACL
determined in this Lemanea luviatilis population for
these two lipid classes lie at opposite extremes, being
the highest (UI = 7.6 and ACL = 38.5) for PC and the
lowest (UI = 2.8 and ACL = 34.0) for DGTS lipid species. Although, the L. luviatilis habitat in our study has
low phosphorus concentrations, nutrients in this turbulent, high–current–velocity habitat are likely to be replenished rapidly through the reduced boundary layer
(maCFarlane & raVen 1985; Sheath & hambrooK
1990). Moreover, emerging evidence (GG unpublished
data) seems to indicate that betaine lipids distribution
mainly relects the algal phylogenetic relationships
rather than its ability to mediate changing environmental conditions.
The modiications of the unsaturation degree
of the lipid acyl chains are important for maintaining
adequate membrane luidity and therefore all the metabolic processes associated to the plasma membrane,
especially for organisms living in extreme environments. Accordingly, Lemanea luviatilis living in lotic,
very–cold streams possesses lipid species with high UI
241
and long acyl chains. The detailed lipid analysis of this
species further demonstrated that red algae are a rich
source of a important w–3 fatty acids such EPA (20:5)
as already been reported in the red alga Porphyridium
cruentum (Cohen 1990) and in several other species.
acknowledgeMents
We are grateful to Leonardo Cerasino, Edmund Mach Foundation, S.
Michele all’Adige (TN, Italy) for making available main–ion & nutrients data on the Lemanea habitat, and to Paolo Gabrielli and Emilie Beaudon, Byrd Polar and Climate Research Center, Columbus,
OH USA, for providing trace elements’ data. Emily Keil is thanked
for sequencing the Lemanea specimen. The results of this study were
presented at the International Workshop on Benthic Algae Taxonomy
(InBAT), June 17th–19th 2015, Museo delle Scienze – MUSE, Trento,
Italy.
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Supplementary material
the following supplementary material is available for this
article:
Table S1. Morphometrical and ecological comparative
study between L. luviatilis and L. fucina in this study and
other relevant literature.
Excel ile. Details of lipidomics and pigments of L. luviatilis in this study.
This material is available as part of the online article
(http://fottea.czechphycology.cz/contents)
© Czech Phycological Society (2016)
Received January 2, 2016
Accepted April 18, 2016