Marie-Laure Guillemin

Species replacement along a linear coastal habitat: phylogeography and speciation in the red alga Mazzaella laminarioides along the south east pacific

BackgroundThe Chilean shoreline, a nearly strait line of coast expanding across 35 latitudinal degrees, represents an interesting region to assess historical processes using phylogeographic analyses. Stretching along the temperate section of the East Pacific margin, the region is characterized by intense geologic activity and has experienced drastic geomorphological transformations linked to eustatic and isostatic changes during the Quaternary. In this study, we used two molecular markers to evaluate the existence of phylogeographic discontinuities and detect the genetic footprints of Pleistocene glaciations among Patagonian populations of Mazzaella laminarioides, a low-dispersal benthic intertidal red seaweed that inhabits along ~3,700 km of the Chilean coastal rocky shore.ResultsThree main genetic lineages were found within M. laminarioides. They are distributed along the Chilean coast in strict parapatry. The deep divergence among lineages suggests that they could be considered putative genetic sibling species. Unexpectedly, genetic breaks were not strictly concordant with the biogeographic breaks described in the region. A Northern lineage was restricted to a broad transition zone located between 30°S and 33°S and showed signals of a recent bottleneck. The reduction of population size could be related to warm events linked to El Niño Southern Oscillation, which is known to cause massive seaweed mortality in this region. To the south, we propose that transient habitat discontinuities driven by episodic tectonic uplifting of the shoreline around the Arauco region (37°S-38°S); one of the most active forearc-basins in the South East Pacific; could be at the origin of the Central/South genetic break. The large beaches, located around 38°S, are likely to contribute to the lineages’ integrity by limiting present gene flow. Finally, the Southern lineage, occupies an area affected by ice-cover during the last glaciations. Phylogeny suggested it is a derived clade and demographic analyses showed the lineage has a typical signature of postglacial recolonization from a northern glacial refugium area.ConclusionsEven if environmental adaptation could have strengthened divergence among lineages in M. laminarioides, low dispersal capacity and small population size are sufficient to generate phylogeographic discontinuities determined by genetic drift alone. Interestingly, our results confirm that seaweed population connectivity over large geographic scales does not rely only on dispersal capacity but also seem to depend highly on substratum availability and population density of the receiving locality.

Differential ecological responses to environmental stress in the life history phases of the isomorphic red alga Gracilaria chilensis (Rhodophyta)

In order to better understand the alternation of generations that characterizes haploid–diploid life cycles, we assessed the existence of ecological differences between the two phases (haploid gametophyte and diploid tetrasporophyte) in Gracilaria chilensis, a rhodophyte with a typical Polysiphonia-type life cycle. We investigated the effect of light intensity and salinity on viability and growth of both phases at different ontogenetic stages: juveniles and adults. In our study, the survival of juvenile gametophytes (n) was higher than the survival of juvenile tetrasporophytes (2n) despite culture conditions; however, low salinity had greater effect on carpospores (2n) than on tetraspores (n). On the other hand, a complex interaction between salinity and light intensity within each life history phase generated observed differences between juvenile growth rates. Low light was shown to trigger early onset of alteration of the holdfast growing pattern. In addition, adult tetrasporophytes showed, despite the conditions, a faster vegetative growth than female and male gametophytes. These differences between phases could have led to the complete dominance of tetrasporophyte fragments of fronds observed in G. chilensis farms. We hypothesize that Chilean fishers could have unknowingly selected for tetrasporophyte thalli during domestication of the species, thus enhancing the natural trend of tetrasporophytes dominance already present in estuarine natural populations of free-floating plants.

DEFENSE EVOLUTION IN THE GRACILARIACEAE (RHODOPHYTA): SUBSTRATE-REGULATED OXIDATION OF AGAR OLIGOSACCHARIDES IS MORE ANCIENT THAN THE OLIGOAGAR-ACTIVATED OXIDATIVE BURST1

Combined phylogenetic, physiological, and biochemical approaches revealed that differences in defense‐related responses among 17 species belonging to the Gracilariaceae were consistent with their evolutionary history. An oxidative burst response resulting from activation of NADPH oxidase was always observed in two of the subgenera of Gracilaria sensu lato (Gracilaria, Hydropuntia), but not in Gracilariopsis and in species related to Gracilaria chilensis (“chilensis” clade). On the other hand, all species examined except Gracilaria tenuistipitata var. liui and Gracilariopsis longissima responded with up‐regulation of agar oligosaccharide oxidase to an challenge with agar oligosaccharides. As indicated by pharmacological experiments conducted with Gracilaria chilensis and Gracilaria sp. “dura,” the up‐regulation of agar oligosaccharide oxidase involved an NAD(P)H‐dependent signaling pathway, but not kinase activity. By contrast, the activation of NADPH oxidase requires protein phosphorylation. Both responses are therefore independent, and the agar oligosaccharide‐activated oxidative burst evolved after the capacity to oxidize agar oligosaccharide, probably providing additional defensive capacity to the most recently differentiated clades of Gracilariaceae. As demonstrated with Gracilaria gracilis, Gracilaria dura, and Gracilariopsis longissima, the different responses to agar oligosaccharides allow for a fast and nondestructive distinction among different clades of gracilarioids that are morphologically convergent. Based upon sequences of the chloroplast‐encoded rbcL gene, this study suggests that at least some of the samples from NW America recorded as Gs. lemanaeiformis are probably Gs. chorda. Moreover, previous records of Gracilaria conferta from Israel are shown to be based upon misidentification of Gracilaria sp. “dura,” a species that belongs to the Hydropuntia subgenus.

Species delimitation and phylogeographic analyses in the Ectocarpus subgroup siliculosi (Ectocarpales, Phaeophyceae).

The genus Ectocarpus (Ectocarpales, Phaeophyceae) contains filamentous algae widely distributed in marine and estuarine habitats of temperate regions in both hemispheres. While E. siliculosus has become a model organism for genomics and genetics of the brown macroalgae, accurate species delineation, distribution patterns and diversity for the genus Ectocarpus remain problematic. In this study, we used three independent species delimitation approaches to generate a robust species hypothesis for 729 Ectocarpus specimens collected mainly along the European and Chilean coasts. These approaches comprised phylogenetic reconstructions and two bioinformatics tools developed to objectively define species boundaries (General Mixed Yule Coalescence Method and Automatic Barcode Gap Discovery). Our analyses were based on DNA sequences of two loci: the mitochondrial cytochrome oxidase subunit 1 and the nuclear internal transcribed spacer 1 of the ribosomal DNA. Our analyses showed the presence of at least 15 cryptic species and suggest the existence of incomplete lineage sorting or introgression between five of them. These results suggested the possible existence of different levels of reproductive barriers within this species complex. We also detected differences among species in their phylogeographic patterns, range and depth distributions, which may suggest different biogeographic histories (e.g., endemic species or recent introductions).

Tracing the Trans-Pacific Evolutionary History of a Domesticated Seaweed (Gracilaria chilensis) with Archaeological and Genetic Data

The history of a domesticated marine macroalga is studied using archaeological, phylogeographic and population genetic tools. Phylogeographic and population genetic analyses demonstrated that the cultivated red alga Gracilaria chilensis colonised the Chilean coast from New Zealand. Combining archaeological observations with phylogeographic data provided evidence that exchanges between New Zealand and Chile have occurred at least before the Holocene, likely at the end of the Last Glacial Maximum (LGM) and we suggest that migration probably occurred via rafting. Furthermore, the remarkably low microsatellite diversity found in the Chilean populations compared to those in New Zealand is consistent with a recent genetic bottleneck as a result of over-exploitation of natural populations and/or the process of domestication. Therefore, the aquaculture of this seaweed, based essentially on clonal propagation, is occurring from genetically depressed populations and may be driving the species to an extinction vortex in Chile.

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.

Phylogeography of Seaweeds in the South East Pacific: Complex Evolutionary Processes Along a Latitudinal Gradient

The coast along the temperate South East Pacific (SEP) presents a simple linear topography with a north-south orientation spanning more than 4600 km. However, environmental heterogeneity associated with two major biogeographic boundaries has been described along the SEP (30–33°S and 42°S). Recent phylogeographic studies of seaweeds revealed the existence of different cryptic species along the SEP coast and that most of the genetic breaks between them are broadly congruent with the biogeographic boundaries. These phylogeographic patterns characterized by genetic discontinuities could be attributed to historical vicariance or to budding speciation. For SEP seaweeds, two major phylogeographic patterns are observed. Endemic species living north of 42°S show complex haplotype networks and an almost complete genetic isolation between populations located only a few kilometres from each other. This extreme genetic patchiness has been related to the combined effects of limited dispersal, reduced population size and high population turnover of these intertidal seaweeds due to stochastic effects of climatic and tectonic catastrophes. On the other hand, species with a range distribution limited to the south of 42°S and inhabiting the area highly affected by the coastal ice cap during the Last Glacial Maximum (LGM), show typical signatures of post-glacial demographic expansion. Finally, molecular studies reveal that several species are recent immigrants from New Zealand, demonstrating the importance of oceanic dispersal in shaping the diversity of the SEP.

Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre- and postzygotic barriers in populations of individuals with varying ploidy levels

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid–diploid life cycles that include the principal genetic model organism for the brown algae. In haploid–diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system.

On the Choice of Linear Regression Algorithms for Biological and Ecological Applications

MARETEC, Instituto Superior Técnico, Universidade Técnica de Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal. Departamento de Ecologia, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rua São Francisco Xavier 524, 20.559-900, Rio de Janeiro, Brazil. Department of Biology, Saint Francis Xavier University, Antigonish, Nova Scotia B2G 2W5, Canada. Universidade Autónoma de Lisboa, Rua de Santa Marta, no 56 1169-023, Lisboa, Portugal. CCMAR, Center of Marine Science, University of Algarve, Campus Gambelas, 8005-139 Faro, Portugal. Instituto de Ciencias Ambientales y Evolutivas, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile. CNRS, Sorbonne Universités, UPMC University Paris VI, UMI 3614, Evolutionary Biology and Ecology of Algae, Station Biologique de Roscoff, CS 90074, Place G. Tessier, 296888 Roscoff, France.

Guidelines for the restoration of the tropical timber tree Anacardium excelsum: first input from genetic data

Translocation of trees has been used as a common method to mediate genetic conservation and restoration of forests. However, very few programs include strategies developed to recover or maintain the genetic diversity of the translocated species. Anacardium excelsum is a tree native to the tropics of America that is extensively used in forestry. In Colombia, restoration of forests through the translocation of native species has regained importance, and A. excelsum has been recently included in the National Strategy for Plant Conservation. Thus, in order to define the level of genetic structure and the level of genetic diversity within certain regions where remnants of the seasonally dry tropical forests (SDTF) of Colombia have been retained, we genotyped 106 trees using nuclear inter-simple sequence repeats (ISSR) and sequenced two non-coding chloroplast loci for these specimens. Our ISSR dataset revealed the existence of a gradient in genetic diversity within A. excelsum with the most diverse remnants encountered in the south of the country, while the localities sampled in the Caribbean coast and in the Chicamocha canyon were less diverse. Chloroplast loci also pointed out the very low genetic diversity of A. excelsum from the Chicamocha canyon and we propose to prioritize this area within future conservation programs. Both chloroplast and nuclear markers supported the existence of genetic divergence between distinct regions of Colombia, uncovering genetic differences between inter-Andean, Caribbean, and Chicamocha canyon A. excelsum remnants. Hence, we advise to choose the provenance of seeds or plants carefully before translocation and to consider minimal mixing of material from different regions when initializing restoration programs for A. excelsum, in Colombia.