Summer temperature dependency of larch budmoth outbreaks revealed by Alpine tree-ring isotope chronologies

Larch budmoth (LBM, Zeiraphera diniana Gn.) outbreaks cause discernable physical alteration of cell growth in tree rings of host subalpine larch (Larix decidua Mill.) in the European Alps. However, it is not clear if these outbreaks also impact isotopic signatures in tree-ring cellulose, thereby masking climatic signals. We compared LBM outbreak events in stable carbon and oxygen isotope chronologies of larch and their corresponding tree-ring widths from two high-elevation sites (1800–2200 m a.s.l.) in the Swiss Alps for the period AD 1900–2004 against isotope data obtained from non-host spruce (Picea abies). At each site, two age classes of tree individuals (150–250 and 450–550 years old) were sampled. Inclusion of the latter age class enabled one chronology to be extended back to AD 1650, and a comparison with long-term monthly resolved temperature data. Within the constraints of this local study, we found that: (1) isotopic ratios in tree rings of larch provide a strong and consistent climatic signal of temperature; (2) at all sites the isotope signatures were not disturbed by LBM outbreaks, as shown, for example, by exceptionally high significant correlations between non-host spruce and host larch chronologies; (3) below-average July to August temperatures and LBM defoliation events have been coupled for more than three centuries. Dampening of Alps-wide LBM cyclicity since the 1980s and the coincidence of recently absent cool summers in the European Alps reinforce the assumption of a strong coherence between summer temperatures and LBM defoliation events. Our results demonstrate that stable isotopes in tree-ring cellulose of larch are an excellent climate proxy enabling the analysis of climate-driven changes of LBM cycles in the long term.     

Host choice promotes reproductive isolation between host races of the larch budmoth Zeiraphera diniana

The chances for sympatric speciation are improved if ecological divergence leads to assortative mating as a by-product. This effect is known in parasites that find mates using host cues, but studies of larch- and pine-feeding races of the larch budmoth (Zeiraphera diniana, Lepidoptera: Tortricidae) suggest it may also occur when mate attraction is via sex pheromones that are independent of habitat. We have previously shown that females releasing pheromones on or near their own host attract more males of their own race than if placed on the alternative host. This host effect would enhance assortative mating provided adults preferentially alight on their native hosts. Here we investigate alighting preferences in natural mixed forest using a novel likelihood analysis of genotypic clusters based on three semidiagnostic allozyme loci. Both larch and pine females show a realized alighting preference for their own host of 86%. The equivalent preferences of males were 79% for the larch race and 85% for the pine race. These preferences are also detectable in small-scale laboratory experiments, where alighting preferences of larch and pine races towards their own hosts were, respectively, 67 and 66% in females and 69 and 63% in males. Pure larch race moths reared in the laboratory had alighting choice similar to moths from natural populations, while hybrids were intermediate, showing that alighting preferences were heritable and approximately additive. The field estimates of alighting preference, coupled with earlier work on mate choice, yield an estimated rate of natural hybridization between sympatric host races of 2.2-3.8% per generation. Divergent alighting choice enhances pheromone-mediated assortative mating today, and is likely to have been an important cause of assortative mating during initial divergence in host use. Because resources are normally 'coarse-grained' in space and time, assortative mating due to ecological divergence may be a more important catalyst of sympatric speciation than generally realized.     

Genomic evidence for divergence with gene flow in host races of the larch budmoth

Ecological divergence in the face of gene flow has recently become implicated as a potentially important cause of speciation and adaptive radiation. Here, we develop a genomic approach to test for divergent selection in sympatric host races of the larch budmoth Zeiraphera diniana (Lepidoptera: Tortricidae). We analysed hundreds of amplified fragment length polymorphism markers in 92 individuals in sympatric and allopatric populations, and in two backcross broods used to map the markers to individual chromosomes. The results directly confirm the existence of natural hybridization and demonstrate strong heterogeneity between chromosomes in terms of molecular divergence between host races (the average level of divergence was FST = 0.216). However, genomic heterogeneity was not found when we analysed divergence between geographically separated populations of the same host race. We conclude that the variance of the level of sympatric divergence among chromosomes is the footprint of divergent selection acting on a few linkage groups, combined with appreciable gene flow that homogenizes between-race variation at the remaining linkage groups. These results, coupled with other recent multilocus analyses of sister species pairs, demonstrate that selection-driven sympatric phase of genetic divergence in the presence of gene flow is a likely feature of speciation.     

The potential for using Larix decidua ring widths in reconstructions of larch budmoth (Zeiraphera diniana) outbreak history: dendrochronological estimates compared with insect surveys

In the Alps, larch (Larix decidua Mill.) forests show periodic discolouration due to larch budmoth (LBM) outbreaks (Zeiraphera diniana Guénée, Lepidoptera: Tortricidae). Tree defoliation causes severe reductions in radial growth, visible in tree rings. This paper aims at reconstructing LBM outbreak history, and critically examining the potential for using dendrochronological data by comparing tree-ring estimates with insect surveys. The occurrence of LBM outbreaks was investigated using 249 cores from larch growing near the timberline in three regions of the French Alps (Briançonnais, Maurienne, and Tarentaise). Years with an abrupt decrease in radial growth (-40%) were considered as negative pointer years reflecting the potential impact of LBM. The comparison with three non-host conifers (Norway spruce, stone and mountain pines) permitted us to distinguish growth reductions in larch due to climatic effects from those due to defoliation by LBM. The dendrochronological data were matched with information reporting conspicuous discolouration in old forestry reports or recovered from systematic field observations. Twenty-two outbreaks are discernible within the period 1800-1983. A peak reduction in larch growth occurred at intervals of 8.86ǃ.01 years in the following years: 1801, 1811, 1820, 1830, 1838, 1846, 1857, 1867, 1875, 1884, 1892, 1901, 1909, 1918, 1926, 1936, 1945, 1953, 1963, 1972, 1980, and 1987. Our objective method based on ring measurements and comparison with non-host trees was compared with qualitative techniques based on the visual detection of conspicuous latewood anomalies. Larch in the Briançonnais (which experiences a continental climate) are infested first, whereas the Tarentaise region exhibits a much weaker impact of LBM. Complete tree recovery was observable 3 years after outbreak peaks.     

Estimating niche width using stable isotopes in the face of habitat variability: a modelling case study in the marine environment

Distributions of stable isotopes have been used to infer an organism's trophic niche width, the 'isotopic niche', and examine resource partitioning. Spatial variation in the isotopic composition of prey may however confound the interpretation of isotopic signatures especially when foragers exploit resources across numerous locations. In this study the isotopic compositions from marine assemblages are modelled to determine the role of variation in the signature of prey items and the effect of dietary breadth and foraging strategies on predator signatures. Outputs from the models reveal that isotopic niche widths can be greater for populations of dietary specialists rather than for generalists, which contravenes what is generally accepted in the literature. When a range of different mixing models are applied to determine if the conversion from δ to p-space can be used to improve model accuracy, predator signature variation is increased rather than model precision. Furthermore the mixing models applied failed to correctly identify dietary specialists and/or to accurately estimate diet contributions that may identify resource partitioning. The results presented illustrate the need to collect sufficiently large sample sizes, in excess of what is collected under most current studies, across the complete distribution of a species and its prey, before attempts to use stable isotopes to make inferences about niche width can be made.     

Studies on estrogen biosynthesis using radioactive and stable isotopes

The conversion of androgens into estrogen involves three distinct generic reactions which are catalyzed by a single P450 enzyme (aromatase or P450(aromatase)). The first step in the process is the conversion of 19-methyl into a hydroxymethyl group which requires NADPH + O2, thus representing the well-known hydroxylation process. The next stage, converting the -CH2OH into -CHO, also requires NADPH + O2 and may be rationalized either through a second hydroxylation reaction producing a gem-diol, CH(OH)2 (which dehydrates to the aldehyde), or via another route. The final stage in the process again uses NADPH + O2, culminating in the release of C-19 as formate. Our extensive studies using precursors containing 2H, 3H, and 18O have shown that the carbonyl oxygen of the 19-aldehyde group is the one that was introduced in the first step as the hydroxyl group. The aldehydic oxygen along with another, from O2, used in the third step of the process, is incorporated into the released formate. It was found that at each stage of the process, oxygen atoms were introduced or transferred as "whole numbers." In light of these data, mechanisms in which H2O is used to promote the C-10-C-19 bond cleavage or those in which the conversion of the 19-oxoandrostenedione into estrogen is considered to occur via the sequence -CHO----(-)CH(OH)2----estrogen are eliminated. In addition, our mechanistic analysis makes it unlikely that 1 beta-, 2 beta-, or 10 beta-hydroxysteroids serve as intermediates in estrogen biosynthesis. We consider a free radical mechanism for the hydroxylation process.     

Separating the climatic signal from tree-ring width and maximum latewood density records

We propose a technique for separating the climatic signal which is contained in two tree-ring parameters widely used in dendroclimatology. The method is based on the removal of the relationship between tree-ring width (TRW) and maximum latewood density (MXD) observed for narrow tree rings from high latitudes. The new technique is tested on data from three larch stands located along the northern timberline in Eurasia. Correlations were calculated between the temperatures of pentads (five consecutive days), TRW chronologies and MXD chronologies calculated according to the standard and proposed methods. The analysis confirms the great importance of summer temperature for tree radial growth and tree-ring formation. TRW is positively correlated with the temperature of four to eight pentads (depending on the region) at the beginning of the growth season, but MXD as obtained by the standard technique is correlated with temperature over a much longer period. For maximum density series from which the relationship between MXD and TRW has been removed (MXD′), there is a clear correlation with temperatures in the second part of the growing season. These results are consistent with the known dynamics of tree-ring growth in high latitudes and mechanisms of tree-ring formation.     

Tracing the influence of larch-bud-moth insect outbreaks and weather conditions on larch tree-ring growth in Engadine (Switzerland).

Tree-ring analysis of insect-defoliated trees has so far been used for detecting past insect outbreaks only. We hypothesize that the impact of the larch-bud-moth (LBM) Zeiraphera dinian outbreaks on the growth of larch Larix decidua in the Engadine (Switzerland) is closely coupled to the spatial development of the outbreak and the ecological characteristics of the respective sites and weather conditions. We tested this hypothesis by reviewing data sets available in the literature and by analysing original data. We monitored LBM population densities and the needle phenology, growth and defoliation of larch over 28 years, i.e. over four outbreak cycles. In addition, information on defoliation patterns covering six earlier outbreaks over 50 years was matched with tree-ring information. Tree-ring chronologies of 18 larch stands were analysed with regard to abrupt growth changes and latewood events. Defoliation induces an immediate reduction in latewood, followed by a reduction in needle length and a significant decrease in radial growth in the subsequent year. We have called this tree-ring pattern the "larch-bud-moth syndrome". A careful analysis of the various parameters of the LBM syndrome for two specific population cycles enabled us to define different interaction patterns between weather conditions and tree growth. These can then be included in climate change models to help disentangle the impact of insect defoliation from that of adverse climatic conditions.     

Effect of ring width, cambial age, and climatic variables on the within-ring wood density profile of Norway spruce Picea abies (L.) Karst.

Studying the effects of dendrometric and climatic variables on within-ring density variations needs flexible and interpretable models. We described the within-ring density profile using a piecewise linear regression and studied its dependence on (1) dendrometric variables such as cambial age (CA) and ring width (RW), and (2) climatic variables. Based on X-ray analysis of 5,191 Norway spruce rings, a six-parameter three-segmented model was fitted on each within-ring density profile. Each model parameter was related to dendrometric and climatic variables using multiple linear regressions. Then, these models were assembled in two models relating the within-ring density profile to (1) RW and CA (model M1), and (2) climatic variables (model M2). M1 showed an R ² of 83.4 % and a residual standard error of 68.5 kg m^?3. Larger rings were associated with a decrease of latewood proportion and mean ring density. Rings with high CA were characterised by high maximum ring density. M2 showed an R ² of 60.9 % and a residual standard error of 94.9 kg m^?3. Warm summers increased the maximum ring density. Years with favourable water status decreased mean ring density. The piecewise linear models allowed the classification of within-ring density profiles in three types. Considering CA and RW led to the most explicative model since RW described many processes such as silviculture or climate. Earlywood density was impacted by water status while latewood density was conditioned by both temperatures and water status. Our approach may be used for the identification of within-ring density fluctuations or to assess the effects of silviculture or global change on the within-ring density profile.     

Estimating the timing of diet shifts using stable isotopes

Stable isotope analysis has become an important tool in studies of trophic food webs and animal feeding patterns. When animals undergo rapid dietary shifts due to migration, metamorphosis, or other reasons, the isotopic composition of their tissues begins changing to reflect that of their diet. This can occur both as a result of growth and metabolic turnover of existing tissue. Tissues vary in their rate of isotopic change, with high turnover tissues such as liver changing rapidly, while relatively low turnover tissues such as bone change more slowly. A model is outlined that uses the varying isotopic changes in multiple tissues as a chemical clock to estimate the time elapsed since a diet shift, and the magnitude of the isotopic shift in the tissues at the new equilibrium. This model was tested using published results from controlled feeding experiments on a bird and a mammal. For the model to be effective, the tissues utilized must be sufficiently different in their turnover rates. The model did a reasonable job of estimating elapsed time and equilibrial isotopic changes, except when the time since the diet shift was less than a small fraction of the half-life of the slowest turnover tissue or greater than 5–10 half-lives of the slowest turnover tissue. Sensitivity analyses independently corroborated that model estimates became unstable at extremely short and long sample times due to the effect of random measurement error. Subject to some limitations, the model may be useful for studying the movement and behavior of animals changing isotopic environments, such as anadromous fish, migratory birds, animals undergoing metamorphosis, or animals changing diets because of shifts in food abundance or competitive interactions.     

The Middle Miocene climatic transition in the Southern Ocean: Evidence of paleoclimatic and hydrographic changes at Kerguelen plateau from planktonic foraminifers and stable isotopes

Middle Miocene (14.8–11.9 Ma) deep-sea sediments from ODP Hole 747A (Kerguelen Plateau, southern Indian Ocean) contain abundant, well-preserved and diverse planktonic foraminiferal assemblages. A detailed study of the climatic and hydrographic changes that occurred in this region during the Middle Miocene Climatic Transition led to the identification of an intense cooling phase (the Middle Miocene Shift). Abundance fluctuations of planktonic foraminiferal species with different paleoclimatic affinities, and oxygen and carbon stable isotopes have been integrated in a multi-proxy approach. Reconstruction of changes in foraminiferal faunal composition and diversity through time were the basis for identification of three foraminiferal biofacies. The most prominent faunal change took place at 13.8 Ma, when a fauna with warm-water affinity (marked by high abundance of Globorotalia miozea group and Globoturborotalita woodi plexus) was replaced by an oligotypic, opportunistic fauna with typical polar characters and dominated by neogloboquadrinids. This faunal change is interpreted as the result of foraminiferal migration from adjacent bioprovinces, caused by modifications in climate and hydrography. A positive 2.0‰ shift in δ¹⁸O (interpreted as the Mi3 event) and a related positive 1.0‰ shift in δ¹³C (corresponding to the CM6 event) accompanied this faunal turnover. These are interpreted to reflect substantial reorganization of Southern Ocean waters, the northward migration of the Polar Front and a strong increase in primary productivity. The second faunal change took place at 12.9 Ma and was characterized by the gradual decrease in abundance of the neogloboquadrinids and the recovery of Globorotalia praescitula/scitula group and Globigerinita glutinata. A positive 1.5‰ shift in δ¹⁸O (interpreted as the Mi4 event) and a concurrent gradual negative shift in δ¹³C accompanied this faunal change, witnessing further modifications of the climate/ocean system. Variations in sea surface temperature, considered as the main factor causing changes of surface hydrography at the Kerguelen Plateau, seem to have been driven by obliquity and long-term eccentricity, thus suggesting a key role played by the astronomical forcing on the evolution of Southern Ocean dynamics during the Middle Miocene. Also an evident 1.2 Myr modulation of the δ¹³C record suggests a main control of the long-term obliquity cycles on the carbon cycle dynamics. Particularly, the Mi3/CM6 events exactly fit with a node of the 1.2 Myr modulation cycles. This confirms the key role played by orbital parameters on high-latitude temperatures and Antarctic ice volume, and indirectly on global carbon burial and/or productivity. This climatic transition was marked also by changes in surface hydrography. From 14.8 to 13.8 Ma an intermediate-strength thermocline controlled by seasonality developed just below the photic zone. Weaker seasonality characterized the interval from 13.8 to 12.9 Ma, when the thermocline became shallower and sharper and favored intermediate-water foraminifers. From 12.9 Ma, seasonality increased again and an intermediate-strength thermocline re-developed.     

Measuring the trophic ecology of ants using stable isotopes

Ants are prominent components of most terrestrial arthropod food webs, yet due to their highly variable diet, the role ants play in arthropod communities can be difficult to resolve. Stable isotope analysis is a promising method for determining the dietary history of an organism, and has the potential to advance our understanding of the food web ecology of social insects. However, some unique characteristics of eusocial organisms can complicate the application of this technique to the study of their trophic ecology. Using stable isotopes of N and C, we investigated levels of intraspecific variation both within and among colonies. We also examined the effect of a common preservation technique on δ¹⁵N and δ¹³C values. We discuss the implications of our results on experimental design and sampling methods for studies using stable isotopes to investigate the trophic ecology of social insects. Received 4 February 2005; revised 23 June 2005; accepted 4 July 2005.     

Correlation of the width of annual tree rings with climatic changes and solar activity

Fluctuations in the width of annual tree rings in the time interval from the eighth century till the present time were analyzed on the basis of dendroclimatic scales. Periods of extremely high and low growth of trees were distinguished, and their correlations with heliogeophysical factors were studied. The major climatic parameters determining the growth of trees are warmth and moisture; a small gain is observed in cold and droughty periods. No correlations between the width of tree rings and the minima and maxima of solar activity cycles were observed with the method used.     

The role of larch budmoth (Zeiraphera diniana Gn.) on forest succession in a larch (Larix decidua Mill.) and Swiss stone pine (Pinus cembra L.) stand in the Susa Valley (Piedmont, Italy)

In the Alps, larch (Larix decidua Mill.) is severely affected by larch budmoth (Zeiraphera diniana Guénée) (LBM) attacks. The impact of these outbreaks on the Swiss stone pine (Pinus cembra L.) and on the dynamic processes acting in subalpine forest stands are still not well known. Dendroecological methods were used in this study to reconstruct past LBM outbreaks in Susa Valley, Piedmont, Italy. The analysis was carried out on 62 cores from larch and 101 cores from stone pine. The length and severity of each outbreak was quantified for both species and for each tree by means of the programme OUTBREAK. The frequency of the outbreaks was determined using singular spectral analysis and superposed epoch analysis was used to test the significance of the associations between outbreaks and tree-ring growth. In order to verify if trees belonging to different age classes are differently affected by LBM, the reconstructed outbreaks are then classified taking into account the cambial age of the tree at the moment of the outbreak. From 1760 to 1999, 19 outbreaks were recorded in the larch chronologies, while only three outbreaks in the stone pine chronologies. The larch growth is strongly influenced by LBM and the identified outbreaks are equally distributed in all the three age classes. On the stone pine the sporadic occurrence of the identified events made difficult any interpretation of the eventual effect of LBM. Our results lead us to argue that LBM has not played an important role both in determining the stone pine growth rate and in influencing the present observed succession from the stage dominated by larch, to a stage dominated by stone pine or by a mixed stone pine-larch forest.     

Effects of nutritional restriction on nitrogen and carbon stable isotopes in growing seabirds

When using stable isotopes as dietary tracers it is essential to consider effects of nutritional state on isotopic fractionation. While starvation is known to induce enrichment of ¹⁵N in body tissues, effects of moderate food restriction on isotope signatures have rarely been tested. We conducted two experiments to investigate effects of a 50–55% reduction in food intake on δ¹⁵N and δ¹³C values in blood cells and whole blood of tufted puffin chicks, a species that exhibits a variety of adaptive responses to nutritional deficits. We found that blood from puffin chicks fed ad libitum became enriched in ¹⁵N and ¹³C compared to food-restricted chicks. Our results show that ¹⁵N enrichment is not always associated with food deprivation and argue effects of growth on diet–tissue fractionation of nitrogen stable isotopes (Δ¹⁵N) need to be considered in stable isotope studies. The decrease in δ¹³C of whole blood and blood cells in restricted birds is likely due to incorporation of carbon from ¹³C-depleted lipids into proteins. Effects of nutritional restriction on δ¹⁵N and δ¹³C values were relatively small in both experiments (δ¹⁵N: 0.77 and 0.41‰, δ¹³C: 0.20 and 0.25‰) compared to effects of ecological processes, indicating physiological effects do not preclude the use of carbon and nitrogen stable isotopes in studies of seabird ecology. Nevertheless, our results demonstrate that physiological processes affect nitrogen and carbon stable isotopes in growing birds and we caution isotope ecologists to consider these effects to avoid drawing spurious conclusions.     

An approach to the use of stable isotopes for DNA sequencing

The sequencing of DNA by current procedures involves the use of radioisotopic or fluorescent labels. We propose that stable isotopes can be used as such labels and that the large number of stable isotopes available would allow multiplexing so that many DNA segments could be sequenced simultaneously. We have developed methods to use 57Fe2O3 to synthesize ferrocene and to attach the ferrocene to the 5' end of oligonucleotides. The 57Fe-labeled M13 universal primer functioned normally in a Sanger sequencing procedure. When a 57Fe-labeled oligonucleotide had migrated on a polyacrylamide gel it was readily located on the dried gel by scanning with resonance ionization spectroscopy (RIS) coupled with mass spectrometry. Using a 57Fe-labeled primer in a PCR reaction a 2000-bp DNA was produced that was detected by RIS on nylon membrane after agarose electrophoresis. The rapid analysis features of RIS coupled with the multispectral multiplexing possibilities of stable isotopes should significantly increase the rate of determination of DNA sequences.     

Avian claw morphometry and growth determine the temporal pattern of archived stable isotopes

Detailed knowledge about claw formation and growth rate is a prerequisite for the interpretation of avian claw stable isotopes, as is commonly done with feather stable isotopes to e.g. infer habitat use, dietary specialisations, and spatial occurrence. In this study, we provide basic information about claw formation and empirical evidence about the time scale of archiving isotopic information to develop a reliable assessment of archived isotopic pattern in claws of passerines. Avian claws grow conically from the tip of the bone of the phalanx. The length of the tip of an avian claw, suitable for stable isotope analysis, is about 42 ± 6.8% (SD) of total linear claw length and can also be estimated from the body mass of a given species. Claw growth rate in adult songbirds typically ranged between 0.03 and 0.05 mm d^?1, but differed between front and back toes, and varied seasonally. From the claw growth rate, the archiving period of a given claw length can be estimated. In long‐distance migrant species, δ¹³C of claws matched δ¹³C of feathers grown during the same period (wintering or breeding period). In Palaearctic‐African migrants sampled in the breeding season, δ¹³C of the distal half of the claw tip reflected the African wintering site, while the proximal half reflected a blend of African and European δ¹³C signatures, as expected. Hence there is empirical evidence that entire claw tips mirror the isotopic environments over longer periods (up to months), and over weeks when parts can be analysed. However any part of a claw contains a blend of material formed at different times due to the claw's conical (i.e. longitudinal and lateral) growth. Avian claws provide a complementary isotope archive for investigations, but its applicability may vary according to the ecology of the study species.