PLoS One. 2017 Feb 24;12(2):e0169182. doi: 10.1371/journal.pone.0169182 

Frequency of chimerism in populations of the kelp Lessonia spicata in Central Chile

Alejandra V. González1 and Bernabé Santelices2 

1Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile.

2Departamento de Ecología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile. Santiago, Chile.

 

Abstract:

Chimerism occurs when two genetically distinct conspecific individuals fuse together generating a single entity. Coalescence and chimerism in red seaweeds has been positively related to an increase in body size, and the consequent reduction in susceptibility to mortality factors, thus increasing survival, reproductive potential and tolerance to stress in contrast to genetically homogeneous organisms. In addition, they showed that a particular pattern of post-fusion growth maintains higher genetic diversity and chimerism in the holdfast but homogenous axes. In Chilean kelps (brown seaweeds), intraorganismal genetic heterogeneity (IGH) and holdfast coalescence has been described in previous research, but the extent of chimerism in wild populations and the patterns of distribution of the genetically heterogeneous thallus zone have scarcely been studied. Since kelps are under continuous harvesting, with enormous social, ecological and economic importance, natural chimerism can be considered a priceless in-situ reservoir of natural genetic resources and variability. In this study, we therefore examined the frequency of IGH and chimerism in three harvested populations of Lessonia spicata. We then evaluated whether chimeric wild-type holdfasts show higher genetic diversity than erect axes (stipe and lamina) and explored the impact of this on the traditional estimation of genetic diversity at the population level. We found a high frequency of IGH (60-100%) and chimerism (33.3-86.7%), varying according to the studied population. We evidenced that chimerism occurs mostly in holdfasts, exhibiting heterogeneous tissues, whereas stipes and lamina were more homogeneous, generating a vertical gradient of allele and genotype abundance as well as divergence, constituting the first time “within- plant” genetic patterns have been reported in kelps. This is very different from the chimeric patterns described in land plants and animals. Finally, we evidenced that IGH affected genetic differentiation among populations, showed lower levels of FST index when we compared holdfast than lamina samples. In the light of this, future studies should evaluate the significance of chimeric holdfasts in their ability to increase kelps resilience, improve restoration and ecosystem service.

PMID: 28234957

 

Supplement:

  1. Brown algae and mankind

Marine algae have been utilized since ancient times, providing a wide variety of products for direct or indirect human use. Traditionally, algae have been used as a food due to their richness in protein, fatty acids, minerals and vitamins. However, a better identification of natural compounds and understanding of their functional properties have been increasing their use in both health and medicinal applications (see review by [1]). Consequently, the worldwide macroalgal production, which on average increases 5.7% every year, is produced from harvesting and aquaculture activities [2]. As a result, more than 18 million tons of macroalgae were produced in 2011; 96% of this comes from aquaculture production, dominated by cultures from Asia. The remaining 4% comes from the direct exploitation of natural populations, most of which is harvested in 28 different countries, with Chile (51.3%) and Norway (19.2%) leading the global catch [2,3]. Specifically, brown algae demand has been increasing for new uses, such as, food for invertebrates, fertilizers, bioremediation, technology of lithium batteries, and for medicinal purposes [1, 3, 4, 5, 6, 7, 8].

In the case of kelps (brown macroalgae), they are dominant organisms found in temperate coastal areas forming seaweed forests, which perform photosynthetic and production functions, contributing to ocean carbon fixation and serving as a habitat for marine life forms [9]. Along the Chilean coast, kelp forests (Lessonia species), in addition to sustaining a broad diversity of marine organisms, are a source of raw materials for several types of industries. Consequently, their annual harvest of up to 400,000 dry tons [10, 11] provides 10% of the total biomass of brown algae worldwide [2, 8, 12]. 

 

  1. Kelp biocompounds and human health

Chilean kelps (Lessonia. spicata, L. berteroana, and L. trabeculata) are traditionally a source of raw materials for the hydrocolloid, food, and cosmetics industries [8, 13]. However, there is also great potential for physiologically active compounds to play an important role in human health. For instance, alginates, fucoidans, galactofucans and laminarans (present on the seaweed thallus) are directly used in therapeutic activities or, indirectly, in the generation of biomaterial for medical purposes (Fig. 1), thus increasing the number of patent applications by pharmaceutical industries. The most novel use is the development of biomaterial for medical purposes including their use in drug/protein delivery platforms, cell-interactive alginates, hydrogel, wound-healing materials, bone-building composites and ex-vivo scaffolds for manipulation of stem-cell growth, with a view to differentiation into particular tissue types [14,15]. From a biomedical point of view, perhaps the most important function is the use of alginates in cell culture to grow pluripotent stem cells, with the potential to transform into any kind of somatic cell, such as those of internal organs. Consequently, this opens up a window of possibilities for many thousands of stem-cell lines currently being built up in biobanks. The use of stored stem cells so far has provided access to perfect models of the genetic illnesses of each patient, and enabled both doctors and pharmaceutical companies to test new drugs far more efficiently and effectively than before [16].

On the other hand, therapeutic uses of kelp biocompounds include antiviral, anthelmintic, antifungal, antibacterial, anticoagulant and anticancer treatments, which act along different pathways. Fucoidans, for example, perform more than one type of anticancer activity such as scavenger -receptor modulation, immune activation, anti-angiogenesis, metastasis blocking, stem-cell mobilization and interference with the SDF1/CXCR4 axis, as well as having anti-oxidant and pro-oxidant effects [17].

Accordingly, understanding the chemical composition and the structural variability of each type of biocompound is key to developing successful therapeutic and biomaterials products. The composition of these polymers, however, depends on several factors that include the choice of algae species [18, 19], the high or low molecular weight fractions, seasonal changes in the abiotic environment [20, 21, 22], population age, geographic location and the part of the plant to be used (e.g., lamina, stipes and holdfasts [23]). In this context, the chimerism with intraorganismal genetic diversity found in L. spicata is probably an additional factor to be considered in order to ensure, for example, the molecular weight fraction, purity, and specificity of bioactive compounds, so as to reduce their variability and interference in any use as a structure function for human health.

 

 

Fig. 1. Diagram representing the polysaccharides extracted from the Chilean Lessonia species with their respective use in therapeutic activities and in the generation of biomaterial for human health. Numbers indicate the bibliographic references.

 

  1. Chimerism in the Kelp Lessonia spicata

In our genetic study, we found that the natural kelp populations (L. spicata, Fig. 2) showed a high frequency (33.3-86.7%) of chimeras, which means the same individual having more than one genotype. Moreover, results showed that chimeric tissues would be located mainly in the holdfast, with genetic diversity decreasing towards the apical portions of the axes. In addition, we found that larger holdfasts are mostly composed of non-related genetic lineages. Thus, a negative correlation (R=-0.66, P<0.01) was found between holdfast diameter and genetic relatedness (QG value, Fig. 3). In the light of these results, the larger chimeric holdfasts can be considered as hotspots of plant genetic diversity with an additional ecological function as a reservoir of genetic diversity. In the same way, there is a positive correlation with habitat provision and larger chimeric holdfasts, since higher abundance of invertebrates are found in the internal cavities of the latter (R=0.43, P<0.003), increasing the ecosystem service provided by L. spicata. At the same time, in socioeconomic terms, larger holdfast sizes are probably related to a greater availability of biomass to be harvested, thereby increasing returns. Therefore, the abundance of chimeric plants, resulting in larger holdfast, has several positive implications (genetic diversity, nursery for invertebrate, and biomass production) in the natural populations of L. spicata.

Taken together, our study suggests that most of the Lessonia catch is a mix between chimeric and non-chimeric plants. However, by understanding the distribution of chimeric tissues in the thallus, it should be possible to increase the purity and specificity of the bio-compound. For example, axes and laminas were genetically more homogenous and are likely to hold more homogeneous chemical compounds. By contrast, holdfasts showing chimeric tissues with high levels of allelic richness do not guarantee the homogeneity of specific bio-compound derived from them.

 

 

Fig. 2. Intertidal kelp forest of Lessonia spicata from Central Chile.

 

Fig 3. Morphological implications of chimeric holdfasts. Relationships between the level of relatedness of samples within holdfasts versus their diameter, R=-0.66, P<0.01.

 

  1. Looking to the future: building chimeras

The knowledge gained from the identification of natural chimeric plants in Lessonia can be transferred via novel approaches to the manipulation of genetically distinct entities to produce chimeras with specific known compounds under laboratory conditions. For example, our previous work evidenced that chimera can be built in laboratory conditions in the five brown macroalgae present along the Chilean coast: Lessonia spicata, L. berteroana, L. trabeculata, Macrocystis pyrifera and Durvillaea antarctica [24]. In all of them, fusion among holdfasts therefore follows a general pattern that occurs due to the morphocytological changes of cells located in the contact zones between holdfasts, the latter being responsible for the somatic fusion of genetically different lineages. A graphic representation of chimeric plants in L. spicata (Fig. 4A) exemplifies, at the microscopic level (Figs. 4B-4D), how tissues in the contact zone undergo morphological modifications. Then epidermal cells in contact areas adopted a polygonal shape and increased in size (see contact zone in the square, Fig. 3B). At ultra-structural level, these cells form de-novo secondary connections (plasmodesmata) between cells from different lineages (white lines in Figs 4C-4D), thus establishing continuity in chimeric holdfasts and direct cytoplasm connections within neighboring cells. The fused and chimeric holdfasts therefore form a single robust, and inseparable entity that responds as a single larger individual.

Successful chimeras can be formed using genetically related and genetically non-related individuals of the Lessonia species (Chilean Patent Application Nº 1827-2017). These methodologies would lead to: a) faster vegetative propagation, b) faster production of plant biomass, c) greater resistance to stress such as higher temperatures, d) and to the building of plants with selected functions.

The production and use of coalescent and chimeric seaweeds has great potential and would allow for the production, for example, of more robust individuals with greater growth, survival, and resistance to stressful conditions [e.g. 25, 26]. In addition, it also avoids the incessant search for a natural “super strain” by other methods, such as producing a genetic hybrid or transgenic strain, which necessitates further evaluation in terms of human safety and environmental risk assessment. In this context, the knowledge of building chimeric plants has great potential for therapeutic activities and biomaterial technology, both having an important role to play in human health. 

 

 

Fig 4. A model representing the genetic composition within the chimeric plants of L. spicata. (A) Thallus of L. spicata composed of five replicate analyses with only two genetic lineages (blue and brown) at the holdfast, basal, medial and lamina levels. (B) Cross section of the chimeric holdfast showing the contact zone for different lineages, the square represent the initial epidermal cells in contact areas which adopted a polygonal shape and increased in size. (C) and (D) Numerous de-novo plamodesmata formed in the modified epidermal cell (white lines) that maintain physiological continuity among different lineages in the holdfast cortex. H1-H5 five replicates of genetically characterized holdfasts. P: plasmodesmata

 

 

Acknowledgments:

This study was supported by PAIFAC-2015-2017 and ENL020/16 (AG); FONDECYT 1120129 (BS). Contact: Alejandra V. González, Ph.D.

Professor of Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago, Chile. Email: apgonzalez@uchile.cl

 

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