BACKGROUND
the release of a massive amount of radioactive materials from the fukushima dai-ichi nuclear power plant  to the surrounding environment on  <dig> march  <dig> and afterwards resulted in large-scale radioactive pollution worldwide and especially severe pollution in the tohoku and kanto districts of japan  <cit> .
both marine and forest ecosystems have been heavily polluted  <cit> , but scientific studies on biological impacts of this accident are still scarce.
yet, such studies are gradually accumulating now, which includes changes in abundance of animals, especially birds and butterflies, in the polluted areas  <cit> .
recently, low blood cell counts have been reported in wild japanese monkeys  <cit> .
in gall-forming aphids, severe morphological abnormalities have been documented from fukushima samples, which are rare from other samples  <cit> .
in some of these studies, insects played an important role as environmental indicators.
coincidentally, another insect, the pale grass blue butterfly, zizeeria maha, has been used to examine the biological effects of the accident  <cit> .
this small butterfly has many advantages over other animals as an environmental indicator  <cit> .
it is a multivoltine insect, having a life cycle of about a month from may to november in the tohoku and kanto districts.
because larvae of this butterfly cannot grow rapidly in winter, approximately six or seven generations occur per year in the tohoku and kanto districts.
this butterfly is completely dependent on a single host plant, oxalis corniculata.
partly because this plant is very small in height, this butterfly species lives on or near the surface of the ground throughout its life stages.
it thus responds strongly to changes in the ground surface environment.
additionally, methods for its rearing in a small laboratory space have been well developed  <cit> .
in this butterfly, morphological abnormalities and reduced wing size have been documented in field-caught adult samples from the polluted areas in  <dig>  <cit> .
moreover, these observations in the field populations have been supported by offspring  rearing experiments in the laboratory, resulting in a high incidence of deaths, morphological abnormalities, and growth retardation  <cit> .
furthermore, external and internal irradiation experiments have reproduced the field observations, including the high mortality rate, high morphological abnormality rate, and reduced wing size, strongly suggesting the causality of the pollution  <cit> .
also in that study, morphological abnormalities detected in the spring and fall of  <dig> were compared, showing a possible accumulation of genetic and physiological damage in the fall of  <dig>  <cit> .
the heritability of abnormal traits by the offspring generations was also demonstrated  <cit> .
however, long-term monitoring of the field populations is critical to correctly understand the impacts on pale grass blue butterfly populations in polluted areas.
following the high mortality and abnormality in  <dig>  <cit> ,  <dig> scenarios may be hypothesized.
first, the high mortality and abnormality are kept for the following years .
second, the populations experience even higher mortality and abnormality, eventually resulting in extinction .
third, the populations gradually gain normality .
temporal data were required to test these hypotheses.
in the present study, we present our 3-year monitoring results of the pale grass blue butterfly in the tohoku and kanto districts from  <dig> to  <dig>  we performed field surveys in the spring and fall every year, mainly visiting the following  <dig> localities: fukushima, motomiya, hirono, iwaki, takahagi , mito, and tsukuba .
we then examined the morphological abnormalities of the collected adults from the field populations , expressed as the adult abnormality rate .
in addition, we obtained the offspring generation  from the collected adults, and we obtained the total abnormality rate , which includes deaths at the larval, prepupal, and pupal stages and the morphological abnormalities of the surviving adults.
the offspring observations provided us with invaluable information on the field populations, including the heritability of abnormalities, and on a natural selection process that has occurred in the polluted environment.figure  <dig>
collection localities for the adult butterfly samples.
a map of japan.
minor collection localities for control samples are shown together with the fukushima dai-ichi nuclear power plant  in red.
distances from the fnpp are indicated by circles.
a map of the tohoku and kanto districts.
the seven major localities from which the adult samples were collected are shown by black dots.
takahagi and kita-ibaraki are treated as a single locality, “takahagi”, in this study because of their proximity.
minor localities are shown by open circles.
the fnpp is shown in red.
distances from the fnpp are indicated by circles.
RESULTS
the dynamics of the ground radiation dose
the ground radiation dose at the collection sites in the  <dig> localities was measured at the time of butterfly sample collection in  <dig> sampling attempts over 3 years.
the ground radiation dose reached its maximum level in the spring of  <dig> and declined afterwards , likely due to fast-decaying radionuclides.
however, even in the fall of  <dig>  considerable levels were detected, especially from fukushima and motomiya.
the level of hirono was also high.
it is important to note that the ground radiation doses in these localities in the fall of  <dig> were still higher than the doses in other localities in the spring of  <dig> figure  <dig>
dynamics of the radioactivity levels on the ground of the  <dig> major collection localities in 2011– <dig>   radioactivity measured with a scintillation counter .
radioactivity measured with a gm counter .
error bars indicate the mean ± se.
data point of takahagi in the fall of  <dig> was not obtained.
the aar dynamics of the field-collected adults
we monitored the aar of field-collected adults from the  <dig> localities in  <dig> sampling attempts over 3 years.
we first plotted the raw aar data from the  <dig> localities on a single graph .
considering that the normal level of aar is below 10% , the aar in the spring of  <dig> was higher than the normal level in hirono, fukushima, and mito.
an increase in the fall of  <dig> was then observed in many localities except tsukuba and mito.
notably, the localities with high levels of pollution, i.e., fukushima and motomiya, showed a clear peak in the fall of  <dig>  approaching 40%.
the localities with intermediate pollution levels, i.e., iwaki, hirono, and takahagi, showed similarly high aars both in the fall of  <dig> and in the spring of  <dig>  in contrast, the locality with the lowest contamination level, tsukuba, peaked later, in the spring of  <dig>  all localities showed normal aar levels in the fall of  <dig> and afterwards.
however, we noticed that mito behaved differently from the rest, at least during the spring and fall of  <dig>  which was apparent in the cluster analysis  and the multidimensional scaling plot .
based on these results, we excluded mito as an outlier from the subsequent analyses.figure  <dig>
dynamics of the adult abnormality rate  of the p generation from  <dig> localities in 2011– <dig>   the aar changes over time in 3 years.
statistically significant differences are indicated between the fall of  <dig> and the fall of  <dig>  after the exclusion of mito.
the total number of individuals collected in the  <dig> localities  and the expected number of generations after the nuclear accident are shown.
cluster analysis with a heat map.
the cluster of fukushima and motomiya  and that of other localities  are indicated.
additionally, the cluster of the fall of  <dig> and the spring of  <dig> and that of other time points are indicated.
mito is positioned as an outlier.
the aar changes of the fm and htit clusters over time.
the aars from other localities that can be considered non-contaminated are shown for comparison.
statistically significant differences among time points in multiple comparisons are indicated .
the total numbers of individuals collected in all localities, including the ones from non-contaminated localities , are indicated.
after excluding mito, we found statistically significant differences between the peak time-point  and the latest time-point  , supporting the visual interpretations of the dynamics discussed above .
the cluster analysis revealed that the  <dig> localities were segregated into two clusters: the fukushima-motomiya cluster  and the cluster including hirono, takahagi, iwaki, and tsukuba  .
the fm cluster corresponded to the inland cities relatively far from the ocean  to the northwest of the fnpp, and the htit cluster corresponded to the coastal cities  along the pacific ocean to the south of the fnpp.
the cluster analysis also revealed two time-point clusters: the cluster containing the fall of  <dig> and the spring of  <dig> and the cluster containing the rest of the time points , suggesting that major biological changes occurred during the fall of  <dig> and the spring of  <dig> in fukushima and motomiya.
based on the clustering results, we then calculated the aar values for the fm and htit clusters of all the combined samples from the included localities, which were plotted against time .
as expected, the fm cluster showed a higher aar than the htit cluster except in the fall of  <dig>  the fm cluster showed a steep decrease in the spring of  <dig> and also in the fall of  <dig>  but the htit cluster showed a large decrease only in the fall of  <dig>  after which the aars were at normal levels in both clusters.
the samples from other localities  that were minimally contaminated  were plotted together with the fm and htit clusters .
using these  <dig> groups, statistically significant differences were found among different time points in multiple comparisons .
because these other localities were at the minimal contamination level, we found that the aars of the normal populations were largely below 10%, ranging from 0% to  <dig> % with a median of  <dig> %.
similar results have been shown in a previous study  <cit> , where the aars of the field-collected samples had a mean value of  <dig> %.
the adult abnormalities were classified into four different categories , and their changes over time were examined in the  <dig> different localities .
overall, it seemed that abnormalities in the appendages occurred earlier, followed by abnormalities in wing color patterns and wing shape, especially in the relatively highly contaminated localities.
the relatively small level of wing-shape abnormalities may be due to the limited mobility of such individuals, resulting in a lower probability of being captured.
a cluster analysis on the wing shape abnormalities revealed two clusters: one cluster included fukushima, motomiya, and hirono , and the other cluster included iwaki, takahagi, and tsukuba  .
the incidence of abnormality in the fmh cluster peaked in the fall of  <dig>  whereas abnormality in the itt cluster peaked in the spring of  <dig> , showing a delay of peaking in the low-contamination localities.figure  <dig>
dynamics of the part-specific adult abnormal rate  of the p generation from  <dig> localities in 2011– <dig>   part-specific aars for wing shape, wing color patterns, appendages, and other parts in  <dig> localities.
cluster analysis with a heat map for the wing-shape aars.
two clusters  are indicated.
dynamics of the wing-shape aars in two clusters.
the fmh cluster and the itt cluster are shown.
part-specific aars for wing shape, wing color patterns, appendages, and other parts in  <dig> localities.
we then plotted the four categories of the abnormalities individually throughout the 3-year period.
the incidence of wing shape abnormality was irregular and sporadic over time .
abnormalities in wing color patterns peaked mainly in the spring of  <dig> , and abnormalities in the appendages peaked mainly in the fall of  <dig> .
the abnormalities of the other parts were quite irregular and sporadic .
wing shape abnormalities may be behaviorally lethal or sublethal, and such abnormal individuals cannot be captured frequently by us in the field, resulting in the sporadic and low-level occurrence.
in contrast, abnormalities in appendages and color patterns may be less lethal, and individuals with such abnormalities can still be captured by us in the field, resulting in the peaks at specific time-points.
this interpretation is in good agreement with the results of the f <dig> generation discussed later.
consistent with these results, the number of individuals caught per minute in the field was low in both the fall of  <dig> and in the spring of  <dig> .
these dynamics should be interpreted with caution because it is likely that the adult density is generally low in the spring and gradually increases toward the summer every year, at least in tokyo  <cit>  and most likely in the entire tohoku and kanto districts.
moreover, the number of individuals caught per minute is also affected by environmental factors including the weather of the day.
nevertheless, the relatively low number of individuals in the fall of  <dig> was notable compared with the number in the fall of  <dig> .
for example, in hirono, we were able to collect only very small number of individuals in the fall of  <dig> and in the spring of  <dig> despite the fine weather.figure  <dig>
dynamics of the number of collected individuals per minute in 2011– <dig>   seven localities over 3 years.
seven localities combined over 3 years.
arrows indicate changes over  <dig> fall points or  <dig> spring points that are comparable to one another.
error bars indicate the mean ± se.
in summary, the radiation levels peaked first in the spring of  <dig>  followed by an increase in the aars and a decrease in the number of individuals in the fall of  <dig>  then, the aar decreased and the number of individuals increased back to normal levels.
this chronological order supports the normalization hypothesis, rejecting the extended mortality hypothesis and the extinction hypothesis .
the tar and aar dynamics of the reared offspring
we next monitored the total ar  of the offspring  generation from the adults caught in the  <dig> localities .
the tar includes the deaths in the larval, prepupal, and pupal stages and the abnormalities in the surviving adults, which could be examined by rearing the offspring in the laboratory.
the tars peaked mostly either in the fall of  <dig> or in the spring of  <dig>  similar to the results of the aar of the parent generation shown above, with a significant difference between the fall of  <dig> and the spring of  <dig>  and between other two time-points.
however, we did not obtain clear clusters in the cluster analysis and the multi-dimensional scaling , suggesting that the tar patterns over time were similar among the localities monitored.
the decrease of the tar was relatively slow: the tar levels decreased back to normal in the spring of  <dig>  taking more time than the aar of the parent generation.
another noteworthy point was that the levels of the tars were rather high.
these results suggest a transgenerational character of abnormalities, at least from the p to f <dig> generations, throughout this time period and also suggest that selection against abnormal individuals occurred in the field throughout this time period.figure  <dig>
dynamics of the abnormality rate  in the offspring  generation in 2011– <dig>   the tar changes of  <dig> localities.
statistically significant differences between two time-points in multiple pairs  or multiple comparisons  are indicated in all panels in this figure.
time points with a lack of data are indicated by blank circles with dotted lines .
the aar changes in  <dig> localities.
the tar, aar, and aar changes in  <dig> localities combined.
we also plotted the aar of the f <dig> generation .
the aars of the f <dig> generation had higher values and peaked more sharply in the fall of  <dig> than the aar of the parent generation in all seven localities.
for comparison, we integrated all  <dig> localities  by considering that all samples were collected from a single tohoku-kanto area, and we plotted the aar of the p and f <dig> generations and the tar of the f <dig> generation together .
all  <dig> ars showed an overall similar pattern: a peak in the fall of  <dig> and a subsequent decrease.
however, it is notable that the tar peak was much higher than the peaks of aars and that the tar peak in the fall of  <dig> extended to the spring of  <dig>  the aars of the p and f <dig> generations were similar to each other in that they both showed a single peak in the fall of  <dig>  but they were clearly different in magnitude; the aar of the f <dig> generation was much higher.
these results confirmed the idea that the wild populations of this butterfly produced many abnormal individuals that were selected out before appearing as viable adults in the field.
as in the previous analysis of the parent generation, the adult abnormalities were classified into  <dig> categories , and the changes in the aars over time were examined .
the levels of aars were especially high for the wing shape in the fall of  <dig>  compared with the wing color patterns , the appendages , and the other parts .
the dynamics of the mortality rate  added further insights into what occurred in the polluted area.
when all  <dig> localities were combined as samples from a single collection area, the mr throughout immature life stages  showed the highest in the spring of  <dig>  scoring approximately 60%, and decreased afterwards to the normal level in the spring of  <dig> .
when  <dig> localities were examined separately, hirono notably scored more than 90% in the fall of  <dig> , suggesting that the large-scale death occurred in hirono at that time.
takahagi, mito, fukushima, and iwaki scored more than 50% in the spring of  <dig> figure  <dig>
dynamics of the mortality rate  in the offspring  generation in 2011– <dig>   dynamics of mr throughout immature life stages  of  <dig> localities combined.
locality-based dynamics of mrs throughout immature life stages.
dynamics of larval, prepupal, and pupal mrs of  <dig> localities combined.
dynamics of larval, prepupal, and pupal mrs per day of  <dig> localities combined.
when the mrs of developing life stages  were examined separately in all  <dig> localities combined, the pupal mr was highest in the spring of  <dig> and decreased sharply afterwards .
when the stage-specific mrs were simply divided by the number of days for each stage, prepupal death was notable in the spring of  <dig> .
the dying stage may be different between  <dig> and  <dig>  the main selection stage was likely the prepupal stage in the spring of  <dig> and then moved to the pupal and larval stages afterwards.
because we reared these larvae, prepupae, and pupae under standard laboratory conditions, where no artificial radionuclides were detected, and in the absence of any predators, the selection pressures against these immature stages were likely to be more severe in the field than in the laboratory.
we then examined the dynamics of the stage-specific mrs in  <dig> different localities .
most locations showed peak larval mortality in the spring of  <dig>  with the exception of hirono, at which larval mortality peaked in the fall of  <dig>  and of motomiya, at which larval mortality peaked in the fall of  <dig> .
at the prepupal stage, the highest level of mortality was found in the spring of  <dig> in most localities, again with the exception of hirono, at which prepupal mortality peaked in the fall of  <dig> .
at the pupal stage, most localities exhibited peak mortality in the spring of  <dig>  again with the exception of hirono, at which mortality peaked in the fall of  <dig> .
correlations of ars with the ground radiation level and with the distance
to investigate the possible causal involvement of radioactive pollution released by the fukushima nuclear accident in the ar dynamics in 2011– <dig>  we examined correlations of ars with the ground radiation dose at the collection sites and with the distance from the fnpp, although a similar analysis has already been performed for the  <dig> data in previous papers  <cit> .
radioactive materials released early, which mainly contained short-lived radionuclides, were dispersed in a concentric fashion, whereas radioactive materials released later, which mainly contained cesium, were dispersed mainly toward the northwestern cities  <cit> .
therefore, our categorization may help to distinguish the effects.
in the p generation, changes in pearson correlation coefficients over the  <dig> time points between the aar level and the ground radiation level and between the aar level and the distance were similar to each other, but with a slight delay in the distance .
correlation was strongest in the fall of  <dig> for the ground radiation and in the spring of  <dig> for the distance, both of which then decreased toward the fall of  <dig> or toward the spring of  <dig> figure  <dig>
correlation analysis of aar, aar, and tar.
dynamics of pearson correlation coefficients  between the aar of the field-collected samples  and the ground radiation dose  or the distance from the fnpp in 2011– <dig>   the aar levels of  <dig> localities in the fall of  <dig>   dynamics of pearson correlation coefficients  between the aar of the laboratory-reared samples  and the ground radiation dose  or the distance from the fnpp in 2011– <dig>   dynamics of pearson correlation coefficients  between the tar of the laboratory-reared samples  and the ground radiation dose  or the distance from the fnpp in 2011– <dig>
unexpectedly, the coefficients then increased again in the spring of  <dig> for the ground radiation and in the fall of  <dig> for the distance.
to understand what occurred, scatter plots were examined .
the relatively high correlation of aar with the ground radiation dose in the fall of  <dig> was disrupted mainly because of the fast decrease of the aars in the high-radiation localities .
by the fall of  <dig>  the aars in the low-radiation localities decreased, recreating relatively high correlation again, but with much lower aars.
similar dynamics, although less clear, were observed in the distance correlation .
to understand the latest state of the aars, we examined the aars of the adult samples that we collected in the fall of  <dig> from  <dig> localities .
the aars were relatively low in most localities but relatively high in motomiya and koriyama and very high in iitate, confirming the origin of the relatively high correlation in the fall of  <dig>
similar dynamics were obtained in the f <dig> generation with the aars  and with the tars , confirming the trends obtained in the p generation.
scatter plots also showed a similar trend .
however, the peak time difference, observed in the p generation, between the ground radiation dose and the distance  was not observed in the f <dig> generation .
discussion
in the present study, we described the spatial and temporal dynamics of the ars of the pale grass blue butterfly in the first 3 years after the fukushima nuclear accident.
the field sampling attempts were conducted in  <dig> localities   <dig> times a year.
there are no long-term study on the biological effects in the fukushima accident from the beginning of the accident covering several localities except this paper, and nothing similar will be published in the future.
importantly, in addition to the aars of the field populations, we recorded the tars and aars of the offspring generation, which were obtained under standard rearing conditions with neither external nor internal radiation exposure from artificial radionuclides.
therefore, we believe that the present study is invaluable to understand biological effects of the accident.
we witnessed the time course in which the disturbed populations regained their normal ar levels.
the ars do not consider the degrees of abnormality in a given individuals, but we believe that the degrees of abnormality parallel the ars over 3 years.
therefore, we assert that the normalization hypothesis, but not the extended mortality hypothesis and the extinction hypothesis, is correct .
because the spatial and temporal dynamics of the ars of the parent  and offspring  generations were largely similar to each other, we assert that these results are reasonably precise and accurate descriptions of the genetic and physiological effects that were accumulated over generations.
in this study, we do not know if the observed transgenerational effects are genetic or physiological.
because maternal effects do not have to be genetic, we do not know if dna of the p generation was damaged.
in any case, it should be noted that if there was no transgenerational effect representing the heritable damage from the parent generation, the tar of the offspring generation would be always at the normal level because the offspring generation was obtained and reared under non-contaminated laboratory conditions.
the experimental reproduction  of the field data  are highly important, especially in those studies that examine a rare field event.
it should also be noted that because we obtained tars by rearing larvae in okinawa using non-contaminated leaves, the actual tars of the field populations in polluted areas would be considerably higher than those of the laboratory-reared samples, considering the continuous and simultaneous external and internal radiation exposures in the field.
indeed, our previous studies demonstrated independent but similar effects of external and internal exposures in terms of deaths, morphological abnormalities, and small wing size in this butterfly  <cit> .
we provided evidence that the butterfly populations were severely disturbed, especially in the fall of  <dig> and the spring of  <dig>  the ar levels in the fall of  <dig> and the spring of  <dig> were notably higher than those at other time points, both in the parent and offspring generations.
cluster analysis revealed fukushima and motomiya to be clustered together based on the aar and fukushima, motomiya, and hirono based on the wing shape abnormalities.
these results imply that radioactive contamination in these  <dig> or  <dig> localities had deleterious impacts on butterflies.
the status of hirono is somewhat ambiguous but understandable in that hirono is located more closely to the fnpp than other localities examined here and thus was more contaminated by high-energy radionuclides  <cit> .
these results also imply that wing shape is likely sensitive to radiation exposure.
it is interesting that the temporal dynamics of the high- and low-pollution localities differed in the magnitude of peak ar but shared both the timing of the ar decline and the level of the steady state  in  <dig>  that is, the population accumulated the adverse effects transgenerationally in the first year but regained normality by the third year in all localities.
earlier achievements of the steady state in the low-pollution localities were not clearly observed.
the reasons for this are not known, but these data compellingly indicate that the butterfly populations in the localities that we surveyed were highly disturbed in  <dig> and regained normality by  <dig>
in examining the mortality rates of the f <dig> generation during the larval, prepupal, and pupal stages, we noticed that the prepupal death rate in the spring of  <dig> was notably high.
this may be considered an acute effect of radiation exposure.
we also noted peaks of the mortality rates of all  <dig> stages in the spring of  <dig> but not in the fall of  <dig>  as might have been expected from the dynamics of aar and aar.
this may be a transgenerational accumulation of damage or may be considered a chronic effect of radiation exposure.
these acute and chronic effects may be independent.
morphological abnormalities in adult individuals, frequently seen in the fall of  <dig>  can be interpreted not to be extremely severe, in that these individuals survived to the adult stage and that the most severe symptom was death, frequently seen in the spring of  <dig>
what is the cause for this transgenerational disturbance widely seen in the tohoku and kanto districts that occurred specifically in 2011?
the present field-based study is necessary, but not sufficient, to prove the causal role of the fukushima dai-ichi npp accident in this disturbance of the pale grass blue butterfly.
nevertheless, we observed high correlations between the ars and the ground radiation dose  and between the ars and the distance from the fnpp.
these results do not prove but clearly suggest the causality of the radioactive pollution to the temporal and spatial ar dynamics, which is even more compelling when we consider the preceding studies on this butterfly  <cit> .
higher correlations of ars with the distance than with the ground radiation dose that were obtained in the f <dig> generation can be understood if the biological impacts by the early burst mainly containing short-lived species were severer than those by the later release mainly containing cesium.
the exceptional behavior of hirono may also be due to the early exposure to high-energy radionuclides because of its proximity to the fnpp   <cit> .
thus, the time course of deaths proceeded from prepupae  in the spring of  <dig> to pupae in the spring of  <dig>  the prepupal stage is short, spanning approximately just one day, but it is a very important stage for metamorphosis.
the impacts of radiation are especially severe at the prepupal stage probably because of highly frequent cell division during metamorphosis with high metabolic activity.
in the case of the chernobyl accident, a long-term study has been conducted using a small mammal, the bank vole  <cit> .
chromosomal aberrations and embryonic lethality did not cease even after  <dig> generations in 10 years  <cit> .
we believe that differences from the butterfly dynamics may be because of differences in generation time, ecological status, amount and species of radionuclides released, and other factors.
assuming that the population disturbance observed above was caused by the radioactive materials from the collapsed fnpp, we propose two possible reasons for the falling phase of the ars: first, due to a decrease in the pollution level itself over time and second, due to adaptive evolution for resistance against radioactive stress.
the first possibility that a decrease in radioactivity levels over 3 years contributed to the recovery of the normal state of the populations appeared to be reasonable.
we believe that the impacts of the early, acute exposure to the fast-decaying radionuclides that existed immediately after the burst  were most likely higher than the chronic exposure to the slow decaying radionuclides that remained for years  <cit> .
furthermore, because the pale grass blue butterfly lives on or near the ground surface, especially at the larval stage, this butterfly is likely to be severely affected not only by γ-rays but also by β-rays  <cit> .
the importance of β-rays was implicated by a high count rate detected by a gm counter in the spring of  <dig> in the present study.
the gm counter’s count rates quickly decreased afterwards.
a similar but milder decrease was observed in the radioactivity level detected by a scintillation counter.
however, it is important to note that the radiation level at the high-pollution localities in the fall of  <dig> was still much higher than the levels at the low-pollution localities in the spring of  <dig>  nevertheless, the ars of all localities showed similar temporal dynamics over 3 years and converged to a similar, normal level in  <dig>  therefore, the decrease of the ars to the normal level cannot solely be attributed to the decrease in radiation levels.
the second possibility states that a decrease of ars was a result of adaptive evolution through natural selection against abnormal adults and radiation-susceptible larvae, prepupae, and pupae in the field.
because the tar and aar of the offspring generation reared under standard conditions with no artificial external and internal radiation exposure were much higher than the aar of the parent generation, the pollution likely acted as a source of strong selective pressure for this butterfly.
only normal and nearly normal individuals could be collected in the field, and this tendency appeared to be higher in more polluted areas.
consistent with this idea, the number of adult individuals caught per minute, which represents the population density, appeared to have decreased dramatically in the fall of  <dig> and the spring of  <dig>  the ar peaks were observed not in the spring of  <dig> but in the fall of  <dig> and the spring of  <dig>  this delay from the time of the accident would be best explained by the biological accumulation of adverse effects, followed by a falling phase that may be explained by adaptive evolution through the process of natural selection.
adaptation to radiation stress has been reported in the literature.
for example, transgenerational developmental responses to radiation have been documented in grasshoppers in chernobyl  <cit> , and possible adaptation to oxidative stress in birds as a consequence of the chernobyl accident has been reported recently  <cit> .
in regions with high natural radiation levels, radiation resistance has been observed in drosophila  <cit>  and other organisms  <cit> .
what is novel in the present study is the real-time documentation of the spatial and temporal dynamics of ongoing evolution.
the migration of animals from low-pollution areas to high-pollution areas may be another issue to consider.
although the mobility of this butterfly species is rather limited  <cit> , it could migrate many kilometers over generations, and the increase of the population  after a decline may partly be explained by immigrants from less affected areas.
however, the contribution of immigrants to the decrease of the ars would likely be small because immigrants from non-polluted areas would also face a high-radiation environment, resulting in high tars of the immigrants, similar to the native residents.
demonstrating the biological effects of long-term low-dose radiation exposure  due to nuclear pollution has scientific and political complications  <cit> .
in the case of the chernobyl accident, a paucity of scientific studies covering the early years after the accident generated confusion about the biological impacts of the accident  <cit> .
the fukushima case was the first opportunity in the history of mankind to rigorously study the biological effects of a large-scale nuclear accident from the very beginning of the accident.
it should be recognized that the results of this type of study are heavily dependent on the time period of a given study and on generation time, radiation resistance or susceptibility, and evolvability of the biological species of interest.
for example, this study retrospectively demonstrated the importance of an accurate description immediately after the nuclear accident to understand the biological impacts of the accident.
in other words, the present study implies that any study that did not cover the early years after a leakage of radioactive materials may have overlooked the real biological impacts because of a relatively quick adaptation process, especially in organisms that have a short-generation time, such as multivoltine insects.
likewise, any study on organisms that have a long generation time, including large mammals, may have also overlooked the biological impacts because the transgenerational effects are difficult to detect.
the pale grass blue butterfly has a short generation time, and the ar peak in the fall of  <dig> corresponds to the fifth generation after the accident.
the rise and fall of the ars was completed in approximately 2 years, in 11– <dig> generations.
by extrapolation, an organism that has a generation time of one year may show the worst effects in  <dig>  assuming that the human generation time is 20 years, the adverse effects in this most intelligent species would increase gradually toward  <dig>  if our results in the butterfly can be applied directly to humans.
if the biological phenomena we report here involve adaptive evolution in the polluted environment, it means that we witnessed the real-time adaptive evolution for radiation resistance in the pale grass blue butterfly.
this evolutionary process is likely driven by natural selection, as suggested by the difference in aars between the parent and offspring generations.
the radioactive pollutants themselves are harmful to organisms, which necessitates natural selection, although it is ironic to call this anthropogenically mediated selection “natural”.
precisely recorded real-time evolution in the field is still rare, but the well-known textbook version of evolution is the case of the peppered moth, biston betularia, which showed industrial melanism  <cit> .
furthermore, a case of interesting real-time evolution has recently been published  <cit> .
it is interesting to note that we previously demonstrated that the pale grass blue butterfly showed evolutionary changes in its color patterns at its northern range margin  <cit> .
this case occurred in geographically different regions and is clearly distinguishable, phenotypically and mechanically, from the fukushima case.
in the former, specific color patterns evolved  <cit> , whereas in the latter, radiation resistance evolved, although the resistance that evolved in the population in the polluted localities should be demonstrated physiologically by laboratory experiments in subsequent studies.
CONCLUSIONS
in this study, we presented the spatial and temporal dynamics of ars in the first 3 years of the fukushima nuclear accident, which clearly suggest the nuclear accident’s causation of the biological changes.
the rise and fall of the ars were recorded, which is likely due not only to the decrease of the radioactive materials in the environment but also to adaptive evolution of the butterfly through natural selection.
our study clearly showed the importance of prompt and continuous field monitoring after a nuclear accident.

 the release of a massive amount of radioactive materials from the fukushima dai-ichi nuclear power plant to the surrounding environment on march and afterwards resulted in large-scale radioactive pollution worldwide and especially severe pollution in the tohoku and kanto districts of japan. both marine and forest ecosystems have been heavily polluted, but scientific studies on biological impacts of this accident are still scarce. yet, such studies are gradually accumulating now, which includes changes in abundance of animals, especially birds and butterflies, in the polluted areas. recently, low blood cell counts have been reported in wild japanese monkeys. in gall-forming aphids, severe morphological abnormalities have been documented from fukushima samples, which are rare from other samples. in some of these studies, insects played an important role as environmental indicators. coincidentally, another insect, the pale grass blue butterfly, zizeeria maha, has been used to examine the biological effects of the accident. this small butterfly has many advantages over other animals as an environmental indicator. it is a multivoltine insect, having a life cycle of about a month from may to november in the tohoku and kanto districts. because larvae of this butterfly cannot grow rapidly in winter, approximately six or seven generations occur per year in the tohoku and kanto districts. this butterfly is completely dependent on a single host plant, oxalis corniculata. partly because this plant is very small in height, this butterfly species lives on or near the surface of the ground throughout its life stages. it thus responds strongly to changes in the ground surface environment. additionally, methods for its rearing in a small laboratory space have been well developed. in this butterfly, morphological abnormalities and reduced wing size have been documented in field-caught adult samples from the polluted areas in. moreover, these observations in the field populations have been supported by offspring rearing experiments in the laboratory, resulting in a high incidence of deaths, morphological abnormalities, and growth retardation. furthermore, external and internal irradiation experiments have reproduced the field observations, including the high mortality rate, high morphological abnormality rate, and reduced wing size, strongly suggesting the causality of the pollution. also in that study, morphological abnormalities detected in the spring and fall of were compared, showing a possible accumulation of genetic and physiological damage in the fall of. the heritability of abnormal traits by the offspring generations was also demonstrated. however, long-term monitoring of the field populations is critical to correctly understand the impacts on pale grass blue butterfly populations in polluted areas. following the high mortality and abnormality in, scenarios may be hypothesized. first, the high mortality and abnormality are kept for the following years. second, the populations experience even higher mortality and abnormality, eventually resulting in extinction. third, the populations gradually gain normality. temporal data were required to test these hypotheses. in the present study, we present our 3-year monitoring results of the pale grass blue butterfly in the tohoku and kanto districts from to we performed field surveys in the spring and fall every year, mainly visiting the following localities: fukushima, motomiya, hirono, iwaki, takahagi, mito, and tsukuba. we then examined the morphological abnormalities of the collected adults from the field populations, expressed as the adult abnormality rate. in addition, we obtained the offspring generation from the collected adults, and we obtained the total abnormality rate, which includes deaths at the larval, prepupal, and pupal stages and the morphological abnormalities of the surviving adults. the offspring observations provided us with invaluable information on the field populations, including the heritability of abnormalities, and on a natural selection process that has occurred in the polluted environment.figure collection localities for the adult butterfly samples. a map of japan. minor collection localities for control samples are shown together with the fukushima dai-ichi nuclear power plant in red. distances from the fnpp are indicated by circles. a map of the tohoku and kanto districts. the seven major localities from which the adult samples were collected are shown by black dots. takahagi and kita-ibaraki are treated as a single locality, “takahagi”, in this study because of their proximity. minor localities are shown by open circles. the fnpp is shown in red. distances from the fnpp are indicated by circles.  the dynamics of the ground radiation dose the ground radiation dose at the collection sites in the localities was measured at the time of butterfly sample collection in sampling attempts over 3 years. the ground radiation dose reached its maximum level in the spring of and declined afterwards, likely due to fast-decaying radionuclides. however, even in the fall of considerable levels were detected, especially from fukushima and motomiya. the level of hirono was also high. it is important to note that the ground radiation doses in these localities in the fall of were still higher than the doses in other localities in the spring of figure dynamics of the radioactivity levels on the ground of the major collection localities in 2011– radioactivity measured with a scintillation counter. radioactivity measured with a gm counter. error bars indicate the mean ± se. data point of takahagi in the fall of was not obtained. the aar dynamics of the field-collected adults we monitored the aar of field-collected adults from the localities in sampling attempts over 3 years. we first plotted the raw aar data from the localities on a single graph. considering that the normal level of aar is below 10%, the aar in the spring of was higher than the normal level in hirono, fukushima, and mito. an increase in the fall of was then observed in many localities except tsukuba and mito. notably, the localities with high levels of pollution, i.e., fukushima and motomiya, showed a clear peak in the fall of approaching 40%. the localities with intermediate pollution levels, i.e., iwaki, hirono, and takahagi, showed similarly high aars both in the fall of and in the spring of in contrast, the locality with the lowest contamination level, tsukuba, peaked later, in the spring of all localities showed normal aar levels in the fall of and afterwards. however, we noticed that mito behaved differently from the rest, at least during the spring and fall of which was apparent in the cluster analysis and the multidimensional scaling plot. based on these results, we excluded mito as an outlier from the subsequent analyses.figure dynamics of the adult abnormality rate of the p generation from localities in 2011– the aar changes over time in 3 years. statistically significant differences are indicated between the fall of and the fall of after the exclusion of mito. the total number of individuals collected in the localities and the expected number of generations after the nuclear accident are shown. cluster analysis with a heat map. the cluster of fukushima and motomiya and that of other localities are indicated. additionally, the cluster of the fall of and the spring of and that of other time points are indicated. mito is positioned as an outlier. the aar changes of the fm and htit clusters over time. the aars from other localities that can be considered non-contaminated are shown for comparison. statistically significant differences among time points in multiple comparisons are indicated. the total numbers of individuals collected in all localities, including the ones from non-contaminated localities, are indicated. after excluding mito, we found statistically significant differences between the peak time-point and the latest time-point, supporting the visual interpretations of the dynamics discussed above. the cluster analysis revealed that the localities were segregated into two clusters: the fukushima-motomiya cluster and the cluster including hirono, takahagi, iwaki, and tsukuba. the fm cluster corresponded to the inland cities relatively far from the ocean to the northwest of the fnpp, and the htit cluster corresponded to the coastal cities along the pacific ocean to the south of the fnpp. the cluster analysis also revealed two time-point clusters: the cluster containing the fall of and the spring of and the cluster containing the rest of the time points, suggesting that major biological changes occurred during the fall of and the spring of in fukushima and motomiya. based on the clustering results, we then calculated the aar values for the fm and htit clusters of all the combined samples from the included localities, which were plotted against time. as expected, the fm cluster showed a higher aar than the htit cluster except in the fall of the fm cluster showed a steep decrease in the spring of and also in the fall of but the htit cluster showed a large decrease only in the fall of after which the aars were at normal levels in both clusters. the samples from other localities that were minimally contaminated were plotted together with the fm and htit clusters. using these groups, statistically significant differences were found among different time points in multiple comparisons. because these other localities were at the minimal contamination level, we found that the aars of the normal populations were largely below 10%, ranging from 0% to % with a median of %. similar results have been shown in a previous study, where the aars of the field-collected samples had a mean value of %. the adult abnormalities were classified into four different categories, and their changes over time were examined in the different localities. overall, it seemed that abnormalities in the appendages occurred earlier, followed by abnormalities in wing color patterns and wing shape, especially in the relatively highly contaminated localities. the relatively small level of wing-shape abnormalities may be due to the limited mobility of such individuals, resulting in a lower probability of being captured. a cluster analysis on the wing shape abnormalities revealed two clusters: one cluster included fukushima, motomiya, and hirono, and the other cluster included iwaki, takahagi, and tsukuba. the incidence of abnormality in the fmh cluster peaked in the fall of whereas abnormality in the itt cluster peaked in the spring of, showing a delay of peaking in the low-contamination localities.figure dynamics of the part-specific adult abnormal rate of the p generation from localities in 2011– part-specific aars for wing shape, wing color patterns, appendages, and other parts in localities. cluster analysis with a heat map for the wing-shape aars. two clusters are indicated. dynamics of the wing-shape aars in two clusters. the fmh cluster and the itt cluster are shown. part-specific aars for wing shape, wing color patterns, appendages, and other parts in localities. we then plotted the four categories of the abnormalities individually throughout the 3-year period. the incidence of wing shape abnormality was irregular and sporadic over time. abnormalities in wing color patterns peaked mainly in the spring of, and abnormalities in the appendages peaked mainly in the fall of. the abnormalities of the other parts were quite irregular and sporadic. wing shape abnormalities may be behaviorally lethal or sublethal, and such abnormal individuals cannot be captured frequently by us in the field, resulting in the sporadic and low-level occurrence. in contrast, abnormalities in appendages and color patterns may be less lethal, and individuals with such abnormalities can still be captured by us in the field, resulting in the peaks at specific time-points. this interpretation is in good agreement with the results of the f generation discussed later. consistent with these results, the number of individuals caught per minute in the field was low in both the fall of and in the spring of. these dynamics should be interpreted with caution because it is likely that the adult density is generally low in the spring and gradually increases toward the summer every year, at least in tokyo and most likely in the entire tohoku and kanto districts. moreover, the number of individuals caught per minute is also affected by environmental factors including the weather of the day. nevertheless, the relatively low number of individuals in the fall of was notable compared with the number in the fall of. for example, in hirono, we were able to collect only very small number of individuals in the fall of and in the spring of despite the fine weather.figure dynamics of the number of collected individuals per minute in 2011– seven localities over 3 years. seven localities combined over 3 years. arrows indicate changes over fall points or spring points that are comparable to one another. error bars indicate the mean ± se. in summary, the radiation levels peaked first in the spring of followed by an increase in the aars and a decrease in the number of individuals in the fall of then, the aar decreased and the number of individuals increased back to normal levels. this chronological order supports the normalization hypothesis, rejecting the extended mortality hypothesis and the extinction hypothesis. the tar and aar dynamics of the reared offspring we next monitored the total ar of the offspring generation from the adults caught in the localities. the tar includes the deaths in the larval, prepupal, and pupal stages and the abnormalities in the surviving adults, which could be examined by rearing the offspring in the laboratory. the tars peaked mostly either in the fall of or in the spring of similar to the results of the aar of the parent generation shown above, with a significant difference between the fall of and the spring of and between other two time-points. however, we did not obtain clear clusters in the cluster analysis and the multi-dimensional scaling, suggesting that the tar patterns over time were similar among the localities monitored. the decrease of the tar was relatively slow: the tar levels decreased back to normal in the spring of taking more time than the aar of the parent generation. another noteworthy point was that the levels of the tars were rather high. these results suggest a transgenerational character of abnormalities, at least from the p to f generations, throughout this time period and also suggest that selection against abnormal individuals occurred in the field throughout this time period.figure dynamics of the abnormality rate in the offspring generation in 2011– the tar changes of localities. statistically significant differences between two time-points in multiple pairs or multiple comparisons are indicated in all panels in this figure. time points with a lack of data are indicated by blank circles with dotted lines. the aar changes in localities. the tar, aar, and aar changes in localities combined. we also plotted the aar of the f generation. the aars of the f generation had higher values and peaked more sharply in the fall of than the aar of the parent generation in all seven localities. for comparison, we integrated all localities by considering that all samples were collected from a single tohoku-kanto area, and we plotted the aar of the p and f generations and the tar of the f generation together. all ars showed an overall similar pattern: a peak in the fall of and a subsequent decrease. however, it is notable that the tar peak was much higher than the peaks of aars and that the tar peak in the fall of extended to the spring of the aars of the p and f generations were similar to each other in that they both showed a single peak in the fall of but they were clearly different in magnitude; the aar of the f generation was much higher. these results confirmed the idea that the wild populations of this butterfly produced many abnormal individuals that were selected out before appearing as viable adults in the field. as in the previous analysis of the parent generation, the adult abnormalities were classified into categories, and the changes in the aars over time were examined. the levels of aars were especially high for the wing shape in the fall of compared with the wing color patterns, the appendages, and the other parts. the dynamics of the mortality rate added further insights into what occurred in the polluted area. when all localities were combined as samples from a single collection area, the mr throughout immature life stages showed the highest in the spring of scoring approximately 60%, and decreased afterwards to the normal level in the spring of. when localities were examined separately, hirono notably scored more than 90% in the fall of, suggesting that the large-scale death occurred in hirono at that time. takahagi, mito, fukushima, and iwaki scored more than 50% in the spring of figure dynamics of the mortality rate in the offspring generation in 2011– dynamics of mr throughout immature life stages of localities combined. locality-based dynamics of mrs throughout immature life stages. dynamics of larval, prepupal, and pupal mrs of localities combined. dynamics of larval, prepupal, and pupal mrs per day of localities combined. when the mrs of developing life stages were examined separately in all localities combined, the pupal mr was highest in the spring of and decreased sharply afterwards. when the stage-specific mrs were simply divided by the number of days for each stage, prepupal death was notable in the spring of. the dying stage may be different between and the main selection stage was likely the prepupal stage in the spring of and then moved to the pupal and larval stages afterwards. because we reared these larvae, prepupae, and pupae under standard laboratory conditions, where no artificial radionuclides were detected, and in the absence of any predators, the selection pressures against these immature stages were likely to be more severe in the field than in the laboratory. we then examined the dynamics of the stage-specific mrs in different localities. most locations showed peak larval mortality in the spring of with the exception of hirono, at which larval mortality peaked in the fall of and of motomiya, at which larval mortality peaked in the fall of. at the prepupal stage, the highest level of mortality was found in the spring of in most localities, again with the exception of hirono, at which prepupal mortality peaked in the fall of. at the pupal stage, most localities exhibited peak mortality in the spring of again with the exception of hirono, at which mortality peaked in the fall of. correlations of ars with the ground radiation level and with the distance to investigate the possible causal involvement of radioactive pollution released by the fukushima nuclear accident in the ar dynamics in 2011– we examined correlations of ars with the ground radiation dose at the collection sites and with the distance from the fnpp, although a similar analysis has already been performed for the data in previous papers. radioactive materials released early, which mainly contained short-lived radionuclides, were dispersed in a concentric fashion, whereas radioactive materials released later, which mainly contained cesium, were dispersed mainly toward the northwestern cities. therefore, our categorization may help to distinguish the effects. in the p generation, changes in pearson correlation coefficients over the time points between the aar level and the ground radiation level and between the aar level and the distance were similar to each other, but with a slight delay in the distance. correlation was strongest in the fall of for the ground radiation and in the spring of for the distance, both of which then decreased toward the fall of or toward the spring of figure correlation analysis of aar, aar, and tar. dynamics of pearson correlation coefficients between the aar of the field-collected samples and the ground radiation dose or the distance from the fnpp in 2011– the aar levels of localities in the fall of dynamics of pearson correlation coefficients between the aar of the laboratory-reared samples and the ground radiation dose or the distance from the fnpp in 2011– dynamics of pearson correlation coefficients between the tar of the laboratory-reared samples and the ground radiation dose or the distance from the fnpp in 2011– unexpectedly, the coefficients then increased again in the spring of for the ground radiation and in the fall of for the distance. to understand what occurred, scatter plots were examined. the relatively high correlation of aar with the ground radiation dose in the fall of was disrupted mainly because of the fast decrease of the aars in the high-radiation localities. by the fall of the aars in the low-radiation localities decreased, recreating relatively high correlation again, but with much lower aars. similar dynamics, although less clear, were observed in the distance correlation. to understand the latest state of the aars, we examined the aars of the adult samples that we collected in the fall of from localities. the aars were relatively low in most localities but relatively high in motomiya and koriyama and very high in iitate, confirming the origin of the relatively high correlation in the fall of similar dynamics were obtained in the f generation with the aars and with the tars, confirming the trends obtained in the p generation. scatter plots also showed a similar trend. however, the peak time difference, observed in the p generation, between the ground radiation dose and the distance was not observed in the f generation. discussion in the present study, we described the spatial and temporal dynamics of the ars of the pale grass blue butterfly in the first 3 years after the fukushima nuclear accident. the field sampling attempts were conducted in localities times a year. there are no long-term study on the biological effects in the fukushima accident from the beginning of the accident covering several localities except this paper, and nothing similar will be published in the future. importantly, in addition to the aars of the field populations, we recorded the tars and aars of the offspring generation, which were obtained under standard rearing conditions with neither external nor internal radiation exposure from artificial radionuclides. therefore, we believe that the present study is invaluable to understand biological effects of the accident. we witnessed the time course in which the disturbed populations regained their normal ar levels. the ars do not consider the degrees of abnormality in a given individuals, but we believe that the degrees of abnormality parallel the ars over 3 years. therefore, we assert that the normalization hypothesis, but not the extended mortality hypothesis and the extinction hypothesis, is correct. because the spatial and temporal dynamics of the ars of the parent and offspring generations were largely similar to each other, we assert that these results are reasonably precise and accurate descriptions of the genetic and physiological effects that were accumulated over generations. in this study, we do not know if the observed transgenerational effects are genetic or physiological. because maternal effects do not have to be genetic, we do not know if dna of the p generation was damaged. in any case, it should be noted that if there was no transgenerational effect representing the heritable damage from the parent generation, the tar of the offspring generation would be always at the normal level because the offspring generation was obtained and reared under non-contaminated laboratory conditions. the experimental reproduction of the field data are highly important, especially in those studies that examine a rare field event. it should also be noted that because we obtained tars by rearing larvae in okinawa using non-contaminated leaves, the actual tars of the field populations in polluted areas would be considerably higher than those of the laboratory-reared samples, considering the continuous and simultaneous external and internal radiation exposures in the field. indeed, our previous studies demonstrated independent but similar effects of external and internal exposures in terms of deaths, morphological abnormalities, and small wing size in this butterfly. we provided evidence that the butterfly populations were severely disturbed, especially in the fall of and the spring of the ar levels in the fall of and the spring of were notably higher than those at other time points, both in the parent and offspring generations. cluster analysis revealed fukushima and motomiya to be clustered together based on the aar and fukushima, motomiya, and hirono based on the wing shape abnormalities. these results imply that radioactive contamination in these or localities had deleterious impacts on butterflies. the status of hirono is somewhat ambiguous but understandable in that hirono is located more closely to the fnpp than other localities examined here and thus was more contaminated by high-energy radionuclides. these results also imply that wing shape is likely sensitive to radiation exposure. it is interesting that the temporal dynamics of the high- and low-pollution localities differed in the magnitude of peak ar but shared both the timing of the ar decline and the level of the steady state in that is, the population accumulated the adverse effects transgenerationally in the first year but regained normality by the third year in all localities. earlier achievements of the steady state in the low-pollution localities were not clearly observed. the reasons for this are not known, but these data compellingly indicate that the butterfly populations in the localities that we surveyed were highly disturbed in and regained normality by in examining the mortality rates of the f generation during the larval, prepupal, and pupal stages, we noticed that the prepupal death rate in the spring of was notably high. this may be considered an acute effect of radiation exposure. we also noted peaks of the mortality rates of all stages in the spring of but not in the fall of as might have been expected from the dynamics of aar and aar. this may be a transgenerational accumulation of damage or may be considered a chronic effect of radiation exposure. these acute and chronic effects may be independent. morphological abnormalities in adult individuals, frequently seen in the fall of can be interpreted not to be extremely severe, in that these individuals survived to the adult stage and that the most severe symptom was death, frequently seen in the spring of what is the cause for this transgenerational disturbance widely seen in the tohoku and kanto districts that occurred specifically in 2011? the present field-based study is necessary, but not sufficient, to prove the causal role of the fukushima dai-ichi npp accident in this disturbance of the pale grass blue butterfly. nevertheless, we observed high correlations between the ars and the ground radiation dose and between the ars and the distance from the fnpp. these results do not prove but clearly suggest the causality of the radioactive pollution to the temporal and spatial ar dynamics, which is even more compelling when we consider the preceding studies on this butterfly. higher correlations of ars with the distance than with the ground radiation dose that were obtained in the f generation can be understood if the biological impacts by the early burst mainly containing short-lived species were severer than those by the later release mainly containing cesium. the exceptional behavior of hirono may also be due to the early exposure to high-energy radionuclides because of its proximity to the fnpp. thus, the time course of deaths proceeded from prepupae in the spring of to pupae in the spring of the prepupal stage is short, spanning approximately just one day, but it is a very important stage for metamorphosis. the impacts of radiation are especially severe at the prepupal stage probably because of highly frequent cell division during metamorphosis with high metabolic activity. in the case of the chernobyl accident, a long-term study has been conducted using a small mammal, the bank vole. chromosomal aberrations and embryonic lethality did not cease even after generations in 10 years. we believe that differences from the butterfly dynamics may be because of differences in generation time, ecological status, amount and species of radionuclides released, and other factors. assuming that the population disturbance observed above was caused by the radioactive materials from the collapsed fnpp, we propose two possible reasons for the falling phase of the ars: first, due to a decrease in the pollution level itself over time and second, due to adaptive evolution for resistance against radioactive stress. the first possibility that a decrease in radioactivity levels over 3 years contributed to the recovery of the normal state of the populations appeared to be reasonable. we believe that the impacts of the early, acute exposure to the fast-decaying radionuclides that existed immediately after the burst were most likely higher than the chronic exposure to the slow decaying radionuclides that remained for years. furthermore, because the pale grass blue butterfly lives on or near the ground surface, especially at the larval stage, this butterfly is likely to be severely affected not only by γ-rays but also by β-rays. the importance of β-rays was implicated by a high count rate detected by a gm counter in the spring of in the present study. the gm counter’s count rates quickly decreased afterwards. a similar but milder decrease was observed in the radioactivity level detected by a scintillation counter. however, it is important to note that the radiation level at the high-pollution localities in the fall of was still much higher than the levels at the low-pollution localities in the spring of nevertheless, the ars of all localities showed similar temporal dynamics over 3 years and converged to a similar, normal level in therefore, the decrease of the ars to the normal level cannot solely be attributed to the decrease in radiation levels. the second possibility states that a decrease of ars was a result of adaptive evolution through natural selection against abnormal adults and radiation-susceptible larvae, prepupae, and pupae in the field. because the tar and aar of the offspring generation reared under standard conditions with no artificial external and internal radiation exposure were much higher than the aar of the parent generation, the pollution likely acted as a source of strong selective pressure for this butterfly. only normal and nearly normal individuals could be collected in the field, and this tendency appeared to be higher in more polluted areas. consistent with this idea, the number of adult individuals caught per minute, which represents the population density, appeared to have decreased dramatically in the fall of and the spring of the ar peaks were observed not in the spring of but in the fall of and the spring of this delay from the time of the accident would be best explained by the biological accumulation of adverse effects, followed by a falling phase that may be explained by adaptive evolution through the process of natural selection. adaptation to radiation stress has been reported in the literature. for example, transgenerational developmental responses to radiation have been documented in grasshoppers in chernobyl, and possible adaptation to oxidative stress in birds as a consequence of the chernobyl accident has been reported recently. in regions with high natural radiation levels, radiation resistance has been observed in drosophila and other organisms. what is novel in the present study is the real-time documentation of the spatial and temporal dynamics of ongoing evolution. the migration of animals from low-pollution areas to high-pollution areas may be another issue to consider. although the mobility of this butterfly species is rather limited, it could migrate many kilometers over generations, and the increase of the population after a decline may partly be explained by immigrants from less affected areas. however, the contribution of immigrants to the decrease of the ars would likely be small because immigrants from non-polluted areas would also face a high-radiation environment, resulting in high tars of the immigrants, similar to the native residents. demonstrating the biological effects of long-term low-dose radiation exposure due to nuclear pollution has scientific and political complications. in the case of the chernobyl accident, a paucity of scientific studies covering the early years after the accident generated confusion about the biological impacts of the accident. the fukushima case was the first opportunity in the history of mankind to rigorously study the biological effects of a large-scale nuclear accident from the very beginning of the accident. it should be recognized that the results of this type of study are heavily dependent on the time period of a given study and on generation time, radiation resistance or susceptibility, and evolvability of the biological species of interest. for example, this study retrospectively demonstrated the importance of an accurate description immediately after the nuclear accident to understand the biological impacts of the accident. in other words, the present study implies that any study that did not cover the early years after a leakage of radioactive materials may have overlooked the real biological impacts because of a relatively quick adaptation process, especially in organisms that have a short-generation time, such as multivoltine insects. likewise, any study on organisms that have a long generation time, including large mammals, may have also overlooked the biological impacts because the transgenerational effects are difficult to detect. the pale grass blue butterfly has a short generation time, and the ar peak in the fall of corresponds to the fifth generation after the accident. the rise and fall of the ars was completed in approximately 2 years, in 11– generations. by extrapolation, an organism that has a generation time of one year may show the worst effects in assuming that the human generation time is 20 years, the adverse effects in this most intelligent species would increase gradually toward if our results in the butterfly can be applied directly to humans. if the biological phenomena we report here involve adaptive evolution in the polluted environment, it means that we witnessed the real-time adaptive evolution for radiation resistance in the pale grass blue butterfly. this evolutionary process is likely driven by natural selection, as suggested by the difference in aars between the parent and offspring generations. the radioactive pollutants themselves are harmful to organisms, which necessitates natural selection, although it is ironic to call this anthropogenically mediated selection “natural”. precisely recorded real-time evolution in the field is still rare, but the well-known textbook version of evolution is the case of the peppered moth, biston betularia, which showed industrial melanism. furthermore, a case of interesting real-time evolution has recently been published. it is interesting to note that we previously demonstrated that the pale grass blue butterfly showed evolutionary changes in its color patterns at its northern range margin. this case occurred in geographically different regions and is clearly distinguishable, phenotypically and mechanically, from the fukushima case. in the former, specific color patterns evolved, whereas in the latter, radiation resistance evolved, although the resistance that evolved in the population in the polluted localities should be demonstrated physiologically by laboratory experiments in subsequent studies.  in this study, we presented the spatial and temporal dynamics of ars in the first 3 years of the fukushima nuclear accident, which clearly suggest the nuclear accident’s causation of the biological changes. the rise and fall of the ars were recorded, which is likely due not only to the decrease of the radioactive materials in the environment but also to adaptive evolution of the butterfly through natural selection. our study clearly showed the importance of prompt and continuous field monitoring after a nuclear accident. 
similar results were obtained not only in the spatiotemporal dynamics of the number of individuals caught per minute but also in the temporal dynamics of the correlation coefficient between the adult abnormality rate and the ground radiation dose or the distance from the power plant. long-term monitoring of the biological impacts of the radioactive pollution caused by the fukushima nuclear accident in march is required to understand what has occurred in organisms living in the polluted areas. these results demonstrated an occurrence and an accumulation of adverse physiological and genetic effects in early generations, followed by their decrease and leveling off at a normal level, providing the most comprehensive record of biological dynamics after a nuclear accident available today. in the offspring generation, the total ars, which include deaths at the larval, prepupal, and pupal stages and morphological abnormalities at the adult stage, peaked either in the fall of or in the spring of with much higher levels than those of the parent field populations, suggesting that high incidence of deaths and abnormalities might have occurred in the field populations. importantly, the elevated ars of the field and offspring populations settled back to a normal level by the fall of and by the spring of respectively. this study also indicates the importance of considering generation time and adaptive evolution in evaluating the biological impacts of artificial pollution in wild organisms. here, we investigated spatial and temporal changes of the abnormality rate in both field-caught adult populations and laboratory-reared offspring populations of the pale grass blue butterfly, zizeeria maha, which has generation time of approximately one month. adaptive evolutionfukushima nuclear accidentlong-term monitoringpale grass blue butterflyradioactive contaminationissue-copyright-statement© the author 2015
 long-term monitoring of the biological impacts of the radioactive pollution caused by the fukushima nuclear accident in march is required to understand what has occurred in organisms living in the polluted areas. here, we investigated spatial and temporal changes of the abnormality rate in both field-caught adult populations and laboratory-reared offspring populations of the pale grass blue butterfly, zizeeria maha, which has generation time of approximately one month. we monitored localities every spring and fall over 3 years.  the adult ars of these localities quickly increased and peaked in the fall of which was not observed in non-contaminated localities. in the offspring generation, the total ars, which include deaths at the larval, prepupal, and pupal stages and morphological abnormalities at the adult stage, peaked either in the fall of or in the spring of with much higher levels than those of the parent field populations, suggesting that high incidence of deaths and abnormalities might have occurred in the field populations. importantly, the elevated ars of the field and offspring populations settled back to a normal level by the fall of and by the spring of respectively. similar results were obtained not only in the spatiotemporal dynamics of the number of individuals caught per minute but also in the temporal dynamics of the correlation coefficient between the adult abnormality rate and the ground radiation dose or the distance from the power plant.  these results demonstrated an occurrence and an accumulation of adverse physiological and genetic effects in early generations, followed by their decrease and leveling off at a normal level, providing the most comprehensive record of biological dynamics after a nuclear accident available today. this study also indicates the importance of considering generation time and adaptive evolution in evaluating the biological impacts of artificial pollution in wild organisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords adaptive evolutionfukushima nuclear accidentlong-term monitoringpale grass blue butterflyradioactive contaminationissue-copyright-statement© the author 2015 