Diets of crustacean zooplankton,inferred from stable carbon and nitrogen isotope analyses,in lakes with varying allochthonous dissolved organic carbon content

We used stable carbon and nitrogen isotope analyses to estimate the relative proportions of three putative food sources (1) algae, (2) allochthonous organic matter (but including also heterotrophic bacteria and green-sulphur bacteria having similar isotopic values) and (3) methane-oxidizing bacteria (MOB) in the diets of crustacean zooplankton in five small boreal lakes representing a gradient of dissolved organic carbon (DOC) concentration from ca. 5 to 40 mg C l⁻¹. The lakes were sampled in May, after establishment of stratification, and again in October during autumnal mixing of the water column. IsoSource mixing model outputs indicated that the proportion of algae in the diets of zooplankton was generally higher in May than in October, and that bacteria contributed to the diets of both cladocerans and copepods. Our results indicate that bacteria, especially MOB, can make an appreciable contribution to zooplankton diets in these small lakes, even in those with relatively low DOC concentrations.     

Zooplankton interactions in an enclosure experiment: insights from stable isotope analyses

1. Density gradients of cladocerans and copepods were generated in an enclosure experiment to compare the impact on the plankton of a filter feeder (Daphnia hyalina × galeata) with that of more selective feeders (calanoid and cyclopoid copepods). The experiment was conducted in situ over 25 days during spring in a mesotrophic lake, Schöhsee, Germany. 2. The plankton community was monitored regularly. Daphniids were able to graze on the phytoplankton present, which mainly consisted of small (<1000 μm³) species, whereas copepods did not show any impact on algae. 3. At the end of the experiment, Daphnia and remaining cyclopoid copepods were harvested and sorted manually, prior to analyses for stable isotopes of carbon and nitrogen. Daphniids from mesocosms stocked purely with differing densities of Daphnia showed little variability in stable isotope values, whereas those that thrived in enclosure bags together with copepods exhibited lower δ¹³C values. 4. The change in Daphniaδ¹³C indicates a change of food sources, modified by the presence of the copepods: the higher the mean abundance of copepods in the enclosures, the more ¹³C‐depleted the daphniids. Increasing abundance of high nucleic acid (HNA) bacteria in the copepod bags may account for the trend in Daphniaδ¹³C via increased grazing on the bacteria themselves, or via grazing on phytoplankton utilising isotopically light CO₂ from respiratory release. 5. Cyclopoid copepod stable isotope signatures were related to Daphnia and copepod abundances in copepod bags, suggesting that cyclopoids preyed on the available zooplankton.     

Multiple origins of sequestrate basidiomes within Entoloma inferred from molecular phylogenetic analyses

The genus Entoloma comprises diverse trophic modes and basidiome morphologies. Although Entoloma includes some sequestrate species, their origins are not clearly understood in relation to phylogenetic position and trophic status. In this study, we collected 34 sequestrate Entoloma specimens in Japan over a 9-y period. Their identities and phylogenetic positions were determined by molecular analyses using three nuclear loci [internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA and RNA polymerase II second LSU (rpb2) gene]. Based on species delimitation of 97 % sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified four sequestrate Entoloma species. Molecular phylogenetic analyses that included all related sequences in the International Nucleotide Sequence Database revealed that the four sequestrate Entoloma species belonged to two major phylogroups. One of the phylogroups was Inocephalus–Cyanula, which is composed only of saprotrophic species. Three of the Japanese sequestrate species, as well as three previously known sequestrate species from other regions, fell into at least two independent clades in this phylogroup, indicating multiple origins of sequestrate forms within this saprotrophic lineage. Another phylogroup, Rhodopolioid, was also shown to include a sequestrate species for the first time. Because the Rhodopolioid phylogroup is composed exclusively of mycorrhizal species (ectomycorrhizal and tuberculate mycorrhizal species), the sequestrate form may also have evolved from a mycorrhizal ancestor. Our results suggest that sequestrate basidiomes have evolved multiple times, irrespective of their trophic status in Entoloma. Finally, based on molecular and morphological characteristics, here we describe two new sequestrate Entoloma species, i.e., Entoloma prismaticum Sasaki, Kinoshita et Nara, sp. nov. and Entoloma hypogaeum Sasaki, Kinoshita et Nara, sp. nov.     

Advances in linking wintering migrant birds to their breeding-ground origins using combined analyses of genetic and stable isotope markers

An enduring problem in avian ecology and conservation is linking breeding and wintering grounds of migratory species. As migratory species and populations vary in the degree to which individuals from distinct breeding locales mix on stop-over sites and wintering grounds, establishing migratory connectivity informs our understanding of population demography and species management. We present a new Bayesian approach for inferring breeding grounds of wintering birds of unknown origins in North America. We incorporate prior information from analysis of genetic markers into geographic origin assignment based upon stable-hydrogen isotope analysis of feathers (δ(2)H(f)), using the Loggerhead Shrike (Lanius ludovicianus). Likely geographic origins derived from analyses of DNA microsatellites were used as priors for Bayesian analyses in which birds were assigned to a breeding-ground origin using their δ(2)H(f) values. As with most applications of Bayesian methods, our approach greatly improved the results (i.e. decreased the size of the potential area of origin). Area of origin decreased by 3 to 5-fold on average, but ranged up to a 10-fold improvement. We recommend this approach in future studies of migratory connectivity and suggest that our methodology could be applied more broadly to the study of dispersal, sources of productivity of migratory populations, and a range of evolutionary phenomena.     

Carbon source accounting for fish using combined DNA and stable isotope analyses in a regulated lowland river weir pool

Determining the source and flow of carbon, energy and nutrients through food webs is essential for understanding ecological connectivity and thus determining the impact of management practices on biodiversity. We combined DNA sequencing, microarrays and stable isotope analyses to test whether this approach would allow us to resolve the carbon flows through food webs in a weir pool on the lower Murray River, a highly impacted, complex and regulated ecosystem in southern Australia. We demonstrate that small fish in the Murray River consume a wide range of food items, but that a significant component of carbon and nitrogen entering the food web during dry periods in summer, but not spring, is derived from nonconventional sources other than in-channel primary producers. This study also showed that isotopic analyses alone cannot distinguish food sources and that a combined approach is better able to elucidate food-consumer dynamics. Our results highlight that a major river ecosystem, stressed by reduced environmental flows, can rapidly undergo significant and previously undetected changes that impact on the ecology of the system as a whole.     

Affinities among anostracan (Crustacea: Branchiopoda) families inferred from phylogenetic analyses of multiple gene sequences

To gain insights into the relationships among anostracan families, molecular phylogenetic analyses were performed on nuclear (28S D1-D3 ribosomal DNA) and mitochondrial (16S rDNA, COI) gene regions for representatives of seven families and an outgroup. Data matrices used in the analyses included 951 base pairs (bp) of aligned sequences for 28S, 465 bp for 16S, and 658 bp (219 amino acids) for COI. Maximum-parsimony and maximum-likelihood methods were used to construct phylogenetic trees, enabling the evaluation of both previous hypotheses of taxonomic relationships among families based on morphology, and of the relative merits of independent versus simultaneous analyses of multiple data sets for phylogeny construction. Data from various combinations of the gene regions produced relatively congruent patterns of phylogenetic affinity. In most analyses, two monophyletic groups were resolved: one cluster included the families Polyartemiidae, Chirocephalidae, Branchinectidae, Streptocephalidae, and Thamnocephalidae, while the other contained the Artemiidae and Branchipodidae. Comparative analyses showed that combining gene regions in a single matrix generally resulted in increased resolution and support for each cluster relative to those obtained from single-gene analyses. Statistical tests demonstrated that morphology-based hypotheses of relationships among families had poorer support than those determined from molecular data, reflecting the homoplasy in characters used to differentiate families.     

Dispersal and phylogenetic diversity of nonmarine picocyanobacteria,inferred from 16S rRNA gene and cpcBA-intergenic spacer sequence analyses

More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.     

Genetic variability and mycohost association of Ampelomyces quisqualis isolates inferred from phylogenetic analyses of ITS rDNA and actin gene sequences

Ampelomyces quisqualis complex is well known as the most common and widespread hyperparasite of the family Erysiphaceae, the cause of powdery mildew diseases. As commercial biopesticide products it is widely used to control the disease in field and plastic houses. Although genetic diversity within Ampelomyces isolates has been previously recognized, a single name A. quisqualis is still applied to all pycnidial intracellular hyperparasites of powdery mildew fungi. In this study, the phylogenetic relationships among Ampelomyces isolates originating from various powdery mildew fungi in Korea were inferred from Bayesian and maximum parsimony analyses of the sequences of ITS rDNA region and actin gene. In the phylogenetic trees, the Ampelomyces isolates could be divided into four distinct groups with high sequence divergences in both regions. The largest group, Clade 1, mostly accommodated Ampelomyces isolates originating from the mycohost Podosphaera spp. (sect. Sphaerotheca). Clade 2 comprised isolates from several genera of powdery mildews, Golovinomyces, Erysiphe (sect. Erysiphe), Arthrocladiella, and Phyllactinia, and was further divided into two subclades. An isolate obtained from Podosphaera (sect. Sphaerotheca) pannosa was clustered into Clade 3, with those from powdery mildews infecting rosaceous hosts. The mycohosts of Ampelomyces isolates in Clade 4 mostly consisted of species of Erysiphe (sect. Erysiphe, sect. Microsphaera, and sect. Uncinula). The present phylogenetic study demonstrates that Ampelomyces hyperparasite is indeed an assemblage of several distinct lineages rather than a sole species. Although the correlation between Ampelomyces isolates and their mycohosts is not obviously clear, the isolates show not only some degree of host specialization but also adaptation to their mycohosts during the evolution of the hyperparasite.     

Danish free-ranging mink populations consist mainly of farm animals: Evidence from microsatellite and stable isotope analyses

Two methods were used to separate free-ranging mink Mustela vison into wild mink and escaped farm mink. Analysis of stable carbon isotopes was performed on teeth and claws of 226 free-ranging mink from two areas in Denmark. A classification based on empirical data resulted in three groups (n=213); 47% were newly escaped farm mink and another 31% had been born in farms and lived in nature for more than ca. 2 months. The remaining 21% may or may not have been born in nature, but they had been free ranging for more than a year and were thus considered wild. A genetic analysis by means of microsatellites was performed on a subsample of the trapped mink (86 individuals) and on 70 farm mink, in order to assess the proportion of escaped farm mink in the free-ranging population. Strong genetic evidence for a high percentage of escaped farm mink in the free-ranging population (86%) was found, in agreement with the carbon isotope results. Both methods can be used to distinguish between farm and wild mink, but whereas microsatellites can only say whether a given mink originated from a farm or not, carbon isotopes can give some more detail on the period of time that a farm mink has been living in natural habitats. The high proportion of escaped farm mink in the Danish nature could have serious implications for the preservation of other vulnerable species and should be carefully considered when designing conservation strategies.     

Speciation in Pyricularia inferred from multilocus phylogenetic analysis

Pyricularia isolates from various host plants were subjected to a multilocus phylogenetic analysis based on rDNA-ITS, actin, β-tubulin, and calmodulin loci. A combined gene tree resolved seven groups with 100 % BS support, suggesting that they are monophyletic groups supported concordantly by all four loci. By incorporating biological and morphological species criteria, each of the seven groups was considered to be a current species. However, phylogenetic relationships among these species were unresolved in the single-gene trees and in the combined tree. Furthermore, the transition from concordance to conflict occurred more than once in the combined gene tree. They were interpreted by assuming that Pyricularia has evolved through repeated species radiation. The transition point other than the current species limit was considered to be the limit of the former species.     

Linking aboveground and belowground food webs through carbon and nitrogen stable isotope analyses

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) have been used for more than two decades in analyses of food web structure. The utility of isotope ratio measurements is based on the observation that consumer δ¹³C values are similar (<1‰ difference) to those of their diet, while consumer δ¹⁵N values are about 3‰ higher than those of their diet. The technique has been applied most often to aquatic and aboveground terrestrial food webs. However, few isotope studies have examined terrestrial food web structure that includes both above- and belowground (detrital) components. Here, we review factors that may influence isotopic signatures of terrestrial consumers in above- and belowground systems. In particular, we emphasize variations in δ¹³C and δ¹⁵N in belowground systems, e.g., enrichment of ¹³C and ¹⁵N in soil organic matter (likely related to soil microbial metabolism). These enrichments should be associated with the high ¹³C (~3‰) enrichment in belowground consumers relative to litter and soil organic matter and with the large variation in δ¹⁵N (~6‰) of the consumers. Because such enrichment and variation are much greater than the trophic enrichment generally used to estimate consumer trophic positions, and because many general predators are considered dependent on energy and material flows from belowground, the isotopic variation in belowground systems should be taken into account in δ¹³C and δ¹⁵N analyses of terrestrial food webs. Meanwhile, by measuring the δ¹³C of key predators, the linkage between above- and belowground systems could be estimated based on observed differences in δ¹³C of primary producers, detritivores and predators. Furthermore, radiocarbon (¹⁴C) measurements will allow the direct estimation of the dependence of predators on the belowground systems.     

Evaluating the utility of stable isotope analyses of archived freshwater sample materials

We evaluated the potential utility of stable isotope analysis of tissues commonly archived by aquatic biologists. Previous studies with chemically preserved samples have shown contradictory results, which present an obstacle for the use of archived sample materials. We tested the effects of ethanol and formalin preservation on zooplankton and of ethanol on benthic macroinvertebrate δ¹³C and δ¹⁵N values. We found that neither formalin nor ethanol had a significant effect on δ¹³C and δ¹⁵N values of preserved zooplankton. Nor did ethanol significantly affect δ¹³C or δ¹⁵N values of macroinvertebrates. However, ethanol preservation slightly, but significantly decreased C:N ratios of both zooplankton and macroinvertebrates, probably reflecting some extraction of lipids. Overall, the effects of preservatives on δ¹³C and δ¹⁵N values that we observed were minor. We also compared δ¹³C and δ¹⁵N values analysed from roach scales and perch operculum bones with those analysed from muscle tissue. Decalcification of scales and operculum bones only slightly improved our comparison to muscle tissue δ¹³C and δ¹⁵N values. Decalcified scales had slightly higher δ¹³C and lower δ¹⁵N values. Similarly, decalcified operculum bones showed slightly increased δ¹³C and decreased δ¹⁵N values to those for fish muscle. Our results confirm that scales and operculum bones can provide a suitable proxy for fish muscle in isotope studies with minor correction. We conclude that various archived sample materials can indeed be used with confidence for historical reconstructions of freshwater food webs by stable isotope analysis. Handling editor: K. Martens     

Genetic divergence and phylogenetic independence of Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) inferred from mitochondrial DNA analyses

Classification of freshwater fish in the subfamily Leuciscinae, Cyprinidae is hampered by complexity or lack of morphological diversity. In this study, analyses based on mtDNA sequences were undertaken to clarify phylogenetic relationships among Far Eastern, North American and European species in the Leuciscinae. Evolutionary rate in cytochrome b gene (Cyt-b) and D-loop sequences appear to be almost constant in Leuciscinae. The topology of phylogenetic trees generated by neighbor-joining (NJ) and maximum likelihood (ML) methods based on Cyt-b gene and D-loop sequences was similar. Five major clades, designated clades 1-5, and a minor clade were discriminated. Most of the Far Eastern, North American and European species were included in the major clades. Clade 1, comprised almost entirely of Far Eastern phoxinins, is monophyletic and greatly diverged from the other species of Leuciscinae. From the present phylogenetic relationships and the previous studies, we present the following hypotheses with respect to the evolutionary history of the Far Eastern phoxinins. The Far Eastern species should be classified into Far Eastern-specific genera, although ichthyologists have still insisted that the species should be included in the European genera. The Far Eastern clade 1 consists of two subclades, including genera Pseudaspius-Tribolodon and Far Eastern Phoxinus species. According to our phylogenetic analyses, Pseudaspius leptocephalus and Tribolodon species should be reclassified into the same genus. On the basis of evolutionary rate in Cyt-b gene in Cyprinidae, it is estimated that the Far Eastern lineage diverged approximately 10-14 million years ago (mya) from the common ancestor of Leuciscinae. It is deduced that speciation of the Far Eastern species occurred until approximately 4 mya, in relation to the formation of the Sea of Japan and the Japanese Islands.     

Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences

Rhizopogon (Basidiomycota, Boletales) is a genus of hypogeous fungi that form ectomycorrhizal associations mostly with members of the Pinaceae. This genus comprises an estimated 100(+) species, with the greatest diversity found in coniferous forests of the Pacific northwestern United States. Maximum parsimony analyses of 54 nuclear ribosomal DNA internal transcribed spacer (ITS) sequences including 27 Rhizopogon and 10 Suillus species were conducted to test sectional relationships in Rhizopogon and examine phylogenetic relationships with the closely related epigeous genus, Suillus. Sequences from 10 Rhizopogon type collections were included in these analyses. Rhizopogon and Suillus were both monophyletic. Rhizopogon section Rhizopogon is not monophyletic and comprised two clades, one of which consisted of two well supported lineages characterized by several long insertions. Rhizopogon sections Amylopogon and Villosuli formed well supported groups, but certain species concepts within these sections were unresolved. Four species from section Fulviglebae formed a strongly supported clade within section Villosuli. Subgeneric taxonomic revisions are presented.     

Size-dependent ontogenetic diet shifts to piscivory documented from stable isotope analyses in an introduced population of largemouth bass

Piscivorous largemouth bass (Micropterus salmoides) have been introduced in several regions outside of their native range in North America, resulting in significant disturbance to native fish communities. This species exhibits an ontogenetic diet shift from zooplanktivory to piscivory as juveniles. An early switch to piscivory allows 0+ bass to increase their growth rate prior to winter, resulting in reduced mortality. However, little is known about the dietary switch at the population level during the first year. We used carbon stable isotope analyses to examine the diets of age 0+ individuals in Lake Izunuma, Japan. The onset of the shift to piscivory occurred at a smaller size than in native or other non-native areas [>40 mm total length (TL)]. We found a positive correlation between TL and δ¹³C throughout summer and autumn. Small individuals had δ¹³C values that were similar to those of zooplankton, whereas large individuals had δ¹³C values that were similar to those of cyprinid prey species. This suggests that the smaller 0+ individuals remain zooplanktivorous until late autumn, whereas the larger individuals shift to piscivory as early as June, soon after the breeding season ends. Our results also suggest that a significant number of 0+ bass failed to switch to piscivory until the winter of their first year, despite the smaller size threshold for the onset of piscivory.     

Integrating stomach content and stable isotope analyses to quantify the diets of pygoscelid penguins

Stomach content analysis (SCA) and more recently stable isotope analysis (SIA) integrated with isotopic mixing models have become common methods for dietary studies and provide insight into the foraging ecology of seabirds. However, both methods have drawbacks and biases that may result in difficulties in quantifying inter-annual and species-specific differences in diets. We used these two methods to simultaneously quantify the chick-rearing diet of Chinstrap (Pygoscelis antarctica) and Gentoo (P. papua) penguins and highlight methods of integrating SCA data to increase accuracy of diet composition estimates using SIA. SCA biomass estimates were highly variable and underestimated the importance of soft-bodied prey such as fish. Two-source, isotopic mixing model predictions were less variable and identified inter-annual and species-specific differences in the relative amounts of fish and krill in penguin diets not readily apparent using SCA. In contrast, multi-source isotopic mixing models had difficulty estimating the dietary contribution of fish species occupying similar trophic levels without refinement using SCA-derived otolith data. Overall, our ability to track inter-annual and species-specific differences in penguin diets using SIA was enhanced by integrating SCA data to isotopic mixing modes in three ways: 1) selecting appropriate prey sources, 2) weighting combinations of isotopically similar prey in two-source mixing models and 3) refining predicted contributions of isotopically similar prey in multi-source models.     

Root of the Eukaryota tree as inferred from combined maximum likelihood analyses of multiple molecular sequence data

Extensive studies aiming to establish the structure and root of the Eukaryota tree by phylogenetic analyses of molecular sequences have thus far not resulted in a generally accepted tree. To re-examine the eukaryotic phylogeny using alternative genes, and to obtain a more robust inference for the root of the tree as well as the relationship among major eukaryotic groups, we sequenced the genes encoding isoleucyl-tRNA and valyl-tRNA synthetases, cytosolic-type heat shock protein 90, and the largest subunit of RNA polymerase II from several protists. Combined maximum likelihood analyses of 22 protein-coding genes including the above four genes clearly demonstrated that Diplomonadida and Parabasala shared a common ancestor in the rooted tree of Eukaryota, but only when the fast-evolving sites were excluded from the original data sets. The combined analyses, together with recent findings on the distribution of a fused dihydrofolate reductase-thymidylate synthetase gene, narrowed the possible position of the root of the Eukaryota tree on the branch leading to Opisthokonta or to the common ancestor of Diplomonadida/Parabasala. However, the analyses did not agree with the position of the root located on the common ancestor of Opisthokonta and Amoebozoa, which was argued by Stechmann and Cavalier-Smith [Curr. Biol. 13:R665-666, 2003] based on the presence or absence of a three-gene fusion of the pyrimidine biosynthetic pathway: carbamoyl-phosphate synthetase II, dihydroorotase, and aspartate carbamoyltransferase. The presence of the three-gene fusion recently found in the Cyanidioschyzon merolae (Rhodophyta) genome sequence data supported our analyses against the Stechmann and Cavalier-Smith-rooting in 2003.     

A single phylogenetic analysis of Bacillus thuringiensis strains and bacilli species inferred from 16S rRNA gene restriction fragment length polymorphism is congruent with two independent phylogenetic analyses

AIMS: To assess the congruence between two earlier independent phylogenetic studies on Bacillus thuringiensis strains and on Bacillus-related species and the single, all-encompassing, phylogenetic tree presented here inferred from the combination of the two earlier datasets. METHODS AND RESULTS: A dendrogram was constructed using a combination of the data from our previous studies on 16S rRNA gene fingerprinting of 86 B. thuringiensis strains and of 77 species of Bacillus and related taxa. It revealed that all B. thuringiensis strains were clustered together in four distinct groups at a DNA similarity rate of 93%, except two serovars, bolivia and finitimus. Four bacilli species, Paenibacillus alvei, P. azotofixans, B. lentus and B. licheniformis, share a DNA similarity rate of 92% with Bt Group IV. CONCLUSIONS: Most, but not all, B. thuringiensis strains could be grouped together based on the DNA similarity rate. They were also very close to some other bacilli species. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined phylogenetic study presented here, inferred from 16S rRNA gene restriction fragment length polymorphism, is congruent with two earlier independent phylogenetic studies, one on B. thuringiensis and the other on bacilli species.