Intraspecific Level Research Articles

Food patch selection by the largest European herbivore

AbstractThe scaling relationship between digestive system and body mass shapes foraging strategies in herbivores. The Jarman–Bell principle and the forage maturation hypothesis (FMH), two of the most important predictions formulated in herbivore foraging ecology, have been used to explain this relationship.We aimed to test these predictions for the largest European herbivore—the European bison-which is highly sexually dimorphic and recognised as a refugee species in non-optimal forest habitats. We conducted our study in the Białowieża Primeval Forest, where the European bison has been recognized as a refugee species. We estimated vegetation quality and biomass along the foraging paths of male and female European bison, and the vegetation biomass at random points in different habitats and months. We found no support for the Jarmann-Bell principle at the intraspecific level in European bison, as males and females foraged on patches of similar crude protein and fibre content. However, females selected for higher biomass. The quality of vegetation on the bison foraging patches showed a significant spatio-temporal variation. It increased from May to July and then gradually decreased. It also increased with the fertility and wetness of the habitats being the highest in wet forests. Mean biomass on European bison foraging patches (131.5 g/m2), was significantly lower than on random plots (210.5 g/m2), which supported the FMH. Our study suggests that in rich forest habitats, the abundant food resources available in summer may not constrain the metabolic rate/gut capacity ratio of either sex, potentially leading to dietary segregation. In contrast to open habitats, where vegetation maturation is less temporally variable, European bison in rich forest habitats may optimise patch selection and energy intake by tracking a habitat- and species-specific changes in the plant phenology. Understanding the relationship between European bison forage selection and forage nutritional constraints in forest habitats may be critical for conservation programmes, as European bison are better adapted to more open habitats and are recognised as refugee species in suboptimal forest habitats.

A straightforward protocol to sample morphological traits of dragonflies and damselflies in the field.

Scarcity of morphological data limits the potential of functional ecology approaches, which rely on traits to elucidate ecological processes. Dragonflies and damselflies (Odonata) are a frequently used ecological model for which, however, only limited morphological data is available. Here, it is presented a field sampling protocol to collect ecologically relevant yet largely unavailable morphological traits of Odonata. The protocol enables the straightforward collection of traits from living individuals directly in the field. Those traits include body mass, wing area and wing loading as well as thorax width, hindwing length and body length. Furthermore, the protocol allows for posterior wing morphometric analyses. The protocol proved to be robust and universally applicable based on testing on roughly half (76) of all European odonate species. The use of this protocol can increase our understanding of odonatan morphology at interspecific and intraspecific levels and assist in developing mechanistic understanding of their ecology.

Effects of overwintering on the transcriptome and fitness traits in a damselfly with variable voltinism across two latitudes

Winter diapause consists of cessation of development that allows individuals to survive unfavourable conditions. Winter diapause may bear various costs and questions have been raised about the evolutionary mechanisms maintaining facultative diapause. Here, we explored to what extent a facultative winter diapause affects life-history traits and the transcriptome in the damselfly Ischnura elegans, and whether these effects were latitude-specific. We collected adult females at central and high latitudes and raised their larvae in growth chambers. Larvae were split into a non-diapausing and post-winter (diapausing) cohort, were phenotyped and collected for a gene expression analysis. At the phenotypic level, we found no difference in survival between the two cohorts, and the post-winter cohort was larger and heavier than the non-winter cohort. These effects were mostly independent of the latitude of origin. At the transcriptomic level, wintering affected gene expression with a small fraction of genes significantly overlapping across latitudes, especially those related to morphogenesis. In conclusion, we found clear effects of diapause on the phenotype but little evidence for latitudinal-specific effects of diapause. Our results showed a shared transcriptomic basis underpinning diapause demonstrated, here, at the intraspecific level and supported the idea of evolutionary convergence of the response to diapause across organisms.

Applying geometric morphometrics in megalopa larval stages: relevance for species distribution and biological invasion studies

Abstract Larval morphology in brachyuran crabs is an essential trait defining survival in the planktonic environment. The present study describes the morphology (carapace and chelipeds) of megalopa larvae in three different species (Percnon gibbesi, Cronius ruber, and Achelous hastatus) using geometric morphometric analysis (gma). The study aim was to compare the observed body patterns at interspecific and intraspecific levels. The results showed a marked interspecific distinction of C. ruber larvae based on the rostral region of carapace and cheliped length. We relate these differences to advantages in life performance, as in other animal models, suggesting that larval morphology can be another factor contributing to the invasiveness of C. ruber. At intraspecific level, we found a high morphological similarity between two distant geographical locations, indicating strong genetic connectivity in the populations of P. gibbesi from the Canary Islands. Our findings advance the importance of larval morphology for species performance during the plankton phase that eventually can determine invasiveness in brachyuran crabs. We suggest future studies focusing on the inter-population comparisons of megalopa morphology at larger spatiotemporal scales.

Evidence of Relationship between X = 9 and X = 7 Groups of <I>Pennisetum</I> Species at Intraspecific Level

No Abstract.

Early development of Acacia longifolia is more severely impacted by water and nutrient stress in invasive than native seedlings

Acacia longifolia (Andrews) Willd. is a legume native to southeast mainland Australia and Tasmania and has two described subspecies: A. l. subsp. longifolia and A. l. subsp. sophorae. The species has been introduced around the world and is considered invasive in several Mediterranean-type climate regions, including in South America, South Africa, and southern Europe. Previous studies comparing native and invasive populations of A. longifolia have focused on its reproductive ecology and population genetics, and little information exists on the species’ early life development and how abiotic factors influence it. Here, we performed a glasshouse experiment to compare the phenotypic responses of native and invasive (in Portugal) A. longifolia seedlings to different levels of water and nutrient availability. We found that seedlings of both subspecies responded similarly to different water and nutrient availability conditions in terms of biomass accumulation, root length, the number of phyllodes produced, phyllode water content, and root-to-shoot ratio. However, compared to native seedlings, invasive seedlings had limited capacity for stress responses. We found that invasive seedlings had lower drought tolerance than native seedlings, and thus the speed of invasion by A. longifolia into drier parts of Portugal may be hindered. Our results also hint of a possible role of seed “imprinting” in this species’ early growth responses, resulting in different resource allocation strategies such as favouring early growth and development over drought resistance in the invaded range. Further studies are required to better understand the species’ abiotic stress responses at the intraspecific level and their relation to its invasiveness.

Low Intraspecific Aggression Level, Cuticular Hydrocarbons, and Polydomy in the Bullet Ant.

Ants use chemical cues known as cuticular hydrocarbons (CHCs) for both intraspecific and interspecific recognition. These compounds serve ants in distinguishing between nestmates and non-nestmates, enabling them to coexist in polydomous colonies characterized by socially connected yet spatially separated nests. Hence, the aim of this study was to investigate the intraspecific aggression level between nestmates and non-nestmates of the bullet ant Paraponera clavata (Fabricius, 1775), analyze and compare their CHCs, and evaluate the occurrence of polydomy in this species. We conducted aggression tests between foragers, both in laboratory and field settings. To identify the chemical profiles, we utilized gas chromatography coupled with mass spectrometry (GC-MS). We marked the foragers found at nest entrances and subsequently recaptured these marked ants to validate workers exchange among nests. Across all nests, a low intraspecific aggression level was observed within the same area. However, a significant difference in aggression correlated to distance between nests. Analysis of the cuticular chemical profile of P. clavata unveiled colony-specific CHCs, both qualitatively and quantitatively. Notably, we observed instances of ants from certain nests entering or exiting different nests. This behavior, in conjunction with the observed low intraspecific aggression despite differences in CHCs suggests polydomy for this species. Polydomy can offer several benefits, including risk spreading, efficient exploitation of resources, potential for colony size increasing and reduced costs associated with foraging and competition.

Towards acoustic monitoring of bees: wingbeat sounds are related to species and individual traits.

Global pollinator decline urgently requires effective methods to assess their trends, distribution and behaviour. Passive acoustics is a non-invasive and cost-efficient monitoring tool increasingly employed for monitoring animal communities. However, insect sounds remain highly unexplored, hindering the application of this technique for pollinators. To overcome this shortfall and support future developments, we recorded and characterized wingbeat sounds of a variety of Iberian domestic and wild bees and tested their relationship with taxonomic, morphological, behavioural and environmental traits at inter- and intra-specific levels. Using directional microphones and machine learning, we shed light on the acoustic signature of bee wingbeat sounds and their potential to be used for species identification and monitoring. Our results revealed that frequency of wingbeat sounds is negatively related with body size and environmental temperature (between-species analysis), while it is positively related with experimentally induced stress conditions (within-individual analysis). We also found a characteristic acoustic signature in the European honeybee that supported automated classification of this bee from a pool of wild bees, paving the way for passive acoustic monitoring of pollinators. Overall, these findings confirm that insect sounds during flight activity can provide insights on individual and species traits, and hence suggest novel and promising applications for this endangered animal group. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Decoding the molecular phylogenetics of ornamental catfishes (siluriformes) of North East India using DNA barcoding approach.

Catfishes (order Siluriformes) are among the most diverse and widely distributed fish groups in the world. They are not only used for human consumption but are also a major part of the ornamental fish trade. Being a Biodiversity Hotspot, the North Eastern Region of India is home to a diverse population of ornamental fishes. Catfishes contain a humongous number of species; in this study, the authors have tried to elucidate the phylogenetic relationship of some important ornamental catfishes found in North East India using DNA barcodes. In this study, we have tried to explore the phylogenetic history of 13 species (41 specimens) of ornamental catfishes spanning 12 genera and 9 families of Siluriformes using DNA barcoding. Pairwise genetic distances using Kimura 2-Parameter (K2P) were calculated at intra-specific and inter-specific levels. A Neighbor-Joining tree was constructed to understand the phylogenetic relationship among the nine different catfish families. All the specimens under this study clustered with their respective species under the same family and formed three sub-clades. However, Olyra longicaudata, belonging to the Bagridae family, did not cluster with other species from the same family. In this study, the authors have suggested a revision of the classification of O. longicaudata back to its original family, Olyridae. In this study, the maximum intraspecific genetic distance of 0.03 and the minimum interspecific genetic distance of 0.14 were observed among the species. Therefore, it is evident that there is a barcoding gap among the species, which helped in the correct identification of the species. Thus, DNA barcoding helped complement the phenetic approach and also revealed a different phylogenetic relationship among the catfishes belonging to the Bagridae family.

Acoustic difference in advertisement calls among two sympatric Boulenophrys species: A confirmatory case to acoustic niche hypothesis and morphological constraint hypothesis.

In anurans, acoustic communication is the most important form of communication at the interspecific and intraspecific levels. Acoustic diagnostic features may be a potential alternative to morphometric and molecular diagnostics. Here, we assessed the variations in advertisement calls between two sympatric species, Boulenophrys leishanensis and Boulenophrys spinata, that share their breeding season and breeding sites. In addition, we investigated any potential relationships between call parameters and body size. We found that the advertisement calls of both species are simple calls. The two species exhibited significant differences in all call parameters. Both B. leishanensis and B. spinata showed a significant negative correlation with their body size on dominant frequency. These differences in call parameters may play an important role in interspecific recognition. Additionally, because intraspecific acoustic variation reflects body size, calls may be relevant for sexual selection. Our study supports the acoustic niche hypothesis and the morphological constraint hypothesis and calls are a valid tool for distinguishing between the two species of Boulenophrys in the field.

Intraspecific and Intrageneric Genomic Variation across Three Sedum Species (Crassulaceae): A Plastomic Perspective.

Sedum is the largest succulent genus in Crassulaceae. Because of predominant maternal inheritance, little recombination, and slow evolution, plastomes can serve as powerful super barcodes for inter- or intra-species phylogenetic analyses. While previous research has focused on plastomes between Sedum species, intra-species studies are scarce. Here, we sequenced plastomes from three Sedum species (Sedum alfredii, Sedum plumbizincicola, and Sedum japonicum) to understand their evolutionary relationships and plastome structural evolution. Our analyses revealed minimal size and GC content variation across species. However, gene distribution at IR boundaries, repeat structures, and codon usage patterns showed diversity at both inter-specific and intra-specific levels. Notably, an rps19 gene expansion and a bias toward A/T-ending codons were observed. Codon aversion motifs also varied, potentially serving as markers for future studies. Phylogenetic analyses confirmed the non-monophyly of Sedum and divided the Acre clade into two groups. Individuals from the same species clustered together, with strong support for the relationships between S. alfredii, S. tricarpum, and S. plumbizincicola. Additionally, S. japonicum clearly affiliates with the Acre clade. This study provides valuable insights into both intra-specific and intra-generic plastome variation in Sedum, as well as overall plastome evolution within the genus.

Between‐ versus within‐species variation in plant–soil feedback relates to different functional traits, but exudate variability is involved at both scales

Abstract Plant–soil feedback—feedback from plant‐induced changes in soil properties to plant fitness—is increasingly shown to drive the maintenance of local plant diversity at both interspecific and intraspecific levels. A robust understanding of the relationships between plant–soil feedback and functional plant traits, which would improve our ability to generalize plant–soil feedback results beyond specific study systems, is, however, still lacking. This is especially true at the intraspecific plant level. We assessed the relationship between plant–soil feedback and several functional traits in 13 co‐occurring grassland species, including 20 genotypes of the dominant grass, Festuca rubra. The traits encompassed various aspects of growth, root properties and root exudate variability. Combining these traits into principal gradients of functional trait variation, we also tested the potential for the conservation and collaboration gradients to explain variation in PSF. Between‐species plant–soil feedback variation was explained by differences in biomass production and exudate composition, as well as contrasting strategies along the collaboration gradient. Within‐species plant–soil feedback variation—that is between Festuca rubra genotypes—was associated with exudate variability, especially contrasting amounts of exuded phenols. Several traits had a significant effect on plant–soil feedback only via their interaction with exudate composition. Overall, PSF was associated with different traits at between‐species versus within‐species levels. Root exudate variability was, however, involved at both diversity levels. Our results put forth the role of root exudation patterns as an important driver of variation in plant–soil feedback. Better integration between research on plant–soil feedback and on root exudation would therefore improve our understanding of the processes—both ecological and evolutionary—supporting the maintenance of plant diversity within grassland communities. Read the free Plain Language Summary for this article on the Journal blog.

Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb.

Abstract. Maruzy A, Susandarini R. 2024. Short Communication: Analysis of simple sequence repeats variations for the assessment of genotypic diversity in Centella asiatica (L.) Urb. Biodiversitas 25: 734-740. Centella asiatica (L.) Urb. is a perennial plant widely known as herbal ingredient in traditional and modern medicine, as well as in the cosmetics industry. This herbaceous species grows in various types of habitat, and is known for its morphological variations among populations, indicating the existence of genotypic variation. In this study, the assessment of genotypic diversity using simple sequence repeats (SSR) was carried out on C. asiatica accessions from 32 populations collected from eight mountains in Central Java. Representative individuals from four populations grow at different altitudes were sampled in each mountain. Amplification of genomic DNA using the SSR primer mCaCIR002 produced a single amplicon of 180 bp. The results of SSR analysis showed that there is high genotypic diversity in C. asiatica which is characterized by variations in the number of microsatellite repeats of (CT)10 to (CT)19. Variations in the number of repeats are mainly caused by insertions and deletions in DNA sequences at the positions 102-145. Haplotype analysis also showed high genetic diversity within and between populations of C. asiatica, indicated by the haplotype diversity of 1.00 in all populations. The haplotype network analysis indicated that there was no gene flow between the four populations at each mountain where C. asiatica populations grow. The results of this study provide evidence for the role of SSR as a suitable molecular marker for studying genotypic diversity at the intraspecific level, and confirming the morphological variability in this species.

Heterothermy use in winter is associated with reduced litter size during following breeding season

Heterothermy is considered to be the most effective energy-saving strategy improving survival under natural conditions. Interspecific studies suggest that this strategy is also associated with reduced reproductive output. Yet little is known about the reproductive consequences of heterothermy use at the intraspecific level and thus its repercussions for microevolutionary processes. Moreover, as yet no study has aimed to test if litter size and juvenile mass are affected by torpor use in wild captured animals under undemanding laboratory conditions. Here we tested the hypothesis that intraspecific variation in heterothermy use is associated with different reproductive successes, being the result of the evolution of distinct life histories. We predicted that heterothermy use in winter negatively correlates with litter size and juvenile body mass during the subsequent breeding season. To test this prediction, we used yellow-necked mice from a population in which individuals consistently differ in their use of heterothermy in winter. We measured body size (head width) and body mass, basal metabolic rate, as well as metabolism and body temperature during fasting-induced torpor in wild caught mice in winter. Phenotyped mice were bred in the subsequent summer selectively – males and females with similar heterothermy characteristics were paired, the most to the least heterothermic. Dam body size, but not basal metabolism, was positively correlated with litter size (but not juvenile mass). However, when accounting for this relationship, litter size was negatively while juvenile mass was positively correlated with the average heterothermy use of a given couple. Our study indicates that heterothermy use correlates with specific life-history strategies arising from a fundamental evolutionary trade-off between survival and reproduction.

Karyological Study of Acanthocephalus lucii (Echinorhynchida): The Occurrence of B Chromosomes in Populations from PCB-Polluted Waters

In this study, we performed a cytogenetic analysis of Acanthocephalus lucii specimens from three sites with different levels of environmental pollution. Standard and fluorochrome staining (CMA3/DAPI), fluorescence in situ hybridization (FISH) with 18S rDNA and histone H3 probes, and silver impregnation were performed. Chromosome complements of 2n = 7/8 (male/female), n = 1m + 2sm + 1a (X), and CMA3-positive bands in all chromosomes were found in all three populations. FISH revealed one 18S rDNA locus on the X chromosome and one locus of H3 histone genes on the first chromosome pair. At the intraspecific level, the populations differed in the presence of supernumerary B chromosomes, which were found in all specimens from Zemplínska Šírava and in 89.4% of specimens from the Laborec River, but not at the reference site. The first two sites are considered to be water bodies with high toxin contamination. Based on this fact, we assume an increased frequency of chromosome breaks leading to the formation of DNA fragments that have the potential to form B chromosomes. The present results add to the very limited data on the organization of multigene families in the genome of Acanthocephala and suggest a possible causal link between water pollution and the occurrence of B chromosomes in fish parasites.

Taxonomic significance of megaspores in some species of Selaginella from Arunachal Pradesh in India.

The present study was carried out to record the color, size, and ornamentation of megaspores in 18 species of the family Selaginellaceae from Arunachal Pradesh using scanning electron microscope (SEM) and light microscope (LM). Electron microscopic study of the spore features revealed the type of ornamentation and microsculptural detail. SEM study on megaspore of Selaginella pentagona (S. pentagona), Selaginella tenuifolia, Selaginella semicordata, and Selaginella chrysorrhizos is presented for the first time. Variation in the megaspore ornamentation is noted at the interspecific level and intraspecific level in some cases. Examination of the megaspores under study found all the megaspores as trilete, with a size ranging from 116 to 560 μm in diameter. Taxonomic key is prepared to differentiate the species. Short descriptions of megaspores are provided and supported by photo plates. New features are recorded for seven species. The study contributes to the separation of species within the genus Selaginella based on the spore feature and brings forward the use of spore as a diagnostic tool in the taxonomy of the genus. These works contribute to the systematic of the family Selaginellaceae and provide useful information in the field of palynology. RESEARCH HIGHLIGHTS: Study on megaspore features of Selaginella species collected from Arunachal Pradesh, using SEM and LM. Taxonomic key is provided for each species based on megaspores features. New megaspore features are recorded for the seven species. The study brings forward the use of spore as a diagnostic tool in the taxonomy of the genus. These works provide useful information in the field of systematic and palynology.

Problem-solving skills are predicted by technical innovations in the wild and brain size in passerines

Behavioural innovations can provide key advantages for animals in the wild, especially when ecological conditions change rapidly and unexpectedly. Innovation rates can be compared across taxa by compiling field reports of novel behaviours. Large-scale analyses have shown that innovativeness reduces extinction risk, increases colonization success and is associated with increased brain size and pallial neuron numbers. However, appropriate laboratory measurements of innovativeness, necessary to conduct targeted experimental studies, have not been clearly established, despite decades of speculation on the most suitable assay. Here we implemented a battery of cognitive tasks on 203 birds of 15 passerine species and tested for relationships at the interspecific and intraspecific levels with ecological metrics of innovation and brain size. We found that species better at solving extractive foraging problems had higher technical innovation rates in the wild and larger brains. By contrast, performance on other cognitive tasks often subsumed under the term behavioural flexibility, namely, associative and reversal learning, as well as self-control, were not related to problem-solving, innovation in the wild or brain size. Our study yields robust support for problem-solving as an accurate experimental proxy of innovation and suggests that novel motor solutions are more important than self-control or learning of modified cues in generating technical innovations in the wild.

Broad-scale perspective on body size-trophic position patterns of freshwater fishes at the intraspecific and community levels from individual-based data in China

As predators are generally larger than their prey, positive body size and trophic position relationships are typically assumed, and these relationships have helped in estimating and predicting the effects of global environmental change on the trophic dynamics of aquatic ecosystems. However, current efforts to confirm the generality of body size-trophic position relationships have focused mainly on interspecific patterns using species-aggregated and averaged data, and little effort has been devoted to assessing how trophic position scales with body size at the intraspecific and community levels as well as exploring its ecological drivers, particularly for fishes in freshwater ecosystems. To fill this gap, we present a broad-scale study of body size-trophic position relationships in freshwater ecosystems at both the intraspecific and community levels using an individual-based body size and isotopic signature dataset that includes 2564 samples of 65 fish species in China. Our results indicate that body size-trophic position patterns at both the intraspecific and community levels can be positive, negative or insignificant. Non-significant patterns predominated at both levels, with slightly more than 30 % showing positive relationships, which highlights that the positive body size-trophic position pattern is not universally prevalent and that body size should be used with caution as a proxy for the trophic position of fishes at both the intraspecific and community levels in freshwater ecosystems. No factor was related to body size-trophic position patterns at the community level, while habitat type, elevation, temperature and fishing pressure were identified as the key determinants of body size-trophic position patterns at the intraspecific level, implying that fishing and climate warming can shift the food-web size structure of freshwater ecosystems by affecting population trophic dynamics.

Polyphenism predicts actuarial senescence and lifespan in tiger salamanders.

Actuarial senescence (called 'senescence' hereafter) often shows broad variation at the intraspecific level. Phenotypic plasticity likely plays a central role in among-individual heterogeneity in senescence rate (i.e. the rate of increase in mortality with age), although our knowledge on this subject is still very fragmentary. Polyphenism-the unique sub-type of phenotypic plasticity where several discrete phenotypes are produced by the same genotype-may provide excellent study systems to investigate if and how plasticity affects the rate of senescence in nature. In this study, we investigated whether facultative paedomorphosis influences the rate of senescence in a salamander, Ambystoma mavortium nebulosum. Facultative paedomorphosis, a unique form of polyphenism found in dozens of urodele species worldwide, leads to the production of two discrete, environmentally induced phenotypes: metamorphic and paedomorphic individuals. We leveraged an extensive set of capture-recapture data (8948 individuals, 24 years of monitoring) that were analysed using multistate capture-recapture models and Bayesian age-dependent survival models. Multistate models revealed that paedomorphosis was the most common developmental pathway used by salamanders in our study system. Bayesian age-dependent survival models then showed that paedomorphs have accelerated senescence in both sexes and shorter adult lifespan (in females only) compared to metamorphs. In paedomorphs, senescence rate and adult lifespan also varied among ponds and individuals. Females with good body condition and high lifetime reproductive success had slower senescence and longer lifespan. Late-breeding females also lived longer but showed a senescence rate similar to that of early-breeding females. Moreover, males with good condition had longer lifespan than males with poor body condition, although they had similar senescence rates. In addition, late-breeding males lived longer but, unexpectedly, had higher senescence than early-breeding males. Overall, our work provides one of the few empirical cases suggesting that environmentally cued polyphenism could affect the senescence of a vertebrate in nature, thus providing insights on the ecological and evolutionary consequences of developmental plasticity on ageing.

Benchmarking classification abilities of novel optical photothermal IR spectroscopy at the single-cell level with bulk FTIR measurements.

Fourier-transform infrared (FTIR) spectroscopy is a simple, fast and inexpensive method with a history of use for bacterial analysis. However, due to the limitations placed on spatial resolution inherent to infrared wavelengths, analysis has generally been performed on bulk samples, leading to biological variance among individual cells to be buried in averaged spectra. This also increases the bacterial load necessary for analysis, which can be problematic in clinical settings where limiting incubation time is valuable. Optical photothermal-induced resonance (O-PTIR) spectroscopy is a novel method aiming to bypass this limitation using a secondary lower wavelength laser, allowing for infrared measurements of a single bacterium. Here, using Staphylococcus capitis, Staphylococcus epidermidis and Micrococcus luteus strains as a model and FTIR as a benchmark, we examined O-PTIR's ability to discriminate single-cell samples at the intergenetic, interspecific and intraspecific levels. When combined with chemometric analysis, we showed that O-PTIR is capable of discriminating different between genera, species and strains within species to a degree comparable with FTIR. Furthermore, small variations in the amide bands associated with differences in the protein structure can still be seen in spite of smaller sample sizes. This demonstrates the potential of O-PTIR for single-cell bacterial analysis and classification.