Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(2): 81-90, 2019 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/cayologia-237 Citation: H. Myoshu, M.A. Iwasa (2019) Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and east- ern Honshu. Caryologia 72(2): 81-90. doi: 10.13128/cayologia-237 Published: December 5, 2019 Copyright: © 2019 H. Myoshu, M.A. Iwasa. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu Hikari Myoshu, Masahiro A. Iwasa* Course in Natural Environment Studies, Graduate School of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan *Corresponding author, e-mail: iwasa.masahiro@nihon-u.ac.jp Abstract. We characterized and categorized the C-band patterns of the house mouse Mus musculus from four areas in Hokkaido and Honshu of the Japanese Islands as a biparental marker. The C-band patterns are categorized as polymorphic, monomor- phic, or intermediate, corresponding to those of the Korean mice, C57BL/6, and F1 hybrid mice bred from the Japanese mice and a laboratory mouse, respectively. The C-band patterns mainly differ between mice from Hokkaido and Honshu. The poly- morphic patterns are shown in mice in Honshu, while the intermediate patterns are shown in mice in Hokkaido, with an exceptional case of a monomorphic pattern found in one locality. In the other localities of Hokkaido and northeastern Honshu, the C-band patterns are not congruent with an estimation by maternal element in our pre- vious study, whereas the congruence is observed in other localities. It is suggested that the characteristics of the Japanese house mice have been formed through complicated processes by different expansions between biparental and maternal elements. Keywords. C-band pattern, Mus musculus, the Japanese house mouse, hybridization, replacement. INTRODUCTION The Japanese house mouse, Mus musculus (Mammalia, Rodentia), has been colonized in the Japanese Islands through artificially complicated processes by overseas mice based on the genetic and morphological analy- ses (Myoshu and Iwasa 2018). According to previous studies of intraspecific variations of mitochondrial DNA (mtDNA) haplotypes, the Japanese house mice have been derived from two lineages as the MUS and CAS types, cor- responding to the subspecific musculus and castaneus, respectively, that currently occur in the Korean Peninsula and southern China (MUS-1c and CAS-1a groups in Suzuki et al. 2013). In addition, the colonization history of the Japanese house mice is estimated as following scenario: mice migrat- ed primarily from southern China or southeastern Asia; secondarily, mice migrated from the Korean Peninsula and replaced the distributions of the former mice, considering the distribution patterns of mtDNA haplotypes 82 Hikari Myoshu, Masahiro A. Iwasa (Yonekawa et al. 1988; Terashima et al. 2006; Nunome et al. 2010, 2013; Suzuki et al. 2013; Kuwayama et al. 2017; Myoshu and Iwasa 2018). Nuclear genome analy- sis has shown evidence of the introgression and replace- ment (Kuwayama et al. 2017). Additionally, many stud- ies suggest recent migrations by stowaway introduction in several areas, including non-port areas (Miyashita et al. 1985; Yonekawa et al. 2000; Tsuda et al. 2001, 2002; Terashima et al. 2006; Nunome et al. 2010; Kodama et al. 2015; Kuwayama et al. 2017; Myoshu and Iwasa 2018). On the other hand, the distribution of nuclear DNA types is not always congruent with that of mtDNA hap- lotypes in the Japanese house mice. The sequence of the musculus lineage is observed in all targeted regions of the nuclear genome, or relatively short segments of the castaneus lineage are observed in some targeted regions, although its samples were obtained from a locality where the CAS type is exclusively observed (Kuwayama et al. 2017). In addition, our previous study (Myoshu and Iwasa 2018) shows that observed external characteristics sometimes do not coincide with the subspecific char- acteristics estimated by the mitochondrial haplotypes. Thus, these incongruences between nuclear traits and mtDNA traits suggest complicated hybridization and/ or replacement process, regarding different progress between biparental and maternal elements. To elucidate the process of replacement by the Korean mice, it is nec- essary to comprehensively investigate the biparental ele- ment by a marker distinguishing the Korean mice. Cytogenetically, variations in the C-band patterns have been well studied in wild house mice (Dev et al. 1973, 1975; Miller et al. 1976; Moriwaki and Minezawa 1976; Ikeuchi 1978; Moriwaki et al. 1985, 1986; Mori- waki 2010; Yonekawa et al. 2012; Myoshu and Iwasa 2016). By evaluating the C-band patterns, we can con- firm whether the Japanese house mice have experienced hybridization and/or replacement with the mice that introduced from northern China and the Korean Pen- insula, or not. According to these previous studies, the C-banding patterns of house mice can be roughly cat- egorized into two patterns. The European, central and southern Asiatic, and laboratory mice show a monomor- phism of C-band sizes in a homologue; almost all chro- mosomes carry smaller centromeric C-bands (hereinaf- ter called a “monomorphic pattern”). On the other hand, northern Chinese and/or Korean mice show a polymor- phism of C-band sizes in a homologue; a few chromo- somes carry larger centromeric C-bands, and most of the residual chromosomes carry no C-band (hereinafter called a “polymorphic pattern”). The C-banding pat- terns of the Japanese house mice are visually categorized as the latter (Dev et al. 1973, 1975; Moriwaki and Min- ezawa 1976; Ikeuchi 1978; Moriwaki et al. 1985, 1986, 2009; Moriwaki 2010; Yonekawa et al. 2012), polymor- phic states of C-bands (Myoshu and Iwasa 2016). The mice in southern China and/or southeastern Asia, which primarily migrated to the Japanese Islands (Suzuki et al. 2013; Kuwayama et al. 2017), shows the former type of C-band pattern (Moriwaki et al. 1986; Yonekawa et al. 2012). In addition, the C-band patterns of F1 offspring reveal the inheritance states of the C-band size, because the C-band size does not vary over a generation (Dev et al. 1973, 1975; Miller et al. 1976). Thus, the C-band pat- tern is a useful marker to comprehensively investigate the biparental element. In this study, we statistically characterized the C-band patterns of house mice from four areas in Hok- kaido and Honshu of the Japanese Islands that we previ- ously analysed for mtDNA haplotypes and morphologi- cal characteristics (Myoshu and Iwasa 2018). According to results of previous mtDNA analysis and the present analysis, we elucidated the migration, hybridization, and replacement processes of maternal and biparental ele- ments in the four areas. MATERIALS AND METHODS Mouse samples Wild-caught mice of the Japanese Islands (Mus mus- culus) were collected (n = 31; Table 1 and Figure 1) in the Sorachi and Iburi areas of Hokkaido (n = 3; HKD1, including BBI and HYK; Table 1, 1 and 2 in Figure 1(a)), the Hidaka area of Hokkaido (n = 8; HKD2, includ- ing MID, KB1, KB2, and NK2; Table 1, 3 to 6 in Fig- ure 1(a)), Iwate and Miyagi Prefectures in Honshu (n = 3; HON1, including SYG, TNS, and FTK; Table 1, 7 to 9 in Figure 1(b)) and Kanagawa and Chiba Prefectures in Honshu (n = 11; HON2, including KZK, KMN, OHB, and CGS; Table 1, 10 to 12 in Figure 1(c)) using Sher- man traps baited with oatmeal. We used the same divi- sion names and abbreviations of areas and localities in this study as in Myoshu and Iwasa (2018). In addition, a wild-caught mouse (M. musculus) collected in Seong- modo Island, neighboring the Korean Peninsula, was used (n = 1; SMD in Table 1; 14 in Figure 1(d)). A labora- tory mouse (C57BL/6, Japan SLC Inc.) was also used for the analysis as a standard. Moreover, hybrid mice from a cross experiment using a female wild-caught mouse from Kanagawa Prefecture (specimen nos.: MAI-1239, 1306 and 1308) and a male C57BL/6N were analyzed (n = 3; Table 1) to confirm intermediate C-banding patterns from their parents. 83Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu C-banding for somatic cells Chromosome preparations were performed from bone marrow cells. Bone marrow cells were cultured in MEM including 15% calf serum containing colchicine (final concentration: 0.025 µg/ml) at 37 ˚C for 40 min. These cells were treated in 0.075 M KCl at 37 ˚C for 20 min as a hypotonic treatment. Subsequently, the cells were fixed with modified Carnoy’s fixative (methanol : acetic acid = 3 : 1) three times. Then air-dried cells were primarily G-banded using the ASG technique (Sumner et al. 1971) to identify each chromosome by Committee of Standardized Genetic Nomenclature for Mice (1972) and Cowell (1984). After destaining using Carnoy’s fixa- tive, the cells were subsequently C-banded using the BSG technique by Summer (1972). Quantification of the C-band pattern Photographs of somatic metaphases were obtained using a digital camera under a microscope (Olym- Table 1. House mouse samples examined in this study. Collecting locality (code*) Specimen No. (sex) Wild caught mice Sorachi and Iburi areas, Hokkaido, Japan (HKD1) Koshunai-cho, Bibai, Hokkaido (BBI, 1) MAI-1919 (f ) Hayakita-tomioka, Abira-cho, Yufursu-gun, Hokkaido (HYK, 2) MAI-1895 (f ), 1915 (m) Hidaka area, Hokkaido Japan (HKD2) Midorimachi, Hidaka-cho, Saru-gun, Hokkaido (MID,3) MAI-1837 (m), 1840 (f ) Kabari, Hidaka-cho, Saru-gun, Hokkaido (KB1, 4) MAI-1916 (m), 1917 (m) Kabari, Hidaka-cho, Saru-gun, Hokkaido (KB2, 5) MAI-1913 (m), 1918 (f ) Bansei, Niikappu-cho, Niikappu-gun, Hokaido (NK2, 6) MAI-2004 (f ), 2016 (m) Northeastern Honshu area, Japan (HON1) Shimoyahagi, Rikuzentakata, Iwate Pref., Honshu (SYG, 7) MAI-1289 (m) Takinosato, Rikuzentakata, Iwate Pref., Honshu (TNS, 8) MAI-1293 (m) Futaki, Sendai, Miyagi Pref., Honshu (FTK, 9) MAI-1843 (m) Central Honshu area, Japan (HON2) Kohzaki, Katori-gun, Chiba Pref., Honshu (KZK, 10) MAI-1991 (m), 1992 (f ) Kameino, Fujisawa, Kanagawa Pref., Honshu (KMN, 11) MAI-1114 (f ), 1120 (f ), 1443 (m), 1444 (f ) Ohba, Fujisawa, Kanagawa Pref., Honshu (OHB, 12) MAI-1239 (f ), 1306 (f ), 1308 (f ), 1309 (m) Akabane, Chigasaki, Kanagawa Pref., Honshu (CGS, 13) MAI-1548 (f ) Korea Seongmodo Is., Ganghwa-gun, Incheon-gwangyeoksi, Korea (SMD, 14) HEG007-97 (m) Laboratory mouse C57BL/6N MAI-1301 (m) Hybrid mice (wild caught mice x Laboratory mouse) MAI-1239 x 1301 MAI-1450 (f ), MAI-1452 (f ) MAI-1306 x 1301 MAI-1379 (f ) MAI-1308 x 1301 MAI-1563 (m) *Code numbers are corresponding to those in Figure 1. Fig. 1. Collection localities of the house mice examined in this study. Locality code numbers correspond to those in Table 1. 84 Hikari Myoshu, Masahiro A. Iwasa pus BX41). To correct errors caused by variations in the extension condition of chromosomes among meta- phase plates, we calculated the relative lengths of the C-bands. Primarily, the boundary between negatively and positively stained regions was identified at the prox- imal region of the long arm in the No. 2 chromosomes according to Myoshu and Iwasa (2016). Subsequently, the distance from the distal end of the long arm to the proximal boundary of the negatively stained region was measured in one of the No. 2 chromosomes as a control length for all relative lengths on its metaphase using Adobe Illustrator CC. In addition, the lengths of all of positively C-banded regions on all chromosomes were measured by the same method. Finally, the relative lengths of the C-bands on each chromosome were cal- culated using following formula: the length of the posi- tively stained C-band region of each chromosome / the control length of the No. 2 chromosome in its metaphase plate. When a positively stained C-band region was not observed in a chromosome or when the entire chromo- some was stained lightly, as seen in the Y chromosome (Figure 2), we considered relative length of the chromo- some to be zero. The relative lengths of C-bands for all chromosomes were calculated in five or more metaphase plates per individual. All of the relative lengths of C-bands were catego- rized as classes by 0.1. The mean number of chromo- somes of each class in an individual was calculated using following formula: the observed number of chro- mosomes included in each class in an individual / the number of observed metaphase plates in an individual. Regarding the set of all mean numbers in each class for an individual as the C-band pattern of the individual, we performed a clustering analysis for the C-band pat- terns of all individuals to estimate analogies among them. We first calculated Euclidian distances using all sets of the mean numbers for each class from all mouse samples. Then we performed a clustering analysis using the ward method based on these distances. RESULTS In the karyotypes of wild-caught mice, the hybrid individuals and C57BL/6, ty pical examples of the C-banded metaphases (samples prepared from wild- caught mice in SMD, HON1, HON2, NK2, and HKD1, and from C57BL/6N and the hybrid mice) were shown in Figure 3. These C-banding patterns showed the pres- ence of chromosomes with null C-bands in the mouse samples without C57BL/6N carrying the Y chromo- some negatively stained by C-banding (Figures 2 and 3). The C-band patterns of the hybrid individual seemed to be inherited from those of the parents (a wild-caught mouse and C57BL/6N) as their intermediate type (Fig- ure 2). In addition, size variations of C-bands seemed to be confirmed in all of the mice excluding C57BL/6 (Fig- ures 2 and 3). The mean numbers of chromosomes with a C-band and with a null C-band were classified into classes based on the relative length; 0.01–0.10, 0.11–0.20, 0.21–0.30, 0.31–0.40, 0.41–0.50, and >0.51 as in Table 2, and the Fig. 2. Typical C-banded karyotypes of a wild-caught mouse (a, MAI-1239), a hybrid mouse (b, MAI-1452), and a C57BL/6N mouse (c, MAI-1301). Stars indicate chromosomes with null C-bands. Asterisks indicate crossing of chromosomes. 85Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu histograms of these mean numbers were indicated in Figure 4. On the basis of our observation and calcu- lation of C-bands on a metaphase plate of C57BL/6 (Table 2 and Figure 3(d), upper histogram in Figure 4), we defined the C-bands (0.01–0.30 in relative length) as “smaller C-bands”. In contrast, we defined the C-bands that is never observed in C57BL/6 (> 0.31 in relative length) as ‘larger C-bands’. Furthermore, the result of clustering analysis showed clear discriminations with three major clades consisting of the HON1/HON2 mice and the SMD mouse, HKD1/HKD2 (without NK2) mice and the hybrid mice, and HKD2 (NK2) mice and C57BL/6 (Figure 5). The HON1/HON2 mice and the SMD mouse in a major clade (Figure 5) showed high frequencies of null C-band chromosomes (the mean numbers ranged from 23.6 to 29.5 per metaphase plate, Table 2). In addition, there were residual chromosomes carrying positively stained C-bands showing variable sizes, including larg- er C-bands with relative lengths of not only 0.31–0.50 but also > 0.51 (sums of the mean numbers of chromo- somes with relative lengths of > 0.31 ranged from 1.4 to 7.0 per metaphase). On the other hand, the mean num- bers of smaller C-bands were lower (sums of the mean numbers of chromosomes with relative lengths of 0.01– 0.30 ranged from 5.2 to 14.4 per metaphase). Of these, smaller C-bands with relative lengths of 0.21–0.30 were observed much more than those with relative lengths of 0.11–0.20 in HON2; however, individuals from HON1 showed the highest number of C-bands with relative lengths of 0.11–0.20 (Table 2 and Figure 4). Moreover, the larger C-bands on the No.2 chromosomes, which were usually observed in the HON1/HON2 mice (Fig- ure 3(b) and 3(c), respectively) were not observed in the SMD mouse (Figure 3(a)). Furthermore, hybrid mice and the HKD1/HKD2 (without NK2) mice belonged to the other major clade (Figure 5). The mean numbers of null C-bands were apparently lower (the mean numbers ranged from 14.2 to 19.4 per metaphase, Table 2) than those from HON1/ HON2 and SMD (the mean numbers ranged from 23.6 to 29.5 per metaphase, Table 2) and higher than those of C57BL/6N and NK2 of HKD2 (the mean numbers ranged from 1.2 to 9.0 per metaphase, Table 2). Addi- tionally, the mean numbers of the smaller C-bands (sums of the mean numbers with relative lengths of 0.01–0.30 ranged from 17.8 to 23.8 per metaphase, Table 2) was also intermediate between those from HON1/ HON2 and SMD (5.2~14.4) and that from C57BL/6N (38.8). Moreover, there was lower number of larger C-bands, at least one chromosome per metaphase (sums of the mean numbers with relative lengths of > 0.31 ranged from 0.4 to 1.0 per metaphase, Table 2). The third major clade consisted of C57BL/6 and mice from NK2 of HKD2 (Figure 5). C57BL/6 showed smaller C-bands in all of the chromosomes (sum of the mean numbers of chromosomes with relative lengths of 0.01–0.30 was 38.8 per metaphase, Table 2). In addition, C57BL/6 showed no larger C-band with relative lengths of > 0.31 and lower numbers of null C-bands (the mean number was 1.2 per metaphase, Table 2). Meanwhile, individuals from NK2 of HKD2 (specimen nos. MAI- 2004 and MAI-2016) carried a pattern similar to that of C57BL/6, especially in terms of the higher number of appearances of smaller C-bands (sums of the mean numbers of chromosomes with relative lengths of 0.01– 0.30 were 36.2 and 30.0 per metaphase in MAI-2004 and MAI-2016, respectively, Table 2). Moreover, they car- ried no more than a chromosome with a larger C-band (sums of the mean numbers of chromosomes with rela- tive lengths of > 0.31 were 0.2 and 1.0 per metaphase in MAI-2004 and MAI-2016, respectively, Table 2) and sev- eral chromosomes with null C-bands (the mean num- bers were 3.6 and 9.0 per cell in MAI-2004 and MAI- 2016, respectively). Fig. 3. Typical examples of C-banded metaphase plates of poly- morphic patterns: Seongmodo Is. (a, HEG007-97), Ohba (b, MAI- 1306), and Takinosato (c, MAI-1293); monomorphic patterns: C57BL/6N (d, MAI-1301) and Niikappu-2 (e, MAI-2004); and intermediate patterns: a hybrid mouse (f, MAI-1379) and Hayakita (g, MAI-1839). 86 Hikari Myoshu, Masahiro A. Iwasa DISCUSSION The polymorphic pattern in two areas geographical- ly isolated in the Japanese Islands, HKD1/HKD2 (with- out NK2) and HON1/HON2, are categorized into two groups (Figure 5), which include the SMD and hybrid mice, respectively. On the other hand, the monomor- phic pattern consists of many smaller C-bands without a null C-band and a larger C-band (Table 2 and Figure 4) and belongs to the cluster including C57BL/6N (Fig- Table 2. Mean numbers of chromosomes with null C-band and C-band classified into each class of relative length. Specimen Null C-band Classification Smaller C-band Relative length of C-band Larger C-band Relative length of C-band 0.01~0.10 0.11~0.20 0.21~0.30 Total 0.31~0.40 0.41~0.50 >0.51 Total HKD1 MAI-1919 19,0 0 10,4 7,6 18,0 2,8 0,2 0 3,0 MAI-1895 18,2 1,4 11,2 7,6 20,2 1,6 0 0 1,6 MAI-1915 18,2 0,2 10,6 7,8 18,6 3,0 0,2 0 3,2 HKD2     MAI-1837 15,8 1,2 13,4 5,6 20,2 2,4 1,4 0 3,8 MAI-1840 17,4 0 9,0 8,8 17,8 2,6 1,4 0 4,0 MAI-1916 19,4 0,8 13,0 5,6 19,4 1,2 0 0 1,2 MAI-1917 18,2 0,4 10,4 8,2 19,0 2,6 0,2 0 2,8 MAI-1913 16,6 0 13,3 7,9 21,2 1,9 0,3 0 2,2 MAI-1918 18,4 0,8 15,6 4,8 21,2 0,2 0,2 0 0,4 MAI-2004 3,6 1,2 31,4 3,6 36,2 0,2 0 0 0,2 MAI-2016 9,0 3,2 23,4 3,4 30,0 0,6 0,4 0 1,0 HON1     MAI-1289 25,0 0 7,4 5,2 12,6 2,0 0,4 0 2,4 MAI-1293 23,6 0,4 10,4 3,6 14,4 1,2 0,6 0,2 2,0 MAI-1843 27,6 0,6 2,8 4,4 7,8 2,6 2,0 0,0 4,6 HON2     MAI-1991 28,0 0 1,0 4,6 5,6 3,4 2,6 0,4 6,4 MAI-1992 27,3 0 3,0 5,0 8,0 3,3 0,9 0,6 4,7 MAI-1114 29,5 0 1,2 4,0 5,2 2,0 1,8 1,5 5,3 MAI-1120 29,2 0,2 0,8 4,2 5,2 3,2 1,5 1,0 5,7 MAI-1443 27,8 0 5,2 5,6 10,8 0,8 0,6 0 1,4 MAI-1444 29,2 0 0 3,8 3,8 2,8 2,6 1,6 7,0 MAI-1239 28,2 0,2 1,8 5,4 7,4 3,0 1,0 0,4 4,4 MAI-1306 24,8 1,3 6,2 3,1 10,7 2,4 1,9 0,2 4,6 MAI-1308 28,0 0,2 3,8 4,2 8,2 2,7 1,0 0,2 3,8 MAI-1309 28,0 0 2,4 3,6 6,0 2,5 2,2 1,3 6,0 MAI-1548 29,2 0 3,6 3,8 7,4 2,8 0,6 0 3,4 Korea     HEG007-97 25,2 0,2 9,6 3,4 13,2 1,0 0,6 0 1,6 Labolatory mouse     MAI-1301 1,2 0,2 34,8 3,8 38,8 0 0 0 0 Hybrid mice     MAI-1450 14,4 0 16,6 7,2 23,8 0,8 0,6 0,4 1,8 MAI-1452 14,4 0,2 13,2 9,0 22,4 1,4 1,0 0,8 3,2 MAI-1379 15,8 1,4 16,2 4,0 21,6 0,8 1,6 0,2 2,6 MAI-1563 14,2 1,6 16,2 4,4 22,2 2,8 0,6 0,2 3,6 87Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu ure 5). Our previous study (Myoshu and Iwasa 2018) indicates the occurrences of three Cytb haplotypes that are confirmed in the same areas of the Japanese Islands, as shown in Table 1. In the areas showing the polymor- phic pattern similar to SMD, only the subspecific cas- taneus (CAS) type and only the subspecific musculus (MUS) type occur in HON1 and HON2, respectively. Additionally, only the CAS type and multiple haplotypes including the CAS, MUS, and the subspecific domes- ticus (DOM) types occur in the area showing the poly- morphic pattern similar to that of hybrid mice, HKD1, and HKD2 without NK2, respectively. On the other hand, not the MUS type but rather the CAS and DOM types occur in NK2 showing the monomorphic pat- tern. According to these results, a concordant combina- tion between biparental C-band pattern and maternal Cytb haplotype is shown in HON2 and HKD2 but is not shown in HON1 and HKD1. Fig. 4. Histograms showing mean numbers of chromosomes without C-bands or with C-bands per metaphase, classified into each class of relative length with 0.01–0.10, 0.11–0.20, 0.21–0.30, 0.31–0.40, 0.41–0.50, and >0.51. 88 Hikari Myoshu, Masahiro A. Iwasa The present C-band results can agree with the find- ing of Cytb properties in HON2/HKD2 and the poten- tial founders, mice in northern China and/or the Korean peninsula (Yonekawa et al. 1988; Terashima et al. 2006; Nunome et al. 2010, 2013; Suzuki et al. 2013), carry the polymorphic C-bands and the MUS type as in the HON2 mice (Myoshu and Iwasa 2018; Figures 3, 4, 5). On the basis of these results, it is a feasible explana- tion from both viewpoints by chromosome and mtD- NA traits (Myoshu and Iwasa 2018; Figures 3, 4, 5) that the HON2 mice have maintained the traits of potential founders. In HKD2, on the basis of a simple inherit- ance of the C-band size (Dev et al. 1975; Figure 2), the hybridization between mice carrying a combination of the monomorphic pattern and the CAS or DOM type, and mice carrying a combination of the polymorphic pattern and the MUS type from northern China and/ or the Korean peninsula is concordantly estimated by both viewpoints from these analyses (Myoshu and Iwasa 2018; Figures 3, 4, 5). The monomorphic pattern of NK2 is in accordance with our previous results showing not only the occurrence of Cytb haplotypes (the CAS and DOM types) but also their morphological character- istics (external body dimensions and coat coloration) (Myoshu and Iwasa 2018). There is a possibility that the distribution in the Japanese Islands of the predominant mice which were introduced from northern China and/ or the Korean Peninsula (Yonekawa et al. 1988; Terashi- ma et al. 2006; Nunome et al. 2010, 2013; Suzuki et al. 2013) has not yet expanded into NK2. However, putting emphasis on their larger head and body length as the subspecies M. musculus domesticus (Myoshu and Iwasa 2018) and the occurrence of the “intact” stowaway hap- lotype of nuclear DNA (Nunome et al. 2010; Kodama et al. 2015) and mtDNA (Yonekawa et al. 2000; Tsuda et al. 2001, 2002) in the Japan Islands, a more feasible expla- nation is that a relatively recent introduction(s) has led stowaway mice to this locality. In the areas with exclusive occurrence of the CAS type from southern China and/or southeastern Asia (Suzuki et al. 2013; Kuwayama et al. 2017), HKD1 and HON1, there are discordances between the both view- points mentioned above (Myoshu and Iwasa 2018; Table 2; Figures 3, 4, 5). In contrast to the Cytb finding, the C-band patterns of the HKD1 mice and the HON1 mice are estimated to have been affected by the introgression from northern Chinese and/or Korean mice carrying the polymorphic patterns (Yoshida and Kodama 1983; Moriwaki et al. 1985, 1986; Moriwaki 2010; Yonekawa et al. 2012). In addition, although the CAS type occurs exclusively in both areas, the C-band patterns are not categorized into the same groups (Figure 5). Specifical- ly, the numbers of null C-bands (23.6–27.6) and small- er C-bands (7.8-14.4) of the HON1 mice are especially more similar to those in the HON2 mice (24.8-29.5 null C-bands; 3.8-10.8 smaller C-bands) than those in the HKD1 mice (18.0-20.2 null C-bands; 18.6-20.2 smaller C-bands). Moreover, the sizes of the smaller C-bands differ between HON1 (more frequent relative length: 0.11-0.20, Table 2 and Figure 4) and HON2 (more fre- quent relative length: 0.21-0.30, Table 2 and Figure 4). These results suggest that the C-band patterns of HON1 and HKD1 are not genetically identical. Several studies using biparental markers, which are the haplotypes on the haemoglobin bate chain (Hbb) locus (Minezawa et al. 1979; Miyashita et al. 1985; Kawashima et al. 1991, 1995; Ueda et al. 1999; Sato et al. 2006, 2008; Yonekawa et al. 2012), eight (Nunome et al. 2010) and seven (Kodama et al. 2015) linked nuclear genes, would provide a suggestion for why the differ- ence in the C-band pattern has been caused in the same mtDNA occurrence areas. According to these previous studies, the distributions of the polymorphic C-band patterns (Moriwaki and Minezawa 1976; Moriwaki et al. 1985, 1986; Yonekawa et al. 2012; Myoshu and Iwasa 2016; Table 1 and Figures 4, 5) overlap the distributions of the p (Minezawa et al. 1979; Miyashita et al. 1985; Kawashima et al. 1995; Ueda et al. 1999; Yonekawa et al. 2012) and the MUS-II haplotype groups (Nunome et al. 2010; Kodama et al. 2015) which are derived from the Fig. 5. Cluster analysis considering the mean numbers of chromo- somes without C-bands or with C-bands in each class. Top values indicate Euclidian distances. 89Differences in C-band patterns between the Japanese house mice (Mus musculus) in Hokkaido and eastern Honshu musculus lineage. The other hapolotype groups, which are recognized as the d (Minezawa et al. 1979; Miyashita et al. 1985; Kawashima et al. 1995; Yonekawa et al. 2012) and the recombinant haplotype groups (Nunome et al. 2010; Kodama et al. 2015) mainly derived from the cas- taneus lineage, have been observed in northern Honshu and Hokkaido localities. However, a survey of the Hbb allele reveals the difference in p frequency between the northeastern area of Honshu (37.0% in Minezawa et al. 1979) and Hokkaido (7.9% in Minezawa et al. 1979). Thus, the introgression of the lineage with MUS-II and p haplotypes, which has been derived from Eurasian mice carrying the polymorphic C-band patterns, has strongly affected the HON1 mice more than the HKD1 mice. In addition, since male mice have larger dispersal areas than female mice (Pocock et al. 2005), expansions of maternal genetic traits would be later than those of the paternal and biparental genetic traits. On the basis of this dispersal pattern, it is estimated that the poly- morphic patterns as a biparental trait primarily expand into populations including both male and female mice with monomorphic patterns and the CAS type, by male mice considering the larger dispersal potential. There- fore, the polymorphic patterns from male mice have less affected mice in HKD1 than in HON1, based on the sign of hybridization in its C-band patterns is signifi- cantly observed in HKD1. In HON1, where the C-band patterns are similar to those in HON2 and SMD, the mice may have been replaced completely by the male mice carrying the polymorphic patterns. Otherwise, the HON1 mice have hybridized and/or maintained the traits of mice carrying a discordant combination, for example the polymorphic pattern and the CAS type, which may have been caused in other places as suggested in Searle et al. (2009), Nunome et al. (2010), Kodama et al. (2015) and Kuwayama et al. (2017). 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