Introduction

Beaked whales (Family Ziphiidae, Odontoceti, Cetacea) include the second largest number of species among toothed whale families. Their preference for deep ocean waters, elusive habits, and long dive capacity1 make beaked whales hard to see and inadequately understood. A total of 22 species are currently recognized in six genera (Berardius, Hyperoodon, Indopacetus, Mesoplodon, Tasmacetus, and Ziphius)2. The genus Berardius has two species, Baird’s beaked whale Berardius bairdii, found in the North Pacific and adjacent waters, and Arnoux’s beaked whale B. arnuxii, found in the Southern Ocean3. Besides the two nominal species, however, whalers’ observations off Hokkaido, northern Japan, have alluded to the occurrence of two groups of Berardius, one being slate-gray form and the other, the black form, which are smaller in body size4,5. Today, slate-gray form is common around Japan, which are traditionally considered as B. bairdii, but black form is rare, and no detailed morphological examinations have been conducted so far. Recent molecular phylogenetic analyses strongly suggest the black and the slate-gray forms in the North Pacific as genetically separate stocks of Berardius6,7, awaiting further work with sufficient morphological data to verify the differences between the two types of Berardius.

Here, we examined black type beaked whale external morphology and skull osteometric data obtained from four specimens including three from Hokkaido and one from the United States National Museum of Natural History (USNM) collection, to highlight the morphological characteristics of the black form after comparison with those of their congeners, B. bairdii and B. arnuxii. The observed unique external characters and skull osteomorphology, coupled with updated molecular phylogeny of Berardius, distinguish the black form as a third Berardius species previously unknown in cetacean taxonomy.

Genus Berardius

Before discussing the above-mentioned subject, it would be useful to summarise what is known about the genus Berardius. Berardius was established by Duvernoy in 18518, who described B. arnuxii based on a specimen collected in New Zealand. The skull and mandibles of this individual are preserved in le Museum Nationalle d’Histoire Naturelle (MNHN) in Paris. Stejneger9 described a similar species of this genus, B. bairdii Stejneger (USNM 20992), as a northern counterpart in 1883; this description was published just a few months earlier than Malm’s10 description of B. vegae, which was later defined as a junior synonym of B. bairdii11. Both specimens were collected from Bering Island. The B. bairdii holotype includes a skull and mandibles, and the B. vegae holotype consisted of broken skull pieces. B. arnuxii and B. bairdii could be good examples of antitropical distribution12.

As summarised by Kasuya5,13, there have been extensive debates on the identities of these two species, because they are very similar except for body size and distribution. B. arnuxii is slightly smaller than B. bairdii. True11 pointed out several characters that are distinct between these two species. However, as the number of specimens increased, most of the characters lost systematic significance, and their validity was disputed14,15. Dalebout et al.16 put an end to this discussion and showed that the two species are genetically distinct and independent. However, morphological discrimination of these two species is not currently well established and we have to rely on molecular results or distribution to discriminate these two species. Ross17 noted that more thorough morphological investigations are needed to distinguish B. bairdii and B. arnuxii.

Berardius skulls are the least asymmetrical and sexually dimorphic among genera of the family Ziphiidae; only the body length of females is slightly larger than that of males. The beak is straight and long. Unlike most other ziphiids, they have two pairs of teeth in the lower jaw. The blowhole slit is unique, with a posteriorly opened arch that is unlike those of all other odontocete groups (e.g. Kasuya18). Although the nasals are large, they do not overhang the superior nares.

History of Berardius in Japan

In 1910, True11 summarised the ziphiid specimens that were preserved and stated “Berardius is the rarest genus, only about fourteen specimens having been collected thus far”. Also in 1910, Andrews visited the Imperial Museum at Tokyo, which is now called the National Museum of Nature and Science (NMNS), to find a B. bairdii skeleton19; this occurred when existence of Berardius in Japan was known to science and, on this historical occasion, B. bairdii was confirmed to correspond to “tsuchi-kujira”20 of Japan. When considering the recognition of B. bairdii in Japan, however, the Japanese name tsuchi-kujira had been used since the early 18th century, and whaling activities have been aimed at this species since then21,22,23,24. Proper comparison and recognition of this species using the Western (or Linnean) systematic scheme took some time after the introduction of modern science from the West, which began in 1868 after the Meiji Restoration. Researchers such as Okada25 incorrectly identified tsuchi-kujira as Hyperoodon rostratus, and this notion was generally accepted in most publications. In 1910, Andrews examined the specimens of tsuchi-kujira (then recognised as H. rostratus) that were exhibited in the Imperial Museum in Tokyo, and identified them as B. bairdii19. He surveyed the locality of this B. bairdii specimen and collected a whole skeleton of this species in Chiba. This event was reported by Nagasawa20 to the Zoological Society of Japan and confirmed the existence of B. bairdii in Japanese waters.

Results

The following description was prepared by Tadasu K. Yamada, Shino Kitamura and Takashi F. Matsuishi.

Systematics

Order CETARTIODACTYLA Montgelard, Catzeflis and Douzery, 199726.

Infraorder CETACEA Brisson, 176227

Parvorder ODONTOCETI Flower, 186428

Family ZIPHIIDAE Gray, 186529

Genus BERARDIUS Duvernoy, 18518

Berardius minimus sp. nov.

(New Japanese name: Kurotsuchikujira)

Etymology

The specific name reflects the smallest body size of physically mature individuals of this species compared with the other Berardius species. Historically, whalers in Hokkaido recognised this species as different from B. bairdii and called them “kuro-tsuchi”, which means black Baird’s beaked whale; however, the colour difference mainly depends on the scar density and is not biologically fundamental (Figs 1 and 2). We therefore chose the most basic difference, the significantly small body size, which is smallest among the congeners, to be reflected in the scientific name.

Figure 1
figure 1

Unidentified beaked whale incidentally caught in Shibetsu, Hokkaido (photo taken by Minako Kurasawa on 20 July 2004, courtesy of Hal Sato).

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Figure 2
figure 2

Unidentified beaked whales sighted in Nemuro strait. Note the short beak, dark body colour, and sparse linear scars (photo taken by Hal Sato on 21 May 2009).

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Holotype

Adult male (NSMT-M35131) skull, mandible, and most of post of postcranial skeleton at National Museum of Nature and Science (NMNS). In addition, tissue samples are also preserved at the NMNS. This specimen, a fairly well decomposed stranded carcass was found on 4 June 2008 (Fig. 3A–C). Upon receiving notice, SNH took action, and Prof. Mari Kobayashi of Tokyo University of Agriculture and her students examined the carcass on-site. The carcass was then buried at a nearby. The whole skeleton was excavated and recovered on 26 and 27 August 2009 by one of us (SNH), Tokyo University of Agriculture, Institute of Cetacean Research, and NMNS.

Figure 3
figure 3

Severely decomposed beaked whale stranded in Kitami, Hokkaido on 4 June 2008. (A) The relatively shorter beak indicates it is not B. bairdii (photo taken by Mari Kobayashi), (B) although the blow hole shape indicates it belongs to Berardius (photo taken by Mari Kobayashi). (C) The general body shape is that of typical ziphiid species. When compared with adult B. bairdii, this specimen is more spindle-shaped.

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Type Locality

Tokoro Town (44°07′14.5N, 144°06′29.6E), Kitami City, Hokkaido, Japan, southern Okhotsk Sea, North Pacific.

Nomenclatural statement

A Life Science Identifier (LSID) was obtained for the new species (B. minimus): urn:lsid:zoobank.org:act:C8D63A76-B1A3-4C67-8440-AFCE08BE32E9, and for this publication: urn:lsid:zoobank.org:pub:52AD3A26-4AE6-42BA-B001-B161B73E5322.

Diagnosis

Berardius minimus differs from all of its congeners by having the following unique characters: remarkably smaller body size of physically mature individuals, proportionately shorter beak, darker body colour subsequent noticeable cookie-cutter shark bites.

External characters

External appearance is mostly known from a male individual found stranded on 10 November 2012 in Sarufutsu, Hokkaido (Fig. 4). Most of the external characters of B. minimus are typical of medium- to large-sized ziphiids, with several discriminating characters, such as the narrow, straight, and longer beak; reverse V-shaped throat grooves; relatively smaller flippers (flipper length is 11.4% of body length on average; range, 7.7–13.4%); small dorsal fin (dorsal fin height is 3.7% of body length on average; range, 3.4–3.9%) located 70% of body length (on average; range, 66.7–71.8%); and tail flukes that lack the median notch. However, the posteriorly opened crescent-shaped blowhole slit indicates Berardius affinity. Additionally, B. minimus has a substantially smaller body size (maximum body length of 6.9 m in physically mature individuals, so far), more spindle-shaped body, and relatively shorter beak, which is approximately 4% of the body length and is not consistent with the morphology of either of the known Berardius species.

Figure 4
figure 4

Fresh carcass of Berardius minimus (male, 662 cm) found stranded on 10 November 2012 in Sarufutsu Hokkaido. (A) Ventral view of the carcass. Note the whole body is almost black except for the faintly white beak. (B) The relatively short beak of the same individual (photos taken by Yasushi Shimizu).

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Body colour is almost black with a pale white portion on the rostrum; this is in contrast to B. bairdii, which is described as “slatish”4 or “slate grey”6,7 or B. arnuxii, which is described as black30 or light grey31. The greyish tone of the B. bairdii body is mainly attributed to the dense healed scars that are probably caused by intraspecific conflicts and/or behaviour. At least in adult and subadult individuals of B. minimus, cookie-cutter shark bites are fairly conspicuous, but not to the extent as usually seen in some other species such as Ziphius cavirostris, Mesoplodon densirostris, and/or Balaenoptera borealis. The darker body colour with almost no scars produces a sharp contrast with the healed cookie-cutter shark bites, which are white and very conspicuous against the black body of B. minimus.

The beak is much shorter than in the other two Berardius species. In B. bairdii, the head proportions are extremely small, and are much smaller than that of B. minimus. Body colour is almost uniformly dark brown with a whiter portion at the tip of rostrum. No white patch on the belly was confirmed in B. minimus. An illustration of an adult male of B. minimus is shown as Fig. 5. At present, we do not know what adult females look like.

Figure 5
figure 5

Illustrations of (A) Berardius minimus, and (B) B. bairdii. The black bars show 1 m. In general appearance, B. minimus resembles a small B. bairdii with a proportionately shorter beak and more spindle-shaped body (drawn by Yoshimi Watanabe, National Museum of Nature and Science).

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External measurements

As mentioned above, the distinctly small body length of physically mature individuals and proportionately shorter beak are the most reliable characters which indicate that the population in question represents a species that was previously not known to science.

Regarding body length, a strong significant difference was found between the body length of male B. bairdii from the Okhotsk Sea (n = 34)32 and mature male B. minimus (n = 4, Table 1) (Welch’s t-test, t = 18.5, P < 0.001).

Table 1 List of Berardius minimus specimens that were stranded or drifting and collected in Hokkaido.
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To confirm relative rostrum-to-body length, Welch’s t-test was also conducted. For B. minimus, four samples in Table 1 were analysed. For B. bairdii, the mean and standard deviations for male B. bairdii in the Okhotsk Sea (n = 29) that appeared in Table 2 of Kishiro32 were used. Rostrum length was standardised by body length, and was 3.62 ± 0.39 SD% (n = 4) for B. minimus and 5.81 ± 0.80 SD% (n = 29) for B. bairdii. Welch’s t-test showed strong significant difference (P = 2.3 × 10−5). Female B. bairdii relative length was 6.27, which is longer than that of males. Note this female was not physically mature. The difference between B. minimus and B. bairdii was obviously larger if the sex-pooled data were used. A strong significant difference was also found between B. minimus and B. bairdii in the Pacific Ocean and Sea of Japan (P < 0.001). Thus, the relative rostrum length of B. minimus was significantly shorter than that of B. bairdii. However, we note that the sample size for both B. minimus and B. arnuxii are extremely small, in contrast to B. bairdii.

Table 2 Mean, standard deviation, and range of each measurement by species.
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Skull morphology

The skull morphology resembles the skulls of both existing Berardius species, but B. minimus has a distinctly shorter rostrum if contrasted to the condylobasal length, and smaller bulla and periotic bone. In general, the sutures are more tightly closed in B. minimus than those in the other Berardius species. In the hyoid bone, thylohyal and basihyal are not fused at all (Fig. 6).

Figure 6
figure 6

Skull of the B. minimus holotype. (A) Dorsal, lateral, and ventral views of the skull. Note the relatively short rostrum. The white bar indicates 10 cm. (B) Anterior and posterior views of the skull. The dorsal view is more triangular, whereas the dorsal views in B. bairdii and B. arnuxii are more pentagonal. The white bar indicates 10 cm. (C) Lingual (inner, upper) and buccal (outer, lower) sides of the left mandible. The white bar indicates 10 cm. (D) Buccal (external) view of the anterior (left) and posterior (right) teeth of the lower jaw. The white bar indicates 1 cm.

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Superior aspect

The following characters are readily recognisable as species-specific. The relative beak length in B. minimus is clearly smallest among the three Berardius species. The B. minimus skull has much tighter sutures compared with those in both B. arnuxii and B. bairdii. The proportional distance of the anterior end of the maxillae from the tip of the rostrum (i.e. premaxillae) relative to condylobasal length of the skull is much smaller in B. minimus (6.93% in NSMT35131) than the two previously known Berardius species (which have a distance of approximately 10%). The inclination of the occipital bone is stronger in B. minimus, and the occipital plane is much wider compared with the other two species. The antorbital notch is proportionately narrower in B. minimus than in B. bairdii but similar to that in B. arnuxii. The B. minimus rostrum has simple tapering contour lines toward the tip, whereas both contour lines of the rostrum are parallel in B. bairdii and B. arnuxii. The lateral border of the orbit, which consists of the maxilla and frontal bones, is almost parallel to the sagittal plane in B. minimus, but is oblique in other two species.

Lateral aspect

The relative rostrum length is obviously shorter in B. minimus, and the B. minimus rostrum also looks much shorter than those of the other two species in side view. The skull height relative to condylobasal length is much larger (0.41–0.44) in B. minimus than those in B. bairdii (0.35–0.40) and B. arnuxii (0.40–0.41). There is stronger inclination of the higher portion of the occipital plane in B. minimus, and the convexity of the occipital plane is stronger in B. minimus. The temporal fossa is the shallowest in B. minimus and the medial wall of the fossa is convex, but is concave in B. bairdii and B. arnuxii.

Posterior aspect

The structure above the temporal fossa is proportionately much larger and higher in B. minimus than those in B. bairdii and B. arnuxii, which gives the impression that the B. minimus skull is rather triangular in the posterior view, whereas those of the other two species are pentagonal.

Anterior aspect

In the frontal view, lateral expansion of the premaxillae at the posterior is prominent, and the posterior margins of both maxillae are clearly visible in B. minimus.

In B. minimus, the height of skull relative to the width is much higher than those of the other Berardius species. The prominential notch and related structure are much higher, more distinct and more rugged in B. bairdii and B. arnuxii.

Teeth

As in the other two Berardius species, B. minimus has two pairs of teeth only at the tip of the lower jaw. The anterior tooth is much larger than the posterior tooth. Teeth dimensions of the holotype are shown in Table 3 (57-1 and 2, 58-1 and 2). In the holotype specimen of B. minimus the pulp cavities are almost closed in all teeth other than the right 2nd tooth, where the pulp cavity is open.

Table 3 Skulls used for craniometry analyses. Twenty-one specimens (10 Berardius bairdii, seven B. arnuxii, and four B. minimus).
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Post cranial skeleton

The vertebral column has proportionately high spinous processes, which is observed in most ziphiid species (Fig. 7). The bone matrix is coarse and porous, and they float on the processing water after internal soft tissue was removed. In the holotype specimen, the vertebral formula is C. 7, Th. 10, L. 10, Ca. 19, making the total count as 46. Among 7 cervical vertebrae, C1–C3 were fused. L4 and L5 are the tallest vertebrae. Ca10 and 11 are so-called ball vertebrae. Ten chevrons were counted. Ribs are in 10 pairs, among which seven pairs are dual-headed with both costovertebral and costotransversal articulations. The remaining three pairs have only one articulation facet which articulate with “transverse” processes of the caudal thoracic vertebrae. No ossified cervical ribs were found. The sternum is composed of five segments.

Figure 7
figure 7

Articulated skeleton of the B. minimus holotype specimen.

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Paired ossified pelvic bones have a lateral surface which is fairly smooth; however, in the medial surface, approximately two-thirds of the total length is an elevated area where the corpus cavernosum penis attaches. Viewed from the dorsal side, the pelvic bones show a very gentle s-shape. No rudimental femur or any additional appendicular bone was collected.

Pectoral appendage

Regrettably, we could not secure all phalangeal bones of the left flipper. On the right side, there are three carpal bones in the proximal row, possibly the Ossa radiale, centrale, and ulnare. In the distal row are another three carpal bones. All five digits have one each metacarpal; the phalangeal formula is 0-5-4-3-2.

Multi-measurement comparison

Table 2 shows the mean, standard deviation, and range of each measurement by species. PCA showed that the contribution of the first principal component (PC1) was 73.9%, and the cumulative contribution reached 90% for PC1-6. Thus, linear discriminant analysis was conducted using PC1-6.

Table 4 shows the linear discriminant coefficients obtained by linear discriminant analysis (LDA). The linear discriminants coefficients of each sample are plotted in Fig. 8. The distribution of the linear discriminants variates was very clearly separated by species.

Table 4 Linear discriminant coefficients obtained by linear discriminant analysis (LDA).
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Figure 8
figure 8

Linear discriminant variates of each sample are plotted. The linear discriminants variates are clearly separated by species. B: Berardius bairdii, A: B. arnuxii, M: B. minimus.

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Genetic considerations

Molecular phylogenetic relationships among three Berardius species were examined using nucleotide sequence variation of the mitochondrial (mt)DNA control region (CR). The 879-bp complete CR sequence data from eight B. minimus specimens (Table 5) (Acc. Nos AB572006-AB572008 from Kitamura et al.6, Acc. Nos LC175771-LC175773 in this study, and Acc. Nos KT936580-KT936581 from Morin et al.7) showed five haplotypes with only 1–4 nucleotide differences without gaps after multiple alignment. Using the CR sequences aligned with 430-bp B. arnuxii sequences (Acc. Nos AF036229 and AY579532 from Dalebout et al.16) excluding gaps, the number of nucleotide differences between B. minimus and its congeners was 18–22 for B. bairdii and 25–29 for B. arnuxii. Thus, the mtDNA nucleotide difference between B. minimus and any of its congeners was much greater than the difference between B. bairdii and B. arnuxii, which is 12–16 nucleotides. The observed CR nucleotide differences supported the distinct position of B. minimus in the Berardius tree constructed from 430-bp sequences using the maximum likelihood method, where B. bairdii and B. arnuxii formed a sister clade (Fig. 9).

Table 5 Individuals and sequences used in this study. SNH: Stranding Network Hokkaido, Hokkaido, Japan; EW: Ehime University es-Bank, Ehime, Japan; NSMT: National Museum of Nature and Science, Ibaraki, Japan.
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Figure 9
figure 9

Maximum likelihood-based molecular phylogenetic relationships among the three Berardius species, with Indopacetus pacificus as the outgroup. See Materials and Methods for details regarding nucleotide sequencing and tree construction.

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Known distribution

As is indicated by the map of localities where B. minimus was found (Fig. 10), their known distribution is very limited and occurs between 40°N and 60°N, and 140°E and 160°W.

Figure 10
figure 10

Berardius minimus localities plotted against the B. bairdii distribution map (shaded area, as described by Kasuya18). Circles show B. minimus localities. The white circle with a black X indicates the B. minimus type locality, whereas the black circle with the white X indicates the B. bairdii type locality.

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Discussion

Kasuya5,18 summarised Hokkaido whalers’ traditional knowledge. The whalers recognised two types of tsuchi-kujira: the ordinary “tsuchi-kujira” (Berardius bairdii) and the darker and smaller “kuro-tsuchi” (black Baird’s beaked whale) or “karasu” (crow). However, it is unclear whether “kuro-tsuchi” and “karasu” are used to describe the same type of whales or each notion represents the different population.

In this study, we described a new species, B. minimus, which corresponds to “kuro-tsuchi”. If “karasu” exists as a third type, it could be a species that is not yet recognised or a Mesoplodon species found in Hokkaido (either M. stejnegeri or M. carlhubbsi). Recognition of these Mesoplodon species around Hokkaido is rather recent; the earliest M. stejnegeri specimen was collected in 198533, and the earliest M. carlhubbsi in 200434. These Mesoplodon species were not recognised as distinct species by whalers or the media until recently.

As was also pointed out by Kasuya18, Fig. 364 and 366 of Heptner35 hinted at the possibility of a Hyperoodon-like whale in the northern Pacific. The animal in the photo was definitely not Berardius. This could be a species of probably about 10-m long with a beak almost like that of Hyperoodon. We suspect this could be an example of an extralimital occurrence of H. ampullatus. Considering the recent sightings of the gray whales in the Mediterranean or in Namibia36,37, the possibility of vagrant individual navigate through the Northwest passage during summer should be studied.

The species we described is rather readily recognisable by people with whale taxonomy experience based on the external characters. The species has an obviously smaller body size, which is 6.3–6.9 m in physically mature individuals, so far we confirmed (Morin et al.7 reported an adult male with 7.3 m body size). Their body size ranges from 9.1–11.1 m in B. bairdii and 8.5–9.75 m in B. arnuxii38. They have a relatively short beak that is approximately 4% of the body length. They have a dark body colour, which is almost uniformly black with noticeable healed cookie-cutter shark bites forming white dots; this impressively contrasts with the much lighter colouration of B. bairdii and likely result from healed scratches and scars that were probably caused by intra-specific struggling and bottom-feeding behaviour.

Osteologically, the small body size of physically mature individuals is the main defining character of B. minimus. Condylobasal length of the skull is 935–1042 mm, in contrast to 1343–1524 mm in B. bairdii and 1174–1420 mm in B. arnuxii9. Skull characters indicate significant influence of size difference, such as tighter bone sutures compared with those of other Berardius species. Skull elements of the brain case are relatively large and conspicuous. The vertebral formula of the type specimen is C. 7, Th. 10, L. 10, Ca. 19 (totalling 46), whereas it is C. 7, Th. 9–11, L. 12–14, Ca. 17–22 (47–52) in B. bairdii and C. 7, Th. 10–11, L. 12–13, Ca. 17–19 (47–49) in B. arnuxii13. Rib count, which reflects the thoracic vertebral count, is 10 in the B. minimus type specimen.

As was mentioned above, when comparing the skull sutures in similarly mature individuals of different species of cetaceans, there is a general tendency that the larger the adult form is the less rigid skull composition is observed. Cetacean facial skull consists loosely articulated bones, including the maxillae, premaxillae and frontals, which are adhered to the mesorostral cartilage pillar on the vomer by connective tissue. It is a physically significant principle where cetaceans swing their rostrum in the water for foraging. It requires tremendous power and the flexibility of the skull structure must ease the stress given to the skull structure. In this context it is quite reasonable that the skull of B. minimus is far more rigidly composed compared to those of the far larger species, such as B. bairdii and B. arnuxii. It means adult size of B. minimus is essentially far smaller than the other two Berardius species.

The molecular biology of B. minimus was previously discussed by Kitamura et al.6, and specific genome characters were only identified in individuals collected from Hokkaido. However, we found a skull with B. minimus characters in the collection of the USNM which was collected from the Unalaska Island in 1943. Additional individuals were detected among the samples collected in the Aleutian area, and further analyses and considerations were conducted and discussed by Morin et al.7. Further detailed analyses on Berardius species in both the northern and southern hemispheres are needed to explain Berardius speciation processes.

The currently recorded B. minimus distribution is very limited and occurs between 40°N and 60°N, and 140°E and 160°W. They have fairly dense cookie-cutter shark (Isistius brasiliensis) bites. The cookie-cutter shark is understood to be a tropical to warm-temperate species and their northern limit in the western North Pacific is reported to be 30°N to 43°N39. However, the southern limit of the B. minimus distribution might extend further south.

Although species identities of B. arnuxii and B. bairdii have been previously debated, we described another species of this genus. However, it is unclear whether B. minimus speciation occurred before or after the antitropical split of B. arnuxii and B. bairdii. Additionally, the area where Berardius speciation took place should be examined in the future.

Methods

Specimens examined

The specimens of this unknown species, which were collected in Hokkaido, are listed in Table 1. No live animals were used for the current research. Observations on the external appearance and morphometrics, observations on the skeleton especially of the skull, skull morphology and measurements and molecular phylogenetic analysis were conducted.

External morphology and measurements

External observations of the five individuals of Berardius minimus (three physically mature males, one subadult female, and a head of one neonate female) were made, and the external morphometrics following previous studies32,40 (Tables 6 and 7) were conducted on four B. minimus (all physically mature males; Table 1). Raw data examination revealed that body length and the ratio of beak length-to-body length significantly differed, and Welch’s t-test was applied to these variables.

Table 6 External measurements of Berardius minimus used for the comparison with B. bairdii.
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Table 7 Measured external morphometrics characters for B. bairdii as described in previous studies32,40.
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Skeletal morphology and measurements of the skull

Observations of the skeleton, especially of the skull, and skull measurements were made for 21 specimens (10 B. bairdii, seven B. arnuxii, and four B. minimus) (Table 2). Specimens are stored at the USNM, NMNS, MNHN, Natural History Museum of London (BMNH), and Museo Acatushún (MA).

Multivariate analysis

To examine the difference between the morphological features among species, a multivariate analysis was conducted. To describe the effect of the difference of body size by species, a principal component analysis (PCA) was conducted for 27 measurements shown in Table 5 for 22 samples (four B. minimus, 10 B. bairdii, seven B. arnuxii) shown in Table 3. For all variables, measured values using this analysis are indicated in bold gothic.

A linear discriminant analysis (LDA) was then conducted to compare species using the scores obtained from the principal component analysis (PCA). Calculations were carried out using “prcomp” and “lda” function in R ver.3.3.141.

Nucleotide sequence analysis and molecular phylogeny

The 18 mtDNA control region (CR) sequences analysed (Table 5) included sequences from three B. minimus specimens (Acc. Nos LC175771-LC175773 for SNH12044, SNH12054, and SNH14016, respectively) and 15 previously reported sequences, which included seven for B. bairdii (Kitamura et al.6, AB571999-AB572005), five B. minimus (Kitamura et al.6, AB572006-AB572008, updated complete sequences August 2016; and Morin et al.7, Acc. Nos KT936580-KT936581), two B. arnuxii (Dalebout et al.16, Acc. Nos AF036229 and AY579532), and one Indopacetus pacificus (Kitamura et al.6, AB572012) as an outgroup. I. pacificus was selected because it belongs to the same family but is in a rather distant genus, which was inferred by a previous CR phylogenetic tree6. All the newly collected samples for the nucleotide sequence analysis and molecular phylogeny were officially transferred to the authors from the original sample holder, the Stranding Network Hokkaido. Nucleotide sequencing of the complete mtDNA CR in the three B. minimus was performed using primer pairs CRL (5′-CAA CAC CCA AAG CTG GAA TTC T-3′)6 and CRH2 (5′-TAG ACA TTT TCA GTG TCT TGC-3′, which was newly designed for this study) for PCR amplification, and CRH (5′-CCA TCG AGA TGT CTT ATT TAA G-3′)6 and LCR (5′-GAC ATC TGG TTC TTA CTT CAG G-3′)42 as internal sequencing primers.

CR sequence alignment was performed using CLUSTAL X43, and the output was inspected by eye following the application of multiple alignment parameters in the program. All CR sequences were adjusted to the short length of the B. arnuxii sequence, 430 bp (Dalebout et al.16, Acc. Nos AF036229 and AY579532), for multiple sequence comparison and molecular phylogenetic analysis.

A molecular phylogenetic tree was constructed with 430-bp mitochondrial CR sequences of all analysed species using the maximum likelihood algorithm in MEGA version 744 based on the Tamura 3-parameter model45 with gamma distribution (parameter = 0.2001), which was suggested to be the best nucleotide substitution model based on a model test in this program. Bootstrap values were calculated by 1,000 replicates46.