Evidence of unidirectional hybridization and second‐generation adult hybrid between the two largest animals on Earth, the fin and blue whales

Abstract Biodiversity in the oceans has dramatically declined since the beginning of the industrial era, with accelerated loss of marine biodiversity impairing the ocean's capacity to maintain vital ecosystem services. A few organisms epitomize the damaging and long‐lasting effects of anthropogenic exploitation: Some whale species, for instance, were brought to the brink of extinction, with their population sizes reduced to such low levels that may have caused a significant disruption to their reproductive dynamics and facilitated hybridization events. The incidence of hybridization is nevertheless believed to be rare, and very little information exists on its directionality. Here, using genetic markers, we show that all but one whale hybrid sample collected in Icelandic waters originated from the successful mating of male fin whale and female blue whale, thus suggesting unidirectional hybridization. We also demonstrate for the first time the existence of a second‐generation adult (male) hybrid resulting from a backcross between a female hybrid and a pure male fin whale. The incidence of hybridization events between fin and blue whales is likely underestimated and the observed unidirectional hybridization (for F1 and F2 hybrids) is likely to induce a reproductive loss in blue whale, which may represent an additional challenge to its recovery in the Atlantic Ocean compared to other rorquals.


| INTRODUC TI ON
From the 12th to the 20th century, the hunt of whales throughout their distribution range led to the near extinction of several species (Gambell, 1993;McVay, 1966), which greatly impacted most of marine ecosystems (Clapham & Link, 2006;Croll, Kudela, & Tershy, 2006) and then compelled drastic changes to conservation strategies. Many species of closely related cetaceans were reduced to population sizes that may have facilitated hybridization due to the rarity of conspecifics, hence increasing the risk | 315 PAMPOULIE Et AL.
of genetic swamping and possibly threatening the persistence of species.
Hybridization among closely related species is an important evolutionary phenomenon (Mallet, 2007) and has been reported in several animal taxa (Schwenk, Brede, & Streit, 2008). Large marine mammals such as fin whale (Balaenoptera physalus) and blue whale (B. musculus) are no exception, with alleged hybrids reported as early as 1887 during commercial whaling operations along the Lapland coast, when Cocks (Cocks, 1887) mentioned the presence of socalled "bastards" among the fin and blue whalers. Another alleged hybrid was reported almost 80 years later off Kodiak Island in 1965 (Doroshenko, 1970). Twenty years later, hybridization between fin and blue whales was eventually demonstrated for the first time using genetic tools (Árnason, Spilliaert, Pálsdóttir, & Árnason, 1991;Bérubé & Aguilar, 1998;Spilliaert et al., 1991): A whale caught in 1983 in Icelandic waters was classified as a hybrid between a fin whale mother and a blue whale father, while two whales caught in 1986 and 1989 were found to be hybrids between a fin whale father and a blue whale mother (Árnason et al., 1991;Spilliaert et al., 1991). A further case was documented in the northwest of Spain in 1984, where a female rorqual was genetically identified as a hybrid between a fin whale father and a blue whale mother (Bérubé & Aguilar, 1998). Baleen whales are elusive and relatively difficult to study, which likely affects reporting of hybrids and introgressive events. Scientists often rely on expensive biopsy sampling cruises, stranded animals, or aboriginal/commercial whaling operation to obtain genetic samples from large marine mammals. While current commercial whaling activities remain a highly controversial issue in conservation fora, they also provide unique and opportunistic access to sample and therefore to crucial biodiversity information. In recent years, commercial whaling operations in Iceland have led to the discovery of three more alleged hybrids, one in 2013 and two in 2018, while one living hybrid is known to regularly visit the Skjálfandi Bay (Northeast Iceland) almost every year since 2012. Here we screen the largest collection of hybrids between the two largest mammals on Earth using 24 microsatellite and one mitochondrial DNA marker (mtDNA), with the aim to verify directionality of hybridization and explore the potential for hybrid fertility.

| Sampling
Fin whale genetic samples were routinely collected during the commercial whaling operation in 2018; 34 samples were randomly selected among the ones exhibiting standard pure phenotypic features of fin whale. The hybrid samples of 2013 and 2018 were also collected during commercial whaling operations in Iceland, while those of 1986 and 1989 were collected during a scientific research project according to a special permit (see https://iwc.int/table_permit). Blue whale samples come from stranded (N = 2) and biopsied (N = 25) individuals. All samples were collected in Icelandic waters ( Figure 1).
For both species, samples were collected <24 hr after stranding, biopsy or death of the animal, put in 96% ethanol and conserved at 5°C until analyses in the laboratory. Information on individual sex and date of capture is presented in supplementary materials when available (Table S1).
The six suspected hybrid samples analyzed during this study (Table 1) include the confirmed hybrids caught in 1986 and 1989, the suspected living hybrid from Skjálfandi Bay, and the hybrids caught in 2013 (n = 1) and 2018 (n = 2). No tissues from the 1983 hybrid or from the fetus found in the 1986 hybrid could be retrieved due to sample loss.
Annealing temperature was 54°C, and 32 PCR cycles were run.
Annealing temperature was 56°C, and 35 PCR cycles were run.
PCRs were performed in a total volume of 10 μl consisting of 2 μl of DNA template (5-20 ng/µl), 0.1 µl Taq DNA polymerase, 1.0 μl of 10× Standard buffer, 0.8 μl dNTP (10 mM), 0.03-0.25 μl reverse and forward primers (100 μM), and dH 2 O up to final volume. The PCR was as follows: a 4-min denaturation at 94°C followed by 32-35 cycles of 94°C denaturing for 30s, 54-58°C annealing for 50s and 68°C extension for 50s, plus a final extension of 7 min at 68°C. Amplified DNA fragments were separated by an ABI 3730 DNA Analyzer and were sized according to the GeneScan™-500LIZ™ Size Standard. Alleles were scored manually with the GeneMapper™ Analysis Software version 4.1.

| Data analysis
Genetic diversity indices of the 24 microsatellite loci including the number of alleles (n), allelic size range (ASR), observed (H O ) and expected (H E ) heterozygosities, and departure from Hardy-Weinberg Equilibrium (HWE) within each samples for each locus were calculated in GENEPOP'007 (Rousset, 2008). Statistical significance for HWE was assessed using exact P-values by Markov chain methods implemented in the same software. A principal component analysis (PCA) was performed using GenAlEX (Peakall & Smouse, 2006, 2012 to visualize relative multilocus genetic differences among parental species and hybrids. Bayesian cluster analysis was performed using the program STRUCTURE (Pritchard, Stephens, & Donnelly, 2000) to assess the genetic relationship of the alleged hybrids to their potential parental species and was used to generate assignment probability of hybrids The analysis of mtDNA sequences was not performed to infer phylogeny but merely to verify the matrilineage of the hybrids, and hence infer directionality of hybridization.

| RE SULTS
Microsatellite loci diversity was usually higher in fin whale than blue whale, and this was reflected both at the mean number of alleles and at the species/loci heterozygosities level (Table 2). A total of 2 microsatellite loci were fixed for the fin whale (CAAA074, TGAA610; Table 2), while 4 were fixed for the blue whale (TGAA610, GT227, TAA023, GATA053; Table 2). Interestingly, some of these fixed microsatellite loci displayed alternative allele in both species, for example, TGAA610-allele 143, GATA053-allele 256, and GT227-allele 122 were only found in blue whale (Table 2). In addition, several microsatellite loci displayed different allelic size range for both species, which make these loci powerful diagnostic markers for the identification of the hybrids ( Table 2).
The PCA clearly separated the two investigated species, the fin and blue whale (Figure 2) with 37.5% of the variation explained by the first axis and 2.8% on the second axis. As expected, the hybrids were in the middle of the ordination, half-way between fin and blue whale data points. The greater spread of fin whale specimens reflects their greater intraspecific variation compared to blue whale.  (Figure 3c), while all other hybrids exhibited a 99% posterior probability of being first-generation hybrids (Figure 3c). We concluded that the 2018 male hybrid was sired by a fin whale and mothered by a first-generation hybrid female (see Table 1).
In addition, these findings highly support a unidirectional hybridization with male fin whales siring female blue whales (Chi 2 test, for F1 hybrids, p = .025 considering our data; p = .059 when all data presented in Table 1 were added).

| D ISCUSS I ON
Hybridization events among fin whale and blue whale have been reported since the 19th century but very little information exists on the directionality of such events and the reproductive status of hybrids. Here, we document a consistent pattern of male fin whales siring female blue whales, and the first occurrence of a second-generation adult hybrid.
Until recently, hybridization among cetacean species has been thought to be a "dead-end" because most hybrids were deemed to be infertile (Bérubé & Palsbøll, 2018). However, the observation of the 1986 hybrid fin × blue whale carrying a fetus in Icelandic waters and more recently of a hybrid between common minke (B. acurostrata) and Antarctic minke (B. bonaerensis) whales carrying a fetus resulting from a backcross mating with a male common minke whale in Norway (Glover et al., 2013) tend to support the idea that first-generation hybrids might be, in certain circumstances, able to breed with one of the parental species. In the present study, the discovery of a second-generation adult hybrid was surprising since only a pregnant hybrid female had been mentioned so far and proofs of living second-generation adult marine mammals are crucially lacking. This discovery supports the fact that hybrids resulting from a successful mating of the two largest mammals on Earth can in some cases reproduce and that their offspring can survive to adulthood.
Fin and blue whales belong to the same genus, Balaenoptera, which diverged during the late Miocene between 10.5 and 7.5 Ma ago (Árnason, Lammers, Kumar, Nilsson, & Janke, 2018) with an estimate time to the most common ancestor of mysticetes in the late Oligocene (Sasaki et al., 2005). Whole-genome sequencing studies investigating hybridization between blue whale and other rorquals confirmed the likely occurrence of ancestral introgression between fin and blue whales (Árnason et al., 2018;Westbury, Petersen, & Lorenzen, 2019); however, more recent, contemporary signatures are likely challenging to detect. Overall, it can be expected that fin x blue whale hybrids will exhibit reduced fitness, preventing backcross with both parental species (Árnason et al., 2018;Westbury et al., 2019). Yet, our discovery of the first second-generation hybrid adult and the previous report of the 1986 pregnant hybrid female  indicate that at least some hybrid fin × blue whales are fertile and can reproduce with both parental species, under certain environmental and demographic scenarios.
The identification of a second-generation hybrid sired by a male fin whale and the observed directionality of hybridization (male fin whales siring female blue whales) might represent a concern for the future of the blue whale. A total of 7 out of the 8 hybrids genetically analyzed (see Table 1 for a summary) so far had a blue whale mother which suggests unidirectional hybridization (Árnason et al., 1991;Bérubé & Aguilar, 1998;Spilliaert et al., 1991). Unidirectional hybridization may occur for different reasons, such as size difference, ecological or behavioral bias, but one of the main potential explanations remains the "sexual selection hypothesis for unidirectional hybridization" (Wirtz, 1999). This hypothesis crucially depends on the abundance of the species involved in the hybridization event and suggests that the females of the rarer species, which initially reject allospecific males from the more common species, will eventually successfully mate with them due to the lack of conspecific males (Wirtz, 1999). Alternatively, the observed unidirectional hybridization could also be due to size constraints and the result of a purely physical/mechanical impossibilities for blue whale male to sire fin whale female. Today, abundance of the fin whales in the whole North Atlantic is estimated to be over 80,000 individuals (Aguilar & García-Vernet, 2018;Pike, Gunnlaugsson, Mikkelsen, Halldórsson, & Víkingsson, 2019;IUCN, 2020) while blue whales abundance estimates vary between 2,100 and 4,000 (Sears & Perrin, 2018).

Considering only the North Atlantic Central Region which includes
Icelandic waters, the estimates are 36,800 (Pike et al., 2019) for the fin whale and 3,000 for the blue whale (Pike et al., 2019). The "sexual selection for unidirectional hybridization" will therefore likely result in the female of the rarest species, the blue whale, to be the maternal species of the hybrids, which coincides with our findings.
In Brownell, 2016). The inherent difficulty of blue whale to recover has been suggested to be due to resource competition (Reeves, Clapham, Brownell, & Silber, 1998) and climate change (Thomas & Brownell, 2016). Hybridization events among these large marine mammals are likely to be underestimated, and while population growth models considering hybridization events might have to be implemented to confirm this, we here raise awareness of an additional potential threat to blue whale population recovery. Blue whale is currently listed as endangered in the IUCN list and its global population remains at a very low level compared to prewhaling status although increase has been detected in the North Atlantic and Antarctic Oceans (Cooke, 2018;Pike et al., 2019;Sigurjónsson & Gunnlaugsson, 1990). A continued or increased hybridization and introgression with fin whale is likely to induce a loss of blue whale population reproductive output, thereby potentially affecting its recovery rate. At present, there are no frequency estimates of hybrids between fin and blue whales (the frequency of hybrids was 2% in our data in 2018, including the living hybrid). If hybridization is frequent and includes animals not visually identifiable from fin or blue whales in surveys, it might lead to the overestimation of population size of both species. This would be however more of concern for blue whale than fin whale, as its population size is about 25 times lower. It is therefore prudent to consider and monitor hybridization, where possible.

CO M PE TI N G I NTER E S TS
"The authors declare no competing interests."

ACK N OWLED G EM ENTS
Special thanks are addressed to Ágústa Sigfúsdóttir and Steinunn Magnúsdóttir for laboratory assistance. We would like to dedicate this study to Dr Rémi Spilliaert, Dr Úlfur Árnason, and Dr Alfreð Árnason who genetically confirmed the first hybrid between these two species, an animal caught in Icelandic waters in 1986. We would like to thank two anonymous referees for their constructive comments which greatly improved the manuscript. We would like to thank Hvalur hf. for giving us the opportunity to conduct this study. All authors discussed the results and implications and commented on the manuscript at all stages.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used during the present study (