Introduction
Beaked whales (ziphiids) are diverse and wide-ranging, yet remain poorly understood. Mesoplodon exemplifies this contradiction: with 16 members, it is the most speciose cetacean genus (Committee on Taxonomy, 2022) but also amongst the most enigmatic large mammals on Earth (Jefferson et al., Reference Jefferson, Pitman and Webber2015). Notable knowledge gaps persist regarding species distributions, abundance estimates and even basic biology, with several species having never been recorded alive (MacLeod et al., Reference MacLeod, Perrin, Pitman, Barlow, Ballance, D'Amico, Gerrodette, Joyce, Mullin and Palka2006; Pitman, Reference Wursig, Thewissen and Kovacs2018). Mesoplodon occurs from cold subpolar latitudes to the tropics, but can be difficult to observe owing to a preference for deep-water habitats (Reeves et al., Reference Reeves, Smith, Crespo and di Sciara2003; MacLeod et al., Reference MacLeod, Perrin, Pitman, Barlow, Ballance, D'Amico, Gerrodette, Joyce, Mullin and Palka2006).
Ramari's beaked whale (Mesoplodon eueu, Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021) is the most recently described mesoplodont with records from South Africa, Australia, and Aotearoa New Zealand. Until recently, M. eueu was thought to be a southern, antitropical population of the North Atlantic True's beaked whale (Mesoplodon mirus True, Reference True1913), but molecular and morphological data now confirm a deep independent history warranting species status (Ross, Reference Ross1969, Reference Ross1984; MacLeod et al., Reference MacLeod, Perrin, Pitman, Barlow, Ballance, D'Amico, Gerrodette, Joyce, Mullin and Palka2006; Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021).
A previously reported stranding of True's beaked whale in Brazil also plausibly represents M. eueu, which may therefore range across the Southern Hemisphere (Souza et al., Reference Souza, Siciliano, Cuenca and Sanctis2005); however, to date no South American records of Ramari's beaked whale have been genetically confirmed. Here, we provide the first such record, based on two stranded individuals from Uruguay, and explore the implications of our new molecular data for the diversity and evolutionary history of the species.
Materials and methods
Our analysis is based on two beaked whales that stranded in Canelones Department, Uruguay, in 2019 (Figure 1). On 27th October 2019, the fresh carcass (Code 2 of Pugliares et al., Reference Pugliares, Bogomolni, Touhey, Herzig and Harry2007) of a 338 cm long female calf/juvenile was found on San Luis Beach (34°46′31.6″ S, 55°35′24.09″ W; Figure 2). Three days later, a 528 cm long adult female washed up on Marindia Beach (34°46′48.19″ S; 55°49′9.41″ W), 24 km west from the first individual. This specimen showed a moderate state of decomposition (Code 3) suggesting that it died some time prior to the stranding (Figure 3).
Figure 1. Location of the Mesoplodon eueu strandings in Canelones department (Uruguay), stars indicate the two stranding sites.
Figure 2. Left lateral view of the head (A) and ventral view (B) of a juvenile female of Mesoplodon eueu (MNHN 8211) stranded on the coast of Uruguay on 27th October 2019.
Figure 3. Dorsolateral view of an adult female Mesoplodon eueu (MNHN 8212) stranded on the coast of Uruguay on 30th October 2019.
Biological information and several photographs were recorded for both specimens. The skulls of both specimens along with muscle, blubber and skin samples and – for the second stranding only – the stomach contents were deposited at the National Museum of Natural History (Montevideo) under registration numbers MNHN 8211 and 8212, respectively. Tissue samples of both specimens were preserved in 95% ethanol and stored in a freezer at −20°C. As far as the position of the carcasses on the beach permitted, body measurements were taken following Norris (Reference Norris1961).
Skull morphometrics
Following Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021) we used eight measurements of the skull and mandible to test whether our individuals more closely resemble M. eueu or M. mirus. Measurements were taken to the nearest mm, adjusted for size by dividing them by the bizygomatic width, and log-transformed prior to further analysis. Finally, we combined our observations with those of Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021) and summarized the entire dataset via principal component analysis (PCA) in PAST 4.03 (Hammer et al., Reference Hammer, Harper and Ryan2001).
DNA extraction, control region sequencing and genomic sequencing
Total genomic DNA was extracted from muscle following Medrano et al. (Reference Medrano, Aasen and Sharrow1990). The control region was sequenced using t-Pro (Dalebout et al., Reference Dalebout, van Helden, Van Waerebeek and Baker1998) and HS-5 primers (Okumura, Reference Okumura2004). The PCR cycling profile was the following: an initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. The amplified products were sequenced by MACROGEN (Seoul, Korea) using the Sanger sequencing method.
The genome of MNHN 8211 was sequenced on a DNBseq sequencing platform (BGITech Solutions, Hong Kong) using a 150 paired-end strategy and low coverage (1x ≈ 2.5 Gb, genome size of Gervais' beaked whale Mesoplodon densirostris; Yuan et al., Reference Yuan, Zhang, Zhang, Liu, Wang, Gao, Hoelzel, Seim, Lv, Lin, Dong, Gao, Yang, Caruso, Lin, da Fonseca, Wang, Wang, Rasmussen, Liu, Zheng, Zhao, Campos, Kang, Iversen, Song, Guo, Guo, Qin, Pan, Xu, Meng, A, Liu, Lee, Liu, Xu, Yang, Fan, Wang and Li2021). Adaptor clean-up and quality filtering were performed by BGI. Mitogenome assembly was carried out using Novoplasty (Dierckxsens et al., Reference Dierckxsens, Mardulyn and Smits2017) with the control region sequence acting as a seed. In addition, we assembled the mitogenome of MNHN 8211 via Bowtie2 (Langmead and Salzberg, Reference Langmead and Salzberg2012), Samtools 1.10 (Li et al., Reference Li, Handsaker, Wysoker, Fennell, Ruan, Homer, Marth, Abecasis and Durbin2009) and Trinity v. 2.6.6 (Grabherr et al., Reference Grabherr, Haas, Yassour, Levin, Thompson, Amit, Adiconis, Fran, Raychowdhury, Zeng, Chen, Mauceli, Hacohen, Gnirke, Rhind, di Palma, Birren, Nusbaum, Lindblad-Toh, Friedman and Regev2011), using the mitogenome of M. eueu (GenBank accession number OK326893) as a reference. Both mitogenome assemblies were congruent and the average coverage of the entire mitochondrial genome was 748. The average Phred scores for MNHN 8211 and 8212 were 48.6 and 57.3, respectively. Control region sequences for both individuals and the annotated mitogenome of MNHN 8211 assembled by Novoplasty were deposited in Genbank as OP994180, OP994181, and OP994173, respectively.
Genetic analyses
We used sequences retrieved from GenBank to generate two data sets: one comprising the mitochondrial control region (CR) of the two stranded specimens, 49 individuals representing various species of Mesoplodon (M. bidens, M. densirostris, M. eueu, M. europaeus, M. ginkgodens, M. grayi, M. mirus, and M. stejnegeri), and two northern bottlenose whales that acted as outgroup; and one comprising the entire mitogenomes of MNHN 8211 and 25 additional individuals representing M. bidens, M. eueu, M. europaeus, M. ginkgodens, and M. mirus (Supplemental Tables S1 and S2).
CR sequences were aligned in Clustal W 1.8 (Thompson et al., Reference Thompson, Higgins and Gibson1994) and then subjected to phylogenetic analysis via Maximum Likelihood (ML) and Bayesian inference (BI). JModelTest v.2.1.17 (Darriba et al., Reference Darriba, Taboada, Doallo and Posada2012) identified TPM3uf + I as the best-fitting nucleotide substitution model based on the Bayesian information criterion (Schwarz, Reference Schwarz1978). The ML analysis was carried out in PhyML 3.1 (Guindon and Gascuel, Reference Guindon and Gascuel2003) using NNI (fast-nearest neighbour interchange search) and 1000 bootstrap pseudoreplicates to estimate branch support. BI was implemented in MrBayes 3.2.7 (Ronquist et al., Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Hohna, Larget, Liu, Suchard and Huelsenbeck2012) on the CIPRES Portal (Miller et al., Reference Miller, Pfeiffer, Schwartz and Miller2010) and comprised two Markov chains in twelve separate runs of 20 million generations. Genetic differentiation between our specimens and other Mesoplodon spp. was assessed by computing pairwise P-distances in MEGA v. 11 (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018).
Divergence times were estimated based on the mitogenomes, including protein-coding genes (split by codon position), transfer and ribosomal RNA genes, and the D-loop region. Substitution models were determined via PartitionFinder2 (Lanfear et al., Reference Lanfear, Frandsen, Wright, Senfeld and Calcott2017) and subsets (Supplemental Table S3) linked with a relaxed clock log normal and a linked Yule tree model. We implemented three calibration points derived from the divergence dating analysis of Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021), (Supplemental Table S4). The analysis was run in BEAST v.2.6.7 (Bouckaert et al., Reference Bouckaert, Vaughan, Barido-Sottani, Duchêne, Fourment, Gavryushkina, Heled, Jones, Kühnert, De Maio and Matschiner2019) in three separate runs of 15 million generations, sampled every 10,000 generations. The results were then combined in Log Combiner (Bouckaert et al., Reference Bouckaert, Vaughan, Barido-Sottani, Duchêne, Fourment, Gavryushkina, Heled, Jones, Kühnert, De Maio and Matschiner2019) after a 10% burn-in, convergence assessed via Tracer v1.7 (Rambaut et al., Reference Rambaut, Drummond, Xie, Baele and Suchard2018), and a Maximum Clade Credibility summary tree calculated via TreeAnnotator (Bouckaert et al., Reference Bouckaert, Vaughan, Barido-Sottani, Duchêne, Fourment, Gavryushkina, Heled, Jones, Kühnert, De Maio and Matschiner2019) with a burn-in of 20%. Finally, we estimated the mitochondrial nucleotide diversity (π) of individuals identified as M. eueu and M. mirus using DnaSP v.6 (Rozas et al., Reference Rozas, Ferrer-Mata, Sánchez-DelBarrio, Guirao-Rico, Librado, Ramos-Onsins and Sánchez-Gracia2017).
Results and discussion
External appearance
Ramaris's and True's beaked whales cannot be consistently distinguished based on their external appearance (Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021). Both share with our specimens the presence of a proportionally small head with a pronounced forehead bulge, a relatively short but distinct beak, a straight mouth line, and a single pair of teeth at the tip of the mandible (Supplementary Figure S1). As is normal in females (Moore, Reference Moore1968), the teeth in MNHN 8211 and 8212 have not erupted.
The overall body colour of both species appears to be one of basic countershading (medium grey on top, lighter grey on the bottom), in addition to a dark patch around the eye and an area of whiter pigmentation that extends from the anus to the genital slit (Mead, Reference Mead, Ridgway and Harrison1989; Pitman, Reference Wursig, Thewissen and Kovacs2018; Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021). The same pattern occurs in our stranded specimens, albeit less clearly so in MNHN 8212 owing to its advanced state of decomposition. In some individuals of M. eueu a pale area stretches from the dorsal fin back towards the flukes (MacLeod, Reference MacLeod, Wursig, Thewissen and Kovacs2018; Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021). A similar patch can be seen in MNHN 8212 but not MNHN 8211 (Figure 4), suggesting ontogenetic variation in coloration patterns as observed in other ziphiids (Mead, Reference Mead, Wursig, Thewissen and Kovacs2009).
Figure 4. Caudal fins of both Mesoplodon eueu specimens stranded, dashed lines indicate the pale area over the peduncle that extends towards the flukes on MNHN 8212 (A) which is absent on MNHN 8211 (B).
Skull morphometrics
The first two principal components of our PCA account for 83.8% of the variance and place our stranded individuals either side of the morphospace occupied by M. eueu (Table 1; Figure 5). Principal component 1 separates MNHN 8211 from all other specimens, with its shorter rostrum and mandibular symphysis likely reflecting its immature state. MNHN 8212 is located roughly halfway between the previously defined morphospace areas of M. eueu and M. mirus. Overall, these results are consistent with – though not conclusive proof of – both of our specimens representing M. eueu.
Table 1. Cranial measurements taken in Mesoplodon eueu specimens stranded in Uruguay
CBL, Condylobasal length; CH, Cranial height; RL, Rostrum length; RW, Rostrum width between antorbital notches; PFW, Premaxillary sac fossa width; PCW, Width across prenarial crest; BZW, Bizygomatic width; ML, Mandible length; SL, Symphysis length.
All measurements are in mm
Figure 5. Principal component analysis of eight cranial and mandibular measurements shows that the two stranded Mesoplodon specimens from Uruguay (MNHN 8211 and 8212) plausibly cluster with M. eueu. Comparative data from Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021).
Genetic analyses
The CR sequences of MNHN 8211 and 8212 are identical, suggesting that they are closely related. We hypothesize that they represent a mother-calf dyad, given that (i) one is an adult female and the other a calf/juvenile, (ii) both stranded within days of each other and in geographical proximity, and (iii) the more advanced decay of the adult (found three days later), which in turn suggests that both may have died at a similar time. Alternatively, they could belong to the same population.
Breeding knowledge of beaked whales is scarce but the lactation period is thought to be of one to several years of duration (MacLeod, Reference MacLeod, Wursig, Thewissen and Kovacs2018). In some species it has been estimated to last from 3 to 5 years, and weaning occurs when the calf approximately doubles its size at birth (MacLeod and D'Amico, Reference MacLeod and D'Amico2006; Feyrer et al., Reference Feyrer, Zhao, Whitehead and Matthews2020). The size at birth varies among species but offspring of some beaked whales range from 190 to 460 cm (Ferguson et al., Reference Ferguson, Higdon, Schmidt, Pomerleau and Matthews2023), specifically for M. mirus a neonate of 220 cm of length has been reported (Ross, Reference Ross1984). Taking this information into account and given the body length recorded on MNHN 8211, we suggest that this individual was still dependent on its mother.
Phylogenetic analysis of the CR data clusters our specimens with Ramari's beaked whales and, consistent with our morphometric results, strongly supports their referral to M. eueu (Figure 6). The specimens thus correspond to the first confirmed record of this species from Uruguay and the Southwest Atlantic. Furthermore, it is probable that the previously reported stranding of True's beaked whale from Southeastern Brazil (Souza et al., Reference Souza, Siciliano, Cuenca and Sanctis2005) may also represent M. eueu, given the currently known geographic distribution for M. mirus and M. eueu and that the latter was described after the stranding recorded in Brazil. This would imply an extension of the distribution range of M. eueu to the tropics.
Figure 6. Bayesian phylogeny of Mesoplodon based on 51 mitochondrial control regions. Values above branches are ML bootstrap support, those below are Bayesian posterior probabilities.
As found previously (Carroll et al., Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021), we recover M. mirus as sister to M. eueu. The P-distance between the Uruguayan individuals and other members of M. eueu is 0.0208 (variance = 0.0086), and that between the same specimens and M. mirus is 0.0432 (variance = 0.0105). Mitogenome analysis dates the last common ancestor of M. eueu to 0.8606 Ma (95% HPD: 0.118–1.7422 Ma) and the divergence of M. eueu from M. mirus to 2.5902 Ma (1.4201–3.9211 Ma; Supplementary Figure S2).
Our divergence time estimates somewhat predate those of Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021) and reveal greater genetic diversity within Ramari's beaked whales than previously thought. This is reflected in the notable genetic distance between our individuals and previously analysed specimens of M. eueu. Specifically, we found a total of 164 substitutions between the mitochondrial genome of MNHN 8211 and the M. eueu sequences of Carroll et al. (Reference Carroll, McGowen, McCarthy, Marx, Aguilar, Dalebout, Dreyer, Gaggiotti, Hansen, van Helden, Onoufriou, Baird, Baker, Berrow, Cholewiak, Claridge, Constantine, Davison, Eira, Fordyce, Gatesy, Greg Hofmeyr, Vidal, Mead, Mignucci-Giannoni, Morin, Reyes, Rogan, Rosso, Silva, Springer, Steel and Olsen2021), compared to 814 substitutions between M. eueu (including MNHN 8211) and M. mirus. With the inclusion of MNHN 8211, the mitochondrial nucleotide diversity of M. eueu now stands at 0.0070 (standard deviation = 0.0031). This is notably higher than in M. mirus (0.0024; SD = 0.0003), confirming the pattern previously described, and suggests a greater effective population size and/or possible population structuring, as seen in other ziphiids like bottlenose whales (Ellegren and Galtier, Reference Ellegren and Galtier2016).
Conclusion
We report the first confirmed record of Ramari's beaked whale from Uruguay and the wider Southwest Atlantic. Our findings expand the range and genetic diversity of this recently described species and suggest possible population structuring. Further samples from across the range of Ramari's beaked whale are needed to test this idea. Identifying a new species of marine mammal in Uruguayan waters calls for further resource allocation both to stranding responses and long-term systematic surveys of the regional coast.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315423000929
Acknowledgements
We thank Alejandro Fallabrino and Diana Szteren for notifying the strandings. We greatly appreciate the help of the Dirección General de Gestión Ambiental of the Intendencia de Canelones for their help in accessing and transporting the specimens. Authors thank Morten Tange Olsen for providing very valuable feedback on the draft manuscript. We appreciate the reviewers' comments that helped improve our manuscript.
Author´s contributions
LF, ER, PL, and MV collected the specimens analysed. NR and MV carried out the genetic and morphological analysis. ELC, MMcG, and FGM made substantial contributions to the conception of the work. All authors interpreted the data and discussed the results. All authors wrote and prepared the draft of the manuscript, and approved the final version of the manuscript.
Competing Interest
None.
Data Availability Statement
The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/, accession numbers are available in the manuscript text and Supplementary material S1.
