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Al-Qaeda's leader ordered the group's Syrian affiliate, the Al-Nusra Front, to end fighting with its jihadist rivals, in an audiotape posted online on Friday.
Ayman al-Zawahiri, in a message to Al-Nusra chief Abu Mohammed al-Jolani, ordered that "all soldiers of the front immediately cease fighting" other jihadist groups.
Instead, he urged him to "devote himself to combat the enemies of Islam, specifically Baathists, Shiites and their allies."
The Baath is the party of Syrian President Bashar al-Assad. Al-Qaeda is a hardline Sunni organisation that considers Shiite Muslims, including the many volunteers from neighbouring Iraq and Lebanon fighting alongside Assad's forces, to be heretics.
Powerful rebel groups in Syria, including Al-Nusra, have been locked in fierce fighting with the Islamic State of Iraq and the Levant since January that has killed thousands of fighters.
ISIL was initially welcomed by other rebels, who have been fighting to topple Assad since 2011, but allegations of brutal abuses against civilians as well as rival opposition fighters sparked a backlash.
Zawahiri was among the critics, and ordered ISIL chief Abu Bakr al-Baghdadi to restrict the group's activities to its home base in Iraq.
In his latest message, he renewed that instruction, urging him to "devote himself to wounded Iraq, which needs you to redouble your efforts" there.