Rehabilitation and release of orphaned Eurasian lynx (Lynx lynx) in Europe: Implications for management and conservation.

Rehabilitation of injured or immature individuals has become an increasingly used conservation and management tool. However, scientific evaluation of rehabilitations is rare, raising concern about post-release welfare as well as the cost-effectiveness of spending scarce financial resources. Over the past 20 years, events of juvenile Eurasian lynx presumably orphaned have been observed in many European lynx populations. To guide the management of orphaned lynx, we documented survival, rehabilitation and fate after the release and evaluated the potential relevance of lynx orphan rehabilitation for population management and conservation implications. Data on 320 orphaned lynx was collected from 1975 to 2022 from 13 countries and nine populations. The majority of orphaned lynx (55%) were taken to rehabilitation centres or other enclosures. A total of 66 orphans were released back to nature. The portion of rehabilitated lynx who survived at least one year after release was 0.66. Release location was the best predictor for their survival. Of the 66 released lynx, ten have reproduced at least once (8 females and 2 males). Conservation implications of rehabilitation programmes include managing genetic diversity in small, isolated populations and reintroducing species to historical habitats. The lynx is a perfect model species as most reintroduced populations in Central Europe show significantly lower observed heterozygosity than most of the autochthonous populations, indicating that reintroduction bottlenecks, isolation and post-release management have long-term consequences on the genetic composition of populations. The release of translocated orphans could be a valuable contribution to Eurasian lynx conservation in Europe. It is recommended to release orphans at the distribution edge or in the frame of reintroduction projects instead of a release in the core area of a population where it is not necessary from a demographic and genetic point of view. Rehabilitation programmes can have conservation implications that extend far beyond individual welfare benefits.

Evolutionary ecology of the bark beetles Ips typographus and Pityogenes chalcographus.

Ips typographus (L.) and Pityogenes chalcographus (L.) (Coleoptera: Curculionidae) are two common bark beetle species on Norway spruce in Eurasia. Multiple biotic and abiotic factors affect the life cycles of these two beetles, shaping their ecology and evolution. In this article, we provide a comprehensive and comparative summary of selected life-history traits. We highlight similarities and differences in biotic factors, like host range, interspecific competition, host colonization, reproductive behaviour and fungal symbioses. Moreover, we focus on the species' responses to abiotic factors and compare their temperature-dependent development and flight behaviour, cold adaptations and diapause strategies. Differences in biotic and abiotic traits might be the result of recent, species-specific evolutionary histories, particularly during the Pleistocene, with differences in glacial survival and postglacial recolonization. Finally, we discuss future research directions to understand ecological and evolutionary pathways of the two bark beetle species, for both basic research and applied forest management.

Effect of Temperature and Photoperiod on Development, Survival, and Growth Rate of Mealworms, Tenebrio molitor.

Insects are a potential substitute for conventional meat and can be part of a sustainable human diet due to their valuable nutrients and relatively low environmental production impact. One species that is already produced for human consumption and livestock feed is the mealworm, i.e., larvae of Tenebrio molitor. Knowledge of the effects of temperature, and particularly photoperiod, on mealworm development is scarce, but crucial for the improvement of rearing. Therefore, the effects of three temperatures (20 °C, 25 °C, and 30 °C), in combination with three photoperiods (long-day-16 h:8 h light:dark; short-day-8 h:16 h light:dark, and constant darkness) on mealworm survival, developmental time, and growth rate were tested. We describe a significant effect of temperature on survival rate, developmental time, and growth rate. Furthermore, significant effects of photoperiod on developmental time and growth rate were found. At 25 and 30 °C and constant darkness, the highest survival and growth rate, along with the shortest developmental time, were observed. Our data can be used to improve the mass rearing of mealworms for an efficient production of food and feed.

Facultative and obligate diapause phenotypes in populations of the European spruce bark beetle Ips typographus.

The bark beetle Ips typographus is the most destructive insect pest in Norway spruce-dominated forests. Its potential to establish multiple generations per year (multivoltinism) is one major trait that makes this beetle a severe pest. Ips typographus enters diapause to adjust its life cycle to seasonally changing environments. Diapause is characterized by developmental and reproductive arrest; it prolongs generation time and thus affects voltinism. In I. typographus a facultative, photoperiod-regulated diapause in the adult stage has been described. In addition, the presence of an obligate, photoperiod-independent, diapause has been hypothesized. The diapause phenotype has important implications for I. typographus voltinism, as populations with obligate diapausing individuals would be univoltine. To test for the presence of different I. typographus diapause phenotypes, we exposed Central and Northern European individuals to a set of photoperiodic treatments. We used two ovarian traits (egg number and vitellarium size) that are associated with gonad development, to infer reproductive arrest and thus diapause. We found a distinct effect of photoperiod on ovarian development, with variable responses in Central and Northern European beetles. We observed obligate diapausing (independent of photoperiod) individuals in Northern Europe, and both facultative (photoperiod-regulated) as well as obligate diapausing individuals in Central Europe. Our results show within-species variation for diapause induction, an adaptation to match life cycles with seasonally fluctuating environmental conditions. As the diapause phenotype affects the potential number of generations per season, our data are the basis for assessing the risk of outbreaks of this destructive bark beetle. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10340-021-01416-w.

Assessment of drivers of spatial genetic variation of a ground-dwelling bird species and its implications for conservation.

In modern wildlife ecology, spatial population genetic methods are becoming increasingly applied. Especially for animal species in fragmented landscapes, preservation of gene flow becomes a high priority target in order to restore genetic diversity and prevent local extinction. Within Central Europe, the Alps represent the core distribution area of the black grouse, Lyrurus tetrix. At its easternmost Alpine range, events of subpopulation extinction have already been documented in the past decades. Molecular data combined with spatial analyses can help to assess landscape effects on genetic variation and therefore can be informative for conservation management. Here, we addressed whether the genetic pattern of the easternmost Alpine black grouse metapopulation system is driven by isolation by distance or isolation by resistance. Correlative ecological niche modeling was used to assess geographic distances and landscape resistances. We then applied regression-based approaches combined with population genetic analyses based on microsatellite data to disentangle effects of isolation by distance and isolation by resistance among individuals and subpopulations. Although population genetic analyses revealed overall low levels of genetic differentiation, the ecological niche modeling showed subpopulations to be clearly delimited by habitat structures. Spatial genetic variation could be attributed to effects of isolation by distance among individuals and isolation by resistance among subpopulations, yet unknown effects might factor in. The easternmost subpopulation was the most differentiated, and at the same time, immigration was not detected; hence, its long-term survival might be threatened. Our study provides valuable insights into the spatial genetic variation of this small-scale metapopulation system of Alpine black grouse.

Testing the potential contribution of Wolbachia to speciation when cytoplasmic incompatibility becomes associated with host-related reproductive isolation.

Endosymbiont-induced cytoplasmic incompatibility (CI) may play an important role in arthropod speciation. However, whether CI consistently becomes associated or coupled with other host-related forms of reproductive isolation (RI) to impede the transfer of endosymbionts between hybridizing populations and further the divergence process remains an open question. Here, we show that varying degrees of pre- and postmating RI exist among allopatric populations of two interbreeding cherry-infesting tephritid fruit flies (Rhagoletis cingulata and R. indifferens) across North America. These flies display allochronic and sexual isolation among populations, as well as unidirectional reductions in egg hatch in hybrid crosses involving southwestern USA males. All populations are infected by a Wolbachia strain, wCin2, whereas a second strain, wCin3, only co-infects flies from the southwest USA and Mexico. Strain wCin3 is associated with a unique mitochondrial DNA haplotype and unidirectional postmating RI, implicating the strain as the cause of CI. When coupled with nonendosymbiont RI barriers, we estimate the strength of CI associated with wCin3 would not prevent the strain from introgressing from infected southwestern to uninfected populations elsewhere in the USA if populations were to come into secondary contact and hybridize. In contrast, cytoplasmic-nuclear coupling may impede the transfer of wCin3 if Mexican and USA populations were to come into contact. We discuss our results in the context of the general paucity of examples demonstrating stable Wolbachia hybrid zones and whether the spread of Wolbachia among taxa can be constrained in natural hybrid zones long enough for the endosymbiont to participate in speciation.

Enterobacter sp. AA26 as a Protein Source in the Larval Diet of Drosophila suzukii.

The Spotted-Wing Drosophila fly, Drosophila suzukii, is an invasive pest species infesting major agricultural soft fruits. Drosophila suzukii management is currently based on insecticide applications that bear major concerns regarding their efficiency, safety and environmental sustainability. The sterile insect technique (SIT) is an efficient and friendly to the environment pest control method that has been suggested for the D. suzukii population control. Successful SIT applications require mass-rearing of the strain to produce competitive and of high biological quality males that will be sterilized and consequently released in the wild. Recent studies have suggested that insect gut symbionts can be used as a protein source for Ceratitis capitata larval diet and replace the expensive brewer's yeast. In this study, we exploited Enterobacter sp. AA26 as partial and full replacement of inactive brewer's yeast in the D. suzukii larval diet and assessed several fitness parameters. Enterobacter sp. AA26 dry biomass proved to be an inadequate nutritional source in the absence of brewer's yeast and resulted in significant decrease in pupal weight, survival under food and water starvation, fecundity, and adult recovery.

Comparative genome sequencing reveals insights into the dynamics of Wolbachia in native and invasive cherry fruit flies.

Wolbachia is a maternally inherited obligate endosymbiont that can induce a wide spectrum of effects in its host, ranging from mutualism to reproductive parasitism. At the genomic level, recombination within and between strains, transposable elements, and horizontal transfer of strains between host species make Wolbachia an evolutionarily dynamic bacterial system. The invasive cherry fruit fly Rhagoletis cingulata arrived in Europe from North America ~40 years ago, where it now co-occurs with the native cherry pest R. cerasi. This shared distribution has been proposed to have led to the horizontal transfer of different Wolbachia strains between the two species. To better understand transmission dynamics, we performed a comparative genome study of the strain wCin2 in its native United States and invasive European populations of R. cingulata with wCer2 in European R. cerasi. Previous multilocus sequence genotyping (MLST) of six genes implied that the source of wCer2 in R. cerasi was wCin2 from R. cingulata. However, we report genomic evidence discounting the recent horizontal transfer hypothesis for the origin of wCer2. Despite near identical sequences for the MLST markers, substantial sequence differences for other loci were found between wCer2 and wCin2, as well as structural rearrangements, and differences in prophage, repetitive element, gene content, and cytoplasmic incompatibility inducing genes. Our study highlights the need for whole-genome sequencing rather than relying on MLST markers for resolving Wolbachia strains and assessing their evolutionary dynamics.

Wolbachia-Mitochondrial DNA Associations in Transitional Populations of Rhagoletis cerasi.

The endosymbiont Wolbachia can manipulate arthropod host reproduction by inducing cytoplasmic incompatibility (CI), which results in embryonic mortality when infected males mate with uninfected females. A CI-driven invasion of Wolbachia can result in a selective sweep of associated mitochondrial haplotype. The co-inheritance of Wolbachia and host mitochondrial DNA can therefore provide significant information on the dynamics of an ongoing Wolbachia invasion. Therefore, transition zones (i.e., regions where a Wolbachia strain is currently spreading from infected to uninfected populations) represent an ideal area to investigate the relationship between Wolbachia and host mitochondrial haplotype. Here, we studied Wolbachia-mitochondrial haplotype associations in the European cherry fruit fly, Rhagoletis cerasi, in two transition zones in the Czech Republic and Hungary, where the CI-inducing strain wCer2 is currently spreading. The wCer2-infection status of 881 individuals was compared with the two known R. cerasi mitochondrial haplotypes, HT1 and HT2. In accordance with previous studies, wCer2-uninfected individuals were associated with HT1, and wCer2-infected individuals were mainly associated with HT2. We found misassociations only within the transition zones, where HT2 flies were wCer2-uninfected, suggesting the occurrence of imperfect maternal transmission. We did not find any HT1 flies that were wCer2-infected, suggesting that Wolbachia was not acquired horizontally. Our study provides new insights into the dynamics of the early phase of a Wolbachia invasion.

Mass-Rearing of Drosophila suzukii for Sterile Insect Technique Application: Evaluation of Two Oviposition Systems.

Drosophila suzukii (Diptera: Drosophilidae) is an invasive pest of a wide range of commercial soft-skinned fruits. To date, most management tactics are based on spraying of conventional and/or organic insecticides, baited traps, and netting exclusion. Interest has been expressed in using the sterile insect technique (SIT) as part of area-wide integrated pest management (AW-IPM) programs to control D. suzukii infestations. Mass-rearing protocols are one of the prerequisites for successful implementation of the SIT. To establish mass-rearing methods for this species, two different egg-collection systems were developed and compared with respect to the number of eggs produced, egg viability, pupa and adult recovery, adult emergence rate, and flight ability. Female flies kept in cages equipped with a wax panel produced significantly more eggs with higher viability and adult emergence rate, as compared to the netted oviposition system. The wax panel system was also more practical and less laborious regarding the collection of eggs. Furthermore, the wax panel oviposition system can be adapted to any size or design of an adult cage. In conclusion, this system bears great promise as an effective system for the mass production of D. suzukii for SIT.

New Data on the Range Expansion of the Thaumetopoea pityocampa (Lepidoptera: Notodontidae) 'ENA clade' in Greece: The Role of Bacterial Endosymbionts.

The pine processionary moth, Thaumetopoea pityocampa (Denis and Schiffermüller), is an important insect in the Mediterranean region, as it defoliates pines and its urticating hairs can cause allergic reactions in humans and animals. Moreover, this species exhibits an interesting genetic structure as recently a distinct East-North African mtDNA lineage ('ENA clade') has been described. This clade has been recently detected in Greek populations where it has currently expanded its range by replacing the 'endemic' T. pityocampa lineages. Here, we report new data on the rapid spread of 'ENA clade' in the Greek island Evoia in only a few years. As the underlying mechanisms of the 'ENA clade' range expansion has not been studied so far, we screened T. pityocampa for an infection with the heritable bacterial endosymbionts Wolbachia (Bacteria: Anaplasmataceae), Cardinium (Bacteria: Bacteroidaceae), Rickettsia (Bacteria: Rickettsiaceae) and Spiroplasma (Bacteria: Spiroplasmataceae). These bacteria can manipulate the reproduction of infected hosts, something that could potentially explain the rapid spread of 'ENA clade' lineage. Therefore, we screened 28 individuals that exhibited T. pityocampa 'ENA clade' and 'endemic' T. pityocampa haplotypes from nine populations scattered all over Greece. None of them was infected with any of the four endosymbionts, suggesting that these bacteria do not cause reproductive manipulations in T. pityocampa lineages and, thus, other factors should be explored in future research efforts.

Host plant-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi.

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host-related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host-related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America.

The Apennines as a cryptic Pleistocene refugium of the bark beetle Pityogenes chalcographus (Coleoptera: Curculionidae).

The Apennine Mountains in Italy are an important biogeographical region and of particular interest in phylogeographical research, because they have been a refugium during Pleistocene glaciation events for numerous European species. We performed a genetic study on the Eurasian bark beetle Pityogenes chalcographus (Linnaeus, 1760), focusing on two Apennine (Italian) and two Central European (Austrian) locations to assess the influence of the Apennines in the evolutionary history of the beetle, particularly during the Pleistocene. We analysed a part of the mitochondrial COI gene and a set of 5470 informative genome-wide markers to understand its biogeography. We found 75 distinct mitochondrial haplotypes, which are structured in three main clades. In general, the Apennine locations harbour a higher number of mitochondrial clades than Central European sites, with one specific clade exclusively detected in the Apennines. Analysis of our genome-wide, multi-locus dataset reveals a clustering of P. chalcographus by geography, with Italian individuals clearly separated from Austrian samples. Our data highlight the significance of the Apennines for the genetic diversity of P. chalcographus and support the hypothesis that this area was an important refugium during unfavourable conditions in the Pleistocene. We discuss additional life-history traits and processes that shaped the evolution of this widespread beetle.

Dynamics of an Ongoing Wolbachia Spread in the European Cherry Fruit Fly, Rhagoletis cerasi (Diptera: Tephritidae).

Numerous terrestrial arthropods are infected with the alphaproteobacterium Wolbachia. This endosymbiont is usually transmitted vertically from infected females to their offspring and can alter the reproduction of hosts through various manipulations, like cytoplasmic incompatibility (CI), enhancing its spread in new host populations. Studies on the spatial and temporal dynamics of Wolbachia under natural conditions are scarce. Here, we analyzed Wolbachia infection frequencies in populations of the European cherry fruit fly, Rhagoletis cerasi (L.), in central Germany-an area of an ongoing spread of the CI-inducing strain wCer2. In total, 295 individuals from 19 populations were PCR-screened for the presence of wCer2 and their mitochondrial haplotype. Results were compared with historic data to understand the infection dynamics of the ongoing wCer2 invasion. An overall wCer2 infection frequency of about 30% was found, ranging from 0% to 100% per population. In contrast to an expected smooth transition from wCer2-infected to completely wCer2-uninfected populations, a relatively scattered infection pattern across geography was observed. Moreover, a strong Wolbachia-haplotype association was detected, with only a few rare misassociations. Our results show a complex dynamic of an ongoing Wolbachia spread in natural field populations of R. cerasi.

Irradiation dose response under hypoxia for the application of the sterile insect technique in Drosophila suzukii.

Treating insects with a lower oxygen atmosphere before and during exposure to radiation can mitigate some of the negative physiological effects due to the irradiation. The irradiation of pupae under oxygen-reduced environment such as hypoxia or anoxia is routinely used in the sterile insect technique (SIT) of some tephritid species as it provides radiological protection. This treatment allows to have the sterile pupae already in sealed containers facilitating the shipment. SIT is an environment friendly control tactic that could be used to manage populations of Drosophila suzukii in confined areas such as greenhouses. The objectives of this study were to assess the effect of irradiation on the reproductive sterility in D. suzukii males and females under low-oxygen atmosphere (hypoxia) and atmosphere conditions (normoxia). Additionally, we assessed the differences in radiological sensitivity of pupae treated under hypoxia and normoxia conditions. Finally, the effect on emergence rate and flight ability of the irradiated D. suzukii adults exposed to doses that induced >99% of sterility were assessed. Pupae needed a 220 Gy irradiation dose to achieve >99% of egg hatch sterility in males irrespective of the atmosphere condition. For females the same level of sterility was achieved already at 75 Gy and 90 Gy for the normoxia and hypoxia treatments, respectively. Radiation exposure at 170 and 220 Gy under the two atmosphere treatments did not have any effect on the emergence rate and flight ability of D. suzukii males and females. Therefore, hypoxia conditions can be used as part of an area-wide insect pest management program applying SIT to facilitate the protocols of packing, irradiation and shipment of sterile D. suzukii pupae.

Limited Genetic Structure of Gypsy Moth Populations Reflecting a Recent History in Europe.

The gypsy moth, Lymantria dispar, a prominent polyphagous species native to Eurasia, causes severe impacts in deciduous forests during irregular periodical outbreaks. This study aimed to describe the genetic structure and diversity among European gypsy moth populations. Analysis of about 500 individuals using a partial region of the mitochondrial COI gene, L. dispar was characterized by low genetic diversity, limited population structure, and strong evidence that all extant haplogroups arose via a single Holocene population expansion event. Overall 60 haplotypes connected to a single parsimony network were detected and genetic diversity was highest for the coastal populations Croatia, Italy, and France, while lowest in continental populations. Phylogenetic reconstruction resulted in three groups that were geographically located in Central Europe, Dinaric Alps, and the Balkan Peninsula. In addition to recent events, the genetic structure reflects strong gene flow and the ability of gypsy moth to feed on about 400 deciduous and conifer species. Distinct genetic groups were detected in populations from Georgia. This remote population exhibited haplotypes intermediate to the European L. dispar dispar, Asian L. dispar asiatica, and L. dispar japonica clusters, highlighting this area as a possible hybridization zone of this species for future studies applying genomic approaches.

Pleistocene climate cycling and host plant association shaped the demographic history of the bark beetle Pityogenes chalcographus.

Historical climatic oscillations and co-evolutionary dependencies were key evolutionary drivers shaping the current population structure of numerous organisms. Here, we present a genome-wide study on the biogeography of the bark beetle Pityogenes chalcographus, a common and widespread insect in Eurasia. Using Restriction Associated DNA Sequencing, we studied the population structure of this beetle across a wide part of its western Palaearctic range with the goal of elucidating the role of Pleistocene glacial-interglacial cycling and its close relationship to its main host plant Norway spruce. Genetic distance among geographic sites was generally low, but clustering analysis revealed three genetically distinct groups, that is, southern, central/south-eastern, and north-eastern locations. Thus, three key P. chalcographus glacial refugia were identified: in the Italian-Dinaric region, the Carpathians, and the Russian plain, shared with its main host. The current phylogeographic signal was affected by genetic divergence among geographically isolated refugia during glacial periods and postglacial re-establishment of genetic exchange through secondary contact, reflected by admixture among genetic groups. Additionally, certain life history traits, like the beetle's dispersal and reproductive behaviour, considerably influenced its demographic history. Our results will help to understand the biogeography of other scolytine beetles, especially species with similar life history traits.

Reproductive Manipulators in the Bark Beetle Pityogenes chalcographus (Coleoptera: Curculionidae)-The Role of Cardinium, Rickettsia, Spiroplasma, and Wolbachia.

Heritable bacterial endosymbionts can alter the biology of numerous arthropods. They can influence the reproductive outcome of infected hosts, thus affecting the ecology and evolution of various arthropod species. The spruce bark beetle Pityogenes chalcographus (L.) (Coleoptera: Curculionidae: Scolytinae) was reported to express partial, unidirectional crossing incompatibilities among certain European populations. Knowledge on the background of these findings is lacking; however, bacterial endosymbionts have been assumed to manipulate the reproduction of this beetle. Previous work reported low-density and low-frequency Wolbachia infections of P. chalcographus but found it unlikely that this infection results in reproductive alterations. The aim of this study was to test the hypothesis of an endosymbiont-driven incompatibility, other than Wolbachia, reflected by an infection pattern on a wide geographic scale. We performed a polymerase chain reaction (PCR) screening of 226 individuals from 18 European populations for the presence of the endosymbionts Cardinium, Rickettsia, and Spiroplasma, and additionally screened these individuals for Wolbachia. Positive PCR products were sequenced to characterize these bacteria. Our study shows a low prevalence of these four endosymbionts in P. chalcographus. We detected a yet undescribed Spiroplasma strain in a single individual from Greece. This is the first time that this endosymbiont has been found in a bark beetle. Further, Wolbachia was detected in three beetles from two Scandinavian populations and two new Wolbachia strains were described. None of the individuals analyzed were infected with Cardinium and Rickettsia. The low prevalence of bacteria found here does not support the hypothesis of an endosymbiont-driven reproductive incompatibility in P. chalcographus.

Turning one into five: Integrative taxonomy uncovers complex evolution of cryptic species in the harvester ant Messor "structor".

Seed harvesting ants are ecosystem engineers that shape vegetation, nutrient cycles, and microclimate. Progress in ecological research is, however, slowed down by poor species delimitation. For example, it has not been resolved to date, how many species the European harvester ant Messor "structor" (Latreille, 1798) represents. Since its first description, splitting into additional taxa was often proposed but not accepted later on due to inconsistent support from morphology and ecology. Here, we took an iterative integrative-taxonomy approach - comparing multiple, independent data sets of the same sample - and used traditional morphometrics, Wolbachia symbionts, mitochondrial DNA, amplified fragment length polymorphism, and ecological niche modelling. Using the complementarity of the data sets applied, we resolved multiple, strong disagreements over the number of species, ranging from four to ten, and the allocation of individuals to species. We consider most plausible a five-species hypothesis and conclude the taxonomic odyssey by redescribing Messor structor, M. ibericus Santschi, 1925, and M. muticus (Nylander, 1849) stat.rev., and by describing two new species, M. ponticus sp.n. and M. mcarthuri sp.n. The evolutionary explanations invoked in resolving the various data conflicts include pronounced morphological crypsis, incomplete lineage-sorting or ongoing cospeciation of endosymbionts, and peripatric speciation - these ants' significance to evolutionary biology parallels that to ecology. The successful solution of this particular problem illustrates the usefulness of the integrative approach to other systematic problems of comparable complexity and the importance of understanding evolution to drawing correct conclusions on species' attributes, including their ecology and biogeography.