PROJECT:


Fingerprinting of genetic diversity and patterns of spatial genetic variation in the endemic tree Cedrus brevifolia (Hook f.) Henry from Cyprus:
implications for its conservation

Over the last four decades genetic inventories became one of the most dynamic areas of natural sciences. The main aim of these inventories is to describe the existing temporal and spatial genetic patterns: these patterns result from the impact of evolutionary factors on the various elements of biodiversity (i.e. ecosystem, species, gene diversity). Population structure and evolutionary processes are main factors influencing the present genetic diversity and genetic structure within populations. Specific conditions of population structures and functions are characterising island populations. Often these populations share many features with endangered species, like small population size and elevated risks of extinction, and they experience different environments than those of their mainland counterparts. As a consequence, island populations are generally characterised by less genetic diversity than mainland populations, are often inbred and may have lowered reproductive fitness.
One of these island populations is the sole natural population of Cedrus brevifolia or Cyprus cedar, an endemic tree species of the Cyprus flora with a long history of presence on the island (first review from Theophrastus 370-287 B.C). The Cyprus cedar belongs to the genus of Cedrus (family Pinaceae) and is known as the short-leaf cedar: it has the shortest needles among all four species of this genus. The range of C. brevifolia has nowadays been limited to only one population due to uncontrolled felling, fires and grazing; such events have been characteristic of east Mediterranean tree populations since the classical times. However, this population is non-uniform and has been restricted to five neighbouring stands in the region of the Tripylos forest. This population corresponds to the Cyprus cedar forest (Cedrosetum brevifolia) and to the habitat type 9590 (European Commission).
The present study aims at determining the genetic variation of C. brevifolia with regard to the spatial genetic structure within and among restricted landscape patches, as well as planted populations of Cyprus cedar. In order to achieve these aims, sampling was performed for each of the five identified patches (Trypilos, Mauroi Kremoi, Sellai tis Ellias, Throni and Exo Milos), as well as for three plantations. A total of 550 individuals from 11 randomly established plots (Trypilos -6 plots, Mauroi Kremoi -2 plots, Sellai tis Ellias -1 plot, Throni -1 plot and Exo Milos -1 plot), and 150 individuals from three plantations were collected. In each of the sampling plots, branches from 50 trees were collected. The plot size and shape depended on the density and distribution of 50 sampled trees collected from each plot.
Investigation of the genetic variation of Cyprus cedar was performed using two types of molecular markers. Biparentally inherited genetic information of C. brevifolia, which is transmitted through seed (and partially through pollen), was studied by nuclear microsatellites (nSSRs). Further, paternally inherited chloroplast genome, which is transmitted through pollen (and partially through seed), was investigated using chloroplast microsatellites (cpSSRs). The analysis of both markers gave an unexpectedly high total genetic diversity (HT) for an endemic island species with restricted dispersal: nSSRs (0.70) and cpSSRs (0.93). In addition, the non-significant deviation from Hardy-Weinberg equilibrium (HWE), and the relatively low inbreeding coefficients (FIS= 0.006), support a primary outcrossing and an approximately random mating system for the Cyprus cedar. These results could explain the absence of a consequent genetic drift in this population, and, further, the fact that during the last two centuries this population has been under natural regeneration that has increased its census size (with overlapping among regenerations). In addition, the migration of this species to the island of Cyprus is an important factor, which possibly influences the high genetic variation of this species, and reinforces the probability of exceptional gene flow from C. libani populations to C. brevifolia.
Remarkable is also the fact that the genetic differentiation among the fragmented patches (ΦCT) was significant for both markers (nSSRs=0.027; cpSSRs=0.099). Although this differentiation among the restricted patches was relatively low, the patches could be identified as separate subpopulations; the genetic differentiation among them may be the effect of restricted gene flow during several generations. Furthermore, the significant genetic differentiation among the sampling plots (ΦST) detected for both markers (nSSRs= 0.052; cpSSRs=0.114), imply different patterns of genetic diversity within and among the sampling plots. This is particularly important, given the relatively small total habitat area of C. brevifolia (approximately 64 km2). Additionally, both markers were employed to study genetic differentiation among the sampling plots, using Nei’s genetic distance and UPGMA cluster analyses. The clustering of plots, based on UPGMA analysis, did not follow the geographical origin of plots. This unclear grouping supports that the identified subpopulations nowadays have resulted from the fragmentation of a previously common population. Nonetheless, the location of Throni (Subpop.4) and Exo Milos (Subpop.5) indicated, for both markers, relatively high differentiation from the other plots, suggesting that these two subpopulations were first isolated from the previously uniform population.
As has been mentioned, the present C. brevifolia subpopulations have originated from a fragmentation event in a previously common population. An effect of this population fragmentation was the biased contribution of each subpopulation to the total diversity. This bias is most probably influenced by the different genetic structure among and within the subpopulations. Remarkably, in this study the fragmentation event showed two different directions of influence: (i) due to the restricted gene contact between some subpopulations the fragmentation acted as catalytic to the genetic erosion and broke up the local genetic structure (i.e. Mauroi Gremoi –Subpop.2, Throni –Subpop.4 and Exo Milos –Subpop.5) and (ii) in some circumstances, fragmentation events did not appear to reduce genetic variation of the patches (subpopulation), since the design of fragmentation (geographical location of restricted patches) support the continuous distribution of gene flow between patches (between Tripylos -Subpop.1 and Sellae tis Ellias –Subpop.3). More assiduous interpretation of these genetic variation patterns illustrates a gradual increase of within plot genetic variation from the north to the south; this pattern of genetic variation becomes more obvious from the observation of cpSSRs, where Subpop.3 (the southern subpopulation) showed the highest genetic variation (HE=0.94) and the highest haplotypic richness (HR=22.36), contrary to Subpop.4 (the western subpopulation) and Subpop.5 (the northern subpopulation), in which the values were HE=0.58/HR=8.88 and HE=0.72/ HR=5.00, respectively. These results suggest that the fragmentation event and the reduction of population size are not the only factors contributing to the genetic divergence among and within the different subpopulations: rather, the geographical location and the environmental factors also influence gene dispersal within this non-continuously distributed population.
Based on zMantel test the significant correlation between geographical and genetic distances for both markers, allows the assumption that the model of Isolation-By-Distance (IBD) can also be applied in this fragmented population, and, hence, gene dispersal in the whole population is related to the distances among the subpopulations. No significant correlation was detected when the analysis was performed including only the sampling plots from the main subpopulation (Tripylos -Subpop.1); this confirms the assumption of restricted gene flow among the subpopulations, and especially among the main subpopulation and the other more isolated subpopulations. The kinship analysis was used for investigating the Spatial Genetic Structure (SGS) at large- and fine-scales. This analysis showed increased detected fingerprinting of spatial genetic aggregation of C. brevifolia trees.
In large-scale analysis, both markers showed similar patterns of SGS. In this analysis both markers showed positive significant autocorrelation (high genetic similarity of tree pairs) up to the distance of 500 m; this significant coefficient for cpSSRs was continued until the distance of 1,500 m. These results are in accordance with the biology of the pine species, which shows extensive dispersal of paternal genome, versus limited biparental genome dispersal. However, the oxymoron of these results is that cpSSRs indicate stronger spatial aggregations (Fij) than nSSRs. This extreme result must be sought in the historical demography and evolution processes of this population: cpSSRs, having half the effective population size than nSSRs and also having a non-recombinant inherited model, result in the presented higher values of kinship coefficients than nSSRs.
At distances larger than 4,500 m negative significant autocorrelation was observed (pairs of trees are genetically less similar). This long distance corresponds to the geographical distance between the main subpopulation (Subpop.1) and the other more isolated subpopulations.
The analysis on fine-scale patterns (intra-plot level analysis) showed all of the four possible combinations (i.e. significant either at cpSSRs or nSSRs, or at both of them, or none of them) of SGS. Noteworthy is that, in five out of the eleven sampling plots, significant SGS were detected in the first distance class (family structure). These results imply a combination of factors influencing gene dispersal (i.e. pollen and seed) in a short distance or in a small group of neighbouring trees. Also, the fact that SGS was detected at cpSSRs, probably indicates that the mating process in some cases among neighbouring trees is not random and/or that the pollination of trees from a few individuals influences the effective pollination of many trees.
Finally, the genetic structure of the investigated plantations supports the existing strategy for plantations of C. brevifolia (seed-sampling, seedling, planting), which follows some principles (i.e. sampling which covers parts of the distribution area, sampling from several individuals). However, despite the high genetic diversity which was observed within the plantations, the inbreeding coefficient was extremely high, implying that the sampled trees were relatively neighbouring and most likely genetically similar.
In conclusion, the conservation of the Cyprus cedar is an important parameter for the sustainable management of the species. In situ conservation must comprise of: (i) static conservation which must aim to protect this population from damaging abiotic and biotic factors (i.e. fires, disease) and (ii) dynamic conservation which must support the regeneration where such support is needed (especially in the isolated subpopulations) by the admixture with seedlings of different origins, or even by technically reinforcing regeneration through the local trees (Throni, Mauroi Kremoi). In addition, ex situ conservation demands the preservation of C. brevifolia in plantations outside and inside its natural habitat, in order to provide generative planting stock. This process of ex situ conservation will represent a “backup system” for stochastic events that may possibly threat the existence of the last remaining population of this species.