Review of the Biological Span of the Earth

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Inquiry Article

Implications of Extreme Life Bridge in Clonal Organisms: Millenary Clones in Meadows of the Threatened Seagrass Neptunegrass oceanica

• Sophie Arnaud-Haond,
• Carlos M. Duarte,
• Elena Diaz-Almela,
• Núria Marbà,
• Tomas Sintes,
• Ester A. Serrão

10

• Published: Feb one, 2012
• https://doi.org/x.1371/journal.pone.0030454

Figures

Abstract

The maximum size and age that clonal organisms can reach remains poorly known, although we practise know that the largest natural clones tin extend over hundreds or thousands of metres and potentially live for centuries. We fabricated a review of findings to date, which reveal that the maximum clone age and size estimates reported in the literature are typically limited past the calibration of sampling, and may grossly underestimate the maximum historic period and size of clonal organisms. A case study presented here shows the occurrence of clones of deadening-growing marine angiosperm Posidonia oceanica at spatial scales ranging from metres to hundreds of kilometres, using microsatellites on 1544 sampling units from a full of 40 locations beyond the Mediterranean Ocean. This analysis revealed the presence, with a prevalence of 3.v to 8.9%, of very large clones spreading over 1 to several (up to xv) kilometres at the different locations. Using estimates from field studies and models of the clonal growth of P. oceanica, we estimated these large clones to be hundreds to thousands of years old, suggesting the evolution of full general purpose genotypes with large phenotypic plasticity in this species. These results, obtained combining genetics, census and model-based calculations, question nowadays noesis and understanding of the spreading capacity and life span of plant clones. These findings call for further research on these life history traits associated with clonality, because their possible ecological and evolutionary implications.

Citation: Arnaud-Haond Southward, Duarte CM, Diaz-Almela E, Marbà Due north, Sintes T, Serrão EA (2012) Implications of Extreme Life Bridge in Clonal Organisms: Millenary Clones in Meadows of the Threatened Seagrass Neptunegrass oceanica. PLoS ONE 7(2): e30454. https://doi.org/10.1371/journal.pone.0030454

Editor: Hans Henrik Bruun, University Copenhagen, Kingdom of denmark

Received: September 5, 2011; Accepted: December 16, 2011; Published: February i, 2012

Copyright: © 2012 Arnaud-Haond et al. This is an open-access article distributed nether the terms of the Creative Eatables Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original writer and source are credited.

Funding: This inquiry was supported past the Foundation BBVA (Kingdom of spain), the European Commission (Grand&MS European union projection; FEDER/POCI 2010, FCT, project NETWORK&DIVSTAB). These funders are national (Portuguese FCT) or European (FP5) agencies funding research projects. T. Sintes acknowledges the Project FIS2007-60327, funded by the Spanish MEC. The funders had no role in report design, information collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have alleged that no competing interests exist.

Introduction

Clonal organisms are present in all living kingdoms and play cardinal roles in the biosphere, including making a significant contribution to global master product [ane], [2]. Despite their importance, withal, our current understanding of their ecology and development is express by the constraints that the clonal life history trait puts on the use of classical ecological and evolutionary approaches. Such methods rely on the assumption that individuals have different genotypes and limited generation times, whereas these partially asexual organisms may accept accomplished an culling optimal evolutionary strategy. Clonal organisms tin potentially recombine genetically as much as necessary to avert the accumulation of deleterious mutations and escape parasites, while being able to maintain the best performing genotypes [3] over time scales far exceeding sexual generation times. In clonal organisms, the maximum life span of a detail genotype is a crucial component of the 'generation fourth dimension' (i.e., the average interval betwixt the birth of a genetic individual and the nativity of its sexually generated offspring) and, therefore, influences all evolutionary processes afflicted by this life history trait. Indeed, the generation time, maximum life span and size reached past clones have considerable implications for the action range of migrate, and the spatial and temporal variation of selection regimes.

Only a few studies have reported estimates of these parameters for clonal organisms, and the largest known clones identified so far, which are fungi [4] and angiosperms [five], [six], [7], extend over hundreds of metres in space and potentially centuries to millennia in fourth dimension. Additionally, shut exam of the sampling strategies used in deriving these estimates strongly suggest that past estimates of the maximum size (and life bridge when related, see also [8]) of clonal organisms accept been limited by the pick of sampling calibration (Tabular array 1, Fig. 1). Sampling strategy can be chosen for different research questions and species, only is typically limited to several hundred metres [4], [six], [9], [10], [11], [12], [xiii], [14], [15], [16], [17], [18], [nineteen], [xx], [21], [22], [23], [24]. A strong relationship tin be seen between the maximum altitude sampled and the maximum estimated clonal size amid these studies (Table ane, Fig. 1), suggesting that greater estimates of maximum clonal size could have been derived had greater sampling scales been used. Well-nigh half of the published reports of very big clones indeed testify maximum clonal sizes corresponding to the size of the sampling area (Tabular array ane). Moreover, a good number of studies on clonal organisms exercise non address the possibility that mutual genotypes occur beyond distant localities, suggesting that, despite their important ecological and evolutionary implications, the true maximum size and life span of clonal organisms are ofttimes overlooked.

Figure ane. Relationship between maximum clonal size detected and distance sampled in studies reporting very large clones.

The solid line shows the fitted regression line of clonal size (Due south, m) over distance (D, m): log10 S = −0.14 (±0.10)+1.15 (±0.26) log10 D (R2 = 0.91, North = 8, p<0.001). The regression intercept and slope practice non differ significantly from 0 and 1, respectively (t-test, p>0.05), thus not rejecting the hypothesis that there is a full general trend for the maximum clonal size detected to exist limited by the maximum distance sampled.

https://doi.org/x.1371/journal.pone.0030454.g001

Seagrasses are clonal marine angiosperms that can form extensive meadows. They support important marine ecosystems that rank amid the most valuable on earth in terms of biodiversity and production, merely are experiencing a worldwide decline [25], [26]. Agreement the factors influencing seagrass ecological and evolutionary dynamics is disquisitional for forward planning to foreclose their decline. Among seagrass species, clones of the Zostera marina and Cymodocea nodosa accept already been shown to achieve large genet sizes (genet: term used in clonal plants to refer to clones) attaining several centuries in age [6], [27]. These 2 species are relatively fast-growing and accept shorter-lived shoots, exhibiting a quicker wheel of growth and death of ramets (term used in clonal plants to refer to shoots), than the Mediterranean endemic Posidonia oceanica. Size and age attained are highly dependent on the species studied and the style of clonal growth. Seagrass clonal growth relies on dichotomous branching and rhizomatic extension through cell division at the rhizome meristems, a growth blueprint that leads to a predictable correlation between clonal size and age within species [28] and which is likely to minimize the probability of spread of somatic mutations, in dissimilarity to other clonal growth models such every bit that of aspen [8].

The endemic Mediterranean seagrass Posidonia oceanica ranks amid the slowest-growing and longest-lived plants in beingness [29], [thirty]. This structural species lacks native competitors and major predators in the littoral marine habitat (0–40 m) it occupies, leading to the development of extensive, monospecific meadows ([29], Fig. 2); however, these are presently declining [30], [31] throughout its range. Previous studies provided evidence that Posidonia oceanica meadows have grown continuously at particular locations for over 6000 years [32] and that clone mates occur at distances upwards to at to the lowest degree 80 metres [nine], [33] that can only exist covered over a minimum of 600 to 700 years of clonal growth (Table 1, come across Fig. S1). Altogether these results suggest that P. oceanica clones can achieve millenary life spans.

Figure 2. Photograph of meadows of Posidonia oceanica.

Pictures (a) illustrating the individual shoots (ramets), (b) hosting the largest (fifteen km) clones detected in this report. Photo by M. San Félix.

https://doi.org/ten.1371/journal.pone.0030454.g002

Here, in an attempt to improve estimates of the spatial scales over which identical P. oceanica clones spread and, thus, estimate their potential age, we tested whether P. oceanica clones were spread across ranges spanning kilometres. We proceeded by deliberately searching for the existence of shared genets among 40 P. oceanica populations across the Mediterranean, using microsatellite markers [34], [35], [36] to place clonal lineages.

Results

We found 902 distinct multi-locus genotypes (mlg) amid the ∼1500 sampling units collected across 40 locations, based on the seven microsatellite markers [34] previously selected for their ability to discriminate among genets from the corresponding mlg [9], [35], [36]. We constitute no show for somatic mutation or scoring errors with the sets of 7 or ix microsatellites analysed [36]. It was non necessary to define any Multi Locus Lineages (mll, [36]) corresponding to genets including slightly distinct Multi Locus Genotypes that would accept diverged through somatic mutation or scoring error, since the statistics supported all unlike mlg'due south to exist derived from different reproductive events (meet beneath for judge of somatic mutation charge per unit).

Amid the 741 pairs of sampling locations, which were separated by upward to 3500 km, no shared mlg were institute for meadows more than 15 km apart. In contrast, in v out of the 21 pairs of locations less than 15 km apart, ane to iv mlg were shared amidst distinct locations, sometimes represented past several (upward to 10 upon xl) sampling units (shoots, usually referred to as ramets for clonal plants, see Fig. 2a and Fig. S1) widespread at each sampling location. In fact, in Formentera Island (Balearic Islands, Spain), up to 18 to 30% of sampled ramets belonged to genets encountered in two localities 15 km apart (Table 2, Fig. three). In four of the v identified population pairs, the probability that the shared mlg occurred as a consequence of independent sexual events □ which would have resulted in singled-out genets sharing the same mlg □ was very depression (p<0.01, and in most cases p<0.001; Tabular array 3). Therefore, the most parsimonious explanation for the presence of identical mlgs in these pairs of locations is that these distant ramets were generated by clonal growth of the same genet. In the fifth case, among sites in Paphos (Cyprus), where p>0.01, the same mlg represented nigh 25% of each sample. Such dominance lowers the statistical power to ascertain clonal membership, and the non-significance may either be due to the occurrence of different undistinguished genets or to low statistical power.

Figure 3. Location of the meadows sampled in Formentera (Balearic Islands, Kingdom of spain).

Map of the location and littoral surface area of the island of Formentera (A, B) The circles signal the sites where the shared genotypes were sampled.

https://doi.org/ten.1371/periodical.pone.0030454.g003

The combination of demographic data [31] with data on clonal diversity [nine] for the populations we studied indicates that the number of genets (distinct mlg) expected in each of the sampling areas (the 80 m×20 k area from which shoots were taken randomly) is very big (fifteen,000 to 795,000, Table two). Bearing this in mind, the fact that we found a large percentage of shared genets in the sample pairs (upwards to 8.9% of sampled genets, Table 2) despite the small sample size collected at each site (0.003 to 0.one% of the ramets present in the sampling surface area, Table ii) indicates that shared genotypes must indeed be very common in meadows less than xv km apart. We estimated that the number of shared genotypes between two sampled areas in Formentera must attain 127,000 to yield the observed percentage of shared mlgsouthward (Table 2), thus supporting the presence of a high prevalence of shared genotypes between these meadows. Although the lower number of shared genotypes in the other pairs of locations renders those estimates subject to higher uncertainties, they also suggest a high charge per unit of shared genets in those meadows, with several thousands to tens of thousands of genets potentially shared between whatsoever 2 meadows less than 15 km apart. Moreover, although no shared genets were detected in the remaining fifteen population pairs (seventy% of all possible pairs) sampled within a spatial scale of 15 km, the number of shared genotypes may still range from 1.5 to 10% of the full number of genotypes present (i.east., up to 80,000 shared genotypes in pairs of sampling areas; Table 2), the limit of detection possible with our sampling endeavor, and nonetheless remain undetected in our samples.

Under a hypothesis of exclusively clonal spread, the models of clonal growth for P. oceanica with an estimate of iv cm.yr⁻¹ atomic number 82 to estimates of clonal growth fourth dimension of nearly 10,000 to several tens of thousands of years for mlg growing across meadows ane to xv km apart. Given the deadening growth rate of P. oceanica, clones that extend over kilometres may be expected to accept accumulated somatic mutations, although none were detected. The apparent lack somatic mutation in the sampling does, nevertheless, concur with results obtained through modelling performed using a rate of genotypic modify lower than p _Thousand = 10^−v (P(mlg _sampled =mlg ₁) = 0.99005 for p _M = 10⁻⁵ and 0.99978 for p _M = 10⁻⁶). Our modelling showed that the exponential nature of seagrass clonal growth leads to a disproportionate numerical dominance of the original mlg, even in the presence of somatic mutation. Such a large numerical advantage of the initial mlg ₁ (Fig. 4) results in a rather depression probability of sampling whatever mutant in our dataset for p _Grand = 10^{−half-dozen} (P(mlg _sampled =mlg ₁)∧400 = 0.1, confronting 0.98 for p _M = ten⁻⁵). The results reported here are, therefore, in understanding with a maximum rate of somatic mutation at microsatellites of most p _µs = 10⁻⁶ to 10⁻⁷ per locus, which is closer to the lower bound estimated for the poplar Populus tremuloides [8] than to that of the western redcedar Thuja plicata [37].

Figure four. Ranked proportions of the genets (X) belonging to an MLG present in the meadows.

from X = one, the original genet, to Ten = 2…n representing the genets derived from somatic mutations, ranked past abundance of ramets. The results are derived from somatic mutations in meadows growing over 400 to 500 years with an overall probability (beyond nine loci) pM = 10-vi. The original ramet, X = one, is the most abundant one, P(X = 1) = 0.99978, whereas the abundance of mutants, P(Ten>1), follow a power-police force decay that strongly depends on the somatic mutation probability.

https://doi.org/10.1371/journal.pone.0030454.g004

Discussion

The finding of clone mates 1 to 15 km apart, with an exceedingly low sampling endeavour (about 1 in 10,000 to i in 100,000 of the shoots sampled in each site) suggests the occurrence of a large number of clones spreading on a scale of kilometres across genetically-differentiated meadows [9], [11]. A clonal spread of P. oceanica on such a scale is impressive and calls for an assessment of potential confounding factors. Homoplasy is extremely unlikely to business relationship for completely identical genotypes over 9 loci. Indeed, a biogeographic study of P. oceanica across its full distribution range, including distances where homoplasy would be more likely, provided no bear witness for its occurrence [9]. In addition, the probability that the spread of genets across km-scale distances can exist accounted for by independent sexual events yielding identical mlg is exceedingly low (Table 3). Three alternative scenarios could business relationship for our observations. The km-scale spread of clones may result from i) clonal growth alone; ii) rhizome fragmentation and dispersal of thousands of shoots beyond these scales; or iii) a combination of both spread through clonal growth beyond big distances and range extension through border dispersal of fragments.

The scenario of a km-range spread achieved exclusively through clonal growth requires that the clones reach a minimum age of about 12,500 years. Applying the aforementioned estimates to the genets shared between the two pairs of meadows, located 7 km autonomously betwixt Formentera and Ibiza and 15 km apart around a cape in Formentera (Fig. 3), yields a minimum historic period estimate between 80,000 and 200,000 years, projecting the origin of the clones well into the tardily Pleistocene. Although there is no biologically compelling reason to exclude this possibility, we consider it to be an unlikely scenario because local bounding main level changes during the last ice age (from −80,000 to −10,000 years) would place these sampling locations on land (the sea was 100 metres below its present level). The dominance of identical clones on both sides of the island may, therefore, exist explained by the occurrence of fifty-fifty older clones that would have been split during glaciation and spread upwards into the newly inundated areas, tracking ocean level rise.

The 2d scenario involves km-range shoot dispersal. Although successful vegetative dispersal through drifting shoots has not been observed in long term surveys of established meadows, information technology has been reported to contribute to nearly 70% of patch recruitment in two colonizing sites over a 4-year study [38]. Dispersal and successful reattachment of globe-trotting shoots is a diffusive process, likely to pass up exponentially with distance. The spread across kilometre spatial scales via this mechanism is, therefore, expected to be a very rare phenomenon, unlikely to be detected with the limited sampling ability of this written report. An extrapolation of our information (Table ii) suggests that km-range drifting of shoots should have occurred almost two,000 to 127,000 times for the pairs of meadows sharing clone mates in order to result in the number of events we institute (Table 2). The thousands of repeated events of drifting shoots post-obit the same trajectories before condign successfully settled requires a sequence of improbable events that renders this scenario implausible. This scenario is specially unlikely when one considers that all pairs of locations sharing genotypes showed a significant level of genetic differentiation that is not compatible with a massive exchange of fragments [9], [11].

A canonical, more parsimonious scenario may include the disengagement of shoots at the edge of meadows and their reattachment at relatively curt distances, generating a stepping-rock model of spread combining clonal growth with vegetative dispersal. Recent evidence of the mosaic nature of Zostera marina meadows [39] suggests that meadows are equanimous of distinct patches resulting from a history of successive local extinction and recolonization by a small set of propagules or shoots within a narrow window of time following local depletion. Although detachment of the incomparably thicker and stronger rhizome of P. oceanica would be considerably less frequent, such events may have punctuated the long-term evolution of meadows and the events of (re)colonization. Large, millenary clones spanning across long distances may thus farther extend through the detachment and subsequent reattachment of shoots, accelerating the spread otherwise reached exclusively through clonal growth. We consider this model equally the most parsimonious with the knowledge available. It implies a less extreme life span than the exclusive clonal growth model, just nevertheless requires long-term clonal growth over thousands years, in both potential source and 'border' locations, for clones to be detected in both samples.

The inference of an extreme historic period of P. oceanica clones is supported past results from a numerical model of clonal growth (described below) examining the spread of somatic mutation within genets and assessing the resulting dynamics of the frequency distribution of genet size over time. Offset, the model confirms the authority of big genets every bit illustrated past a power-law distribution of genet size within the meadow when genets approach 500 years of age (the limit of the model; Fig. iv) conforming to the observations made in twoscore meadows of P. oceanica examined here). Additionally, the model supports a probability of occurrence of somatic mutation lower than ten⁻⁶, which would explain the lack of mutants detected in the sampling, and which underlines the persistent numerical advantage of the original mlg over slightly distinct ones emerging through somatic mutations.

Finally, these results are consistent with the phylogeny of the Posidonia genus obtained through sequencing of distinct genes from the mitochondrial, chloroplastic and nuclear genomes [40]. The consummate lack of polymorphism within P. oceanica across its whole distribution range at all tested genes including ITS, and its very depression difference from its Australian counterparts, atomic number 82 to an judge of evolutionary rates among the slowest reported for herbaceous plants. The highly conserved genetic structure of the Posidonia genus suggests depression mutation rates and long generation time of genets [41], [42] every bit a likely caption for such low genetic divergence.

In this report, we found evidence supporting the occurrence of extremely large clones (upwards to 15 km) and life spans of thousands to tens of thousands of years. These P. oceanica genets would have coped with environmental variation over long periods of time and are soon distributed in microhabitats kilometres autonomously. They would, therefore, be expected to display large phenotypic plasticity. Reaching such an old age has been suggested to have at least two major evolutionary implications for a clonal lineage [43], [44]. One is the lack or limited accumulation of deleterious mutations that would otherwise somewhen lead to extinction: the and then chosen "Muller's ratchet", which is predicted under certain population parameters. Our results suggest that this accumulation might be limited or hampered due to large population size [45], also as by the persistent dominance of the original genotype nether a pure government of clonal growth demonstrated by the present model (Fig. 4). The other is a potentially high phenotypic plasticity providing long-lived individuals with the chapters to survive in a changing surround.

Two evolutionary models have been proposed to explain the ubiquity of clonal organisms: the General-Purpose Genotype model [46] and the Frozen Niche Variation model [47]. The first explains the ubiquity of clonal organisms by the power to retain the near competent genotypes over time (i.e., "the factor complexes well-nigh co-adapted to the prevailing conditions [48]"), when "a single event of outcrossing may destroy such genic association [48]". The second model explains the co-occurrence of diverse sexual and asexual genotypes past their adaptation to narrow, non-overlapping, specialist niches. Both theories have been supported past empirical observations on different organisms. The results reported hither advise that there is phenotypic plasticity in particular genotypes, allowing adaptation to microhabitats spreading over kilometres and to fluctuating mid or long term ecology conditions; this would support positive selection of "general purpose genotypes" [46]. Such a mechanism would, in turn, favour the absence of recombination and, therefore, of sexual reproduction, once an optimal genotype was reached. This is supported by straight observations of very infrequent sexual reproduction in P. oceanica [38], [49], suggesting that molecular estimates of the proportion of sexually derived recruitment [9], [35] are likely to correspond to the integration of rare successful sexual episodes during narrow windows of fourth dimension rather than frequent or nowadays day sexual output, equally already reported past Balestri and Lardicci [50]. Although genet diversity has been shown to enhance short term response and ecosystem resistance of Zostera marina in experimental plots [51], [52], low genet diversity accompanying large clone size coincides with improved chapters to respond to disturbances, such equally an increased organic load imposed past fish farming on natural P. oceanica meadows [53], [54]. This farther supports the hypothesis of phenotypic plasticity associated with large clonal size and quondam age. Nonetheless, fifty-fifty though such phenotypic plasticity possibly evolved across millennia, information technology may well be challenged by the unprecedented charge per unit of environmental modify imposed by electric current global climate change [55], including temperature increase and ocean acidification, and recent anthropogenic pressure on coastal areas resulting in changes in water quality, eutrophication, and nutrient load, particularly in seagrass meadows [56].

Conclusions

The finding of P. oceanica clones (i.e., genets) that are extreme in size (km-sized) and age (multi-millenary quondam) across the Mediterranean indicates that some meadows are the effect of ecological and evolutionary processes integrated over long fourth dimension scales. Fourth dimension scales such as these are in a stark contrast to the current rapid and astute impact caused directly or indirectly by human pressure on this species. Indeed, the ancient meadows of P. oceanica are failing at a rate several hundred-fold faster (virtually v%.yr⁻¹, [31], [55]) than the charge per unit over which they spread when forming [28], [57], a situation that this slow growing, long-lived species is poorly capable of recovering from.

Critical screening of the literature and the results presented hither suggest that a systematic search for temporal persistence of clones has non yet been attempted for most species, and that extreme potential to persist over big spatial and temporal scales may exist a common characteristic associated with clonality. This finding, if confirmed by farther specific efforts to exam for large clonal size, would take important implications for ecological and evolutionary theory, as all basic parameters in evolutionary environmental. Effective population size and consistent drift, migration, mutation and pick would be strongly influenced by the potentially extreme generation overlap caused by life spans reaching thousands to peradventure tens of thousands of years. Our results underline the need to improve our cognition of the life span of clonal organisms, as some assumptions underlying classical population models may non exist met by taxa with ancient and big clonal lineages, such as the Mediterranean seagrass Posidonia oceanica.

Materials and Methods

Sampling

P. oceanica samples were taken at full of 40 sites across the Mediterranean basin from Spain to Cyprus by collecting about 40 shoots at randomly selected coordinates inside a 1600 thousand^two (80×xx m) area at each site [9], [11]. The basal meristematic section of the leaves was removed and preserved in silica crystals until analysis. 20 vi of these meadows were analysed in previous studies on biogeography [9] and the furnishings of aquaculture installations on P. oceanica meadows [11]; the other 14 were added in the nowadays study (Tabular array S1). Sampling locations were separated by approximately ane to 3500 km.

All 40 sampling quadrats were located in non-privately-owned or specifically protected areas, then no specific permits were required for the field studies. Posidonia oceanica is not considered equally an endangered (its status is 'to the lowest degree concern' in the IUCN list) or protected species, although the meadows it constitutes are a protected habitat.

Genotyping

Genomic DNA was isolated following a standard CTAB extraction process [58]. Each shoot or grouping of continued shoots sampled together were treated as a "sampling unit" (SU), or ramet. All meadows were analysed with the well-nigh efficient combination [35] of seven dinucleotide nuclear markers [34], Po4-3, Po5, Po5-10, Po5-39, Po5-40, Po5-49 and Po 15, as described in [9]. When identical genotypes were found across sites with a probability college than 1% of existence issued from distinct sexual reproduction events rather than from clonal growth (see here-below), two additional loci, Po4-36 and Po5-61 [34], were genotyped in club to ascertain genet membership with higher confidence.

Methods used for clone (genet) discrimination

When the same genotype was detected more once, the probability that the samples actually originated from distinct reproductive events (i.east., from split genets) was estimated [twenty], [59] using the software GenClone [60], taking into account Wright's inbreeding coefficient estimated for each locus [60].

The possible occurrence of somatic mutations or scoring errors leading to slightly distinct mlg really derived from a unmarried reproductive event, i.e., from one unmarried clone, was besides tested for. This screening aimed to identify, if they occurred, Multi Locus Lineages (mll), including slightly singled-out mlg originated from the aforementioned zygote, as mentioned above [nine], [36].

Genotypic richness

Genotypic richness was estimated from the number of ramets sampled (N) and the number of multilocus genotypes detected (G), as suggested by Dorken et al. [61]: (3)

Demographic estimates

In each meadow studied for demography, the number of shoots was counted inside three replicated quadrats, varying between 0.09 m² and one yard^two in area depending on the sites (so as to include at least 100 shoots quadrat⁻¹). The but exception was 1 station influenced by the presence of aquaculture cages (El Campello, Spain), where the quadrat was ten thousand² [11]. More details on the demographic estimates are available in Marbá et al. [31].

Estimates of shared genets and type Ii errors: likelihood of undetected existence of shared genets

An approximation of the total number of genets in the expanse sampled was obtained as follows, with S the estimated number of shoots in the expanse sampled: allowing an estimate of the expected percentage of these sampled % Thousand_sampled as: For each pair of populations, the observed number of genets shared among pairs of samples (Gs-samples) was then used to estimate the full percentage of genets shared : The likelihood of occurrence of undetected shared genets amongst all pairs of localities separated by less than xv kilometres was estimated. For each meadow pair, an guess was made of the maximum possible percentage of shared genets, past considering that the addition of 1 single genet to one of the samples may allow the ascertainment of a new, shared, previously undetected genet. The gauge of the maximum percentage of shared genets between the meadows that may however remain undetected in our sampling was, therefore, considered for each pair of meadows i and j with G_i and Yard_j genets: (iv)

Modelling of clonal growth and spread of somatic mutations

We modified an existing model of P. oceanica clonal growth [28], [62], [63] to derive quantitative expectations of the number of somatic mutations we would look to exist detected and to compare these estimates with the data bachelor on the genetic structure of meadows. More specifically, the model was modified to introduce the occurrence of random somatic mutations and to study their quantitative spread within a genet across extended fourth dimension spans (500 years). The model is initiated with a seed, growing to occupy space through clonal growth. Each new shoot has a probability p _M to modify its genotype with respect to its neighbour forth the rhizome. The model tracks the fate of all individual shoots and rhizome meristems produced during the clone's development to yield a spatially-explicit representation of the clone at various fourth dimension steps [63]. At the aforementioned time, the model output contains descriptors of the changes over time in the average number of surviving shoots, internal shoot density, patch radial expansion and growth charge per unit, too as the distribution probability and abundance of the unlike genotypes arising from somatic mutations during clonal growth. In order to derive a bourgeois estimate of age through clonal growth modelling, the highest spring estimate of spatial clonal growth rate estimated for P. oceanica was used (6 cm.year-one), and the maximum altitude was divided past 2, assuming a starting point for clonal spread in the midpoint between the two near afar ramets sharing the same mlg in the sampling.

The expected mutation rate at microsatellites for mitotic divisions may be ten to hundred fold lower than the mutation charge per unit per sexual generation in higher eukaryotes [64]. Estimates of the charge per unit of somatic mutations are nevertheless scarce overall, highly variable between different organisms and still mostly simply available for copse, with values ranging from four.10^−three to half-dozen. 10⁻⁴ in the western redcedar Thuja plicata [37] and 4.10⁻⁵ and 6.10⁻⁷ for the poplar Populus tremuloides [8]. Nosotros explored a range of three orders of magnitude in somatic mutation with overall probability of genotypic change p _Thou = 5. 10⁻³, 10⁻⁴, 10^−v and 10^−vi, respective to a rate of somatic mutation of about μ _s = 1.5×10⁻³ to 10⁻⁷ per locus for the 7 to ix loci used to discriminate identical mlg. The amount of ramets for the original multi locus genotype (mlg ₁) compared to ones that were slightly dissimilar due to somatic mutations (mlg _x) were estimated for all rates in patches that had been growing for 100 to 500 years (p₁). The well-nigh dominant genets detected in real samples of 40 shoots were sampled between 5 and x times and no somatic mutations were detected with a ready of seven to nine loci. The probability of sampling only the initial mlg _i in all 40 meadows where each dominant mlg was on average sampled 10 times was roughly estimated as p₁∧400. The ranked abundances of mlg ₁ and the mlg derived from this initial ane through somatic mutations were so built in order to illustrate the dominance of mlg _ane (from which the sampling probability p₁ is derived) depending on the probability of genotypic change p _M used to run the model. Finally, p₁∧400 was estimated in society to test whether the p _G and respective somatic mutation rate μ _{due south} were compatible with the lack of ascertainment of somatic mutation in the set up of samples analysed in this work, which independent about 1500 sampling units analysed in this work.

Supporting Information

Effigy S1.

Mapping of clones extent and distribution. Multiple occurrence of large genets mapped in adjacent meadows is detailed in (a) and (b) Amathous ST3 and ST5 (for which shared genet is labelled as 4 and 19, respectively), (c) Es Castell (where the largest clone was encountered), and (d) Los Genoveces.

https://doi.org/10.1371/journal.pone.0030454.s001

(DOC)

Acknowledgments

We thank R. Martínez, R. Santiago, and East. Alvarez for aid with field work, S. Teixeira, Thou.S. van de Vliet, Due south.I. Massa and T. Aires for assistance with laboratory work, three anonymous referees for useful comments on the first version of this manuscript, and Grand. Sán Félix for the photographs. We also give thanks Chiliad. Valero, F Alberto, N. Bierne and F. Viard for useful discussions.

Author Contributions

Conceived and designed the experiments: SAH CMD NM EAS. Performed the experiments: SAH NM EDA. Analyzed the data: SAH TS CMD. Contributed reagents/materials/assay tools: SAH CMD TS NM EAS. Wrote the paper: SAH CMD.

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