Chloroplast genomes as a tool to resolve red algal phylogenies: a case study in the Nemaliales

Research article Open Access Chloroplast genomes as a tool to resolve red algal phylogenies: a case study in the Nemaliales Joana F. Costa1Email author, Showe-Mei Lin2, Erasmo C. Macaya3, 4, Cindy Fernández-García5 and Heroen Verbruggen1 BMC Evolutionary BiologyBMC series – open, inclusive and trusted201616:205 https://doi.org/10.1186/s12862-016-0772-3 ©  The Author(s). 2016 Received: 31 May 2016Accepted: 28 September 2016Published: 10 October 2016 Abstract Background Obtaining strongly supported phylogenies that permit confident taxonomic and evolutionary interpretations has been a challenge in algal biology. High-throughput sequencing has improved the capacity to generate data and yields more informative datasets. We sequenced and analysed the chloroplast genomes of 22 species of the order Nemaliales as a case study in the use of phylogenomics as an approach to achieve well-supported phylogenies of red algae. Results Chloroplast genomes of the order Nemaliales are highly conserved, gene-dense and completely syntenic with very few cases of gene loss. Our ML estimation based on 195 genes recovered a completely supported phylogeny, permitting re-classification of the order at various taxonomic levels. Six families are recognised and the placement of several previously contradictory clades is resolved. Two new sub-orders are described, Galaxaurineae and Nemaliineae, based on the early-branching nature and monophyly of the groups, and presence or absence of a pericarp. Analyses of subsets of the data showed that >90 % bootstrap support can be achieved with datasets as small as 2500 nt and that fast and medium evolving genes perform much better when it comes to resolving phylogenetic relationships. Conclusions In this study we show that phylogenomics is an efficient and effective approach to investigate phylogenetic relationships. The six currently circumscribed Nemaliales families are clustered into two evolutionary lineages with strong statistical support based on chloroplast phylogenomic analyses. The conserved nature of red algal chloroplast genomes is a convenient and accessible source of data to resolve their ancient relationships. Keywords Plastid genomesChloroplast phylogenomicsRed algaeNemalialesConserved genomes Background Molecular phylogenies are the cornerstone of biodiversity and evolutionary research but many phylogenetic relationships are contradictory or not known with certainty, for example due to low statistical support. One of the major challenges in designing phylogenetic studies is to decide how much molecular data is needed to achieve a satisfactory result [1, 2]. High throughout sequencing (HTS) techniques have made the acquisition of multilocus datasets easy, even for non-model organisms. Their use has become common practice for comparative and phylogenetic analyses of entire genomes (phylogenomics). This provides us with insights not only into phylogenetic relationships but also how other features of the genome (e.g. genome synteny, gene loss, intergenic regions) evolve. While obtaining eukaryotic nuclear genomes still presents significant challenges in terms of sequencing coverage, assembly and annotation [3], for photosynthetic organisms there is a more accessible alternative - their plastid genomes. Chloroplast genomes are an attractive option for phylogenomic studies for various reasons. First of all, they are present in multiple copies in each cell, therefore chloroplast DNA (cpDNA) data is easily obtained from bulk DNA extractions. Additionally, they are relatively small in size (~100-190 kb) and exhibit low variability in gene content and (in some groups) gene arrangement [4], meaning that assembly and annotation are straightforward. Furthermore, the non-recombinant nature of plastids makes them a good tool when inferring ancient phylogenetic relationships [5]. The oldest eukaryotic fossil is believed to be a red alga (Bangiomorpha pubescens) dated as being 1.2 billion years old [6]. However resolving the red algal tree of life has been challenging with the relationships between Florideophyceae – the most diverse class of red algae – especially difficult to resolve. While the five florideophyte subclasses are well supported, within these lineages many early-branching nodes are yet to be resolved [1, 7, 8]. A study comparing ten red algae chloroplast genomes showed the potential of plastid phylogenomics to unravel relationships among red algal classes and their constituent lineages [9]. Among the eukaryotes with primary plastids (Archaeplastida), red algal chloroplast genomes are the most conserved and have the highest gene content [9, 10]. This is a potential perfect combination of features to resolve the ancient relationships among red algal groups: a conserved architecture of the genome, which simplifies data processing, and a high number of genes that are likely to hold enough phylogenetic signal. A persistent problem within the Florideophyceae is found in the order Nemaliales. The Nemaliales belong to the subclass Nemaliophycideae, one of the earliest branching clades within the florideophytes. Molecular clocks suggest that the Nemaliales diverged from other Nemaliophycideae lineages approximately 200 Ma ago [8]. There are 276 species of Nemaliales currently described [11] distributed across 34 genera and 6 families. The Liagoraceae is the most species-rich family, followed by the Scinaiaceae and Galaxauraceae. Three monogeneric families have been recently recognised: the re-instated Nemaliaceae and the new Yamadaellaceae and Liagoropsidaceae [12]. However, the phylogenetic relationships among these six families have not been resolved with confidence and we lack a comprehensive reference phylogeny for the group. The placement of Scinaiaceae has been contradictory in previous studies [12–14] and the relationships between the remaining families have low support [12]. The early branches within the Liagoraceae also have low support [12, 15]. Analyses of whole cpDNA genomes greatly improved phylogenetic resolution in the green plant lineage [16–20]. Despite its promising features, chloroplast phylogenomics has not been widely applied to resolve phylogenetic relationships among red algae. The Nemaliales are a good model to assess the utility of plastid phylogenomics in red algae - an old photosynthetic group with ambiguous phylogenetic relationships. This study aims to (1) characterize chloroplast genomes of Nemaliales, (2) use the data to reconstruct a well-supported phylogenetic tree and, based on these results, (3) revise the classification of the order and (4) evaluate the utility of chloroplast phylogenomics in red algae. Our approach consists of high-throughput sequencing of a phylogenetically diverse set of Nemaliales species, comparison of genomes across the group, and phylogenetic analyses of the complete dataset and subsets thereof. Methods Taxon sampling and sequencing We selected 1 (...truncated)