Incipient ecological speciation between successional varieties of a dominant tree involves intrinsic postzygotic isolating barriers

Whereas disruptive selection imposed by heterogeneous environments can lead to the evolution of extrinsic isolating barriers between diverging populations, the evolution of intrinsic postzygotic barriers through divergent selection is less certain. Long‐lived species such as trees may be especially slow to evolve intrinsic isolating barriers. We examined postpollination reproductive isolating barriers below the species boundary, in an ephemeral hybrid zone between two successional varieties of the landscape‐dominant Hawaiian tree, Metrosideros polymorpha, on volcanically active Hawai'i Island. These archipelago‐wide sympatric varieties show the weakest neutral genetic divergence of any taxon pair on Hawai'i Island but significant morphological and ecological differentiation consistent with adaptation to new and old lava flows. Cross‐fertility between varieties was high and included heterosis of F₁ hybrids at the seed germination stage, consistent with a substantial genetic load apparent within varieties through low self‐fertility and a lack of self‐pollen discrimination. However, a partial, but significant, barrier was observed in the form of reduced female and male fertility of hybrids, especially backcross hybrids, consistent with the accumulation of genetic incompatibilities between varieties. These results suggest that partial intrinsic postzygotic barriers can arise through disruptive selection acting on large, hybridizing populations of a long‐lived species.     

Strong asymmetry in the relative strengths of prezygotic and postzygotic barriers between two damselfly sister species

One of the longest debates in biology has been over the relative importance of different isolating barriers in speciation. However, for most species, there are few data evaluating their relative contributions and we can only speculate on the general roles of pre- and postzygotic isolation. Here, we quantify the absolute and cumulative contribution of 19 potential reproductive barriers between two sympatric damselfly sister species, Ischnura elegans and I. graellsii, including both premating (habitat, temporal, sexual and mechanical isolation) and postmating barriers (prezygotic: sperm insemination success and removal rate, oviposition success, fertility, fecundity; postzygotic: hybrid viability, hybrid sterility and hybrid breakdown). In sympatry, total reproductive isolation between I. elegans females and I. graellsii males was 95.2%, owing mostly to a premating mechanical incompatibility (93.4%), whereas other barriers were of little importance. Isolation between I. graellsii females and I. elegans males was also nearly complete (95.8%), which was caused by the cumulative action of multiple prezygotic (n= 4, 75.4%) and postzygotic postmating barriers (n= 5, 7.4%). Our results suggest that premating barriers are key factors in preventing gene flow between species, and that the relative strengths of premating barriers is highly asymmetrical between the reciprocal crosses.     

Mitochondrial genomes of the green macroalga Ulva pertusa (Ulvophyceae,Chlorophyta): novel insights into the evolution of mitogenomes in the Ulvophyceae

To further understand the trends in the evolution of mitochondrial genomes (mitogenomes or mtDNAs) in the Ulvophyceae, the mitogenomes of two separate thalli of Ulva pertusa were sequenced. Two U. pertusa mitogenomes (Up1 and Up2) were 69,333 bp and 64,602 bp in length. These mitogenomes shared two ribosomal RNAs (rRNAs), 28 transfer RNAs (tRNAs), 29 protein‐coding genes, and 12 open reading frames. The 4.7 kb difference in size was attributed to variation in intron content and tandem repeat regions. A total of six introns were present in the smaller U. pertusa mtDNA (Up2), while the larger mtDNA (Up1) had eight. The larger mtDNA had two additional group II introns in two genes (cox1 and cox2) and tandem duplication mutations in noncoding regions. Our results showed the first case of intraspecific variation in chlorophytan mitogenomes and provided further genomic data for the undersampled Ulvophyceae.     

Synergistic effects of HSE and LTR elements from hsp70 gene promoter of Ulva prolifera (Ulvophyceae,Chlorophyta) upon temperature induction1

Besides heat stress, the 70 kDa heat shock proteins (HSP70s) have been shown to respond to cold stress. However, the involved cis‐acting elements remain unknown. The hsp70 gene from the green macroalga Ulva prolifera (Uphsp70) has been cloned, from which one heat shock element HSE and one low‐temperature‐responsive element LTR were found in the promoter. Using the established transient expression system and quantitative GUS assay, a series of element deletion experiments were performed to determine the functions of HSE and LTR in response to temperature stress. The results showed that under cold stress, both HSE and LTR were indispensable, since deletion leads to complete loss of promoter activity. Under heat stress, although the HSE could respond independently, coexistence with LTR was essential for high induced activity of the Uphsp70 promoter. Therefore, synergistic effects exist between HSE and LTR elements in response to temperature stress in Ulva, and extensive bioinformatics analysis showed that the mechanism is widespread in algae and plants, since LTR coexists widely with HSE in the promoter region of hsp70. Our findings provide important supplements to the knowledge of algal and plant HSP70s response to temperature stress. We speculated that for algal domestication and artificial breeding, HSE and LTR elements might serve as potential molecular targets to temperature acclimation.     

Conspecificity of the model organism Ulva mutabilis and Ulva compressa (Ulvophyceae,Chlorophyta)

As one of the most abundant and ubiquitous representatives of marine and brackish coastal macrophytobenthos communities, the genus Ulva is not only an important primary producer but also of ecological and morphogenetic interest to many scientists. Ulva mutabilis became an important model organism to study morphogenesis and mutualistic interactions of macroalgae and microorganisms. Here, we report that our collections of Ulva compressa Linnaeus (1753) from Germany are conspecific with the type strains of the model organism U. mutabilis Føyn (1958), which were originally collected at Olhão on the south coast of Portugal and have from that time on been maintained in culture as gametophytic and parthenogenetic lab strains. Different approaches were used to test conspecificity: (i) comparisons of vegetative and reproductive features of cultured material of U. mutabilis and German U. compressa demonstrated a shared morphological pattern; (ii) gametes of U. compressa and U. mutabilis successfully mated and developed into fertile sporophytic first‐generation offspring; (iii) molecular phylogenetics and species delimitation analyses based on the Generalized Mixed Yule‐Coalescent method showed that U. mutabilis isolates (sl‐G[mt+]) and (wt‐G[mt‐]) and U. compressa belong to a unique Molecular Operational Taxonomic Unit. According to these findings, there is sufficient evidence that U. mutabilis and U. compressa should be regarded as conspecific.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

The contribution of multiple barriers to reproduction between edaphically divergent lineages in the Amazonian tree Protium subserratum (Burseraceae)

Disentangling the strength and importance of barriers to reproduction that arise between diverging lineages is central to our understanding of species origin and maintenance. To date, the vast majority of studies investigating the importance of different barriers to reproduction in plants have focused on short‐lived temperate taxa while studies of reproductive isolation in trees and tropical taxa are rare. Here, we systematically examine multiple barriers to reproduction in an Amazonian tree, Protium subserratum (Burseraceae) with diverging lineages of soil specialist ecotypes. Using observational, molecular, distributional, and experimental data, we aimed to quantify the contributions of individual prezygotic and postzygotic barriers including ecogeographic isolation, flowering phenology, pollinator assemblage, pollen adhesion, pollen germination, pollen tube growth, seed development, and hybrid fitness to total reproductive isolation between the ecotypes. We were able to identify five potential barriers to reproduction including ecogeographic isolation, phenological differences, differences in pollinator assemblages, differential pollen adhesion, and low levels of hybrid seed development. We demonstrate that ecogeographic isolation is a strong and that a combination of intrinsic and extrinsic prezygotic and postzygotic barriers may be acting to maintain near complete reproductive isolation between edaphically divergent populations of the tropical tree, P. subserratum.     

Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding N:P dynamics in Ulva lactuca (Chlorophyta)

Dissolved inorganic phosphorus (DIP ) is an essential macronutrient for maintaining metabolism and growth in autotrophs. Little is known about DIP uptake kinetics and internal P‐storage capacity in seaweeds, such as Ulva lactuca (Chlorophyta). Ulva lactuca is a promising candidate for biofiltration purposes and mass commercial cultivation. We exposed U. lactuca to a wide range of DIP concentrations (1–50 μmol · L^?1) and a nonlimiting concentration of dissolved inorganic nitrogen (DIN ; 5,000 μmol · L^?1) under fully controlled laboratory conditions in a “pulse‐and‐chase” assay over 10 d. Uptake kinetics were standardized per surface area of U. lactuca fronds. Two phases of responses to DIP ‐pulses were measured: (i) a surge uptake (V_S ) of 0.67 ± 0.10 μmol · cm^?2 · d^?1 and (ii) a steady state uptake (V_M ) of 0.07 ± 0.03 μmol · cm^?2 · d^?1. Mean internal storage capacity (ISC_P ) of 0.73 ± 0.13 μmol · cm^?2 was calculated for DIP . DIP uptake did not affect DIN uptake. Parameters of DIN uptake were also calculated: V_S  = 12.54 ± 1.90 μmol · cm^?2 · d^?1, V_M  = 2.26 ± 0.86 μmol · cm^?2 · d^?1, and ISC_N  = 22.90 ± 6.99 μmol · cm^?2. Combining ISC and V_M values of P and N, nutrient storage capacity of U. lactuca was estimated to be sufficient for ~10 d. Both P and N storage capacities were filled within 2 d when exposed to saturating nutrient concentrations, and uptake rates declined thereafter at 90% for DIP and at 80% for DIN . Our results contribute to understanding the ecological aspects of nutrient uptake kinetics in U. lactuca and quantitatively evaluating its potential for bioremediation and/or biomass production for food, feed, and energy.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

A new Eocene fossil of the genus Phycopeltis (Ulvophyceae,Chlorophyta)

A fossil of the aerophytic green algal genus Phycopeltis (Trentepohliaes, Ulvophyceae) dated to 35 Ma, is reported from the Pikopiko Fossil Forest, Southland, New Zealand. Previous reports of fossilized Phycopeltis have been subsequently synonymized with fungi by other authors; however, our specimen is not vulnerable to their criticisms. Inflated cells present in two approximately concentric rings are interpreted as gametangia, with irregular structures resembling the gametangial pores of modern material; sporophytic material is absent. The fossil resembles the modern disc‐forming species P. novae‐zelandiae, P. expansa, and P. arundinacea. The limited material available prevents the assignation of a specific epithet, but the habit and dimensions of the fossil clearly fall within those of modern representatives of the genus. Its single cell thickness throughout, absence of distinct melanization, and larger size demonstrate that it is not a fungal shield. The specimen constitutes arguably the most convincing fossil belonging to Trentepohliales, and the first unambiguously for the genus Phycopeltis. It is consistent in age with other known fossils of the order that, when combined with molecular evidence, suggests a terrestrial radiation far more recent than that of land plants.     

Species divergence in field crickets: genetics,song, ecomorphology,and pre‐ and postzygotic isolation

Studies that simultaneously estimate levels of species divergence in genetics, reproductive and ecological traits, and pre‐ and postzygotic isolation are relatively rare. Here we compare levels of divergence in three allopatric sister species of field crickets. We compare divergence in both nuclear and mitochondrial DNA, male song, female ovipositor length, levels of pre‐ and postzygotic isolation, and male versus female contributions to prezygotic isolation. Taken together, our data show the accumulation of a multitude of potential reproductive isolating barriers if secondary contact were to become established. Furthermore, ecological and behavioural prezygotic isolation appear significantly more advanced than postzygotic isolation, with prezygotic isolation due to female behaviour exceeding that due to male behaviour.     

Interaction of Photoprotective and Acclimation Mechanisms in Ulva rigida (Chlorophyta) in Response to Diurnal Changes in Solar Radiation in Southern Chile

Species of the genus Ulva (Chlorophyta) are regarded as opportunistic organisms, which efficiently adjust their metabolism to the prevailing environmental conditions. In this study, changes in chlorophyll‐a fluorescence‐based photoinhibition of photosynthesis, electron transport rates, photosynthetic pigments, lipid peroxidation, total phenolic compounds, and antioxidant metabolism were investigated during a diurnal cycle of natural solar radiation in summer (for 12 h) under two treatments: photosynthetically active radiation (PAR: 400–700 nm) and PAR+ ultraviolet (UV) radiation (280–700 nm). In the presence of PAR alone, Ulva rigida showed dynamic photoinhibition, and photosynthetic parameters and pigment concentrations decreased with the intensification of the radiation. On the other hand, under PAR+UV conditions a substantial decline up to 43% was detected and an incomplete fluorescence recovery, also, P‐I curve values remained low in relation to the initial condition. The phenolic compounds increased their concentration only in UV radiation treatments without showing a correlation with the antioxidant activity. The enzimatic activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased over 2‐fold respect at initial values during the onset of light intensity. In contrast, catalase (CAT) increased its activity rapidly in response to the radiation stress to reach maxima at 10 a.m. and decreasing during solar. The present study suggests that U. rigida is capable of acclimating to natural radiation stress relies on a concerted action of various physiological mechanisms that act at different times of the day and under different levels of environmental stress.     

Flip‐flop organization in the chloroplast genome of Capsosiphon fulvescens (Ulvophyceae,Chlorophyta)

To better understand organelle genome evolution of the ulvophycean green alga Capsosiphon fulvescens, we sequenced and characterized its complete chloroplast genome. The circular chloroplast genome was 111,561 bp in length with 31.3% GC content that contained 108 genes including 77 protein‐coding genes, two copies of rRNA operons, and 27 tRNAs. In this analysis, we found the two types of isoform, called heteroplasmy, were likely caused by a flip‐flop organization. The flip‐flop mechanism may have caused structural variation and gene conversion in the chloroplast genome of C. fulvescens. In a phylogenetic analysis based on all available ulvophycean chloroplast genome data, including a new C. fulvescens genome, we found three major conflicting signals for C. fulvescens and its sister taxon Pseudoneochloris marina within 70 individual genes: (i) monophyly with Ulotrichales, (ii) monophyly with Ulvales, and (iii) monophyly with the clade of Ulotrichales and Ulvales. Although the 70‐gene concatenated phylogeny supported monophyly with Ulvales for both species, these complex phylogenetic signals of individual genes need further investigations using a data‐rich approach (i.e., organelle genome data) from broader taxon sampling.     

Freshwater population of Ulva flexuosa (Ulvaceae,Chlorophyta) as a food source for great pond snail: Lymnaea stagnalis (Mollusca,Lymnaeidae)

Cosmopolitan species of the genus Ulva (Ulvaceae; Chlorophyta) that populate the littoral zone of marine habitats constitute a staple diet for a variety of organisms, particularly snails, shellfish, polychaetes, and birds. Occurrence of Ulva species (e.g., U. flexuosa and U . prolifera) has also been observed in freshwater inland ecosystems that have no contact with saline water. However, the influence of the development of macroalgal mats of Ulva on indigenous organisms in limnic ecosystems has not been established. This study investigates the trophic relationships between Ulva flexuosa and one species of snail from freshwater habitats in central Europe. During the summer, the great pond snail (Lymnaea stagnalis) consumed Ulva as a source of nutrition even when other algae and plants were available. Lymnaea stagnalis consumed an average of 100 mg of Ulva thalli per day. This level of biomass exceeded the consumption of an alternative food source, the shoots of Elodea canadensis. Ulva thalli are more actively consumed by great pond snails than Elodea shoots, and this is expressed in terms of the differences of biomass consumption. It was also observed that the interior of the monostromatic tubular thalli of Ulva flexuosa serves as a protective shelter for juvenile great pond snails.     

Molecular and morphological diversity of Narragansett Bay (RI,USA) Ulva (Ulvales,Chlorophyta) populations

Macroalgal bloom‐forming species occur in coastal systems worldwide. However, due to overlapping morphologies in some taxa, accurate taxonomic assessment and classification of these species can be quite challenging. We investigated the molecular and morphological characteristics of 153 specimens of bloom‐forming Ulva located in and around Narragansett Bay, RI, USA. We analyzed sequences of the nuclear internal transcribed spacer 1 region (ITS1) and the chloroplast‐encoded rbcL; based on the ITS1 data, we grouped the specimens into nine operational taxonomic units (OTUs). Eight of these OTUs have been previously reported to exist, while one is novel. Of the eight OTUs, all shared sequence identity with previously published sequences or differed by less than 1.5% sequence divergence for two molecular markers. Previously, 10 species names were reported for Ulva in Rhode Island (one blade and nine tube‐forming species) based upon morphological classification alone. Of our nine OTUs, three contained blade‐forming specimens (U. lactuca, U. compressa, U. rigida), one OTU had a blade with a tubular stipe, and six contained unbranched and/or branched tubular morphologies (one of these six, U. compressa, had both a blade and a tube morphology). While the three blade‐forming OTUs in Narragansett Bay can frequently be distinguished by careful observations of morphological characteristics, and spatial/temporal distribution, it is much more difficult to distinguish among the tube‐forming specimens based upon morphology or distribution alone. Our data support the molecular species concept for Ulva, and indicate that molecular‐based classifications of Ulva species are critical for proper species identification, and subsequent ecological assessment or mitigation of Ulva blooms.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

Differential toxic effects of Ulva lactuca (Chlorophyta) on the herbivorous gastropods,Littorina littorea and L. obtusata (Mollusca)

Members of the genus Ulva are widespread and abundant in intertidal and shallow subtidal areas but there are conflicting data regarding susceptibility to herbivory. While some studies have documented that Ulva spp. were favored by a diversity of marine herbivores, other work has revealed herbivore deterrence. We investigated grazing and growth rates of the littorinid species, Littorina littorea and L. obtusata, when offered Fucus vesiculosus, Ascophyllum nodosum, Ulva lactuca, and Chondrus crispus, highlighting distinctive vulnerabilities to toxic effects of U. lactuca. Ulva lactuca was the preferred food of L. littorea, while L. obtusata showed no grazing on this ephemeral algal species. In contrast, F. vesiculosus was highly preferred by L. obtusata. Although L. littorea demonstrated a grazing preference for U. lactuca, growth rate of this gastropod species was nearly 3× greater when fed F. vesiculosus, suggesting a non‐lethal, negative effect of U. lactuca on L. littorea with long‐term exposure. Mortality of L. obtusata ranged from 0% to 100% when held in the presence of various Ulva densities for 1 week, and Ulva exudate depressed herbivory of this gastropod. We conclude that the water‐soluble, toxic exudate produced by U. lactuca in response to herbivory had allelochemical properties, and may contain a cleavage product (acrylic acid) of dimethylsulfoniopropionate or reactive oxygen species (i.e., H₂O₂). Observed differences in susceptibility to Ulva toxicity by the littorinid species may be related to generalist versus specialist feeding and habitat strategies.     

Ulva lactuca and U. flexuosa (Chlorophyta,Ulvophyceae) cultivation in Brazilian tropical waters: recruitment,growth, and ulvan yield

Ulva spp. are used in a wide range of commercial applications, including bioremediation, food, bioenergy, pharmaceuticals, and agriculture. The sulfated polysaccharide ulvan obtained from Ulva spp. is of interest for triggering plant defenses against disease. However, the cultivation of Ulva spp. is still in its infancy. This study verified the feasibility of cultivating Ulva lactuca and Ulva flexuosa at two sites on the tropical Brazilian coast. We investigated the following: (a) methods to induce sporulation, (b) comparison of seeding ropes inoculated in vitro versus seeding at sea over 40 days, (c) production and harvest cycles at 15 and 30 days, (d) growth productivity of U. flexuosa at sea and in outdoor tanks, and (e) comparison of ulvan yields from biomass cultivated in tanks and the sea. High nutrient treatment was the most efficient method to induce sporulation (7,540?±?3,133 spores m^?1). Sea-based cultivation of U. flexuosa was only successful at one site. Seeding of ropes in vitro was more efficient than seeding at sea (0.31?±?0.20 g m^?2 day^?1), and 15-day harvest cycles were most efficient (20.1?±?1.8 % day^?1; 0.46?±?0.11 g m^?2 day^?1). Despite differences in plant growth in tanks (27.9?±?4.4 % day^?1) and at sea (20.1?±?1.8 % day^?1), the dry biomass and ulvan yields (17.7?±?5.0 %) did not differ between these systems. Cultivation of U. flexuosa was feasible at sea using in vitro seeding with a production cycle of 15 days in Brazilian tropical waters and tanks with high irradiance and enriched seawater.