Using classification tree analysis to reveal causes of mortality in an insect population

• 1 Invasive species pose significant threats to native and managed ecosystems. However, it may not always be possible to perform rigorous, long‐term studies on invaders to determine the factors that influence their population dynamics, particularly when time and resources are limited. We applied a novel approach to determine factors associated with mortality in larvae of the sawfly Profenusa thomsoni Konow, a leafminer of birch, and a relatively recent invader of urban and rural birch forests in Alaska. Classification tree analysis was applied to reveal relationships between qualitative and quantitative predictor variables and categorical response variables in a large data set of larval mortality observations.
• 2 We determined the state (living or dead) of sawfly larvae in samples of individual leaves. Each leaf was scored for variables reflecting the intensity of intra‐specific competition and leaf quality for leafminers, year of collection and degree‐days accumulated were recorded for each sample. We explored the association of these variables with larval state using classification tree analysis.
• 3 Leafminer mortality was best explained by a combination of competition and resource exhaustion and our analysis revealed a possible advantage to group feeding in young larvae that may explain previously observed patterns of resource overexploitation in this species. Dead larvae were disproportionately found in smaller leaves, which highlights the potential effect of competition on mortality and suggests that smaller‐leaved species of birch will better able to resist leafminer damage.
• 4 We show that classification tree analysis may be useful in situations where urgency and/or limited resources prohibit traditional life‐table studies.

     

Induced resistance in mountain birch: defence against leaf-chewing insect guild and herbivore competition

Summary The effect of leaf damage simulating the feeding of early season insect herbivore species, e.g. Epirrita autumnata, to mountain birch, Betula pubescens ssp. tortuosa, on the performance of insect larvae was studied with eleven leaf-chewing sawfly species. I found variation in the results that was due to short- and long-term inducible responses and to the phenology of herbivore species. In general, early and mid-season species were more strongly affected by induced reactions than late-season species. This finding is in accordance with earlier results but I could show that the persistance of induced reactions rather than the influence of timing of damage is responsible for the result. The growth of the larvae of mid-season sawfly species was affected by both short- and long-term induced reactions. This result shows that early season species may escape short-term induced reactions of mountain birch in current year but may not avoid long-term effects. It is supposed that seasonal deterioration of leaf quality either masks the effects of induced defences or late-season species are better adapted to low-quality leaves. Some species show variation in their response to induced defence in different years. This may be due to yearly differences in induced reactions as well as to species-specific responses. Induced defence reactions may play a role in competitive interactions between herbivore species in leaf-chewing guild of mountain birch.     

Diversity of birch sawfly responses to seasonally atypical diets

Most insect herbivores are specialised on a particular plant taxon. To have a better understanding of host shift functions and consequences for insect herbivores, it is essential to gather more information on the effects of variation in host quality on specialists across species and environments. We examined the effects of seasonally atypical food on mortality, developmental time, and final body mass of six sawfly species (Hymenoptera: Symphyta) feeding on the foliage of mountain birch (Betula pubescens ssp. czerepanovii), whose pooled larval feeding periods form a gradient and cover the growing season. Insect phenology was manipulated so that the larvae of early-season species would feed on atypically mature leaves and mid- or late-season species would feed on atypically young leaves of their major host plant. Mortality increased dramatically for all species when the larval feeding schedule was advanced or delayed. This indicates a high degree of specialisation not only on a particular host but also to its phenological phases. The main cause of mortality on novel food was a rejection of the diet by the young larvae and their subsequent starvation. An interesting observation was that late-season species showed this response on nutritious young foliage. The effects of seasonally atypical diets on larval development and growth were species-specific and milder than the effects on mortality. Interestingly, for those individuals that accepted it, atypical food seemed to be most beneficial for species appearing at both ends of the seasonal gradient, which might be related to a wider exposure to variable food quality in natural conditions compared with other species. The diversity of responses to atypical food among closely related herbivore species with overlapping feeding periods on the same host plant is the most crucial finding of this study.     

Diet and growth of a leaf-shredding caddisfly in southern Appalachian streams of contrasting disturbance history

Diet and growth of leaf-shredding caddisfly larvae, Pycnopsyche spp.,were examined in streams draining a reference catchment and a 16-year-oldclear-cut (disturbed) catchment at Coweeta Hydrologic Laboratory insouthwestern North Carolina, USA. The objective was to explain why shredderproduction is higher in the disturbed streams despite the larvae having lessfood (i.e., leaves) available. We predicted larvae would grow faster onfast-decaying leaf material representative of the disturbed streams. Larvaeconsumed mostly leaf detritus in three streams draining each catchment overthree seasons (fall, winter, and spring), which showed larvae did notconsume higher quality foods (e.g., algae and animal material) in disturbedstreams. When fed 2-month-old conditioned black birch (Betula lenta L.) (afast-decaying leaf species) and white oak (Quercus alba L.) (a slow-decayingleaf species) leaves in the laboratory, larvae grew significantly faster onthe birch leaves. However, when larvae were fed the same leaf types after3-months conditioning, larvae grew significantly faster on oak leaves. Afield growth experiment conducted for 42 d using mixed-species leaf dietsrepresentative of each catchment and initially conditioned for 2 monthsfound that Pycnopsyche grew significantly better on the diet representativeof the reference catchment. The reference diet contained more oak leaveswhich apparently became a more acceptable food as the experiment proceeded.High shredder production in the disturbed streams could not be explained byhigh Pycnopsyche growth rates on fast-decaying leaves. Instead, larvae grewbetter on leaves that were apparently conditioned optimally regardless ofconditioning rate.     

SEASONALLY VARYING DIET QUALITY AND THE QUANTITATIVE GENETICS OF DEVELOPMENT TIME AND BODY SIZE IN BIRCH FEEDING INSECTS

Abstract Genetic variance‐covariance structures (G), describing genetic constraints on microevolutionary changes of populations, have a central role in the current theories of life‐history evolution. However, the evolution of Gs in natural environments has been poorly documented. Resource quality and quantity for many animals and plants vary seasonally, which may shape genetic architectures of their life histories. In the mountain birch‐insect herbivore community, leaf quality of birch for insect herbivores declines profoundly during both leaf growth and senescence, but remains stable during midsummer. Using six sawfly species specialized on the mountain birch foliage, we tested the ways in which the seasonal variation in foliage quality of birch is related to the genetic architectures of larval development time and body size. In the species consuming mature birch leaves of stable quality, that is, without diet‐imposed time constraints for development time, long development led to high body mass. This was revealed by the strongly positive phenotypic and genetic correlations between the traits. In the species consuming growing or senescing leaves, on the other hand, the rapidly deteriorating leaf quality prevented the larvae from gaining high body mass after long development. In these species, the phenotypic and genetic correlations between development time and final mass were negative or zero. In the early‐summer species with strong selection for rapid development, genetic variation in development time was low. These results show that the intuitively obvious positive genetic relationship between development time and final body mass is a probable outcome only when the constraints for long development are relaxed. Our study provides the first example of a modification in guild‐wide patterns in the genetic architectures brought about by seasonal variation in resource quality.     

An entomopathogenic fungus and nematode prove ineffective for biocontrol of an invasive leaf miner Profenusa thomsoni in Alaska

A non-native invasive sawfly, the amber-marked birch leaf miner Profenusa thomsoni (Konow), was first detected in south-central Alaska in 1996 and is now widely distributed throughout urban and wild birch trees in Alaska. Impacts have been considered primarily aesthetic because leaf miners cause leaves of birch trees (Betula spp.) to senesce prematurely, but the leaf miners likely also reduce birch vigour and thereby increase susceptibility to diseases and other insects. We tested the ability of commercially available biological control agents to control P. thomsoni. The entomopathogenic fungus Beauveria bassiana (Bals.-Criv.) Vuillemin GHA strain and the entomopathogenic nematode Steinernema carpocapsae (Weiser) were applied in aqueous suspension to the soil/litter surface beneath infested birch trees in Alaska at one site in 2007 and 2008 and two sites in 2010. There was no evidence the fungus or nematode controlled P. thomsoni. Instead, there was evidence the fungus increased the density of this pest insect at two sites, likely by reducing its predators. As tested, B. bassiana and S. carpocapsae do not appear effective as biological controls of P. thomsoni.     

Plant phenology-mediated indirect effects: The gall midge opens the phenological window wider for a leaf beetle

We examined whether larvae of the gall midge Rabdophaga rigidae (Diptera: Cecidomyiidae) can modify the seasonal dynamics of the density of a leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae), by modifying the leaf flushing phenology of its host willow species, Salix serissaefolia and Salix eriocarpa (Salicaceae). To test this, we conducted field observations and a laboratory experiment. The field observations demonstrated that the leaf flushing phenology of the willows and the seasonal dynamics of the beetle density differed between shoots with stem galls and shoots without them. On galled shoots of both willow species, secondary shoot growth and secondary leaf production were promoted; consequently, leaf production showed a bimodal pattern and leaf production periods were 1 to 2 months longer than on non‐galled shoots. The adult beetle density on galled shoots was thus enhanced late in the season, and was found to change seasonally, synchronizing with the production of new leaves on the host willow species. From the results of our laboratory experiment, we attributed this synchrony between adult beetle density and willow leaf flush to beetles’ preference to eat new leaves rather than old. Indeed, beetles consumed five times more of the young leaves when they were fed both young and old leaves. These results indicate that stem galls indirectly enhance the adult beetle density by enhancing food quality and quantity late in the beetle‐feeding season. We therefore conclude that midge galls widen the phenological window for leaf beetles by extending the willows’ leaf flush periods.     

Oviposition preference and larval performance in the exotic birch-leafmining sawfly Profenusa thomsoni

This study used experiments at several spatial scales to determine whether (1) intraspecific competition occurs among larvae of the leafmining sawfly Profenusa thomsoni (Konow) (Hymenoptera: Tenthredinidae) on birch (Betula spp.), (2) oviposition site preferences of P. thomsoni maximize offspring performance, and (3) early‐season damage by external folivores or the leafminer Fenusa pumila Leach (Hymenoptera: Tenthredinidae) affects oviposition preferences or larval performance of P. thomsoni. Larval P. thomsoni competed at natural densities; survival and weight of larvae were reduced under crowded conditions. Despite this, females of P. thomsoni tended to lay eggs on leaves already bearing eggs from other females and discriminated only weakly among leaves of different sizes on a branch. Both damage by F. pumila and artificial damage to leaves early in the season decreased survival of P. thomsoni larvae on the same branch, and ovipositing P. thomsoni females avoided damaged leaves but not other leaves on the same branch. In general, oviposition choices by P. thomsoni reduced larval survival. Possible reasons for the lack of a strong preference–performance relationship in P. thomsoni are discussed.     

Host specificity and host recognition in a chemically-defended herbivore, the tenthredinid sawfly Rhadinoceraea nodicornis

The sawfly Rhadinoceraea nodicornis Konow (Hymenoptera: Tenthredinidae) is a member of a closely related group of species, the tribe Phymatocerini, which feed on the Liliales and Ranunculales. It is known to sequester steroid alkaloids from its host plants, species in the genus Veratrum (Liliales: Melanthiaceae), and to use them as a defence against predators. There are known chemical relationships between the hosts of R. nodicornis and hosts of related sawfly species. We tested whether the R. nodicornis larvae would accept hosts of closely- and more distantly-related sawflies, but found that they accepted only plant species in the genus Veratrum. This specificity was apparently innate, as it was independent of early larval experience. A feeding bioassay showed that the steroid alkaloids from Veratrum nigrum were phagostimulatory for R. nodicornis larvae, suggesting that they may be involved in host recognition. We discuss the possibility that the evolution of recognition of specific compounds may represent the mechanism of host radiation within the Phymatocerini.     

Effects of host-plant shift on immune and other key life-history traits of an eruptive Geometrid, Epirrita autumnata (Borkhausen)

Abstract.  1. Population density of Epirrita autumnata (Lepidoptera: Geometridae) reaches outbreak densities regularly in northernmost Scandinavia. During these outbreak years, the most abundant host species, the mountain birch ( Betula pubescens ssp. czerepanovii ), is regularly exhausted, although larvae may rescue themselves from starvation by using alternative host species.
2. In this paper, the effects of the shift of host species on the immune defence and other life-history traits of E. autumnata were investigated, and possible consequences for population dynamics were briefly discussed. Moth larvae were reared on the leaves of the main host, mountain birch, until larvae reached their third instar. After this, larvae were allocated randomly to five treatments: larvae were either allowed to finish larval stage on the mountain birch or were shifted onto four alternative host species that are typical species for the area.
3. As expected, the host species had a major effect on fitness traits: body weight, development, and survival rate of the moths. The pupal weight was lower and development rates slower on the three alternative host species, Salix myrsinifolia Salisb., Vaccinium uliginosum L., and Betula nana L., than on the main host, mountain birch.
4. The immunity was, however, the same or better on the alternative hosts than on the main host. The immunity and pupal weights were negatively related, suggesting a trade-off between body size and immunocompetence.
5. The decreased body size and fecundity of E. autumnata during outbreak years may be partly due to the shift to alternative host species whereas the host-plant species probably does not affect markedly the rate of parasitism.     

Latitudinal patterns in the phenological responses of leaf colouring and leaf fall to climate change in Japan

Aim  To estimate the potential effect of global climate change on the phenological responses of plants it is necessary to estimate spatial variations at larger scales. However, previous studies have not estimated latitudinal patterns in the phenological response directly. We hypothesized that the phenological response of plants varies with latitude, and estimated the phenological response to long-term climate change using autumn phenological events that have been delayed by recent climate change.
Location  Japan.
Methods  We used a 53-year data set to document the latitudinal patterns in the climate responses of the timing of autumn leaf colouring and fall for two tree species over a wide range of latitudes in Japan (31 to 44° N). We calculated single regression slopes for leaf phenological timing and air temperature across Japan and tested their latitudinal patterns using regression models. The effects of latitude, time and their interaction on the responses of the phenological timings were also estimated using generalized linear mixed models.
Results  Our results showed that single regression slopes of leaf phenological timing and air temperature in autumn were positive at most stations. Higher temperatures can delay the timing of leaf phenology. Negative relationships were found between the phenological response of leaves to temperature and latitude. Single regression slopes of the phenological responses at lower latitudes were larger than those at higher latitudes.
Main conclusions  We found negative relationships between leaf phenological responsiveness and latitude. These findings will be important for predicting phenological timing with global climate change.     

Attraction of willow warblers to sawfly‐damaged mountain birches: novel function of inducible plant defences?

Plants wounded by invertebrate herbivores emit volatile compounds which invertebrate predators and parasitoids can utilize in locating herbivore prey or hosts. We studied the possibility that an analogical phenomenon might operate between plants and avian insectivores. We show that foliar damage by sawfly larvae on the mountain birch led a passerine bird (willow warbler) to prefer intact branches from trees with introduced larvae over intact branches from control trees. Besides olfaction, the UV vision of birds offers a possible mechanism, as some herbivore‐inducible leaf compounds, e.g. surface flavonoids, have UV spectral maxima well within the range of birds’ UV vision.     

Communication of leaf suitability by gregarious eastern tent caterpillars (Malacosoma americanum)

ABSTRACT. 1. Previous work has shown that leaf age affects recruitment trail marking by eastern tent caterpillars ( Malacosoma americanum Fabr.). Young leaves of host plants elicit trail marking to a greater degree than mature leaves.
2. Experiments were conducted to establish the relationship between the differential behavioural responses of larvae to young and mature leaves and the suitability of foliage for larval growth and survival. Foliage of black cherry ( Prunus serotina Ehrh.), a typical rosaceous host plant, was used for this comparison.
3. Larvae preferred young leaves to mature leaves in choice tests, and marked more to young leaves than to mature leaves in no-choice tests.
4. Mature leaves supported adequate growth through two larval instars of rearing, but thereafter were unsuitable for growth. Larvae fed mature leaves had lower pupal weight, poorer survival, and grew less efficiently than larvae fed young leaves.
5. The results support the hypothesis that the trail communication system of eastern tent caterpillars is an adaptation to efficiently locate leaves which are favourable for larval growth and survival.     

Experimental simulations of climate change induced mismatch in oak and larval development rates impact indicators of fitness in a declining woodland moth

Consequences of climate change-driven shifts in the relative timing of spring activities of interacting species are insufficiently understood, especially for insects. We use a controlled experiment which simulates a trophic mismatch scenario in which lepidopteran larvae predominately feed on older leaves due to foliage developing faster than larvae growth rates. As a case study our experiment uses Orthosia cerasi, which is a widespread but declining woodland moth whose UK declines appear to be driven by warming temperatures. In the control experiment larvae are fed young oak Quercus robur leaves (bud burst stages six and seven), whilst in the treatment newly emerged larvae are fed young leaves but then gradually transition to feed on older leaves (post bud burst stage seven). We assess impacts on duration of the larval stage, pupal size and overwintering duration and survival. Larvae in the phenological mismatch treatment had a longer larval period, and smaller and lighter pupae. Larval diet did not carry over to influence emergence dates as earlier pupation of control larvae was balanced by an equivalent increase in the duration of the pupal stage. Increased time spent as larvae could increase predation rates from avian predators, whilst slowing the seasonal decline in food availability for those bird species. Reduced pupal size and weight are indicators of lower fecundity in emerging adults. Notably, we find that adults emerging from the mismatch treatment exhibited greater rates of abnormal vestigial wing development, which is likely to further reduce fitness. Trophic mismatches in which caterpillars have reduced availability of young leaves may thus contribute to the population declines observed in many woodland moth species due to increased mortality at larval stages, and adverse effects of early life conditions that reduce the reproductive success of emerging adults.     

Preference for protected feeding sites by larvae of the willow-feeding leaf beetle Galerucella lineola

1. Free-living insects are often thought of as more vulnerable to environmental hazards than concealed insects, such as galling or mining insects. The possibility that larvae of the free-living leaf beetle Galerucella lineola seek out existing plant structures and thereby become partly concealed was explored.
2. Neonate larvae of G. lineola frequently feed in rolled-in margins of young leaves of their host plant, Salix viminalis . In addition to nutritional benefits from feeding on young leaves, larvae may also gain protection against adverse weather conditions and general predators by feeding in the leaf rolls. Field and laboratory experiments were conducted to test these hypotheses.
3. Artificial shelters were constructed and cohorts of neonate larvae were placed on experimental plants. In all experiments, larvae preferred to feed in shelters, even when shelters were constructed on mature leaves.
4. In one of the experiments, fewer larvae disappeared when shelters were provided. In a predator exclusion experiment, however, no differences in predator-inflicted mortality on G. lineola were found between shelter-containing shoots and control shoots.
5. A laboratory experiment showed increased protection from desiccation when shelters were present; growth rate was higher for larvae feeding on plants with shelters.
6. Thus, free-living insects may not always be as exposed to environmental hazards as is often assumed. In particular, young larvae may take advantage of preformed structures on their host plant and feed in a concealed microhabitat. Because mortality, in general, is high during early instars, shelter-seeking behaviour may increase survival significantly. The existence of preformed shelters may therefore be a plant characteristic that should be considered when exploring the environmental risks associated with the free-living habit.     

Decline in gypsy moth (Lymantria dispar) performance in an elevated CO2 atmosphere depends upon host plant species

Plant species differ broadly in their responses to an elevated CO₂ atmosphere, particularly in the extent of nitrogen dilution of leaf tissue. Insect herbivores are often limited by the availability of nutrients, such as nitrogen, in their host plant tissue and may therefore respond differentially on different plant species grown in CO₂-enriched environments. We reared gyspy moth larvae (Lymantria dispar) in situ on seedlings of yellow birch (Betula allegheniensis) and gray birch (B. populifolia) grown in an ambient (350 ppm) or elevated (700 ppm) CO₂ atmosphere to test whether larval responses in the elevated CO₂ atmosphere were species-dependent. We report that female gypsy moths (Lymantria dispar) reared on gray birch (Betula populifolia) achieved similar pupal masses on plants grown at an ambient or an elevated CO₂ concentration. However, on yellow birch (B. allegheniensis), female pupal mass was 38% smaller on plants in the elevated-CO₂ atmosphere. Larval mortality was significantly higher on yellow birch than gray birch, but did not differ between the CO₂ treatments. Relative growth rate declined more in the elevated CO₂ atmosphere for larvae on yellow birch than for those on gray birch. In preference tests, larvae preferred ambient over elevated CO₂-grown leaves of yellow birch, but showed no preference between gray birch leaves from the two CO₂ atmospheres. This differential response of gypsy moths to their host species corresponded to a greater decline in leaf nutritional quality in the elevated CO₂ atmosphere in yellow birch than in gray birch. Leaf nitrogen content of yellow birch dropped from 2.68% to 1.99% while that of gray birch leaves only declined from 3.23% to 2.63%. Meanwhile, leaf condensed tannin concentration increased from 8.92% to 11.45% in yellow birch leaves while gray birch leaves only increased from 10.72% to 12.34%. Thus the declines in larval performance in a future atmosphere may be substantial and host-species-specific.     

Phenology of nutritional differences between new and mature leaves and its effect on caterpillar growth

Abstract. 1. We determined the phenology of the shrub Spiraea latifolia Ait. Bork. (Rosaceae), which has indeterminate shoot growth, and the effects of phenological changes in leaf quality on growth rate of the early-spring feeding buckmoth caterpillars, Hemileuca lucina Hy. Edw. (Saturniidae).
2. Leaves, regardless of whether they were newly expanded or several weeks old, were tougher later in the growth season (mid-June) than similarly aged leaves collected earlier; correspondingly, water and nitrogen content for leaves of all ages declined through the larval period. By July, newly expanded leaves had no more nitrogen than mature leaves.
3. Relative growth rate of third instar larvae fed new leaves or a mixture of new and mature leaves in early June was higher than that of those fed mature leaves, and efficiency of conversion of digested food to biomass was higher for larvae fed new leaves than for those fed mature leaves or a mixture.
4. In another experiment, larvae were reared on new leaves through the fourth instar and then fed a diet of new, mature or a combination of new and mature leaves, a regimen that was similar to the phenologies of both plants and caterpillars in the field. There was no difference in time to pupation or pupal weights among these treatments.     

Sources of variation in rapidly inducible responses to leaf damage in the mountain birch-insect herbivore system

Summary Studies on rapidly inducible resistance in trees against insect herbivores show substantial variation in the strength of responses. Here we report the results of a study which examined causes of this variation. We bioassayed the quality of leaves of two developmental phases (young vs. mature) of the mountain birch Betula pubescens ssp. tortuosa by measuring the growth of two instars of Epirrita autumnata larvae. We used only short shoot leaves from trees of a natural stand, uniform in size and age. Damage was caused by larvae and artificial tearing of leaf lamina, varying the scale and time. We separated seasonal changes in plants from instar-dependent effects of the animals by testing experimental larvae in two subsequent growth trials. We found that only larval-made damage induced responses in leaves that made the leaves significantly poorer quality for the test larvae. Artificial damage induced only weak responses, and artificial canopy-wide damage even caused slight improvement of leaf quality. Cumulative leaf damage did not strengthen birch responses. Leaves that were in the expansion phase responded to damage while fully-expanded, mature leaves showed no response. The pattern of responses indicated that there might be physiological constraints: small-scale damage induced resistance against the larvae but largescale damage did not. Prevalent weather conditions might have modified these responses. Larvae of two instars and sexes, of low- and high-density populations responded to leaf damage similarly. However, prior experience of larvae with the host plant may have affected subsequent larval performance. Variation in rapidly inducible responses in birches was caused by plant characters rather than by test animals.     

Willow leaf traits affecting host use by the leaf-gall-forming sawfly

Gall-forming insects usually have very restricted host ranges, but plant traits affecting patterns of host use have rarely been examined. The sawfly Phyllocolpa sp. (Hymenoptera: Tenthredinidae) forms leaf-roll galls on three of seven Salix (Salicaceae) species that occur together on riverbanks in central Japan. We have attempted to explain this host-use pattern by invoking three plant traits: synchrony of leaf flush with the oviposition period of the sawfly, intrinsic leaf quality as a potential larval food, and leaf morphology. Two Salix species frequently used by the sawfly, Salix eriocarpa and Salix pierotii, had similar leaf traits suitable for larval survival. The third species, Salix serissaefolia, was used relatively less often and the sawfly frequently stopped laying eggs on the plant during oviposition, suggesting ovipositional selection. S. serissaefolia had the smallest leaves, and survival of sawfly larvae was lower on S. serissaefolia than on S. eriocarpa and S. pierotii, because of gall destruction, by other herbivorous insects, and leaf-size restrictions. Among the four unused species, Salix chaenomeloides had a late leaf-flush phenology, Salix gracilistyla had inferior leaf quality, and Salix gilgiana had linear leaves; these seemed to be critical factors for non-use. Salix subfragilis was also unused, but the reason for this could not be explained by the three leaf traits studied.     

HPLC analysis of leaf surface flavonoids for the preliminary classification of birch species

Flavonoid aglycones found on the surfaces of birch (Betula spp.) leaves may constitute up to 10% of the dry weight of the leaf. A facile extraction and HPLC procedure has been developed that can be used for the preliminary classification of birch species according to the patterns of their leaf surface flavonoids. The procedure involves no complex sample preparation steps, and is able to provide HPLC chromatograms from fresh leaves in less than 30 min. If necessary, leaves do not even need to be removed from the tree. Since the genus Betula is taxonomically complex and separation of different birch species can be problematic, the developed method was applied to 15 Betula species and four sub-species of Betula pendula Seven of the studied species were classified as B. pubescens and eight as B. pendula-type birches. The remaining four species did not belong to either of these two classes on account of their unique pattern of external flavonoids. The difference between the leaf surface flavonoid composition of B. pubescens and B. pendula type birch species was unambiguously clear, and the developed method could reliably distinguish between the two species. Whilst leaf surface flavonoids can be valuable chemotaxonomic markers, they classify birch species differently from morphological markers. Birch species with diploid chromosome sets did not contain any of the flavanones that were present in the leaves of other species. The close relationship between the occurrence of some flavonoid aglycones and the ploidy level of Betula species suggests that these chemotaxonomic markers may be useful both in taxonomic and phylogenetic analyses.