RECOVERY AND GENETIC DIVERSITY OF THE INTERTIDAL KELP LESSONIA NIGRESCENS (PHAEOPHYCEAE) 20 YEARS AFTER EL NIÑO 1982/831

Massive mortality in kelp beds of the Pacific coasts of North and South America was caused by the rise in surface seawater temperature during the El Niño Southern Oscillation (ENSO) event of 1982/83, the strongest in the four and half previous centuries. In northern Chile a stretch of 600 km of coastline showed massive mortality of the intertidal kelp species Lessonia nigrescens Bory, of which only a few individuals managed to survive. Kelps and their associated biodiversity recovered but kelp beds re-colonization in general was variable in time and space seemingly very slow along northern Chilean coasts. Here we show, effectively, that northward re-colonization advanced less than 60 km in 20 years. Conversely, kelp beds of the Northern Hemisphere recovered 300 km in only six months after the same ENSO event. Genetic diversity in the two most affected populations of L. nigrescens shows half of the heterozygosity and polymorphism with respect to that observed in six non affected populations. In addition, geographically separated populations seem highly isolated as evidenced by high and significant fixation indices (all F_ST values over 0.4).     

Microsatellites of <Emphasis Type="BoldItalic">Laminaria digitata</Emphasis> tested in <Emphasis Type="BoldItalic">Lessonia nigrescens</Emphasis>: Evaluation and improvement of cross amplification between kelps of two different families

The use of primers designed originally to amplify DNA for one species in a different one can save time and resources, particularly for microsatellite loci. Microsatellite amplification improvements across two kelp families are reported, where loci originally described in Laminaria digitata (Laminariaceae) were tested in Lessonia nigrescens) was observed in two localities affected by massive mortality events. Nei’s distances among five populations presented similar patterns to those of 30 multilocus dominant loci (RAPD) evaluated in the same localities. Although some success might be achieved in cross-species microsatellite amplifications, the strong mutations detected between these two Laminarian families suggests that better results of cross-amplifications should be expected at much lower taxonomic levels. Thus, although more expensive, construction of new gene libraries is strongly recommended.     

Artificial induction of sporulation in Lessonia (Phaeophyta, Laminariales)

The effects of three physical variables on sporulation, germination and gametophyte survival were evaluated on two species of Laminariales at Coquimbo, Chile (30° S). Parameters were evaluated using reproductive fronds of Lessonia nigrescens Bory and Lessonia trabeculata Villouta and Santelices with treatment variables including type of pre-rinse, dehydration, and temperature. Although the response was slightly different in the two species, it was found that pre-rinsing the blades with tap water produced marked sporulation without affecting germination and survival. Minor effects were observed with other variables, including positive effects of lower temperature and negative effects of longer dehydration periods. The high commercial value of these two species has promoted much interest in their artificial propagation in Chile. This revised version was published online in June 2006 with corrections to the Cover Date.     

Temperature effects on gametophyte life-history traits and geographic distribution of two cryptic kelp species

A major determinant of the geographic distribution of a species is expected to be its physiological response to changing abiotic variables over its range. The range of a species often corresponds to the geographic extent of temperature regimes the organism can physiologically tolerate. Many species have very distinct life history stages that may exhibit different responses to environmental factors. In this study we emphasized the critical role of the haploid microscopic stage (gametophyte) of the life cycle to explain the difference of edge distribution of two related kelp species. Lessonia nigrescens was recently identified as two cryptic species occurring in parapatry along the Chilean coast: one located north and the other south of a biogeographic boundary at latitude 29-30°S. Six life history traits from microscopic stages were identified and estimated under five treatments of temperature in eight locations distributed along the Chilean coast in order to (1) estimate the role of temperature in the present distribution of the two cryptic L. nigrescens species, (2) compare marginal populations to central populations of the two cryptic species. In addition, we created a periodic matrix model to estimate the population growth rate (λ) at the five temperature treatments. Differential tolerance to temperature was demonstrated between the two species, with the gametophytes of the Northern species being more tolerant to higher temperatures than gametophytes from the south. Second, the two species exhibited different life history strategies with a shorter haploid phase in the Northern species contrasted with considerable vegetative growth in the Southern species haploid stage. These results provide strong ecological evidence for the differentiation process of the two cryptic species and show local adaptation of the life cycle at the range limits of the distribution. Ecological and evolutionary implications of these findings are discussed.     

IDENTIFICATION OF CRYPTIC SPECIES IN THE LESSONIA NIGRESCENS COMPLEX (PHAEOPHYCEAE,LAMINARIALES)1

The kelp Lessonia nigrescens Bory is the most ecologically and economically important seaweed in rocky intertidal and shallow subtidal habitats along the temperate Pacific South American coasts. Recent molecular studies suggest the existence of two lineages, one (northern lineage) from 17° S to 30° S and a second (central lineage) from 29° S to 41° S. To identify and name these lineages we performed morphological, nomenclatural and field studies. Four external and three internal anatomical traits permitted a morphological separation of the two lineages. The internal structure of both lineages was different from the isolectotype of Lessonia nigrescens. It is therefore concluded that the name Lessonia nigrescens should not be used for the Chilean material. Chordaria spicata Suhr appears as the oldest available name for the central lineage, while Lessonia berteroana Montagne is the oldest name for the northern lineage. In both cases, the type material consisted of small‐sized, apical branches of larger plants. The new combination Lessonia spicata (Suhr) Santelices is proposed for the central lineage and we reinstate Lessonia berteroana for the northern lineage. Laminaria scissa Suhr is reduced to synonym of L. spicata. Representative specimens of Lessonia nigrescens were not found during new visits to its type locality in Cape Horn and along Chile. Future studies should verify the status of this species.     

PLASTID FATTY ACID BIOSYNTHESIS IN THE DIATOMS NITZSCHIA ALBA AND NITZSCHIA LAEVIS

Among the co-dominant molecular markers, microsatellite loci have a number of advantages in population genetic studies. However, the different methods to hunt these markers are expensive, time consuming, and they require sophisticated laboratory equipment. Using in one species the microsatellites primers originally described for another one saves time and reduces costs. Examples in the literature revealed that microsatellites described for Gracilaria gracilis from France have not worked for other members of the Gracilariales. The results were not very promising, at least for Gracilaria chilensis from Chile. In this study, a number of microsatellite loci described for Laminaria digitata (Laminariaceae) from France were amplified and sequenced in two Lessonia (Lessoniaceae) species from Chile. Preliminary results show a partial conservation of both, flanking and tandem repeat regions. Some polymorphism has also been detected in Lessonia spp. The higher molecular affinity (conservation of primer sites) observed in species belonging to different families of brown algae, respect to that observed among species of the same genus in the red algae, is surprising. Such a result is strikingly counterintuitive when observing the morphological disparity among the Laminariales, respect to the similarity observed in the Gracilariales. It also reminds one of an earlier discussion by Gary Saunders about "When a family is not a family".     

Demography and feeding behavior of the kelp crab Taliepus marginatus in subtidal habitats dominated by the kelps Macrocystis pyrifera or Lessonia trabeculata

We studied the population and feeding ecology of the kelp crab Taliepus marginatus in subtidal kelp forests dominated by either of two morphologically different kelp species (Macrocystis pyrifera or Lessonia trabeculata) in northern Chile. The sizes and abundances of T. marginatus differed between the two kelp habitats. Kelp crabs were more abundant in the M. pyrifera forest than in the L. trabeculata forest. Size‐frequency distributions showed that juvenile and immature crabs were more common in the M. pyrifera forest than in the L. trabeculata forest, where reproductive adults predominated. The smaller crabs in the M. pyrifera habitat also consumed a higher proportion of kelp tissues than the larger crabs in the L. trabeculata habitat, which had a higher proportion of animal food in their diet. In both kelp forests, individuals of T. marginatus showed a similar pattern of nocturnal feeding over a 24‐h period, consuming more food at night than during the day. The more complex and dense forests of M. pyrifera appear to present better nursery habitats for juvenile kelp crabs than the more open and less dense forests dominated by L. trabeculata. These results suggest that the role of the two kelp habitats for T. marginatus varies during the life cycle of the kelp crabs, with M. pyrifera tending to have nursery function and L. trabeculata being more suitable as a reproductive habitat.     

Micropopulation differentiation in phenol content and susceptibility to herbivory in the Chilean kelp Lessonia nigrescenss (Phaeophyta,Laminariales)

Micropopulation differences in phenol content between intertidal and subtidal individuals of the kelp Lessonia nigrescens were found. Subtidal plants showed: (1) significantly higher phenol content than intertidal individuals, in vegetative and reproductive tissues, (2) intra-plant differences, with higher content in apical frond tissues, (3) higher resistance to consumption by herbivorous fishes. The microscopic progeny of subtidal plants showed the same trend as adult plants: (1) haploid spores from subtidal plants had higher phenol content than spores from intertidal individuals, and (2) the microscopic sporophytes derived from subtidal spores and gametophytes were less consumed by herbivorous snails (Tegula tridentata) than those derived from intertidal plant propagules. No increase in phenol content was detected after mechanical injury to experimental fronds, or after transplantation to the subtidal environment.In addition to the absence of inducible responses, the different phenol content between intertidal and subtidal individuals, in adult diploid plants and also in the haploid progeny, suggests that both environments differ someway enough to fix the mentioned features on the plants of Lessonia nigrescens. It is likely that the differences in herbivory between the two distributional extremes contributed to the observed pattern.     

Recruitment of the intertidal kelp Lessonia nigrescens Bory in northern Chile: successional constraints and opportunities

The strong 1982/83 El Niño event caused local extinction in populations of the low intertidal kelp Lessonia nigrescens Bory on northern Chilean coasts. The kelp has partially recolonized, but its recovery has apparently been low. This study documents the effect of biological factors that potentially decrease the velocity of kelp recolonization in northern Chile. A removal experiment showed that encrusting coralline algae, which dominate the lower intertidal, significantly reduce the recruitment of L. nigrescens in the presence of herbivores. Epithallium shedding by encrusting corallines is the most probable cause for this inhibition process. On the other hand, grazing on encrusting corallines by the chiton Enoplochiton niger (Barnes), a large-sized herbivore (10–20 cm length), could also affect kelp recruitment. The intestinal content of E. niger, where encrusting corallines are the main item (84.2%), revealed the presence of L. nigrescens. Among the natural substrata on which kelp recruits, measured at five different localities, the coarsely-branched alga Corallina officinalis (Decaisne) Kützing reached the highest frequency, despite its extremely low cover (< 1%) in the field. This suggests that settlement on turfs of C. officinalis allows the kelp to escape from herbivory, thus facilitating its recruitment. The roles of timing of kelp recruitment and seasonal grazing are also discussed.     

Long Term Variability in the Structure of Kelp Communities in Northern Chile and the 1997–98 ENSO

This is the first study on the south eastern Pacific coast of South America which details long term, interannual variability in the structure of subtidal rocky-bottom kelp-dominated communities before, during, and after the El Niño Southern Oscillation (ENSO) event of 1997–1998 in northern Chile (23^°S). The temporal patterns of the main components of these ecosystems, which included Macrocystis integrifolia, Lessonia trabeculata, echinoids and asteroids, were evaluated seasonally between 1996 and 2004. M. integrifolia demonstrated high interannual variability in temporal patterns of abundance. The 1997–1998 ENSO did not significantly modify the temporal patterns of Macrocystis, although local extinction of M. integrifolia beds occurred during negative thermal anomalies in 1999–2000 (La Niña event), facilitating the establishment of urchin dominated “barren grounds”. The abundance of Lessonia trabeculata showed little temporal variability, and this species dominated the deeper regions of the kelp assemblage (8–13 m depth). The structure of the kelp communities in the study area is regulated by a trophic cascade which modulates alternation between kelp dominated areas and sea urchin barrens. In this context, frequent and intense upwelling of cold water high in nutrients favors the establishment and persistence of kelp assemblages. During ENSO, coastal upwellings can mitigate superficial warming of coastal water and increase the nutrient concentration in the water column. Superficial warming during the 1997–1998 ENSO induced spawning by different species of echinoderms, which resulted in major recruitment of these species during 1999. Top-down events, such as the decrease in densities of the asteroids after the 1997–1998 ENSO event, favored increases in densities of benthic grazers, which caused significant decreases in abundance of M. integrifolia. The re-establishment of the adult fraction of the carnivore (starfish) guild coincided with a decrease in the density of sea urchins and thus re-establishment of the kelp. In the temperate south eastern Pacific, oceanographic events, which act on different spatial-temporal scales, trigger trophic cascades that act at local levels, producing interannual variability in the structure of kelp communities. On the other hand, considering the high macroinvertebrate diversity associated with kelp assemblages, the transitions between kelp-dominated areas and sea urchin barrens do not appear to significantly affect the biodiversity of these assemblages of benthic invertebrates.     

Spatial and short-term variability of larval,post-larval and macrobenthic assemblages associated with subtidal kelp forest ecosystems in Central Chile

Identifying patterns of spatial and temporal variability in the composition of communities associated with kelp forests is critical to understand the functioning of this productive, yet vulnerable ecosystem. We used a suite of sampling methods (light attraction and airlift devices) to evaluate the variability of larval, post-larval and macrobenthic assemblages associated with kelp forests (Lessonia trabeculata) in Central Chile (30° to 33°S). Pelagic collections identified two assemblages: early-life stages and emerging macrobenthos, with the later contributing three quarters to the total abundance regardless of the source of illumination (permanent or flashing). Field experiments showed that moon phases affected the structure and composition of the samples. Surveys carried out during new moon showed the highest abundances and taxonomic richness of emergent assemblages. However, species composition varied in both assemblages depending on the moon phase. Although the pelagic assemblages collected at sites with contrasting upwelling intensity did not show differences in community structure, differences in composition were evident for early-life stages. The relationship between pelagic and benthic collections indicated that four decapod crustaceans were represented at both larval and early juvenile stages; however, only the high abundances and densities of Paraxanthus barbiger allowed for estimations of benthic-pelagic coupling. For this species, larval abundances and benthic juvenile densities demonstrated contrasting local and regional patterns, suggesting a decoupling between pelagic and benthic environments. These findings highlight the differential variability in smaller components of kelp forests, but also suggest that post-settlement processes may be driving biological interactions through these highly productive and complex environments.     

A comparison between two populations of Lessonia trabeculata (Phaeophyta: Laminariales) microscopic stages

Lessonia trabeculata is the most important brown seaweed in northern Chilean subtidal environments. It usually grows in areas with high to moderately high water movement, and only occasionally in sheltered habitats. These different hydrodynamic environments produce morphologically and physiologically distinct plants. The purpose of this study was to determine probable differences in reproductive features between two L. trabeculata populations exposed to different water movement intensities. The zoospore production, settlement, germination, gametophyte fertility and survival were studied.Zoospore release and spore attachment capacity were higher from blades from El Francés Beach, the locality with higher water movement energy. Germination at 24 hours was higher in the germlings from El Francés Beach than those from La Herradura Bay. However, after 4 days of culture germination rates were similar. Average survival rates of gametophytes were similar for both populations. After 20 days culturing gametophytes from El Francés Beach fertilization was observed, while in samples from La Herradura Bay the sporophytes were seen at 27d.The results suggest a higher reproductive vitality in plants of Lessonia trabeculata living in an environment with an active water movement, than plants growing in a sheltered environment.     

Ancestors of white clover ( Trifolium repens L.), as revealed by isozyme polymorphisms

Isozymes were used to study the putative ancestors of white clover ( Trifolium repens L.). Ten enzymes were examined, and 18 loci were resolved via starch-gel electrophoresis for accessions representing T. repens, Trifolium isthmocarpum, Trifolium nigrescens, Trifolium occidentale and Trifolium uniflorum, in addition to two more distantly related species, Trifolium alpinum and Trifolium purseglovei. Nei's genetic identities indicate that T. uniflorum and T. nigrescens are the closest relatives of T. repens. The isozyme data thus support a hypothesis that the two genomes of the tetraploid T. repens could have been derived from hybridization between T. nigrescens and T. uniflorum. This conclusion is further supported by shared alleles between T. repens, T. nigrescens and T. uniflorum. However, the origin of T. repens is somewhat obscured by the presence of shared alleles between T. repens and both T. occidentale and T. isthmocarpum, suggesting that introgression of genes from the latter two species into T. repens may also have taken place. High values of genetic identity are shared between T. occidentale with T. nigrescens and T. uniflorum, also indicative of introgression. Alternatively the presence of shared alleles among the five species may reflect their recent common ancestry.     

Microsatellite markers for the intertidal kelp Postelsia palmaeformis (Heterokontophyta; Laminariales)

Postelsia palmaeformis (Ruprecht) is an intertidal brown alga belonging to the ecologically and economically important group of seaweeds commonly referred to as kelp (Order Laminariales). Six polymorphic microsatellite loci were identified for this monotypic genus. The number of alleles ranged from two to five, with observed heterozygosities ranging from 0.00 to 0.704.     

Variation in Patterns of Metal Accumulation in Thallus Parts of Lessonia trabeculata (Laminariales; Phaeophyceae): Implications for Biomonitoring

Seaweeds are well known to concentrate metals from seawater and have been employed as monitors of metal pollution in coastal waters and estuaries. However, research showing that various intrinsic and extrinsic factors can influence metal accumulation, raises doubts about the basis for using seaweeds in biomonitoring programmes. The thallus of brown seaweeds of the order Laminariales (kelps) is morphologically complex but there is limited information about the variation in metal accumulation between the different parts, which might result in erroneous conclusions being drawn if not accounted for in the biomonitoring protocol. To assess patterns of individual metals in the differentiated parts of the thallus (blade, stipe, holdfast), concentrations of a wide range of essential and non-essential metals (Fe, Cr, Cu, Zn, Mn, Pb, Cd, Ni and Al) were measured in the kelp Lessonia trabeculata. Seaweeds were collected from three sampling stations located at 5, 30 and 60 m from an illegal sewage outfall close to Ventanas, Chile and from a pristine location at Faro Curaumilla. For the majority of metals the highest concentrations in bottom sediment and seaweed samples were found at the site closest to the outfall, with concentrations decreasing with distance from the outfall and at control stations; the exception was Cd, concentrations of which were higher at control stations. The patterns of metal concentrations in different thallus parts were metal specific and independent of sampling station. These results and the available literature suggest that biomonitoring of metals using seaweeds must take account of differences in the accumulation of metals in thallus parts of complex seaweeds.     

Inshore spawning grounds of the squid Doryteuthis gahi suggest the consistent use of defoliated kelp Lessonia trabeculata in Central Chilean waters

Coastal spawning grounds of the squid Doryteuthis gahi were identified for the first time at three sites in Central Chile during 2014 and 2015. A total of 15 egg masses were collected from between 10 and 15?m depth and brought to the laboratory for evaluating capsular, embryonic and when possible, paralarval characters. Egg capsules from all spawning areas were similar in size (～25?mm in length) and number of embryos per capsule (～15). Egg-laying patterns, in addition to field observations, suggest that relatively small D. gahi may be using shallow waters in semi-protected environments to reproduce and spawn. Additionally, the differential use of healthy versus defoliated kelp Lessonia trabeculata suggests that egg-laying females selectively choose substrata that limit mechanical damage to the fragile egg capsules and that also allow for adequate water flow, which could be reduced in a mass of kelp fronds. These findings highlight the opportunistic behaviour of D. gahi which use overgrazed L. trabeculata, a condition that is widespread in areas with high herbivory pressure.     

A Marine Strain of Flavobacteriaceae Utilizes Brown Seaweed Fucoidan

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) with a yield of about 3.8% dry weight. To isolate enzymes that degrade fucoidan, we first screened marine bacteria for their ability to utilize fucoidan, and isolated one strain of Flavobacteriaceae from seawater that could do this. Phylogenetic analysis of the 16S ribosomal DNA sequence suggested that this strain appeared to belong to a new genus, and was tentatively named Fucobacter marina. The strain utilized L-fucose (17%), D-mannose (91%), D-galactose (46%), and D-glucuronic acid (66%) in the fucoidan from K. crassifolia. The strain partially utilized fucoidan from 2 other seaweeds that belong to the order Laminariales, Undaria pinnatifida (10%) and Lessonia nigrescens (48%).     

Disentangling the links between habitat complexity and biodiversity in a kelp‐dominated subantarctic community

Habitat complexity is one of the most important factors modulating species diversity. This feature comprises several interrelated attributes, such as number, size, and spatial arrangement of complexity‐forming elements. However, the separate and joint effects of these attributes on diversity and community structure are still not well understood. Here, we assess the relationships between several structural‐complexity attributes of the subantarctic kelp Lessonia flavicans and species richness, total abundance, and structure of kelp‐associated macrobenthic communities. We predicted that longer thalli and larger holdfasts favor greater species richness and total abundance of invertebrate organisms. To test the prediction, an observational sampling program was established in two sites of the Strait of Magellan. Uni‐ and multivariate analyses revealed both positive and negative effects of kelp structural‐complexity attributes on diversity. Holdfast diameter and maximum frond length, followed by thallus wet weight, had the strongest positive fits to species richness and total abundance; the number of stipes, on the other hand, was negatively associated with both response variables. Longer fronds were associated with greater abundances of spirorbid polychaetes. Larger holdfasts supported larger abundances of Nereididae and Terebelidae polychaetes and the limpet Nacella mytilina. Contrarily, kelps with longer fronds and more stipes supported fewer amphipods. In this way, we demonstrate that different dimensions of habitat complexity can have contrasting effects on diversity and community structure, highlighting the fundamental role of multiple dimensions of kelp habitat complexity for local biodiversity.     

Fine-scale genetic structure and fire-created habitat patchiness in the Australian allodapine bee, Exoneura nigrescens (Hymenoptera: Apidae)

Fire promotes an abundance of nest sites for the stem nesting bee Exoneura nigrescens, which remain viable for approximately 10 years. The finite duration of nesting substrate and localized fire events suggest that migration should minimize genetic structure among suitable habitat patches. Exoneura nigrescens was sampled from 7 localities with a known fire history in southwestern Victoria, Australia. Individual bees were genotyped at 8 microsatellite loci and genic and genotypic analyses applied to examine genetic structure among burn patch localities, within burn patches, and within colonies. Despite relatively short-term availability of nesting substrates, remarkably fine-scale genetic structure was observed both among burn patches and within burn patches. The spatial distribution of relatedness shows a strong pattern of isolation-by-distance at geographic distances to 35 km, suggesting that genetic partitioning among burn patches is, at least in part, a result of dispersal ability. Genetic structure within burn patches includes colonies consisting of close kin with genic partitioning among nests. Relatedness structure within colonies suggests that polygamy, multiple breeding pairs, and a lack of inbreeding typifies the mating system.     

LAMINARIOCOLAX SP. (PHAEOPHYCEAE) ASSOCIATED WITH GALL DEVELOPMENTS IN LESSONIA NIGRESCENS (PHAEOPHYCEAE)1

As part of a long‐term study on the biology and ecology of the intertidal kelp Lessonia nigrescens Bory, we report on the occurrence of gall development on this alga, identified the possible causal agent, and assessed the extent of the phenomenon in two wild stands of the host. Our results showed that galls affecting natural populations of L. nigrescens were associated with the infection by a filamentous brown algal endophyte of the genus Laminariocolax. Assignment to Laminariocolax of the endophytes isolated from cultured gall tissue was based on the (i) high internal transcribed spacer 1 (ITS1) sequence similarity and phylogenetic relationship between the Chilean isolates and several species of the genus Laminariocolax endophytic in other kelps, (ii) reproductive and vegetative features of the endophyte in culture, and (iii) anatomical agreement of fully developed galls of Lessonia with those described for other kelp galls caused by endophytic members of Laminariocolax. Unequivocal identification at the species level of the endophytes infecting Lessonia, however, awaits further studies.