Impact of persistent barrier to gene flow and catastrophic events on red algae evolutionary history along the Chilean coast.

Historical vicariance events, linked to the existence of stable physical barriers to gene flow, generate concordant genetic breaks in co-distributed species while stochastic processes (e.g., costal uplift) could cause species-specific genetic breaks as a result of local strong demographic bottlenecks or extinction. In Chile, previous studies show that the area of the 30°S-33°S could correspond to a stable barrier to gene flow that have affected the genetic structure of various algae and marine invertebrates. Here we sequenced two organellar genes (COI and rbcL) in four taxonomically accepted co-distributed red seaweeds species characterized by a low dispersal potential: Mazzaella laminarioides, M. membranacea, Asterfilopsis disciplinalis, and Ahnfeltiopsis vermicularis. Our results revealed the existence of ten strongly differentiated linages in the taxa studied. Strong genetic breaks, concordant in both space and time (divergence estimated to have occurred some 2.9-12.4 million years ago), were observed between taxa distributed across the 33°S. Conversely, in the Central/South part of the Chilean coast, the localization of the genetic breaks/sub-structure observed varied widely (36°S, 38°S, 39°S, and 40°S). These results suggest that a major historical vicariance event has modeled the genetic structure of several Chilean marine organisms in the north of the Chilean coast during the mid-Miocene, while more recent stochastic events and genetic drift could be the driving forces of genetic divergence/structuration in the central-southern part of the coast.

Pre-domestication bottlenecks of the cultivated seaweed Gracilaria chilensis.

Gracilaria chilensis is the main cultivated seaweed in Chile. The low genetic diversity observed in the Chilean populations has been associated with the over-exploitation of natural beds and/or the founder effect that occurred during post-glacial colonization from New Zealand. How these processes have affected its evolutionary trajectory before farming and incipient domestication is poorly understood. In this study, we used 2232 single nucleotide polymorphisms (SNPs) to assess how the species' evolutionary history in New Zealand (its region of origin), the founder effect linked to transoceanic dispersion and colonization of South America, and the recent over-exploitation of natural populations have influenced the genetic architecture of G. chilensis in Chile. The contrasting patterns of genetic diversity and structure observed between the two main islands in New Zealand attest to the important effects of Quaternary glacial cycles on G. chilensis. Approximate Bayesian Computation (ABC) analyses indicated that Chatham Island and South America were colonized independently near the end of the Last Glacial Maximum and emphasized the importance of coastal and oceanic currents during that period. Furthermore, ABC analyses inferred the existence of a recent and strong genetic bottleneck in Chile, matching the period of over-exploitation of the natural beds during the 1970s, followed by rapid demographic expansion linked to active clonal propagation used in farming. Recurrent genetic bottlenecks strongly eroded the genetic diversity of G. chilensis prior to its cultivation, raising important challenges for the management of genetic resources in this incipiently domesticated species.

Differential Frond Growth in the Isomorphic Haploid-diploid Red Seaweed Agarophyton chilense by Long-term In Situ Monitoring.

Conditional differentiation between haploids and diploids has been proposed to drive the evolutionary stability of isomorphic biphasic life cycles. The cost of producing and maintaining genetic information has been posed as a possible driver of this conditional differentiation. Under this hypothesis, haploids benefit over diploids in resource-limited environments by halving the costs of producing and maintaining DNA. Spared resources can be allocated to enhance survival, growth or fertility. Here we test in the field whether indeed haploids have higher growth rates than diploids. Individuals of the red seaweed Agarophyton chilense, were mapped and followed during 2 years with 4-month census intervals across different stands within the Valdivia River estuary, Chile. As hypothesized, haploids grew larger and faster than diploids, but this was sex-dependent. Haploid (gametophyte) females grew twice as large and 15% faster than diploids (tetrasporophytes), whereas haploid males only grew as large and as fast as the maximum obtained by diploids in summer. However, haploid males maintained their maximum sizes and growth rates constant year-round, while diploids were smaller and had lower growth rates during the winter. In conclusion, our results confirm the conditional differentiation in size and growth between haploids and diploids but also identified important differences between males and females. Besides understanding life cycle evolution, the dynamics of A. chilense frond growth reported informs algal farmers regarding production optimization and should help in determining best planting and harvesting strategies.

Comparative phylogeography of two Agarophyton species in the New Zealand archipelago.

Molecular studies have reported the coexistence of two species of Agarophyton in New Zealand: the newly described A.transtasmanicum with an apparently restricted distribution to some sites in the North Island, and the more widespread A.chilense. Here, we compared the distribution, genetic diversity, and structure of both Agarophyton species throughout the archipelago using sequences of the nuclear Internal Transcribed Spacer 2 (ITS2) marker. Agarophyton chilense's distribution was continuous and extensive along the North and South Islands, Stewart Island, and Chatham Island, and the genetic clusters were mostly concordant with boundaries between biogeographic regions. In contrast, specimens of A.transtasmanicum were collected in four sites broadly distributed in both the North and South Islands, with no clear spatial structure of the genetic diversity. Populations, where the species co-occurred, tended to display similar levels in genetic diversity for the two species. Demographic inferences supported a postglacial demographic expansion for two A.chilense genetic clusters, one present in the South Island and the eastern coast of the North Island, and the other present in northern South Island. A third genetic cluster located on the western coast of the North Island had a signature of long-term demographic stability. For A.transtasmanicum, the skyline plot also suggested a postglacial demographic expansion. Last, we developed a new molecular tool to quickly and easily distinguish between the two Agarophyton species, which could be used to ease future fine-scale population studies, especially in areas where the two species coexist.

Differentiation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio.

BACKGROUND: Algal isomorphic biphasic life cycles alternate between free-living diploid (tetrasporophytes) and haploid (dioicious gametophytes) phases and the hypotheses explaining their maintenance are still debated. Classic models state that conditional differentiation between phases is required for the evolutionary stability of biphasic life cycles while other authors proposed that the uneven ploidy abundances observed in the field are explained by their cytological differences in spore production. RESULTS: We monitored the state and fate of individuals of the red seaweed Gracilaria chilensis periodically for 3 years in five intertidal pools from two sites with distinct conditions. We tested for differentiation in fecundity and spore survival among the gametophyte males and females (haploids) and the tetrasporophytes (diploids). We tested for the influence of fecundity and spore survival on the observed uneven ploidy abundances in recruits. The probability of a frond becoming fecund was size-dependent, highest for the haploid males and lowest for the haploid females, with the diploids displaying intermediate probabilities. Fecund diploids released more tetraspores than carpospores released by the haploid females. Spore survival depended on ploidy and on the local density of co-habiting adult fronds. An advantage of diploid over haploid germlings was observed at very low and very high adult fronds densities. CONCLUSIONS: Neither spore production nor spore survival determined the highly variable ploidy ratio within G. chilensis recruits. This result invalidates the hypothesis of natural cytological differences in spore production as the only driver of uneven field ploidy abundances in this species. Diploid spores (carpospores) survived better than haploid spores (tetraspores), especially in locations and time periods that were associated with the occurrence of strong biotic and abiotic stressors. We hypothesise that carpospore survival is higher due to support by their haploid female progenitors passing-on nutrients and chemical compounds improving survival under stressful conditions.

Haploid females in the isomorphic biphasic life-cycle of Gracilaria chilensis excel in survival.

BACKGROUND: Conditional differentiation is one of the most fundamental drivers of biodiversity. Competitive entities (usually species) differ in environmental or ecological niche enabling them to co-exist. Conditional differentiation of haploid and diploid generations is considered to be a requirement for the evolutionary stability of isomorphic biphasic life-cycles and the cause for the natural occurrence of both phases at uneven abundances. Theoretically, stage dependent survival rates are the most efficient way to explain conditional differentiation. RESULTS: We tested for conditional differentiation in survival rates among life stages (haploid males, haploid females, and diploids) of Gracilaria chilensis, an intertidal red alga occurring along the Chilean shores. Therefore, the fate of individuals was followed periodically for 3 years in five intertidal pools and, for the first time in isomorphic red algae, a composite model of the instantaneous survival rates was applied. The results showed the survival dependency on density (both competition and Allee effects), fertility, age, size, season and location, as well as the differentiation among stages for the survival dependencies of these factors. The young haploid females survived more than the young of the other stages under Allee effects during the environmentally stressful season at the more exposed locations, and under self-thinning during the active growth season. Furthermore, fertile haploid females had a higher survival than fertile haploid males or fertile diploids. CONCLUSIONS: Here, we show a survival advantage of haploids over diploids. The haploid females probably optimize their resource management targeting structural and physiological adaptations that significantly enhance survival under harsher conditions. In a companion paper we demonstrate a fertility advantage of diploids over haploids. Together, the survival and fertility differentiation support the evolution and prevalence of biphasic life-cycles.

Correction: Linear-In-The-Parameters Oblique Least Squares (LOLS) Provides More Accurate Estimates of Density-Dependent Survival.

[This corrects the article DOI: 10.1371/journal.pone.0167418.].

Linear-In-The-Parameters Oblique Least Squares (LOLS) Provides More Accurate Estimates of Density-Dependent Survival.

Survival is a fundamental demographic component and the importance of its accurate estimation goes beyond the traditional estimation of life expectancy. The evolutionary stability of isomorphic biphasic life-cycles and the occurrence of its different ploidy phases at uneven abundances are hypothesized to be driven by differences in survival rates between haploids and diploids. We monitored Gracilaria chilensis, a commercially exploited red alga with an isomorphic biphasic life-cycle, having found density-dependent survival with competition and Allee effects. While estimating the linear-in-the-parameters survival function, all model I regression methods (i.e, vertical least squares) provided biased line-fits rendering them inappropriate for studies about ecology, evolution or population management. Hence, we developed an iterative two-step non-linear model II regression (i.e, oblique least squares), which provided improved line-fits and estimates of survival function parameters, while robust to the data aspects that usually turn the regression methods numerically unstable.

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID-DIPLOID RED ALGA GRACILARIA CHILENSIS(1).

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.