Bichir microRNA repertoire suggests a ray-finned fish affinity of Polypteriforme

Highlights

• A total of 228 known miRNA were identified in the bichir genome.

• A total of 107 novel miRNAs were identified.

• MiRNAs in bichir provides evidence for their affinity of ray-finned fishes.

Abstract

The phylogenetic position of Polypteriforme (bichirs) remains elusive, despite extensive research both on morphological and molecular datasets. Unfortunately morphological cladistic analyses and molecular phylogenetic analyses had reached conflicting conclusions, as Polypteriformes were either grouped with lobe-finned fishes (Sarcopterygii) or ray-finned fishes (Actinopterygii), or even classified as their own group, the Brachiopterygii. In this study, we applied a third independent source of datasets, the presence versus absence of microRNAs, to re-investigate the phylogenetic relationship of bichirs. Through deep sequencing of small RNA library, we showed that bichirs should be grouped into ray-finned fishes rather than lobe-finned fishes. Phylogenetic analysis confirmed that bichirs were placed as the most basal member of the ray-finned fishes. Bichirs shared five unique microRNA families with teleostei, which were not found in any other species investigated to date. Bichirs have also retained three of five microRNAs that were previously deemed to be lost exclusively in teleostei. Furthermore, we report more than one hundred novel microRNAs that are unique to bichir. The identification of microRNAs in bichir provides overwhelming evidence for their affinity of ray-finned fishes. The sarcopterygian-like characteristics such as lobed fins, paired ventral lungs, and external gills in juveniles should not be considered as derived traits shared with those of sarcopterygians.

Introduction

The phylogenetic position of polypteriform fishes (bichirs and reedfish) has received intensive attention over the past 150 years. Both morphological and molecular approaches had been applied to resolve this; however these two approaches had reached conflicting conclusions. The earliest morphological analyses conducted in the 1860s concluded that bichirs were sarcopterygians and they were thus grouped into the suborder Crossopterygii together with lungfish, coelacanth and fossil rhipidistians (Tree 1; Fig. 1A) (Huxley, 1861, Cope, 1871). Later morphological analyses classified bichirs as basal actinopterygians (Tree 3; Fig. 1C) (Goodrich, 1928, Gardiner, 1973, Schaeffer, 1973, Patterson, 1982, Lauder and Liem, 1983, Gardiner and Schaeffer, 1989, Nelson, 1994) or even a distinct subclass of Osteichthyes, the Brachiopterygii (Tree 2; Fig. 1B) (Jessen, 1973, Nelson, 1973, Jarvik, 1981, Bjerring, 1985). On the other hand, although the majority of molecular phylogenetic studies have favored the view that bichirs are the most basal member of the ray-finned fishes (Normark et al., 1991, Le et al., 1993, Noack et al., 1996, Venkatesh et al., 2001, Inoue et al., 2003), there are also studies that reported alternative phylogenetic trees (Rasmussen and Arnason, 1999, Rocco et al., 2004), albeit with low confidence. The difficulties to assess the phylogenetic position of bichirs are mainly that their fossil evidence can only extend back to the Eocene (Greenwood, 1974, Carroll, 1988) and that these fishes exhibit a lot of primitive (e.g. cartilaginous skeleton, ganoid scales, and the intestine with a spiral valve) and sarcopterygian-like characteristics. For example, their jaw structure is more similar to that of the tetrapods than that of the teleosts, and their pectoral fins with lobed base covered with scales are extremely similar to those of lobe-finned fishes. Bichirs also possess highly vascularized paired lungs and a pair of external gills in juveniles, which are not seen in any other ray-finned fishes, except in some species of lungfish (Robinson, 2011). These conflicting characteristics are so interesting that some researchers are still arguing that Polypteriformes are morphologically too distinct from actinopterygians to be classified as member of this group, and should be placed into sarcopterygians or at least more closely related to sarcopterygians than to actinopterygians (Jessen, 1973, Nelson, 1973, Jarvik, 1981, Bjerring, 1985).

Here, we attempt to reassess the problematic classification of bichirs through analysis of bichir's microRNAs (miRNAs) repertoire, an independent source of molecular dataset. miRNA sequences, which have been successfully applied to many metazoan phylogenetic studies (Sperling et al., 2009, Heimberg et al., 2010, Sperling et al., 2010, Campbell et al., 2011, Philippe et al., 2011, Rota-Stabelli et al., 2011, Sperling et al., 2011, Wiegmann et al., 2011, Helm et al., 2012, Lyson et al., 2012, Pisani et al., 2012, Fromm et al., 2013, Peterson et al., 2013, Tarver et al., 2013), have five properties making them among the most reliable phylogenetic markers: (i) identification of novel miRNAs does not necessarily require fully sequenced genome sequence, (ii) new lineage-specific miRNA families are continually added to metazoan genomes through time which makes them ideal as phylogenetic markers, (iii) low levels of secondary miRNA loss, (iv) rarity of substitutions to the mature miRNA sequence, and (v) almost impossible scenario of convergent evolution of miRNAs (Wheeler et al., 2009, Tarver et al., 2013). Therefore, miRNAs should provide an alternative and valuable evidence for the evolutionary position of Polypteriformes. In the present study, we constructed a small RNA library from five pooled organs (heart, brain, liver, kidney, and spleen) of bichir (Polypterus senegalus) and conducted high-depth next generation sequencing. We found that bichir shares five unique miRNA families with teleostei and none with sarcopterygians; this provides the strongest support for the Actinopterygii affinity for Polypteriformes.