The study evaluates the prevalence of sex-reversed or intersex individuals in free-living birds.The authors start with the premise (in the abstract) that “avian sex determination processes are more fluid than previously thought, challenging the reliability of traditional sexing methods that rely solely on external morphology, internal anatomy, or genetic markers. These methods, when used in isolation, fail to identify sex-reversed or intersex individuals, potentially overlooking their impact on population dynamics.”
Therefore, the study compared internal (mainly presences of testes, ovary, both) and external morphological phenotypic characteristics with the presence of sex-linked molecular markers (using PCR) in 5 common, free-living Australian bird species from 4 orders. The study is based on a total of 498 specimens. It found sex-reversed individuals (at the gonadal level) in all 5 species, with a frequency of 3 – 6 %. That is, in the sex-reversed individuals the identified gonadal morphological phenotype (a single ovary or left and right testis) did not match the genetic/chromosomal (ZW female or ZZ male). From these results authors conclude that sex-reversal is common and widespread in wild birds; they discuss the significance and implications for conservation biology and environmental evaluation.

I commend the authors for tackling the interesting and important question of disruption of sex determination/differentiation (i.e. establishing a proper link between genetic and phenotypic sex) in wild birds. The data are properly analyzed and the paper generally well written, but I have concerns with the presentation (include critical additional information about specimen collection sites and gonadal condition) and discussion of the data that need to be addressed. Some results such as reported measurements of gonads need clarification (see specific comments). Revision, in particular of the conceptual background of phenotypic sex, and an expansion of references to sex determination and differentiation, would strengthen the paper.

The authors’ aim was to identify individuals in which genetic/chromosomal sex and phenotypic sex do not match. Genetic sex was assigned using amplified sex-specific DNA markers extracted from liver tissue of diseased or euthanized birds (males two distinct bands (ZZ) females one (ZW) band) that were submitted to wildlife hospitals (see below). The study is based on a reasonable sample size comprised of a total of 498 specimens (between 63 to 127 individuals for the species).
The recorded phenotypic sex characteristic was primarily gonadal anatomy (presence of a single ovary versus presence of a left and a right testis, as shown in photographs of Fig 1.).
It is stated “Following convention, sex reversal is used here to describe birds in which at least the gonads but possibly other sexually dimorphic characteristics appear discordant with the genetic sex.” Discordance (also termed sex-reversed or intersex) was assigned when an individual’s genetic sex (as identified by the sex chromosome complement; ZZ, ZW bands from PCR) did not match the observed gonadal phenotype anatomy (paired testes or single ovary). This classification scheme revealed non-discordant males (i.e. normal males – 2 testes), non-discordant females (normal females - ovary), and discordant females in all species; discordant males were found in two of the species. Discordant females presented testes that were similar in anatomy and size to those of non-discordant (normal) males; a non-discordant male in a single species had an ovary and a distended oviduct). Some genetic females had ambiguous gonads, with a testis on the right side and an ovotestis on the left (intersex). Two genetic females had both testicular and ovarian structures.
No major differences in dimorphic external morphological sex characteristics such as size (bill, wing, tarsus) were found. Sexually dimorphic plumage color is not reported (see below).

General concerns/questions/comments:
1) The study relies on specimens that were submitted to wildlife hospitals and subsequently died naturally or were euthanized because they did not recover. This implies that all sampled specimens were ill or injured. How might this bias of sampling influence the rates of intersexes that were found? Might discordant individuals be more likely to become ill or injured. If so, the rates of intersexes might be inflated.
2) Given the aim of the study (identifying inter-sexes in wild populations of birds) one wonders why no information is provided about the origin of the birds, other than that they were submitted to animal hospitals on the Sunshine Coast, Brisbane and Gold Coast. Can the authors provide information about the habitats (rural, urban, algricultural ...) where the birds were found and in which condition they were in when submitted?
3) In the description of the 5 species, please include information on external sexual dimorphic differences (including plumage coloration etc.).
4) Apparently, gonads were only examined in situ but not excised and preserved. Why not? Preserved gonads could be very useful for histological examinations of structure/anatomy and the abundance/differentiation of germ cells (for example seminiferous tubules, Sertoli/Leydig cells, spermatocytes in testes; yolky and pre-vitellogenic follicles, atretic follicles). This information would allow evaluation of gonadal functionality in non-discordant (and discordant) individuals and describe the seasonal reproductive state (see next).
5) It is stated that specimens were collected during the reproductive season. Please provide more detail about the seasonality of reproduction in the 5 species to allow the reader to judge in how far seasonal breeding could have influenced gonadal state and results? See also specific comment below.
6) Throughout the manuscript you use language that implies that phenotypic sex characteristics are strictly binary. I recommend considering the recently emerging perspective in the biological sciences that dimorphic sexual phenotypic traits are bimodal rather that binary. Surely, gonadal phenotype presents either as testes (with sperm) or ovary (with eggs) in birds, but there certainly is variation within the sexes with regard to gonadal hormone levels and internal and external secondary sex characteristics (see below).
7) There is extensive literature on the mechanistic processes of sex determination and sexual differentiation in birds that might be included in the introduction and discussion (see below). See also reports on avian gynandromorphs.
8) The study would benefit if the introduction to the topic and discussion were expanded to include recent changes in perspectives of phenotypic sex in relation to causation and function. The authors might consult and include some of the references below and develop and present a broader background and perspective of their research. The following mechanistic and conceptual publications might be considered/included:
Hirst CE, Major AT, Ayers KL, Brown RJ, Mariette M, Sackton TB, Smith CA. Sex Reversal and Comparative Data Undermine the W Chromosome and Support Z-linked DMRT1 as the Regulator of Gonadal Sex Differentiation in Birds. Endocrinology. 2017 Sep 1;158(9):2970-2987. doi: 10.1210/en.2017-00316. PMID: 28911174.
Kuroiwa, A. (2018). Sex Determination and Differentiation in Birds. In: Kobayashi, K., Kitano, T., Iwao, Y., Kondo, M. (eds) Reproductive and Developmental Strategies. Diversity and Commonality in Animals. Springer, Tokyo. https://doi.org/10.1007/978-4-431-56609-0_19
Agate RJ, Grisham W, Wade J, Mann S, Wingfield J, Schanen C, Palotie A, Arnold AP (2003) Neural, not gonadal, origin of brain sex differences in a gynandromorphic finch. Proc Natl Acad Sci U S A 100:4873–4878
Arnold AP, Chen X, Link JC, Itoh Y, Reue K (2013) Cell-autonomous sex determination outside of the gonad. Dev Dyn 242(4):371. https://doi.org/10.1002/dvdy.23936.
Fusco, G., Minelli, A. Descriptive versus causal morphology: gynandromorphism and intersexuality. Theory Biosci. 142, 1–11 (2023). https://doi.org/10.1007/s12064-023-00385-1
J F McLaughlin, Kinsey M Brock, Isabella Gates, Anisha Pethkar, Marcus Piattoni, Alexis Rossi, Sara E Lipshutz (2023). Multivariate Models of Animal Sex: Breaking Binaries Leads to a Better Understanding of Ecology and Evolution, Integrative and Comparative Biology, Volume 63, Issue 4, October 2023, Pages 891–906, https://doi.org/10.1093/icb/icad027

Specific comments:
Line 26: please change “determine” to “identify” in order to reserve “determine” to the biological process of “sex determination”.
Abstract: please define “sex-reversed” and “intersex” before using it here.
Line 66 – 71: please consider adding here that hormonal regulation of sexual dimorphic phenotype can come in two ways: 1) (permanent) organization of phenotype in the embryo by gonadal hormones and 2) (transient and reversible) activation of sex differences in the adult when gonads are hormonally active. This is the classical concept of organizational versus activational action of hormones in sexual differentiation. The concept also applies to the next two paragraph in your discussion. See the following 2 references among many about the concept:
Arnold AP. 2009. The organizational-activational hypothesis as the foundation for a unified theory of sexual differentiation of all mammalian tissues. Horm Behav. 2009 May;55(5):570-8. doi: 10.1016/j.yhbeh.2009.03.011. PMID: 19446073; PMCID: PMC3671905.
Adkins-Regan E. (2012). Hormonal organization and activation: evolutionary implications and questions. General and comparative endocrinology 176: 279-85.
Line 134 - : please add some relevant information about the sexual dimorphic traits (plumage, behavior) of the 5 selected species.
Line 140 -: here it might be very useful to include information on seasonality of breeding in the 5 species. It would allow the reader to evaluate how “time of death and processing. …. during the spring and summer…” relates to life history stage (seasonal breeding) and gonadal state.
Please provide information on where specimens were found.
Line 145: were dimorphic plumage color characteristics evaluated?
Line 176: please be more specific and replace “phenotype” with “gonadal sex”.
Line 185: what do you mean with “juvenile gonads”? Please specify? How were juvenile/immature birds defined and identified? See also comment above on seasonal breeding.
Line 222 and Table 2: What are the testis measurements? Mass (g), length/width (mm), left/right testis, combined measurements?
Line 231: what are the reported measurements for the ovary? Mass, length? Follicle diameter/mass?
Line 235: ovary-like testis = ovotestis in the literature?
Line 234: why do you not present and information on dimorphic plumage coloration which often is affected by in ovo or adult hormone levels and disruption?
Line 272: what do you mean with “atypical gonadal makeup”. Please specify. Were these gonads regressed (not enlarged) due to immaturity or seasonal regression.
Line 279: here it would have been nice to include histological data on structure of tubules, spermatozoa etc.
Line 295: what are “masses of ovary-like tissue”. Please specifywhy and how the tissue resembles ovarian tissue? Presence of follicles, yolky material. I recommend that you somewhere describe (maybe when presenting Fig. 1) how non-discordant (normal) ovaries and testes look like.
Line 300: why do you not provide any information on sexual dimorphic traits other than size? How about plumage/bill color? Such traits are critical in sexual signaling and mate choice.

The authors have adequately addressed my comments/concerns in their revision.