Elsevier

Gene

Volume 350, Issue 2, 9 May 2005, Pages 117-128
Gene

The first complete chloroplast genome sequence of a lycophyte, Huperzia lucidula (Lycopodiaceae)

https://doi.org/10.1016/j.gene.2005.01.018Get rights and content

Abstract

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8× depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373 bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,657 bp. Gene order is more similar to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperzia chloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophyte chloroplast genome data also enable a better reconstruction of the basal tracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, inferred amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.

Introduction

Green plants are an old group dating back about 1 billion years (Mishler, 2000). There are about half a million extant species (Mishler, 2000), including the main primary energy producers in both terrestrial and aquatic ecosystems. Reconstructing the pattern and processes of the evolution of this large and diverse group is imperative, yet challenging. Arguably, the fastest growing front in these efforts is the rapid growth in genome sequencing, which has ignited the fields of comparative and evolutionary genomics (Normile, 2001). Although large scale phylogenetic analyses of complete eukaryotic nuclear genomes are just beginning, many phylogenomic studies of the much smaller organellar genomes are complete or underway. Most of this work has been on animal mitochondrial genomes (Boore, 1999), of which over 400 species are currently represented in public databases. More recently, chloroplast genomes have been sequenced from several clades of green plants and these genomes have been found to contain considerable amounts of phylogenetically useful data (Lemieux et al., 2000).

The chloroplasts of green plants are descendents of cyanobacteria that established an endosymbiotic relationship with a primitive eukaryote. Although many proteins necessary for chloroplast functioning are imported from the cytoplasm, chloroplasts have retained their own, now diminished, genome (Stoebe et al., 1999), along with systems for expressing these genes. Across green plants, there is a high degree of consistency in chloroplast genome structure and in gene content and arrangement (Palmer and Stein, 1986). However, these features vary sufficiently among lineages to provide useful characters for phylogenetic reconstruction. Such genome-level characters have proven to be especially robust indicators of evolutionary relatedness due to their complexity and low frequency of reversal (Helfenbein and Boore, 2004).

Comparing complete chloroplast genome sequences also enables a reconstruction of events, such as gene transfers between intracellular compartments (i.e., nucleus, chloroplast, mitochondrion), and a better understanding of the evolutionary processes that account for the features of today's chloroplast genomes. Unfortunately, as of the beginning of 2004, there are still only 25 complete chloroplast genomes published and many critical clades remain unrepresented. Here we describe the first of a series of complete chloroplast genome sequences selected to fill important phylogenetic gaps, initially focusing on land plants. Currently, complete chloroplast genomes are available from each of the three main bryophyte lineages (a hornwort, a moss, and a liverwort), 2 ferns, 2 gymnosperms, and 13 angiosperms. These taxa represent the bulk of phylogenetic diversity, but no chloroplast genome sequence has been published for any lycophyte. This is somewhat surprising because the best evidence that the lycophytes are sister to remaining extant vascular plants comes from the observation of a 30 kb inversion in the chloroplast genome, detected by restriction-site mapping studies (Raubeson and Jansen, 1992). Here we describe (1) the first complete chloroplast genome sequence of a lycophyte (Huperzia lucidula (Michx.) Trevis.); (2) a novel method of providing chloroplast genome-enhanced material from which to obtain the sequence; and (3) the unique aspects of the genome. We also present phylogenetic analyses based on amino acid sequences and DNA sequences extracted from published land plant chloroplast genomes plus that of H. lucidula. Furthermore, we explore the use of genome structure to infer land plant phylogeny.

Section snippets

Preparation and DNA sequencing

Vegetative material of H. lucidula was collected from Balsam Gap Overlook, NC (USA). A voucher specimen (Renzaglia #3200) is deposited at the University of California Herbarium at Berkeley (UC). Purified fractions of intact chloroplasts of H. lucidula were collected by fluorescence-activated cell sorting (FACS). One hundred milligrams of fresh leaf tissue was placed on ice in a sterile plastic Petri dish containing 1.0 mL of an organelle isolation solution containing 0.33 M sorbitol, 50 mM

Results

Our overarching goal is to resolve the phylogeny of green plants using a wide range of data including sequences of organellar genomes (http://ucjeps.berkeley.edu/TreeofLife/). Many of the taxa of interest are rare or have small or unicellular body plans, so traditional methods of organelle isolation, such as sucrose gradients, are not feasible because of tissue quality or quantity. However, PCR-based methods and cloning mean that even a small amount of DNA would suffice. We used a taxon, H.

Discussion

We first focus on our, albeit limited, phylogenetic inferences before discussing the issues associated with details of data and analysis. We present several different analyses of land plant phylogeny using both sequence data (DNA and protein) and data from genome structure. This has been made possible by the addition of a new chloroplast genome sequence from a previously unsampled clade of land plants. Any phylogenetic inference depends very much on the type of analysis used. We have performed

Acknowledgments

Thanks to Karen Renzaglia and James Donaldson for plant material, Tori Yamamoto for technician assistance with the organelle isolation, Jessica Roper for lab assistance, Stacia Wyman for help with DOGMA, Andrea Warnick for drawing the genome map, and Ashwin Manne for help with data analysis. Thanks to Lee Bjerregaard, Jeff Palmer, Alice Bain, and two anonymous reviewers for comments on an earlier version of the manuscript. This research was supported by the Green Tree of Life grant from the

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