ploS One. 2016; 11(7): e0159545.
Published online 2016 Jul 21. doi: 10.1371/journal.pone.0159545
PMCID: PMC4956250
Kenneth M Olsen, Editor
Abstract
Dalmatian sage (Salvia officinalis
L., Lamiaceae) is a well-known aromatic and medicinal Mediterranean
plant that is native in coastal regions of the western Balkan and
southern Apennine Peninsulas and is commonly cultivated worldwide. It is
widely used in the food, pharmaceutical and cosmetic industries.
Knowledge of its genetic diversity and spatiotemporal patterns is
important for plant breeding programmes and conservation. We used eight
microsatellite markers to investigate evolutionary history of indigenous
populations as well as genetic diversity and structure within and among
indigenous and cultivated/naturalised populations distributed across
the Balkan Peninsula. The results showed a clear separation between the
indigenous and cultivated/naturalised groups, with the cultivated
material originating from one restricted geographical area. Most of the
genetic diversity in both groups was attributable to differences among
individuals within populations, although spatial genetic analysis of
indigenous populations indicated the existence of isolation by distance.
Geographical structuring of indigenous populations was found using
clustering analysis, with three sub-clusters of indigenous populations.
The highest level of gene diversity and the greatest number of private
alleles were found in the central part of the eastern Adriatic coast,
while decreases in gene diversity and number of private alleles were
evident towards the northwestern Adriatic coast and southern and eastern
regions of the Balkan Peninsula. The results of Ecological Niche
Modelling during Last Glacial Maximum and Approximate Bayesian
Computation suggested two plausible evolutionary trajectories: 1) the
species survived in the glacial refugium in southern Adriatic coastal
region with subsequent colonization events towards northern, eastern and
southern Balkan Peninsula; 2) species survived in several refugia
exhibiting concurrent divergence into three genetic groups. The insight
into genetic diversity and structure also provide the baseline data for
conservation of S. officinalis genetic resources valuable for future breeding programmes.
Introduction
For
thousands of years, people have gathered plant and animal resources for
their needs, resulting in changes to genetic structure of populations
over the course of cultivation and domestication. This process is
particularly manifested in crop species used for food [1], but is less evident in medicinal and aromatic plants (MAP), which are still harvested primarily from wild populations [2, 3]. Nevertheless, impacts on MAP intra-specific genetic diversity can occur through overharvesting in natural environments [4, 5]
or through population genetic bottlenecks caused by collection of seeds
from a limited number of wild plants that are subsequently used to
found cultivated populations [1, 6].
In either case, the need for comprehensive surveys of genetic diversity
in natural and cultivated MAP populations is an imperative for
efficient conservation efforts, breeding programmes and agricultural
production.
The reductions of gene diversity in
domesticated plants vary across species and have usually been examined
in crop plants such as soybean [7], maize [8] and wheat [9]. Domestication bottleneck processes reduce neutral genetic diversity across the entire genome [7, 9]; the strength of such a bottleneck is determined by duration and effective population size [10].
One of the key questions relating to the evolutionary processes
underlying domestication and cultivation of plant species concerns the
identity and geographic origin of populations [11] as well as the tempo and mode of domestication (e.g., single or multiple origin events) [10].
Furthermore, the cultivation of plants in proximity to their natural
environment can induce introgressive hybridization between domesticated
forms and their wild relatives, thereby impacting the initial loss of
genetic diversity [1, 12].
Additionally, similarities in habitat and climate conditions can foster
the naturalization of cultivated plants, thus expanding their influence
on natural populations and surrounding biodiversity [13–16].
Dalmatian sage (Salvia officinalis L.) is an outcrossing, insect-pollinated, perennial subshrubby plant of the family Lamiaceae. The genus Salvia is one of the largest genera in the family, with nearly 1,000 species distributed worldwide [17, 18]. Recent molecular phylogenetic studies revealed the non-monophyly of the genus [19–21] and the inclusion of the type species S. officinalis within the monophyletic clade I (Salvia sensu stricto; [20]). Salvia officinalis
is naturally distributed throughout the coastal region of the western
Balkan and central and southern Apennine Peninsulas, where it grows
abundantly on dry calcareous rocky soil [22–24].
The species is a well-known aromatic Mediterranean plant and has been
widely cultivated since ancient times for medicinal, culinary and
ornamental purposes. Extracts of S. officinalis have been shown to exhibit antioxidant [25, 26], anti-inflammatory [27, 28], fungicidal and bactericidal [29–31], virucidal [32], antispasmatic [33], antidiabetic [34], gastroprotective [35] and anti-obesity [36]
activity. The leaves are broadly used for aromatization in the food
industry, and the plant has recently become popular as an ornamental
garden plant [37], with several cultivars developed for this purpose.
Despite
the medicinal, historical and cultural importance of Dalmatian sage,
molecular data describing the population genetics and phylogeography of
the species are scarce. The majority of previous studies focused on
discovery and characterisation of bioactive compounds [38–41] and assessment of essential oil content and composition in relation to collecting site [42–46], environmental conditions [42, 47, 48], season [42], physiological stage (i.e., time of harvest; [49]), plant parts used for the extraction of essential oil [42, 50], soil mineral fertilization [51], drying procedure [52], and extraction [53] and distillation methods [54].
Random Amplified Polymorphic DNA (RAPD) [55, 56] and Amplified Fragment Length Polymorphism (AFLP) [57]
fingerprinting were used to analyse the genetic diversity and structure
of natural populations distributed in Croatia and Bosnia and
Herzegovina. Both marker types revealed high variability within the
populations, while genetic differentiation among populations showed a
pattern of isolation by distance. The highest genetic diversity was
found in populations from central part of eastern coast of the Adriatic
Sea, while the highest frequency down-weighted marker values were found
in the northernmost populations and the southernmost inland population.
Recently, a plastid DNA phylogeographic study based on eight Balkan
populations confirmed the natural origin of four disjunct inland
populations and revealed the presence of inland and southern coastal
lineages [58].
However, no studies have yet investigated the genetic diversity of wild
populations across the whole Balkan area as well as the genetic
diversity of cultivated and naturalised populations.
Microsatellites
are molecular markers widely used in germplasm conservation, genetic
diversity analysis, studies of genetic relationships, genetic mapping,
DNA fingerprinting and marker-assisted breeding [59–61].
Due to their abundance, high polymorphism, codominance, stability and
suitability for automated analysis, microsatellites provide an accurate
outline of the genetic structure of populations and can be used to
determine plants origin and phylogeographic history. The isolation and
characterization of specific S. officinalis microsatellite loci was recently provided by the Molecular Ecology Resources Primer Development Consortium et al. [62], Radosavljević et al. [63] and Radosavljević et al. [64].
The
main objectives of this study were to analyse demographic history,
genetic diversity and population structure in wild, naturalised and
cultivated populations of S. officinalis on the Balkan
Peninsula using eight microsatellite makers. We assessed the relative
levels of genetic diversity of natural populations compared to planted
populations in proximity and discuss the extent of genetic diversity
reduction that has occurred in naturalised and cultivated populations.
In order to reconstruct the species demographic history, and to better
understand contemporary genetic structure of wild populations, Maximum
Entropy Method along with Approximate Bayesian Computation were
implemented. As an endemic as well as economically important plant
species, knowledge of population genetics and demographic history of S. officinalis is of great importance for the effective conservation and utilization of the wild germplasm.
