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Communication

Towards the Conservation of Monumental Taxus baccata L. Trees of Thasos Island: Genetic Insights

by
Ermioni Malliarou
1,†,
Evangelia V. Avramidou
2,*,†,
Georgios D. Ranis
3 and
Diamantis I. Bountis
4
1
Forest Research Institute, ELGO-DIMITRA, Vasilika, 57006 Thessaloniki, Greece
2
Laboratory of Forest Genetics and Biotechnology, Institute of Mediterranean Forest Ecosystems, ELGO-DIMITRA, Terma Alkmanos, Ilisia, 11528 Athens, Greece
3
Forest Service of Thasos, A. Thelogiti 2, Thasos Town, 64004 Limenas, Greece
4
Private Forest Office, Pier de Vabez and Theagenous, Thasos Town, 64004 Limenas, Greece
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2023, 14(12), 2384; https://doi.org/10.3390/f14122384
Submission received: 23 October 2023 / Revised: 1 December 2023 / Accepted: 4 December 2023 / Published: 6 December 2023
(This article belongs to the Special Issue Vitality of Forest Ecosystems through Genetic, Epigenetic, Physiology and Biodiversity Exploitation)
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Abstract

:
Taxus baccata L. is a tertiary relict, long-lived, wind-pollinated dioecious tree species found throughout Europe. In the rocky mountains of Thasos island, monumental old trees create a unique area of natural beauty. In recent times, the need to implement conservation measures for key endangered species such as Taxus baccata has intensified. Exploring the genetic diversity of the species is a prerequisite for successful forest management decisions aimed at conservation. In this study, 28 monumental trees from two natural populations of Thasos were investigated using eight Simple Sequence Repeat markers in order to assess the levels of genetic diversity and genetic differentiation within the individuals, to estimate the degree of inbreeding and the effective population size of each population, and to discuss the impact this study has on conservation efforts for the species. Although the population size was small (14 individuals per population), the results showed moderate to high genetic diversity parameters. The mean expected heterozygosity was He = 0.649 and the number of effective alleles was Ne = 3.270 for both populations. Moreover, allelic richness (AR = 3.395) was high, indicating a variable genetic pool which is probably a result of a past established expansion of the species in the area. The results of the present study present a unique genetic pool harbored by specific trees, which is an important advantage for ensuring their conservation and resistance against biotic and abiotic threats. Our study paves the way towards conservation measures, which can be prioritized as follows: (a) in situ conservation, (b) seed bank establishment, and (c) in vitro propagation in order to secure future resilience and sustainability of the species.

1. Introduction

Forest species are a unique element of the biodiversity of natural ecosystems and play an important role in their existence and functioning. Within populations, there is high genetic variation, while among populations, it is low in relation to other plant species. This is due to their effective population size and extensive gene flow over long geographic distances [1,2].
The Mediterranean region is one of the regions with the greatest plant biodiversity in the world [3,4,5]. This is due to several factors, such as the complex geological history of the region, the cyclical climate changes that have occurred and are occurring, and the long-term impact of human activities that have formed the current landscape [6]. Due to human interventions, many habitats have been reduced in size, several have been fragmented, and, in some cases, there has been a loss of habitats [7]. The genetic consequences that arise in plants that have a limited range of distribution are a reduction in their genetic diversity and an increase in inbreeding, which can lead to a reduced reproductive capacity of species [8]. It is therefore imperative that conservation measures are taken for these species to preserve their genetic diversity and potential adaptive values for their long-term conservation [9,10]. While there have been many studies on the partitioning of genetic diversity in widespread forest species [11], very few studies have studied the partitioning of genetic diversity in forest species that grow sparsely [12]. Interestingly, even fewer studies have been devoted to the development of these species growing in temperate regions than those growing in tropical regions [13].
The yew (Taxus baccata L.) is a tertiary relict and long-lived forest species that is pollinated with the help of the wind and is found throughout Europe [14]. It is a species that grows in a wide range of territories and climate conditions and for this reason has a wide natural distribution with high ecological importance. Its natural distribution extends from North Africa to Scandinavia and from the Iberian Peninsula to the Caspian Sea (Figure 1). The yew plant has a wide range of pharmaceutical applications [15,16,17,18] and is valuable for its wood [19]. Some of the reasons that have led to a decline in yew forests as well as individual specimens are its slow growth, its weak competitiveness, the high quality of its wood, its uses in medical science but also its leaves which are toxic to animals [19,20]. The Species Survival Commission [21] has recognized it as an endangered species, as its populations have declined significantly; in many cases, they have become isolated and there are several areas where they have become extinct [22,23,24,25]. Its genetic diversity is structured both at the local [26,27] and the species-wide [28] scales. The high environmental heterogeneity of the species, combined with a reduced gene flow among populations of the species, could contribute to adaptive divergence for several genes of interest [29]. The yew is a forest species of particular interest for the study of its potentially adaptive genetic variation.
In recent years, a significant decrease in the number of natural populations of the species has been observed in different parts of Europe [11]. This decline is primarily due to two factors: long-term human impacts and climate change. Additionally, other factors that contribute to this decline are the lack of natural regeneration due to overgrazing and the reduced competitive ability of the species in relation to other plant species [25,30,31,32]. Nowadays, most of its remaining populations are small and fragmented, contributing to increased inbreeding and the potential for genetic drift. In addition, its extinction risk is high compared to that of homosexual species [33,34]. In conclusion, the species is endangered; for this reason, it has been given priority status in many in situ and ex situ conservation and restoration programs.
Many studies use Simple Sequence Repeats as a tool for conservation and in breeding programs of agricultural plants [35,36,37,38,39,40] and forest tree species [41,42,43]. In the present study, we used a set of Simple Sequence Repeats (SSR) to investigate the genetic diversity of monumental Taxus baccata tree populations on Thasos. Our objectives were (1) to determine the level of genetic diversity and genetic differentiation within individuals, (2) to estimate the degree of inbreeding of each population as well as the effective population size, and (3) to discuss the implications that this study has on efforts to conserve the species, more specifically these unique populations and individuals on the island of Thasos.

2. Materials and Methods

2.1. Study Area

Greece is located at the southeastern tip of Europe and is a major biodiversity hotspot [44] because it is a biogeographical barrier of distribution of many forest species and maintains important genetic pools of these species [41]. The island of Thasos is in the northern Aegean Sea, extending from 24°30′ to 24°48′ E and 40°33′ to 40°49′ N. Its climate is cool and humid Mediterranean with a mean annual precipitation of 860 mm and a mean annual temperature of 15.4 °C. Thasos was affected by large and extensive forest fires in the period 1984–2022 [45], with the most significant fires occurring in the last decade, when 20,000 ha of Pinus brutia Ten. and P. nigra J.F. Arnold forests burned [45,46]. Fires affect all parts of an ecosystem and most of the time lead to its degradation [47]. Thasos has a volcanic origin, with abundant limestone and marble [48]. Besides Pinus brutia and P. nigra, which are the main forest species of the island, there is also a rich plant community of Quercus coccifera L., Phillyrea latifolia L., Pistacia terebinthus L., P. lentiscus L., Arbutus unedo L., A. andrachne L., Myrtus communis L., and Taxus baccata, as well as shrubs (e.g., Erica arborea L., E. manipuliflora Salisb., Cistus creticus L., C. salviifolius L., Paliurus spina-christi Mill., Calicotome villosa (Poir.) Link, etc.). In the present study, populations of monumental old Taxus baccata trees growing in rocky, inaccessible locations and at an altitude of 1200 m were studied. This study was carried out in collaboration with the National Forestry Department of Thasos, which helped in the identification and collection of the samples as well as in providing information about the specific populations and the climatic and soil conditions prevailing on the island.

2.2. Plant Material

Sample collection was carried out from 28 trees (14 trees per population) from the two natural populations of Thasos island (indicative coordinates for population A: X: 560023, Y: 4504731 and B: X: 560603, Y: 4504011). All yew individuals growing on the island were selected due to the small sample size. The trees were old, monumental trees, as shown in Figure 2. Fresh needles were collected from each tree and placed in bags bearing a unique number and stored in a deep freezer to preserve their genetic material.

2.3. Molecular Analysis

DNA extraction from the fresh needles was performed with Macherey Nagel Plant kit (Düren, North Rhine-Westphalia, Germany) according to manufacturer’s protocol. In the present study, a total of eight primers were used to study the genetic variation of yew, namely Tax23, Tax26, Tax36, Tax86, and Tax92, according to [49], and ABRII-TB50, ABRII-TB56, and ABRII-TB58, according to [50] (Table 1). These primers were selected based on their polymorphic content and after an initial screening of a larger number of 25 primers [49]. PCR reactions were performed in a Biorad thermal cycler. The conditions applied for polymerase chain reactions (PCRs) were in agreement with corresponding papers [51,52]. Furthermore, 1 μL of PCR product was added to 10 μL of HiDi™ formamide (Applied Biosystems, Waltham, MA, USA) and 0.15 μL of 500 LIZ Size Standard (Applied Biosystems). Capillary electrophoresis was performed on an ABI3730 DNA Analyzer (Applied Biosystems, Waltham, MA, USA). GeneMapper v4.1 software (Life Technologies, Carlsbad, CA, USA) was used to analyze the results.

2.4. Statistical Analysis

The software GeneAlex vs. 6.5 was used for the analysis of the results [51]. Studying variations within populations, the effective number of alleles (Ne), expected (He) and observed heterozygosity (Ho), as well as Shannon’s Information Index were calculated using the software Genepop 4.7.0 [52]. Allelic richness (AR) and the number of private alleles (PAR) were calculated with HP-RARE 1.1 [53]. Inbreeding coefficient (Fis) for each locus was calculated with the FSTAT software version 1.2 [54]. The frequency of null alleles was also calculated with ML-Null software version 1 [55]; finally, Polymorphic Information Content (PIC) was calculated with the program Cervus [56].
The software program GENEPOP was used to test for genotypic disequilibrium between all pairs of loci. Furthermore, the GenAlEx software version 6.5 program was used for the hierarchical distribution of genetic variation (AMOVA) within and among populations. In addition, Principal Coordinate Analysis (PcoA) was also performed using the same program. Moreover, the number of migrants (Nm) was estimated based on the private allele method and Nei’s unbiased genetic distance and significance among populations were calculated [57]. Finally, the MEGA software version 11 was used to construct a phylogenetic tree according to neighbor joining distances [58].

3. Results

The results obtained in this study are presented below. A total of 74 alleles were obtained from the eight SSR loci used (Table 2). The number of alleles per locus (Na) ranged from 4 (ABRII-TB56) to 16 (Tax36) and the effective number of alleles (Ne) ranged from 1.96 (ABRII-TB56) to 5.49 (Tax36), with a mean value of 3.27 per locus. Although the number of trees per population was small, the effective number of alleles was high. In addition, expected heterozygosity (He) within the population was 0.65 for all SSR loci studied. The Fis studied ranged from −0.75 (ABRII-TB56) to 0.587 (Tax86), with a mean value of 0.235 alleles per locus. It was also observed that the degree of inbreeding was not high, although individuals in each population were few, indicating the maintenance of genetic diversity. The mean value of genetic differentiation (Fst) was 0.056, with the highest value of 0.117 for ABRII-TB50 loci and the lowest value of 0.016 for Tax23 loci. Finally, the mean value of the null alleles frequency (fn) was 0.202 (Table 2).
The overall mean value of allelic richness (AR) was 3.395 and the corresponding mean value of private allelic richness (PAR) was 1.04 (Table 3). The PIC ranged from 0.375 (TB58) to 0.823 (Tax36) and is presented in Table 3.
The results showed that genetic differentiation between the populations was high (Fst = 0.225, p > 0.05). The AMOVA test proved that differentiation among the populations was high (10%) and 90% of the variation was at the within-population level (Table 4). Furthermore, the level of gene flow (Nm) was measured to be 2.25 individuals per generation between the populations.
Additionally, Principal Coordinate Analysis (PcoA) explained 43.45% of the genetic variation, providing grouping for the two populations (Figure 3).
Finally, neighbor joining distances are presented in Figure 4 and also show the grouping of individuals according to their population.

4. Discussion

Recently, the need to take measures to properly manage monumental trees has become more and more imperative. The reason is that these trees host unique genetic pools. In view of the ongoing climate change and human interventions in forest ecosystems, the protection and preservation of monumental trees is considered necessary. These trees host unique genetic pools which are an important asset for ensuring the conservation and resilience of both them and their ecosystems against biotic and abiotic threats. The yew is a valuable tree for humans due to its widespread use in many areas of human life. In recent years, it has been recognized as an endangered species due to its low competitive ability and high wood quality which led to a decline in and shrinking of its populations around the world.
Despite the small size of the two populations studied (14 trees per population), the results of genetic diversity parameters were comparable to those of other studies. It was observed that the mean allelic richness value (AR) was 3.395, and mean expected heterozygosity (He = 0.649) was quite similar to Spanish (He = 0.509–0.661) [28] and Polish populations [59] (He = 0.737–0.865) of T. baccata (He = 0.509–0.661). Furthermore, the results of the present study had slightly lower values than [60], a study which studied 31 populations from Poland (mean: AR = 4.2; He = 0.738), but this could be explained by the small size of our populations. The same authors [60] also explain the high intra-population genetic variation is due to specific life history traits of Taxus baccata such as dioecy, wind-assisted pollination, longevity (up to 1000 years), and participation in late successional stages [61]. Previous studies using RAPD markers and isoenzymes also indicated high levels of genetic differentiation of Taxus baccata [22,60,62,63,64,65].
In another study of 93 Western T. baccata populations [27], contrasting patterns of genetic structure were found at different spatial scales: a small proportion of the observed variation was attributed to regional difference. Moreover, in the same study [27], populations in central Europe and northern Iberian Peninsula were less divergent compared to populations from Mediterranean Iberia and North Africa. Regarding population differentiation, even though only two populations were studied herein, the average value of Fst was high (0.225, p > 0.05) and similar to the Spanish populations studied in [26] (Fst = 0.227). Fst is the level of population differentiation, and it was shown to be relatively high in our study; that may be due to forest fragmentation which probably occurred in the area. This phenomenon of forest fragmentation has also been observed on Montseny mountain [28].
Taxus baccata is a long-lived dioecious gymnosperm tree and in theory, the level of genetic variation in Taxus should be relatively high within populations and relatively low between populations [65]. Indeed, in the present study, it was found that 90% of the genetic variation was within populations and only 10% was among populations. Similar results (84% and 16%) were obtained in the study of [29] and in [64], where 11% of variation was found among and 89% within populations. Moreover, similar results were obtained in the morphometric studies of [66], where sexual dimorphism was studied in Serbia, and in a study of seven populations from the northwestern part of the Balkan Peninsula where variability was also found and was correlated with geographic structure [67].
Monumental old trees of the species Taxus baccata occupy rocky, inaccessible sites of Thasos and could further indicate a past forest fragmentation of the area, due to fires, human interventions, as well as climate change. There is also evidence that over the last 4000 years, European populations of the species have gradually declined in many areas of its natural range [14,68]. Until today, no management measures have been taken to protect and conserve Taxus baccata on the island of Thasos; but, now that there are only a few trees of the species left, immediate measures must be implemented. According to the results of this paper, these monumental trees should first be protected and preserved “in situ”. This method can be implemented in collaboration with the Forest Service, which will be responsible for the protection of these trees, either by fencing them or by frequent patrols in the area. In situ conservation will aim to preserve their genetic pool (which has been found to have moderate to high indices of genetic diversity) and the natural regeneration of the species and preserve the unique natural landscape of the island of Thasos. Moreover, an ex situ conservation program should be implemented for those populations that are defined as marginal populations of the species and contain a unique genetic pool. In the present study, ex situ conservation is deemed necessary because the remaining individuals are few and at any moment are in danger of disappearing due to environmental and other factors. Ex situ conservation may include a seed collection or an in vitro propagation protocol to successfully preserve the genetic pool of these unique trees, as natural disasters such as forest fires are common on the island and drought events, which have been intense in the last decade, can cause the extinction of the species on the island and the loss of valuable genetic material alongside it. The collection of seeds can be done in collaboration with the Forestry Service and after the seeds have been collected, the procedures for their storage must be followed and checks for germination should be carried out at regular intervals so that it can be replaced immediately.
In conclusion, it is known that isolated/marginal populations contain important information for the conservation and resilience of species in extreme/special environments and climatic conditions. This makes the need to protect and preserve them due to climate change more urgent than ever.

5. Conclusions

In this paper, the unique monumental and isolated trees of Taxus baccata growing on the island of Thasos were studied in order to calculate and evaluate their genetic diversity pool so as to contribute to further decision-making on measures to protect and preserve them. In Greece, the natural distribution of yew trees may be considered as one of the ultimate refugia of the species in the European continent and can be nominated as a marginal population.
Monumental trees from two populations of Taxus baccata were evaluated using microsatellite markers in order to investigate the future adaptability and resilience of the species. Briefly, and despite the small number of samples and populations, we found that inbreeding was low, genetic differentiation was high, and allelic richness was high, indicating unique genetic pools for the species. Conservation and protection measures should be taken immediately and be a high priority for these unique trees as their unique genetic pool has been shown to be highly variable. Towards this axis, we could prioritize and propose the following protection and conservation measures for the Taxus baccata species: (a) in situ protection and conservation measures, (b) seed collection establishment and storage, and (c) in vitro propagation of these monumental trees in order to ensure the future resilience and sustainability of the species in Greece. Future research should also focus on exploring yew populations on other islands with past forest fragmentation events.

Author Contributions

Conceptualization, E.V.A. and G.D.R.; methodology, E.V.A., D.I.B. and E.M.; software, E.M.; validation, E.V.A., D.I.B. and E.M.; formal analysis, E.V.A.; investigation, G.D.R. and E.V.A.; data curation, E.M.; writing—original draft preparation, E.M. and E.V.A.; writing—review and editing, all authors; visualization, E.V.A. and D.I.B.; supervision, E.V.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We acknowledge Ms Panagiota Giannouxou for providing supplementary information about the populations and collaboration and Konstantinos Akritidis Koutsioumpas, Chrysa-Ioanna Charitatou-Typaldou, and Anna Metaxa for the extraction of DNA and laboratory assistance.

Conflicts of Interest

Author Diamantis I. Bountis was employed by the company Private Forest Office. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Distribution map of Taxus baccata L. (EUFORGEN, 2016, https://www.euforgen.org/species/taxus-baccata/, accessed on 3 December 2023). Blue dots in the map indicate natural populations of Taxus baccata.
Figure 1. Distribution map of Taxus baccata L. (EUFORGEN, 2016, https://www.euforgen.org/species/taxus-baccata/, accessed on 3 December 2023). Blue dots in the map indicate natural populations of Taxus baccata.
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Figure 2. Indicative photos of the monumental trees of Thasos island.
Figure 2. Indicative photos of the monumental trees of Thasos island.
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Figure 3. Principal Coordinate Analysis (PCoA) explaining 43.45% of the genetic variation for the two populations A and B.
Figure 3. Principal Coordinate Analysis (PCoA) explaining 43.45% of the genetic variation for the two populations A and B.
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Figure 4. Phylogenetic tree constructed with neighbor joining distances.
Figure 4. Phylogenetic tree constructed with neighbor joining distances.
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Table 1. Locus, motif, and size range of amplified PCR products of the eight microsatellite primers.
Table 1. Locus, motif, and size range of amplified PCR products of the eight microsatellite primers.
LocusCitationMotifSize Range (bp)Annealing Temperature °C
Tax23[51](GT)21153–18360
Tax26[51](GT)30208–28460
Tax36[51](GT)25126–26260
Tax86[51](GT)34152–30460
Tax92[51](GT)24160–28060
ABRII-TB50[52](TC)8-(CT)13270–33060
ABRII-TB56[52](TG)6-(CTAGAT)2204–27060
ABRII-TB58[52](CTATCC)2-(TCTGTA)2206–23060
Table 2. Characteristics of eight SSR loci across two Taxus baccata L. populations.
Table 2. Characteristics of eight SSR loci across two Taxus baccata L. populations.
LocusNaNeIHoHeFstFisfnPIC
Tax2372.4571.0310.2500.5910.0160.5770.0060.540
Tax2683.2971.2710.7140.6910.038−0.0330.410.667
Tax36165.4941.8630.6070.8110.0350.2520.0960.823
Tax86113.7411.4290.2860.6930.0920.5870.0000.733
Tax92154.4671.6800.6790.7720.0810.1210.0360.541
ABRII-TB5072.3380.9690.2500.5640.1170.5570.0430.819
ABRII-TB5641.9600.6830.8570.4900.020-0.7500.9870.568
ABRII-TB5862.4050.9660.2500.5840.0500.5720.0380.375
Na (number of alleles), Ne (number of effective alleles), I (Shannon’s information index), Ho (observed heterozygosity), He (expected heterozygosity), Fst (fixation index), Fis (inbreeding coefficient), fn (null allele frequency), and Polymorphic Information Content (PIC).
Table 3. Genetic diversity parameters at the population level based on eight SSR loci.
Table 3. Genetic diversity parameters at the population level based on eight SSR loci.
PopulationNeHeIARPARFst
A3.2990.6631.2703.481.130.199
B3.2410.6361.2033.310.950.251
Mean3.2700.6491.2363.3951.040.225
Ne (number of effective alleles), He (expected heterozygosity), I (Shannon’s information index), AR (allelic richness), PAR (private allelic richness), Fst (fixation index).
Table 4. Analysis of molecular variance (AMOVA) for 28 individuals in two populations of Taxus baccata.
Table 4. Analysis of molecular variance (AMOVA) for 28 individuals in two populations of Taxus baccata.
SourcedfSSMSEst. Var.%ΦST
Among Pops117.89317.8930.778100.067
Within Pops26181.9296.9976.99790
Total27199.821 7.776100
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Malliarou, E.; Avramidou, E.V.; Ranis, G.D.; Bountis, D.I. Towards the Conservation of Monumental Taxus baccata L. Trees of Thasos Island: Genetic Insights. Forests 2023, 14, 2384. https://doi.org/10.3390/f14122384

AMA Style

Malliarou E, Avramidou EV, Ranis GD, Bountis DI. Towards the Conservation of Monumental Taxus baccata L. Trees of Thasos Island: Genetic Insights. Forests. 2023; 14(12):2384. https://doi.org/10.3390/f14122384

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Malliarou, Ermioni, Evangelia V. Avramidou, Georgios D. Ranis, and Diamantis I. Bountis. 2023. "Towards the Conservation of Monumental Taxus baccata L. Trees of Thasos Island: Genetic Insights" Forests 14, no. 12: 2384. https://doi.org/10.3390/f14122384

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