The genus Metrosideros Banks ex Gaertn. (Myrtaceae) is an emblematic genus of trees of the Pacific islands, being present on most high Pacific islands where it is particularly abundant in mountain and ridge vegetation. In Hawai‘i, ‘ōhi‘a lehua (M. polymorpha Gaudich.) is an ecologically dominant tree with high intraspecific diversity (eight varieties) and four satellite species that is emerging as a model for studies of speciation in trees (Stacy et al., 2014). Low levels of variation in plastid (Percy et al., 2008) and ribosomal (Wright et al., 2000) DNA sequences and the occurrence of homoplasy in nuclear microsatellites (Harbaugh et al., 2009) have hampered understanding of Metrosideros' evolutionary history. Single-copy nuclear DNA sequences are likely to provide the necessary intermediate level of variation to improve understanding of the evolution of Metrosideros.
Metrosideros is part of a larger clade (BKMMST; Biffin et al., 2010) that comprises Backhousieae, Kanieae, Myrteae, Metrosidereae, Syzygieae, Tristanieae, and Cloezia Brongn. & Gris. This clade encompasses considerable morphologic diversity within the Myrtaceae family, including the two fleshy-fruited groups. The first group is the tribe Syzygieae, chiefly the genus Syzygium P. Browne ex Gaertn. (ca. 1000 species) including clove (S. aromaticum (L.) Merr. & L. M. Perry) and mountain apple (S. malaccense (L.) Merr. & L. M. Perry). The second group, Myrteae, comprises ca. 47 genera, including the large Eugenia L. (ca. 1100 species) and guava (Psidium guajava L.). Whether these two tribes are sister groups, and therefore whether fleshy fruits evolved once or twice in Myrtaceae, is still unclear. Therefore, phylogenetically useful markers are desirable for this entire group to answer such questions and to resolve systematic issues.
METHODS AND RESULTS
We obtained a pooled, partial transcriptome library from leaf and floral buds (fixed in the field in RNA later [QIAGEN, Germantown, Maryland, USA] and stored at −80°C) of four Hawaiian Metrosideros taxa: M. rugosa A. Gray (O‘ahu), M. tremuloides (A. Heller) P. Knuth (O‘ahu), M. polymorpha var. incana (H. Lév.) H. St. John (Big Island of Hawai‘i), and M. polymorpha var. newellii (Rock) H. St. John (Big Island). Total RNA was isolated with the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany), and mRNA were poly A selected using the Dynabeads mRNA Purification Kit (Life Technologies, Carlsbad, California, USA). cDNA libraries were prepared and normalized following the Roche-FLX Titanium cDNA Rapid Library Preparation Method Manual (Roche, Basel, Switzerland). RNA isolation, cDNA synthesis, and 454 sequencing were done at the University of Arizona Genetics Core Laboratory. The 454 run provided 715,065 reads with an average length of 401 bp. Adapters, ribosomal RNA, and low-quality and low-complexity sequences were removed/trimmed using SeqClean ( http://sourceforge.net/projects/seqclean/), and each taxon was assembled separately by the TGI Clustering tools (TGICL; Pertea et al., 2003) using default settings. We searched for single-copy genes reported by Duarte et al. (2010) in the Metrosideros transcriptome through BLAST searches. We designed primers flanking each side of one intron for about 80 genes with Primer3 (Rozen and Skaletsky, 2000), setting the optimal annealing temperature to 60°C (minimum 59°C, maximum 61°C) and Max Poly X to 3, and using default settings for other parameters. We report here loci that should be of potential use for phylogenetic studies in Myrtaceae, i.e., loci with relatively long amplicons (>500 bp) and successful amplifications across a range of species. We tested the primers on a single accession of M. polymorpha var. glaberrima (H. Lèv.) H. St. John, P. guajava, Syzygium sandwicense (A. Gray) Müll. Berol., and Melaleuca quinquenervia (Cav.) S. T. Blake. Three tribes of the BKMMST clade were thus represented, including the two fleshy-fruited tribes, and also the more distantly related tribe Melaleuceae.
Characterization of 10 sing1e-copy nuclear markers designed to amplify in Metrosideros and fleshy-fruited Myrtaceae. Statistics on polymorphism are given for markers successfully sequenced in a sampling including all four of Metrosideros polymorpha var. glaberrima, Syzygium sandwicense, Psidium guajava, and Melaleuca quinquenervia.
Fresh or frozen leaves were homogenized with Lysing Matrix A tubes in the FastPrep-24 instrument for 40 s at 4.0 m/s (MP Biomedicals, Santa Ana, California, USA). DNA was extracted from the lysate using a NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany). Extracted DNA was precipitated with 100% ethanol and 3 M sodium acetate (pH 5.2), vacuum dried, washed with 70% ethanol, and resuspended in elution buffer (Macherey-Nagel). The nuclear regions were amplified using the following mix: 11.3 µL of H2O, 4 µL of GoTaq 5× Buffer (Promega Corporation, Madison, Wisconsin, USA), 2 µL of MgCl2 25 mM, 1 µL of bovine serum albumin (BSA) 10 mg/mL, 0.4 µL of dNTP 1.25 mM, 0.2 µL of each primer 10 µM, 0.1 µL of GoTaq Flexi DNA polymerase 5 U/µL (Promega), and 0.8 µL of DNA template. The following amplification program was used: 2 min at 94°C, 38 cycles of 1 min at 94°C, 1 min at 63°C, 1 min at 72°C (1 min 30 s when the fragment exceeded 1000 bp), and a final extension of 5 min at 72°C. PCR products were sequenced directly at the Core Genetics Laboratory at the University of Hawai‘i Hilo. DNA sequences were aligned with MUSCLE within MEGA 6.06 (Tamura et al., 2013).
We identified 10 genes that were successfully amplified in Metrosideros as well as Syzygium, Psidium, or Melaleuca; six worked in all four genera tested (Table 1). Direct sequencing was straightforward with a single exception. For one locus, MeNu61, the accession of Melaleuca appears to be heterozygous with the presence of two alleles differing by at least one indel; we could not obtain a clear direct sequence and did not attempt cloning. Alignment was straightforward in most genes, but somewhat difficult for MeNu18 and partly ambiguous for Psidium in MeNu47 because of a large indel. The cumulative number of polymorphic sites per gene across the four genera was high (>80) for most genes. We also identified microsatellites, e.g., (CT)13 and (CT)19 in MeNu18 for M. polymorpha and S. sandwicense, respectively, and (TAAA)11 in MeNu79 for P. guajava. The levels of variation and usefulness of these microsatellites remain to be tested.
The 10 markers described here should be useful for investigations of the relationships within the BKMMST clade of Myrtaceae that includes all of the fleshy-fruited Myrtaceae. The level of polymorphism is promising for population genetic studies as well as for the resolution of phylogenetic relationships among genera of the tribe Myrteae and among species of large genera such as Syzygium or Psidium. They may also be useful in more distantly related Myrtaceae as indicated by their successful amplification in Melaleuca. Their usefulness at broader scales (e.g., across the entire family) may be limited by difficulty in alignment and homoplasy. Finally, they may facilitate discovery of microsatellites for population genetic studies in Myrtaceae.
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 The authors thank H. Issar (University of Arizona Genetics Core Laboratory, Tucson, Arizona) and A. Veillet (Core Genetics Laboratory, University of Hawai‘i at Hilo, Hilo, Hawai‘i) for technical assistance, and the Hawai‘i State Department of Land and Natural Resources–Division of Forestry and Wildlife for facilitating the collection of plant specimens. Funding was provided through the National Science Foundation (DEB 0954274).