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Cryptogamie, Bryologie, 2016, 37 (2): 211-223
©2016 Adac. Tous droits réservés
doi/10.7872/cryb/v37.iss2.2016.211
Sphagnum centrale and S. palustre from
Mediterranean basin: a comparison with conspeci!c
North American populations by microsatellite analysis
David CRESPO PA RDO*, Simonetta GIORDANO, Maria Cristina SORRENTINO
&Valeria SPAGNUOLO
Dipartimento di Biologia, Universitàdegli Studi di Napoli Federico II, Complesso
Universitario Monte S. Angelo, Vi a Cinthia 4 - 80126 Napoli, Italy.
Abstract –Twenty nine South European specimens of Sphagnum centrale,S. palustre,
S. papillosum and S. magellanicum were studied with 15 microsatellite markers. In contrast
with eastern North American populations, our analysis showed a genetic overlapping between
S. centrale and S. palustre in mixed populations. Moreover, Mediterranean species showed a
genetic richness (total number of alleles) higher than that calculated in conspeci!c American
samples. As Mediterranean Sphagnum bogs are remnant populations, microsatellites could
well work for selecting source populations in order to recover Mediterranean peatlands.
Genetic richness / Mediterranean peatland conservation / peat mosses / relict populations
INTRODUCTION
Peatlands are ecosystems with a great importance in the global climate
since they !x large amounts of carbon. These systems cover about 3% of the Earth
land surface (Yu et al., 2011). Even if peatlands are distributed worldwide, the
largest areas are in North America and North Eurasia, whereas in South Europe they
are in regression (Vasander et al., 2003; Yu et al., 2011). Indeed peatlands are
protected by the European Council Habitat Directive (Council Directive 92/43/EEC
of 21 May 1992 on the conservation of natural habitats and of wild fauna and "ora).
One primary objective of nature conservation is the maintenance of genetic
diversity, which implies the need to acquire information about intraspeci!c genetic
variation in populations of endangered/vulnerable species (Reed & Frankham, 2003).
The outcomes of population studies based on molecular markers should be taken
into account for planning strategies aimed at conservation strategies. The usefulness
of population genetics in biodiversity conservation, in particular for plants, has been
indeed recognized since the end of ‘80s (Avise, 1994).
At present, many projects aim to restore degraded peatlands due to habitat
loss and/or exploitation; the efforts are mainly focused on the establishment of the
best environmental conditions (see for instance Robroek et al., 2009). Nevertheless,
in most cases genetic imprint of the species and/or populations are not taken in
account, although the use of molecular methods (see Crespo Pardo et al., 2014, for
a review) can provide data for the application of different recovery procedures.
*Corresponding author: david.crespo@hotmail.es
212 D. Crespo Pardo, S. Giordano, M.C. Sorrentino & V. Spagnuolo
The scarceness of Sphagnum in South Europe makes unrealistic the use of
close populations to recover the relict peatlands. Some authors have criticized
reintroductions because they often involve genotype translocations across different
geographical areas (Edmands, 2006). In fact, especially in predominantly sel!ng
populations, organisms tend to be highly locally adapted, with a strong linkage
disequilibrium, which would be broken by introducing new non-adapted genotypes,
extraneous to local genetic pools, with the risk of outbreeding depression (Fischer
& Matthies, 1997; Edmands, 2006; Johnson & Shaw, 2015). However, Krishnamurthy
& Francis (2012), suggest the calculation of genetic distances between source and
receiving populations, in order to overcome this problem.
Sphagnum occupies the most basal position in moss phylogenies, holding a
highly conserved genome; accordingly, sequences for the internal transcribed spacers
of the nuclear ribosomal DNA can be easily aligned across its species (e.g. Shaw et al.,
2003). Furthermore, microsatellite primers that were developed for one group of closely
related Sphagnum species amplify homologous loci across the whole genus (Shaw et
al., 2008). By contrast, systematic studies sensu lato, mainly carried out by
microsatellites, highlight the occurrence of interspeci!c hybridization, molecular
divergence between disjunct populations, cryptic speciation and introgression, all
phenomena demonstrating an ongoing molecular evolution in peat mosses, in striking
contrast to the traditional idea of living fossils, frequently used to tag these plants (e.g.
Shaw et al., 2003, 2014; Karlin et al., 2008, 2009; Ricca et al., 2008, 2011).
Genetic approach in conservation biology is directed towards assisting in
resolution of taxonomic uncertainties. This effort is seriously compromised by the
lack of an agreed de!nition for species in biodiversity conservation. For instance the
relationship between Sphagnum centrale C. Jens. and S. palustre L. has been
extensively considered in the past years, since the morphological identi!cation
between these taxa is often dif!cult (Daniels & Eddy, 1985; Anderson & Ammann,
1991), and both taxa from North American populations were differentiated by a
microsatellite analysis (Karlin et al., 2010).
South European population of Sphagnum have been poorly studied so far,
in relation to biodiversity and conservation aspects. Terracciano et al. (2012) studied
the genetic variability in Italian populations of S. palustre with inter simple sequence
repeat (ISSR) markers. They found a higher genetic variation in the northern
populations, which have greater possibility of gene exchange with larger Northern-
European populations. However, they suggested local extinction risk for all the Italian
populations due to their very small size and the vulnerability of those peatlands.
This study aims to: i) investigate the genetic structure of populations of
Sphagnum centrale and S. palustre in South Europe by microsatellites; ii) compare
them to conspeci!c eastern North American populations; iii) clarify the relationships
between the two taxa in the studied populations. The results are discussed in relation
to management and conservation implications.
MATERIALS AND METHODS
Sampling
A total of 29 samples were analysed: 9 samples of S. palustre from Croatia,
Italy and Spain,11 of S. centrale from Bulgaria, Italy, Serbia and Turkey, 5 of
Microsatellite analysis of Sphagnum centrale and S. palustre from Mediterranean basin 213
S. papillosum Lindb. from Greece, Italy and Spain, and 4 of S. magellanicum Brid.
from Italy and Spain. All species, (the three !rst of which are allopolyploids and the
last one haploid), are included in the Section Sphagnum. Although the present study
aimed to investigate on a molecular ground the relationships between S. centrale and
S. palustre from Mediterranean basin, some samples of S. magellanicum and
S. papillosum were also included in the study, in order to validate the analysis.
Sphagnum palustre and S. centrale were regarded as distinct species, according to
Crum (1984) and Karlin et al. (2010). Species names and their acronyms used in
this work, origin information, and Herbarium data are reported in Table 1.
All the specimens were herbarium samples from AY DN, BP, NAP, SANT,
Herbarium Orto Botanico Universitádella Calabria (Table 1).
Table 1. Provenance and herbarium information of the samples analysed of the four Sphagnum species
studied.Acronym legend: CN = S. centrale;MG=S. magellanicum;PL=S. palustre;PP=S. papillosum;
BG = Bulgaria; ES = Spain: GR = Greece, HR = Croatia; IT = Italy; RS = Serbia; TR = Turkey)
Sphagnum species
(acronym sample used) Provenance Herbarium and accession code
S. centrale (CN-BG) Bulgaria, Vitosha Mt. NAP 30/2000
S. centrale (CN-IT1) Italy, Piemonte, Lagoni di
Mercurago NAP 12/2009
S. centrale (CN-IT2) Italy, Ve neto, Vigo di Cadore NAP 103/2009
S. centrale (CN-IT3) Italy, Piemonte, Va l d’ Ala NAP 154/1999
S. centrale (CN-IT4) Italy, Piemonte, Va l d’ Ala NAP 155/1999
S. centrale (CN-IT5) Italy, Piemonte, Va lle di ViùNAP 170/1999
S. centrale (CN-IT6) Italy, Piemonte, Va ldieri NAP 132/2010
S. centrale (CN-IT7) Italy, Piemonte, Va ldieri NAP 133/2010
S. centrale (CN-RS1) Serbia, Mt Vrtop BP 9536/S
S. centrale (CN-RS2) Serbia, Okruglica, Vlasina lake BP 9474/S
S. centrale (CN-TR) Turkey, Trabzon, AğaçbaşıYayla AYDN 6074-S
S. magellanicum (MG-ES1) Spain, Vall de Conangles (Lleida) SANT, Bryo 2407-A
S. magellanicum (MG-ES2) Spain, Xistral (Lugo) SANT, Bryo 2408-A
S. magellanicum (MG-ES3) Spain, P.to del Tremedal (Avila) SANT, Bryo 2409-A
S. magellanicum (MG-IT) Italy, Piemonte, Valdieri NAP 49/2010
S. palustre (PL-ES1) Spain, Galicia, Abadin SANT, Col. Carlos Real 155
S. palustre (PL- ES2 Spain, Galicia, Muras SANT, Col. Carlos Real 296
S. palustre (PL- ES3) Spain, Galicia, Ourol SANT, Col. Carlos Real 383
S. palustre (PL-HR1) Croatia, Gorski kotar Mts BP 9534/S
S. palustre (PL-HR2) Croatia, Gorski kotar Mts BP 9531/S
S. palustre (PL-IT1) Italy, Lazio, Posta Fibreno NAP 179/2010
S. palustre (PL-IT2) Italy, Toscana, Lago di Sibolla NAP 57/2006
S. palustre (PL-IT3) Italy, Piemonte, Lagoni di
Mercurago NAP 128/2006
S. palustre (PL-IT4) Italy, Cosenza, Parco Monte
Caloria
Herbarium Orto Botanico
Universitádella Calabria 1202
S. papillosum (PP-ES1) Spain, Galicia, Cervo SANT, Col. Carlos Real 379
S. papillosum (PP-ES2) Spain, Galicia, Muras SANT, Col. Carlos Real 162
S. papillosum (PP-ES3) Spain, Galicia, Abadin SANT, Col. Carlos Real 154
S. papillosum (PP-GR) Greece, Vo ras Mts. BP 9523/S
S. papillosum (PP-IT) Italy, Piemonte, Lagoni di
Mercurago NAP 129/2006
214 D. Crespo Pardo, S. Giordano, M.C. Sorrentino & V. Spagnuolo
Microsatellite analysis
DNA was extracted following the protocols described in Te rracciano et al.
(2012). Primer sequences and microsatellite characteristics for the 15 markers
analysed in this study are described by Shaw et al. (2008). The 15 microsatellite
markers, numbered as in Shaw et al. (2008), are: 1, 3, 4, 5, 7, 9, 10, 12, 14, 16, 17,
18, 19, 20, 22, 28, 29, 30.
Microsatellites were ampli!ed in 8 µl multiplexed reactions, each targeting a
set of three loci. Primer sets were arrayed for multiplexing according to expected
fragment sizes (for non overlapping ampli!cation products) and alternating
"uorophores. Each primer pair included a forward primer "uorescently labeled with
HEX or 6-FAM (Integrated DNA Technologies, Coralville, IA). Multiplexing was
accomplished using a Qiagen Multiplex PCR kit (Valencia, CA), scaled for smaller
reactions, but otherwise used according to the manufacturer’s recommendations. Five
to 20 ng of genomic DNA in 3 μl H2O served as template in each reaction. A standard
thermocycling regime was implemented for all primer sets, with no additional
optimization. This consisted of an initial denaturation and hot-start activation at 95°C
for 15 min, then 30 cycles of 94°C for 30 sec, 54°C for 90 sec and 72°C for 60 seconds.
A!nal extension at 60°C for 30 min was performed. PCR products were diluted in
sterile water, and 1.2 µl of the dilution was mixed with GS500 size standard and Hi-Di
TM Formamide (Applied Biosystems, Foster City, CA) for electrophoresis on anABI
3730 sequencer. Size determinations and genotype assignments were made using
GeneMarker 1.30 software (Softgenetics, State College, PA).
Genetic analyses
Fragment sizes were coded as alleles. All statistical calculations were
performed using GenAlEx v 6.1 (Peakall & Smouse, 2006). The Analysis of
Molecular Variance (AMOVA, Excof!er et al., 1992) was calculated using the
Codominant-Genotypic genetic distance option.
The proportion of shared alleles (POSA; Bowcock et al., 1994) was
calculated using Microsatellite Analyzer 4.05 (Dieringer & Schlötterer, 2003). POSA
ranges from 0 to 1, with 1 indicating that the two species are genetically identical.
The Principal Coordinates Analysis (PCoA) was performed as described in Smouse
& Peakall (1999). Nei’s Genetic Identity (Nei, 1972, 1978) among populations was
calculated as well.
Scanning Electron Microscope (SEM)
SEM observations were performed to rule out any possible morphological
misidenti!cation between Sphagnum centrale and S. palustre (Daniels & Eddy,
1985; Anderson & Ammann, 1991). Moss samples (see Ta ble 1, acronyms PL-IT3
and CN-IT1) from Lagoni di Mercurago (Northern Italy, 45°733’ 900’’N; 8°550’
220’’ E) were chosen because that was the only site where the two species are
sympatric. Shoots of Sphagnum centrale and S. palustre were !xed with 3%
glutaraldehyde for 24 h at 4°C, post-!xed in 2% OsO4 in 0.1 M phosphate buffer
(pH 6.8) at 4°C for other 24 h; afterwards, shoots were thoroughly washed in
phosphate buffer, cut into small pieces (3-5 mm), mounted on stubs and observed
humid under an environmental scanning electron microscope FEI QUANTA 200
working in low vacuum conditions.
Microsatellite analysis of Sphagnum centrale and S. palustre from Mediterranean basin 215
RESULTS
Microsatellite analyses
The number of amplicons per markers varied from 4 (marker number 3) to
24 (marker 10). Allele frequencies are shown in Table 2. The proportion of shared
alleles between Sphagnum centrale and S. palustre ranged from 0.06 (marker 22) to
0.70 (marker 29). The mean value for the 15 markers was 0.33.
The expected heterozygosity for Sphagnum centrale,S. palustre,
S. papillosum and S. magellanicum was 0.697, 0.605, 0.320 and 0.351 respectively,
whereas the observed heterozygosity was 0.800, 0.706, 0.200, 0.000. The
heterozygosity levels for each marker for S. centrale and S. palustre are given in
Table 3.
PCoA clearly separates the four species, with the exception of Sphagnum
centrale and S. palustre, slightly overlapping; in particular, three samples of
S. palustre -those from Italy, (PL-IT1, Pl-IT2, PL-IT3) –grouped with S. centrale
from Bulgaria, Italy and Serbia (CN-BG, CN-IT1, CN-RS1, CN-RS2) (Fig. 1). An
AMOVA for the four species showed a percentage of variation within species of
68% and 32% among species (PhiPT=0.324; p=0.001). An AMOVA between
S. centrale and S. palustre was performed as well. The percentage of variation within
species was 82% and 18% between species (PhiPT = 0.178; p = 0.0002). The genetic
distances (Fst) between the four species, all signi!cant (p< 0.05), and the Nei’s
Genetic Identity are given in Ta ble 4; as expected, the lowest divergence (lowest
Fst) and the highest Nei’s Genetic Identity were observed between S. central and
palustre.
Ax i s 2
Axis1
Principal Coordinates (1 vs 2)
S. palustre
S. centrale
S. papillasum
S. magellanicum
Fig. 1. Principal Coordinates Analysis of Sphagnum centrale,S. magellanicum,S. palustre and
S. papillosum genotypes. The !rst two principal coordinates comprise the 26.47% and 22.37% of the
variation, for a cumulative total of 48.84%.
216 D. Crespo Pardo, S. Giordano, M.C. Sorrentino & V. Spagnuolo
Table 2. Allele frequency and proportion of shared alleles (POSA) for 15 microsatellite markers in
Sphagnum centrale and S. palustre. Marker numbers are those used by Shaw et al. (2008)
Marker Allele S.
centrale
S.
palustre POSA Marker Allele S.
centrale
S.
palustre POSA
1242 –0.20 0.39 14 227 0.08 –0.21
243 0.04 0.25 229 –0.13
244 0.25 –231 0.13 –
245 0.08 0.05 238 0.04 –
250 0.17 0.20 245 0.08 –
251 0.13 0.30 17 153 0.08 –0.33
252 0.04 –155 0.58 –
254 0.04 –156 –0.20
255 0.08 –158 0.17 0.30
256 0.04 –160 –0.15
261 0.13 –161 0.08 0.20
3165 0.88 0.40 0.53 162 0.08 0.15
167 –0.10 18 96 0.22 0.57 0.51
168 0.13 0.40 126 –0.14
169 –0.10 138 0.11 –
4175 –0.33 0.34 139 0.11 –
176 0.11 –153 0.56 0.29
177 0.06 –19 243 0.05 –0.15
180 0.17 0.25 245 0.05 –
182 0.11 –253 0.20 –
183 0.06 0.17 254 –0.06
186 0.110.25 255 0.10
189 0.11 –256 –0.06
195 0.22 –260 0.15 –
198 0.06 –261 0.05 0.11
5184 –0.10 0.42 262 0.05 –
185 –0.05 263 0.05 0.44
186 –0.05 264 0.05 0.33
187 0.13 0.05 265 0.10 –
188 –0.10 266 0.10 –
189 0.08 0.10 267 0.05 –
190 0.08 0.10 20 272 0.17 –0.16
191 0.13 0.15 273 0.17 –
192 –0.05 278 –0.06
193 0.04 –282 0.17 –
194 0.08 0.10 284 0.04 –
196 0.08 –287 –0.22
197 –0.10 288 0.08 0.11
199 0.08 –289 –0.33
200 0.04 –290 –0.06
201 0.17 –291 0.08 0.17
202 0.04 –292 –0.06
203 –0.05 293 0.17 –
205 0.04 –298 0.08 –
9153 0.110.06 303 0.04 –
170 0.09 –22 87 –0.06 0.49
Microsatellite analysis of Sphagnum centrale and S. palustre from Mediterranean basin 217
Table 2. Allele frequency and proportion of shared alleles (POSA) for 15 microsatellite markers
in Sphagnum centrale and S. palustre. Marker numbers are those used by Shaw et al. (2008)
(continued)
Marker Allele S.
centrale
S.
palustre POSA Marker Allele S.
centrale
S.
palustre POSA
174 –0.56 90 0.04 –
175 0.09 –91 0.08 –
178 0.09 –96 0.04 –
180 0.05 –98 0.13 0.31
181 –0.22 99 0.04 –
182 0.32 –100 0.04 –
183 0.05 –101 0.17 0.13
185 0.09 –102 0.04 –
186 0.05 –103 0.08 –
189 0.09 –104 0.29 0.19
190 0.09 0.06 106 0.04 0.19
192 –0.06 110 –0.13
10 179 0.05 0.10 28 221 0.13 0.10 0.27
209 –0.05 223 0.08 0.20
210 0.09 –224 0.04 0.20
212 0.09 –226 0.08 –
213 0.09 –228 0.08 –
214 0.05 –231 –0.05
215 0.05 0.10 233 –0.20
216 0.05 0.05 234 –0.10
217 0.05 –235 –0.10
218 0.09 –237 0.13 –
219 0.05 –239 0.25 –
220 0.05 –241 0.08 –
222 –0.10 242 0.04 –
224 –0.10 244 0.08 0.05
227 –0.05 29 181 0.05 –0.70
229 –0.10 189 0.05 –
230 0.18 –190 0.20 0.15
231 –0.05 192 0.05 0.10
232 –0.10 193 0.30 0.25
233 –0.05 195 0.25 0.50
234 –0.15 199 0.10 –
239 0.09 –30 131 0.09 –0.25
244 0.09 –135 0.05 –
258 –0.05 137 –0.06
14 192 0.08 –0.21 138 0.41 0.25
206 0.08 –139 0.14 –
2110.04 –140 –0.19
212 0.04 0.25 141 –0.25
214 0.13 0.50 143 0.09 –
217 0.04 0.13 144 –0.25
219 0.17 –147 0.09 –
220 0.04 –153 0.05 –
225 0.04 –154 0.09 –
218 D. Crespo Pardo, S. Giordano, M.C. Sorrentino & V. Spagnuolo
SEM observations
SEM observations indicate that no morphological misidenti!cation occurred
in the mosses sampled at Lagoni di Mercurago, where Sphagnum centrale and
S. palustre are sympatric. Abaxial phyllidium surface (Figs 2 and 4) is not diagnostic,
showing in both species hyalocysts of similar size, with numerous pores, mainly
located between V-shaped wall thickenings. However, the two species can be
distinguished according to the different shape of their chlorocysts, very narrow and
barrel-shaped in S. centrale (Figs 5 and 6), triangular-shaped and protruding towards
the adaxial surface of the phyllidium in S. palustre (Fig. 3).
DISCUSSION
Conservation of the biodiversity is one of the main focus in the framework
of the environmental biology; knowledge and classi!cation of the organisms are
fundamental aspects to promote conservation policy. Research attention is mainly
converged on vulnerable organisms and habitats that are exposed to a concrete
extinction risk.
Bogs and fens are relict habitats in Southern Europe, and therefore are
worthy of attention, especially because they may keep a noticeable fraction of the
Markers
1345910 14 17 18 19 20 22 28 29 30
S. centrale N117 911101011118 9111111910
Na 10 2912 10 13 13 5412 911107 8
Ho 0.55 0.00 0.44 0.64 0.30 0.50 0.55 0.00 0.00 0.56 0.36 0.55 0.64 0.110.60
S. palustre N9 95989797887997
Na 54412 5 13 4 53576845
Ho 1.00 0.00 0.40 0.89 0.13 0.78 0.00 0.110.00 0.00 0.50 0.71 0.67 0.44 0.86
Total N20 16 14 20 18 19 18 20 15 17 19 18 20 18 17
Na 11 410 19 14 24 147 514 14 13 14 7 12
Table 3. Number of samples (N), number of alleles (Na) and observed heterozygosity (Ho) at
each locus for Sphagnum centrale and S. palustre for 15 microsatellite markers, numbered as in
Shaw et al. (2008)
Fst/GI S. centrale S. magellanicum S. palustre S. papillosum
S. centrale 0.000/1.000
S. magellanicum 0.310/0.145 0.000/1.000
S. palustre 0.151/0.398 0.323/0.184 0.000/1.000
S. papillosum 0.306/0.196 0.509/0.095 0.373/0.078 0.000/1.000
Table 4. Pairwise population Fst values/Nei’s Genetic Identity (GI) matrix between Sphagnum
centrale,S. magellanicum,S. palustre and S. papillosum
Microsatellite analysis of Sphagnum centrale and S. palustre from Mediterranean basin 219
biodiversity, as shown in the present study. However, the identi!cation of peat
mosses, and particularly Sphagnum species, possesses some problems due to their
high phenotypic plasticity (Stenøien et al., 2014; Szurdoki et al., 2014); mosses
growing in wet habitats exhibit high morphological variability in response to water
level "uctuations and quality changes, among other environmental conditions
(Hedenäs, 1996). Another factor affecting morphological variability and confusing
the species classi!cation of peat mosses is hybridization (Cronberg & Natcheva,
2002; Flatberg et al., 2006; Natcheva & Cronberg, 2007). Sphagnum centrale and
S. palustre are regarded as allopolyploid taxa, having one parental species in common
(Karlin et al., 2010).
Figs 2-6. SEM micrographs of Sphagnum palustre (NAP 128/2006) and S. centrale (NAP 12/2009).
2. S. palustre abaxial phyllidium surface. 3. S. palustre phyllidium cross section showing triangular
chlorocysts. 4. S. centrale abaxial phyllidium surface. 5. S. centrale phyllidium cross section. 6. Detail
of the S. centrale phyllidium cross section showing narrow, barrel-shaped chlorocysts (ch = chlorocyst;
hy = hyalocyst).
220 D. Crespo Pardo, S. Giordano, M.C. Sorrentino & V. Spagnuolo
Previous studies in other Sphagnum sections (i.e. within the S. subsecundum
Nees in Sturm complex) have pointed out the differences between American and
European populations (Shaw et al., 2008). In line with this study, our results contrast
with those reported by Karlin et al. (2010), who found that, in eastern North
American populations, S. centrale and S. palustre formed two distinct entities on the
basis of microsatellite analysis. They found a between-species molecular variance of
29% and a between-species Nei’s Genetic Identity of 0.608. These values remarkably
contrast with ours (18% of the total variance between species and Nei’s Genetic
Identity of 0.398); despite the lower genetic identity, in our case the two taxa are
less divergent, and this is in agreement with their partial overlapping in the PCoA.
However, in agreement with the results by Karlin et al. (2010), taxa other than
S. centrale and S. palustre, formed distinct clusters in American as well as in
Mediterranean populations. The pattern of heterozygosity is more complex than
previously reported (Karlin et al., 2010). Sphagnum centrale and S. palustre from
eastern North America very frequently showed !xed heterozygosity, which support
hybrid origin for these diploid species; in Mediterranean samples !xed heterozygosity
is only observed once (at locus 1 for S. palustre). Fixed heterozygosity occurs in
allopolyploid taxa, and indicates a disomic inheritance for S. centrale and S. palustre
from eastern North America; a predominance of disomic inheritance was also
calculated in our conspeci!c samples (heterozigosity observed in S. centrale and
S. palustre 0.800 and 0.706, respectively). American samples showed higher levels
of heterozygosity than Mediterranean ones; this result could be related to the small
population size, or to ongoing genetic drift in Mediterranean populations. However,
this conclusion is not consistent with the results from POSA analysis, which indicate
a higher genetic richness of Mediterranean populations according to the higher
number of alleles over all loci found in these populations, despite the lower number
of loci analysed (!fteen versus seventeen). Even comparing the loci in common
showing very low levels of variability (loci 3 and 17), Mediterranean populations
seem to have a higher genetic richness; in fact, S. centrale and S. palustre from
eastern North America have a single allele in common at marker 3, versus four (one
American allele plus other three) alleles found in Mediterranean conspeci!c taxa.
Similarly, at locus 17, only two alleles were observed in American samples versus
seven (two American alleles plus other !ve) alleles found in South European ones.
These results indicate an overall higher genetic richness in the Mediterranean
populations, and suggest that, after the last glaciations, the eastern North America
populations have recruited from Mediterranean populations. Mediterranean basin is
considered a hot spot of biodiversity, including genetic diversity in mosses, e.g.,
Pleurochaete squarrosa (Brid.) Lindb. (Grundmann et al., 2008; Spagnuolo et al.,
2007, 2009); this !nding is probably related to the role of refuge area for animals
and plants of the Mediterranean region during the last ice ages. The retention of an
ancient polymorphism, re"ecting the ancient larger population sizes, could provide
a feasible explanation (Van der Velde & Bijlsma, 2003; Spagnuolo et al., 2009),
despite the lower size of these relict populations compared to those from Northern
Europe and eastern North America. Even if in some cases the number of shared
alleles (Table 2) between both taxa was very low (see loci 9, 10 and 30), the
percentage of shared alleles in relation to their total number was overall comparable
to that observed in eastern North American populations (23% versus 25%).
According to the lower divergence and the partial overlapping between the
diploid mosses Sphagnum centrale and S. palustre from Mediterranean area, and to
the absence of !xed heterozygosity in both taxa, a between-species cross scenario
may be hypothesized in the past in the study area. It is possible that the two taxa,
Microsatellite analysis of Sphagnum centrale and S. palustre from Mediterranean basin 221
able to hybridize in the past, lost this capacity while diverging in time. Indeed, the
partial overlapping between the two species in sympatric Italian population, coupled
to the dif!cult morphological identi!cation (Daniels & Eddy, 1985; Anderson &
Ammann, 1991) pushed us to observe morphological characters for ruling out any
possible misidenti!cation. A comparison of sympatric Italian samples by SEM
showed triangular chlorocysts projecting on adaxial surface in S. palustre phyllidium,
and barrel shaped chlorocysts in S. centrale, indicating a correct identi!cation of
mixed Italian populations (Figs 3, 5 and 6).
To our knowledge this is the !rst contribution addressing the management
and conservation of Mediterranean Sphagnum populations by microsatellite analysis;
the high genetic richness and the role as genetic reservoir are good reasons for their
conservation. The management of the endangered South-European Sphagnum
populations should take in account two critical aspects: i) a proper in situ conservation
of the endangered wetlands; and ii) the increment of the population size and genetic
diversity. Whereas the application of the legislation devoted to conservation is a key
issue in the !rst, the choice of the closest population as a source for the transplant
is decisive for the success of reintroduction programs, in order to reduce the risk of
outbreeding depression. To maximize the accomplishment of the reintroduction
programs in plants, Krishnamurthy & Francis (2012), have suggested to calculate
the genetic distances with barcoding tools, but the highly conserved nature of the
Sphagnum genome makes impossible the sequence approach in practice. Instead,
microsatellites, used in plants for conservation purposes (Reunova et al., 2014),
have proved their suitability to assess the distinction between Sphagnum populations
of different origin, and therefore, could be potentially used to calculate between-
population genetic distances in order to individuate source populations for recovery
of Mediterranean peatlands.
Acknowledgements. We would thank all collectors, and donors of Sphagnum
herbarium samples analysed in the present work: Beáta Papp, Carlos Real Rodriguez,
Domenico Puntillo, Luca Miserere, Mesut Kirmaci, Raina Natcheva. This paper is dedicated
to Stefano Terracciano, who !rst planned the idea of analyzing Sphagnum from Mediterranean
peatlands, for conservation purposes.
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