Description of the karyotypes of Stejneger's beaked whale (Mesoplodon stejnegeri) and Hubbs’ beaked whale (M. carlhubbsi)

Abstract The genus Mesoplodon (Cetacea: Odontoceti: Ziphiidae) is one of the few cetacean genera with the karyotype 2n = 42. The 2n = 42 karyotype of M. europaeus and M. carlhubbsi is largely consistent with the general cetacean karyotype 2n = 44, although other 2n = 42 karyotypes do not exhibit clear homologies with the general cetacean karyotype. Therefore, the chromosomes of Mesoplodon species may be the key to understanding cetacean karyological evolution. In the present study, the male karyotypes of M. stejnegeri and M. carlhubbsi were examined. In both species, the diploid number of the male karyotype was 42. Both species had the following characteristics: 1) a huge subtelocentric X chromosome with a large C-block; 2) a small metacentric Y chromosome; 3) nucleolus organizer regions (NORs) in the terminal regions of a large autosome and one or two small metacentric autosomes; 4) small metacentric autosomes; 5) large submetacentric and subtelocentric autosomes; 6) less accumulated C-heterochromatin in the centromeric region; and 7) heteromorphism in C-heterochromatin accumulation between homologues. Characteristics 1 and 3 are peculiar to only the karyotypes of Mesoplodon species, whereas characteristics 4, 5, 6, and 7 are also found in the species with the general cetacean karyotype 2n = 44.

Two diploid chromosome numbers are known in the order Cetacea: 2n = 44 and 2n = 42 (Árnason, 1974). Most cetaceans have the karyotype 2n = 44, and many authors have pointed out the uniformity in chromosome morphology and banding pattern among cetaceans with this karyotype (e.g., Árnason, 1974, 1980Duffield et al., 1991). The karyotype 2n = 42 has been described in only seven species: Eubalaena glacialis (Pause et al., 2006), Balaena mysticetus (Jarrell, 1979), Physeter macrocephalus (Árnason and Benirschke, 1973), Kogia breviceps (Árnason and Benirschke, 1973), Ziphius cavirostris (Benirschke and Kumamoto, 1978), Mesoplodon europaeus (Árnason et al., 1977) and M. carlhubbsi (Árnason et al., 1977). According to Árnason and Benirschke (1973) and Árnason (1974), the 2n = 42 karyotypes in P. macrorhynchus and K. breviceps do not exhibit clear homologies with the general cetacean karyotype 2n = 44. On the other hand, the 2n = 42 karyotypes of M. europaeus and M. carlhubbsi are largely in agreement with the general cetacean karyotype (Árnason et al., 1977). Therefore, the chromosomes of Mesoplodon species are of great interest when considering karyological evolution in the order Cetacea. However, the chromosomes of only two out of 15 Mesoplodon species are known. The Y chromosomes of this genus are also still unknown. The lack of knowledge on the chromosomes of the Mesoplodon species is due to the difficulty in collecting living cells from these animals because of their deep sea habitat and in identifying species due to their similar external morphology (Jefferson et al., 2008).
We obtained living cells from males of the Stejneger's beaked whale M. stejnegeri and the Hubbs' beaked whale M. carlhubbsi stranded in Japan. The present study provides the first description of the male karyotypes of the M. stejnegeri and M. carlhubbsi.
A male Mesoplodon stejnegeri (NSMT-M 42578), which stranded in Niiya-cho, Sakaiminato-shi, Tottori prefecture, Japan, on March 25, 2014, and a male M. carlhubbsi (SNH15011), which stranded in Samani-cho, Hokkaido, Japan, on April 14, 2015, were examined. Both species were identified based on external morphology and tooth shape (Figure 1). The adult male M. stejnegeri is characterized by a dark gray body, a head sloping gently down to the beak, and a tusk of which the leading edge is nearly straight and the pointed tip situates almost inline on the superior extension of this leading edge. The adult male M. carlhubbsi has a tusk of which the leading edge continues to a shoulder-like curve and the tip is found well behind the leading edge. The whole body is almost dark gray with white portions on the tip of the beak and on a bulged frontal region of the head.
Small pieces of the intercostal muscle from M. stejnegeri and cartilage pieces from the pectoral fin tip of the M. carlhubbsi were sampled within 24 hours of their respective deaths and preserved at 4 o C until use. The pieces were cultivated in a culture medium (AmnioMAX TM -II Complete medium, Gibco ® , Life Technology Inc., New York) at 37 o C, 5% CO 2 . The early-passage cells were incubated in hypotonic solution (0.075M KCl) at 37 o C for 18 min after the addition of Colcemid (KaryoMAX ® COL-CEMID ® Solution, Gibco ® , Life Technology Inc., NY) and incubation at 37 o C for 1-2 h. The cells treated with hypotonic solution were fixed with modified Carnoy's solution (1:3 acetic acid methanol). C-banding was performed using the barium hydroxide-saline-Giemsa (BSG) method of Sumner (1972). Gbanding was also conducted according to the technique of Burgos et al. (1986) with some modifications in times. The slide was dried at 95 o C for 23 min. The dried slides were immersed in 0.0125% trypsin (2.5% Trypsin (10X), Gibco ® , Life Technology) for 7 s, then in 70% ethanol. The slides were treated with 2SSC at 60 o C for 10 min and stained with 4% Giemsa (KaryoMAX ® Giemsa Stain Improved R66 Solution "Gurr",Gibco ® , Life Technology) for 8 min. Nucleolus organizer regions (NORs) were stained using the one-step method of Howell and Black (1980). We observed a total of 27 cells (conventional karyotype, 18; C-banding, 9) and 17 cells (conventional, 7; C-banding, 4; G-banding, 4; NOR, 2) for M. stejnegeri and M. carlhubbsi, respectively. The chromosomes were identified as proposed by Levan et al. (1964).
The males of M. stejnegeri and M. carlhubbsi had the same diploid number of chromosomes (2n = 42) but differed in chromosomal morphology (Figures 2 and 3). The karyotype of M. stejnegeri comprised 12 metacentric, four submetacentric, two subtelocentric, and two acrocentric autosomal pairs and subtelocentric X and metacentric Y chromosomes. The karyotype of M. carlhubbsi comprised 12 metacentric, five submetacentric, and three acrocentric autosomal pairs and subtelocentric X and metacentric Y chromosomes. In both karyotypes, the metacentric autosomes were all small and the submetacentric and subtelocentric autosomes were relatively large. These characteristics are also common throughout the general cetacean karyotype 2n = 44 (Árnason, 1974).
The C-banding karyotypes of both species were characterized by C-heterochromatin accumulation (Figures 2b  and 3b). The total lengths of the C-heterochromatic regions of M stejnegeri and M. carlhubbsi represented 28.4% and 17.8%, respectively, of the total lengths of all chromosomes in the hypothetical female haploid set (autosomes + XX). In another Mesoplodon species, M. europaeus, the C-banding positive regions occupied 17% of all chromatic regions (Árnason et al., 1977). According to Árnason (1974), in general, the degree of C-heterochromatin accumulation appears to be greater in mysticetes (around 25%) than in odontocetes (12-15%). The degree of C-hetero- 804 Kurihara et al. chromatin accumulation in Mesoplodon species is similar to that in mysticetes rather than that in odontocetes. Furthermore, notably, M. stejnegeri and M. carlhubbsi had a large X chromosome with a huge C-block in the long arm. Similar characteristics were also reported in M. europaeus and M. carlhubbsi by Árnason et al. (1977). This characteristic is considered a peculiarity of the Mesoplodon species karyotype, because it is not found in other cetaceans, e.g., Stenella clymene (Árnason, 1980), Phocoena phocoena (Árnason, 1980), Physeter macrocephalus (Árnason, 1981a), and Pontoporia blainvillei (Heinzelmann et al., 2008). C-banding karyotypes of M. stejnegeri and M. carlhubbsi also possessed characteristics identical to those of the general cetacean karyotype 2n = 44 described by Árnason (1974): less accumulated C-heterochromatin in the centromeric region and heteromorphism in the C-banding pattern, as shown in ST1 and ST2 of M. stejnegeri ( Figure 2b) and M4 of M. carlhubbsi (Figure 3b). The Y chromosome was small, with its whole body strongly stained in both M. stejnegeri and M. carlhubbsi. On the other hand, some differences in C-banding pattern were found between M. stejnegeri and M. carlhubbsi. Whereas M. stejnegeri had large C-blocks in ST1 and ST2 ( Figure  2b), M. carlhubbsi did not (Figure 3b). Interstitial C-bands were found in SM3, SM5, A1, and A3 in M. carlhubbsi, but only in A2 in M. stejnegeri. Therefore, it is considered that interspecific variation in chromosomal morphology among Mesoplodon species appears to be caused by C-heterochromatin accumulation.
The G-banding karyotype of M. carlhubbsi exhibited heteromorphisms in SM5 (Figure 3c). The distal G-band positive region of the long arm of SM5 was larger in one of the homologues (Figure 3c). This heteromorphism was in agreement with the C-banding pattern and was found in all cells examined (Figures 3b and c).
The NOR-banding karyotype of M. carlhubbsi was obtained on the same slide as that used for the conventional karyotype ( Figure 3d). NOR regions were found at the telomeric positions in both the long and short arms of SM1 and at the telomeric positions in the short arms of M11 and M12. Although NORs were not stained for M. stejnegeri, a chromosome association was found in one cell, indicating the presence of the NOR regions (Figure 4). A small metacentric autosome and a large subtelocentric autosome (ST1) were attached at the terminal positions of their short arms. It is known that M. europaeus has two NOR pairs, one on a large and one on a small autosomal pair (Árnason, 1981b). Therefore, the presence of NORs on a large autosomal pair and on the one or two small autosome pairs would be common throughout Mesoplodon species. As mentioned by Árnason (1981b), NORs on the terminal region of the smaller autosomes were also identical to the general cetacean karyotype (2n = 44).
In the present study, the male karyotypes of two whales (M. stejnegeri and M. carlhubbsi) were clarified. It was confirmed that the karyotypes of Mesoplodon species have some peculiarities, and their 2n = 42 karyotype possesses some characteristics identical to those of the general Karyotype of Mesoplodon species 805