Macroparasites at peripheral sites of infection are major and dynamic modifiers of systemic antimicrobial pattern recognition responses

Immune defences and the maintenance of immunological homeostasis in the face of pathogenic and commensal microbial exposures are channelled by innate antimicrobial pattern recognition receptors (PRRs) such as toll‐like receptors (TLRs). Whilst PRR‐mediated response programmes are the result of long‐term host‐pathogen or host–commensal co‐evolutionary dynamics involving microbes, an additional possibility is that macroparasitic co‐infections may be a significant modifier of such interactions. We demonstrate experimentally that macroparasites (the model gastrointestinal nematode, Heligmosomoides) at peripheral sites of infection cause substantial alteration of the expression and function of TLRs at a systemic level (in cultured splenocytes), predominantly up‐regulating TLR2, TLR4 and TLR9‐mediated cytokine responses at times of high standing worm burdens. We consistently observed such effects in BALB/c and C57BL/6 mice under single‐pulse and trickle exposures to Heligmosomoides larvae and in SWR and CBA mice under single‐pulse exposures. A complementary long‐term survey of TLR2‐mediated tumour necrosis factor‐alpha responses in wild wood mice (Apodemus sylvaticus) was consistent with substantial effects of macroparasites under some environmental conditions. A general pattern, though, was for the associations of macroparasites with TLR function to be temporally dynamic and context‐dependent: varying with different conditions of infection exposure in the field and laboratory and with host genetic strain in the laboratory. These results are compelling evidence that macroparasites are a major and dynamic modifier of systemic innate antimicrobial responsiveness in naturally occurring mammals and thus likely to be an important influence on the interaction between microbial exposures and the immune system.     

Does pollution influence small mammal diet in the field? A metabarcoding approach in a generalist consumer

Mammals are mainly exposed to trace metals (TMs) via consuming contaminated food. Several studies have demonstrated relationships between metal concentrations in food and in animal tissues. However, potential effects of TMs on feeding behaviour of wildlife have been poorly documented under field conditions, despite experimental evidence showing that food selection is impacted by resource contamination. Here, we test the hypothesis that the diet of a generalist rodent, the wood mouse (Apodemus sylvaticus), is altered by soil TM contamination in the field. Wood mice were sampled in spring and in autumn along a gradient of soil contamination in the surroundings of a former smelter located in northern France. Available resources in the field were inventoried, and the diet of the animals was analysed using DNA “metabarcoding.” We demonstrated that (a) relationship between the resource richness in the diet and their richness in the field was altered by soil metal contamination. Wood mice specialized their diet along the gradient of soil metal contamination for both plant and invertebrate resources in spring. We also showed that (b) preference for Salicaceae, a plant family accumulating metals, decreased when soil contamination increased. These results suggest that environmental TM pollution could act as a force modulating trophic interactions in terrestrial food webs, thereby affecting wildlife exposure to contaminants by trophic route.     

Adaptive latitudinal trends in the mandible shape of Apodemus wood mice

Aim Size and shape of the mandible are investigated across the latitudinal range of the European wood mouse (Apodemus sylvaticus), in order to address the relative importance of genetic structure, insularity, and geographical gradient in patterning morphological variation. Results are compared with those on two Asiatic species of wood mice, A. argenteus and A. speciosus. Location The European wood mouse is sampled by a set of trapping localities including both, islands and mainland populations, as well as the four genetic groups identified in previous studies. The localities cover a latitudinal gradient from 55° N to 36° N. Methods Different Fourier methods are applied to the outlines of mandibles and their results compared in the case of A. sylvaticus. All provide similar results and allow a quantification of the size and shape variations across the geographical range of the European wood mouse. Using the method allowing for the best reduction of the informative data set, a comparison of the European wood mouse with the two Asiatic species was performed. Results Within the European wood mouse A. sylvaticus, a strong latitudinal gradient in mandible shape overrides the influence of insularity and genetic structure. Yet, random morphological divergence in insular conditions can be identified as a secondary process of shape differentiation. Size displays no obvious pattern of variation, neither with insularity or latitude. A comparison with two other species of wood mice suggests that a similar latitudinal gradient in mandible shape exists in different species, mandibles being flatter in the north and wider in the south. Main conclusion The latitudinal gradient in mandible shape observed in the three species of wood mice is interpreted as an intraspecific adaptive response to gradual changes in feeding behaviour.     

Linking community assembly and structure across scales in a wild mouse parasite community

Understanding what processes drive community structure is fundamental to ecology. Many wild animals are simultaneously infected by multiple parasite species, so host–parasite communities can be valuable tools for investigating connections between community structures at multiple scales, as each host can be considered a replicate parasite community. Like free‐living communities, within‐host–parasite communities are hierarchical; ecological interactions between hosts and parasites can occur at multiple scales (e.g., host community, host population, parasite community within the host), therefore, both extrinsic and intrinsic processes can determine parasite community structure. We combine analyses of community structure and assembly at both the host population and individual scales using extensive datasets on wild wood mice (Apodemus sylvaticus) and their parasite community. An analysis of parasite community nestedness at the host population scale provided predictions about the order of infection at the individual scale, which were then tested using parasite community assembly data from individual hosts from the same populations. Nestedness analyses revealed parasite communities were significantly more structured than random. However, observed nestedness did not differ from null models in which parasite species abundance was kept constant. We did not find consistency between observed community structure at the host population scale and within‐host order of infection. Multi‐state Markov models of parasite community assembly showed that a host's likelihood of infection with one parasite did not consistently follow previous infection by a different parasite species, suggesting there is not a deterministic order of infection among the species we investigated in wild wood mice. Our results demonstrate that patterns at one scale (i.e., host population) do not reliably predict processes at another scale (i.e., individual host), and that neutral or stochastic processes may be driving the patterns of nestedness observed in these communities. We suggest that experimental approaches that manipulate parasite communities are needed to better link processes at multiple ecological scales.     

Tick infestation of small mammals in an English woodland

Tick infestations on small mammals were studied from April to November, 2010, in deciduous woodland in southern England in order to determine whether co‐infestations with tick stages occurred on small mammals, a key requirement for endemic transmission of tick‐borne encephalitis virus (TBEV). A total of 217 small mammals was trapped over 1,760 trap nights. Yellow‐necked mice (Apodemus flavicollis) made up the majority (52.5%) of animals, followed by wood mice (A. sylvaticus) 35.5% and bank voles (Myodes glareolus) 12%. A total of 970 ticks was collected from 169 infested animals; 96% of ticks were Ixodes ricinus and 3% I. trianguliceps. Over 98% of ticks were larval stages. Mean infestation intensities of I. ricinus were significantly higher on A. flavicollis (6.53 ± 0.67) than on A. sylvaticus (4.96 ± 0.92) and M. glareolus (3.25 ± 0.53). Infestations with I. ricinus were significantly higher in August than in any other month. Co‐infestations with I. ricinus nymphs and larvae were observed on six (3.6%) infested individuals, and fifteen small mammals (8.9%) supported I. ricinus – I. trianguliceps co‐infestations. This work contributes further to our understanding of European small mammal hosts that maintain tick populations and their associated pathogens, and indicates that co‐infestation of larvae and nymph ticks does occur in lowland UK. The possible implications for transmission of tick‐borne encephalitis virus between UK ticks and small mammals are discussed.     

Mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> sequences resolve the taxonomy of field mice (<Emphasis Type="Italic">Apodemus</Emphasis>) in the western Balkan refugium

Apodemus sylvaticus stankovici, described from the topographically rough landscape of the western Balkan glacial refugium, was recently proposed as being either a junior synonym of Apodemus flavicollis or a species on its own right. To untangle this taxonomic vagueness, we sequenced complete cytochrome b gene in 28 field mice collected at 12 locations in the mountains of Bosnia and Herzegovina, Montenegro, western Macedonia and northern Greece. Samples yielded 27 new haplotypes which clustered into two distinct groups. One of these clades also included the reference haplotype of A. flavicollis, while another cluster emerged as being identical with the reference sample for A. sylvaticus. As is common in Apodemus, both species retrieved in our analysis were characterized by low levels of intraspecific variation (0.4–0.9%) as opposed to a high level of differentiation between them (8.0–10.0%); therefore, the taxonomic classification of our material was without doubt. We found no evidence regarding the presence of an additional cryptic species in the mountains of the western Balkans. The very similar values of genetic variability in the two species imply their common evolutionary history of a long-term coexistence in the western Balkan refugium.     

Seasonal variation in the resting metabolic rate of male wood mice Apodemus sylvaticus from two contrasting habitats 15 km apart

Diurnal and nocturnal resting metabolic rates of winter- and summer-acclimatized adult male wood mice Apodemus sylvaticus from two adjacent populations, 15 km apart, were measured. One population lived in deciduous woodland, and experienced a narrower daily range of temperatures than the second population, which inhabited maritime sand-dunes. Ambient temperature and body mass had significant effects on the resting metabolism of mice, excluding winter-acclimatized sand-dune animals where only temperature explained significant amounts of the observed variation. Only in this latter group could a thermoneutral zone be determined, with a lower critical temperature of ca. 25 °C and resting metabolism of 0.155 W. Nocturnal resting metabolic rates were significantly greater than diurnal levels. Winter acclimatization was associated with reductions in thermal conductance and resting metabolism, thus minimizing energy expenditure at rest. Site differences in thermoregulatory strategies were only found in winter, thermal conductances remained similar but mice from the sand-dunes had significantly lower metabolic rates than those from the woodland. Winter acclimatization in wood mice was influenced by factors in addition to photoperiod. Intra-specific and individual variations in resting metabolism, as shown in this study, potentially have a pronounced effect on the daily energy expenditure of a free-living animal. Accepted: 6 September 1996     

Molecular identification of three sympatric species of wood mice (Apodemus sylvaticus,A. flavicollis,A. alpicola) in western Europe (Muridae: Rodentia)

The woodmouse (Apodemus sylvaticus) and yellow‐necked fieldmouse (Apodemus flavicollis) are sympatric and even syntopic in many regions throughout their European range. Their field discrimination on the basis of external characters is a real challenge for many fields of research. The problem is even more complicated in the Alpine chain where they live sympatrically with a third similar species: A. alpicola. A rapid and simple method is proposed to discriminate the three species in processing field‐collected biopsies as well as ethanol‐preserved museum samples.     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

Ranging behaviour and time budgets of male wood mice Apodemus sylvaticus in different habitats and seasons

Radiotelemetry was used to measure the range areas, activity patterns and time budgets of 21 adult male wood mice (Apodemus sylvaticus) between May 1991 and August 1992. The study investigated variation in range, total distance travelled, speed of movement and time budgets between wood mice in the nonbreeding and breeding seasons in a deciduous woodland (n = 8 and 6 respectively). We also examined habitat differences by estimating these same parameters for wood mice inhabiting maritime sand-dunes in the breeding season (n = 7). Insufficient males of an appropriate mass for radiotracking were captured to study the sand-dune mice in the nonbreeding season. Significant variation was found across both season and site. In the breeding season, in woodland, range areas were 5 times larger than during the nonbreeding season. Wood mice on the sand-dunes exploited ranges 28 times greater than their woodland counterparts. The pattern of variation in range area was parallelled by significant differences in total distances and average speeds travelled per night. Diurnal activity, c. 60 min day⁻¹, was frequently recorded, at both sites, but only, in the breeding season, which was attributed to the need to forage in order to maintain energy balance. The comparatively lower availability of food on the sand-dunes was considered the main factor explaining the greater range area, total distance moved, speed travelled and level of activity of animals at this site. Received: 11 September 1995 /Accepted: 12 August 1996     

Different patterns of retinal cone topography in two genera of rodents,Mus and Apodemus

Recently, we have reported the peculiar topographic separation of shortwave- and middlewave-sensitive (S and M) cones in the retina of the common house mouse (Mus musculus) and in a number of inbred laboratory mouse strains derived from the same species. In an attempt to follow the phylogeny of the complementary cone fields, we have investigated the retina of other mouse-like rodents. Two monoclonal anti-visual pigment antibodies, OS-2 and COS-1, specific to the S and M cones, respectively, have been used to identify the two cone types. Immunocytochemistry on retinal sections and on whole-mount preparations have shown that, as in the house mouse, the two cone types in the mound builder mouse (Mus spicileugus) occupy opposite halves of the retina. In contrast, in the wood mouse (Apodemus sylvaticus), both cone types are scattered uniformly across the whole retinal surface. Another distinguishing feature between the two genera is the frequency of the S cones. Whereas their density in the Mus species is above 7 000/mm² in the S-field, the maximum density of the S cones in A. sylvaticus is one order of magnitude smaller. In another species of this genus (the herb field mouse, A. microps), the S cones are completely missing.     

The diet of the Alpine mouse Apodemus alpicola in the Swiss Alps

In this study, we used faecal analysis to determine the diet of the alpine mouse Apodemus alpicola in the field for the first time. This species consumes mainly insects in spring and fruits and seeds of small herbaceous dicotyledons in summer and autumn. Compared to the two congeneric species A. flavicollis and A. sylvaticus, which also occur in the Alps, the diet of A. alpicola is rich in contrasts, with a very pronounced difference between spring and summer. The absence of tree seeds also suggests a mostly terrestrial behaviour. We explain these facts by the specificity of the habitat of A. alpicola: the extreme weather conditions produce great seasonal changes in the vegetation, and the rocky ground favours the adaptation to rock climbing rather than to arboreal living.

Zusamenfassung

Nahrungszusammensetzung bei der Alpenwaldmaus Apodemus alpicola in den Schweizer AlpenDie Nahrungszusammensetzung der Alpenwaldmaus Apodemus alpicola wurde erstmals an Wildfängen und anhand von Kotanalysen qualitativ und quantitativ untersucht. Die Alpenwaldmaus konsumierte im Frühjahr hauptsächlich Insekten, im Sommer und Herbst Früchte und Samen kleiner Dicotyledonen der Krautschicht. Verglichen mit der Gelbhalsmaus A. flavicollis und der Waldmaus A. sylvaticus, die ebenfalls in den Alpen vorkommen, ist die Nahrungspalette von A. alpicola kontrastreicher, mit größeren Unterschieden zwischen Frühjahrs- und Sommerkost. Wir erklären dies durch extreme Klimabedingungen, die zu ausgeprägten jahreszeitlichen Änderungen in der Vegetation führen. Das Fehlen von Baumsamen läßt auf ein vorwiegend terrestrisches Futtersuchverhalten schließen. Große Hinterfüße und langer Schwanz sind offenbar Anpassungen ans Felsklettern und nicht an arboricoles Verhalten.     

Influence of age, sex, and sexual activity on trace element levels and antioxidant enzyme activities in field mice (Apodemus sylvaticus and Mus spretus)

The influence of age, gender and sexual activity on both hepatic levels of some trace elements (Fe, Cu, Zn, Mn, Se) and the activities of glutathione-S-transferase (GST) and superoxide dismutase (SOD) was investigated in Wood mice (Apodemus sylvaticus) and Algerian mice (Mus spretus). Animals were taken from a riverside community of an unpolluted area of central Portugal. Adult A. sylvaticus presented the highest hepatic mean concentrations of Cu and Mn, whereas adult M. spretus had the highest Fe concentration in the liver. Moreover, an influence of age on the contents of Fe, Zn, and Mn has been observed in A. sylvaticus, whereas in M. spretus an influence of gender and sexual activity was only detected on Zn levels. In contrast, enzyme activities were not influenced by the studied variables, despite a tendency for an increase in SOD activity in sexually active M. spretus. GST activity was species dependent, whereas SOD activity was similar between species. These findings were analyzed regarding the relationship of both essential trace elements and the two antioxidant enzymes with physiological and metabolic pathways related to life cycles in the two species of mice. Results enhanced the understanding of A. sylvaticus and M. spretus as biological models, allowing their future use as bioindicators of environmental toxicity.     

Discrimination of the sibling species Apodemus flavicollis and A. sylvaticus (Rodentia,Muridae)

Karyotyping and several molecular methods have allowed successful identification of two morphologically similar wide-ranging Western Palearctic species, the yellow-necked field mouse Apodemus flavicollis (Melchior, 1934) and the long-tailed wood mouse A. sylvaticus (Linnaeus, 1758), but reliable species diagnosis on the basis of morphometric characters is particularly problematic. Although they are easily morphologically distinguishable in Central and Northern Europe, this is not the case in southern parts of their distribution areas. Despite that, we have successfully discriminated A. flavicollis and A. sylvaticus from Serbia (Southern Europe) using geometric and traditional morphometric methods on a data set for ventral crania of specimens previously genotyped by the Inter Simple Sequence Repeat-PCR (ISSR-PCR). Discrimination power of applied approaches was more or less similar. The majority of our results were consistent with those obtained for specimens collected across the Czech Republic (Central Europe). Morphological differences observed herein, as well as those already reported between A. flavicollis and A. sylvaticus from the central and northern parts of their distribution areas, could be the outcome of their biology, i.e. ecological discrepancies, different assumed evolutionary scenarios considering biogeography, phylogeny, history and ontogeny.     

Limited influence of local and landscape factors on finescale gene flow in two pond‐breeding amphibians

Dispersal and gene flow within animal populations are influenced by the composition and configuration of the landscape. In this study, we evaluated hypotheses about the impact of natural and anthropogenic factors on genetic differentiation in two amphibian species, the spotted salamander (Ambystoma maculatum) and the wood frog (Lithobates sylvaticus) in a commercial forest in central Maine. We conducted this analysis at two scales: a local level, focused on factors measured at each breeding pond, and a landscape level, focused on factors measured between ponds. We investigated the effects of a number of environmental factors in six categories including Productivity, Physical, Land Composition, Land Configuration, Isolation and Location. Embryos were sampled from 56 spotted salamander breeding ponds and 39 wood frog breeding ponds. We used a hierarchical Bayesian approach in the program GESTE at each breeding pond and a random forest algorithm in conjunction with a network analysis between the ponds. We found overall high genetic connectivity across distances up to 17 km for both species and a limited effect of natural and anthropogenic factors on gene flow. We found the null models best explained patterns of genetic differentiation at a local level and found several factors at the landscape level that weakly influenced gene flow. This research indicates multiscale investigations that incorporate local and landscape factors are valuable for understanding patterns of gene flow. Our findings suggest that dispersal rates in this system are high enough to minimize genetic structuring and that current forestry practices do not significantly impede dispersal.     

False phylogenies on wood mice due to cryptic cytochrome-b pseudogene

The phylogeny and phylogeography of the Old World wood mice (subgenus Sylvaemus, genus Apodemus, Muridae) are well-documented. Nevertheless, the distributions of species, such as A. fulvipectus and A. ponticus remain dubious, as well as their phylogenetic relationships with A. sylvaticus. We analysed samples of Apodemus spp. across Europe using the mitochondrial cytochrome-b gene (cyt-b) and compared the DNA and amino-acid compositions of previously published sequences. The main result stemming from this study is the presence of a well-differentiated lineage of Sylvaemus including samples of various species (A. sylvaticus, A. fulvipectus, A. ponticus) from distant locations, which were revealed to be nuclear copies of the mitochondrial cyt-b. The presence of this cryptic pseudogene in published sequences is supported by different pathways. This has led to important errors in previous molecular trees and hence to partial misinterpretations in the phylogeny of Apodemus.     

Estimating occupancy dynamics for large‐scale monitoring networks: amphibian breeding occupancy across protected areas in the northeast United States

Regional monitoring strategies frequently employ a nested sampling design where a finite set of study areas from throughout a region are selected and intensive sampling occurs within a subset of sites within the individual study areas. This sampling protocol naturally lends itself to a hierarchical analysis to account for dependence among subsamples. Implementing such an analysis using a classic likelihood framework is computationally challenging when accounting for detection errors in species occurrence models. Bayesian methods offer an alternative approach for fitting models that readily allows for spatial structure to be incorporated. We demonstrate a general approach for estimating occupancy when data come from a nested sampling design. We analyzed data from a regional monitoring program of wood frogs (Lithobates sylvaticus) and spotted salamanders (Ambystoma maculatum) in vernal pools using static and dynamic occupancy models. We analyzed observations from 2004 to 2013 that were collected within 14 protected areas located throughout the northeast United States. We use the data set to estimate trends in occupancy at both the regional and individual protected area levels. We show that occupancy at the regional level was relatively stable for both species. However, substantial variation occurred among study areas, with some populations declining and some increasing for both species. In addition, When the hierarchical study design is not accounted for, one would conclude stronger support for latitudinal gradient in trends than when using our approach that accounts for the nested design. In contrast to the model that does not account for nesting, the nested model did not include an effect of latitude in the 95% credible interval. These results shed light on the range‐level population status of these pond‐breeding amphibians, and our approach provides a framework that can be used to examine drivers of local and regional occurrence dynamics.     

Cryptic species of a water hyacinth biological control agent revealed in South Africa: host specificity,impact, and thermal tolerance

The discovery that cryptic species are more abundant than previously thought has implications for weed biological control, as there is a risk that cryptic species may be inadvertently released with consequences for the safety of the practice. A cryptic species of a biological control agent released for the control of the invasive alien macrophyte, water hyacinth, Eichhornia crassipes (C. Mart.) Solms. (Pontederiaceae), was recently discovered in South Africa. The two species were considered a single species prior to genetic analysis and interbreeding experiments. The original biological control agent retains the name Eccritotarsus catarinensis (Carvalho) (Heteroptera: Miridae) whereas the new species has been described as Eccritotarsus eichhorniae Henry. In this study, we compared the host specificity, efficacy, and thermal physiologies of the two species. The host specificity of the two species within the Pontederiaceae was very similar and both are safe for release in South Africa. Comparison of the per capita impact of the two species indicated that E. eichhorniae was the more damaging species but this is likely to be influenced by temperature, with E. catarinensis being more effective under lower temperatures and E. eichhorniae being more effective under higher temperatures. Releasing the correct species for the thermal environment of each release site will improve the level of control of water hyacinth in South Africa. This example highlights the need to keep populations of biological control agents from different native range collection localities separate, and to screen for host specificity and efficacy.     

Phylogeny of Gracilariaceae (Rhodophyta): evidence from plastid and mitochondrial nucleotide sequences

Gracilariaceae are mostly pantropical red algae and include ~230 species in seven genera. Infrafamilial classification of the group has long been based on reproductive characters, but previous phylogenies have shown that traditionally circumscribed groups are not monophyletic. We performed phylogenetic analyses using two plastid (universal plastid amplicon and rbcL) and one mitochondrial (cox1) loci from a greatly expanded number of taxa to better assess generic relationships and understand patterns of character distributions. Our analyses produce the most well‐supported phylogeny of the family to date, and indicate that key characteristics of spermatangia and cystocarp type do not delineate genera as commonly suggested. Our results further indicate that Hydropuntia is not monophyletic. Given their morphological overlap with closely related members of Gracilaria, we propose that Hydropuntia be synonymized with the former. Our results additionally expand the known ranges of several Gracilariaceae species to include Brazil. Lastly, we demonstrate that the recently described Gracilaria yoneshigueana should be synonymized as G. domingensis based on morphological and molecular characters. These results demonstrate the utility of DNA barcoding for understanding poorly known and fragmentary materials of cryptic red algae.     

Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.