Phylogenetic analysis classifies A. gambiae non-LTRs into two new clades and eight previously defined clades. The two new clades, Loner and Outcast, are in bold. Shown here is the Neighbor-Joining tree constructed using alignment of approximately 260 amino acids of the RT domain from non-LTR retrotransposons of A. gambiae, A. aegypti, and divergent non-mosquito and non-insect species. The tree was rooted using RTs of three prokaryotic group II introns (not shown, Malik, Burke, and Eickbush 1999). Maximum parsimony was also used, and it produced a similar phylogenetic tree (not shown). Confidence of the groupings was estimated using 500 bootstrap replications for both methods. Each Arabic numeral at the base of a node is the bootstrap value that represents percent of times out of 500 bootstrap re-samplings that branches were grouped together at a particular node. The first and second numbers at a particular node represent the bootstrap values derived from Neighbor-Joining and maximum parsimony analysis, respectively. Only the values for the major groupings (clades) that are above 50% are shown. The scale at bottom left indicates amino acid divergence. The names of elements from previously established clades are given, but names of new A. gambiae non-LTR families are omitted to save space (see table 1 and Appendix A for family name). Previously reported A. gambiae non-LTRs in the tree are Q, T1, RT1, and RT2 (Besansky 1990; Besansky et al. 1992; Besansky, Bedell, and Mukabayire 1994). Non-LTRs from other mosquito species are MosquI, Lian, JAM1, and JuanA from Aedes aegypti (Mouches, Bensaadi, and Salvado 1992; Warren, Hughes, and Crampton 1997; Tu, Isoe, and Guzova 1998; Tu and Hill 1999). Amino acid sequences from previously reported non-LTRs used in this phylogenetic analysis were obtained from an alignment produced by Malik, Burke, and Eickbush (1999). Accession numbers with corresponding positions for other sequences are as follows: Fugu1 (AJ459419, 1929–1123); Zebra2 (AL591210, 72154–72957); Zebra3 (AL831768, 51653–52456). Representatives of the recently described Ingi and Rex1 clades are not included in the tree. We performed phylogenetic analysis including representatives of the above two clades, and the resulting groupings were unchanged (not shown)