Download Population genetic diversity of the endemic Sardinian newt

BIOLOGICAL
CONSERVATION
Biological Conservation 119 (2004) 263–270
www.elsevier.com/locate/biocon
Population genetic diversity of the endemic Sardinian newt
Euproctus platycephalus: implications for conservation
Roberta Lecis
a
a,*
, Ken Norris
b
Lab.Genetica, Istituto Nazionale Fauna Selvatica, Via Ca Fornacetta 9, 40064 Ozzano dellÕEmilia (Bo), Italy, Via Cagna n.66, 09126, Cagliari, Italy
b
School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, UK
Received 27 June 2003; received in revised form 20 November 2003; accepted 21 November 2003
Abstract
The Sardinian mountain newt Euproctus platycephalus, endemic to the island of Sardinia, (Italy), is considered a rare and
threatened species and is classed as critically endangered by IUCN. It inhabits streams, small lakes and pools on the main mountain
systems of the island. Threats from climatic and anthropogenic factors have raised concerns for the long-term survival of newt
populations on the island. MtDNA sequencing was used to investigate the genetic population structure and phylogeography of this
endemic species. Patterns of genetic variation were assessed by sequencing the complete Dloop region and part of the 12SrRNA,
from 74 individuals representing four different populations. Analyses of molecular variance suggest that populations are significantly differentiated, and the distribution of haplotypes across the island shows strong geographical structuring. However, phylogenetic analyses also suggest that the Sardinian population consists of two distinct mtDNA groups, which may reflect ancient
isolation and expansion events. Population structure, evolutionary history of the species and implications for the conservation of
newt populations are discussed.
Ó 2003 Elsevier Ltd. All rights reserved.
Keywords: Control region; Critically endangered; Management units; Phylogeography; Sardinian brook salamander
1. Introduction
Characterizing genetic diversity at the molecular level
has been applied to a wide range of species conservation
problems (Hoelzel and Dover, 1994). One of the main
practical applications of conservation genetics is the
identification of Evolutionary Significant Units (ESUs)
and Management Units (MUs) within species and among
populations. As defined by Moritz (1994), ESUs are
geographically discrete populations which have evolved
separately for a substantial period of time, being reciprocally monophyletic at mitochondrial DNA, and
showing significant frequency differences of nuclear alleles. MUs are appropriate units for implementing shortterm conservation measures, being populations with
significant divergence of allele frequencies at nuclear
or mitochondrial loci (Moritz, 1994), which indicates
*
Corresponding author. Tel.: +39-0516512257/3282779966.
E-mail address: [email protected] (R. Lecis).
0006-3207/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocon.2003.11.011
some degree of short-term demographic independence
(Sherwin et al., 2000).
Amplification and direct sequencing of highly polymorphic regions of the mitochondrial genome provide a
potentially rich source of variation for investigating the
molecular population structure within species and the
phylogeny of intraspecific lineages (Wenink et al., 1993).
There are numerous recent examples of mtDNA studies
applied to the conservation of endangered amphibians,
through investigations of genetic variability and population structure (Murphy et al., 2000; Shaffer et al.,
2000), patterns of gene flow (Barber, 1999) and phylogeography (McGuigan et al., 1998; Bos and Sites,
2001). We report here an intraspecific investigation
based on the nucleotide sequences of Sardinian newtsÕ
control region.
The mountain newt Euproctus platycephalus
(Urodela, Amphibia), endemic to the island of Sardinia, Italy, is listed in Appendix II/Annex II of the Bern
Convention (1998) and is classed as critically endangered by IUCN (2000). It is also protected by the
264
R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
Regional Law n 23/1998, but should receive special
conservation status in Sardinia and nationally, being
probably the rarest and most threatened of all European salamanders (Grossenbacher cited by Andreone
and Luiselli, 2000). The other two species belonging to
the genus, Euproctus montanus and Euproctus asper,
are also endemics, inhabiting, respectively, the Corsican and the Pyrenean mountains. A mtDNA study
investigating phylogenetic relationships between the
three species of the genus Euproctus, found the Sardinian and Corsican newts more closely related to each
other than to the Pyrenean newt (Caccone et al., 1997).
Populations of E. platycephalus are found in all major
mountain systems of Sardinia: Sette Fratelli, Gennargentu and Limbara. Prior to our studies (Lecis and
Norris, 2004a,b), scarce information existed on the
geographic distribution and habitat ecology of the species, which is usually described as a fully aquatic newt
inhabiting streams, pools and small lakes on the eastern
side of the island (Rimpp, 1998). The range of Sardinian
newts has been shrinking and the population size declining in the past two decades (Puddu et al., 1988;
Colomo, 1999). This decline could be due to loss and
fragmentation of newt habitat, caused primarily by a
prolonged climatic drought which has involved the
whole island (Regione Sardegna, 2000).
An understanding of the genetic variation of Sardinian
newt populations is crucial for formulating conservation
strategies and management proposals. The geographical
isolation and small size of some populations of E.
platycephalus and its aquatic lifestyle (Colomo, 1999;
Voesenek and van Rooy, 1984) make genetically structured populations very likely. Recent work with other
Urodeles has shown substantial genetic divergence and
geographical structuring among salamander populations
(Alexandrino et al., 2000; Murphy et al., 2000). This
paper reports on a mtDNA sequence-based study designed to assess the population structure and phylogenetic relationships of Sardinian newt populations across
their entire range. The aim of the study is to investigate
patterns of genetic variation in E. platycephalus in order
to evaluate implications for its conservation.
2. Materials and methods
2.1. Study sites and sample collection
During 1999 and 2000, a total of 74 newts were
sampled from 8 localities across the species known
range, the purpose being to get samples from the three
major mountain systems on the island (Table 1).
Streams and pools inhabited by newts were selected
through field surveys. As shown in Fig. 1, a total of 11
individuals were sampled in the north of Sardinia, 27
were sampled in the centre east (Supramonte mountains,
eastern ridges of the Gennargentu), 18 in the central
mountains of Gennargentu, and 18 individuals in the
south of the island, Sette Fratelli mountains. Table 1
shows in detail study sites and number of samples collected. Samples within each population originated from
the same river catchments.
All individuals were sampled using either a toe-clipping technique (Sutherland, 1996) or by clipping a tiny
bit of the tail. In both cases, the digit or the tail tip regrows in a short period of time (Griffiths, 1996). Sex,
total and snout-vent length of individuals, description of
sampling sites and environmental parameters (water
temperature, relative humidity, water pH) were recorded
(Lecis and Norris, 2004b). Tissue samples were taken
from newts caught by hand or using fishing nets in
streams and pools. Animals were released into the pool
as close as possible to where they were found after the
data were collected and the tissue sample taken. These
were preserved in 95% ethanol and stored at 4 °C until
their use for DNA extraction.
2.2. DNA extraction and primer selection
Total DNA was extracted from tissue samples using a
standard phenol/chlorophorm method and Proteinase K
(10 mg/ml; Kirby, 1990). Four primers were selected
from Steinfartz et al. (2000), and one from a set of
universal primers designed primarily for mitochondrial
DNA (Kocher et al., 1989). In 2000, three new specific
primers were designed directly on the Dloop sequence of
Table 1
E. platycephalus study sites and number of samples collected
N
Site
Locality
1
2
3
4
5
6
7
8
Rio Suergiu Mannu
Rio Monte Gattu
Rio Guventu
Roa Paolinu
Rio Lardai
Pischina Ortaddala
Rio Pisciaroni
Lettodifica
M.te Sette Fratelli (south)
M.te Sette Fratelli (south)
M.te Sette Fratelli (south)
Gennargentu (centre)
Gennargentu (centre)
Gennargentu, Supramonte (centre.east)
M.te Limbara (north)
Gallura (north)
N refers to the localities in Fig. 1.
No. samples 1999
1
4
7
20
2
1
No. samples 2000
10
3
5
6
7
8
R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
265
2.3. Polymerase chain reaction (PCR) protocols
The PCR profile was defined according to the Tm of
the primers and the length of the expected PCR products
(Hillis et al., 1997), as follows: first denaturation at 94 °C
for 5 min, 30 cycles of denaturation at 94 °C (1 min),
annealing at 50–55 °C (1 min) and extension at 72 °C (1
min), final extension at 72 °C for 5 min. The reactions
were performed in 10 ll volumes with: 5 lM F Primer 0.5
ll, 5 lM R Primer 0.5 ll, 1Mm dNTPs 1 ll, 50 Mm
MgCl2 0.3 ll, 10 Taq buffer 1 ll, distilled water 4.6 ll
(variable), Taq DNA polymerase (5 U/ll) 0.1 ll, DNA 2
ll. When required, PCR was optimised adjusting the final concentration of MgCl2 . PCR reactions were performed by a Hybaid PCRExpress thermal cycler.
BioTaqTM DNA Polymerase was used at the required
concentration in each reaction. Before sequencing, PCR
products were checked by electrophoresis in 0.8% agarose in TBE buffer with ethidium bromide, and the bands
visualized using ultraviolet illumination at 360 nm.
2.4. DNA sequencing
Fig. 1. Sampling localities for E. platycephalus (for number of samples
collected in each site, see Table 1): 1, Rio Suergiu Mannu; 2, Rio
Monte Gattu; 3, Rio Guventu; 4, Roa Paolinu; 5, Rio Lardai; 6,
Pischina Ortaddala; 7, Rio Pisciaroni; 8, Lettodifica. The big circles
show the distribution of the 22 newt haplotypes across the island
(haplotypes are named after the first individual falling into the group).
Below, the number of individuals for each mtDNA type. E1 27 + 6, R3
3, M7 3, R2 2, M6 2, M5 1, E51 1, L4 4, L8 3, L11 1, Gu1 1 + 3, E46 1,
E45 1, G3 4, E34 2, G15 2, G14 2, E21 1, E32 1, E38 1, G19 1, G12 1.
E. platycephalus. One of these (SarEu1-H) in particular
has been successfully used for both amplifying and sequencing the 50 -end of the control region. Table 2 lists
all primers used in this study.
PCR products were cycle sequenced using the ABI
PrismÒ BigDyeTM Terminator Cycle Sequencing Ready
Reaction Kits (PE Biosystems), following the protocol
suggested by the manufacturer. The sequence reaction
recipe and the sequencing profile are as follows: DNA
template (PCR product) 2 ll, dilution buffer 3 ll, ready
reaction mix 2 ll, primer 1 ll, water to 20 ll; 30 cycles of
denaturation at 96 °C (10 s), annealing at 50 °C (5 s),
extension at 60 °C (4 min), (change of T° ¼ 1 °C/sec.).
Sequence reactions were precipitated by adding 2.5
volumes of 95% ethanol and 1/10 volume of sodium
acetate (pH 4.6), centrifuging for 10 min. at 4 °C, removing the ethanol and repeating the procedure with
200 ll of 70% ethanol. Sequence products were stored at
)20 °C, wrapped in foil, until their use for acrylamide
electrophoresis. This was performed using an ABI Prism
377 Sequencer, by the Plant Science Sequence Service
(University of Reading). Both the L and H strands of
Table 2
List of primers tried and used (sequence in bold) during this study; their nucleotide sequence, melting Temperature (Tm ), domain of the mtDNA in
which their sequence falls (position) and source. Letters L and H refer to the light and heavy strands. All primers were obtained by MWG AG
Biotech
Primer name
H1478
L-pro-ML
H12S1-ML
E.platyc.-L
E.platyc.-H
SarEu-L
SarEu1-H
SarEu2-H
Sequence
0
0
5 -tgactgcagagggtgacgggcggtgtgt-3
50 -ggcacccaaggccaaaattct-30
50 -caaggccaggaccaaaccttta-30
50 -ggcccatgatcaacagaact-30
50 -gctggcacgagatttaccaa-30
50 -gtcaaataacccaacaggag-30
50 -tcgtgtactgataagacgga-30
50 -ctgtcttagcattttcagtgc-30
Tm (°C)
Position
Source
72.4
59.8
60.3
57.3
57.3
58.5
58
59
12SrRNA
tRNA-Pro
12SrRNA
Dloop
12SrRNA
Cyt b
Dloop
tRNA-Phe
Kocher et al. (1989)
Steinfartz et al. (2000)
Steinfartz et al. (2000)
Steinfartz et al. (2000)
Steinfartz et al. (2000)
This study
This study
This study
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R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
the amplified products were sequenced for all samples. Sequence electropherograms were edited using
Chromas version 1.43 (http//www.technelysium.com.au/
chromas.html).
2.5. Sequence analysis
Sequences were aligned and consenses were produced
using Clustal X (http://www.igbmc.u-strasbg.fr/). Sequence ambiguities were resolved by comparing complementary strands. The identity of the consensus
sequences was investigated and confirmed using BLAST
(http://www.ncbi.nlm.nih.gov/BLAST/). Genetic structure and variation were investigated using ARLEQUIN
version 2.000 (Schneider et al., 2000; http://anthro.unige.
ch/arlequin), used to compute haplotype diversity (H),
nucleotide diversity ðpÞ, and infer population genetic
structure by analysis of variance (AMOVA), calculating
F-statistics (Wright, 1965). Levels of significance of Fstatistics were determined through 1023 random permutation replicates. The AMOVA approach in Arlequin
takes into account the number of mutations between
molecular haplotypes (Excoffier et al., 1992). Phylogeny
was investigated using PAUP 4.0 beta version (Swofford,
1999; http://www.sinauer.com). Parsimony and distance
methods were used to infer phylogenetic relationships of
haplotypes from the Dloop, tRNA-Phe and 12SrRNA
combined data set. Parsimony trees were constructed and
the strict consensus tree generated from 100 MP trees.
Distances were estimated using the Kimura-2-parameter
distance method (Kimura, 1980). The robustness of each
phylogeny was assessed by implementing bootstrap
analysis consisting of 1000 replicates.
3. Results
The 50 end of the sequenced region show a greater
number of variable sites, while the less variability was
found in conserved blocks between pair position 300 and
420 (Dloop) and 680 and 860 (30 -end of Dloop and
12SrRNA). Estimates of haplotypic diversity (gene diversity) was ranging from 0 (centre-east) to 0.9216 ±
0.0391 (south) across the populations, with a value of
0.7927 ± 0.0479 for the entire data set. A test of neutrality
(Tajima, 1989) indicated no evidence of a departure from
a standard neutral genealogy in a panmictic population
ðD ¼ 0:73377; P > 0:1Þ.
3.2. Population genetic structure
Overall, there is a highly significant geographical
structuring in E. platycephalus haplotype distribution.
The centre-east fixed mitochondrial type is also found in
the centre population, where other six distinct and unique haplotypes are represented. One haplotype in the
south population is shared with one individual from the
north, while the remaining nine south and five north
mtDNA types are all unique to the geographical area
where they are found (Fig. 1).
All AMOVA analyses resulted in significant structure.
The analysis of variance yielded highly significant values
of Fst (0.37517), revealing that a relevant proportion of
the sequence variation was distributed among populations. Significant structuring was also observed in the
pairwise comparisons between populations (Table 3).
The least divergence (Fst ¼ 0.10492) was observed between north and south populations, while the genetically
most distant populations (Fst ¼ 0.72253) were the north
and the centre-east ones. As expected, pairwise values for
the centre-east population, characterized by a fixed
haplotype, are the highest in the table. The lack of differentiation between north and south populations probably results from the presence of one shared haplotype.
3.1. Sequence variation
3.3. Phylogeographic relationships
A total of 915 bp was sequenced from 74 individuals,
representing 4 populations of the Sardinian newt from
north, centre, centre-east and south of the island. The
sequence obtained consists of approximately 700 bp of
the control region, 70 bp of tRNA-Phe and 145 bp belonging to 12SrRNA. Phylogenetic and population
genetic structure analyses were performed on the combined data set (Dloop, tRNA-Phe and part of
12SrRNA).
Sequence analysis revealed 22 unique haplotypes,
based on 19 polymorphic sites. Variable sites include 14
transitions, 2 transversions and 3 indels. Sequence divergence ranged from 0.11% to 1.85%. Nucleotide diversity was 0.0045 ± 0.0025 (0.45%) for the whole set of
sequences. On average, base composition was A 32%, T
31.6%, C 20.8% and G 15.5%.
In order to compare all the sequences under a phylogeographic perspective, a phylogenetic tree was constructed using the neighbour-joining algorithm and the
Kimura 2-parameter distance (Fig. 2). Parsimony analysis gave a similar result (observation of a consensus from
Table 3
Population pairwise FSTs
Centre-east
Centre
North
South
Computing conventional F-statistics from haplotype frequencies
Centre-east 0.00000
Centre
0.42656
0.00000
North
0.72253
0.15651
0.00000
South
0.60063
0.11438
0.10492
0.00000
R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
267
Fig. 3. Unrooted Maximum parsimony genealogical tree of 22 mtDNA
Dloop haplotypes from E. platycephalus (Length ¼ 20, C.I. ¼ 0.80).
Percent bootstrap replication scores >500 are indicated on each
branch.
the mtDNA lineages of the two haplotype clades of E.
platycephalus would have diverged between one and two
million years ago, therefore sometimes during the
Pleistocene.
Fig. 2. Unrooted neighbour-joining tree of E. platycephalus haplotypes, constructed using Kimura 2-parameter distances and midpoint
rooting. Haplotypes are coded with letters corresponding to geographic areas (N, north; S, south; C, centre; CE, centre east).
100 equally parsimonious trees, Fig. 3). From both trees,
it is clear that Sardinian newtsÕ haplotypes fall into two
strongly supported groups (1000 bootstrap replicates in
the MP tree). Each of these clades comprises haplotypes
found in the north, centre and south of the island. Despite
the highly significant geographical structuring in E.
platycephalus haplotype distribution, the presence of the
two clades suggests a more complicated phylogeographic
picture.
North, south and centre haplotypes appear quite
clustered in clade B, while clade A is characterized by a
less distinct inner structure (Fig. 2). Haplotype E1, fixed
for the centre-east population and also present in the
centre populations, is found in clade A, genetically very
close to other centre and south haplotypes (M6, M7,
E51, E32). Most of the north haplotypes belong to clade
B, apart from L4 and the sample L3, which represents
the shared haplotypes between north and south (Gu1).
Based on the mtDNA clock of 0.8% sequence divergence per million year for the Dloop of salamanders
(Steinfartz et al., 2000), it is possible to construct an
approximate time frame for the splitting of the different
groups identified. Using the estimated substitution rate,
4. Discussion
4.1. Dloop in salamanders and population structuring
As discussed by Steinfartz et al. (2000), the control
region (the most variable part of the mitochondrial genome in many taxa) is found to be comparatively slow
evolving in Urodeles. This is probably due to the lack of
some hypervariable segments which are apparently lost
in salamander Dloop, resulting overall much shorter
than, for example, mammalian Dloop (Steinfartz et al.,
2000). Nevertheless, variation in the mtDNA control
region has revealed remarkable levels of genetic structuring in the endemic newt E. platycephalus and is useful
to describe the relationships between newt populations
and discuss conservation implications.
The distribution of the newts haplotypes revealed by
sequence analysis shows a high geographical structuring
across Sardinia. The analysis of variance indicates that
37.5% of sequence variation is distributed among newt
populations, and 62.5% within them. The monomorphy
of the centre-east population brings as a consequence a
general increase in the pairwise Fsts values (Table 3)
between this population and the others. Pairwise Fsts
among north, centre and south populations appear
lower than expected, due to this effect.
In general, the geographical structure found in the
haplotype distribution (implying a degree of isolation
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R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
among populations from north, centre and south), does
not appear particularly supported by AMOVA pairwise
results and by the phylogenetic trees, where individuals
from the north, centre and south of the island do not
cluster as independently as expected (Figs. 2 and 3). As
discussed by Neigel and Avise (1986) and Avise (2000),
the observed polyphyletic pattern of maternal genealogies and the relatively high number of haplotypes found
in all but one population (Supramonte) would be expected – under neutrality assumption – when the effective population size is much greater than the number of
generations since founding. In the case of E. platycephalus, this could suggest that either foundings are recent
events or effective population sizes are large.
4.2. Phylogeographic relationships and molecular clock
Few studies exist on the intraspecific phylogenetic
structure and its association with geography over the
native range of newts and salamanders (Moritz et al.,
1992; Phillips, 1994; Alexandrino et al., 2000; Tarkhnishvili et al., 2000). An understanding of the biogeography of the Sardinian mountain newt throughout its
range has very important implications for the conservation of the species. The estimated sequence divergence for
salamander Dloop suggests an approximate time frame
for the events originating the detected haplotype clades
(Fig. 2). However, further genetic studies using both
mtDNA and nuclear markers (such as microsatellite
loci), with greater sampling throughout the distribution
of E. platycephalus, are needed to confirm any hypothesis.
Following a molecular clock and using clock calibrations for the Dloop in salamanders (0.8% sequence
divergence pMY; Steinfartz et al., 2000), the distinction
of two clades observed in E. platycephalus was found
corresponding to a genetic isolation of approximately
one to two million years ago. Given the time frame involved, the major climatic and environmental changes
that occurred during the Pleistocene (1.8 million years to
11,000 years ago) appear to have determined the evolutionary history of Sardinian newts.
In the Northern Hemisphere, Pleistocene glaciations
have had a major influence on the evolutionary history
of most species (Hewitt, 1996). The biogeography of
many species in Europe suggests that their population
structure was influenced by the quaternary climatic oscillations that have lead to glaciation events (Taberlet
et al., 1998; Steinfartz et al., 2000).
The apparent split of the two clades in E. platycephalus
could have been caused by adverse climatic conditions
during the Pleistocene, such as cold and dry glacial periods. These might have contributed to a south-north separation of populations (or generally to the isolation of
populations in two refugia), originating the two major
groups of haplotypes. A subsequent period of better climatic and hydrological conditions, typical of warm and
humid interglacial periods, could have promoted a secondary wide range expansion with migration of individuals, various re-colonization events and the consequent
presence of haplotypes from both clades all over the island. Following this hypothesis, the actual phylogeographic picture within the species might have been caused
by a repeated process of population contractions and expansions, originating from two opposite climatic scenarios. Founder effect or strong population bottlenecking,
followed by isolation, might have originated the fixed
haplotype characteristic of the centre-east population.
4.3. Gene flow and population isolation
The mountain systems where Sardinian newt populations were sampled (Limbara, Gennargentu and Sette
Fratelli mountains) can be considered as three isolated
patches in the range inhabited by E. platycephalus.
Currently isolated montane areas may have been connected transiently in the past. Sardinia used to be covered
by forests over most of its territory, and it is likely that in
the recent past a more capillary and widespread network
of mountainous streams were connected to each other
and to the major rivers. The period between 5000 and
1000 years BC was characterized by a warm and humid
climate with frequent precipitation, maintaining forest
cover on most of the island territory (Serra, 1980).
In the last 2500 years, the combination of fires, stock
breeding, human-induced deforestation, and consequent
desertification of the island, have gradually changed the
landscape and the climate in Sardinia (Serra, 1980;
Pungetti, 1995). More recently, the reduction in rainfall
and the change in climate which have dramatically reduced the water flow on the island (Regione Sardegna,
2000), and the consequent drainage of many water
courses could have increased the already existent isolation between mountainous areas.
Given that E. platycephalus is never found far from
water (Puddu et al., 1988), it is unlikely that newt populations are now interconnected by gene flow among
sampled areas. As most amphibians exist in metapopulations (Alford and Richards, 1999; Marsh and Trenham, 2001), newt populations in localized mountain
ranges (i.e., Sette Fratelli in the south) may comprise
metapopulations that could be interconnected in wet
years, but this needs to be documented. Finer genetic
markers and a finer spatial scale would be required to
investigate patterns of dispersal over a network of
streams and to quantify current gene flow between newt
populations.
4.4. Conservation and management of populations of E.
platycephalus
The maintenance of genetic variation is a major objective of most species conservation plans (Avise and
R. Lecis, K. Norris / Biological Conservation 119 (2004) 263–270
Hamrick, 1996). From the results obtained, it is possible
to draw a number of inferences on the conservation of
Sardinian newts. Loss and fragmentation of suitable
habitat, caused primarily by drought, and also by predation and competition with introduced species (such as
trout) and anthropogenic pressure could have reduced
populations of E. platycephalus in Sardinia (Colomo,
1999; Read, 1998; Rimpp, 1998). Therefore the longterm survival of this species requires the conservation of
as many genetic stocks as feasible for management.
Although there is no evidence of monophyly at
the mitochondrial level, newt populations inhabiting
streams on different mountain regions across the island
appear recently genetically isolated and possess unique
mtDNA types. This level of genetic structuring would
justify a differential management of various stocks on
Sardinia. Based on the evidence of different allele frequencies, the populations in the north, centre and south
of the island could be considered as three distinct
management units (MU; Moritz, 1994b). Given that the
environment does not change drastically among mountain systems and that E. platycephalus is threatened by
the same possible factors across all its range, distinct
MUs could be overall subjected to similar conservation
measures, although their genetic distinctiveness must
be considered in any re-location or translocation of
individuals.
Conservation management of E. platycephalus should
also aim to expand population ranges and patches of
suitable habitat for Sardinian newts, and population
numbers. Within each MU, interconnection and gene
flow between different populations inhabiting neighbouring streams should be promoted, instead of aggravating the isolation already existent among populations
(due to geological, hydrological and climatic conditions).
Conservation planning should take into account the
historical and current biogeographic structuring of
populations in the two major clades and in the north,
centre and south of the island. As individuals representing the two clades are found in each sampled location, genetic diversity and evolutionary history would be
conserved by protecting each individual population. The
mountain system isolate (Sette Fratelli, Gennargentu
and Supramonte, Limbara) could be used as the appropriate geographical scale for population monitoring,
and, within each one, multiple sites should be monitored. Although the mountain systems where Sardinian
newts occur are all under some degree of natural protection (Monte Limbara and Sette Fratelli being Regional Parks and Gennargentu will be a National Park),
only real implementation of conservation practices focused on this endemic species would be adequate to
protect the major components of genetic diversity of E.
platycephalus in the three areas. Practical conservation
management measures should include long-term monitoring of a sample of populations (estimating popula-
269
tion size and abundance, ecological requirements and
habitat correlates of distribution), protection of stream
habitats (also through some degree of fishing and
tourism control), creation (or implementation) of bioreserves around the main stream and river systems inhabited by newts (Lecis and Norris, in press).
Given the distinctiveness and apparently low adaptive
potential of the centre-east population, a conservation
goal should be to manage this population in order to
maintain its current apparent demographic health
(Sotgiu, 1996), giving the whole area of Supramonte and
Golfo di Orosei special conservation concern. This area
has been already recommended for the creation of a
biogenetic reserve by previous studies, on the basis of
herpetological surveys (Voesenek and van Rooy, 1984;
Voesenek et al., 1987).
Acknowledgements
We thank Ettore Randi and Massimo Pierpaoli from
Istituto Nazionale per la Fauna Selvatica (INFS, Bologna, Italy) for helping throughout the molecular analyses, and Sebastian Steinfartz (University of Cologne),
for providing primer sequences at the beginning of the
project.
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