The first record of An. belenrae in Japan
Our surveys from 2001 to 2015 revealed a significant change in the distribution range of the An. hyrcanus group in Hokkaido reported in the 1980s [3–6], including the first record of An. belenrae in Japan. Two larvae collected in the Kushiro Wetland in 2004 were tentatively named An. sinensis Kushiro strain, based solely on the morphological characteristics of the emerged adults. However, phylogenetic trees constructed using ITS2 sequence revealed that this An. sinensis Kushiro strain formed a robust clade that was clearly different from the clades of An. sinensis and other Anopheles species. Interestingly, the ITS2 sequence of the Kushiro strain was not identical to that of the An. sinensis strains collected in southern Japan, outside Hokkaido but to that of An. belenrae, a new strain reported in South Korea in 2005 . The Kushiro strain could confidently be included in the An. belenrae cluster because of the absence of intraspecific divergence as mentioned above. This species was consistently found in the Kushiro Wetland after the first detection in 2004. In contrast, An. belenrae was not found outside Hokkaido in our 15-year nationwide survey. Thus, we concluded that this species is restricted to the Kushiro Wetland in Hokkaido.
The Kushiro Wetland is the largest marshland/wetland in Japan and is located in the Kushiro Plain. The Kushiro Wetland has been the focus of nature conservation efforts since before World War II, was registered as a Ramsar site in 1980 and designated as a national park in 1987. It is also famous for being the breeding ground for Japanese Cranes, Grus japonensis, and many other wild birds and a protected area for natural monuments, birds, and animals; thus, land development is strictly regulated. In South Korea, An. belenrae is found in the northern part of the country near the border with North Korea [24, 38, 39]. In China, An. belenrae is reportedly distributed in Shandong and Liaoning Provinces in northeast China, facing the Korean Peninsula . These areas are not only geographically close to Japan, but may also have similarities in climate, vegetation, and some environmental factors with the Kushiro Wetland. However, further investigation is needed to compare the morphological characteristics of Japanese and Korean An. belenrae, and to determine the distribution of this species in locations outside Hokkaido in Japan. We hope that ecological and evolutionary factors impacting the emergence of An. belenrae will be elucidated with the development of molecular biological technology.
No information of An. sinensis from Hokkaido
The next noteworthy finding was the disappearance of An. sinensis from Hokkaido. In previous surveys, An. sinensis was generally distributed throughout Hokkaido [3–6] (Fig. 3). Although it is often found in the same larval habitat as An. lesteri, it is thought to occur more frequently in developed paddy fields and swamps . In the 2000s, we did not find any An. sinensis in the habitat of An. lesteri, nor did we find any new sources or habitats (Fig. 4). It is possible that the larval habitat of An. sinensis changed drastically in the 20-year period between the previous studies [3–6] and this current study. For example, in the 1949  and 1976  surveys, four members of the An. hyrcanus group were detected in northeastern Hokkaido, around Rubeshibe (Fig. 3). At that time, there were paddy fields all over the district, and forestry and horse-logging were the main industries. In recent times however, the horse-logging industry has declined drastically, and the paddy fields have been replaced with upland crops. Furthermore, neither An. sinensis nor An. sineroides was found in this area, around Ozora, during our survey (Fig. 4). It is highly likely that the changes in vegetation and industry have affected the distribution of anopheline mosquitoes.
There may be other reasons for the disappearance of An. sinensis from Hokkaido. The classification of organisms was mainly based on morphological keys until the 1990s. Although adults of An. belenrae can be separated morphologically from those of An. lesteri, An. sinensis and other species , it was likely that An. belenrae and An. sinensis could not be differentiated morphologically. Therefore, it should be noted that An. belenrae may have been classified as An. sinensis. The results from the ITS2 sequences in this study revealed that these two species were genetically the closest related. In addition, the pairwise interspecific distance in mitochondrial genomes calculated by each fragment showed minor or no difference between An. sinensis, An. belenrae and An. kleini . Phylogenetic analysis of COI indicated that ancient hybridizations probably occurred among these three closely related species , making differentiation with the COI sequence improbable. To address this problem, we tried to extract DNA and decipher the nucleotide sequence from age-old, dried specimens previously classified as An. sinensis collected in Hokkaido [3–6]. However, no new information could be obtained from these specimens. We hope that techniques for genetic analysis using age-old specimens will be developed as soon as possible.
Anopheles engarensis in Japan
Anopheles engarensis is also a species whose distributional range has reduced in Hokkaido. This species, first described in Engaro-cho (North-eastern Hokkaido) in 1977,  was also found in Monbetsu, Kushiro, and Obihiro until 1984 , suggesting a wide distribution in Hokkaido [3–6] (Fig. 3). However, our surveillance found this species to be restricted to western and southern Hokkaido (Fig. 4). In addition, this species was also collected in northern Tohoku, Akita Prefecture, suggesting a southward shift presumably due to changes in the environment, including the climate of larval habitats. In terms of classification, An. engarensis was recognized as a new species in the An. hyrcanus group only after its chromosomal structure was determined to be different from An. sinensis . This was because of the high morphological similarity between the two species. Indeed, the only distinguishing feature was the unique number of clasper movements of An. engarensis males during artificial mating, a common method for laboratory maintenance of anopheline mosquitoes .
In general, ITS2 is known to have high interspecific and low intraspecific variability; however, extensive intraspecific variations have been reported in anopheline mosquitoes. For instance, ITS2 intraspecific variations ranged from 0.2–19.0% for the Latin American anophelines . In the An. hyrcanus group, the average intraspecific distance was 0.3%, but no intraspecific variations were observed in An. belenrae . These results suggest that the ITS2 spacer is a good marker for differentiating between members of the An. hyrcanus group. In this study, there were no intraspecific variations in the An. belenrae, An. engarensis, and An. sineroides strains from Hokkaido. However, there was significant intraspecific variation between the nine An. engarensis strains from Hokkaido and the Daisen55 strain from Akita Prefecture. The genetic distance of 4.7% was considerably greater than the 0.22% intraspecific variation in the An. sinensis strains from Vietnam, South Korea, and Japan. We inferred that the Daisen55 An. engarensis strain was not introduced from Hokkaido but inhabited the Tohoku region independently. On the other hand, in species groups consisting of recently diverged members, such as the An. gambiae complex, the interspecific differences in ITS2 were reported to be minor, ranging from 0.4–1.6% . It is possible that An. engarensis is a recently diverged lineage.
Anopheles lesteri in Hokkaido
In Rubeshibe Hokkaido, at least seven cases of falciparum malaria were recorded between 1946 and 1947. An unsuccessful survey was conducted to determine the vector mosquitoes involved in the transmission although An. sinensis and An. sineroides were collected . During the falciparum malaria epidemic in the vicinity of Guangdong City, China, around 1942, the transmission was inferred to have involved An. lesteri and not An. sinensis. This inference was based on results from field investigations and subsequent infection experiments with Plasmodium falciparum . Based on this inference, it was suggested but never confirmed that An. lesteri may have been involved in the outbreak of falciparum malaria in Rubeshibe, Hokkaido. In terms of distribution, An. lesteri which was initially thought to be restricted to western Islands of Japan, such as the Kyushu Island , was also found in various areas of Honshu mainland of Japan , Hokkaido , Okinawa Island, and Yaeyama Islands . The present survey confirmed that An. lesteri is still widely distributed in Hokkaido (Figs. 3 and 4). At the start of our survey in 2001, we noticed female mosquitoes collected in Ozora, Hokkaido, to have an intense affinity for human blood. These female mosquitoes were therefore considered, and subsequently confirmed, to be An. lesteri based on the reported high anthropophilic nature of An. lesteri relative to An. sinensis and other members of the An. hyrcanus group [12, 47]. We therefore expected to easily collect An. lesteri in subsequent surveys in Hokkaido.
In our study, the ITS2 intraspecific distance in An. lesteri ranged from 0–9.44%. These values suggest that An. lesteri is a highly divergent species when An. lesteri type B (specimen 1) and type C (specimen 2) from South Korea are included in this species. Since the ITS2 distance of this species varies even within Hokkaido, there is a possibility that An. lesteri includes crypto-species. In defining this species, it is necessary to analyze both the COI barcoding region and the ITS2 region. Moreover, a large number of specimens, collected outside Hokkaido, will be necessary. In a previous study, a short interspecific distance of 7.2% was observed between An. kleini and An. engarensis . We obtained similar ITS2 distances of 6.47% and 8.67% between An. kleini and our An. engarensis specimens from Hokkaido and An. kleini and An. engarensis Daisen55 strain, respectively. Although these results may provide validation that An. kleini is a synonym of An. engarensis, further analysis is required. We also presented evidence that An. anthropophagus and An. lesteri were conspecific, based on the ITS2 divergence between them. Our results based on interspecific comparisons of ITS2 divergence may also support previous reports that An. belenrae and An. sinensis are genetically distinct [24, 25], and An. anthropophagus is a conspecific species of An. lesteri [34, 48].