To field one man in a women's football team may be regarded as unfortunate. To field two looks like carelessness – or blatant cheating.

This is the accusation levelled at Equatorial Guinea, the tiny west African country, with a population of 650,000.

Several rival teams in the recent African women's championship complained that two of its players – the captain, Genoveva Anonma, and a striker, Salimata Simpore – had one Y chromosome too many.

Equatorial Guinea, one of the smallest countries in Africa, saw off opposition including the hosts, South Africa, on their way to the final and qualification for the women's World Cup.

But now African football's governing body is investigating claims that one player who lined up in this month's final is a man. It is not clear whether this refers to Anonma or Simpore.

The inquiry follows a complaint by the Nigerian Football Federation over "the gender status" of a player. "The protest is on," a spokesman, Robinson Okosun, said. "We have lodged our protest with Caf [the Confederation of African Football]."

Caf said today: "The protest has been registered and submitted to the competent committee, Caf sports medicine committee. [We] will provide further information on this case as soon as some other details emerge."

It added: "The player in question featured for the Equatorial Guinean team at the tournament held from October 31 to November 14 in South Africa."

Nigeria beat Equatorial Guinea, the defending champions, 4-2 in the final. Both go through to the World Cup in Germany next June and July.

Other teams, including Cameroon and Ghana, had expressed doubts over Anonma and Simpore during the tournament. But Cameroon's football federation said it had not made a complaint to Caf.

"No, we're not thinking of filing a case against Equatorial Guinea," its spokesman, Junior Binyam, said. "We'll wait to see what the outcome is and then we will know what to do."

The doubts had prompted Equatorial Guinea's football federation, Feguifoot, to deny that it was cheating and blame a "campaign of defamation".

Feguifoot said: "Accusations about the supposed presence of men are totally unfounded. [The allegations] are by groups of people that watch with pessimism the progress made by Equatorial Guinean soccer."

Anonma, who plays for the German club USV Jena, reportedly told the BBC that she had already been gender tested, which she found "offensive".

There was support today from the South Africa team, who lost to Equatorial Guinea in the semi-final. Fran Hilton-Smith, the South Africa manager, said: "I think they are probably intersex and they think they are girls. That's the aspect that needs to be investigated.

"Fifa has to come up with some specific medical gender tests to establish whether these players are intersex. If they have 100% testosterone that definitely gives them an advantage. They shouldn't be banned but they should be helped."

Equatorial Guinea were also accused of fielding men in 2008 when they won the African women's championship, beating the overwhelming favourites, Nigeria, in the semi-finals. They are still the only country other than Nigeria to win the title.

Fifa's executive committee approved new guidelines on gender verification only last month following the high-profile athletics case of the South African 800 metres world champion, Caster Semenya.

Semenya, who was also accused of being a man, was subjected to gender tests and did not compete for 11 months.

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