Mechanisms underlying turgor regulation in the estuarine alga Vaucheria erythrospora (Xanthophyceae) exposed to hyperosmotic shock

Aquatic organisms are often exposed to dramatic changes in salinity in the environment. Despite decades of research, many questions related to molecular and physiological mechanisms mediating sensing and adaptation to salinity stress remain unanswered. Here, responses of Vaucheria erythrospora, a turgor‐regulating xanthophycean alga from an estuarine habitat, have been investigated. The role of ion uptake in turgor regulation was studied using a single cell pressure probe, microelectrode ion flux estimation (MIFE) technique and membrane potential (E_m) measurements. Turgor recovery was inhibited by Gd³⁺, tetraethylammonium chloride (TEA), verapamil and orthovanadate. A NaCl‐induced shock rapidly depolarized the plasma membrane while an isotonic sorbitol treatment hyperpolarized it. Turgor recovery was critically dependent on the presence of Na⁺ but not K⁺ and Cl^? in the incubation media. Na⁺ uptake was strongly decreased by amiloride and changes in net Na⁺ and H⁺ fluxes were oppositely directed. This suggests active uptake of Na⁺ in V. erythrospora mediated by an antiport Na⁺/H⁺ system, functioning in the direction opposite to that of the SOS1 exchanger in higher plants. The alga also retains K⁺ efficiently when exposed to high NaCl concentrations. Overall, this study provides insights into mechanisms enabling V. erythrospora to regulate turgor via ion movements during hyperosmotic stress.     

Evaluating the capacity of plant DNA barcodes to discriminate species of cotton (Gossypium: Malvaceae)

Although two plastid regions have been adopted as the standard markers for plant DNA barcoding, their limited resolution has provoked the consideration of other gene regions, especially in taxonomically diverse genera. The genus Gossypium (cotton) includes eight diploid genome groups (A–G, and K) and five allotetraploid species which are difficult to discriminate morphologically. In this study, we tested the effectiveness of three widely used markers (matK, rbcL, and ITS2) in the discrimination of 20 diploid and five tetraploid species of cotton. Sequences were analysed locus‐wise and in combinations to determine the most effective strategy for species identification. Sequence recovery was high, ranging from 92% to 100% with mean pairwise interspecific distance highest for ITS2 (3.68%) and lowest for rbcL (0.43%). At a 0.5% threshold, the combination of matK+ITS2 produced the greatest number of species clusters. Based on ‘best match’ analysis, the combination of matK+ITS2 was best, while based on ‘all species barcodes’ analysis, ITS2 gave the highest percentage of correct species identifications (98.93%). The combination of sequences for all three markers produced the best resolved tree. The disparity index test based on matK+rbcL+ITS2 was significant (P < 0.05) for a higher number of species pairs than the individual gene sequences. Although all three barcodes separated the species with respect to their genome type, no single combination of barcodes could differentiate all the Gossypium species, and tetraploid species were particularly difficult.     

Red And far‐red regulation of filament movement correlates with the expression of phytochrome and FHY1 genes in Spirogyra varians (Zygnematales,Streptophyta)1

Spirogyra filaments show unique photomovement that differs in response to blue, red, and far‐red light. Phototropins involved in the blue‐light movement have been characterized together with downstream signaling components, but the photoreceptors and mechanical effectors of red‐ and far‐red light movement are not yet characterized. The filaments of Spirogyra varians slowly bent and aggregated to form a tangled mass in red light. In far‐red light, the filaments unbent, stretched rapidly, and separated from each other. Mannitol and/or sorbitol treatment significantly inhibited this far‐red light movement suggesting that turgor pressure is the driving force of this movement. The bending and aggregating movements of filaments in red light were not affected by osmotic change. Three phytochrome homologues isolated from S. varians showed unique phylogenetic characteristics. Two canonical phytochromes, named SvPHY1 and SvPHY2, and a noncanonical phytochrome named SvPHYX2. SvPHY1 is the first PHY1 family phytochrome reported in zygnematalean algae. The gene involved in the transport of phytochromes into the nucleus was characterized, and its expression in response to red and far‐red light was measured using quantitative PCR. Our results suggest that the phytochromes and the genes involved in the transport system into the nucleus are well conserved in S. varians.     

Multiple ice‐binding proteins of probable prokaryotic origin in an Antarctic lake alga,Chlamydomonas sp. ICE‐MDV (Chlorophyceae)

Ice‐associated algae produce ice‐binding proteins (IBPs) to prevent freezing damage. The IBPs of the three chlorophytes that have been examined so far share little similarity across species, making it likely that they were acquired by horizontal gene transfer (HGT). To clarify the importance and source of IBPs in chlorophytes, we sequenced the IBP genes of another Antarctic chlorophyte, Chlamydomonas sp. ICE‐MDV (Chlamy‐ICE). Genomic DNA and total RNA were sequenced and screened for known ice‐associated genes. Chlamy‐ICE has as many as 50 IBP isoforms, indicating that they have an important role in survival. The IBPs are of the DUF3494 type and have similar exon structures. The DUF3494 sequences are much more closely related to prokaryotic sequences than they are to sequences in other chlorophytes, and the chlorophyte IBP and ribosomal 18S phylogenies are dissimilar. The multiple IBP isoforms found in Chlamy‐ICE and other algae may allow the algae to adapt to a greater variety of ice conditions than prokaryotes, which typically have a single IBP gene. The predicted structure of the DUF3494 domain has an ice‐binding face with an orderly array of hydrophilic side chains. The results indicate that Chlamy‐ICE acquired its IBP genes by HGT in a single event. The acquisitions of IBP genes by this and other species of Antarctic algae by HGT appear to be key evolutionary events that allowed algae to extend their ranges into polar environments.     

Phylogeography of the freshwater red alga B atrachospermum viride‐brasiliense (Rhodophyta,Batrachospermales)

Phylogeography of B atrachospermum viride‐brasiliense was investigated using two mitochondrial regions: the cox2‐3 spacer and the barcode region of cox1 gene. Eighty‐seven individuals were analyzed from nine stream segments throughout its distribution in Brazil. Ten cox2‐3 spacer and nine cox1 haplotypes were observed among the individuals studied (87 vs. 43, respectively). Divergences among haplotypes were relatively low (≤2.4% for cox2‐3 and ≤1.8% for cox1). Most locations have a single haplotype, whereas only two locations had two haplotypes for both markers. The haplotype network for cox2‐3 showed a phylogeographic trend from the south towards the southeast with haplotypes from the southeast more closely related. For cox1 a trend from the southeast spreading towards the south and north was revealed, with the southern haplotypes more closely associated. Results clearly indicated that B . viride‐brasiliense represents a single species and the phylogeographic pattern consisted of a closely connected group of haplotypes from southern and southeastern Brazil. Levels of intra‐ and inter‐population variation were similar for the two markers with slightly higher values for cox2‐3. The trend observed in this study is similar to that in other members of Batrachospermales with little variation within a stream segment (one or two haplotypes) and more distant haplotypes showing higher divergences. This pattern could be attributed to the fact that colonization of a site might be rare by a single event with subsequent proliferation of the population. The geographic distribution of B . viride‐brasiliense was interpreted according to the biogeographic models proposed for South America being limited to three morpho‐climatic domains or biogeographic provinces: tropical Atlantic rainforest, sub‐tropical rainforest and cerrado (Brazilian savannah).     

Effective population size as a driver for divergence of an antimicrobial peptide (Hymenoptaecin) in two common European bumblebee species

Social insects are the target of numerous pathogens. This is because the high density of closely‐related individuals frequently interacting with each other enhances the transmission and establishment of pathogens. This high selective pressure results in the rapid evolution of immune genes, which might be counteracted by a reduced effective population size (N_e) lowering the effectiveness of selection. We tested the effect of N_e on the evolutionary rate of an important immune gene for the antimicrobial peptide Hymenoptaecin in two common central European bumblebee species: Bombus terrestris and Bombus lapidarius. Both species are similar in their biology and are expected to be under similar selective pressures because pathogen prevalence does not differ between species. However, previous studies indicated a higher N_e in B. terrestris compared to B. lapidarius. We found high intraspecific variability in the coding sequence but low variability for silent polymorphisms in B. lapidarius. Estimates of long‐ and short‐term N_e were three‐ to four‐fold higher N_e in B. terrestris, although the species did not differ in census population sizes. The difference in N_e might result in less efficient selection and suboptimal adaptation of immune genes (e.g. hymenoptaecin) in B. lapidarius, and thus this species might become less resistant and more tolerant, turning into a superspreader of diseases.     

Patterns of genetic diversity of the cryptogenic red alga Polysiphonia morrowii (Ceramiales,Rhodophyta) suggest multiple origins of the Atlantic populations

The red alga Polysiphonia morrowii, native to the North Pacific (Northeast Asia), has recently been reported worldwide. To determine the origin of the French and Argentine populations of this introduced species, we compared samples from these two areas with samples collected in Korea and at Hakodate, Japan, the type locality of the species. Combined analyses of chloroplastic (rbcL) and mitochondrial (cox1) DNA revealed that the French and Argentine populations are closely related and differ substantially from the Korean and Japanese populations. The genetic structure of P. morrowii populations from South Atlantic and North Atlantic, which showed high haplotype diversity compared with populations from the North Pacific, suggested the occurrence of multiple introduction events from areas outside of the so‐called native regions. Although similar, the French and Argentine populations are not genetically identical. Thus, the genetic structure of these two introduced areas may have been modified by cryptic and recurrent introduction events directly from Asia or from other introduced areas that act as introduction relays. In addition, the large number of private cytoplasmic types identified in the two introduced regions strongly suggests that local populations of P. morrowii existed before the recent detection of these invasions. Our results suggest that the most likely scenario is that the source population(s) of the French and Argentine populations was not located only in the North Pacific and/or that P. morrowii is a cryptogenic species.     

Habitat patch size alters the importance of dispersal for species diversity in an experimental freshwater community

Increased dispersal of individuals among discrete habitat patches should increase the average number of species present in each local habitat patch. However, experimental studies have found variable effects of dispersal on local species richness. Priority effects, predators, and habitat heterogeneity have been proposed as mechanisms that limit the effect of dispersal on species richness. However, the size of a habitat patch could affect how dispersal regulates the number of species able to persist. We investigated whether habitat size interacted with dispersal rate to affect the number of species present in local habitats. We hypothesized that increased dispersal rates would positively affect local species richness more in small habitats than in large habitats, because rare species would be protected from demographic extinction. To test the interaction between dispersal rate and habitat size, we factorially manipulated the size of experimental ponds and dispersal rates, using a model community of freshwater zooplankton. We found that high‐dispersal rates enhanced local species richness in small experimental ponds, but had no effect in large experimental ponds. Our results suggest that there is a trade‐off between patch connectivity (a mediator of dispersal rates) and patch size, providing context for understanding the variability observed in dispersal effects among natural communities, as well as for developing conservation and management plans in an increasingly fragmented world.     

Photosynthetic activity in two heteromorphic life‐history stages of a freshwater red alga,Thorea gaudichaudii (Thoreales) from Japan,in response to an irradiance and temperature gradient

We determined the effect of irradiance and temperature on the photosynthesis of two heteromorphic life‐history stages of an endangered freshwater red alga, Thorea gaudichaudii (Thoreales) by laboratory and field measurements. Net oxygenic photosynthesis–irradiance models of macroscopic and microscopic life‐history stages revealed similar low irradiance‐adapted responses, with a compensation irradiance (E_c) of 6.71 and 2.56 μmol photons m^?2 s^?1 (4.30–9.13 and 0.13–7.19, 95% Bayesian prediction interval, BPI) and saturating irradiance (E_k) of 26.6 and 30.0 μmol photons m^?2 s^?1 (19.0–37.4 and 12.1–63.0, BPI), respectively. A temperature‐dependent model of net photosynthesis and dark respiration in macroscopic and microscopic stages also showed similar temperature responses, and the gross photosynthetic rate (GP_max), 3.54 and 6.34 μg O₂ g_ww^?1 min^?1 (3.10–3.99 and 5.31–8.21, BPI), was highest at 32.1 and 35.7°C (29.8–34.0 and 29.5–48.6, BPI). The maximum quantum yields (F _v/F _m) in macroscopic and microscopic stages were also similar in response with respect to temperature; however, it was somewhat steady at low temperatures with the highest value of 0.54 and 0.62 (0.54–0.55 and 0.61–0.63, BPI) at 17.8 and 15.0°C (16.7–18.8 and 12.3–17.1, BPI). The effective quantum yield (Φ _PSII) in macroscopic and microscopic stages was also negatively correlated with irradiance, which decreased after 12 h of continuous exposure to 50 (low) and 1000 (high) μmol photons m^?2 s^?1 at 12 and 22°C. Large declines of Φ _PSII and subsequent failure of F _v/F _m recovery were particularly enhanced at high irradiance, signifying photoinhibition. Diurnal change of Φ _PSII and incident irradiance of the macroscopic stage under the field measurement revealed the midday depression of Φ _PSII; however, there was little direct sunlight due to shading by the trees, and algae were occurring in the shaded locations in the freshwater spring.     

Growth,condition, and maturity schedules of an estuarine fish species change in estuaries following increased hypoxia due to climate change

Understanding challenges posed by climate change to estuaries and their faunas remains a high priority for managing these systems and their communities. Freshwater discharge into a range of estuary types in south‐western Australia between 1990 and 2015 is shown to be related to rainfall. This largely accounts for decreases in discharge in this microtidal region being more pronounced on the west coast than south coast, where rainfall decline was less. Results of an oxygen‐balance model imply that, as demonstrated by empirical data for the Swan River Estuary, declines in discharge into a range of estuary types would be accompanied by increases in the extent of hypoxia. In 2013–15, growth and body condition of the teleost Acanthopagrus butcheri varied markedly among three permanently open, one intermittently‐open, one seasonally‐closed and one normally‐closed estuary, with average time taken by females to reach the minimum legal length (MLL) of 250 mm ranging from 3.6 to 17.7 years. It is proposed that, in a given restricted period, these inter‐estuary variations in biological characteristics are related more to differences in factors, such as food resources and density, than to temperature and salinity. The biological characteristics of A. butcheri in the four estuaries, for which there are historical data, changed markedly between 1993–96 and 2013–15. Growth of both sexes, and also body condition in all but the normally‐closed estuary, declined, with females taking between 1.7 and 2.9 times longer to attain the MLL. Irrespective of period, body condition, and growth are positively related. Age at maturity typically increased between periods, but length at maturity declined only in the estuary in which growth was greatest. The plasticity of the biological characteristics of A. butcheri, allied with confinement to its natal estuary and ability to tolerate a wide range of environmental conditions, makes this sparid and comparable species excellent subjects for assessing estuarine “health.”     

A polyphasic approach to the delimitation of diatom species: a case study for the genus Pinnularia (Bacillariophyta)

Diatoms are one of the most abundant and arguably the most species‐rich group of protists. Diatom species delimitation has often been based exclusively on the recognition of morphological discontinuities without investigation of other lines of evidence. Even though DNA sequences and reproductive experiments have revealed several examples of (pseudo)cryptic diversity, our understanding of diatom species boundaries and diversity remains limited. The cosmopolitan pennate raphid diatom genus Pinnularia represents one of the most taxon‐rich diatom genera. In this study, we focused on the delimitation of species in one of the major clades of the genus, the Pinnularia subgibba group, based on 105 strains from a worldwide origin. We compared genetic distances between the sequences of seven molecular markers and selected the most variable pair, the mitochondrial cox1 and nuclear encoded LSU rDNA, to formulate a primary hypothesis on the species limits using three single‐locus automated species delimitation methods. We compared the DNA‐based primary hypotheses with morphology and with other available lines of evidence. The results indicate that our data set comprised 15 species of the P. subgibba group. The vast majority of these taxa have an uncertain taxonomic identity, suggesting that several may be unknown to science and/or members of (pseudo)cryptic species complexes within the P. subgibba group.     

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [³²P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.     

Morphology of the crustose coralline alga Pseudolithophyllum muricatum (Corallinales,Rhodophyta) responds to 30 years of ocean acidification in the Northeast Pacific

As the process of ocean acidification alters seawater carbon chemistry, physiological processes such as skeletal accretion are expected to become more difficult for calcifying organisms. The crustose coralline red algae (Corallinales, Rhodophyta) form an important guild of calcifying primary producers in the temperate Northeast Pacific. The morphology of important ecological traits, namely, skeletal density and thallus thickness near the growing edge, was evaluated in Pseudolithophyllum muricatum (Foslie) Steneck & R.T. Paine, the competitively dominant alga within this guild. P. muricatum shows a morphological response to increased ocean acidification in the temperate Northeast Pacific. Comparing historical (1981–1997) and modern (2012) samples from the field, crust thickness near the growing edge was approximately half as thick in modern samples compared with historical samples, while crust calcite density showed no significant change between the two sample groups. Morphological changes at the growing edge have important consequences for mediating competitive interactions within this guild of algae, and may affect the role of crustose coralline algal beds as hosts to infaunal communities and facilitators of recruitment in many invertebrate and macroalgal species.     

Overall grazing tolerance index (overall GTI) is not an ideal predictor for describing a single‐species tolerance to grazing

Plants' pattern of compensatory growth is often used to intuitively estimate their grazing tolerance. However, this tolerance is sometimes measured by the overall grazing tolerance index (overall GTI), which assumes that tolerance is a multivariate linear function of various underlying mechanisms. Because the interaction among mechanisms is not independent, the grazing tolerance expression based on overall GTI may be inconsistent with that based on compensatory growth. Through a manipulative field experiment from 2007 to 2012, we measured the responses of 12 traits of Elymus nutans to clipping under different resource availabilities in an alpine meadow and explored the compensatory aboveground biomass and the overall GTI to assess the possible differences between the two expressions of tolerance. Our results showed that these two expressions of tolerance were completely opposite. The expression based on overall GTI was over‐compensatory and did not vary with clipping and resource availability, while the expression based on compensatory aboveground biomass was under‐compensatory and altered to over‐compensation after fertilization. The over‐expression of highly variable traits with extremely high negative mean GTI to defoliation damage, the influence of random errors contained in traits considered, and the doubling weight of functional redundant traits greatly inflated the overall GTI, which leads to the inconsistency of the two tolerance expressions. This inconsistency is also associated with the different determining mechanisms of the two tolerance expressions. Our data suggest that plants' grazing tolerance is not a multivariate linear function of traits or mechanisms that determine grazing tolerance; the overall GTI is only a measure of traits' variability to defoliation damage. Our findings highlight that the tolerance of E. nutans mainly depends on the response of traits with lower variability to defoliation, and the overall GTI is not an ideal predictor for describing a single‐species tolerance to grazing.     

An assessment of species limits of the South American mouse genus Oligoryzomys (Rodentia,Cricetidae) using unilocus delimitation methods

Oligoryzomys, as currently understood is formed by 25 living species, is the most diverse genus of the tribe Oryzomyini of the New World subfamily Sigmodontinae of cricetid rodents. Nonetheless, the species richness of Oligoryzomys seems to be an underestimate, given some species complex has been proposed in previous studies, at the time that large geographic areas remain to be sampled, and several taxonomic forms have not been assessed with contemporary approaches. In this study, we present a new assessment of the species diversity of Oligoryzomys based on multiple unilocus species delimitation methods (ABGD, BPP, PTP, GMYC and b GMYC), using 665 cytb gene sequences as evidence (532 gathered from Genbank and 133 obtained in this study). We sampled representatives of almost all currently known species of Oligoryzomys, at the time that extending the geographic coverage to the Central Andes, a large area that was largely unrepresented in previous studies. Phylogenetic relationships, based on a non‐redundant alignment, were inferred via maximum likelihood and Bayesian inference; an ultrametric tree, used in species delimitation analyses, was obtained using multiple secondary calibration points. Results of species delimitation methods are discussed at the light of previous knowledge (e.g., taxonomic history and geographic provenance of samples in relation to type localities) and the morphological assessments of some specimens. Results of the distinct delimitation methods are mostly congruent, being BPP and PTP the most sensible to estimate species delimitation, allowing us to suggest that Oligoryzomys is composed of 30 lineages of species level. Of these, 22 correspond to forms currently considered species; some of these include in their synonymy some forms currently considered valid species (e.g., yatesi would be a synonym of longicaudatus). The remaining eight lineages are candidate species that need to be further evaluated. This study, by advancing taxonomic hypothesis that should be further tested in future studies, constitutes a stepping‐stone for upcoming taxonomic and biogeographic studies centred on Oligoryzomys.     

Pollen competition between two sympatric Orchis species (Orchidaceae): the overtaking of conspecific of heterospecific pollen as a reproductive barrier

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post‐pollination pre‐zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.