Sex Determination in Platypus and Echidnas: Multiple Sex Chromosomes and the Emergence of the AMHY Gene

Among vertebrates, sex determination systems occur in several different forms. Placental mammals and marsupials employ the XY system, birds use the ZW system, and reptiles and amphibians exhibit a wide array of mechanisms with distinct evolutionary origins. Within mammals, however, the monotremes—represented by the platypus (Ornithorhynchus anatinus) and the echidnas (family Tachyglossidae)—stand apart as a particularly unusual lineage. These animals diverged from other mammals approximately 188 million years ago during the early Jurassic period.

Earlier studies revealed that male platypuses possess an extraordinary sex chromosome system consisting of five X chromosomes and five Y chromosomes (5X + 5Y), while females carry five pairs of X chromosomes (5X + 5X). In the short-beaked echidna (Tachyglossus aculeatus), males have five X chromosomes and four Y chromosomes (5X + 4Y), whereas females again possess five X pairs (5X + 5X). During male meiosis, these chromosomes form a remarkable chain structure in which ten chromosomes align head-to-tail through pseudoautosomal regions. The arrangement follows an alternating pattern of X and Y chromosomes: X₁–Y₁–X₂–Y₂–X₃–Y₃–X₄–Y₄–X₅–(Y₅).

This chain segregates to produce two types of sperm: one carrying X₁X₂X₃X₄X₅ and the other carrying Y₁Y₂Y₃Y₄(Y₅). Such a system is fundamentally different from the familiar mammalian XY mechanism and lacks the canonical mammalian sex-determining gene SRY found on the Y chromosome.

Platypus karyotype（圖片來源：Rens W et al. (2007)，採用 CC BY 2.0 授權。）

Short-beaked echidna karyotype（圖片來源：Rens W et al. (2007)，採用 CC BY 2.0 授權。）

Chain of sex chromosomes during male meiosis （圖片來源：Marshall Graves JA .(2016)，採用 CC BY-NC-ND 4.0 授權）

Comparisons between the platypus and echidna genomes show that certain chromosomal regions functioning as sex chromosomes in the platypus correspond to autosomes in echidnas, and vice versa. This indicates that extensive chromosomal rearrangements—including translocations and recombination events—occurred after the divergence of these lineages. Current models suggest that the ancestor of monotremes probably possessed fewer sex chromosomes than are seen today. Over evolutionary time, segments of ancestral sex chromosomes were translocated onto autosomes, converting some autosomes into additional sex chromosomes and producing the complex multi-chromosome system observed in living species.

A key question then arises: how do monotremes determine sex in the absence of the SRY gene? Recent research has focused on a well-known vertebrate signaling molecule, anti-Müllerian hormone (AMH). In placental mammals, AMH is not itself the sex-determining gene. Instead, after testes form, Sertoli cells secrete AMH to suppress development of the Müllerian ducts—the embryonic structures that would otherwise develop into the uterus and oviducts.

In several other vertebrates, however, AMH or a male-specific variant of this gene functions directly as the primary sex-determining factor. An example occurs in the Patagonian pejerrey (Odontesthes hatcheri), where a Y-linked copy of the gene controls male development. Monotremes appear to employ a similar strategy. In the platypus, a male-specific copy of the AMH gene is located on the fifth Y chromosome, while in the echidna it occurs on the third Y chromosome. This Y-linked version is known as AMHY and is considered the most likely candidate for the monotreme sex-determining gene. The corresponding X-linked homolog, AMHX, is located on the first X chromosome in both species.

Müllerian duct (pink): (A) bipotential gonad; (B) male gonad (+SRY); (C) female gonad（圖片來源：Teixeira J et al (2008)，採用 CC BY 3.0 授權）

To understand how AMHY and AMHX evolved from the ancestral AMH gene, genomic analyses have revealed that a chromosomal inversion occurred in the common ancestor of monotremes. This inversion separated the original alleles into distinct genomic contexts, placing one copy on the emerging Y chromosome while leaving the other on the X chromosome. As a result, the two genes followed different evolutionary trajectories. AMHX retained a gene arrangement and regulatory environment highly similar to those of other tetrapods, whereas AMHY underwent extensive structural reorganization and accumulated large numbers of repetitive sequences. These differences suggest that AMHY was relocated into a new regulatory landscape that facilitated the evolution of a novel function.

Proportions of exons (blue) and introns (gray) of the AMH gene in different species（圖片來源：Shearwin-Whyatt L et al. (2025)，採用 CC BY-NC-ND 4.0 授權）

At the sequence level, AMHX and AMHY share roughly 62% similarity in DNA sequence and about 52% similarity at the protein level. This divergence is considerably greater than the similarity observed when comparing the same gene between platypus and echidna, implying that the split between the two versions occurred very early—likely more than one hundred million years ago. Despite this divergence, both proteins retain the conserved structural motifs characteristic of the transforming growth factor-β (TGF-β) family. Structural modeling indicates that their three-dimensional configurations remain highly similar, suggesting that both proteins can still interact with the AMH receptor and activate downstream signaling pathways, though their biological roles may have shifted.

The decisive factor appears to lie not in protein structure but in gene expression. In developing echidna embryos, the AMHY gene is expressed from the earliest stages of the bipotential gonad and is restricted exclusively to males. In contrast, AMHX is expressed in both male and female gonads.

In placental mammals, the transcription factor SRY activates SOX9 in the developing male gonad, which subsequently induces expression of the AMH gene. In echidnas, however, both SOX9 and AMHX proteins are already present in early gonadal tissue of both sexes. Developmental studies further show that the transcription factors DMRT1 and GATA1 are expressed in male gonads during early differentiation and bind to the promoter region of the AMHY gene, enhancing its expression. In female embryos, these genes show little or no expression. This regulatory configuration suggests that monotremes may retain a sex-determination network more reminiscent of early mammalian ancestors rather than the system used by placental mammals today.

Taken together, the chromosomal architecture, genomic data, and developmental expression patterns indicate that monotreme sex determination is not merely a biological curiosity. Instead, it represents an evolutionary window into an earlier stage of mammalian history—one that reveals how sex determination may have operated before the emergence of the SRY gene in therian mammals.

Author: Shui-Ye You

Reference:

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