POPULATIONS OF MONARCH BUTTERFLIES WITH DIFFERENT MIGRATORY BEHAVIORS SHOW DIVERGENCE IN WING MORPHOLOGY

The demands of long‐distance flight represent an important evolutionary force operating on the traits of migratory species. Monarchs are widespread butterflies known for their annual migrations in North America. We examined divergence in wing morphology among migratory monarchs from eastern and western N. America, and nonmigratory monarchs in S. Florida, Puerto Rico, Costa Rica, and Hawaii. For the three N. American populations, we also examined monarchs reared in four common environment experiments. We used image analysis to measure multiple traits including forewing area and aspect ratio; for laboratory‐reared monarchs we also quantified body area and wing loading. Results showed wild monarchs from all nonmigratory populations were smaller than those from migratory populations. Wild and captive‐reared eastern monarchs had the largest and most elongated forewings, whereas monarchs from Puerto Rico and Costa Rica had the smallest and roundest forewings. Eastern monarchs also had the largest bodies and high measures of wing loading, whereas western and S. Florida monarchs had less elongated forewings and smaller bodies. Among captive‐reared butterflies, family‐level effects provided evidence that genetic factors contributed to variation in wing traits. Collectively, these results support evolutionary responses to long‐distance flight in monarchs, with implications for the conservation of phenotypically distinct wild populations.     

Lack of genetic differentiation between monarch butterflies with divergent migration destinations

Monarch butterflies are best known for their spectacular annual migration from eastern North America to Mexico. Monarchs also occur in the North American states west of the Rocky Mountains, from where they fly shorter distances to the California Coast. Whether eastern and western North American monarchs form one genetic population or are genetically differentiated remains hotly debated, and resolution of this debate is essential to understand monarch migration patterns and to protect this iconic insect species. We studied the genetic structure of North American migratory monarch populations, as well as nonmigratory populations in Hawaii and New Zealand. Our results show that eastern and western migratory monarchs form one admixed population and that monarchs from Hawaii and New Zealand have genetically diverged from North American butterflies. These findings suggest that eastern and western monarch butterflies maintain their divergent migrations despite genetic mixing. The finding that eastern and western monarchs form one genetic population also suggests that the conservation of overwintering sites in Mexico is crucial for the protection of monarchs in both eastern and western North America.     

Associations between host migration and the prevalence of a protozoan parasite in natural populations of adult monarch butterflies

1. Monarch butterflies Danaus plexippus (L.) (Lepidoptera: Nymphalidae) are susceptible to infection by the obligate protozoan parasite Ophryocystis elektroscirrha (McLaughlin and Myers) (Apicomplexa: Neogregarinida). Because monarchs form resident and migratory populations in different parts of the world, this host–parasite system provides the opportunity to examine how variation in parasite prevalence relates to host movement patterns. 2. Parasite prevalence was evaluated using 14 790 adult monarchs captured between 1968 and 1997. Comparison of three populations in North America indicated that parasite prevalence is associated negatively with host dispersal distances. A continuously breeding, nonmigratory population in southern Florida showed high prevalence (over 70% heavily infected). The western population migrates moderate distances to overwintering sites on the Pacific Coast and has intermediate prevalence (30% heavily infected). The eastern migratory population, which travels the longest distance to Mexican overwintering sites, has exhibited less than 8% infection throughout the past 30 years. 3. Variation in parasite loads within North American migratory populations was investigated to determine whether the prevalence of heavy infection and average parasite loads declined during migration or overwintering. Average parasite loads of summer‐breeding adults in western North America decreased with increasing distance from overwintering sites. This suggests that heavily infected monarchs are less likely to remigrate long distances in spring. No differences in the frequency of heavily infected adults were found among eastern or western North American monarchs throughout the overwintering period, however, suggesting that this parasite does not affect overwintering mortality. 4. Changes in the prevalence of monarchs with low parasite loads demonstrate that spore transfer occurs during migration and overwintering, possibly when adult butterflies contact each other as a result of their clustering behaviour. 5. This study of geographical and temporal variation in O. elektroscirrha among populations of D. plexippus demonstrates the potential role of seasonal migration in mediating interactions between hosts and parasites, and suggests several mechanisms through which migratory behaviour may influence parasite prevalence.     

Monarch butterflies reared under autumn-like conditions have more efficient flight and lower post-flight metabolism

1. Many migratory animals undergo physiological and behavioural changes to prepare for and sustain long-distance movements. Because insect migrations are common and diverse, studies that examine how migratory insects meet the energetic demands of long-distance movements are badly needed. 2. Monarch butterflies (Danaus plexippus) migrate up to 4000 km annually from eastern North America to wintering sites in central Mexico. Autumn generation monarchs undergo physiological and behavioural changes in response to environmental cues to initiate migration. In particular, exposure to cooler temperatures and shorter day lengths in early autumn causes monarchs to enter the hormonally induced state of reproductive diapause. 3. This study examined differences in flight-associated metabolic rate (MR) and flight performance metrics for monarchs experimentally reared under autumn-like conditions (typically experienced before the southward migration) relative to monarchs reared under summer-like conditions. 4. Adult monarchs reared under autumn-like conditions showed lower post-flight MRs, greater flight efficiency, and lower measures of reproductive activity relative to monarchs reared under summer-like conditions. Increases in post-flight metabolism were associated with monarch body weight, age, and flight velocity. 5. These findings suggest that a trans-generational shift in flight energetics is an important component of the monarch's complex migratory syndrome, and that physiological changes that accompany reproductive diapause facilitate energy conservation during flight.     

Parasites hinder monarch butterfly flight: implications for disease spread in migratory hosts

Monarch butterflies (Danaus plexippus) are parasitized by the protozoan Ophryocystis elektroscirrha throughout their geographical range. Monarchs inhabiting seasonally fluctuating environments migrate annually, and parasite prevalence is lower among migratory relative to non‐migratory populations. One explanation for this pattern is that long‐distance migration weeds out infected animals, thus reducing parasite prevalence and transmission between generations. In this study we experimentally infected monarchs from a migratory population and recorded their long‐distance flight performance using a tethered flight mill. Results showed that parasitized butterflies exhibited shorter flight distances, slower flight speeds, and lost proportionately more body mass per km flown. Differences between parasitized and unparasitized monarchs were generally not explained by individual variation in wing size, shape, or wing loading, suggesting that poorer flight performance among parasitized hosts was not directly caused by morphological constraints. Effects of parasite infection on powered flight support a role for long‐distance migration in dramatically reducing parasite prevalence in this and other host–pathogen systems.     

Directional and stabilizing selection on wing size and shape in migrant and resident monarch butterflies, Danaus plexippus (L.), in Cuba

The majority of migrant monarchs (Danaus plexippus) from the eastern USA and south‐eastern Canada migrate to Mexico; however, some of them migrate to Cuba. Cuban migrants hatch in south‐east Canada and eastern USA, and then engage in a southern trip of 4000 km to this Caribbean island. In Cuba, these migrants encounter resident monarchs, which do not migrate, and instead move between plant patches looking for nectar, mating partners and host plants. These differences in flight behaviour between migrant and resident Cuban monarchs may have resulted in different selective pressures in the wing size and shape. Two modes of selection were tested, directional and stabilizing. In addition, wing condition was compared between these two groups. Monarchs were collected for 4 years in Cuba and classified as resident or migrant using two independent techniques: Thin‐layer chromatography and stable hydrogen and stable carbon isotope measurements. Wing size was measured and wing condition was rated in the butterflies. Fourier analysis and wing angular measurements were used to assess wing shape differences. Migrants have significantly longer wings than residents, thus supporting the action of directional selection on wing size. In addition, directional selection acts on wing shape; that is, migrant females differ significantly from resident females in their wing angles. However, the results do not support the action of stabilizing selection: there was no significant variance between migrant and resident monarchs in their wing size or shape. Also, migrant females and males differed in wing condition as a result of differences in flight behaviour. In conclusion, eastern North American monarchs offer a good opportunity to study the selective pressures of migration on wing morphology and how different migratory routes and behaviours are linked to wing morphology and condition. © 2007 The Linnean Society of London, Biological Journal of the Linnean Society, 2007, 92 , 605–616.     

Do Healthy Monarchs Migrate Farther? Tracking Natal Origins of Parasitized vs. Uninfected Monarch Butterflies Overwintering in Mexico

Long-distance migration can lower parasite prevalence if strenuous journeys remove infected animals from wild populations. We examined wild monarch butterflies (Danaus plexippus) to investigate the potential costs of the protozoan Ophryocystis elektroscirrha on migratory success. We collected monarchs from two wintering sites in central Mexico to compare infection status with hydrogen isotope (δ ²H) measurements as an indicator of latitude of origin at the start of fall migration. On average, uninfected monarchs had lower δ ²H values than parasitized butterflies, indicating that uninfected butterflies originated from more northerly latitudes and travelled farther distances to reach Mexico. Within the infected class, monarchs with higher quantitative spore loads originated from more southerly latitudes, indicating that heavily infected monarchs originating from farther north are less likely to reach Mexico. We ruled out the alternative explanation that lower latitudes give rise to more infected monarchs prior to the onset of migration using citizen science data to examine regional differences in parasite prevalence during the summer breeding season. We also found a positive association between monarch wing area and estimated distance flown. Collectively, these results emphasize that seasonal migrations can help lower infection levels in wild animal populations. Our findings, combined with recent declines in the numbers of migratory monarchs wintering in Mexico and observations of sedentary (winter breeding) monarch populations in the southern U.S., suggest that shifts from migratory to sedentary behavior will likely lead to greater infection prevalence for North American monarchs.     

Biological Observations of Monarch Butterfly Behavior at a Migratory Stopover Site: Results from a Long-term Tagging Study in Coastal South Carolina

Like most migratory species, monarch butterflies (Danaus plexippus) must stop frequently during their long southward migration to rest and refuel, and the places where they stop are important for the success of the migration. The behavior of monarch butterflies at migratory stopover sites has never been examined in detail. Here we present results of a long-term study of monarchs at one stopover site in coastal South Carolina where over 12,000 monarchs have been captured, measured and tagged (with numbered stickers to track recovery rates) over 13 years. Only 3 monarchs (0.023%) were recovered at the monarchs’ overwintering sites in Mexico, which is consistent with other tagging studies on the eastern coast. The migration season was longer at this site than at inland locations and monarchs continued to be captured in November and December, when most monarchs had already arrived at the overwintering areas in Mexico. In addition, there were 94 monarchs captured between Jan 1 and Mar 15, indicating that some monarchs overwinter at this site. Of all monarchs captured during the migration season, 80% were captured while nectaring and 10% while roosting. Others were basking, resting, flying and even mating. The sex ratio was male biased by three to one in all behavior categories except those captured mating. Roosting and nectaring monarchs had fresher wings than those in other behavior categories, suggesting that these are younger individuals. There were 13 observations of females ovipositing on non-native Asclepias curassavica during the fall months, which speaks to the potential for this plant to pull monarchs out of the migratory pool. Aside from these insights, this study also serves as an example of the potential that monarch tagging studies have to advance scientific understanding of monarch migration.     

Genomic differentiation and patterns of gene flow between two long‐tailed tit species (Aegithalos)

Patterns of heterogeneous genomic differentiation have been well documented between closely related species, with some highly differentiated genomic regions (“genomic differentiation islands”) spread throughout the genome. Differential levels of gene flow are proposed to account for this pattern, as genomic differentiation islands are suggested to be resistant to gene flow. Recent studies have also suggested that genomic differentiation islands could be explained by linked selection acting on genomic regions with low recombination rates. Here, we investigate genomic differentiation and gene‐flow patterns for autosomes using RAD‐seq data between two closely related species of long‐tailed tits (Aegithalos bonvaloti and A. fuliginosus) in both allopatric and contact zone populations. The results confirm recent or ongoing gene flow between these two species. However, there is little evidence that the genomic regions that were found to be highly differentiated between the contact zone populations are resistant to gene flow, suggesting that differential levels of gene flow is not the cause of the heterogeneous genomic differentiation. Linked selection may be the cause of genomic differentiation islands between the allopatric populations with no or very limited gene flow, but this could not account for the heterogeneous genomic differentiation between the contact zone populations, which show evidence of recent or ongoing gene flow.     

A genomic island linked to ecotype divergence in Atlantic cod

The genomic architecture underlying ecological divergence and ecological speciation with gene flow is still largely unknown for most organisms. One central question is whether divergence is genome‐wide or localized in ‘genomic mosaics’ during early stages when gene flow is still pronounced. Empirical work has so far been limited, and the relative impacts of gene flow and natural selection on genomic patterns have not been fully explored. Here, we use ecotypes of Atlantic cod to investigate genomic patterns of diversity and population differentiation in a natural system characterized by high gene flow and large effective population sizes, properties which theoretically could restrict divergence in local genomic regions. We identify a genomic region of strong population differentiation, extending over approximately 20 cM, between pairs of migratory and stationary ecotypes examined at two different localities. Furthermore, the region is characterized by markedly reduced levels of genetic diversity in migratory ecotype samples. The results highlight the genomic region, or ‘genomic island’, as potentially associated with ecological divergence and suggest the involvement of a selective sweep. Finally, we also confirm earlier findings of localized genomic differentiation in three other linkage groups associated with divergence among eastern Atlantic populations. Thus, although the underlying mechanisms are still unknown, the results suggest that ‘genomic mosaics’ of differentiation may even be found under high levels of gene flow and that marine fishes may provide insightful model systems for studying and identifying initial targets of selection during ecological divergence.     

Reevaluation of a classic phylogeographic barrier: new techniques reveal the influence of microgeographic climate variation on population divergence

We evaluated the mtDNA divergence and relationships within Geomys pinetis to assess the status of formerly recognized Geomys taxa. Additionally, we integrated new hypothesis‐based tests in ecological niche models (ENM) to provide greater insight into causes for divergence and potential barriers to gene flow in Southeastern United States (Alabama, Florida, and Georgia). Our DNA sequence dataset confirmed and strongly supported two distinct lineages within G. pinetis occurring east and west of the ARD. Divergence date estimates showed that eastern and western lineages diverged about 1.37 Ma (1.9 Ma–830 ka). Predicted distributions from ENMs were consistent with molecular data and defined each population east and west of the ARD with little overlap. Niche identity and background similarity tests were statistically significant suggesting that ENMs from eastern and western lineages are not identical or more similar than expected based on random localities drawn from the environmental background. ENMs also support the hypothesis that the ARD represents a ribbon of unsuitable climate between more suitable areas where these populations are distributed. The estimated age of divergence between eastern and western lineages of G. pinetis suggests that the divergence was driven by climatic conditions during Pleistocene glacial–interglacial cycles. The ARD at the contact zone of eastern and western lineages of G. pinetis forms a significant barrier promoting microgeographic isolation that helps maintain ecological and genetic divergence.     

Patterns of parasitism in monarch butterflies during the breeding season in eastern North America

1. Migratory behaviour can result in reduced prevalence of pathogens in host populations. Two hypotheses have been proposed to explain this relationship: (i) ‘migratory escape’, where migrants benefit from escaping pathogen accumulation in contaminated environments; and (ii) ‘migratory culling’, where the selective removal of infected individuals occurs during migration. 2. In the host–parasite system between the monarch butterfly (Danaus plexippus Linn.) and its obligate protozoan parasite Ophryocystis elektroscirrha (OE), there is evidence to support both hypotheses, particularly during the monarchs' autumn migration. However, these processes can operate simultaneously and could vary throughout the monarchs' annual migratory cycle. Assessing the relative strength for each hypothesis has not previously been done. 3. To evaluate both hypotheses, parasite infection prevalence was examined in monarchs sampled in eastern North America during April–September, and stable isotopes (δ²H, δ¹³C) were used to estimate natal origin and infer migration distance. There was stronger support for the migratory escape hypothesis, wherein infection prevalence increased over the breeding season and was higher at southern latitudes, where the breeding season tends to be longer compared with northern latitudes. Little support was found for the migratory culling hypothesis, as infection prevalence was similar whether monarchs travelled shorter or longer distances. 4. These results suggest that migration allows individuals to escape parasites not only during the autumn, as shown in previous work, but during the monarchs' spring and summer movements when they recolonise the breeding range. These results imply a potential fitness advantage to monarchs that migrate further north to exploit parasite‐free habitats.     

Longer wings for faster springs – wing length relates to spring phenology in a long‐distance migrant across its range

In migratory birds, morphological adaptations for efficient migratory flight often oppose morphological adaptations for efficient behavior during resident periods. This includes adaptations in wing shape for either flying long distances or foraging in the vegetation and in climate‐driven variation of body size. In addition, the timing of migratory flights and particularly the timely arrival at local breeding sites is crucial because fitness prospects depend on site‐specific phenology. Thus, adaptations for efficient long‐distance flights might be also related to conditions at destination areas. For an obligatory long‐distance migrant, the common nightingale, we verified that wing length as the aerodynamically important trait, but not structural body size increased from the western to the eastern parts of the species range. In contrast with expectation from aerodynamic theory, however, wing length did not increase with increasing migration distances. Instead, wing length was associated with the phenology at breeding destinations, namely the speed of local spring green‐up. We argue that longer wings are beneficial for adjusting migration speed to local conditions for birds breeding in habitats with fast spring green‐up and thus short optimal arrival periods. We suggest that the speed of spring green‐up at breeding sites is a fundamental variable determining the timing of migration that fine tune phenotypes in migrants across their range.     

Inversions and the origin of behavioral differences in cod

How does adaptation manage to occur in the face of overwhelming gene flow? One popular idea is that the suppression of recombination, for example the fixation of a chromosomal inversion, can maintain linkage disequilibrium between groups of locally adapted alleles that would otherwise be degraded by gene flow. This idea has captured the imagination of many geneticists and evolutionary biologists, but we still have only a basic understanding of its general importance. In this issue of Molecular Ecology, Kirubakaran et al. ( 2016 ) examine the role of recombination suppression in a particularly fascinating example of adaptation in the face of gene flow: the evolution of migratory differences between interbreeding populations of cod. Along the north coast of Norway, two types of cod breed in the near‐shore waters: a ‘stationary’ form that lives near the coast year round, and a ‘migratory’ form that lives far offshore and only returns to the coast to breed. Using a combination of approaches, Kirubakaran et al. ( 2016 ) deftly demonstrate that the migratory form has completely fixed two adjacent inversions containing a suite of genes closely connected to migratory behaviour and feeding differences. This work provides an excellent example of how recombination suppression can facilitate adaptive divergence, and helps us understand the geographic and temporal scales over which genomic structural variation evolves.     

Regional climate on the breeding grounds predicts variation in the natal origin of monarch butterflies overwintering in Mexico over 38 years

Addressing population declines of migratory insects requires linking populations across different portions of the annual cycle and understanding the effects of variation in weather and climate on productivity, recruitment, and patterns of long‐distance movement. We used stable H and C isotopes and geospatial modeling to estimate the natal origin of monarch butterflies (Danaus plexippus) in eastern North America using over 1000 monarchs collected over almost four decades at Mexican overwintering colonies. Multinomial regression was used to ascertain which climate‐related factors best‐predicted temporal variation in natal origin across six breeding regions. The region producing the largest proportion of overwintering monarchs was the US Midwest (mean annual proportion = 0.38; 95% CI: 0.36–0.41) followed by the north‐central (0.17; 0.14–0.18), northeast (0.15; 0.11–0.16), northwest (0.12; 0.12–0.16), southwest (0.11; 0.08–0.12), and southeast (0.08; 0.07–0.11) regions. There was no evidence of directional shifts in the relative contributions of different natal regions over time, which suggests these regions are comprising the same relative proportion of the overwintering population in recent years as in the mid‐1970s. Instead, interannual variation in the proportion of monarchs from each region covaried with climate, as measured by the Southern Oscillation Index and regional‐specific daily maximum temperature and precipitation, which together likely dictate larval development rates and food plant condition. Our results provide the first robust long‐term analysis of predictors of the natal origins of monarchs overwintering in Mexico. Conservation efforts on the breeding grounds focused on the Midwest region will likely have the greatest benefit to eastern North American migratory monarchs, but the population will likely remain sensitive to regional and stochastic weather patterns.     

Local adaptation does not lead to genome‐wide differentiation in lava flow lizards

Adaptation can occur with or without genome‐wide differentiation. If adaptive loci are linked to traits involved in reproductive isolation, genome‐wide divergence is likely, and speciation is possible. However, adaptation can also lead to phenotypic differentiation without genome‐wide divergence if levels of ongoing gene flow are high. Here, we use the replicated occurrence of melanism in lava flow lizards to assess the relationship between local adaptation and genome‐wide differentiation. We compare patterns of phenotypic and genomic divergence among lava flow and nonlava populations for three lizard species and three lava flows in the Chihuahuan Desert. We find that local phenotypic adaptation (melanism) is not typically accompanied by genome‐wide differentiation. Specifically, lava populations do not generally exhibit greater divergence from nonlava populations than expected by geography alone, regardless of whether the lava formation is 5,000 or 760,000 years old. We also infer that gene flow between lava and nonlava populations is ongoing in all lava populations surveyed. Recent work in the isolation by environment and ecological speciation literature suggests that environmentally driven genome‐wide differentiation is common in nature. However, local adaptation may often simply be local adaptation rather than an early stage of ecological speciation.     

Integrative use of spatial, genetic, and demographic analyses for investigating genetic connectivity between migratory, montane, and sedentary caribou herds

Genetic differentiation is generally assumed to be low in highly mobile species, but this simplistic view may obscure the complex conditions and mechanisms allowing genetic exchanges between specific populations. Here, we combined data from satellite-tracked migratory caribou (Rangifer tarandus), microsatellite markers, and demographic simulations to investigate gene flow mechanisms between seven caribou herds of eastern Canada. Our study included one montane, two migratory, and four sedentary herds. Satellite-tracking data indicated possibilities of high gene flow between migratory herds: overlap of their rutting ranges averaged 10% across years and 9.4% of females switched calving sites at least once in their lifetime. Some migratory individuals moved into the range of the sedentary herds, suggesting possibilities of gene flow between these herds. Genetic differentiation between herds was weak but significant (FST=0.015): migratory and montane herds were not significantly distinct (FST all9) than vice-versa (4Nm all<5), which suggests migratory herds had a demographic impact on sedentary herds. Demographic simulations showed that an effective immigration rate of 0.0005 was sufficient to obtain the empirical FST of 0.015, while a null immigration rate increased the simulated FST to >0.6. In conclusion, the weak genetic differentiation between herds cannot be obtained without some genetic exchanges among herds, as demonstrated by genetic and spatial data.     

Phylogeography of the Chinese endemic freshwater crab Sinopotamon acutum (Brachyura,Potamidae)

Phylogeographical inferences based on multiple molecular markers from different modes of inheritance (i.e. mtDNA and nucDNA) can help to explore drivers of current species divergence over different timescales and allow a deeper understanding of evolutionary processes. River systems and mountains, owing to their drainage networks and complex topography, are often a high priority when inferring external causes of phylogeographical patterns. Here, we selected the Chinese endemic freshwater crab Sinopotamon acutum inhabiting drainage networks across the Qinling–Daba Mountains, a watershed that divides northern from southern fauna in China, as a model species (1) to investigate whether river networks and mountain systems act as barriers in shaping the phylogeography of freshwater crabs and (2) to test the impact of historic environments and ecological habitats on crab distribution. Phylogenetic tree and network analyses based on mtDNA revealed divergence between eastern and western populations, but microsatellite DNA did not recover this. Frequent and male‐biased gene flow is evident. The adjacent tributaries, such as Weihe River 1 and Hanjiang River 1, although isolated by Qinling Mts, have low genetic variation in mtDNA. In addition, microsatellite DNA showed low variation between all rivers. Frequent gene flow, genetic variation and mitochondrial genetic structure in S. acutum indicated that the Qinling–Daba Mountains have had no effect on divergence in this species; instead, hydrological networks across these mountains serve as a continuous migration corridor for these crabs. Glacial refugia are probably responsible for the initial isolation of the eastern and western clades, and local adaptations after dispersal have further driven divergence. Cytonuclear discordance in phylogeographical patterns may be attributable to ongoing male‐biased gene flow.     

Genome-wide nuclear data confirm two species in the Alpine endemic land snail Noricella oreinos s.l. (Gastropoda,Hygromiidae)

The Austrian endemic land snail species Noricella oreinos (formerly Trochulus oreinos) occurs in the Northeastern Calcareous Alps at high elevations. Two morphologically highly similar subspecies N. o. oreinos and N. o. scheerpeltzi have been described. First analyses of mitochondrial and nuclear marker sequences indicated a high genetic divergence between them. In the present study, we aimed to assess gene flow between the two subspecies which should help to re-evaluate their taxonomic status. Sequence data and amplified fragment length polymorphism (AFLP) markers of 255 Noricella specimens covering the whole distribution range were analyzed. A clear geographic separation was found within the potential contact zone, the Haller Mauern mountain range. Samples of all western sites were part of the clade representing N. o. scheerpeltzi and almost all samples from the eastern sites clustered with N. o. oreinos. However, within two sampling sites of the eastern Haller Mauern, a few individuals possessed a COI sequence matching the N. o. oreinos clade whereas at the ITS2 locus they were heterozygous possessing the alleles of both taxa. Contrary to the ITS2 results indicating historical and/or ongoing hybridization, AFLP analyses of 202 individuals confirmed a clear separation of the two taxa congruent with the mitochondrial data. Although they occur on the same mountain range without any physical barrier, no indication of ongoing gene flow between the two taxa was found. Thus, we conclude that the two taxa are separate species N. oreinos and N. scheerpeltzi.