BACKGROUND
the circa  <dig> chloroplast genomes  of green algae sequenced to date have revealed a remarkable array of genomic architectures, including a wide range of genome sizes, gene repertoires and arrangements, and nucleotide compositions  <cit> .
many green algal cpdnas have a quadripartite structure also found in land plants, characterized by the presence of two copies of a large inverted repeat sequence separating a small and a large single-copy region.
although this architecture is believed to be ancestral in the green algae, many species  do not have a quadripartite structure  <cit> .
complete cpdna sequences are only available for three species of ulvophyceae , including the marine flagellate oltmannsiellopsis viridis , the freshwater microfilamentous pseudendoclonium akinetum , and the marine siphonous species bryopsis hypnoides   <cit> .
the cpdnas of oltmannsiellopsis  and pseudendoclonium  share the quadripartite architecture, and a similar large complement of genes , but differ in size due to different intron numbers .
the  <dig>  kb cpdna of b. hypnoides differs in several aspects from the two other species of ulvophyceae.
it does not feature a quadripartite architecture, and it includes  <dig> trna genes that are not present in any other green algal cpdna.
another peculiarity of the b. hypnoides cpdna is the presence of multimeric forms of the cpdna, including monomers, dimers, trimers, tetramers, and higher-order multimers, which were detected by pulsed-field gel electrophoresis and southern blot methods  <cit> .
<dig> genes were reported in the cpdna of b. hypnoides but our preliminary analysis indicated a higher number of genes, as also suggested by others  <cit> .
apart from completely sequenced cpdnas, partial chloroplast genome data in the bryopsidales has long been available for codium fragile  <cit>  and caulerpa sertularoides  <cit>  through southern hybridization analysis of restriction fragments.
in addition partial dna sequence data is available for caulerpa filiformis  <cit> .figure  <dig>
phylogenetic relationships among the main clades of green algae, focusing on the core chlorophyta.
the tree is a composite of accepted relationships based on molecular phylogenetic evidence from different studies  <cit> .
uncertain or conflicting relationships are indicated by polytomies or question marks.
numbers in square brackets indicate the number of  complete cpdnas available to date.
clades currently classified as ulvophyceae are in green, and trebouxiophyceae are in yellow.
the relationships within the core chlorophyta have recently been evaluated based on multigene datasets  <cit> , which is relevant in the light of comparative genomics.
although monophyly of the ulvophyceae was supported in a 10-gene phylogeny   <cit> , chloroplast  multigene phylogenetic analyses generally failed to recover the ulvophyceae as a clade.
for example, phylogenetic analysis of  <dig> cp genes recovered caulerpa  as more closely related to chlorella  than to the other two ulvophycean taxa in the phylogeny   <cit> , and a phylogeny inferred from  <dig> cp genes indicated a relationship between bryopsis and chlorophyceae  <cit> .
phylogenomic analyses with increased taxon sampling  suggested a relationship between oltmannsiellopsis and tetraselmis , and like in the two previous studies did not provide support for a monophyletic ulvophyceae  <cit> .
in addition, cp multigene phylogenies have rejected monophyly of the trebouxiophyceae, which was shown to fall apart in at least two main clades  <cit> .
in general, relationships among the major lineages of core chlorophyta remain poorly supported.
as illustrated in figure  <dig>  complete data on chloroplast genomes is scarce in the ulvophyceae compared to other green algal lineages, with data lacking for several important clades, including the cladophorales, dasycladales, and trentepohliales.
this paper focusses on the siphonous marine green algae bryopsis and tydemania, two members of the order bryopsidales  <cit> .
species in this order form thalli that are composed of a single, giant cell, containing millions of nuclei, chloroplasts and mitochondria that move about freely by cytoplasmic streaming.
bryopsis includes more than  <dig> species, all characterized by morphologically simple feather-like thalli, mostly only a few cm tall.
the genus is distributed worldwide in tropical to temperate seas.
tydemania forms more complex and larger thalli composed of whorls of siphons arising from a branched main axis.
the genus includes a single species, t. expeditiones, that is found in the marine tropical indo-west pacific.
the order bryopsidales includes two main clades, bryopsidineae and halimedineae.
bryopsis is a member of the bryopsidineae, which also includes derbesia and codium, and some other, less known genera.
tydemania is a member of the halimedineae, which also includes some of the better known siphonous green seaweeds such as caulerpa and halimeda.
the placement of the bryopsidales in the class ulvophyceae has been implied by 18s data and nuclear multi-gene phylogenetic analysis  <cit> , but as mentioned above, analyses based on cp genes have casted doubt on the affinity of siphonous green algae with other clades of ulvophyceae  <cit> .
in this paper we report on the complete cpdnas of bryopsis plumosa and tydemania expeditiones, and a re-annotated cpdna of b. hypnoides.
we compared these genomes with previously published cpdnas in the core chlorophyta to gain a better understanding of the evolution of chloroplast genomes in this group, and to assess relationships among the main clades of core chlorophyta.
RESULTS
cpdna assembly, size and organization
for both bryopsis plumosa and tydemania expeditiones, assemblies yielded a single cpdna sequence, which could be closed into a circle by an overlap of more than  <dig> base pairs.
both assemblies showed high overall read coverage, and as a result did not contain any ambiguous regions .
for b. plumosa,  <dig>  reads of a total of  <dig>  million mapped to the cpdna with a mean coverage of 430× .
the 4× coverage was 3 bp long and situated in the 3’-end of the cyst gene, and was spanned by paired-end reads.
for t. expeditiones,  <dig>  million reads of a total of  <dig>  million mapped to the cpdna with a mean coverage of  <dig> × .
the circular cpdnas of b. plumosa  and t. expeditiones  consist of  <dig>  bp and  <dig>  bp, respectively.
this is smaller than most published cpdnas of free-living species of core chlorophyta, but similar to those found in pedinomonas minor , marvania geminata , pseudochloris wilhelmii , and planctonema lauterbornii   <cit> .
most prasinophytes have even smaller cpdnas, ranging between 64-86 kb, with the exception of nephroselmis, which has larger chloroplast genomes   <cit> .
unexpectedly, the cpdna of b. plumosa is much smaller than the  <dig>  bp cpdna of b. hypnoides.
this size difference is mainly a result of large intergenic spacers , totaling  <dig>  bp or  <dig> % of the cpdna in b. hypnoides .
intergenic spacers in b. plumosa and t. expeditiones account for only  <dig> % and  <dig> % of the total cpdna.
nevertheless, some long intergenic spacer regions are present in both genomes.
in b. plumosa, the largest intergenic spacer was  <dig>  bp long.
this region contains a few orfs , none of which showed significant homology to known proteins .
in t. expeditiones, a large spacer region  contains ten orfs , none with significant homology to known proteins .
gc content of the two genomes falls within the limits of other ulvophycean and green algal cpdnas   <cit> .figure  <dig>
gene map of the chloroplast genome of
bryopsis plumosa
.
the  <dig>  bp genome contains  <dig> unique genes, including three ribosomal rna genes,  <dig> transfer rna genes, and  <dig> protein coding genes.
genes shown on the outside of the circle are transcribed counterclockwise.
annotated genes are colored according to the functional categories shown in the legend bottom left.
the red arcs indicate gene regions of putative bacterial origin.
gene map of the chloroplast genome of
tydemania expeditiones
.
the  <dig>  bp genome contains  <dig> unique genes, including three ribosomal rna genes,  <dig> transfer rna genes, and  <dig> protein coding genes.
genes shown on the outside of the circle are transcribed counterclockwise.
annotated genes are colored according to the functional categories shown in the legend bottom left.
the red arcs indicate gene regions of putative bacterial origin.
summary of the
bryopsis plumosa
and
tydemania expeditiones
cpdnas and comparison with other ulvophycean cpdnas
species
cpdna size
gc content
gene number
protein coding genes/orfs
d
rrna genes
trna genes
% coding
b
intergenic space
intron
c
intron
b
intron number
quadripartite structure
reference
b. plumosa
t. expeditiones
b. hypnoides
a
p. akinetum
o. viridis
are-annotated in this study .
bincluding intronic open reading frames .
cexcluding intronic orfs.
donly those orfs are included, which were found to have a significant blastp result .
the b. plumosa and t. expeditiones cpdnas both lack a large inverted repeat, similar to the situation found in b. hypnoides  <cit> .
the lack of a quadripartite architecture in bryopsidales had earlier been implied based on southern hybridization analysis of restriction fragments in codium fragile and caulerpa sertularoides  <cit> .
most plastid genomes have a quadripartite structure where a set of large inverted repeats  divides the genome into two single-copy regions.
although the quadripartite structure is believed to be ancestral in viridiplantae, loss of the inverted repeat has occurred multiple times in different green algal lineages  <cit> .
gene content
the b. plumosa cpdna contains  <dig> unique genes, including  <dig> protein coding genes,  <dig> trna genes, and three rrna genes .
in addition to expected, conserved plastid protein coding genes, several freestanding orfs were identified that did not show any relationship with known plastid genes, but instead showed close similarity with bacterial genes .
the large orf <dig>  showed close similarity to bacterial rhs-family proteins.
a similar but slightly smaller orf  was also found in the re-annotated cpdna of b. hypnoides .
rhs genes are a family of composite genes that occur in several gram-negative bacteria, including some cyanobacteria.
their broad phylogenetic distribution has been explained by a complex evolutionary history of horizontal transfers and independent gene losses  <cit> .
despite their wide occurrence, the function of rhs genes remains elusive  <cit> .
other orfs of putative bacterial origin include a gene encoding dna polymerase with maturase-specific domain , and two orfs  showing close similarity to uncharacterized bacterial proteins.
orf <dig>  orf <dig> and orf <dig> are grouped in a 13 kb region of the cpdna, while orf <dig> is situated elsewhere .table  <dig>
comparison of trna genes in the two
bryopsis
species,
tydemania expeditiones
and two other species of ulvophyceae
b. hyp
b. plu
t. exp
p. aki
o. vir
trnma
total
53
27
28
27
27
unique
40
26
28
27
27
athe trnm gene situated in the large trna region in b. hypnoides showed similarity  with land plant nuclear encoded trna-met initiator  genes.
numbers indicate gene copy.
an asterisks indicates that the gene or gene copy is situated in the large trna region in b. hypnoides .
b. hyp = bryopsis hypnoides, b. plu = b. plumosa, t. exp = tydemania expeditiones, p. aki = pseudendoclonium akinetum, o. vir = oltmannsiellopsis viridis.
comparison of protein coding genes content among core chlorophyta.
pd = pedinophyceae.
<dig> genes that are shared among the  <dig> cpdnas are not included: atpa, b, e, f, h, cema, clpp, ftsh, petb, d, g, l, psaa, b, c, j, psba, b, c, d, e, f, h, i, j, k, l, m, n, t, z, rbcl, rpl <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  rps <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  tufa, ycf <dig>  ycf <dig>  ycf <dig>  rpoa, b, c <dig>  c <dig>  rrf, rrl, rrs, and  <dig> trna genes .
ftsh and ycf <dig> are present in c. reinhardtii as orf <dig> and orf <dig>  respectively, and ycf <dig> is present in c. vulgaris as orf <dig> ).
ycf <dig> is present in c. vulgaris as orf <dig> .
data sources: b. hypnoides  <cit> , p. akinetum  <cit> , o. viridis  <cit> , c. vulgaris  <cit> , p. kessleri, o. solitaria, p. minor  <cit> , c. subellipsoidea  <cit> , l. terrestris  <cit> , a. obliquus  <cit> , d. salina  <cit> , v. carteri  <cit> , g. pectorale  <cit> , c. reinhardtii  <cit> , o. cardiacum  <cit> , f. terrestris  <cit> , s. leibleinii  <cit> , s. helveticum  <cit> .
freestanding orfs in
bryopsis plumosa
and
tydemania expeditiones
cpdnas
species orf number
length
closest blast hit
blastp e-value
note
b. plumosa orf3
b. plumosa orf4
b. plumosa orf5
b. plumosa orf7
t. expeditiones orf3
t. expeditiones orf8
t. expeditiones orf9
t. expeditiones orf13
t. expeditiones orf <dig>  orf15
t. expeditiones orf16
ato verify whether homologous genes were present in green algae, blastp searcher were performed with organisms constrained to viridiplantae .
most genes found in b. plumosa, with the exception of ycf <dig> and orf <dig>  were also found in b. hypnoides  <cit>  .
however, the cpdna of b. hypnoides contains a region of about 6 kb including  <dig> trna genes, ten of which have not been found in any other green algal cpdna  <cit> .
this large trna region is not present in b. plumosa, nor is the set of unique trna genes .
as in b. plumosa, several orfs in b. hypnoides showed high similarity to bacterial genes, including a putative rhs gene, and genes containing domains commonly found in mobile elements, such as reverse transcriptase, endonuclease and maturase .
reverse transcriptase proteins catalyze the reverse transcription of rna to complementary dna, and have a key role in the mobility of retroviruses, retrotransposons, and organellar group ii introns  <cit> .
endonuclease-like proteins catalyze cleavage of retrotransposons before transcription  <cit> , and are also involved in mobility and homing of introns  <cit> .
maturase-like proteins are best known from bacterial and organellar introns, where they aid in intron splicing  <cit> .
the t. expeditiones cpdna contains  <dig> unique genes, including  <dig> protein coding genes, three rrna genes, and  <dig> trna genes .
in addition to the conserved set of chloroplast genes, several freestanding orfs were found coding for bacterial or intron related genes .
orf <dig> contains a maturase-specific domain, which are usually found within intronic orfs.
two overlapping orfs  are related to bacterial c- <dig> cytosine-specific dna methyltransferase genes, a family of genes coding for enzymes that catalyzes the methyl-transfer reaction in the process of dna methylation, and in bacteria are often associated with restriction/modification systems, which function as a defense against infection of bacteria by bacteriophages  <cit> .
dna methyltransferases have up till now not been found in plastid genomes.
however, the identification of dna methyltransferase genes in organelles is not unprecedented as a gene of this nature has been identified in the mitochondrial genome of the streptophyte klebsormidium, which was seen as a possible remnant of viral infection  <cit> .
a region of about  <dig> kp contains four freestanding orfs showing close similarity to bacterial genes involved in mobile functions.
orf <dig> showed high similarity to bacterial transposase and contains an integrase core domain.
transposases and integrases catalyze the movement and integration of dna copies to new locations within and between genomes  <cit> .
orf <dig> and orf <dig> showed similarity to bacterial dna polymerase, containing a dna polymerase family a domain.
family a polymerases fill dna gaps that arise during dna repair, recombination and replication, and are found primarily in prokaryotes  <cit> .
orf <dig> showed high similarity to phage/plasmid dna primases, which are known from phages and plasmids of bacteria and archaea  <cit> .
the presence of these four orfs indicates that the  <dig> kp region may be a mobile element of bacterial origin.
the presence of bacterial genes  in both the bryopsis and tydemania cpdnas, and absent in other green algae, suggest that these genes have been acquired through horizontal gene transfer.
transfer of genes to plastid genomes is only rarely observed but some clear instances are known  <cit> .
in the oedogonium chloroplast genome, the unprecedented finding of int  and dpob  was seen as evidence of horizontal transfer, possibly from a mitochondrial genome donor  <cit> .
in the cpdna of nephroselmis olivacea, two large regions  are believed to have been acquired by lateral transfer from a bacterial donor based on a deviant base composition, the lack of genes typically found in cpdnas, and the presence of an orf showing similarity to phage associated dna primases  <cit> .
horizontal gene transfer from bacteria to plastids has also been demonstrated in other groups of algae, including red algae  <cit> , dinoflagellates  <cit> , haptophytes and cryptophytes  <cit> .
it is relevant to note that siphonous green algae  are known to harbor intracellular bacterial communities, with some bacteria showing close associations with the host .
based on fluorescence in situ hybridization, bacteria in bryopsis were found in the vacuole or in the cytoplasmic layer of the siphonous cells, sometimes closely adhered to the chloroplast membrane  <cit> .
these intracellular bacteria may facilitate gene transfer from the endobiontic bacteria to the host genome, possibly via vectors such as bacteriophages.
the two bryopsis cpdnas share nearly identical gene repertoires with t. expeditiones, except for rpl <dig>  which is present in b. plumosa and b. hypnoides but was not found in t. expeditiones.
in addition, several of the orfs discussed above were uniquely found in b. plumosa or t. expeditiones .
a number of genes in bryopsis and/or tydemania, including psbm, rpl <dig>  and rpl <dig>  were found to be quite divergent from core chlorophytan orthologs based on visual inspection of amino acid alignments and branch lengths of resulting phylogenetic trees .
the petl gene is present in two identical copies in b. plumosa .
also b. hypnoides contains two copies of petl, but here they differ in nucleotide composition as well as in length: a 96 bp copy identical to that of b. plumosa, and a 105 bp copy that is divergent from the bryopsidalean orthologs .
tydemania expeditiones contains a single 96 bp copy of petl.
a comparison of gene repertoires between b. plumosa, t. expeditiones and  <dig> published core chlorophytan cpdnas is shown in figure  <dig>  a total of  <dig> genes are shared among the  <dig> cpdnas.
a large proportion of genes are shared among species of ulvophyceae and trebouxiophyceae, while most chlorophyceae have a smaller gene repertoire .
notably, several genes are shared between the bryopsidales and trebouxiophyceae, but are absent from pseudendoclonium and/or oltmannsiellopsis.
these include three genes encoding subunits of protochlorophyllide reductase , trna ile-lysidine synthetase  and hypothetical protein ycf <dig>  only one gene, the organelle division inhibitor factor, mind, was present in pseudendoclonium, oltmannsiellopsis, and trebouxiophyceae but absent in bryopsidales.
trna-lysidine synthase , also known as ycf <dig>  is present in all ulvophyceae and trebouxiophyceae, but absent from chlorophyceae .
in t. expeditiones, this gene seems to be pseudogenized by a stop codon at amino acid position  <dig>  from the start methionine.
a similar situation was found in caulerpa filiformis , which contains a tils pseudogene with a frame shift at about the same position  <cit> .
in b. plumosa and b. hypnoides the gene is interrupted by a 71 bp, at-rich  insertion at amino acid position  <dig>
introns
in b. plumosa  <dig> introns are present in  <dig> genes, comprising  <dig> %  of the total cpdna .
four introns were identified as group i introns, and five as group ii introns, while for four introns the class could not be determined with certainty.
in t. expeditiones  <dig> introns are present in  <dig> genes, comprising  <dig> %  of the total cpdna.
seven introns were identified as group i introns, and two as group ii introns, while the class of two introns could not be determined with certainty.
several of the introns in b. plumosa and t. expeditiones  are commonly found in orthologous genes of other green algae.
others are only rarely found in green algae or have not yet been observed in plastids in general.
these are discussed below.table  <dig>
distribution and characteristics of introns in
bryopsis plumosa
and
tydemania expeditiones
gene
b. plu
t. exp
length
insertion positions
a
at content
intronic orf
class
atpf
ccsa
psaa
psba
psbb
psbc
psbt
rpl5
rpl23
rps19
ycf3
aintron insertion site positions correspond to the nucleotide immediately preceding the intron.
insertion sites in genes coding for proteins and the trna are given relative to the corresponding genes in mesostigma viride cpdna ; insertion sites in the rrna genes are given relative to the 16s and 23s rrna genes of escherichia coli .
bcontains two laglidadg homing endonuclease domains.
rt = reverse transcriptase, im = intron maturase, lhe = laglidadg homing endonuclease.
the atpa gene in b. plumosa contains two introns.
the first was identified as a group i intron, which is commonly found in this gene in other green algae  <cit> .
the second intron was identified as a putative group ii intron , which has until now only been found in the atpa gene of volvox carteri.
the psba gene in t. expeditiones has a large intron containing two overlapping orfs: one encoding reverse transcriptase and intron maturase, the other encoding n-terminal domain of reverse transcriptase.
the majority of psba introns in green algae are group i introns.
thus far, only one other green algal psba group ii intron has been identified, in floydiella terrestris  <cit> .
the psbc intron in t. expeditiones was also identified as a group ii intron based on the presence of an orf encoding reverse transcriptase and intron maturase.
introns are common in green algal psbc genes, but are all group i introns.
psbc group ii introns have thus far only been found in euglenoids  <cit> .
the rpl <dig> and rps <dig> introns in b. plumosa are putative group ii introns .
thus far, rpl <dig> introns have only been found in b. hypnoides.
plastid rps <dig> introns were until now only known in euglenoids   <cit>  and the red alga porphyridium purpureum   <cit> .
these introns ranged between 95-216 bp in size, which is much smaller than the intron found in b. plumosa.
at-rich introns  of which the class could not be determined with certainty were found in several genes, including atpf, ccsa, psbt and rpl <dig>  atpf introns are also present in some charophytic green algae, where they have been identified as group ii introns  <cit> .
introns in plastid rpl <dig> were until now only known in euglenoids  and b. hypnoides.
the length of the bryopsis rpl <dig> intron falls within the range of those found in euglenoids , and the high at-content is similar to the euglenoid introns .
b. plumosa contains an at-rich intron in the psbt gene.
introns in this gene have thus far only been found in euglenoids .
another at-rich intron was found in the ccsa gene of b. plumosa and t. expeditiones, which is unexpected as this gene lacks introns in all other published plastids.
similar to the other at-rich introns, the class could not be determined because we failed to identify conserved structural motives.
the trnl gene in bryopsis and tydemania contains a group i intron.
this intron has been assumed to have been present in the common ancestor of cyanobacteria and chloroplasts because it is found across a wide diversity of cyanobacteria and plastids where it is conserved in position, secondary structure and primary sequence  <cit> .
in green algae, this intron is present in several charophytes, and core chlorophyta, but is absent in the published cpdnas of prasinophytes  <cit>  and the ulvophytes pseudendoclonium and oltmannsiellopsis  <cit> .
the absence from the early diverging prasinophytes implies multiple losses of the trnl intron.
repeats
the b. plumosa cpdna contains three relatively large inverted repeats  and one large tandem repeat .
ir <dig>  duplicates part of the ccsa intron in an intergenic region between rpoc <dig> and rpob.
ir <dig>  duplicates trng, and ir <dig>  duplicates petl and part of psaj.
a tandem repeat of  <dig> × 265 bp duplicates part of psbk and part of ycf <dig> figure  <dig>
sequence repeats  in
bryopsis plumosa
and
tydemania expeditiones
cpdnas.
arrows indicate the direction of the repeats.
protein coding genes are indicated in green, trna genes in purple and rrna genes in red.
in t. expeditiones several repeats are present within cdss: rpoc <dig> contains two inverted repeats of  <dig> and 25 bp long, rpoc <dig> contains a tandem repeat of  <dig> × 38 bp, and rps <dig> contains a tandem repeat of  <dig> × 15 bp .
the intergenic spacer region between orf <dig> and trnl contains a short inverted repeat and a tandem repeat of  <dig> × 9 bp.
in addition, b. plumosa and t. expeditiones contain numerous short inverted repeats near the 3’end of genes or overlapping with the 3’end of genes .
short irs in the 3’untranslated regions  of mrnas are common in organellar genomes where they serve as rna-processing signals  <cit> .
in b. plumosa and t. expeditiones, several of these short irs partially overlap with the 3’-end of the cdss , a condition also found in bacteria where hairpins preceding the stop codon are involved in termination of transcription by rna polymerase at rho-independent sites, known as intrinsic termination  <cit> .
synteny
the mauve alignments between the two bryopsis cpdnas and between b. plumosa and t. expeditiones  visualize locally collinear blocks , representing homologous regions of sequences that do not contain any major rearrangements.
the syntenic structure of the b. plumosa cpdna is similar to that of b. hypnoides, separated by  <dig> rearrangements as calculated by the double cut and join  analysis.
when excluding all trna genes from the analysis, the dcj distance is reduced to  <dig>  this large difference is mainly due to numerous inversions of trna genes, and the absence of the large trna region in b. plumosa.
the cpdnas of the two bryopsis species and tydemania are more rearranged with respect to each other, with a dcj distance of  <dig> .
this high dissimilarity in synteny is not surprising as high variability in cpdna architecture has also been observed among species of chlorophyceae and trebouxiophyceae, including between congeneric taxa  <cit> .
even higher number of rearrangements separate the cpdnas of bryopsidales and pseudendoclonium and oltmannsiellopsis .figure  <dig>
alignments between the chloroplast genomes of
bryopsis plumosa
,
b. hypnoides
, and
tydemania expeditiones
.
the mauve algorithm  <cit>  was used to align the cpdnas between b. plumosa and b. hypnoides
, and between b. plumosa and t. expeditiones
.
corresponding colored boxes are locally collinear blocks , which represent homologous regions of sequences that do not contain any major rearrangements.
inside each block a sequence identity similarity profile is shown.
inverted lcbs are presented as blocks below the center line.
annotations are shown above and below the lcbs: protein coding genes as white boxes, trna genes in green, rrna genes in red and short repeats in pink.
lowered position of a box indicates inverted orientation.
the asterisk indicates the large trna region in b. hypnoides.
several, but not all, gene clusters conserved in the ulvophyceae and trebouxiophyceae  are also conserved in bryopsis and tydemania.
one notable exception is the peta-petl-petg cluster, which is absent in the bryopsidales .
on the other hand, some gene clusters shared in the bryopsidales are not present in any of the four analyzed ulvophyceae and trebouxiophyceae species .figure  <dig>
comparison of conserved gene clusters.
comparison of gene clusters conserved between at least two of the four depicted species, representing different ulvophycean and trebouxiophycean lineages  with gene order found in the bryopsidales .
comparison of gene clusters conserved in bryopsidales with gene order found in four species, representing different ulvophycean and trebouxiophycean lineages.
black connected circles indicate gene clusters.
grey circles indicate genes that are located elsewhere on the cpdna.
white circles indicated genes that are missing from the cpdna.
phylogenomic analyses
the phylogenetic trees resulting from the analyses of the 79-gene dataset are summarized in figure  <dig>  the topology of the trees are in general agreement with published chloroplast phylogenies of chlorophyta  <cit> .
among prasinophytes, the position of pycnococcus provasolii differs from the analyses of lemieux et al.
<cit> , but this may be attributable to a difference in tree rooting.
the core chlorophyta are comprised of several well supported main clades, including the pedinophyceae, chlorellales, the core trebouxiophyceae, chlorophyceae and two clades of ulvophyceae.
the relationships among these lineages are generally poorly supported, which contrasts with previous analyses that did not include members of the bryopsidales  <cit> .
although the phylogenies recovered two separate ulvophyceae clades, the relationship between the two is poorly supported and therefore our analyses are inconclusive with respect to monophyly of the class.
the unstable phylogenetic position of the bryopsidales is also apparent when comparing published chloroplast multi-gene phylogenies, which revealed different relationships depending on gene and taxon sampling: a sister relationship between bryopsis and chlorophyceae  <cit> , between caulerpa  and chlorella  <cit> , or between the core trebouxiophyceae and a bryopsidales + dasycladales + trentepohliales clade  <cit> .figure  <dig>
phylogenetic trees of chlorophyta inferred from a 79-gene dataset.
bayesian majority rule tree showing all compatible partitions, inference from the protein alignment of  <dig> concatenated chloroplast genes .
node support is given as bayesian posterior probabilities and maximum-likelihood  bootstrap values of the protein analyses , and the nucleotide analyses ; values < <dig>  and  <dig>  respectively, are not shown; asterisks indicated full support in both the bayesian and ml analyses.
bayesian tree inference from the nucleotide alignment  of  <dig> concatenated chloroplast genes .
node support is given as bayesian posterior probabilities and maximum-likelihood  bootstrap values, and asterisks indicate full support in both analyses.
the phylogenetic trees resulting from the analyses of the 50-gene dataset are summarized in figure  <dig>  the 50-gene dataset differs from the 79-dataset mainly in the inclusion of species of trentepohliales and dasycladales  and tetraselmis .
overall, the 50-gene phylogenies recovered the same main clades as the 79-gene phylogenies, in addition to two extra clades of ulvophyceae , and tetraselmis.
similar to the 79-gene phylogenies, the relationships among the main clades of core chlorophyta  received little statistical support, and therefore the monophyly of the ulvophyceae cannot be confirmed nor rejected by our data.
the phylogenies inferred from the nucleotide alignment  recovered the same main clades as the protein analyses, but the topology and branch support differs in several aspects, including a well-supported trentepohliales + dasycladales clade, and a different phylogenetic position of tetraselmis.figure  <dig>
phylogenetic trees of chlorophyta inferred from a 50-gene dataset.
bayesian majority rule tree showing all compatible partitions, inference from the protein alignment of  <dig> concatenated chloroplast genes .
node support is given as bayesian posterior probabilities and maximum-likelihood  bootstrap values of the protein analyses , and the nucleotide analyses ; values < <dig>  and  <dig>  respectively, are not shown; asterisks indicated full support in both the bayesian and ml analyses.
bayesian tree inference from the nucleotide alignment  of  <dig> concatenated chloroplast genes .
node support is given as bayesian posterior probabilities and maximum-likelihood  bootstrap values, and asterisks indicate full support in both analyses.
the relationships within the ulvophyceae and the monophyly of the class has been the subject of a longstanding debate  <cit> .
phylogenies based on nuclear ribosomal dna data have remained ambiguous and often recovered two distinct clades: the oltmannsiellopsidales + ulvales-ulotrichales clade, and a clade consisting of trentepohliales, cladophorales, bryopsidales and dasycladales   <cit> .
similarly, multigene chloroplast data have failed to provide support for monophyly of the ulvophyceae  <cit> .
conversely, phylogenetic analyses based on  <dig> genes  recovered the class as a monophyletic group with strong support  <cit> .
these analyses also supported the division of the two above mentioned distinct ulvophycean clades.
within the tcbd clade, the bryopsidales and dasycladales were recovered as sister lineages with high support.
the division of two distinct ulvophycean clades was also supported by independent molecular data, such as the distribution of elongation factor genes  <cit> , and a non-canonical nuclear genetic code  <cit> .
it is important to note that several important clades that were present in the analyses of cocquyt et al.
<cit>  and Škaloud et al.
<cit> , such as the cladophorales, scotinosphaerales, blastophysa, and ignatius-clade, were not included in our study, which may have an important effect on phylogenetic reconstruction.
a notable difference between our phylogenies and the ones of fučíková et al.
<cit>  is the position of oltmannsiellopsis and tetraselmis.
oltmannsiellopsis is currently classified as an ulvophyte based on morphological and molecular data  <cit> .
tetraselmis is a member of the chlorodendrophyceae and has, in nuclear ribosomal-based phylogenies, been recovered as an early branching clade of the core chlorophyta  <cit> .
in contrast, chloroplast multi-gene analyses have recovered a clade uniting tetraselmis and oltmannsiellopsis, which was sister to the ulvales-ulotrichales  <cit>  or branched early in the radiation of the core chlorophyta  <cit> .
our analyses recovered oltmannsiellopsis as sister to the ulvales-ulotrichales , similar to  <cit> , while the position of tetraselmis remained ambiguous, branching early in the core chlorophyta in the protein tree , or branching in the vicinity of ulvophycean lineages in the nucleotide-based phylogeny .
it should be noted that gene sampling in tetraselmis was low, which may explain its unstable phylogenetic position.
an accurate phylogenetic reconstruction of the core chlorophyta, and in particular the ulvophyceae will require a richer sampling of taxa and genes.
as has been shown in the trebouxiophytes, expanding taxon sampling has greatly aided in resolving phylogenetic relationships  <cit> .
compared with other groups of core chlorophyta, the ulvophyceae are still underrepresented in chloroplast phylogenomic studies , and for several important clades chloroplast genomic data is lacking altogether .
although chloroplast genomes contain large amounts of genetic information, a combination of plastid and nuclear genomic data may be needed to test monophyly of the ulvophyceae, resolve relationships within the ulvophyceae, and among the main clades of core chlorophyta.
CONCLUSIONS
the gene dense chloroplast genomes of bryopsis plumosa and tydemania expeditiones are amongst the smallest chloroplast genomes in the core chlorophyta.
comparison of the cpdna of bryopsis plumosa with the previously published cpdnas of ulvophyceae highlighted some exceptional differences in genome size, gene content, and gene order, illustrating the remarkable plasticity of green algal chloroplast genomes, even between congeneric species.
the cpdnas of b. plumosa, b. hypnoides and t. expeditiones contain dna regions that contain open reading frames showing no affinity to conserved plastid genes, but instead having significant similarity to bacterial genes.
these genes include rhs-family genes, and several genes involved in mobile functions, such as transposases, integrases, dna polymerases, and phage/plasmid dna primases.
another unexpected finding was the presence of two orfs in t. expeditiones showing close similarity to bacterial dna methyltransferases, a family of genes that have up till now not been found in plastid genomes.
the presence of dna regions including genes with clear bacterial affinity suggests that these regions may have been acquired through horizontal dna transfer from bacterial donors.
siphonous green algae are characterized by giant tubular cells that contain millions of nuclei and plastids, in addition to diverse bacterial communities.
it is imaginable that these horizontal dna transfers have been facilitated by the occurrence of bacteria residing inside the host cell.
our data adds to the scarce knowledge of horizontal transfer of bacterial dna to plastid genomes.

 the circa chloroplast genomes of green algae sequenced to date have revealed a remarkable array of genomic architectures, including a wide range of genome sizes, gene repertoires and arrangements, and nucleotide compositions. many green algal cpdnas have a quadripartite structure also found in land plants, characterized by the presence of two copies of a large inverted repeat sequence separating a small and a large single-copy region. although this architecture is believed to be ancestral in the green algae, many species do not have a quadripartite structure. complete cpdna sequences are only available for three species of ulvophyceae, including the marine flagellate oltmannsiellopsis viridis, the freshwater microfilamentous pseudendoclonium akinetum, and the marine siphonous species bryopsis hypnoides. the cpdnas of oltmannsiellopsis and pseudendoclonium share the quadripartite architecture, and a similar large complement of genes, but differ in size due to different intron numbers. the  kb cpdna of b. hypnoides differs in several aspects from the two other species of ulvophyceae. it does not feature a quadripartite architecture, and it includes trna genes that are not present in any other green algal cpdna. another peculiarity of the b. hypnoides cpdna is the presence of multimeric forms of the cpdna, including monomers, dimers, trimers, tetramers, and higher-order multimers, which were detected by pulsed-field gel electrophoresis and southern blot methods. genes were reported in the cpdna of b. hypnoides but our preliminary analysis indicated a higher number of genes, as also suggested by others. apart from completely sequenced cpdnas, partial chloroplast genome data in the bryopsidales has long been available for codium fragile and caulerpa sertularoides through southern hybridization analysis of restriction fragments. in addition partial dna sequence data is available for caulerpa filiformis.figure phylogenetic relationships among the main clades of green algae, focusing on the core chlorophyta. the tree is a composite of accepted relationships based on molecular phylogenetic evidence from different studies. uncertain or conflicting relationships are indicated by polytomies or question marks. numbers in square brackets indicate the number of complete cpdnas available to date. clades currently classified as ulvophyceae are in green, and trebouxiophyceae are in yellow. the relationships within the core chlorophyta have recently been evaluated based on multigene datasets, which is relevant in the light of comparative genomics. although monophyly of the ulvophyceae was supported in a 10-gene phylogeny, chloroplast multigene phylogenetic analyses generally failed to recover the ulvophyceae as a clade. for example, phylogenetic analysis of cp genes recovered caulerpa as more closely related to chlorella than to the other two ulvophycean taxa in the phylogeny, and a phylogeny inferred from cp genes indicated a relationship between bryopsis and chlorophyceae. phylogenomic analyses with increased taxon sampling suggested a relationship between oltmannsiellopsis and tetraselmis, and like in the two previous studies did not provide support for a monophyletic ulvophyceae. in addition, cp multigene phylogenies have rejected monophyly of the trebouxiophyceae, which was shown to fall apart in at least two main clades. in general, relationships among the major lineages of core chlorophyta remain poorly supported. as illustrated in figure  complete data on chloroplast genomes is scarce in the ulvophyceae compared to other green algal lineages, with data lacking for several important clades, including the cladophorales, dasycladales, and trentepohliales. this paper focusses on the siphonous marine green algae bryopsis and tydemania, two members of the order bryopsidales. species in this order form thalli that are composed of a single, giant cell, containing millions of nuclei, chloroplasts and mitochondria that move about freely by cytoplasmic streaming. bryopsis includes more than species, all characterized by morphologically simple feather-like thalli, mostly only a few cm tall. the genus is distributed worldwide in tropical to temperate seas. tydemania forms more complex and larger thalli composed of whorls of siphons arising from a branched main axis. the genus includes a single species, t. expeditiones, that is found in the marine tropical indo-west pacific. the order bryopsidales includes two main clades, bryopsidineae and halimedineae. bryopsis is a member of the bryopsidineae, which also includes derbesia and codium, and some other, less known genera. tydemania is a member of the halimedineae, which also includes some of the better known siphonous green seaweeds such as caulerpa and halimeda. the placement of the bryopsidales in the class ulvophyceae has been implied by 18s data and nuclear multi-gene phylogenetic analysis, but as mentioned above, analyses based on cp genes have casted doubt on the affinity of siphonous green algae with other clades of ulvophyceae. in this paper we report on the complete cpdnas of bryopsis plumosa and tydemania expeditiones, and a re-annotated cpdna of b. hypnoides. we compared these genomes with previously published cpdnas in the core chlorophyta to gain a better understanding of the evolution of chloroplast genomes in this group, and to assess relationships among the main clades of core chlorophyta.  cpdna assembly, size and organization for both bryopsis plumosa and tydemania expeditiones, assemblies yielded a single cpdna sequence, which could be closed into a circle by an overlap of more than base pairs. both assemblies showed high overall read coverage, and as a result did not contain any ambiguous regions. for b. plumosa, reads of a total of million mapped to the cpdna with a mean coverage of 430×. the 4× coverage was 3 bp long and situated in the 3’-end of the cyst gene, and was spanned by paired-end reads. for t. expeditiones, million reads of a total of million mapped to the cpdna with a mean coverage of ×. the circular cpdnas of b. plumosa and t. expeditiones consist of  bp and  bp, respectively. this is smaller than most published cpdnas of free-living species of core chlorophyta, but similar to those found in pedinomonas minor, marvania geminata, pseudochloris wilhelmii, and planctonema lauterbornii. most prasinophytes have even smaller cpdnas, ranging between 64-86 kb, with the exception of nephroselmis, which has larger chloroplast genomes. unexpectedly, the cpdna of b. plumosa is much smaller than the  bp cpdna of b. hypnoides. this size difference is mainly a result of large intergenic spacers, totaling  bp or % of the cpdna in b. hypnoides. intergenic spacers in b. plumosa and t. expeditiones account for only % and % of the total cpdna. nevertheless, some long intergenic spacer regions are present in both genomes. in b. plumosa, the largest intergenic spacer was  bp long. this region contains a few orfs, none of which showed significant homology to known proteins. in t. expeditiones, a large spacer region contains ten orfs, none with significant homology to known proteins. gc content of the two genomes falls within the limits of other ulvophycean and green algal cpdnas.figure gene map of the chloroplast genome of bryopsis plumosa. the  bp genome contains unique genes, including three ribosomal rna genes, transfer rna genes, and protein coding genes. genes shown on the outside of the circle are transcribed counterclockwise. annotated genes are colored according to the functional categories shown in the legend bottom left. the red arcs indicate gene regions of putative bacterial origin. gene map of the chloroplast genome of tydemania expeditiones. the  bp genome contains unique genes, including three ribosomal rna genes, transfer rna genes, and protein coding genes. genes shown on the outside of the circle are transcribed counterclockwise. annotated genes are colored according to the functional categories shown in the legend bottom left. the red arcs indicate gene regions of putative bacterial origin. summary of the bryopsis plumosa and tydemania expeditiones cpdnas and comparison with other ulvophycean cpdnas species cpdna size gc content gene number protein coding genes/orfs d rrna genes trna genes % coding b intergenic space intron c intron b intron number quadripartite structure reference b. plumosa t. expeditiones b. hypnoides a p. akinetum o. viridis are-annotated in this study. bincluding intronic open reading frames. cexcluding intronic orfs. donly those orfs are included, which were found to have a significant blastp result. the b. plumosa and t. expeditiones cpdnas both lack a large inverted repeat, similar to the situation found in b. hypnoides. the lack of a quadripartite architecture in bryopsidales had earlier been implied based on southern hybridization analysis of restriction fragments in codium fragile and caulerpa sertularoides. most plastid genomes have a quadripartite structure where a set of large inverted repeats divides the genome into two single-copy regions. although the quadripartite structure is believed to be ancestral in viridiplantae, loss of the inverted repeat has occurred multiple times in different green algal lineages. gene content the b. plumosa cpdna contains unique genes, including protein coding genes, trna genes, and three rrna genes. in addition to expected, conserved plastid protein coding genes, several freestanding orfs were identified that did not show any relationship with known plastid genes, but instead showed close similarity with bacterial genes. the large orf showed close similarity to bacterial rhs-family proteins. a similar but slightly smaller orf was also found in the re-annotated cpdna of b. hypnoides. rhs genes are a family of composite genes that occur in several gram-negative bacteria, including some cyanobacteria. their broad phylogenetic distribution has been explained by a complex evolutionary history of horizontal transfers and independent gene losses. despite their wide occurrence, the function of rhs genes remains elusive. other orfs of putative bacterial origin include a gene encoding dna polymerase with maturase-specific domain, and two orfs showing close similarity to uncharacterized bacterial proteins. orf orf and orf are grouped in a 13 kb region of the cpdna, while orf is situated elsewhere.table comparison of trna genes in the two bryopsis species, tydemania expeditiones and two other species of ulvophyceae b. hyp b. plu t. exp p. aki o. vir trnma total 53 27 28 27 27 unique 40 26 28 27 27 athe trnm gene situated in the large trna region in b. hypnoides showed similarity with land plant nuclear encoded trna-met initiator genes. numbers indicate gene copy. an asterisks indicates that the gene or gene copy is situated in the large trna region in b. hypnoides. b. hyp = bryopsis hypnoides, b. plu = b. plumosa, t. exp = tydemania expeditiones, p. aki = pseudendoclonium akinetum, o. vir = oltmannsiellopsis viridis. comparison of protein coding genes content among core chlorophyta. pd = pedinophyceae. genes that are shared among the cpdnas are not included: atpa, b, e, f, h, cema, clpp, ftsh, petb, d, g, l, psaa, b, c, j, psba, b, c, d, e, f, h, i, j, k, l, m, n, t, z, rbcl, rpl rps tufa, ycf ycf ycf rpoa, b, c c rrf, rrl, rrs, and trna genes. ftsh and ycf are present in c. reinhardtii as orf and orf respectively, and ycf is present in c. vulgaris as orf ). ycf is present in c. vulgaris as orf. data sources: b. hypnoides, p. akinetum, o. viridis, c. vulgaris, p. kessleri, o. solitaria, p. minor, c. subellipsoidea, l. terrestris, a. obliquus, d. salina, v. carteri, g. pectorale, c. reinhardtii, o. cardiacum, f. terrestris, s. leibleinii, s. helveticum. freestanding orfs in bryopsis plumosa and tydemania expeditiones cpdnas species orf number length closest blast hit blastp e-value note b. plumosa orf3 b. plumosa orf4 b. plumosa orf5 b. plumosa orf7 t. expeditiones orf3 t. expeditiones orf8 t. expeditiones orf9 t. expeditiones orf13 t. expeditiones orf orf15 t. expeditiones orf16 ato verify whether homologous genes were present in green algae, blastp searcher were performed with organisms constrained to viridiplantae. most genes found in b. plumosa, with the exception of ycf and orf were also found in b. hypnoides. however, the cpdna of b. hypnoides contains a region of about 6 kb including trna genes, ten of which have not been found in any other green algal cpdna. this large trna region is not present in b. plumosa, nor is the set of unique trna genes. as in b. plumosa, several orfs in b. hypnoides showed high similarity to bacterial genes, including a putative rhs gene, and genes containing domains commonly found in mobile elements, such as reverse transcriptase, endonuclease and maturase. reverse transcriptase proteins catalyze the reverse transcription of rna to complementary dna, and have a key role in the mobility of retroviruses, retrotransposons, and organellar group ii introns. endonuclease-like proteins catalyze cleavage of retrotransposons before transcription, and are also involved in mobility and homing of introns. maturase-like proteins are best known from bacterial and organellar introns, where they aid in intron splicing. the t. expeditiones cpdna contains unique genes, including protein coding genes, three rrna genes, and trna genes. in addition to the conserved set of chloroplast genes, several freestanding orfs were found coding for bacterial or intron related genes. orf contains a maturase-specific domain, which are usually found within intronic orfs. two overlapping orfs are related to bacterial c- cytosine-specific dna methyltransferase genes, a family of genes coding for enzymes that catalyzes the methyl-transfer reaction in the process of dna methylation, and in bacteria are often associated with restriction/modification systems, which function as a defense against infection of bacteria by bacteriophages. dna methyltransferases have up till now not been found in plastid genomes. however, the identification of dna methyltransferase genes in organelles is not unprecedented as a gene of this nature has been identified in the mitochondrial genome of the streptophyte klebsormidium, which was seen as a possible remnant of viral infection. a region of about kp contains four freestanding orfs showing close similarity to bacterial genes involved in mobile functions. orf showed high similarity to bacterial transposase and contains an integrase core domain. transposases and integrases catalyze the movement and integration of dna copies to new locations within and between genomes. orf and orf showed similarity to bacterial dna polymerase, containing a dna polymerase family a domain. family a polymerases fill dna gaps that arise during dna repair, recombination and replication, and are found primarily in prokaryotes. orf showed high similarity to phage/plasmid dna primases, which are known from phages and plasmids of bacteria and archaea. the presence of these four orfs indicates that the kp region may be a mobile element of bacterial origin. the presence of bacterial genes in both the bryopsis and tydemania cpdnas, and absent in other green algae, suggest that these genes have been acquired through horizontal gene transfer. transfer of genes to plastid genomes is only rarely observed but some clear instances are known. in the oedogonium chloroplast genome, the unprecedented finding of int and dpob was seen as evidence of horizontal transfer, possibly from a mitochondrial genome donor. in the cpdna of nephroselmis olivacea, two large regions are believed to have been acquired by lateral transfer from a bacterial donor based on a deviant base composition, the lack of genes typically found in cpdnas, and the presence of an orf showing similarity to phage associated dna primases. horizontal gene transfer from bacteria to plastids has also been demonstrated in other groups of algae, including red algae, dinoflagellates, haptophytes and cryptophytes. it is relevant to note that siphonous green algae are known to harbor intracellular bacterial communities, with some bacteria showing close associations with the host. based on fluorescence in situ hybridization, bacteria in bryopsis were found in the vacuole or in the cytoplasmic layer of the siphonous cells, sometimes closely adhered to the chloroplast membrane. these intracellular bacteria may facilitate gene transfer from the endobiontic bacteria to the host genome, possibly via vectors such as bacteriophages. the two bryopsis cpdnas share nearly identical gene repertoires with t. expeditiones, except for rpl which is present in b. plumosa and b. hypnoides but was not found in t. expeditiones. in addition, several of the orfs discussed above were uniquely found in b. plumosa or t. expeditiones. a number of genes in bryopsis and/or tydemania, including psbm, rpl and rpl were found to be quite divergent from core chlorophytan orthologs based on visual inspection of amino acid alignments and branch lengths of resulting phylogenetic trees. the petl gene is present in two identical copies in b. plumosa. also b. hypnoides contains two copies of petl, but here they differ in nucleotide composition as well as in length: a 96 bp copy identical to that of b. plumosa, and a 105 bp copy that is divergent from the bryopsidalean orthologs. tydemania expeditiones contains a single 96 bp copy of petl. a comparison of gene repertoires between b. plumosa, t. expeditiones and published core chlorophytan cpdnas is shown in figure  a total of genes are shared among the cpdnas. a large proportion of genes are shared among species of ulvophyceae and trebouxiophyceae, while most chlorophyceae have a smaller gene repertoire. notably, several genes are shared between the bryopsidales and trebouxiophyceae, but are absent from pseudendoclonium and/or oltmannsiellopsis. these include three genes encoding subunits of protochlorophyllide reductase, trna ile-lysidine synthetase and hypothetical protein ycf only one gene, the organelle division inhibitor factor, mind, was present in pseudendoclonium, oltmannsiellopsis, and trebouxiophyceae but absent in bryopsidales. trna-lysidine synthase, also known as ycf is present in all ulvophyceae and trebouxiophyceae, but absent from chlorophyceae. in t. expeditiones, this gene seems to be pseudogenized by a stop codon at amino acid position from the start methionine. a similar situation was found in caulerpa filiformis, which contains a tils pseudogene with a frame shift at about the same position. in b. plumosa and b. hypnoides the gene is interrupted by a 71 bp, at-rich insertion at amino acid position introns in b. plumosa introns are present in genes, comprising % of the total cpdna. four introns were identified as group i introns, and five as group ii introns, while for four introns the class could not be determined with certainty. in t. expeditiones introns are present in genes, comprising % of the total cpdna. seven introns were identified as group i introns, and two as group ii introns, while the class of two introns could not be determined with certainty. several of the introns in b. plumosa and t. expeditiones are commonly found in orthologous genes of other green algae. others are only rarely found in green algae or have not yet been observed in plastids in general. these are discussed below.table distribution and characteristics of introns in bryopsis plumosa and tydemania expeditiones gene b. plu t. exp length insertion positions a at content intronic orf class atpf ccsa psaa psba psbb psbc psbt rpl5 rpl23 rps19 ycf3 aintron insertion site positions correspond to the nucleotide immediately preceding the intron. insertion sites in genes coding for proteins and the trna are given relative to the corresponding genes in mesostigma viride cpdna; insertion sites in the rrna genes are given relative to the 16s and 23s rrna genes of escherichia coli. bcontains two laglidadg homing endonuclease domains. rt = reverse transcriptase, im = intron maturase, lhe = laglidadg homing endonuclease. the atpa gene in b. plumosa contains two introns. the first was identified as a group i intron, which is commonly found in this gene in other green algae. the second intron was identified as a putative group ii intron, which has until now only been found in the atpa gene of volvox carteri. the psba gene in t. expeditiones has a large intron containing two overlapping orfs: one encoding reverse transcriptase and intron maturase, the other encoding n-terminal domain of reverse transcriptase. the majority of psba introns in green algae are group i introns. thus far, only one other green algal psba group ii intron has been identified, in floydiella terrestris. the psbc intron in t. expeditiones was also identified as a group ii intron based on the presence of an orf encoding reverse transcriptase and intron maturase. introns are common in green algal psbc genes, but are all group i introns. psbc group ii introns have thus far only been found in euglenoids. the rpl and rps introns in b. plumosa are putative group ii introns. thus far, rpl introns have only been found in b. hypnoides. plastid rps introns were until now only known in euglenoids and the red alga porphyridium purpureum. these introns ranged between 95-216 bp in size, which is much smaller than the intron found in b. plumosa. at-rich introns of which the class could not be determined with certainty were found in several genes, including atpf, ccsa, psbt and rpl atpf introns are also present in some charophytic green algae, where they have been identified as group ii introns. introns in plastid rpl were until now only known in euglenoids and b. hypnoides. the length of the bryopsis rpl intron falls within the range of those found in euglenoids, and the high at-content is similar to the euglenoid introns. b. plumosa contains an at-rich intron in the psbt gene. introns in this gene have thus far only been found in euglenoids. another at-rich intron was found in the ccsa gene of b. plumosa and t. expeditiones, which is unexpected as this gene lacks introns in all other published plastids. similar to the other at-rich introns, the class could not be determined because we failed to identify conserved structural motives. the trnl gene in bryopsis and tydemania contains a group i intron. this intron has been assumed to have been present in the common ancestor of cyanobacteria and chloroplasts because it is found across a wide diversity of cyanobacteria and plastids where it is conserved in position, secondary structure and primary sequence. in green algae, this intron is present in several charophytes, and core chlorophyta, but is absent in the published cpdnas of prasinophytes and the ulvophytes pseudendoclonium and oltmannsiellopsis. the absence from the early diverging prasinophytes implies multiple losses of the trnl intron. repeats the b. plumosa cpdna contains three relatively large inverted repeats and one large tandem repeat. ir duplicates part of the ccsa intron in an intergenic region between rpoc and rpob. ir duplicates trng, and ir duplicates petl and part of psaj. a tandem repeat of × 265 bp duplicates part of psbk and part of ycf figure sequence repeats in bryopsis plumosa and tydemania expeditiones cpdnas. arrows indicate the direction of the repeats. protein coding genes are indicated in green, trna genes in purple and rrna genes in red. in t. expeditiones several repeats are present within cdss: rpoc contains two inverted repeats of and 25 bp long, rpoc contains a tandem repeat of × 38 bp, and rps contains a tandem repeat of × 15 bp. the intergenic spacer region between orf and trnl contains a short inverted repeat and a tandem repeat of × 9 bp. in addition, b. plumosa and t. expeditiones contain numerous short inverted repeats near the 3’end of genes or overlapping with the 3’end of genes. short irs in the 3’untranslated regions of mrnas are common in organellar genomes where they serve as rna-processing signals. in b. plumosa and t. expeditiones, several of these short irs partially overlap with the 3’-end of the cdss, a condition also found in bacteria where hairpins preceding the stop codon are involved in termination of transcription by rna polymerase at rho-independent sites, known as intrinsic termination. synteny the mauve alignments between the two bryopsis cpdnas and between b. plumosa and t. expeditiones visualize locally collinear blocks, representing homologous regions of sequences that do not contain any major rearrangements. the syntenic structure of the b. plumosa cpdna is similar to that of b. hypnoides, separated by rearrangements as calculated by the double cut and join analysis. when excluding all trna genes from the analysis, the dcj distance is reduced to this large difference is mainly due to numerous inversions of trna genes, and the absence of the large trna region in b. plumosa. the cpdnas of the two bryopsis species and tydemania are more rearranged with respect to each other, with a dcj distance of. this high dissimilarity in synteny is not surprising as high variability in cpdna architecture has also been observed among species of chlorophyceae and trebouxiophyceae, including between congeneric taxa. even higher number of rearrangements separate the cpdnas of bryopsidales and pseudendoclonium and oltmannsiellopsis.figure alignments between the chloroplast genomes of bryopsis plumosa, b. hypnoides, and tydemania expeditiones. the mauve algorithm was used to align the cpdnas between b. plumosa and b. hypnoides, and between b. plumosa and t. expeditiones. corresponding colored boxes are locally collinear blocks, which represent homologous regions of sequences that do not contain any major rearrangements. inside each block a sequence identity similarity profile is shown. inverted lcbs are presented as blocks below the center line. annotations are shown above and below the lcbs: protein coding genes as white boxes, trna genes in green, rrna genes in red and short repeats in pink. lowered position of a box indicates inverted orientation. the asterisk indicates the large trna region in b. hypnoides. several, but not all, gene clusters conserved in the ulvophyceae and trebouxiophyceae are also conserved in bryopsis and tydemania. one notable exception is the peta-petl-petg cluster, which is absent in the bryopsidales. on the other hand, some gene clusters shared in the bryopsidales are not present in any of the four analyzed ulvophyceae and trebouxiophyceae species.figure comparison of conserved gene clusters. comparison of gene clusters conserved between at least two of the four depicted species, representing different ulvophycean and trebouxiophycean lineages with gene order found in the bryopsidales. comparison of gene clusters conserved in bryopsidales with gene order found in four species, representing different ulvophycean and trebouxiophycean lineages. black connected circles indicate gene clusters. grey circles indicate genes that are located elsewhere on the cpdna. white circles indicated genes that are missing from the cpdna. phylogenomic analyses the phylogenetic trees resulting from the analyses of the 79-gene dataset are summarized in figure  the topology of the trees are in general agreement with published chloroplast phylogenies of chlorophyta. among prasinophytes, the position of pycnococcus provasolii differs from the analyses of lemieux et al., but this may be attributable to a difference in tree rooting. the core chlorophyta are comprised of several well supported main clades, including the pedinophyceae, chlorellales, the core trebouxiophyceae, chlorophyceae and two clades of ulvophyceae. the relationships among these lineages are generally poorly supported, which contrasts with previous analyses that did not include members of the bryopsidales. although the phylogenies recovered two separate ulvophyceae clades, the relationship between the two is poorly supported and therefore our analyses are inconclusive with respect to monophyly of the class. the unstable phylogenetic position of the bryopsidales is also apparent when comparing published chloroplast multi-gene phylogenies, which revealed different relationships depending on gene and taxon sampling: a sister relationship between bryopsis and chlorophyceae, between caulerpa and chlorella, or between the core trebouxiophyceae and a bryopsidales + dasycladales + trentepohliales clade.figure phylogenetic trees of chlorophyta inferred from a 79-gene dataset. bayesian majority rule tree showing all compatible partitions, inference from the protein alignment of concatenated chloroplast genes. node support is given as bayesian posterior probabilities and maximum-likelihood bootstrap values of the protein analyses, and the nucleotide analyses; values < and respectively, are not shown; asterisks indicated full support in both the bayesian and ml analyses. bayesian tree inference from the nucleotide alignment of concatenated chloroplast genes. node support is given as bayesian posterior probabilities and maximum-likelihood bootstrap values, and asterisks indicate full support in both analyses. the phylogenetic trees resulting from the analyses of the 50-gene dataset are summarized in figure  the 50-gene dataset differs from the 79-dataset mainly in the inclusion of species of trentepohliales and dasycladales and tetraselmis. overall, the 50-gene phylogenies recovered the same main clades as the 79-gene phylogenies, in addition to two extra clades of ulvophyceae, and tetraselmis. similar to the 79-gene phylogenies, the relationships among the main clades of core chlorophyta received little statistical support, and therefore the monophyly of the ulvophyceae cannot be confirmed nor rejected by our data. the phylogenies inferred from the nucleotide alignment recovered the same main clades as the protein analyses, but the topology and branch support differs in several aspects, including a well-supported trentepohliales + dasycladales clade, and a different phylogenetic position of tetraselmis.figure phylogenetic trees of chlorophyta inferred from a 50-gene dataset. bayesian majority rule tree showing all compatible partitions, inference from the protein alignment of concatenated chloroplast genes. node support is given as bayesian posterior probabilities and maximum-likelihood bootstrap values of the protein analyses, and the nucleotide analyses; values < and respectively, are not shown; asterisks indicated full support in both the bayesian and ml analyses. bayesian tree inference from the nucleotide alignment of concatenated chloroplast genes. node support is given as bayesian posterior probabilities and maximum-likelihood bootstrap values, and asterisks indicate full support in both analyses. the relationships within the ulvophyceae and the monophyly of the class has been the subject of a longstanding debate. phylogenies based on nuclear ribosomal dna data have remained ambiguous and often recovered two distinct clades: the oltmannsiellopsidales + ulvales-ulotrichales clade, and a clade consisting of trentepohliales, cladophorales, bryopsidales and dasycladales. similarly, multigene chloroplast data have failed to provide support for monophyly of the ulvophyceae. conversely, phylogenetic analyses based on genes recovered the class as a monophyletic group with strong support. these analyses also supported the division of the two above mentioned distinct ulvophycean clades. within the tcbd clade, the bryopsidales and dasycladales were recovered as sister lineages with high support. the division of two distinct ulvophycean clades was also supported by independent molecular data, such as the distribution of elongation factor genes, and a non-canonical nuclear genetic code. it is important to note that several important clades that were present in the analyses of cocquyt et al. and Škaloud et al., such as the cladophorales, scotinosphaerales, blastophysa, and ignatius-clade, were not included in our study, which may have an important effect on phylogenetic reconstruction. a notable difference between our phylogenies and the ones of fučíková et al. is the position of oltmannsiellopsis and tetraselmis. oltmannsiellopsis is currently classified as an ulvophyte based on morphological and molecular data. tetraselmis is a member of the chlorodendrophyceae and has, in nuclear ribosomal-based phylogenies, been recovered as an early branching clade of the core chlorophyta. in contrast, chloroplast multi-gene analyses have recovered a clade uniting tetraselmis and oltmannsiellopsis, which was sister to the ulvales-ulotrichales or branched early in the radiation of the core chlorophyta. our analyses recovered oltmannsiellopsis as sister to the ulvales-ulotrichales, similar to, while the position of tetraselmis remained ambiguous, branching early in the core chlorophyta in the protein tree, or branching in the vicinity of ulvophycean lineages in the nucleotide-based phylogeny. it should be noted that gene sampling in tetraselmis was low, which may explain its unstable phylogenetic position. an accurate phylogenetic reconstruction of the core chlorophyta, and in particular the ulvophyceae will require a richer sampling of taxa and genes. as has been shown in the trebouxiophytes, expanding taxon sampling has greatly aided in resolving phylogenetic relationships. compared with other groups of core chlorophyta, the ulvophyceae are still underrepresented in chloroplast phylogenomic studies, and for several important clades chloroplast genomic data is lacking altogether. although chloroplast genomes contain large amounts of genetic information, a combination of plastid and nuclear genomic data may be needed to test monophyly of the ulvophyceae, resolve relationships within the ulvophyceae, and among the main clades of core chlorophyta.  the gene dense chloroplast genomes of bryopsis plumosa and tydemania expeditiones are amongst the smallest chloroplast genomes in the core chlorophyta. comparison of the cpdna of bryopsis plumosa with the previously published cpdnas of ulvophyceae highlighted some exceptional differences in genome size, gene content, and gene order, illustrating the remarkable plasticity of green algal chloroplast genomes, even between congeneric species. the cpdnas of b. plumosa, b. hypnoides and t. expeditiones contain dna regions that contain open reading frames showing no affinity to conserved plastid genes, but instead having significant similarity to bacterial genes. these genes include rhs-family genes, and several genes involved in mobile functions, such as transposases, integrases, dna polymerases, and phage/plasmid dna primases. another unexpected finding was the presence of two orfs in t. expeditiones showing close similarity to bacterial dna methyltransferases, a family of genes that have up till now not been found in plastid genomes. the presence of dna regions including genes with clear bacterial affinity suggests that these regions may have been acquired through horizontal dna transfer from bacterial donors. siphonous green algae are characterized by giant tubular cells that contain millions of nuclei and plastids, in addition to diverse bacterial communities. it is imaginable that these horizontal dna transfers have been facilitated by the occurrence of bacteria residing inside the host cell. our data adds to the scarce knowledge of horizontal transfer of bacterial dna to plastid genomes. 
the cpdnas of b. plumosa and t. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core chlorophyta. the presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae. horizontal gene transferintronsmobile elementsphylogenomicsplastid genomeviridiplantaeissue-copyright-statement© the author 2015 we present the complete cpdnas of bryopsis plumosa and tydemania expeditiones, as well as a re-annotated cpdna of b. hypnoides, which was shown to contain a higher number of genes than originally published. a 13 kb region was identified with several freestanding open reading frames of putative bacterial origin, including a large orf closely related to bacterial rhs-family genes. the cpdna of b. hypnoides differs from that of b. plumosa mainly in the presence of long intergenic spacers, and a large trna region. the cpdna of t. expeditiones is  bp long and contains unique genes. little is known about the diversity of chloroplast genomes in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. the cpdna of b. plumosa is  bp long and contains unique genes. as in b. plumosa, several regions were identified with orfs of possible bacterial origin, including genes involved in mobile functions, and orfs showing close similarity with bacterial dna methyltransferases.
 species of bryopsidales form ecologically important components of seaweed communities worldwide. these siphonous macroalgae are composed of a single giant tubular cell containing millions of nuclei and chloroplasts, and harbor diverse bacterial communities. little is known about the diversity of chloroplast genomes in this group, and about the possible consequences of intracellular bacteria on genome composition of the host. we present the complete cpdnas of bryopsis plumosa and tydemania expeditiones, as well as a re-annotated cpdna of b. hypnoides, which was shown to contain a higher number of genes than originally published. chloroplast genomic data were also used to evaluate phylogenetic hypotheses in the chlorophyta, such as monophyly of the ulvophyceae.  both dnas are circular and lack a large inverted repeat. the cpdna of b. plumosa is  bp long and contains unique genes. a 13 kb region was identified with several freestanding open reading frames of putative bacterial origin, including a large orf closely related to bacterial rhs-family genes. the cpdna of t. expeditiones is  bp long and contains unique genes. as in b. plumosa, several regions were identified with orfs of possible bacterial origin, including genes involved in mobile functions, and orfs showing close similarity with bacterial dna methyltransferases. the cpdna of b. hypnoides differs from that of b. plumosa mainly in the presence of long intergenic spacers, and a large trna region. chloroplast phylogenomic analyses were largely inconclusive with respect to monophyly of the ulvophyceae, and the relationship of the bryopsidales within the chlorophyta.  the cpdnas of b. plumosa and t. expeditiones are amongst the smallest and most gene dense chloroplast genomes in the core chlorophyta. the presence of bacterial genes, including genes typically found in mobile elements, suggest that these have been acquired through horizontal gene transfer, which may have been facilitated by the occurrence of obligate intracellular bacteria in these siphonous algae. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords horizontal gene transferintronsmobile elementsphylogenomicsplastid genomeviridiplantaeissue-copyright-statement© the author 2015 