Late changes in spliceosomal introns define clades in vertebrate evolution

Abstract

The evolutionary origin of spliceosomal introns has been the subject of much controversy. Introns are proposed to have been both lost and gained during evolution. If the gain or loss of introns are unique events in evolution, they can serve as markers for phylogenetic analysis. We have made an extensive survey of the phylogenetic distribution of seven spliceosomal introns that are present in Fugu genes, but not in their mammalian homologues; we show that these introns were acquired by actinopterygian (ray-finned) fishes at various stages of evolution. We have also investigated the intron pattern of the rhodopsin gene in fishes, and show that the four introns found in the ancestral chordate rhodopsin gene were simultaneously lost in a common ancestor of ray-finned fishes. These changes in introns serve as excellent markers for phylogenetic analysis because they reliably define clades. Our intron-based cladogram establishes the difficult-to-ascertain phylogenetic relationships of some ray-finned fishes. For example, it shows that bichirs (Polypterus) are the sister group of all other extant ray-finned fishes.

Figure 1

Phylogenetic distribution of spliceosomal introns. The phylogenetic tree is based on Nelson’s classification of fishes (28). A plus sign (+) indicates presence and a minus sign (−) indicates absence of intron. An asterisk (∗) indicates that no PCR fragment was obtained, presumably because of the large size of the intron. The letter “a” indicates a failure to clone by PCR because of the highly variable coding sequence in this region of RAG1. Data from previous studies (Gh4a, refs. and ; Mll25a, ref. ; Dyst6a and Dyst10a, ref. ; Mhc2a, refs. , , and ; RAG1a and RAG1b, B. Peixoto and S.B., unpublished work and refs. and ; Rhod, refs. , , and 25) are enclosed in parentheses. Gh4a, (15); Mll25a, (16); Dyst6a and Dyst10a, (14); Mhc2a, (13); RAG1a and RAG1b, (21); Rhod, rhodopsin gene (+ represents presence of four introns and − represents absence of all four introns). The rhodopsin gene from the primitive lamprey (Agnatha) contains four introns (23) (not shown) as in the mammalian rhodopsin. RAG1a intron was previously reported to be absent from the rainbow trout (Oncorhynchus mykiss) (30), but our PCR products from the rainbow trout and the brown trout (data not shown) contained this intron.

Figure 2

Cladogram showing the phylogenetic relationships of fishes, inferred by using the presence or absence of introns as character states. The numbers in the boxes represent introns cloned by us (1, rhodopsin; 2, RAG1b; 3, RAG1a; Gha4a; 5, Mll25a; 6, Dyst10a; 7, Dyst6a; and 8, Mhc2a). B is an extension of the Division Teleostei from A.

Figure 3

Introns that contain sequences corresponding to their 5′ flanking exons. Coding sequences are shown in uppercase letters and intron sequences in lowercase letters. Intron sequences are aligned with their 5′ flanking exons to show the codon sequences (underlined) in the intron. Nucleotide sequences are numbered above the line. Amino acid sequences are shown in single-letter codes.