Short Communication
Gene discordance in phylogenomics of recent plant radiations, an example from Hawaiian Cyrtandra (Gesneriaceae)

https://doi.org/10.1016/j.ympev.2013.05.003Get rights and content

Highlights

  • We developed 18 new primer pairs to amplify single-copy nuclear genes in Cyrtandra.

  • We tested the concordance of 14 loci for the reconstruction of Hawaiian Cyrtandra phylogeny.

  • We found four discordant loci out of 14.

  • We conclude that accurate phylogenies will require a high number of independent loci.

Abstract

Resolving species relationships within recent radiations requires analysis at the interface of phylogenetics and population genetics, where coalescence and hybridization may confound our understanding of relationships. We developed 18 new primer pairs for nuclear loci in Cyrtandra (Gesneriaceae), one of the largest plant radiations in the Pacific Islands, and tested the concordance of 14 loci in establishing the phylogenetic relationships of a small number of Hawaiian species. Four genes yielded tree topologies conflicting with the primary concordance tree, suggesting plastid capture and horizontal transfer via hybridization. Combining all concordant genes yielded a tree with stronger support and a different topology from the total-evidence tree. We conclude that a small number of genes may be insufficient for accurate reconstruction of the phylogenetic relationships among closely related species. Further, the combination of genes for phylogenetic analysis without preliminary concordance tests can yield an erroneous tree topology. It seems that the number of genes needed for phylogenetic analysis of closely related species is significantly greater than the small numbers commonly used, which fail to isolate coalescence, introgression and hybridization.

Introduction

The generation of unprecedented volumes of DNA sequences has led to novel fields in evolutionary biology, such as phylogenomics (Delsuc et al., 2005, Philippe et al., 2005), that involve the use of a large number of markers across the genome to increase the number of informative characters and therefore provide greater resolution for phylogenetic analyses. Although phylogenomics has been used more often at broad taxonomic scales, it has potential for the investigation of species complexes or young radiations as a bridge between population genetics and phylogenetics. Resolving relationships within young radiations is difficult; even in such cases where divergence is sufficient to minimize gene flow among species, incomplete lineage sorting will often create conflicting tree topologies among loci. Further, in such cases where introgression or hybrid speciation has occurred, markers from different parts of the genome could yield conflicting signals (Howarth and Baum, 2005). As such, the evolution of such groups may be better represented as a network (Huson and Bryant, 2006). Phylogenomics could have a significantly greater potential than single-gene (or few-gene) approaches for solving these problems (Delsuc et al., 2005).

Phylogenomics has yet to contribute significantly to fine-scale plant phylogenies, despite the need for improved resolution over the traditional plastid gene and more recent nuclear gene approaches. Plastid markers have been popular in plant phylogenetics because universal primers are widely available for their amplification, their haploid state facilitates combination for analysis, their physical linkage promotes congruency, and they rarely recombine (Marshall et al., 2001). This is in contrast with nuclear genes where recombination can be common even within loci (Kelly et al., 2010, Pillon et al., 2009). Despite these advantages, plastid capture seems to be common in plants (Rieseberg and Soltis, 1991) and indicates that caution should be used when inferring phylogenies among closely related species based on plastid sequences. Mitochondrial genes in contrast have been little used in plants because of their low level of sequence variation and the shortcomings associated with their uniparental mode of inheritance. Combining sequences from a large number of loci from one organelle (including plastome “phylogenomics”) has gained popularity as it allows greater precision in phylogenetic reconstruction; however, there is no associated increase in accuracy due to the tight linkage of organelle genes. Lastly, nuclear ribosomal markers, including ITS, are other popular phylogenetic markers that also have limitations (Alvarez and Wendel, 2003), including complex concerted evolution. To date, plastid and ribosomal markers have been by far the most commonly used markers in plant phylogenetics (Alvarez and Wendel, 2003, Hughes et al., 2006). In light of the above limitations, it has been argued that multiple nuclear genes should lead to the most accurate solution in phylogenetics (Cronn et al., 2003). However, not only are nuclear gene sequences more difficult to acquire due to the scarcity of universal primers, they also require more intensive and careful analyses, e.g. presence of gene recombination, deep-coalescence time, complex combination of multiple loci, etc. (Joly and Bruneau, 2006, Moody and Rieseberg, 2012, Pillon et al., 2009). Furthermore, multiple unlinked genes may have different evolutionary histories (Small et al., 2004). Thus, when using multiple independent genes for phylogenetic analysis, it is important to first test for concordance among them (Baum, 2007, Degnan and Rosenberg, 2009).

Species complexes are of particular interest for evolutionary studies as they represent ongoing speciation and of particular importance for conservation biology as they often include rare taxa. The young Hawaiian flora is well known for its species-rich radiations, and species delineation within groups is often difficult. Cyrtandra (Gesneriaceae) is one of the most diverse plant genera in the Pacific Islands and the second largest plant radiation in the Hawaiian Islands, with over 53 endemic species. Cyrtandra comprises mostly understory shrubs with relatively uniform white flowers and fleshy fruits; most variation among species occurs in inflorescence structure and vegetative characters. They represent a monophyletic group on the archipelago Hawai‘i with a crown age of 4.4 My (Clark et al., 2009), consistent with a first colonization onto Kaua‘i, the oldest and westernmost of the main islands. Individual Cyrtandra species are usually restricted to a single island, and hybridization is considered common (Smith et al., 1996). Almost all species of the genus investigated cytologically (36 species from Peninsular Malaya to Hawai‘i) have 2n = 34 chromosomes (Möller et al., 2013), and there is no indication of polyploidy in the group. Its high species diversity but low ecological diversity suggests that Cyrtandra has undergone a non-adaptive radiation. Next-generation sequencing offers the possibility to identify many novel nuclear single-copy genes in non-model organisms (Zimmer and Wen, 2012). By developing such markers for Hawaiian Cyrtandra, we aimed to evaluate how phylogenomics can help to elucidate relationships within a species complex and how much data are needed for this purpose.

Section snippets

Development of novel markers

We obtained a pooled, partial transcriptome library from leaf and floral buds [fixed in the field in RNA later (QIAGEN)] for two Hawaiian Cyrtandra: C. longifolia (Kaua‘i) and a purported hybrid C. hawaiiensis × calpidicarpa (O‘ahu). RNA isolation, cDNA synthesis and 454 sequencing were done at the University of Arizona Genetics Core Lab. We conducted BLAST searches of the 400 most highly expressed genes in Arabidopsis (C. Fizames, pers. comm.) against our data in CLC DNA Workbench (using default

Results

Eleven loci (Appendix A and B) were successfully sequenced in all accessions along with the nuclear ribosomal ETS and ITS loci as well as the plastid locus psbA-trnH. Two loci were apparently duplicated (Cyrt6 and Cyrt13), and one (Cyrt15) was duplicated in all Hawaiian accessions but not in the accession from Vanuatu. Amplification was most problematic for longer fragments, and sequencing was difficult for loci that included introns. We came across multiple occurrences of heterozygotes with

Discussion

The lack of congruence among genes observed in this study strongly suggests that spurious relationships may be commonly inferred from single-gene phylogenies, as has been noted previously in Gossypium (Cronn et al., 2003). In our study of six species of Cyrtandra, four out of 14 genes yielded topologies that were incongruent with the primary concordance tree. Soltis et al. (2008) observed that polymorphic sites can affect phylogenetic placement of hybrids, but: (1) our network analyses do not

Acknowledgments

The authors wish to thank the following for facilitating the collection of plant specimens: DLNR DOFAW, DLNR State Parks Division, K. Cassell and & J. Lau. The sample from Vanuatu was collected during the expedition Santo 2006 (IRD/MNHN/Pro-Natura). We thank H. Issar (University of Arizona Genetics Core), and A. Veillet (Hilo Genetics Core Facilities) for technical assistance, M. Lebrun and C. Fizames for information on nuclear genes, C. Ané, S. Geib and L. Barrabé for help and advice with data

References (30)

  • J. Heled et al.

    Bayesian inference of species trees from multilocus data

    Molecular Biology and Evolution

    (2010)
  • D.G. Howarth et al.

    Genealogical evidence of homoploid hybrid speciation in an adaptive radiation of Scaevola (Goodeniaceae) in the Hawaiian islands

    Evolution

    (2005)
  • C.E. Hughes et al.

    From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction

    Philosophical Transactions of the Royal Society of London B

    (2006)
  • D.H. Huson et al.

    Application of phylogenetic networks in evolutionary studies

    Molecular Biology and Evolution

    (2006)
  • D.H. Huson et al.

    Computing recombination networks from binary sequences

    Bioinformatics

    (2005)
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