Sex-by-Sequencing Reveals the First Two Species of Marine Mammals with XXY Chromosomes

Clinicians refer to human XXY’s as XXY Males or more frequently ‘People with Klinefelter’s Syndrome (they say only males can have Klinefelter’s as it denotes a disease of the testes, though oddly KS is not always present among XXY’s) Yet for as long as they have known about XXY “Males” they have also known about XXY Females, those who are phenotypically female but have never publicly acknowledged their existence. Instead they refer to XXY Females as XY females (AIS girls) and talk of their Y chromosome as being a non active SRY. But for the life of them, they have not been able to explain XXY’s who are phenotypical and genotypical females and who, like XX females who are also P+G are capable of conceiving children of their own. As more women become involved in science we are beginning to see life and it’s diversity for what it truly is.

We identified three specimens representing two different cetacean species that had external female morphological traits, Y chromosome haplotypes, and ratios of ZFX:ZFY haplotypes that were above the 1:1 value expected for genetic males. These results provide the first evidence of XXY aneuploidy in cetaceans. Investigation of the reproductive tract of one specimen, a True’s beaked whale (Mesoplodon mirus), revealed an intersex phenotype; despite having external characteristics typically diagnostic for the female sex, a penis and testes were present. Our results suggest that intersex phenotypes may be associated with XXY aneuploidy, and that this phenomenon may be underestimated due to it not being detectable by qualitative assays for determining sex

Anthony L. Einfeldt, Dara N. Orbach, and Laura J. Feyrer
Department of Biology, Dalhousie University, 1355 Oxford Street, Halifax, Nova Scotia B3H 4J1, Canada (ALE, DNO, LJF)

When phenotypic information is available, discrepancies between molecular and morphological results for sex determination are typically attributed to human errors in field observations, sample labelling, or technical errors in molecular methods. This overlooks the possibility that apparently failed assays and mismatches between genotype and phenotype may be of biological importance

Genetic sex determination assays assume that individuals are either XY males or XX females, which can be false at the level of phenotype or genotype. At the phenotypic level, intersex individuals with gonads, sex hormones, or genitalia that do not conform to the definitions of male or female sex characteristics have been documented across a growing number of taxa, including fish, amphibians, reptiles, and mammals. In cetaceans, individuals with both male and female gonads or genital phenotypes have been identified in fin whales, and short-beaked common dolphins. There is little known about the developmental factors influencing these conditions, but phenotypic variations have previously been attributed to hormonal disturbance during early pregnancy and androgen expression failure.

At the genotypic level, individuals that are not strictly XX or XY have been genetically confirmed in many wild and domestic terrestrial mammals (e.g., horses, cats, dogs, tiger). Due in part to the difficulty of identifying and characterizing variations in sex chromosomes, the range of the diversity and effects on sexual phenotype are not well known. In humans, variation in copy number of sex-chromosome-linked genes can result from incomplete segregation (non-disjunction) during meiosis I, meiosis II, or post-zygotic mitosis, and the most common sex chromosome aneuploidy present in humans is the XXY genotype (Jacobs and Strong 1959. Individuals with this aneuploidy often exhibit male secondary sexual characteristics with some feminization, due to the presence of the testes-determining gene SRY. Mosaicism of multiple cell lines with different karyotypes occurs in ~15–20% of human XXY males, but is likely underestimated. In domesticated mammals, the majority of Y chromosome abnormalities are in mosaic form, with different cell types carrying different combinations of Y and X aneuploidies.

Establishment of non-invasive molecular methods to detect sex chromosome copy numbers would provide more sensitive, efficient, and
cost-effective means to understand the extent of sex chromosome anomalies in live animals and wild populations, reduce uncertainties regarding technical or human errors, and support investigations on the causes of atypical sexual development.

Two of the three anomalous individuals we detected were northern bottlenose whales. One individual was harvested during commercial whaling in 1971 in the Davis Strait, and had previously been identified as female based on melon form and genital phenotype characteristics by whalers. This individual had also been identified as female based on the absence of banding in a gel electrophoresis-based assay for the SRY gene
as part of a study by Dalebout et al. (2006). However, electrophoresis-based sex assays are prone to Type II error, especially in old and highly degraded DNA, which may have led to incorrect sex genotyping when this individual was initially assayed. The second anomalous northern bottlenose whale was encountered during surveys in the Davis Strait in 2018. This individual was identified in the field as a juvenile female based on melon morphology.

The presence of a Y chromosome in both of these individuals is at odds with their externally female genotypes, and with most presence–absence-based sex assays would be attributed to observer or laboratory error. However, the high ZFX:ZFY ratios that we detected in both these individuals support
an alternative hypothesis that these whales have XXY aneuploidy. This could explain the apparent mismatch between genotype and phenotype, as sex chromosome aneuploidies. Two of the three anomalous individuals we detected were northern bottlenose whales. One individual was harvested during commercial whaling in 1971 in the Davis Strait, and had previously been identified as female based on melon form and genital phenotype characteristics by whalers. This individual had also been identified as female based on the absence of banding in a gel electrophoresis-based assay for the SRY gene as part of a study by However, electrophoresis-based sex assays are prone to Type II error, especially in old and highly degraded DNA., which may have led to incorrect sex genotyping when this individual was initially assayed.

The second anomalous northern bottlenose whale was encountered during surveys in the Davis Strait in 2018. This individual was identified in the field as a juvenile female based on melon morphology. The presence of a Y chromosome in both of these individuals is at odds with their externally female genotypes, and with most presence–absence-based sex assays would be attributed to observer or laboratory error. However, the high ZFX:ZFY ratios that we detected in both these individuals support an alternative hypothesis that these whales have XXY aneuploidy.

This could explain the apparent mismatch between genotype and phenotype, as sex chromosome aneuploidies in other mammals affect sexual development. Further testing of this hypothesis was not possible, as neither whale was available for morphological inspection, and direct observation of sex chromosomes requires fresh tissue undergoing meiosis.

The hypothesis that XXY aneuploidy is associated with an external female phenotype in beaked whales was supported by the third anomalous whale. This True’s beaked whale had external female characteristics, ZFX–ZFY and SRY genes, and a high ZFX:ZFY ratio. It stranded in the Magdalen Islands in 2017 and its reproductive tract was excised and preserved frozen. Despite having external traits that are typically sex-diagnostic for female cetaceans (two pronounced mammary slits and one long continuous urogenital slit), this individual had an intersex phenotype with internal traits typical of males (a penis, testes,and pelvic bones positioned at the crurae of the penis). While the phenomenon of mammary slits in male cetaceans has not to our knowledge been published in the scientific literature, we are aware of anecdotes of its occurrence in other cases. We suggest that it occurs infrequently, has not been well documented, and is worth considering whether other such individuals may be intersex.

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