The Contribution of Genetic and Genomic Tools in Diversity Conservation: The Case of Endemic Plants of Greece

: The conservation of endemic plant species has come into the global spotlight, not only because of their contribution to biodiversity but also their vulnerability and high extinction risk due to environmental and anthropogenic threats. Based on these developments, it is now essential to monitor and protect these species by applying integrated conservation strategies, especially in view of climate change, which is one of the most severe threats to plants. Genetic and genomic tools provide new potential in assessing and quantifying genetic diversity and thus can be utilized to devise conservation strategies and contribute to biodiversity conservation efforts. Greece comprises a plant biodiversity hotspot in the Mediterranean Basin with a wide variety of rare, threatened, and endemic plant taxa. In this review, we examine several cases where a broad spectrum of genetic tools has been utilized so far in the diversity assessment and conservation management of Greek Endemic Plants (GEPs). Following an extensive database search, we have identified and included in our final data collection 19 studies concerning 32 GEPs for which molecular markers have been used for the determination of population genetic structure and diversity assessment, while at the same time, the research outcomes have been taken into consideration for conservation management. The limited application of genetic and genomic tools in GEP management is demonstrated, while the significance of implementing a comprehensive conservation strategy that will integrate genetic analyses and the data derived therein is also highlighted.


Introduction
Genetic diversity is regarded as the cornerstone of all conservation efforts, since it is essential for evolutionary adaptation and for species' ability to survive long term [1,2].Genetic diversity underpins populations' resistance and ability to adapt to environmental change.At the same time, a reduction in reproductive fitness is often associated with genetic diversity loss [3].Inbreeding, genetic drift, restricted gene flow, and small population size all contribute to a reduction in genetic diversity and, thus, to an increased risk of extinction [4,5].
Conserving plant diversity at genetic and species levels is vital because plants are the basis of human health and well-being, economic welfare, food safety and security, ecosystem stability, and other important areas required for the wellness of individuals and human societies [6,7].For example, the phenotypic selection of plant species and genotypes with favorable traits for cultivation and human nutrition led to the domestication of crop species, which is the foundation of the current food supply [8][9][10].It is evident that food depends on plant genetic diversity, and if it collapses, a major threat to ecosystems as well as humankind will arise.Thus, the continuing decline in plant diversity will have a greater impact on human society than any other type of biodiversity loss [11].
Even if the significance of plant diversity is indubitable worldwide, plants are severely threatened due to various causes including overexploitation, habitat loss, fragmentation, degradation, invasive species, pollution, and generally unsustainable land, water, and energy use [12,13].To make matters worse, climate change, responsible for long-term alterations in global or regional climate patterns, is one of the biggest threats to plant species around the world.It can cause alteration, destruction, or even loss of habitats, subsequently leading to a loss of natural populations (or even entire species) and eventually to a loss of genetic diversity [14][15][16][17].
In the case of a plant species occurring naturally and exclusively in a specific geographic area, the loss of its genetic potential could be irreversible, and these rare species are more likely to have specifically unique traits (linked to specific genes/alleles) that could be further exploited in the future [12,18,19].Plants restricted to one region and found in no others are characterized as endemic.Narrow endemism refers to a common form of rarity used to describe very small areas of distribution of taxa [20][21][22].A very important group of organisms is the extremely narrow endemics, which usually occur in one or very few populations (five or fewer) [23].Most endemics are characterized by a small population size and short reproduction capacity, require specific habitat conditions, and possess a limited ability to cope with radical environmental change and exposure to extreme climatic conditions.They can also be collected and overexploited by humans for a variety of reasons.Therefore, endemic plant species are more vulnerable than widespread ones to anthropogenic threats and/or natural changes [24].The conservation of endemics has become a global priority, making their monitoring and management a primary need [24,25].
Endemic plant species hold a higher extinction risk.In situ and ex situ conservation methods for endemic plant species are the two major approaches that could be undertaken to support the protection and preservation of these species [17].In situ conservation is one of the most suitable methods to apply because it preserves the original genetic material within the geographical centers of biodiversity under conditions that allow plant species to evolve [26][27][28][29].Ex situ conservation enables the preservation of plant genetic diversity outside its natural habitats and can be applied by using different approaches such as germplasm preservation in gene banks and/or botanical gardens, cryopreservation, plant tissue culture, DNA storage, pollen storage, etc., according to each species' particular characteristics and needs.In situ and ex situ conservation methods should be combined for a more comprehensive and successful approach to conservation management [27].
Maintaining genetic diversity is one of the central points of conservation biology.It promotes fitness and long-term survival of populations and guarantees the adaptive potential of species to respond to environmental changes [30].Because it is not possible to conserve all sites of biodiversity, conservation biologists must regularly decide how many and which populations are necessary to be sampled to have the highest probability of conserving the greatest amount of genetic diversity.According to Neel (2008), three basic perspectives should be taken into consideration for conserving the maximum amount of within-species genetic diversity: "(1) maintaining the range of diversity within taxa, required to provide variation in ecological functioning as well as the raw material for future evolutionary potential, (2) preventing excessive levels of inbreeding within populations to ensure that inbreeding depression does not increase species extinction probabilities, and (3) maintaining movement and communication among populations" [31].Genetic diversity can be assessed in conservation actions by mapping genetic management units, evolutionarily significant units, or populations with known alleles or haplotypes in selected reserves [31][32][33].However, the amount of genetic variation required for conservation is still an open question.Neel and Cummings (2003), using empirical data from four rare plant taxa, demonstrated that in the absence of genetic diversity data, it is essential to retain 53-100% of the populations sampled to capture all alleles and 20-64% of all populations to reliably represent heterozygosity and meet the genetic diversity conservation criteria adopted by the Center for Plant Conservation (1991) [33,34].Minimizing the overall genetic relatedness within populations (i.e., pedigree-based kinship breeding) significantly reduces the pace at which diversity is lost and becomes the method of choice to manage small populations [35,36].Especially for small, isolated populations, conservation genetics facilitated empirical insights into how population subdivision and small population size are linked with inbreeding and the increased action of genetic drift, which both lead to a loss of genetic diversity and subsequently to elevated extinction risk [37].
The objective in sampling should be to include at least one copy of the 95% of alleles that occur in the target population at frequencies greater than 0.05 [38].Sample sizes of 160 to 250 plants of a random-mating population are required to capture alleles at frequencies of 0.05 or higher in each of 150 loci, with a 90%-95% probability [39].However, when the required probability for conserving alleles at different loci increases or the frequency of a rare allele drops to 1%, larger sample sizes are required [40].Narrow and extremely narrow endemic plant species usually comprise small populations; therefore, the amount of existing genetic diversity is lower compared to widespread taxa that form large populations.Genetic diversity in small populations for both neutral alleles and those subjected to balancing selection is diminished, primarily due to five main reasons: (1) genetic drift fixes alleles more rapidly, (2) loci subjected to weak selection tend to be neutral, (3) mutation-selection equilibrium is lower, (4) the finite population size of balanced polymorphisms depends on the equilibrium frequency, and (5) balancing selection does not prevent the loss of genetic diversity [5].For example, in small populations, an allele is considered to be effectively neutral if its selection coefficient is less than about I/2N e (N e = effective population size) [41].Thus, population size is a major determinant of genetic diversity for all loci and characters in small populations and species of conservation concern.
Recent technological advances in the field of molecular biology have resulted in an increase in the number of genetic and genomic tools employed to measure genetic diversity and provided novel opportunities to meet conservation challenges and halt biodiversity loss.Knowledge of genetics (study of genes, their roles in inheritance, and their effects) combined with the recently developed field of genomics (study of the genome, i.e., all the genes of an organism, as well as their interactions with each other and with the organism's environment) will contribute to a more effective conservation strategy for threatened species [42,43].Firstly, genetic information could be used to minimize inbreeding and loss of genetic diversity, while genetic data help to resolve taxonomic problems, define management units within species, detect hybridization, define sites, and choose the best populations for reintroduction, eventually improving the overall understanding of species biology.Additionally, the assessment of genetic structure will help reveal populations with genetic issues, while information about gene flow would be useful to determine possible translocations and population movements [5].
Genomics can bring a new perspective on effective conservation strategies, especially for small populations, with detailed evaluations of the genetic history and status of populations that have been previously conserved either in situ or ex situ [44].Genomic tools offer the opportunity to identify allelic variants and thus guide the selective insertion of unrepresented lines to boost genetic diversity while minimizing similarity.Additionally, they can be used to detect potentially valuable rare alleles and haplotypes and are essential for preserving genetic diversity to identify the most suitable individuals for genetic rescue [44,45].Conventional gene banks can be enriched with genomic information to improve the management of the collections by the identification of duplicates and sampling gaps [46].Adaptation ability and specific loci underlying this adaptation in certain species can be evaluated and pinpointed by applying genomic technologies.The combination of genomic sequencing data and phenotypic data may be very effective in identifying genetic variation in specific traits that increase fitness in particular environments for adaptation to future climates [47].The adaptive potential obtained by genomics and other approaches can be included in risk assessments to improve the implementation of legislation made specifically for endangered species [48].
In the present review, we examine the variety of genetic tools employed in conservation management and their contribution to defining conservation prioritization for endemic plants.Greece was selected as a case study since it is one of the ten regional biodiversity hotspots of the Mediterranean Basin and hosts a wide range of rare, threatened, and endemic plant taxa [49,50].Additionally, we aim to investigate how genetic data are utilized in conservation strategies for Greek Endemic Plants (GEPs hereafter).

Genetic/Genomic Tools Used in Conservation
Genetic tools have evolved over the last few decades, revolutionizing conservation research and giving us the ability to measure and quantify genetic diversity [51].Molecular markers have been used to estimate population-level parameters (e.g., heterozygosity, effective population size, bottleneck events, inbreeding, polymorphism at different levels); to understand patterns of gene flow, movement across landscapes, and processes, such as hybridization, contemporary versus historic genetic isolation, reproductive patterns, social structure, and life cycles of threatened species; and to estimate the accumulation of deleterious mutations [43].
Biochemical markers and allozyme electrophoresis have been employed to study genetic variation at a genome-wide level, determine the genetic structure of populations, and gauge evolutionary histories and relationships between different species [52][53][54][55].With the advent of DNA sequencing techniques and polymerase chain reaction (PCR), several types of molecular markers have been developed to assess polymorphisms at the DNA level.These markers are either locus-specific or widely distributed in the genome.Among them, simple sequence repeats (SSRs), also known as microsatellites, have been effectively deployed to assess the genetic structure of natural populations and the genetic stability of long-term-maintained germplasm [56].SSRs are commonly used because of their preferential association with lowcopy regions of plant genomes, their high levels of polymorphism, and the codominant mode of inheritance [57][58][59][60][61].Other molecular markers effectively used in conservation genetics are random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeats (ISSRs), cleaved amplified polymorphic sequences (CAPs), sequence-characterized amplified region (SCAR), sequence-related amplified polymorphism (SRAP), and start codon-targeted polymorphism (SCoT) [62][63][64][65][66][67][68].More recently, with the advent of genomic technologies and their successful employment in delineating plant genomes (nuclear and chloroplast DNA), single-nucleotide polymorphisms (SNPs) are widely used in conservation studies to measure genetic diversity and assess the genetic structure of populations [69,70].
Over the past decade, new genomic approaches have been introduced and applied in the field of conservation.Next-generation sequencing (NGS) methods allowed high-throughput sequencing and large-scale multigene genotyping to be applied in nearly any organism at both DNA and RNA levels, including natural populations of rare or difficult-to-study species, to give new insights into population dynamics and highlight new opportunities in the analysis and interpretation of large genomic datasets [42,[71][72][73].Multiple genomic techniques provide sequence data on a reduced representation of the genome, such as the transcriptome or a preselected set of loci targeted with primers or hybridization probes [74].Many NGS related applications, such as reduced-representation sequencing (e.g., RADseq), transcriptome sequencing, and whole-genome sequencing (WGS) of population pooled samples or individuals, are now applied in conservation [75][76][77].The massive amount of data generated by NGS allows for testing complex demographic scenarios, linking changes in the number of effective alleles (A e ) back to the more distant past, and examining the impact of migration and inbreeding across the genome.WGS produces data from every part of the genome, and it is increasingly feasible for most taxa [78].Eventually, diversity estimates at loci related to expressed quantitative traits (quantitative-trait loci, QTLs) may help predict adaptive genetic variation and evolutionary potential [79,80].
The use of genetic tools is integral to the development of conservation plans for threatened species.However, the incorporation of genetic data into conservation management is not an easy task, although efforts have been attempted to define 'management units'-population units, identified within species to help guide management and conservation [81].In the late 1980s, the concept of evolutionarily significant units (ESUs) based on the level of genetic divergence was suggested [82][83][84][85][86][87].The identification of ESUs has been used to determine which populations should be conserved separately.ESUs are often recognized because they are highly genetically differentiated, presumably because they are likely to be on different evolutionary (and potentially adaptive) trajectories [85].The level of genetic divergence appropriate for defining ESUs is not necessarily straightforward.The main goal is to favor genetic distinctness without promoting the effects of genetic drift on generating genetic divergence [87].Regardless of this, the ESU concept can lead to practical outcomes, such as defining putative cryptic species adapted to different environmental conditions [88].

The Studied Area
Greece is one of the ten hotspots defined for the conservation of plant biodiversity in the Mediterranean area, being the eastern center of endemism together with Turkey [49,50].Compared to the flora of other European countries, Greece is one of the most species-rich, hosting more than 7000 native plant taxa, with ca.20% being endemic [89][90][91].The long and intricate paleogeographical history of the Greek area and its complex topography including ca. 8000 islands and islets and ca.4800 mountain tops contributed to the high diversity and endemism levels [92][93][94][95][96][97][98].

Methods
Despite the intense floristic surveys and recognition of the high number of endemics in Greece, little is known about the genetic diversity of GEPs and the contribution of genetic data to their conservation.Here, we present a comprehensive review of the available case studies regarding genetic diversity and its implication for the conservation management of GEPs.Greek endemics are defined as plants distributed exclusively in the 13 phytogeographical regions of Greece established by Phitos et al. (1995) and Strid and Tan (1991) [92,99].A list of GEPs with their respective characteristics is given in Table 1.In order to find relevant studies reporting the use of genetic tools for GEPs, we searched various databases.All data for the current review were collected, analyzed, and summarized from the published literature with the use of different sources of scientific search engines such as SpringerLink, SciFinder, Scopus, Web of Science, PubMed, Google Scholar, ScienceDirect, and Wiley Online.For this bibliometric investigation, several terms were used such as "endemic plant conservation", "endemic plants conservation genetics", etc.Finally, we selected only those dealing with the genetic structures of populations located strictly within Greece and excluded pure phylogenetic studies along with other studies with no conservation interpretation and meaning.Our final dataset includes 19 studies for 32 GEPs (see Table 1).The taxonomic nomenclature follows Plants of the World Online (POWO) and the Flora of Greece web (FoG).Information on the following has been derived mainly from the Flora of Greece web (FoG), Atlas of the Aegean Flora, and relevant literature that is referenced in Table 1 [100].
For each GEP, we have considered the following:

Insights about the Studied GEPs
Our search identified 19 research articles published between 1997 and 2024, corresponding to 32 Greek endemic taxa (29 species and three subspecies).Some of those taxa were analyzed several times, while in many studies, more than one distinct taxon was analyzed.The plant families that include the majority of GEPs are Asteraceae (6), Liliaceae (6), Caryophyllaceae (4), Lamiaceae (4), and Brassicaceae (3) (the number of included taxa is in parentheses) (Figure 1A).Following the same trend as families, the genera Centaurea and Tulipa comprise the highest number of GEPs with six taxa for each genus.Minuartia includes three species, Sideritis and Crocus comprise two taxa each, whereas the remaining genera include one taxon each (Figure 1B).

Insights about the Molecular Markers and Methods
The markers used to assess the genetic diversity of GEPs were SSRs (42%), DNA barcoding (either nuclear or chloroplast regions) (32%), AFLPs (26%), and ISSRs (21%), while RAPDs, RFLPs, REMAPs, and isozymes were used only in one study each.SSR markers, Most GEPs are found only on island(s) (17 taxa); 10 of them occur only on the mainland and 5 taxa are found both on insular and continental regions (Figure 1C).The most widespread taxon across Greece among the studied GEPs is Abies cephalonica, which is distributed in nine out of thirteen phytogeographical regions, followed by Crocus cartwrightianus occurring in six phytogeographical regions.The remaining taxa are found in one to two phytogeographical regions.The richest phytogeographical region in GEPs is Kriti-Karpathos; almost half of them (i.e., 15 taxa) were found in this region, while 9 taxa were found in Peloponnisos (Figure 1D).This is related to the known biodiversity hotspots in Greece, which include the mountains of Crete and Peloponnisos in the highest ranks [98].
In order to explore the altitudinal range of GEPs, we defined five altitude range classes as suggested by Georghiou and Delipetrou (2010) [121].Our findings are nearly in accordance with the results of the authors cited above since most GEPs occur in the first (0-600 m) but also the second zone (600-1000 m) (23 taxa in each zone).A gradual fall in higher altitudes is observed as well as an extreme downfall in the last zone (2000-3000 m) where only three GEPs could be found (Figure 1E).Only Centaurea raphanina subsp.raphanina and Tulipa cretica can be found in all altitudinal zones.
Concerning the type of substrate that GEPs prefer, it is evident that more than 50% of them grow on limestone (Figure 1F).Ten taxa have no preferences on substrates, whereas there are some edaphic specialists in the list such as Minuartia dirphya and Odontharrhena lesbiaca, which grow only on serpentines.These results are similar to those for Mediterranean Narrow Endemics.Medail and Baumel (2018) mentioned that 41% of these narrow endemics grow on limestone substrate, but they also found taxa highly specialized to specific substrates (gypsum, ultramafic, or volcanic rocks) [122].
The types of habitats have been categorized based on Dimopoulos et al. ( 2013) [89].Ten taxa are found in more than one type.However, cliffs, rocks, walls, ravines, and boulders host nine GEPs, while Xeric Mediterranean Phrygana and grasslands follow with four taxa (Figure 1G).
All GEPs are biennial/perennial apart from Saponaria jagelii, which is an annual species.Concerning the life form, most of the examined GEPs are Hemicryptophytes (11 taxa) or Geophytes (10), followed by Chamaephytes (8 taxa) (Figure 1H).Compared to the results for Mediterranean Narrow Endemics, they have the same pattern in the life cycle, but concerning life forms, Chamaephytes and then Hemicryptophytes predominate [122].
The karyological data retrieved for the studied GEPs presented various chromosome numbers (2n = 16 to 2n = 48), whereas the chromosome number is unknown for four taxa (Figure 1I).All GEPs with known chromosome numbers are diploids except Tulipa doerfleri, which is triploid (3×).For Tulipa goulimyi and T. orphanidea, two more ploidy levels (3X, 4X) have been recorded in addition to diploidy.
Based on the IUCN criteria, 72% of the studied GEPs have been evaluated, resulting in nineteen taxa being placed in threat categories (Critically Endangered = 7; Endangered = 7; Vulnerable = 5), two taxa accessed as Near Threatened and two listed as Least Concern (Figure 1J).Additionally, 18 of the studied GEPs are included in the Red Data Books of Rare and Threatened Plants of Greece and 19 are included in the Presidential Decree 67/81 [99,102].

Insights about the Molecular Markers and Methods
The markers used to assess the genetic diversity of GEPs were SSRs (42%), DNA barcoding (either nuclear or chloroplast regions) (32%), AFLPs (26%), and ISSRs (21%), while RAPDs, RFLPs, REMAPs, and isozymes were used only in one study each.SSR markers, which were the most common markers for population genetics in the 1990s and the 2000s, are the most preferable marker system for the conservation of GEPs, with the number of SSR loci used varying from 3 to 10 for each study.
Regarding the analytical methods used, almost all studies were based on statistics describing genetic variance (F statistics, AMOVA) often complemented by multivariate analyses (PCoA, clustering, etc.).By examining molecular methods, we observed that studies dealing with GEPs were based almost entirely on "low-cost" markers such as SSRs, ISSRs, and AFLPs, while the combination of two separate marker systems was preferred by several authors.However, no study using NGS technology was found.
As regards the SSR markers used, Parducci et al. (2001) studied four Abies species from the Mediterranean, focusing on the Sicilian endemic A. nebrodensis; however, they included four populations of the Greek endemic A. cephalonica in their study [103].In A. cephalonica, 98% of the cpSSR variation detected was within populations, while among populations, the variation was almost zero (fixation index (F ST ) = 0.012, P = 0.143).Nuclear and chloroplast SSRs were used to study population structure and gene flow among seven Cretan populations of the Aegean endemic plant species Brassica cretica, revealing exceptionally high levels of population differentiation (overall F ST = 0.628 and 1.000, respectively) and relatively low within-population diversity (overall within-population gene diversity (H S ) = 0.211 and 0.000, respectively) [105].Valli et al. (2021) also used nuSSR markers to study the rare and threatened species endemic to Zakynthos Island (Ionian Islands), Asperula naufraga, combined with other conservation methods [104].The genetic results showed low heterozygosity within sub-populations and a significant departure from Hardy-Weinberg equilibrium, which, combined with the small population size, suggested an increased threat of genetic diversity loss.Additionally, SSR markers have been used in three endemic Limonium species in the Ionian Islands (Valli et al. 2024, accepted), but this study has not been published yet; thus, it is not included in this review.
The DNA barcoding method was applied to Sideritis syriaca subsp.syriaca and Greek endemic tulips (Tulipa bakeri, T. cretica, T. doerfleri, T. goulimyi, T. hageri, T. orphanidea sensu lato) to provide, among other objectives, valuable guidance for targeted conservation efforts, while several nuclear and chloroplast regions (which could possibly act as DNA barcodes) were used for the Cretan endemic tree, Zelkova abelicea, to identify the unique genetic resources of the species and set priorities for its conservation.Paschalidis et al. (2024) mentioned that DNA barcoding was proven to be a valid technique for the discrimination of the S. syriaca subsp.syriaca genotype, contributing to taxonomy, morphology, and conservation studies [116].Samartza et al. (2024) used nuDNA and cpDNA regions for 15 wild-growing Greek tulip species to facilitate conservation and sustainable utilization efforts [117].The authors cited above provided the first step towards future conservation plans for native tulips of Greece, but more data are necessary to clarify their taxonomic status before prioritizing species and key areas for conservation strategies in Greece and beyond, or prior to their sustainable utilization.
AFLP markers were used by Sarrou et al. (2022) combined with phytochemical methods to evaluate the present status of Sideritis euboea and the inter-and intra-population diversity in the three mountains of its distribution range [115].The analysis revealed differences between the populations concerning their polymorphism and genetic homogeneity, resulting in a clear clustering of the main three populations corresponding to the three mountains.The other studies, which used AFLPs, also included additional methods.
The genetic diversity and population structure were examined using ISSR markers for five Greek endemics.For the Greek edaphic endemic Odontorrhena lesbiaca (=Alyssum lesbiacum), more than 96% of the markers were found to be polymorphic [112].The extremely narrow and rare Greek island endemics Aethionema retsina, Allium iatrouinum, Convolvulus argyrothamnos, and Saponaria jagelii displayed moderate genetic diversity based on ISSRs [68].
RAPD markers were used only by Psaroudaki et al. (2015), who investigated the genetic structure of 11 edible herbs grown in the wild of eastern Crete; among them is the Cretan endemic Centaurea raphanina subsp.raphanina [107].Isozymes were used to study genetic variation in the Cretan endemic Cyclamen creticum, resulting in high population diversity, high inbreeding rates in natural populations, and low levels of population differentiation [110].
The combination of two different marker systems was employed by several authors.Fineschi et al. (2002) used data from nuDNA and cpDNA regions (PCR-RFLP) and SSR markers for two relic tree species, Zelkova abelicea from Crete and Z. sicula from Sicily, highlighting the genetic differentiation between the two species being characterized by different haplotypes [118].Augustinos et al. (2014) used SSR and Retrotransposon Microsatellite Amplified Polymorphism (REMAP) markers for endemic Minuartia species [111].REMAPs revealed a significant amount of genetic variation at the population and species levels compared to the cpSSRs.It is noteworthy to mention that REMAPs were first applied by Kalendar et al. (1999) and then successfully used for the determination of genetic relationships, germplasm identification, and the assessment of genetic variation in several plant species [123][124][125][126][127]. López-Vinyallonga et al. (2015), studying a group of endemic Centaurea to continental Greece, used cpDNA sequences and nuSSRs [106].Their results combined with biogeographical data showed that levels of genetic variability are greater than those expected for species with populations consisting of few individuals, suggesting that there has been gene flow and introgression between species that are isolated today, in response to altitudinal variations that track quaternary climatic oscillations.Georgescu et al. (2016) applied nuDNA and cpDNA regions and AFLP markers to identify hybrids among the three species of Phlomis growing in Greece [114].Both methods showed marker-dependent capacity in the identification of chemotypes and hybrids.Combined genetic analysis with SSRs and AFLPs was performed for Crocus species, which demonstrated significant genetic variation within populations compared with low genetic variation between populations, suggesting substantial gene flow between populations [109].However, Larsen et al. (2015) mentioned that these methods failed to separate examined samples completely at the species level [109].Stathi et al. (2020), focusing on an endangered GEP and a wild relative of the cultivated Cicer arietinum, used ISSRs and AFLPs to determine the levels and structure of genetic variability [108].Based on their findings, medium to high genetic diversity at the population level was indicated.Papaioannou et al. (2020) used SSR markers together with High-Resolution Melting (HRM) and successfully discriminated all studied Origanum species and many of the population samples/genotypes [113].The interspecific relationships derived from the genetic similarity results were consistent, although the phylogenetic applicability of the method should be further tested.
So far, the molecular markers used in most of the studies examined, concerning the assessment of the genetic diversity of GEPs, were highly polymorphic, neutral, codominant, easily amplified, and low-cost, such as SSRs.These markers are still considered to be practical and powerful tools to reveal higher levels of genetic diversity and provide greater precision in conservation biology, since they can detect differences even among closely related species.Nevertheless, the general molecular type of markers such as RAPDs, AFLPs, and ISSRs was also preferred because apart from their low cost, they do not require previous knowledge of the genome of the studied species.Plastid (chloroplast) DNA has been proven to be a useful tool, mainly for phylogenetic purposes and the resolution of taxonomic uncertainties but is almost always combined with nuclear markers due to the maternal inheritance of the cpDNA.On the other hand, in the examined studies, DNA sequencing of specific genes was to a greater extent related to DNA barcoding.The application of NGS methods has not yet been established in conservation studies to any endemic taxon of the Greek flora, primarily due to the lack of reference genomes but also because of the technical complexity, the high cost, and the high level of bioinformatics required for the data analysis.

Insights about Conservation
The studies that were included in the present review have incorporated conservation implications either directly or indirectly.Most of them suggest taking specific conservation measures for the studied taxa, based on results from genetic analyses, usually corroborated by additional conservation methods.
Fineshi et al. (2002) revealed the strong genetic differentiation between the two remaining European Zelkova species, highlighting the conservation value of these marginal populations [118].They proposed ex situ conservation measures (e.g., pollen and seed banks, artificial clonal stands) to conserve the existent gene pools of Z. sicula and Z. abelicea and to facilitate the possibility of their reintroduction in the future when appropriate habitat becomes available.Christe et al. (2014), in accordance with the results of the previous study, added that although Z. abelicea covers a relatively small geographical area, it is highly genetically diverse within each of the four mountain massifs where it occurs, representing separate genetic units [120].
A study on Odontarrhena lesbiaca focused on providing initial guidance for the development of successful management and conservation measures.Adamidis et al. (2014) suggested considering watersheds and ecosystem types where the species grows as management units, and also ex situ conservation and restoration through collecting representative seed samples from different habitats [112].Based on the genetic results, it was suggested to keep one specific population separate from the other three central populations, to avoid any loss of genetic diversity and to preserve the character of the locally adapted populations.
The study by Augustinos et al. ( 2014) is important for developing conservation management plans for the three threatened Minuartia species [111].They suggested prioritizing the genetically unique populations for in situ protection and establishing a micro-reserve for M. wettsteinii.For M. dirphya in situ conservation actions, such as the protection of its entire habitat, the establishment of a micro-reserve and population reinforcement through artificial breeding and germplasm from the single known population are recommended.The relatively high genetic differentiation detected among M. parnonia populations, as well as the low levels of gene flow, indicate that at least those exhibiting higher levels of genetic diversity should be considered as separate management units and should have priority for in situ conservation actions.Moreover, genetically similar individuals can be used as population reinforcements to increase the current levels of genetic diversity in populations with low amounts of variation.
The data obtained by Psaroudaki et al.Based on genetic data, López-Vinyallonga et al. (2015) recommended ensuring the protection of at least one of the populations of Centaurea chrysocephala from Meteora, while the other populations would already be "conserved" [106].
The findings of Sarrou et al. (2015) support the distinctiveness of the main populations of Sideritis euboea from the three mountains, suggesting that all three populations should be considered as distinct units for conservation management, with Mt.Ochi's population being firstly prioritized [115].Overall, the Mt.Ochi population of S. euboea showed the highest genetic uniformity, which is expected as it is comparatively the most geographically isolated of the three main populations studied, though it is the one with the lowest percentages of polymorphism.
Stathi et al. (2020) considered genetic analysis to be a valuable tool for the implementation of an integrated in situ and ex situ conservation scheme approach for activating management programs for Cicer graecum [108].Future conservation plans should prioritize specific populations characterized by high genetic diversity belonging to different clusters.Furthermore, Stathi et al. (2020) suggest creating a stock of preserved propagules representing the entire genetic diversity of C. graecum, ensuring its use in future chickpea breeding programs and allowing future reintroduction or even relocation projects to be developed [108].2021) recommended a combination of in situ and ex situ measures, such as population reinforcement through artificial breeding, seed bank conservation, collecting germplasm, and establishing a micro-reserve for all four examined GEPs [68].
They highlighted the increased survival risk for narrow endemic species characterized by small population size and low to moderate genetic diversity, which are often linked to reduced fitness.The pressure of environmental changes makes things worse for these species.However, even threatened taxa may not become extinct due to environmental change before their genetic diversity is seriously depleted due to inbreeding depression [128].The findings of Kougioumoutzis et al. (2021) clearly show that the studied taxa display heterozygosity values resembling those of mixed or outcrossing reproductive species with low to moderate inbreeding levels [68].This might give them a fighting chance against climate change if they are not simultaneously faced with habitat degradation (as in the case of Allium iatrouinum) and other human-induced threats, such as overgrazing, trampling, and increased touristic activity.Convolvulus argyrothamnos seems to be in a slightly better state, mainly as a result of the inaccessibility of most of its individuals, but this does not mean that it is not showing signs of increased conservation concern (e.g., small population size, low genetic diversity).Samartza et al. (2024), providing DNA barcodes for six endemic tulips but also all the wild-growing tulips of Greece, offer new insights into their genetic distinctiveness that may be useful for future conservation and sustainable utilization efforts [117].However, additional data are necessary to accurately circumscribe and characterize the genetic relatedness, taxonomic distinctiveness, and phylogenesis of such wild and valuable tulip germplasm.
On the other hand, several of the studied publications do not suggest specific conservation measures, but their data could be easily integrated and used for this purpose [103,105,109,110,113,114,116].
All the examined studies related to the conservation management of GEPs clearly suggest that genetic data are an important factor in setting conservation priorities and devising conservation management strategies.It is evident that after the year 2000, there has been an effort by researchers to use genetic information to identify populations of concern, define management units, and/or define sites/populations for reintroduction.Similar studies from endemic plants in the Mediterranean, but also worldwide, have highlighted the importance of genetic data in conservation plans.For example, population genetic analysis of Jacobaea auriculata, an Iberian gypsohalophytic endemic, was conducted as it is a focal species, resulting in the determination of Relevant Genetics Units for Conservation, aiming to preserve the maximum genetic diversity and the selection of the most suitable populations from where seeds were collected for the future creation of new populations or the reinforcement of existing ones [129].These kinds of studies could be considered evaluations of genetic diversity before the reintroduction of a threatened species.Moreover, post-reintroduction genetic analysis using Arenaria grandiflora as a case study produced very promising results and showed that reintroduced populations could maintain increased genetic diversity long term [130].

Conclusions
In the Mediterranean Basin, conservation strategies comprise vital efforts to maintain the high floristic richness of the region.Genetic and genomic tools offer new approaches in the development of conservation strategies, but they have been applied in a limited range of research, especially regarding endemic plant species.It is crucial to consider and include the results of genetic analyses in applied conservation programs, notably since the greater part of the taxa of the studied Greek Endemic Plants in this review have been listed in threat categories according to the IUCN criteria.Conservation genetics give the ability to efficiently study genetic factors in nature, which is important for quantifying and mitigating threats to wildlife populations and gaining a more thorough comprehension of the extent of biodiversity change in order to develop more potent and integrated conservation plans.Filling the gaps is a massive and laborious task from a global perspective which is a prerequisite for establishing efficient conservation strategies.Our review has certainly proven that there are a lot of gaps to be filled in Greek flora and a lot of challenges for conservation geneticists to face.

(
Figure 1J).Additionally, 18 of the studied GEPs are included in the Red Data Books of Rare and Threatened Plants of Greece and 19 are included in the Presidential Decree 67/81 [99,102].
(2015) could contribute to the selection of in situ conservation of genetic resources and protected areas [107].The species examined in this study could be indicative of the capacity of Crete genetic resources for edible plants, such as Centaurea raphanina subsp.raphanina, with potential economic value.
Valli et al. (2021) combined the monitoring of demographic and reproductive parameters with genetic diversity analysis[104].According to their findings, the threat category (based on the IUCN criteria) of Asperula naufraga should be uplisted to Critically Endangered accompanied by effective in situ and ex situ conservation measures.Kougioumoutzis et al. ( Number of known populations and putative extinct populations; (h) IUCN categories according to the IUCN Red List of threatened taxa; (i) Inclusion in National Legislation, i.e., Presidential Decree 67/81 and Red Data Books of Rare and Threatened Plants of Greece [99,102].

Table 1 .
Greek Endemic Plants included in the current review and relevant information.Abbreviations for phytogeographical regions: EAe: East Aegean Islands;