Background
==========

The chloroplast (cp) genome has long been a focus of research in plant molecular evolution and systematics due to its small size, high copy number, conservation and extensive characterization at the molecular level \[[@B1]\]. More recently, with technical advances in DNA sequencing, the number of completely sequenced cp genomes has grown rapidly. Aside from providing information on genome structure, gene content, gene order and nucleotide composition, complete cp genome sequences also offer a unique opportunity to explore the evolutionary changes of the genome itself. In general, cp genomes are structurally highly conserved across land plants. However, structural rearrangements, e.g. gene loss, inverted repeat (IR) loss or expansion and inversion, do occur in certain lineages and have been shown to be extremely informative in resolving deep phylogenetic relationships because they may exhibit less homoplasy than sequence data \[[@B1]\]. For example, a 30-kb inversion shared by all vascular plants except lycopsids identifies the lycopsids as the basal lineage in the vascular plants \[[@B2]\]. Two inversions and an IR expansion can be used to clarify basal nodes in the leptosporangiate ferns \[[@B3],[@B4]\].

Currently, one limiting factor in comparative chloroplast genomics is the sparse taxon sampling in spore-bearing land plants. The representation of genome sequencing almost always favors plants of economic interest \[[@B5]\]. Complete cp genomes have been sequenced for more than one hundred seed plants. Amongst these, more than 10 completed sequences each are from cereals (13), crucifers (12) and conifers (12) respectively (see Additional file [1](#S1){ref-type="supplementary-material"}). But for other land plants, excluding seed plants, only 10 cp genome sequences have been achieved in total, of which only 3 are from ferns prior this study (see Additional file [1](#S1){ref-type="supplementary-material"}). For further insights into the evolutionary dynamics of cp genome organization, more data from plant species representative of other crucial evolutionary nodes is needed \[[@B5]\].

Ferns (monilophytes), with more than 10,000 living species, are the most diverse group of seed-free vascular plants \[[@B6],[@B7]\]. Previous studies have uncovered considerable genomic rearrangements in fern cp genomes, but the details and exact series of these events have not yet been fully characterized \[[@B3],[@B4],[@B8]\]. The completed cp genome sequence of the polypod fern *Adiantum capillus-veneris*shows some unusual features not seen in vascular plants before, including tRNA gene losses, which had only been observed in cp genomes of non-photosynthetic plants \[[@B9],[@B10]\]. For example, a putative *tRNA-selenocysteine*(*tRNA-Sec*) gene in *Adiantum*\[[@B10]\] replaces the typical *trnR-CCG*gene. Unfortunately, because *Adiantum*is the only sequenced representative of leptosporangiates, the most diverse fern lineage, it is difficult to tell which characteristics are unique to *Adiantum*or diagnostic of a much larger clade. Therefore, complete cp genome data from more fern clades are necessary to better resolve these issues.

As part of an effort to shed more light on the cp genome evolution in ferns, we have sequenced the complete plastid genome of a scaly tree fern *Alsophila spinulosa*(ab. *Alsophila*) (Cyatheaceae). This taxon was chosen because it is an easily available representative of an ancient lineage -- tree ferns, for which no cp genome has been sequenced before. In addition to tree ferns, heterosporous and polypod ferns are the other two main lineages within the \"core leptosporangiates\" \[[@B11]\]. The three major lineages of \"core leptosporangiates\" were thought to have originated from a Late Triassic diversification \[[@B11]\]. Recent phylogenetic studies further demonstrated a sister relationship between tree ferns and polypods \[[@B6],[@B11],[@B12]\]. After the Late Triassic diversification, polypods remarkably re-diversified along with angiosperms in the Cretaceous \[[@B6],[@B11]\]. Similarly, the scaly tree ferns (Cyatheaceae) also radiated very recently and diversified at an exceptionally high rate \[[@B13]\]. A comparison of the complete cp genome sequences between *Alsophila*and the polypod fern *Adiantum*will aid interpretation of unusual characters observed in *Adiantum*, such as some missing and novel genes \[[@B9],[@B10]\].

Moreover, sequences of all four published fern cp genomes (including that of *Alsophila*) will enable more detailed comparisons of the organization and evolution of the chloroplast genomes in ferns. Our comparative analyses corroborate that fern cp genomes have undergone substantial changes in gene orders during evolution: two main rearrangements contribute to major differences between \"higher\" and basal ferns. In addition, the comparisons also identify some unique characteristics in the *Alsophila*cp genome including a novel tRNA, interesting pseudogenes and a highly repeated 565-bp-region spanning one endpoint of an ancient inversion.

Results and Discussion
======================

General Features
----------------

The chloroplast (cp) genome of *Alsophila spinulosa*\[GenBank: [FJ556581](FJ556581)\] is 156,661 base pairs (bp) with a large single copy (LSC) region of 86,308 bp separated from a 21,623-bp small single copy (SSC) region by two inverted repeats (IRs), each of 24,365 bp (Figure [1](#F1){ref-type="fig"}). The genome is the largest amongst the four sequenced fern cp genomes (Table [1](#T1){ref-type="table"}), but is smaller than previous estimates of other Cyatheaceae species, e.g. *Alsophila bryophila*(165 kb), *Cyathea furfuracea*(179.2 kb) and *Sphaeropteris cooperi*(164.3 kb), using the mapping method \[[@B14]\]. When the IR is considered only once, the *Alsophila*cp genome contains 117 genes, encoding 85 proteins, 4 rRNAs and 28 tRNAs (Table [1](#T1){ref-type="table"}). Pseudogenes of *ycf66*and *trnT-UGU*were also detected in this genome (Figure [1](#F1){ref-type="fig"}). More than half of the *Alsophila*cp genome is composed of coding regions (92,691 bp, 59.17%) with the protein-coding regions accounting for the major portion (81,111 bp, 51.77%) followed by rRNA genes (9,086 bp, 5.80%) and tRNA genes (2,494 bp, 1.59%) (counting both IRs).

###### 

Comparison of general features of fern chloroplast genomes

                      *Alsophila spinulosa*   *Adiantum capillus-veneris*   *Psilotum nudum*   *Angiopteris evecta*
  ------------------- ----------------------- ----------------------------- ------------------ ----------------------
  Total Length (bp)   156661                  150568                        138829             153901
   GC content (%)     40.43                   42.01                         36.03              35.48
  LSC Length (bp)     86308                   82282                         84617              89709
   GC content (%)     39.62                   40.82                         33.63              33.66
  SSC Length (bp)     21623                   21392                         16304              22086
   GC content (%)     37.85                   37.10                         29.97              33.05
  IR Length (bp)      24365                   23447                         18954              21053
   GC content (%)     43.02                   46.33                         44.00              40.65
  Number of gene^a^   117                     117                           118                121
   Protein gene       85                      84                            81                 85
   rRNA gene          4                       4                             4                  4
   tRNA gene          28                      29^b^                         33                 32

^a^The genes in IRs were considered only once

^b^*trnSeC*was counted here according to the annotation in GenBank and the reference \[[@B10]\]

![**Gene map of the *Alsophila spinulosa*chloroplast genome**. Thick black lines on inner cycle indicate the inverted repeats (IRA and IRB) which separate the genome into the large (LSC) and small (SSC) single copy regions. Genes shown on the inside of the circle are transcribed counterclockwise and those on the outside clockwise. Gene boxes are color coded by functional group as shown in the key. Asterisks denote genes with introns. Ψ represents pseudogene. Nucleotide positions are numbered starting at the boundary of IRA and LSC, with position 1 in the intron of *ndhB*. The circle of hashmarks indicates the location of direct and inverted repeats detected by REPuter \[[@B28]\].](1471-2148-9-130-1){#F1}

The *Alsophila*cp genome has an overall GC content of 40.43%, which is lower only than *Adiantum capillus-veneris*amongst the four sequenced fern cp genomes (Table [1](#T1){ref-type="table"}) and is the fourth highest amongst sequenced land plant cp genomes (see Additional file [1](#S1){ref-type="supplementary-material"}). Like other land plants \[[@B15],[@B16]\], GC content is unevenly distributed across the *Alsophila*cp genome by location, functional group and codon position. The GC content in rRNA genes (55.18%) and tRNA genes (54.55%) is much higher than in protein coding regions (40.87%). The GC percentage in IRs is the highest (Table [1](#T1){ref-type="table"}), reflecting the high GC content of rRNA genes. Amongst the protein genes, photosynthetic genes possess the highest GC content (43.85%), followed by genetic system genes (40.80%), whilst NADH genes have the least (39.54%). The GC content also varies by codon position with the first (47.75%) \> second (40.94%) \> third (33.91%) position in turn.

The start codons of 85 protein genes were inferred by comparisons with previously annotated land plant cp genomes. Sixty-three of these genes start with AUG, 20 with ACG and 2 with GUG (*psbC*and *rps12*). An ACG codon may be restored to a canonical start codon (AUG) by RNA editing, whereas a GUG initiation codon has been reported in other cp genomes \[[@B17],[@B18]\]. Inferring translation start positions based only on genome sequences is merely hypothetical \[[@B10]\]. Future determination of sequences from complementary DNA (cDNA) and/or proteins will help to substantiate the putative translation start positions as well as RNA editing sites.

There are in total 27,046 codons in all protein coding regions (including coding regions in both IRs) (Table [2](#T2){ref-type="table"}), representing the total coding capacity of the *Alsophila*cp genome; of these, 2771 (10.25%) are for leucine, 2365 (8.74%) for serine, 2154 (7.96%) for isoleucine, and 1847 (6.83%) for glycine. One third of the total codons are represented by these four amino acids. The codon usage of the *Alsophila*cp genome reflects an apparent AT bias. Most codons end in A or U (66.13%). As shown in figure [2](#F2){ref-type="fig"}, both codon numbers and RSCU (Relative Synonymous Codon Usage) values are negatively correlated with codon GC content (represented by the number of G+C in a given codon). It appears that nucleotide composition bias has a significant influence on codon usage.

###### 

Total numbers of each codon detected in all putative protein coding regions in the *Alsophila spinulosa*chloroplast genome, indicated with tRNAs for which genes have been identified

  -----------------------------------------------------------------------------
  AA      Codon   Number   tRNA           AA    Codon   Number   tRNA
  ------- ------- -------- -------------- ----- ------- -------- --------------
  Phe     UUU     740                     Ser   UCU     612      

  Phe     UUC     539      trnF-GAA       Ser   UCC     390      trnS-GGA

  Leu     UUA     787                     Ser   UCA     499      trnS-UGA

  Leu     UUG     577      trnL-CAA       Ser   UCG     268      

  Tyr     UAU     669                     Cys   UGU     204      

  Tyr     UAC     267      trnY-GUA       Cys   UGC     91       trnC-GCA

  Ter     UAA     82                      ter   UGA     52       

  Ter     UAG     27                      Trp   UGG     435      trnW-CCA

  Leu     CUU     471                     Pro   CCU     389      

  Leu     CUC     237                     Pro   CCC     312      trnP-GGG

  Leu     CUA     479      trnL-UAG       Pro   CCA     344      trnP-UGG

  Leu     CUG     220                     Pro   CCG     185      

  His     CAU     407                     Arg   CGU     397      trnR-ACG(×2)

  His     CAC     192      trnH-GUG(×2)   Arg   CGC     144      

  Gln     CAA     580      trnQ-UUG       Arg   CGA     314      trnR-UCG

  Gln     CAG     247                     Arg   CGG     170      

  Ile     AUU     1051                    Thr   ACU     530      

  Ile     AUC     488      trnI-GAU(×2)   Thr   ACC     294      trnT-GGU

  Ile     AUA     615      trnI-CAU       Thr   ACA     394      

  Met     AUG     543      trnfM-CAU\     Thr   ACG     208      
                           trnM-CAU                              

  Asn     AAU     938                     Ser   AGU     431      

  Asn     AAC     332      trnN-GUU(×2)   Ser   AGC     165      trnS-GCU

  *Lys*   AAA     883                     Arg   AGA     497      trnR-UCU

  *Lys*   AAG     472                     Arg   AGG     232      

  Val     GUU     563                     Ala   GCU     734      

  Val     GUC     216                     Ala   GCC     262      

  Val     GUA     564      trnV-UAC       Ala   GCA     428      trnA-UGC(×2)

  Val     GUG     236                     Ala   GCG     204      

  Asp     GAU     885                     Gly   GGU     683      

  Asp     GAC     241      trnD-GUC       Gly   GGC     191      trnG-GCC

  Glu     GAA     1006     trnE-UUC       Gly   GGA     658      trnG-UCC

  Glu     GAG     460                     Gly   GGG     315      
  -----------------------------------------------------------------------------

![**Correlations between codon usage and codon GC content in the *Alsophila spinulosa*cp genome**. Codon usage is represented by total number (A) and RSCU (Relative Synonymous Codon Usage) (B) of each codon; codon GC content is indicated by the number of G+C in given codon. Each point represents one of the 59 degenerate codons. Pearson correlations shown in the figure are all significant at p \< 0.001. A, correlation between total number and GC content of each codon; B, correlation between RSCU value and GC content of each codon.](1471-2148-9-130-2){#F2}

Gene order
----------

The *Alsophila*cp genome shares three key inversions with other ferns relative to bryophytes (Figure [3](#F3){ref-type="fig"}): 1) a 30-kb inversion at the beginning of LSC (close to IRA) \[[@B2]\]; 2) an approximately 3 kb inversion involving *trnT*, *psbD*, *psbC*, *trnS*, *psbZ*and *trnG*\[[@B8],[@B10],[@B19]\]; and 3) a minor inversion containing a single gene *trnD-GUC*. The first of these inversions is also shared by all vascular plants except lycophytes, whereas the latter two are restricted to ferns.

![**Expected rearrangements in the evolution of fern cp genomes**. Genes are represented by boxes extending right or left of the base-line according to the direction of transcription. Each colored gene segment shows the same gene order region among the seven land plants cp genomes. The boxes highlighted in red denote the inversion of *trnD-GUC*. Excluding *Alsophila spinulosa*, the unchanged tRNA anti-codon is abbreviated in the other six cp genomes. The genes that are missing in one or more cp genomes are shown in purple. The tRNA-leu (CAA/UAA) gene between *rps4*and *ndhJ*is indicated in bold italic type. The pseudogene is denoted by ψ. A, details are shown in the following Figure 5; B, hypothetical pathways to explain this rearrangement are illustrated in the following Figure 6.](1471-2148-9-130-3){#F3}

To our knowledge, the *trnD-GUC*inversion has not been previously documented. Three conserved and consecutive tRNA genes, *trnD-GUC*, *trnY-GUA*and *trnE-UUC*, have been identified in all land plant cp genomes. Excluding ferns, the three genes have the same directions of transcription. However, in ferns *trnD*is inverted relative to *trnY*and *trnE*(Figure [3](#F3){ref-type="fig"}). The simplest interpretation of this change is a single minor inversion involving only *trnD*. Based on current data, it remains unknown whether the 3-kb inversion or the *trnD*inversion occurred first in ferns.

Overall, the *Alsophila*cp genome shows a high degree of synteny with the previously sequenced cp genome of *Adiantum*(Figure [4A](#F4){ref-type="fig"}). In contrast, there exist striking differences between *Alsophila*and *Angiopteris*(Figure [4B](#F4){ref-type="fig"}) as well as between *Alsophila*and *Psilotum*(Figure [4C](#F4){ref-type="fig"}). A set of complex rearrangements in the IRs, involving a rare duplication of *psbA*gene, was found in \"higher\" ferns using physical mapping \[[@B3],[@B4]\]. The IR gene orders of \"higher\" ferns, such as *Adiantum*, *Cyathea*and *Polystichum*, are highly rearranged in comparison to that of basal leptosporangiate *Osmunda*\[[@B3],[@B4],[@B20]\]. Complete cp genome data from *Angiopteris*, *Psilotum*, *Adiantum*and *Alsophila*detail these rearrangements. The IR gene order in *Alsophila*appears to be the same as that in *Adiantum*, while *Angiopteris*and *Psilotum*have the *Osmunda*gene order. To explain the complex rearrangements, a \"two inversions\" hypothesis was proposed \[[@B20]\]. Figure [5](#F5){ref-type="fig"} illustrates the great gene order changes within these rearrangements and the updated version of the \"two inversions\" model incorporating gene order data from the *Alsophila*and *Angiopteris*cp genomes. Recently, Wolf and Roper \[[@B9]\] indicated that the two major inversions did occur in turn and the second inversion (Figure [5](#F5){ref-type="fig"}, Inversion II) took place on the branch leading to the common ancestor of the heterosporous fern clade and its sister group. Thus, it seems reasonable to hypothesize that the *Adiantum*gene order represents a common feature of the three lineages within core leptosporangiates (including heterosporous ferns, tree ferns and polypod ferns).

![**Comparisons of the gene order of *Alsophila spinulosa*cp genome with other ferns**. Comparison by using online zPicture software <http://zpicture.dcode.org/>\[[@B44]\]. Points along the positive slope are in the same orientation in both genomes, whereas points along the negative slope indicate sequences that are oriented in opposite directions. A, the two groups of points that fall along the negative slope in the upper right corner represent the sequences of IRs. B and C, ellipses denote the complex rearrangements in IRs and arrows indicate the gene order change between *rpoB*and *psbZ*.](1471-2148-9-130-4){#F4}

![**The \"two inversions\" model for IR rearrangements in fern cp genomes**. Genes are represented by boxes extending above or below the base-line according to the direction of transcription. The genes that are absent in the *Alsophila*cp genomes are shown in purple: these loci are merely hypothetical in putative intermediates since the course of their loss is unclear. The pseudogene of *trnT-UGU*is represented in red and indicated by ψ. The tRNA anti-codon is abbreviated in putative intermediates.](1471-2148-9-130-5){#F5}

Interestingly, in the *Adiantum*cp genome, an intron-containing *trnT-UGU*was identified between *trnR-ACG*and *ndhB*(Table [3](#T3){ref-type="table"}) \[[@B10]\]. The *Alsophila*cp genome possesses no intact intron-containing *trnT*. However, two fragments that are similar to the two exons within the *Adiantum trnT*were annotated as a *ΨtrnT-UGU*in this study (Table [3](#T3){ref-type="table"}). This new *trnT*or *ΨtrnT*is just at one endpoint of the Inversion II (Figure [5](#F5){ref-type="fig"}). Therefore, the generation of intron-containing *trnT-UGU*may be associated with the IR rearrangements.

###### 

Comparison of gene contents of fern chloroplast genomes

  -------------------------------------------------------------------------------------------------------------------------------
  Gene^a^                         *Alsophila spinulosa*   *Adiantum capillus-veneris*   *Psilotum nudum*   *Angiopteris evecta*
  ------------------------------- ----------------------- ----------------------------- ------------------ ----------------------
  tRNA gene                                                                                                

   *trnR-CCG*(*rbcL-accD*)        *trnR-UCG*              *trnSeC*                      ●                  ●

   *trnL-CAA*(*ndhB*Exon2 3\')    ○                       ○                             ●                  ●

   *trnL-UAA*(*rps4-ndhJ*)        *trnL-CAA*              *trnL-CAA*                    ●                  ●

   *trnK-UUU*(*MatK*)             ○                       ○                             ●                  ●

   *trnS-CGA*(*psbK-psbI*)        ○                       ○                             ●                  ●

   *trnT-UGU*(*rps4-ndhJ*)\       ○                       ○                             ●                  ●
  (without intron)                                                                                         

   *trnT-UGU*(*ndhB*Exon2 3\')\   ▲                       ●                             ○                  ○
  (with one intron)                                                                                        

   *trnV-GAC*(*rrn16-rps12*)      ○                       ○                             ●                  ●

   *trnG-UCC*(*ycf12-atpA*)       ●                       ●                             ●                  ○

  Protein gene                                                                                             

   *psaM*                         ●                       ○                             ●                  ●

   *ycf66*                        ▲                       ○                             ○                  ●

   *chlB*                         ●                       ●                             ○                  ●

   *chlL*                         ●                       ●                             ○                  ●

   *chlN*                         ●                       ●                             ○                  ●

   *rps16*                        ●                       ●                             ○                  ●

   *ycf1*                         ●                       ●                             ●                  ▲
  -------------------------------------------------------------------------------------------------------------------------------

^a^Only the genes that are missing in one or more cp genomes are shown. The loci of tRNA genes are denoted by their neighbor protein genes. A filled/open circle denotes the presence/absence of a gene. A filled triangle indicates a pseudogene.

*Alsophila*and *Adiantum*share another rearranged region between *rpoB*and *psbZ*in LSC relative to *Angiopteris*and *Psilotum*(Figure [3](#F3){ref-type="fig"}). For the latter two, gene order in this region is \"***rpoB***-*trnC*-*petN*-*psbM*-*trnD*-*trnY*-*trnE-****trnG***-***psbZ***\", whereas in *Alsophila*and *Adiantum*it is \"***rpoB***-*trnD*-*trnY*-*trnE*-*psbM*-*petN*-*trnC*-***trnG***-***psbZ***\" (Genes with boldface are unchanged) (Figure [3](#F3){ref-type="fig"}). Roper *et al*. \[[@B8]\] noted that this gene order change is not caused by a single inversion. Two alternative pathways may account for this rearrangement (Figure [6](#F6){ref-type="fig"}), but more data are needed to determine the order of the two inversions.

![**Two hypothetical pathways to explain rearrangement between *rpoB*and *psbZ***. Genes are represented by boxes, the colors of which are consistent with Figure 3. \*In *Angiopteris evecta ycf66*has an intact ORF, but in *Alsophila spinulosa*it is a pseudogene.](1471-2148-9-130-6){#F6}

Gene content
------------

A total of 117 different genes are present in the *Alsophila*cp genome (Table [1](#T1){ref-type="table"}). This gene content is similar to that of most land plants \[[@B21]\]. However, there are some interesting differences amongst the four sequenced fern cp genomes (Table [3](#T3){ref-type="table"}). The *Alsophila*cp genome possesses the least number of tRNA genes due to 5 missing tRNA genes in comparison to basal ferns (*Psilotum nudum*and *Angiopteris evecta*). Its protein gene number is equal to that of *Angiopteris*, but higher than that of both *Adiantum*and *Psilotum*. Details of these differences are discussed below.

### Novel tRNA gene

A unique *trnR-UCG*gene, encoding tRNA-Arg, is found between *rbcL*and *accD*in the *Alsophila*cp genome (Figure [1](#F1){ref-type="fig"}; Table [3](#T3){ref-type="table"}). Another type of tRNA-Arg gene *trnR-CCG*resides in the same locus in non-flowering land plants including *Angiopteris*\[[@B8]\] and *Psilotum*\[[@B19]\]. In *Adiantum*, an apparent tRNA gene is annotated as *trnSeC*\[[@B10]\]. It is uncertain whether the occurrence of *trnR-UCG*in the *Alsophila*cp genome represents a unique feature for this species or is an apomorphy for a larger clade such as Cyatheaceae or tree ferns. To address this question, we collected all fern *rbcL*-*accD*intergenic sequences deposited in GenBank and examined the tRNA genes within them using ARAGORN \[[@B22]\]. The results indicate that *trnR-UCG*is restricted to tree ferns, whereas *trnR-CCG*is widespread in non-core leptosporangiates and basal ferns (Table [4](#T4){ref-type="table"}). However, neither *trnR-UCG*nor the *trnR-CCG*gene is identified at this locus in polypod ferns. Therefore, the existence of *trnR-UCG*may reflect a putative molecular apomorphy of tree ferns.

###### 

tRNA genes in fern *rbcL*-*accD*intergenic spacer sequences^a^

  Order^b^                  Family                  Number of sequences   tRNA gene^c^
  ------------------------- ----------------------- --------------------- --------------
  Cyatheales (tree ferns)   Cyatheaceae             140                   *trnR-UCG*
                            Dicksoniaceae           6                     *trnR-UCG*
                            Lophosoriaceae          1                     *trnR-UCG*
                            Hymenophyllopsidaceae   1                     *trnR-UCG*
  Gleicheniales             Dipteridaceae           1                     *trnR-CCG*
                            Gleicheniaceae          2                     *trnR-CCG*
  Hymenophyllales           Hymenophyllaceae        84                    *trnR-CCG*
  Osmundales                Osmundaceae             18                    *trnR-CCG*
  Marattiales               Marattiaceae            1                     *trnR-CCG*
  Psilotales                Psilotaceae             1                     *trnR-CCG*
  Ophioglossales            Ophioglossaceae         1                     *trnR-CCG*

^a^All sequence data were obtained from GenBank at March 30, 2009;

^b^Group names at ordinal level follow Smith et al. \[[@B7]\];

^c^tRNA genes were identified by using ARAGORN v1.2 <http://130.235.46.10/ARAGORN/>\[[@B22]\]

Sequence alignment indicates that trnR-UCG and trnR-CCGs have quite similar primary sequences with 44 of 74 nucleotides unchanged across 7 representative land plants (Figure [7A](#F7){ref-type="fig"}). In addition, the *Adiantum*trnSeC shares 51, 41 and 40 identical nucleotides with the *Alsophila*trnR-UCG, the *Psilotum*trnR-CCG and the *Angiopteris*trnR-CCG respectively (Figure [7A](#F7){ref-type="fig"}). Due to their similarities and conserved loci, we propose that *Alsophila trnR-UCG*, *Adiantum trnSeC*as well as *trnR-CCG*s in other land plants are orthologous. Tree fern trnR-UCG can transfer arginine even though its anticodon alters from CCG to UCG. However, *Adiantum*trnSeC has undergone major changes: 1) its anticodon is UCA (unmatchable for an Arg codon), and 2) it contains up to 18 nucleotide differences relative to all other land plant trnR genes (Figure [7A](#F7){ref-type="fig"}). Our findings imply that the *trnR-UCG*is derived from the *trnR-CCG*by the alteration of one anticodon base; then the *Adiantum trnSeC*evolves from the *trnR-UCG*by altering one anticodon base further, becoming a *trnR-UCG*pseudogene (Figure [7B](#F7){ref-type="fig"}). If this is the case, the *Adiantum trnSeC*should be annotated as *ΨtrnR*. Sugiura and Sugita \[[@B23]\] argued that the *trnR-CCG*is not essential for plastid function although it is conserved in non-flowering plants. The evolutionary scenario of *trnR-CCG*in ferns (Figure [7B](#F7){ref-type="fig"}) tends to support this view.

![**Comparisons of the sequences and putative secondary structures of trnR-CCG, trnR-UCG and trnSeC (tRNA-selenocysteine)**. A, primary sequences of *Alsophila spinulosa*trnR-UCG, *Adiantum capillus-veneris*trnSeC and trnR-CCGs from other six land plants. Dashed rectangle indicates anticodons. B, the putative secondary structures of *Angiopteris evecta*trnR-CCG,*Alsophila*trnR-UCG and *Adiantum*trnSeC. Dashed ellipses indicate anticodons.](1471-2148-9-130-7){#F7}

At the locus between *rps4*and *ndhJ*, the *Alsophila*and *Adiantum*cp genomes encode a *trnL-CAA*(tRNA-Leu) rather than a *trnL-UAA*gene (Table [3](#T3){ref-type="table"}). However, they lose another *trnL-CAA*gene (Table [3](#T3){ref-type="table"}), which is found at the 3\' downstream of *ndhB*in almost all other land plant plastid genomes. Consequently, *Alsophila*and *Adiantum*only possess the *trnL-CAA*, whereas the *Angiopteris*and *Psilotum*cp genomes contain both the *trnL-UAA*and the *trnL-CAA*. In the *Adiantum*chloroplast, the missing trnL-UAA could be provided for the heavily used UUA codon by a partial C-to-U edit in the trnL-CAA anticodon \[[@B24]\]. Since the UUA is also a preferred leucine codon for the *Alsophila*cp genome (RSCU = 1.70), the same editing event might occur in the *Alsophila*chloroplast as well.

### Missing tRNA gene

Only 28 tRNA genes are encoded in the *Alsophila*cp genome, whereas 29, 32 and 33 are annotated in *Adiantum*, *Angiopteris*and *Psilotum*, respectively (Table [1](#T1){ref-type="table"}). For cp genomes, it is believed that a set of 30 tRNA species is sufficient for the translation of chloroplast mRNAs \[[@B25]\]. In the *Angiopteris*and *Psilotum*chloroplasts, tRNAs can read all codons by using two-out-of-three and wobble mechanisms \[[@B26]\]. However, in *Alsophila*and *Adiantum*chloroplasts, both lysine codons lack a corresponding tRNA-Lys (encoded by *trnK*) (Table [2](#T2){ref-type="table"}; Table [3](#T3){ref-type="table"}). The loss of *trnK*suggests cytosolic tRNAs may be imported into chloroplasts, despite a lack of experimental evidence \[[@B27]\]. As an incidental consequence of the *trnK*loss, the *matk*open reading frame (ORF) is not nested in the *trnK*intron (Figure [1](#F1){ref-type="fig"}).

Apart from the *trnK*and the *trnL-CAA*, the *Alsophila*cp genome also shares other 3 tRNA gene losses, including the *trnS-CGA*, the *trnV-GAC*and the *trnT-UGU*(intron-free), with *Adiantum*relative to basal ferns *Angiopteris*and *Psilotum*(Table [3](#T3){ref-type="table"}). The shared absence of tRNA genes between *Alsophila*and *Adiantum*suggests that they may derive from a common ancestor.

### Protein genes

The *Alsophila*cp genome contains a *psaM*gene encoding photosystem I reaction center subunit M. This gene has been detected in *Psilotum*\[[@B19]\] and *Angiopteris*\[[@B8]\], but not in *Adiantum*\[[@B10]\] (Table [3](#T3){ref-type="table"}). Besides ferns, *psaM*also exists in bryophytes, lycophytes and gymnosperms, but not in angiosperms, implying its independent loss in ferns and angiosperms \[[@B10]\]. *Alsophila*and *Adiantum*represent tree ferns and polypods, respectively. Due to their sister relationship, we speculate that the loss of *psaM*in ferns occurred after the split of polypods and tree ferns.

A putative pseudogene of *ycf66*is identified in the *Alsophila*cp genome (Figure [1](#F1){ref-type="fig"}; Table [3](#T3){ref-type="table"}). The 5\' ends of its two exons are both destroyed. In the four sequenced fern cp genomes, only *Angiopteris*contains an intact *ycf66*gene \[[@B8]\]. For other land plants, this gene only occurs in *Marchantia polymorpha*(liverworts), *Physcomitrella patens*subsp.*patens*(mosses),*Syntrichia ruralis*(mosses) and *Huperzia lucidula*(lycophytes). The findings suggest that *ycf66*is lost independently in multiple clades of land plants including hornworts, ferns and seed plants.

Inversion Endpoint as Hotspot for Repeats
-----------------------------------------

A total of 133 pairs of repeats (≥ 30 bp) were identified in the *Alsophila*cp genome by using REPuter \[[@B28]\], of which 106 are direct and 27 are inverted repeats. This number of repeats is less than are found in some highly rearranged cp genomes (e.g. *Trachelium caeruleum*) but more than are present in unrearranged ones (e.g. *Nicotiana tabacum*) \[[@B29],[@B30]\]. Up to 66 direct repeats (no inverted repeat) are restricted to a region spanning only 565 bp (153,682--154,246 bp in IRA or 88,724--89,288 bp in IRB) between *trnR-ACG*and *ψtrnT-UGU*in the IRs (Figure [1](#F1){ref-type="fig"}). The GC content of this 565-bp-region (35.93%) is lower than that of IRs and the overall GC content of the whole genome. Detailed sequence analyses revealed that this region is composed of tandem iterations of 11 similar segments ranging from 40 to 58 bp (Figure [8](#F8){ref-type="fig"}). The core repeated motif is AAAATCCTAGTAGTTAgaGCTTTATCcaGGGtaTaGgACT (the lowercase letters denote variable bases) with variant lengths of heads and/or tails (Figure [8](#F8){ref-type="fig"}).

![**Repetitive units within the highly repeated 565-bp sequence**. The sequence from 88,724 to 89,288 bp in IRB is shown in this figure. The numbers on the right hand side indicate the lengths of each segment.](1471-2148-9-130-8){#F8}

In contrast to *Alsophila*, dispersed repeats (≥ 30 bp) are rare in the *Adiantum*cp genome, with only 5 short inverted repeats (30--36 bp); and none of these occurs between the *trnR-ACG*and the *trnT-UGU*. In the *Alsophila*cp genome, the length of the intergenic region between *trnR-ACG*and *ψtrnT-UGU*is 1467 bp, whereas in *Adiantum*it is 913 bp, the difference being 554 bp. We noted that this length is very similar to that of the highly repeated 565-bp-region, and speculate that the difference is caused by the presence of the highly repeated region. To test this hypothesis, we extracted the sequence from *trnR-ACG*to *ψtrnT-UGU*in *Alsophila*and from *trnR-ACG*to *trnT-UGU*in *Adiantum*. The sequence alignment indicates that the highly repeated 565-bp-region is indeed lost in the *Adiantum*cp genome (Figure [9](#F9){ref-type="fig"}).

![**Alignment of the sequence from *trnR-ACG*to *ψtrnT-UGU*in *Alsophila*and corresponding region in *Adiantum***. Comparison by using online zPicture software with ECR criteria of ≥ 100 bp and ≥ 70% identity <http://zpicture.dcode.org/>\[[@B44]\]. Similarities between aligned regions are shown as average percent identity. The cluster of arrows denotes the location of the highly repeated 565-bp-region in the *Alsophila*cp genome. \*, an intact *trnT-UGU*was identified in *Adiantum*, while a *trnT-UGU*pseudogene was found in *Alsophila*.](1471-2148-9-130-9){#F9}

In the *Alsophila*cp genome, the location of the highly repeated 565-bp-region is exactly at the endpoint of the second inversion of the IR rearrangements (Figure [5](#F5){ref-type="fig"}, Inversion II). Dispersed repeated sequences have been reported from several cp genomes. These are associated with numerous DNA rearrangements, particularly inversions \[[@B31]-[@B33]\]. In extensively rearranged cp genomes, the endpoints of rearranged gene clusters are usually flanked by repeated sequences \[[@B29],[@B30],[@B34]\]. If repeat-mediated recombination is the major mechanism generating inversions in cp genomes \[[@B35],[@B36]\], the preservation of repeats would destabilize the genome structure. After inversions, the repeats should be deleted to guarantee genome stability (like the situation in *Adiantum*). The repeats observed at the endpoint of the ancient inversion (Figure [5](#F5){ref-type="fig"}, Inversion II) may be a vestige of recent rearrangement(s) that are undiscovered. The existence of these repeats implies that the region is a potential hotspot for genomic reconfiguration.

Conclusion
==========

In this study, we present the first complete cp genome sequence from a tree fern and provide a comprehensive comparative analysis of cp genomes in ferns. The cp genome size of *Alsophila*is larger than that of *Adiantum, Psilotum*and *Angiopteris*. Besides 117 genes, two pseudogenes *Ψycf66*and *ΨtrnT-UGU*are also detected in the *Alsophila*cp genome. An intact *ycf66*is identified in *Angiopteris*, while an intron-containing *trnT-UGU*is found in *Adiantum*. Based on the findings, we speculate that *Ψycf66*reflects an intermediate during *ycf66*gene loss, and the genesis of *trnT-UGU*may be associated with the IR rearrangements. A *trnR-UCG*gene was detected between *rbcL*and *accD*in *Alsophila*, and this seems to be a molecular apomorphy of tree ferns. In the *Adiantum*cp genome, the *trnR-UCG*gene degenerates to a pseudogene. The *Alsophila*cp genome shares several unusual characteristics with the previously sequenced *Adiantum*(a polypod fern) cp genome, such as five missing tRNA genes and two major rearranged regions. These common characters probably derive from their common ancestor. In the *Alsophila*cp genome, a highly repeated 565-bp-region, which is composed of tandem iterations of 11 similar segments, occurs at one endpoint of an ancient inversion, but it is not detected in the genome of *Adiantum*. Nonetheless, the origin and function of these repeats remain to be characterized in future studies.

Methods
=======

Genome sequencing and assembly
------------------------------

Young leaves of *Alsophila spinulosa*were collected from a plant growing in the greenhouse in Wuhan Botanical Garden, Chinese Academy of Sciences. A voucher specimen was deposited at Wuhan Botanical Garden. Total DNA was extracted using the CTAB-based method \[[@B37]\]. The cp genome was amplified using polymerase chain reaction (PCR). In brief, the coding sequences were extracted from known chloroplast genomic sequences of three ferns \[GenBank: [NC_003386](NC_003386), [NC_008829](NC_008829) and [NC_004766](NC_004766)\], three bryophytes \[GenBank: [NC_001319](NC_001319), [NC_005087](NC_005087) and [NC_004543](NC_004543)\] and one lycophyte \[GenBank: [NC_006861](NC_006861)\] according to their annotations in GenBank. PCR primers were developed from alignments of the above coding sequences. Overlapping regions of each pair of adjacent PCR fragments exceeded 150 bp. We did not clone two inverted repeats (IRs) separately, but designed primers to amplify the regions spanning the junctions of LSC/IRA, LSC/IRB, SSC/IRA and SSC/IRB. Using these primers, we covered the entire cp genome of *Alsophila*with PCR products ranging in size from 500 bp to 5 kb. All PCR reactions were performed using TaKaRa LA taq (TaKaRa Bio Inc, Shiga, Japan). Amplified cp genome fragments were cloned into TaKaRa pMD19-T plasmids (TaKaRa Bio Inc, Shiga, Japan), which were then used to transform *E. coli*DH5α. Multiple (≥ 6) clones were randomly selected and commercially sequenced using ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). For long fragments (\> 1.4 kb), walking primers were designed based on acquired sequences and used for sequencing remaining sequences step by step. Gap regions (caused by unsuccessful PCR amplification or failed primer walking sequencing) were amplified using primers that flank the gaps, then cloned and sequenced as above. From the individual reads we excluded vector, primer and low-quality sequences, then we assembled the reads using Phrap \[[@B38]\]. Since automated assembly methods cannot distinguish two IRs, we input the reads as two parts and acquired two large contigs, with each contig including one IR and its adjacent partial large and small single copy (LSC and SSC) regions. Then the two large contigs were manually assembled into the complete circular genome sequence. Inverted repeats were identified through alignment of the final complete genome sequence against itself via Blast 2 sequences at the National Center for Biotechnology Information \[[@B39]\]. We accumulated 1,415,559 bp sequences, which is about 9-fold coverage.

Annotation and related study
----------------------------

Annotation of the *Alsophila*cp genome was performed using DOGMA (Dual Organellar GenoMe Annotator) \[[@B40]\]. Genes that were undetected by DOGMA, such as *ycf1*, *ycf2*, *rps16*, *ndhF*, *ndhG*and *matK*, were identified by Blastx <http://blast.ncbi.nlm.nih.gov/Blast.cgi>. From this initial annotation, putative starts, stops, and intron positions were determined by comparisons with homologous genes in other cp genomes and by considering the possibility of RNA editing, which can modify the start and stop positions. tRNA genes were annotated using DOGMA and ARAGORN v1.2 <http://130.235.46.10/ARAGORN/>\[[@B22]\], and then confirmed by ERPIN <http://tagc.univ-mrs.fr/erpin/>\[[@B41]\] and TFAM Webserver v1.3 \[[@B42]\]. The circular gene map of the *Alsophila*cp genome was drawn by GenomeVx \[[@B43]\] followed by manual modification.

Synteny among fern cp genomes was analyzed and visualized by using online zPicture software <http://zpicture.dcode.org/>\[[@B44]\].

Examination of GC content
-------------------------

Overall GC content was calculated for 118 land plant plastid genomes (see Additional file [1](#S1){ref-type="supplementary-material"}). For the *Alsophila*cp genome, GC content was farther determined for three groups of genes, protein-coding genes (85), rRNA genes (4) and tRNA genes (28), respectively. For protein-coding genes, GC content was calculated for the entire gene and the first, second and third codon positions, respectively. Protein-coding genes were partitioned into three main functional groups: photosynthetic genes, genetic system genes and NADH genes. GC content of the three groups of genes was then determined. The genes included in each of these three groups were: (1) photosynthetic genes (*rbcL*, *atp\**, *pet\**, *psa\**and *psb\**); (2) genetic system genes (*rpl\**, *rps\**, *rpo\**, *clpP*, *infA*and *matK*); and (3) NADH genes (*ndh\**).

Dispersed repeats
-----------------

Direct and inverted repeats in the *Alsophila*and *Adiantum*\[GenBank: [NC_004766](NC_004766)\] cp genomes were determined by using REPuter \[[@B28]\] at a repeat length ≥ 30 bp with a Hamming distance of 3. The entire genome was used to detect repeats in order to map them in both copies of the IR, but numbers of repeats were based on results from only one IR copy.

List of abbreviations
=====================

cp: chloroplast; IR: inverted repeat; LSC: large single copy; SSC: small single copy; RSCU: relative synonymous codon usage.

Authors\' contributions
=======================

LG participated in the conception of this study, carried out part of the genome sequencing, performed all sequence analyses, annotated the genome, generated tables and figures, and drafted the manuscript. XY and YXY participated in genome sequencing. YJS and TW conceived and supervised the project, contributed to the interpretation of the data and prepared the manuscript. All authors read and approved the final manuscript.

Supplementary Material
======================

###### Additional file 1

**Additional Table 1**. a list of published complete chloroplast sequences in land plants.

###### 

Click here for file

Acknowledgements
================

We give special thanks to Paul G. Wolf for his constructive comments and suggestions. We are grateful to Bobby A Brown and George Littlejohn for significantly improving English, to Ling-Ling Gao, Cynthia Gleason and Su-Min Guo for helpful comments on the manuscript, and to Chen Lu and Gong Xiao for providing valuable assistance on the use of computer hardware and software. This work was supported by the \"100 Talent Project\"of the Chinese Academy of Sciences (Grant No.: 0729281F02), the \"Outstanding Young Scientist Project\" of the Natural Science Foundation of Hubei Province, China (Grant No.: O631061H01) and the Open Project of the State Key Laboratory of Biocontrol, China (Grant No.: 2007-01). We also gratefully acknowledge the valuable comments by three anonymous reviewers.
