Filipe Alberto

Standardizing methods to address clonality in population studies

Although clonal species are dominant in many habitats, from unicellular organisms to plants and animals, ecological and particularly evolutionary studies on clonal species have been strongly limited by the difficulty in assessing the number, size and longevity of genetic individuals within a population. The development of molecular markers has allowed progress in this area, and although allozymes remain of limited use due to their typically low level of polymorphism, more polymorphic markers have been discovered during the last decades, supplying powerful tools to overcome the problem of clonality assessment. However, population genetics studies on clonal organisms lack a standardized framework to assess clonality, and to adapt conventional data analyses to account for the potential bias due to the possible replication of the same individuals in the sampling. Moreover, existing studies used a variety of indices to describe clonal diversity and structure such that comparison among studies is difficult at best. We emphasize the need for standardizing studies on clonal organisms, and particularly on clonal plants, in order to clarify the way clonality is taken into account in sampling designs and data analysis, and to allow further comparison of results reported in distinct studies. In order to provide a first step towards a standardized framework to address clonality in population studies, we review, on the basis of a thorough revision of the literature on population structure of clonal plants and of a complementary revision on other clonal organisms, the indices and statistics used so far to estimate genotypic or clonal diversity and to describe clonal structure in plants. We examine their advantages and weaknesses as well as various conceptual issues associated with statistical analyses of population genetics data on clonal organisms. We do so by testing them on results from simulations, as well as on two empirical data sets of microsatellites of the seagrasses Posidonia oceanica and Cymodocea nodosa. Finally, we also propose a selection of new indices and methods to estimate clonal diversity and describe clonal structure in a way that should facilitate comparison between future studies on clonal plants, most of which may be of interest for clonal organisms in general.

Isolation by oceanographic distance explains genetic structure for Macrocystis pyrifera in the Santa Barbara Channel.

Ocean currents are expected to be the predominant environmental factor influencing the dispersal of planktonic larvae or spores; yet, their characterization as predictors of marine connectivity has been hindered by a lack of understanding of how best to use oceanographic data. We used a high‐resolution oceanographic model output and Lagrangian particle simulations to derive oceanographic distances (hereafter called transport times) between sites studied for Macrocystis pyrifera genetic differentiation. We build upon the classical isolation‐by‐distance regression model by asking how much additional variability in genetic differentiation is explained when adding transport time as predictor. We explored the extent to which gene flow is dependent upon seasonal changes in ocean circulation. Because oceanographic transport between two sites is inherently asymmetric, we also compare the explanatory power of models using the minimum or the mean transport times. Finally, we compare the direction of connectivity as estimated by the oceanographic model and genetic assignment tests. We show that the minimum transport time had higher explanatory power than the mean transport time, revealing the importance of considering asymmetry in ocean currents when modelling gene flow. Genetic assignment tests were much less effective in determining asymmetry in gene flow. Summer‐derived transport times, in particular for the month of June, which had the strongest current speed, greatest asymmetry and highest spore production, resulted in the best‐fit model explaining twice the variability in genetic differentiation relative to models that use geographic distance or habitat continuity. The best overall model also included habitat continuity and explained 65% of the variation in genetic differentiation among sites.

High and distinct range-edge genetic diversity despite local bottlenecks.

The genetic consequences of living on the edge of distributional ranges have been the subject of a largely unresolved debate. Populations occurring along persistent low latitude ranges (rear-edge) are expected to retain high and unique genetic diversity. In contrast, currently less favourable environmental conditions limiting population size at such range-edges may have caused genetic erosion that prevails over past historical effects, with potential consequences on reducing future adaptive capacity. The present study provides an empirical test of whether population declines towards a peripheral range might be reflected on decreasing diversity and increasing population isolation and differentiation. We compare population genetic differentiation and diversity with trends in abundance along a latitudinal gradient towards the peripheral distribution range of Saccorhiza polyschides , a large brown seaweed that is the main structural species of kelp forests in SW Europe. Signatures of recent bottleneck events were also evaluated to determine whether the recently recorded distributional shifts had a negative influence on effective population size. Our findings show decreasing population density and increasing spatial fragmentation and local extinctions towards the southern edge. Genetic data revealed two well supported groups with a central contact zone. As predicted, higher differentiation and signs of bottlenecks were found at the southern edge region. However, a decrease in genetic diversity associated with this pattern was not verified. Surprisingly, genetic diversity increased towards the edge despite bottlenecks and much lower densities, suggesting that extinctions and recolonizations have not strongly reduced diversity or that diversity might have been even higher there in the past, a process of shifting genetic baselines.

Patch definition in metapopulation analysis: a graph theory approach to solve the mega-patch problem.

The manner in which patches are delineated in spatially realistic metapopulation models will influence the size, connectivity, and extinction and recolonization dynamics of those patches. Most commonly used patch-definition methods focus on identifying discrete, contiguous patches of habitat from a single temporal observation of species occurrence or from a model of habitat suitability. However, these approaches are not suitable for many metapopulation systems where entire patches may not be fully colonized at a given time. For these metapopulation systems, a single large patch of habitat may actually support multiple, interacting subpopulations. The interactions among these subpopulations will be ignored if the patch is treated as a single unit, a situation we term the mega-patch problem. Mega-patches are characterized by variable intra-patch synchrony, artificially low inter-patch connectivity, and low extinction rates. One way to detect this problem is by using time series data to calculate demographic synchrony within mega-patches. We present a framework for identifying subpopulations in mega-patches using a combination of spatial autocorrelation and graph theory analyses. We apply our approach to southern California giant kelp (Macrocystis pyrifera) forests using a new, long-term (27 years), satellite-based data set of giant kelp canopy biomass. We define metapopulation patches using our method as well as several other commonly used patch delineation methodologies and examine the colonization and extinction dynamics of the metapopulation under each approach. We find that the relationships between patch characteristics such as area and connectivity and the demographic processes of colonizations and extinctions vary among the different patch-definition methods. Our spatial-analysis/graph-theoretic framework produces results that match theoretical expectations better than the other methods. This approach can be used to identify subpopulations in metapopulations where the distributions of organisms do not always reflect the distribution of suitable habitat.

Synchrony in dynamics of giant kelp forests is driven by both local recruitment and regional environmental controls

Populations of many species display spatially synchronous fluctuations in abundance. Synchrony is most commonly attributed to three processes: factors that influence recruitment (e.g., dispersal, early survival), large-scale environmental variability, and spatially autocorrelated trophic interactions. However it is often difficult to link population synchrony to a specific dominant process, particularly when multiple synchronizing forces are operating. We utilized a new satellite-based data set of giant kelp (Macrocystis pyrifera) canopy biomass to examine population synchrony in southern California kelp forests on spatial scales ranging from 50 m to 300 km and temporal scales ranging from 1 to 11 years. We examined the relationship between synchrony and distance for adult kelp populations, kelp recruits, sea urchin abundance (a major grazer of kelp), and environmental variables known to influence kelp population dynamics. Population synchrony in giant kelp decreased with distance between populations: an initial rapid exponential decrease between 50 m and 1.3 km was followed by a second, large-scale decrease between distances of 1.3 km and 172 km. The 50-m to 1.3-km spatial scale corresponded to the scales of synchrony in the abundance of sea urchins and young kelp recruits, suggesting that local drivers of predation and recruitment influence small-scale synchrony in kelp populations. The spatial correlation patterns of environmental variables, particularly wave height, were similar to the synchrony-distance relationship of kelp populations from 1.3 km to 172 km, suggesting that regional environmental variability, i.e., the Moran effect, was the dominant process affecting synchrony at larger spatial scales. This two-step pattern in the relationship between kelp biomass synchrony and distance was apparent in each of the 11 years of our study. Our results highlight the potential for synthesizing approaches from both landscape and population ecology in order to identify the multiple processes that generate synchrony in population dynamics.

Seascape drivers of Macrocystis pyrifera population genetic structure in the northeast Pacific

At small spatial and temporal scales, genetic differentiation is largely controlled by constraints on gene flow, while genetic diversity across a species distribution is shaped on longer temporal and spatial scales. We assess the hypothesis that oceanographic transport and other seascape features explain different scales of genetic structure of giant kelp, Macrocystis pyrifera. We followed a hierarchical approach to perform a microsatellite‐based analysis of genetic differentiation in Macrocystis across its distribution in the northeast Pacific. We used seascape genetic approaches to identify large‐scale biogeographic population clusters and investigate whether they could be explained by oceanographic transport and other environmental drivers. We then modelled population genetic differentiation within clusters as a function of oceanographic transport and other environmental factors. Five geographic clusters were identified: Alaska/Canada, central California, continental Santa Barbara, California Channel Islands and mainland southern California/Baja California peninsula. The strongest break occurred between central and southern California, with mainland Santa Barbara sites forming a transition zone between the two. Breaks between clusters corresponded approximately to previously identified biogeographic breaks, but were not solely explained by oceanographic transport. An isolation‐by‐environment (IBE) pattern was observed where the northern and southern Channel Islands clustered together, but not with closer mainland sites, despite the greater distance between them. The strongest environmental association with this IBE pattern was observed with light extinction coefficient, which extends suitable habitat to deeper areas. Within clusters, we found support for previous results showing that oceanographic connectivity plays an important role in the population genetic structure of Macrocystis in the Northern hemisphere.

Connectivity structures local population dynamics: a long-term empirical test in a large metapopulation system

Ecological theory predicts that demographic connectivity structures the dynamics of local populations within metapopulation systems, but empirical support has been constrained by major limitations in data and methodology. We tested this prediction for giant kelp Macrocystis pyrifera, a key habitat-forming species in temperate coastal ecosystems worldwide, in southern California, USA. We combined a long-term (22 years), large-scale (~500 km coastline), high-resolution census of abundance with novel patch delineation methods and an innovative connectivity measure incorporating oceanographic transport and source fecundity. Connectivity strongly predicted local dynamics (well-connected patches had lower probabilities of extinction and higher probabilities of colonization, leading to greater likelihoods of occupancy) but this relationship was mediated by patch size. Moreover, the relationship between connectivity and local population dynamics varied over time, possibly due to temporal variation in oceanographic transport processes. Surprisingly, connectivity had a smaller influence on colonization relative to extinction, possibly because local ecological factors differ greatly between extinct and extant patches. Our results provide the first comprehensive evidence that southern California giant kelp populations function as a metapopulation system, challenging the view that populations of this important foundation species are governed exclusively by self-replenishment.

Genetic and experimental evidence for a mixed-age, mixed-origin bank of kelp microscopic stages in southern California

Laboratory studies have demonstrated that the microscopic stages of kelps can rapidly resume development from a delayed state. Like terrestrial seeds or aquatic resting eggs, banks of delayed kelp stages may supplement population recovery after periods of stress, playing an important role for kelp populations that experience adult sporophyte absences due to seasonal or interannual disturbances. We found that removing the microscopic stages from natural rock substratum could prevent the appearance of juvenile kelp sporophytes for three months and the establishment of a diverse kelp assemblage for over four months within a southern California kelp forest. Juveniles were observed within one month in plots where microscopic stages were left intact, which may confer an advantage for the resulting sporophytes as they attain larger sizes before later recruiting neighbors. Microsatellite diversity was high (expected heterozygosity HE approximately 0.9) for juveniles and adults within our sites. Using a microsatellite-based parentage analysis for the dominant kelp, Macrocystis pyrifera, we estimated that a portion of the new M. pyrifera sporophyte recruits had originated from their parents at least seven months after their parents had disappeared. Similar delay durations have been demonstrated in recent laboratory studies. Additionally, our results suggest that zoospore dispersal distances > 50 m may be supported by including additional microsatellite loci in the analysis. We propose a mixed-age and, potentially, a mixed-origin bank of M. pyrifera gametophytes promotes maximal genetic diversity in recovering populations and reduces population genetic subdivision and self-fertilization rates for intact populations by promoting the survival of zoospores dispersed > 10 m and during inhospitable environmental conditions.

Reproductive strategies and isolation-by-demography in a marine clonal plant along an eutrophication gradient.

Genetic diversity in clonal organisms includes two distinct components, (i) the diversity of genotypes or clones (i.e. genotypic richness) in a population and (ii) that of the alleles (i.e. allelic and gene diversity within populations, and differentiation between populations). We investigated how population differentiation and genotypic components are associated across a gradient of eutrophication in a clonal marine plant. To that end, we combined direct measurements of sexual allocation (i.e. flower and seed counts) and genotypic analyses, which are used as an estimator of effective sexual reproduction across multiple generations. Genetic differentiation across sites was also modelled according to a hypothesis here defined as isolation‐by‐demography, in which we use population‐specific factors, genotypic richness and eutrophication that are hypothesized to affect the source‐sink dynamics and thus influence the genetic differentiation between a pair of populations. Eutrophic populations exhibited lower genotypic richness, in agreement with lower direct measurements of sexual allocation and contemporaneous gene flow. Genetic differentiation, while not explained by distance, was best predicted by genotypic richness and habitat quality. A multiple regression model using these two predictors was considered the best model (R2 = 0.43). In this study, the relationship between environment and effective sexual–asexual balance is not simply (linearly) predicted by direct measurements of sexual allocation. Our results indicate that population‐specific factors and the isolation‐by‐demography model should be used more often to understand genetic differentiation.

Looking into the black box: simulating the role of self-fertilization and mortality in the genetic structure of Macrocystis pyrifera

Patterns of spatial genetic structure (SGS), typically estimated by genotyping adults, integrate migration over multiple generations and measure the effective gene flow of populations. SGS results can be compared with direct ecological studies of dispersal or mating system to gain additional insights. When mismatches occur, simulations can be used to illuminate the causes of these mismatches. Here, we report a SGS and simulation‐based study of self‐fertilization in Macrocystis pyrifera, the giant kelp. We found that SGS is weaker than expected in M. pyrifera and used computer simulations to identify selfing and early mortality rates for which the individual heterozygosity distribution fits that of the observed data. Only one (of three) population showed both elevated kinship in the smallest distance class and a significant negative slope between kinship and geographical distance. All simulations had poor fit to the observed data unless mortality due to inbreeding depression was imposed. This mortality could only be imposed for selfing, as these were the only simulations to show an excess of homozygous individuals relative to the observed data. Thus, the expected data consistently achieved nonsignificant differences from the observed data only under models of selfing with mortality, with best fits between 32% and 42% selfing. Inbreeding depression ranged from 0.70 to 0.73. The results suggest that density‐dependent mortality of early life stages is a significant force in structuring Macrocystis populations, with few highly homozygous individuals surviving. The success of these results should help to validate simulation approaches even in data‐poor systems, as a means to estimate otherwise difficult‐to‐measure life cycle parameters.