Abstract
Targeted enrichment and sequencing of hundreds of nuclear loci for phylogenetic reconstruction is becoming an important tool for plant systematics and evolution. Annonaceae is a major pantropical plant family with 110 genera and ca. 2,450 species, occurring across all major and minor tropical forests of the world. Baits were designed by sequencing the transcriptomes of five species from two of the largest Annonaceae subfamilies. Orthologous loci were identified. The resulting baiting kit was used to reconstruct phylogenetic relationships at two different levels using concatenated and gene tree approaches: a family wide Annonaceae analysis sampling 65 genera and a species level analysis of tribe Piptostigmateae sampling 29 species with multiple individuals per species. DNA extraction was undertaken mainly on silicagel dried leaves, with two samples from herbarium dried leaves. Our kit targets 469 exons (364,653 bp of sequence data), successfully capturing sequences from across Annonaceae. Silicagel dried and herbarium DNA worked equally well. We present for the first time a nuclear gene-based phylogenetic tree at the generic level based on 317 supercontigs. Results mainly confirm previous chloroplast based studies. However, several new relationships are found and discussed. We show significant differences in branch lengths between the two large subfamilies Annonoideae and Malmeoideae. A new tribe, Annickieae, is erected containing a single African genus Annickia. We also reconstructed a well-resolved species-level phylogenetic tree of the Piptostigmteae tribe. Our baiting kit is useful for reconstructing well-supported phylogenetic relationships within Annonaceae at different taxonomic levels. The nuclear genome is mainly concordant with plastome information with a few exceptions. Moreover, we find that substitution rate heterogeneity between the two subfamilies is also found within the nuclear compartment, and not just plastomes and ribosomal DNA as previously shown. Our results have implications for understanding the biogeography, molecular dating and evolution of Annonaceae.
Highlights
Targeted enrichment followed by high throughput sequencing of hundreds or even thousands of loci for phylogenetic reconstruction is becoming a golden standard in plant evolutionary biology (Cronn et al, 2012; Xi et al, 2013; Mariac et al, 2014; Barrett et al, 2016)
The trinity analyses recovered a total of 297,193 raw contigs for Marsypopetalum littorale, 342,592 for Sapranthus microcarpus, 102,025 for M. myristica, 194,497 for Monanthotaxis whytei and 164 635 Mkilua fragrans
We found similar high levels of node support in our Piptostigmateae trees when compared to the Annonaceae analyses, despite the reduced evolutionary distance between taxa (Figure 6A). 85% of branches had local posterior probabilities (LPP) values >0.9
Summary
Targeted enrichment followed by high throughput sequencing of hundreds or even thousands of loci for phylogenetic reconstruction is becoming a golden standard in plant evolutionary biology (Cronn et al, 2012; Xi et al, 2013; Mariac et al, 2014; Barrett et al, 2016). Once the baits hybridize to the targeted regions, the rest of the genome is discarded, and only the regions of interest are sequenced (Grover et al, 2012) increasing the read depth of the regions of interest This is in contrast to earlier approaches where the focus lay on recovering complete genomes (e.g., Staats et al, 2013). The difficult step concerns the identification of the targeted regions and the design of the baits This relies on genome-wide information such as transcriptomes or full genomes to identify low copy orthologous genes useful for phylogenetic inference. These data are not always available for non-model groups or clades, especially for tropical lineages. Increased availability of transcriptomes across angiosperms (Wickett et al, 2014) and lowered sequencing costs allow access to more genomic resources useful in non-model groups
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