Morphological and Molecular Identification of Ulva spp. (Ulvophyceae; Chlorophyta) from Algarrobo Bay, Chile: Understanding the Composition of Green Tides.

Green algae blooms of the genus Ulva are occurring globally and are primarily attributed to anthropogenic factors. At Los Tubos beach in Algarrobo Bay along the central Chilean coast, there have been blooms of these algae that persist almost year-round over the past 20 years, leading to environmental, economic, and social issues that affect the local government and communities. The objective of this study was to characterize the species that form these green tides based on a combination of ecological, morpho-anatomical, and molecular information. For this purpose, seasonal surveys of beached algal fronds were conducted between 2021 and 2022. Subsequently, the sampled algae were analyzed morphologically and phylogenetically using the molecular markers ITS1 and tufA, allowing for the identification of at least five taxa. Of these five taxa, three (U. stenophylloides, U. uncialis, U. australis) have laminar, foliose, and distromatic morphology, while the other two (U. compressa, U. aragoensis) have tubular, filamentous, and monostromatic fronds. Intertidal surveys showed that U. stenophylloides showed the highest relative coverage throughout the seasons and all intertidal levels, followed by U. uncialis. Therefore, we can establish that the green tides on the coast of Algarrobo in Chile are multispecific, with differences in relative abundance during different seasons and across the intertidal zone, opening opportunities for diverse future studies, ranging from ecology to algal biotechnology.

Expansion of marine pollution along the coast: Negative effects on kelps and contamination transference to benthic herbivores?

Heavy metals and polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants that frequently co-occur in coastal environments. These contaminants can have negative impacts on the health and stability of marine and coastal ecosystems, affecting both the organisms themselves and the humans who consume them. A coastal industrial park in central Chile, housing a coal thermal power plant and other industrial activities, contributes to such pollution of coastal waters; however, neither the spatial alongshore distribution of heavy metals and PAHs, nor an assessment of their ecological effects on the biota have been systematically documented to date. In this paper, we present evidence regarding the direct negative effect of contamination by heavy metals and PAHs on the early life stages of kelps-being extremely harmful to their population persistence near highly polluted sites-as well as the indirect effects of their transference through the food web to higher trophic levels, leading to negative consequences for the feeding intake, growth, fertility, and larval development of marine herbivores that consume the contaminated seaweed. Likewise, the dispersion of contaminants by ocean currents can exacerbate the effects of pollution, having an adverse influence on marine ecosystem health even at sites far from the pollution source. Therefore, it is necessary to investigate the distribution patterns and extent of pollution along the coast to understand the impact of heavy metals and PAHs pollution on seaweed populations and the food web. It is considered critical for the development of effective environmental policies and regulations to protect these ecosystems and the people who depend on them.

Desiccation Stress Tolerance in Porphyra and Pyropia Species: A Latitudinal Analysis along the Chilean Coast.

One of the most important factors regulating the distribution and abundance of seaweeds is desiccation, triggered mainly by tidal changes and climatic variation. Porphyra and Pyropia species have evolved multiple strategies to tolerate desiccation stress; however, how these tolerance strategies differ in these species inhabiting different latitudes is still unknown. In this context, we analyzed, in situ, the physiological responses of these species (collected from 18° S to 41° S along the Chilean coast) to desiccation stress using biochemical and molecular analyses. The hyper-arid terrestrial climate of northern Chile, with high evaporation and lack of constant rain determines a very steep increase in desiccation stress in the upper intertidal during low tide for these species. Accordingly, the results showed that, in comparison with the southernmost populations, the Porphyra/Pyropia species from the north zone of Chile (18°-30° S) exhibited higher contents of lipoperoxide and carbonyls (1.6-1.9 fold) together with higher enzymatic activities, including ascorbate peroxidase, catalase, peroxiredoxin, and thioredoxin (2-3-fold). In addition, a substantial expression of cat, prx, and trx transcripts during desiccation was demonstrated, mainly in the northernmost populations. These results provide evidence of (i) significant activation of antioxidant enzymes and transcripts (principally cat and prx); (ii) participation of phenolic antioxidant compounds as a highly plastic physiological strategy to cope with desiccation; and (iii) the activation of the tolerance responses was affected by species latitudinal distribution. Thus, for the first time, this study integrated the biochemical and genetic responses of diverse Porphyra/Pyropia species to better understand their physiological dynamics of tolerance over a wide latitudinal range.

Transfer of Pollutants from Macrocystis pyrifera to Tetrapygus niger in a Highly Impacted Coastal Zone of Chile.

PAHs and heavy metals are characteristic pollutants in urbanized coastal areas, especially those with industrial activity. Given this context and the ability of Macrocystis pyrifera to drift when detached and provide trophic subsidy in coastal systems, we analyzed the potential transfer of pollutants to the herbivore Tetrapygus niger, through diet, in an industrialized coastal zone in Central Chile (Caleta Horcón) and characterized the impacted zone using diverse polluted ecotoxicological indices. For this purpose, a culture experiment was conducted where M. pyrifera individuals from Algarrobo (control site) were cultivated in Caleta Horcón and then used as food for T. niger. The contents of both PAHs and heavy metal contents were subsequently determined in algal tissue and sea urchin gonads as well as in the seawater. The results show that algae cultivated in Caleta Horcón had higher concentrations of naphthalene (NAF) compared to those from a low industrial impact zone (Algarrobo) (2.5 and 1.8 mg kg-1, respectively). The concentrations of Cu, As, and Cd were higher in Caleta Horcón than in Algarrobo in both M. pyrifera and T. niger. For all metals, including Pb, higher concentrations were present in T. niger than in M. pyrifera (between 5 and 798 times higher). Additionally, as indicated by the toxicological indices MPI (0.00804) and PLI (10.89), Caleta Horcón is highly contaminated with metals compared to Algarrobo (0.0006 and 0.015, respectively). Finally, the bioconcentration factor (BCF) and trophic transfer factor (TTF) values were greater than one in most cases, with values in Caleta Horcón exceeding those in Algarrobo by one or two orders of magnitude. This study provides evidence that Caleta Horcón is a highly impacted zone (HIZ) compared to Algarrobo, in addition to evidence that the biomagnification of certain pollutants, including the possible responses to contaminants, are apparently not exclusively transferred to T. niger through diet.

Negative Consequences on the Growth, Morphometry, and Community Structure of the Kelp Macrocystis pyrifera (Phaeophyceae, Ochrophyta) by a Short Pollution Pulse of Heavy Metals and PAHs.

The study of pollution effects in the marine environment has become important in recent decades, and the exposure to simultaneous pollutants has become especially relevant. Indeed, the study of key organisms, such as ecosystem engineers, can show a broader view of the effects of pollutants. In this context, we evaluate in situ the effects of a short (7-day) pollution pulse of combined solutions of heavy metals and polycyclic aromatic hydrocarbons (PAHs) (Cu + PAHs, Cd + PAHs, Cu + Cd, and Cu + Cd + PAHs) on the development and morphological features of Macrocystis pyrifera sporophytes over a period of 90 days. Additionally, we determined the effects on the community structure associated with this kelp. This study evidenced a smaller number of blades and a decreased size of blades and holdfasts, as well as the death of individuals exposed to a secondary mix of metals (Cu + Cd) and a tertiary mix of pollutants (Cu + Cd + PAHs). Regarding the effects on the accompanying fauna, low richness and diversity were registered. M. pyrifera grazers, according to the mixture of pollutants, were either absent or diminished. These results show that the pulse of contamination in the early stages of M. pyrifera negatively affects its development and morphometry, as well as its role as an ecosystem engineer, due to a negative alteration in the species composition.

Ingestion of contaminated kelps by the herbivore Tetrapygus niger: Negative effects on food intake, growth, fertility, and early development.

Macrocystis pyrifera reaches distant areas after detachment, accumulate heavy metals, and serve as trophic subsidy. In this context, effects on both adults and larvae of Tetrapygus niger fed with polluted kelps were determined by assessing growth, fertility, and early larval development. Results revealed that sea urchins fed with polluted kelps from highly impacted zone (HIZ) showed a lower growth (3.6% gained weight) and gamete release (358 cells mL-1) than those fed with non-impacted kelps (NIZ) (19.3% and 945 cells mL-1). The HIZ treatment showed a developmental delay in comparison to NIZ, accounted mainly by the abundance of malformed 2-arm pluteus larvae (10-15%) during most of the culture. Malformed 4-arm pluteus larvae showed a constant increase, reaching 37% at the end of the culture. Thus, the pollutants ingested by sea urchins can be transferred to their offspring and cause negative effects in their early development, categorizing M. pyrifera as a pollutant carrier.

Synergistic, antagonistic, and additive effects of heavy metals (copper and cadmium) and polycyclic aromatic hydrocarbons (PAHs) under binary and tertiary combinations in key habitat-forming kelp species of Chile.

Heavy metals (HMs) and polycyclic aromatic hydrocarbons (PAHs) are persistent toxicants in coastal environments. Notably, in comparison to individual metal toxicity, knowledge about the effects of HMs and PAHs mixtures on kelps remains scarce. Accordingly, we performed in vitro experiments to determine the individual and combined effects of Cu, Cd, and PAHs on spore release, settlement, and germination on Macrocystis pyrifera and Lessonia spicata, two key-habitat forming kelp species of the coast of the Valparaíso Region in Chile. This region concentrates highly polluting industries, mainly due to unrestrained mining and fossil-fuel energy production. Single Cu, Cd, and PAHs treatments included concentrations in the ranges 5-200, 0.125-2000, and 0.05-100 µg/L, respectively, and a toxic-free treatment. Cu, Cd, and PAHs concentrations causing 20-50% (IC20, IC50) arrested spore release, settlement, and germination were determined, and the results shown in both species that single Cu, Cd, and PAHs IC20 values were generally lower on spore release than on spore settlement and germination, probably due to the absence of a cell wall in spores compared to later stages. Binary equitoxic IC20s mixture treatments changed from an antagonistic response to another with a greater inhibitory effect on spore release, from hour 1 to 7, whereas in IC50 treatments, the response was always antagonistic. The tertiary IC20 mixture of Cu+Cd+PAHs produced generally an antagonistic effect. Remarkably, all IC20 equitoxic mixture treatments showed a synergistic response on spore settlement in both kelps, suggesting that these toxicants are extremely harmful to kelp population persistence near highly polluted sites.

Coastal pollution from the industrial park Quintero bay of central Chile: Effects on abundance, morphology, and development of the kelp Lessonia spicata (Phaeophyceae).

The industrial park of Quintero Bay (QB) in the central coast of Chile was established in the 1960s, presents high levels of pollution due to the industrial activity, and it is known as one of the five Chilean "sacrifice zones". Lessonia spicata is the most important habitat-forming kelp species in the intertidal along the central and south shores of Chile, and currently there are no morphometric and population studies of L. spicata (or other seaweed species) nor studies about the effects of pollution on its development in QB and neighbouring sites. In this context, the aims of this study were (i) to register the abundance and morphological features of L. spicata populations from Ventanas, Horcón and Cachagua (sites with different pollution histories and located only up to 40 km from the QB); ii) to determine the heavy metals (HMs) concentration in seawater and marine sediments; and (iii) to evaluate in vitro the effects of exposure to seawater from the three sampling sites on spore release and early developmental stages, up to the juvenile sporophyte. Results showed that the chronically exposed Ventanas kelp population had the smallest adult individuals in comparison with the other sites. Ventanas and Horcón registered high HMs concentration in the seawater and marine sediments exceeding the international permissible limits (e.g in seawater Cu 20-859 µg L-1; sediments Cu > 50,000 µg kg-1). Unexpectedly in Cachagua, a site often considered unpolluted, high concentrations of Cu and As were also registered in the seawater (859 and 1,484 µg L-1, respectively) and of As in marine sediments (20,895 µg kg-1). Exposure of gametophytes to the seawater from Ventanas resulted in a developmental delay compared to the other treatments; however, low sporophyte production was determined in all treatments. Our results indicate that QB, more notably Ventanas, induce highly negative effects on individual development, and consequently on seaweed populations, which suggest a long-term negative impact on the community structure of these marine zones. Furthermore, the high concentrations of HMs reported here at Cachagua suggest a recent expansion of pollution along the central coast of Chile, evidencing effects on the marine ecosystem health even on sites far from the pollution source.

Non-Random Distribution and Ecophysiological Differentiation of Pyropia Species (Bangiales, Rhodophyta) Through Environmental Gradients.

Recently 18 Bangiales seaweed species were reported for the Chilean coast, including Pyropia orbicularis and Pyropia variabilis (large [LM] and green [GM] morphotypes). Porphyra/Pyropia spp. occur mainly in the upper intertidal where desiccation stress is triggered by tidal fluctuations. However, the influence of environmental and ecophysiological variables and seasonal differences on Porphyra/Pyropia (microhabitats) intertidal distributions is unknown. Accordingly, we determined (i) the effect of environmental variables (temperature [T], relative humidity [RH], and photosynthetically active radiation [PAR]) and season on distribution, and (ii) physiological (cellular activity and lipid peroxidation [LPX]) and molecular responses (antioxidant enzymes expression at biochemical and transcript level) to desiccation stress in both Pyropia species and morphotypes (common garden experiment, on flat rocky platforms). Multivariate analyses of coverage and abundance in relation to environmental variables revealed a significant effect of temperature on P. variabilis GM distribution, GM dominating almost exclusively on rocky walls (where lowest PAR and T values but maximum RH were registered). Conversely, Pyropia orbicularis and Pyropia variabilis LM were found in high abundance on flat rocky platforms in summer, LM and GM also dominating flat rocky platforms in winter and spring. LPX and catalase activity did not differed among species in summer, while in winter activity and transcription of cat were higher in P. orbicularis than P. variabilis. Results suggest that tolerance to environmental stresses such as temperature could regulate the occurrence of P. variabilis GM on rocky walls; conversely, abundances of P. variabilis and P. orbicularis on flat rocky platforms would be also regulated by other abiotic and/or biotic factors.

Genetic and morphological differentiation of Porphyra and Pyropia species (Bangiales, Rhodophyta) coexisting in a rocky intertidal in Central Chile.

A recent molecular taxonomic study along the Chilean coast (18° S-53° S) described 18 candidate species of bladed Bangiales of which only two were formally described. Few studies focused on local genetic and morphological diversity of bladed Bangiales and attempted to determine their intertidal distribution in contrasting habitats, and none were performed in Chile. To delimit intertidal distributions of genetic species, 66 samples of bladed Bangiales were collected at Maitencillo (32° S) in four zones: a rocky platform, a rocky wall, and two boulders zones surrounded by sandy and rocky bottoms, respectively. These samples were identified based on sequences of the mitochondrial COI and chloroplast rbcL markers. We also collected 87 specimens for morphological characterization of the most common species, rapidly assessing their putative species identity using newly developed species-diagnostic (PCR-RFLP) markers. Eight microscopic and two macroscopic morphological traits were measured. We described and named three of four species that predominate in Maitencillo (including Pyropia orbicularis): Pyropia variabilis Zapata, Meynard, Ramírez, Contreras-Porcia, sp. nov., Porphyra luchea Meynard, Ramírez, Contreras-Porcia sp. nov., and Porphyra longissima Meynard, Ramírez, Contreras-Porcia, sp. nov. With the exception of Po. longissima restricted to boulders surrounded by sandy bottom, and a morphotype of Py. variabilis restricted to rocky walls, the other species/morphotypes have overlapping intertidal distributions. Except for Po. longissima, which is clearly differentiated morphologically (longest and thinnest blades), we conclude that morphology is not sufficient to differentiate bladed Bangiales. Our findings underscore the importance of refining our knowledge of intrinsic and environmental determinants on the distribution of bladed Bangiales.

Expression profile of desiccation tolerance factors in intertidal seaweed species during the tidal cycle.

MAIN CONCLUSION: The transcriptional modulation of desiccation tolerance factors in P. orbicularis explains its successful recuperation after water deficit. Differential responses to air exposure clarify seaweed distribution along intertidal rocky zones. Desiccation-tolerant seaweed species, such as Pyropia orbicularis, can tolerate near 96% water loss during air exposure. To understand the phenotypic plasticity of P. orbicularis to desiccation, several tolerance factors were assessed by RT-qPCR, Western-blot analysis, and enzymatic assays during the natural desiccation-rehydration cycle. Comparative enzymatic analyses were used to evidence differential responses between P. orbicularis and desiccation-sensitive species. The results showed that during desiccation, the relative mRNA levels of genes associated with basal metabolism [trehalose phosphate synthase (tps) and pyruvate dehydrogenase (pdh)] were overexpressed in P. orbicularis. Transcript levels related to antioxidant metabolism [peroxiredoxin (prx); thioredoxin (trx); catalase (cat); lipoxygenase (lox); ferredoxin (fnr); glutathione S-transferase (gst)], cellular detoxification [ABC transporter (abc) and ubiquitin (ubq)], and signal transduction [calmodulin (cam)] increased approximately 15- to 20-fold, with the majority returning to basal levels during the final hours of rehydration. In contrast, actin (act) and transcription factor 1 (tf1) transcripts were down-regulated. ABC transporter protein levels increased in P. orbicularis during desiccation, whereas PRX transcripts decreased. The antioxidant enzymes showed higher specific activity in P. orbicularis under desiccation, and sensitive species exhibited enzymatic inactivation and scarce ABC and PRX protein detection following prolonged desiccation. In conclusion, the reported findings contribute towards understanding the ecological distribution of intertidal seaweeds at the molecular and functional levels.

Phylogeographic structure in benthic marine invertebrates of the southeast Pacific coast of Chile with differing dispersal potential.

The role of dispersal potential on phylogeographic structure, evidenced by the degree of genetic structure and the presence of coincident genetic and biogeographic breaks, was evaluated in a macrogeographic comparative approach along the north-central coast of Chile, across the biogeographic transition zone at 30°S. Using 2,217 partial sequences of the mitochondrial Cytochrome Oxidase I gene of eight benthic invertebrate species along ca. 2,600 km of coast, we contrasted dispersal potential with genetic structure and determined the concordance between genetic divergence between biogeographic regions and the biogeographic transition zone at 30°S. Genetic diversity and differentiation highly differed between species with high and low dispersal potential. Dispersal potential, sometimes together with biogeographic region, was the factor that best explained the genetic structure of the eight species. The three low dispersal species, and one species assigned to the high dispersal category, had a phylogeographic discontinuity coincident with the biogeographic transition zone at 30°S. Furthermore, coalescent analyses based on the isolation-with-migration model validate that the split between biogeographic regions north and south of 30°S has a historic origin. The signatures of the historic break in high dispersers is parsimoniously explained by the homogenizing effects of gene flow that have erased the genetic signatures, if ever existed, in high dispersers. Of the four species with structure across the break, only two had significant albeit very low levels of asymmetric migration across the transition zone. Historic processes have led to the current biogeographic and phylogeographic structure of marine species with limited dispersal along the north-central coast of Chile, with a strong lasting impact in their genetic structure.

Phylogeographic analyses of the 30 degrees S south-east Pacific biogeographic transition zone establish the occurrence of a sharp genetic discontinuity in the kelp Lessonia nigrescens: vicariance or parapatry?

Phylogeographic studies are lacking in the Southern Hemisphere, and in particular in the south-eastern Pacific. To infer the possible scenario for the debated biogeographic transition zone located at 30-33 degrees S along the Chilean coast, we investigated whether there is a concordance between the phylogeographic pattern and the biogeographic transition in the intertidal kelp Lessonia nigrescens whose distribution is continuous across this transition zone. Using a combination of four makers located in the three genomic compartments (chloroplast, mitochondria and nucleus), we showed the presence of two main divergent lineages, possibly cryptic species. There was an exact match of the phylogeographic break with the 30 degrees S biogeographic transition zone, suggesting a common origin. The combined information given by the multilocus approach and by the population analysis suggested the occurrence of a budding speciation, with a northward range expansion.

Phylogenetic and biogeographic relationships of the mouse opossum Thylamys (Didelphimorphia, Didelphidae) in southern South America.

Nucleotide sequence data from the mitochondrial cytochrome b gene were used to evaluate the phylogenetic relationships among mouse opossum species of the genus Thylamys. Based on approximately 1000 bp in five of the six species of the genus and including different localities for some of the species, we concluded that T. macrura from the subtropical forests of eastern Paraguay is the most primitive taxon. Subsequent radiation of the genus is explained mainly via founder effect speciation. This evolutionary scenario would account for the speciation of T. pusilla, T. venusta, T. pallidior, and T. elegans in the Chaco, southern Bolivia and northern Argentina, the Andean Altiplano, the Coastal Desert of Chile, and coastal Perú, respectively. Calibration of a molecular clock set the Pleistocene as the period for the differentiation of Thylamys species. The molecular results confirm the strong genetic connection between populations that inhabit the "pre-cordillera" of northern Chile (T. pallidior) and the canyons that run through the Atacama Desert to the lowlands in northern Chile. Our results confirm the occurrence of two Thylamys species in Chile, T. pallidior and T. elegans, within and south to the Atacama Desert, respectively.