Reconstructing the colonization history of lost wolf lineages by the analysis of the mitochondrial genome
Graphical abstract
Introduction
The grey wolves (Canis lupus) originally inhabited major parts of the Northern hemisphere, and are sometimes referred as the most widely distributed wild terrestrial mammal (Aggarwal et al., 2007, Ellegren et al., 1996). Up to 37 subspecies are recognized (Wilson and Reeders, 2005) though their taxonomy has long been the subject of debate. However, eliminations of populations have been reported in many areas of their previous range (Ellegren et al., 1996, Wilson and Reeders, 2005). Wolves in Japan, which became extinct approximately 100 years ago, are one of the most mysterious subspecies among them. Due to the rapidity of the decline, little information is known about their ecology and behaviour in their natural habitat. The number of bone specimens is limited, and less than 10 stuffed specimens are currently available (Ishiguro et al., 2009, Ishiguro et al., 2010). Therefore, the taxonomic status of wolves in Japan has long been controversial (Ishiguro et al., 2009).
Two lineages of wolves are known from Japan (Fig. 1). Japanese or Honshu wolves (C. l. hodophilax) once inhabited central and southern parts of Japan. Because of the smaller body size than continental grey wolves and some distinguishing osteological characters, they are sometimes treated as an independent species, C. hodophilax (Imaizumi, 1970). Another subspecies, Ezo or Hokkaido wolves (C. l. hattai) were only found in the northern island of Hokkaido and neighbouring small islands. As documented in other places (e.g., Berger et al., 2001), wolves in Japan must have played an important role in the ecosystem as large terrestrial predators. Their population size at that time is unknown, but skeletal remains found at some archaeological sites (Ishiguro et al., 2009) indicate that Japanese wolves had a wide distribution since the Jomon Period (10,000 to 250 BC). Wolves are mentioned in classical Japanese literature (Walker, 2005); they were familiar animals to local people, in particular, farmers and were sometimes worshipped by them as a god. Conflict between local people and wolves appeared to be rare until around 300 years ago, when rabies was first described in Japan (Walker, 2005). Japan opened up to foreign trade in 1854 after hundreds of years of isolation and adopted many Western customs, including livestock farming. Hunting and poisoning of wolves over the next several decades, along with severe weather in some years, led to a drastic decline in the number of wolves; Ezo wolves went extinct between 1880 and 1890, followed by Japanese wolves in the 1900s (Ishiguro et al., 2009, Ishiguro et al., 2010, Walker, 2005).
Advances in molecular genetics have enabled the study of DNA from ancient samples, such as museum specimens and bones from archaeological sites (Pääbo et al., 2004). In the present study, we sequenced the complete mitochondrial genome (16,562–16,731 bp) of six Japanese wolves and two Ezo wolves from ancient specimens and compared them with published complete mitochondrial DNA (mtDNA) sequences of wolves. Extracting and sequencing the ancient mitochondiral genome are easier than the nuclear genome mainly because of higher copy numbers in each cell. The total number of ancient mitochondrial genome sequences has increased rapidly over the last decade (Ho and Gilbert, 2010). The use of mtDNA has several advantages including maternal inheritance, absence of recombination, and high substitution rate. Complete mtDNA sequences have a potential for more reliable phylogenetic reconstruction than short fragments of mtDNA. In particular, analyses of complete mtDNA sequences including ancient DNA samples have been shown to be useful for phylogenetic reconstruction within a species or between closely related species (e.g., Gilbert et al., 2008, Lindqvist et al., 2010, Thalmann et al., 2013). The objectives of the present study are to clarify the phylogenetic status of Japanese wolves and Ezo wolves and to reconstruct the history of their colonization of the Japanese archipelago.
Section snippets
Complete mtDNA sequence generation
DNA was extracted from bones stored in several museums or personal collections throughout Japan (Table 1) according to the methods described by Okumura et al. (1999) and Ishiguro et al. (2009). The criteria for the authenticity of ancient DNA (Hofreiter et al., 2001) were applied, except that we use only a single extract from each specimen to keep valuable specimens as intact as possible, and that we did not send samples to a second laboratory to reproduce the results. The outer layers of bone
Phylogenetic status of Japanese and Ezo wolves
We successfully determined complete mitochondrial genome sequences for six Japanese wolf samples and two Ezo wolf samples (Table 1). The DNA sequences have been deposited in GenBank (AB499818-AB499823). We also determined complete mitochondrial genome sequences of two modern dogs which had the control region sequences similar to Japanese wolves (Kishu and Siberian husky, D83611 and D83637 in Ishiguro et al., 2009). These two sequences have also been deposited in GenBank (AB499816 and AB499817).
Discussion
Our phylogenetic analysis based on the complete mitochondrial genome clarified the origin and phylogenic status of two types of extinct wolves in Japan. Japanese or Honshu wolves are phylogenetically distinct from other grey wolf populations, suggesting their long time separation in the Japanese archipelago. To confirm this, further sampling of wolves in East and Southeast Asia is necessary. By contrast, Ezo or Hokkaido wolves are likely to have arrived at Japan relatively recently. Although a
Acknowledgments
The present study was financially supported by KAKENHI (Grant-in-Aid for Scientific Research (B), 23405040, 2010–2013 to NI; and Grant-in-Aid for Scientific Research (C), 22570087, 2010–2012 to SM).
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