microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate

Abstract

Hagfish and lampreys are the only living representatives of the jawless vertebrates (agnathans), and compared with jawed vertebrates (gnathostomes), they provide insight into the embryology, genomics, and body plan of the ancestral vertebrate. However, this insight has been obscured by controversy over their interrelationships. Morphological cladistic analyses have identified lampreys and gnathostomes as closest relatives, whereas molecular phylogenetic studies recover a monophyletic Cyclostomata (hagfish and lampreys as closest relatives). Here, we show through deep sequencing of small RNA libraries, coupled with genomic surveys, that Cyclostomata is monophyletic: hagfish and lampreys share 4 unique microRNA families, 15 unique paralogues of more primitive microRNA families, and 22 unique substitutions to the mature gene products. Reanalysis of morphological data reveals that support for cyclostome paraphyly was based largely on incorrect character coding, and a revised dataset is not decisive on the mono- vs. paraphyly of cyclostomes. Furthermore, we show fundamental conservation of microRNA expression patterns among lamprey, hagfish, and gnathostome organs, implying that the role of microRNAs within specific organs is coincident with their appearance within the genome and is conserved through time. Together, these data support the monophyly of cyclostomes and suggest that the last common ancestor of all living vertebrates was a more complex organism than conventionally accepted by comparative morphologists and developmental biologists.

Fig. 1.

The two competing hypotheses. Either lampreys are more closely related to hagfish than they are to gnathostomes, making Cyclostomata monophyletic (on the left), or lampreys are more closely related to gnathostomes than they are to hagfish, making Cyclostomata paraphyletic (on the right).

Fig. 2.

Phylogenetic distribution of all miRNA families analyzed in chordates (see Dataset S2 for data matrix and Fig. S2 for complete phylogenetic analysis). Cyclostomes share four miRNA families not found in any other animal species investigated to date, and a maximum parsimony analysis supports the monophyly of Cyclostomata. Note that miRNA families specific to a single species are not indicated, but losses of more primitive families are indicated. Of particular interest is the number of miRNA families acquired in the stem lineage leading to the vertebrate crown group.

Fig. 3.

The presence of paralogues of more primitive miRNA families and conserved nucleotide substitutions both support the monophyly of Cyclostomata. Shown is miR-19 as an example of a group of miRNAs that shows both conserved nucleotide substitutions (19a; Top, bold) with respect to the other paralogue(s) (19b and 19c; Middle and Bottom) and the possession of a paralogue (miR-19c) not present in any known gnathostome (Dataset S1 has the complete description of both paralogues and nucleotide substitutions supporting cyclostome monophyly). Cmi, Callorhinchus milii; Dre, Danio rerio; Hsa, Homo sapiens; Lpl, Lampetra planeri; Mgl, Myxine glutinosa; Pma, Petromyzon marinus.

Fig. 4.

miRNA expression profile of seven different lamprey organs. Only the top 10 highest expressed miRNAs (Dataset S4) are shown, and each specific miRNA is given a distinct color for all pie charts. Below each pie chart is the expression pattern of the highest expressed gene in the lamprey library in the zebrafish (54)—note the concordance between the lamprey and zebrafish for all organs queried except for the heart (Bottom). Pma, Petromyzon marinus; Dre, Danio rerio; Mmu, Mus musculus.