Detection of sex chromosomal aneuploidy (<scp>XXY</scp>) in a southern right whale (<i>Eubalaena australis</i>) using read depth and coverage from whole genome sequence data

Most mammals inherit two copies of each chromosome, one from each parent. Mistakes can occur during meiosis that result in an incorrect number of chromosomes being present in an offspring, known as chromosomal aneuploidies. Chromosomal aneuploidies are one of the main causes of early miscarriages in humans (Hassold & Jacobs, 1984; Petracchi et al., 2007). The few aneuploidies that are not immediately fatal can cause a number of health and fitness complications (Orr et al., 2015; Skuse et al., 2018; Visootsak & Graham, 2006). Aneuploidies in individuals can be complete and present in all cells, or they may be present in mosaic forms, where not all cells of an individual are affected (Raudsepp & Chowdhary, 2016; Skuse et al., 2018). Some of the most common aneuploidies in viable offspring involve the sex chromosomes and can be present in many different varieties including XXY-syndrome (Klinefelter syndrome), X-chromosome trisomy, and X-chromosome monosomy (Turner syndrome) (Skuse et al., 2018). In species of mammals where sex chromosome aneuploidies have been documented, many individuals carrying sex chromosomal aneuploidies are sterile, or display intersex characteristics (Einfeldt et al., 2019; Raudsepp & Chowdhary, 2016; Visootsak & Graham, 2006). In wildlife, the most commonly reported presentation of aneuploidy in the sex chromosomes is XXY, which have been reported in a diverse number of mammals including shrew (Rannala & Yang, 2017), horse (Iannuzzi et al., 2004), tiger (Suedmeyer et al., 2003), baboon (Dudley et al., 2006), beaked whales (Einfeldt et al., 2019), and more. Karyotyping samples is no longer standard practice in most labs, and in the absence of phenotypic data, sex determination is largely genetically based. Standard genetic sex determination in cetaceans has typically involved amplifying the SRY gene from the Y-chromosomes and/or a homologous region on the X- and Y-chromosomes (ZFX/ZFY). If regions present on the Y-chromosome amplify, the individual is considered male, and if not, individuals are considered female. Routine genetic sexing over the past few decades has therefore overlooked the possibility of sex chromosomal aneuploidies. With a shift to next-generation sequencing and larger genome-wide data sets becoming common, the use of read depth across sex chromosomes compared to autosomes is increasing (Cabrera et al., 2022; Hansen et al., 2022; Liu et al., 2023; Peralta et al., 2024). Using read depth of a gene associated with the zinc finger protein locus (ZFX/ZFY), XXY aneuploidy was detected in True's beaked whale (Mesoplodon mirus) and northern bottlenose whale (Hyperoodon ampullatus; Einfeldt et al., 2019). In one individual, morphological assessments could be performed and the XXY genotype was also associated with intersex phenotype (with external genitalia having female characteristics, while reduced testes and penis were present; Einfeldt et al., 2019). In a recent study using whole genome sequence data of right whales (Eubalaena spp.; Crossman et al., 2023), there was a disconnect between the sex determined by traditional genetic sexing and read depth within one sample (Eau10b). Amplification of a region on the Y-chromosome identified the individual as a male (Figure 1a) but mean read depth on the X-chromosome as compared to the autosomes when mapped to a scaffold-level southern right whale assembly (https://www.dnazoo.org) was not suggestive of the individual only having one copy of the X-chromosome (C.A.C., unpublished data; Figure 1b). Here I present a more detailed analysis of this individual and report the first documented case of sex chromosomal aneuploidy (XXY) in a southern right whale (Eubalaena australis). Whole genome sequence data from five southern right whales (two males, two females, and the individual in question) collected in 1989 off Peninsula Valdéz, Argentina, were available from a previous study (Crossman et al., 2023; SRA accession numbers: Eau283 – SRR29386322; Eau7 – SRR29386332; Eau9c – SRR29386331; Eau017 – SRR29386329; Eau10b – SRR29386330). As no information on individual sex was available, the sexes of these individuals were determined based on the standard sexing methods and read depth data described above and depicted in Figure 1. I dropped low quality reads and trimmed poor quality leading and/or trailing bases with Trimmomatic v.039 (Bolger et al., 2014). Paired reads were mapped to a high-quality blue whale (Balaenoptera musculus) genome assembly that includes both X- and Y-chromosomes (GenBank accession number GCF_009873245.2) with bwa-mem v.0.7.17 (Li, 2013; Li & Durbin, 2009). The alignments were sorted and merged across sequencing lanes using samtools v1.16 (Li et al., 2009) and duplicate reads were marked with picard v.2.26.3. Sex of individuals can be determined by comparing coverage and depth across the sex chromosomes relative to the autosomes. While males should have nearly 100% coverage across all autosomes and both sex chromosomes, females should exhibit near 0% coverage on the Y-chromosome (allowing some buffer for poor mapping of genomic regions such as pseudoautosomal regions, PAR, that have high homology between the X- and Y-chromosomes; Mangs & Morris, 2007). I estimated coverage across each chromosome using samtools coverage and required mapping quality to be above 30 for an alignment to be included. Read depth can also provide an insight into the sex of an individual. Both males and females should have two copies of all autosomes. Females should also have two copies of the X-chromosome and therefore read depth on the X should be very similar to that across the autosomes, and they should have hardly any reads mapping to the Y. In contrast, males should have a single copy of both sex chromosomes and therefore their read depth should be approximately half of that reported for the autosomes. I estimated depth across each chromosome for each individual using samtools depth, again only including reads with a mapping quality greater than 30. Einfeldt et al. (2019) proposed sex chromosome aneuploidies may be evident from comparing the ratio of ZFX:ZFY fragments. ZFX/ZFY is a standard primer set used for sex identification in cetaceans (e.g. Konrad et al., 2017) where one set of primers amplifies a homologous 94-bp fragment on both the X- and Y-chromosome (if present) and the resulting amplicons only differ in a single base at a restriction site. To test if the ratio of ZFX:ZFY fragments confirmed our findings of sex chromosomal aneuploidy, I used amplicon sequencing to amplify the ZFX/ZFY region from the five southern right whale samples on a MiSeq Micro v3 2 × 150 run. Poor quality reads were dropped and paired end reads were merged using fastp (Chen et al., 2018). The amplicon data were processed through the GT-Seq pipeline (www.github.com/GTseq/GTseq-Pipeline) to obtain the number of ZFX and ZFY fragments in each individual. The known male and female southern right whales clearly demonstrated the expected patterns in read depth (the mean number of reads mapping to a single position) and coverage (the proportion of a chromosome or region to which reads map). In females, the read depth on the X-chromosome was similar to that of the autosomes and they exhibited a very low depth of reads mapping to the Y-chromosome (Figure 2a). Likewise, females had near 100% coverage across the autosomes and X-chromosome and few reads mapping to the Y-chromosome (Figure 2b). Read depth in both the X- and Y-chromosome was approximately half that of the autosomes in known males (Figure 2a). Males had nearly 100% coverage across the autosomes and both sex chromosomes (Figure 2b). The anomalous individual displayed nearly 100% coverage across all autosomes and both sex chromosomes, suggesting the individual does in fact have a Y-chromosome (Figure 2b). However, mean depth on the X-chromosome was very close to that of the autosomes, and the mean depth of the Y-chromosome was less than 50% of the mean autosomal depth (Figure 2a). The known females had one or no ZFY reads called and the known males demonstrated a near 1:1 ZFX:ZFY ratio between the fragments (1.08:1 or 1.33:1; Figure 2c). The anomalous individual had a ZFX:ZFY ratio of 3.54:1 (Figure 2c). Together, the coverage, read depth and ZFX:ZFY ratios all suggest the anomalous southern right whale (Eau10b) has two copies of an X-chromosome as well as a Y-chromosome. Einfeldt et al. (2019) demonstrated the use ZFX:ZFY ratios with amplicon sequencing to identify XXY genotypes. Here, I confirm this method will work for identifying aneuploidy in another species of cetacean and demonstrate that comparing read depth and coverage in autosomes vs the sex chromosomes can provide even stronger evidence of chromosomal aneuploidies. While this method will help to identify aneuploidies, it may still be difficult to tease apart complete aneuploidy versus mosaicism in individuals. Intersex phenotypes have been reported in many species of cetacean (e.g., fin whale, Balaenoptera physalus: Bannister, 1963; beluga, Delphinapterus leucas: De Guise et al., 1995; short-beaked common dolphin, Delphinus delphis: Murphy et al., 2011; bowhead whale, Balaena mysticetus: Tarpley et al., 1995) and Einfeldt et al. (2019) speculate that XXY aneuploidy may be involved and may therefore be much more widespread than previously thought. Unfortunately, close phenotypic observations are not always possible for each individual, so other tools need to be considered. Screening all wildlife samples that are being analyzed with whole genome sequencing, RADseq or even GTSeq (including a ZFX:ZFY amplicon) for chromosomal aneuploidies should become standard practice to not only document novel cases, but also to prevent biases that could be inadvertently introduced by intersex individuals. While likely still rare, this phenomenon is likely more common throughout the animal kingdom than once thought and as we sequence more individual genomes, it is important that the possibility of intersex genotypes be considered. I wish to thank Dr. Timothy Frasier for helpful comments and suggestions during the analysis and preparation of this manuscript. Samples were originally collected by Judith Perkins and Dr. Michael Moore under permit. C.A.C. was supported by an NSERC CGS-D scholarship. Carla Crossman: Conceptualization; formal analysis; writing – original draft. The whole genome sequence data (raw fastq) are archived in NCBI's Sequence Read Archive under BioProject https://www.ncbi.nlm.nih.gov/bioproject/PRJNA914998. The ZFX/ZFY fragments were processed alongside other samples as part of a larger GT-Seq project. The fasta sequences of the ZFX/ZFY amplicons are saved in a Github repository alongside the code used for the analyses (https://github.com/carlacrossman/SRW_Aneuploidy). The author declares no competing interests with this study.

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