I've written about Two-Spirit identities on AH before here and here. As you will see in the linked discussions, there are many more types of Two Spirit identities than the type you've identified, including transmasculine Two Spirits, and transfeminine Two Spirits in consensual adult relationships with cis men.

Just a polite heads up - afab (aka 'assigned female at birth') is a better term to use than 'biologically female', and likewise amab instead of 'biologically male'. I appreciate that 'biologically female' was not meant in a malicious or offensive way and I'm not suggesting any kind of prejudice on your part, just that the history of nonbinary and gender-diverse people overlaps quite heavily with the history of intersex people and in turn with the history of eunuchs and other non-cishet expressions of sexuality. Thus, many people occupying a third gender space were not eg 'biologically male' to start with even if they were assigned male at birth. It might seem like nit picking, but insisting on discrete binary sexes while also affirming a more expansive view of gender is going to get into difficulties with different cultural ideas of gender and sex fast.

To clarify, are you only talking about Two-Spirit and third gender peoples as they were before European contact? Because many such as hijra still exist today and would be the primary sources of information on their histories. These are still living peoples.

I am writing as someone who identifies as third gender, of European descent, who also studies third gendered peoples from my lineage (as an artist and folk practitioner).

I think that any attempts to frame historical identities in modern terms is a fools errand. Also, it is important to recognize that third gender peoples do still exist in the world in a range of cultures (including those you've listed). It is best to speak to or read sources written by people in those cultures and communities that continue to recognize a culturally linked third gender (ie. two-spirit peoples are very much part of the current modern culture of many North American Indians).

Another consideration is who is writing the history. A lot of historical documents are written by cis-men (male bodied persons who identify as a gender they were assigned at birth). The interpretations of historians do not equal the actual identities of those they are writing about.

There are also assigned female at birth (AFAB) people who are third gender in Europe. The only identity I'm aware of are the so-called "sworn virgins" of eastern europe: https://scholarworks.iu.edu/journals/index.php/aeer/article/download/687/780/2952

But yes, a lot of the third gender peoples mentioned in sources are assigned male at birth (AMAB).

One example, from my own research, are the femminiello and the galli. The former is a third gender identity related to the Neapolitan culture in Italy. The latter is an ancient third gender identity associated with spiritual practices in the ancient Mediterranean. They are similar, but very different given the context of time. The femminiello can be important members of the community, but they have often been relegated to the fringes of society even so.

Another thing to keep in mind with third gender roles in society is that third gender people have been heavily persecuted over the past 2000+ years. So people have had to do what they must to survive. For example, many third gender persons are now associated with poverty and sex work - this is because of the ways society has treated them and the roles they've been deemed "acceptable" to uphold in modern society.

Below are a number of links to sources. Unfortunately none are primary because I do not have access to such sources, but some do cite primary sources. Also, it should be noted that these sources are not written by the people they discuss (third gender persons) and therefore cannot be taken as accounts of how these people identified themselves. I'm providing these sources for information on the galli and femminiello - so the contrast between ancient and modern views can be seen. But the ancient views are coming only from outsiders and historians.

Ultimately, identities cannot be assumed to remain static over long stretches of time. It is also insensitive to assume that two-spirit and other culturally based third gender identities are analogous to westernized and fully modern ideas of gender and gender identity. Modern identities have a different foundation and basis than cultural identities that have existed for thousands of years. Furthermore, modern ideas of non-binary and gender fluid identities do not always align with third gender identities. Many people today reject any gender binary and therefore can view third gender as maintaining the binary. It's a very complex matter even when looking only at modern people.

​

Sources on Galli and Femminiello so that one may compare ancient attitudes with modern ones:

Modern interview with femminiello - https://medium.com/@kwilsonwriter/femminielli-napoletani-aead72d3ad92

Modern blog about femminiello - http://www.naplesldm.com/femm.php

Modern article about femminiello and connections to the Black Madonna and Cybele - https://roadsandkingdoms.com/2015/femminiello-pride/

Article on the ancient galli: http://www.glbtqarchive.com/ssh/galli_S.pdf

Journal article on the ancient galli with primary citations/quotes - https://journal.fi/scripta/article/download/67211/27509

Short article on the galli - https://www.english-heritage.org.uk/learn/histories/lgbtq-history/the-galli/

Article that discusses the galli in relation to ancient Roman opinions and art - https://www.research.manchester.ac.uk/portal/files/27309091/POST-PEER-REVIEW-PUBLISHERS.PDF