Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
DOI: 10.1007/s13131-014-0445-2
http://www.hyxb.org.cn
E-mail: [email protected]
Phylogenomic analysis of transcriptomic sequences of
mitochondria and chloroplasts of essential brown algae
(Phaeophyceae) in China
JIA Shangang1,3†, WANG Xumin1,3†, LI Tianyong2, QIAN Hao2, SUN Jing1,3,4, WANG Liang1,3,4, YU
Jun1,3, REN Lufeng1,3, YIN Jinlong1, LIU Tao2*, WU Shuangxiu1,3*
1
CAS Key Laboratory of Genome Sciences and Information, Beijing Key Laboratory of Genome and
Precision Medicine Technologies, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing
100101, China
2 College of Marine Life Science, Ocean University of China, Qingdao 266003, China
3 Beijing Key Laboratory of Functional Genomics for Dao-di Herbs, Beijing Institute of Genomics, Chinese
Academy of Sciences, Beijing 100101, China
4 University of Chinese Academy of Sciences, Beijing 100049, China
Received 22 March 2013; accepted 13 August 2013
©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014
Abstract
The chloroplast and mitochondrion of brown algae (Class Phaeophyceae of Phylum Ochrophyta) may have
originated from different endosymbiosis. In this study, we carried out phylogenomic analysis to distinguish
their evolutionary lineages by using algal RNA-seq datasets of the 1 000 Plants (1KP) Project and publicly
available complete genomes of mitochondria and chloroplasts of Kingdom Chromista. We have found that
there is a split between Class Phaeophyceae of Phylum Ochrophyta and the others (Phylum Cryptophyta and
Haptophyta) in Kingdom Chromista, and identified more diversity in chloroplast genes than mitochondrial
ones in their phylogenetic trees. Taxonomy resolution for Class Phaeophyceae showed that it was divided
into Laminariales-Ectocarpales clade and Fucales clade, and phylogenetic positions of Kjellmaniella crassifolia, Hizikia fusifrome and Ishige okamurai were confirmed. Our analysis provided the basic phylogenetic
relationships of Chromista algae, and demonstrated their potential ability to study endosymbiotic events.
Key words: Phaeophyceae, brown algae, Chromista, phylogenetic trees, mitochondrion, chloroplast
Citation: Jia Shangang, Wang Xumin, Li Tianyong, Qian Hao, Sun Jing, Wang Liang, Yu Jun, Ren Lufeng, Yin Jinlong, Liu Tao, Wu
Shuangxiu. 2014. Phylogenomic analysis of transcriptomic sequences of mitochondria and chloroplasts of essential brown algae
(Phaeophyceae) in China. Acta Oceanologica Sinica, 33(2): 94–101, doi: 10.1007/s13131-014-0445-2
1 Introduction
Algae comprise a large number of most diverse unicellular
and multi-cellular taxa that virtually populate all the ecosystems on Earth. It covers a large variety of about 20 taxonomic groups. The best-known ones are red, green, brown, and
golden algae, diatoms, glaucophytes, raphidophytes, cryptophytes, haptophytes, chlorarachniophytes, dinoflagellates and
euglenids (Katz, 2012). Kingdom Chromista, nominated by
Cavalier-Smith, comprises of Cryptophyta, Heterokonta and
Haptophyta, which contain chlorophyll a/c and four membranes surrounding the plastids and indicate the presence of a
secondary endosymbiotic event (Medlin et al., 1995). Pigments
and chlorophyll make them appear in brown or golden color.
Many Chromista algae are of great significance. For example,
brown algae (Class Phaeophyceae of Order Ochrophyta of Heterokonta) are a large group of mostly marine multicellular algae
and are one of the most productive ecosystems in the world (Silberfeld et al., 2010), from microscopic filaments (for example,
Ectocarpus) to the giant structurally-complex thalli of kelps. All
of the estimated brown algal 1 811 species, belonging to ca. 285
genera, exhibit many unusual and interesting metabolic, developmental and cell-biological features (Cho et al., 2004; Wynne
and Loiseaux, 1976).
The origins of mitochondria and plastids, the most important organelles in algae, fundamentally explain the evolutionary nature of eukaryotes, which draw much attention from the
researchers all over the world. It was suggested that plastid/
chloroplast, the algal photosynthetic organelle, may originate
directly from a cyanobacterium-like prokaryote via primary endosymbiosis about 1 to 1.5 billion years ago (Rodríguez-Ezpeleta et al., 2005). Based on the recent hypothesis, there are three
major host lineages, red algae, green plants, and glaucophytes,
whose plastids have two bounding membranes (McFadden,
2001). In addition, the second endosymbiosis refers to the engulfment of red algae (Green, 2011) or green algae by eukaryotic hosts, and their plastids finally have three or four bounding membranes. In contrast, algal mitochondria experienced a
different evolutionary history. Gray et al. (2001) concluded that
mitochondria originated from α-proteobacterial ancestor, entered eukaryotes long before plastids and co-evolved together
Foundation item: The National Natural Science Foundation of China under contract Nos 31140070, 31271397 and 41206116; the algal transcriptome sequencing was supported by 1KP Project (www.onekp.com).
*Corresponding author, E-mail: [email protected], [email protected]
†Contributed equally.
95
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
with their host. So mitochondrial DNA can show evolutionary
history of the nuclear genome (Burger and Nedelcu, 2012).
The current knowledge on the origins of chloroplast and
mitochondrion is mostly determined based on the shared
conserved regions or genes in the algal chloroplastic and mitochondrial genomes, respectively. DNA sequences are widely
used to study on the algal phylogenetic relationships, such as,
small subunit (SSU) (Tan and Druehl, 1994) and large subunit
(LSU) of rDNA (Phillips et al., 2008) in nucleus, rbcL, psaA and
psbA (Cho et al., 2004) in plastids, and Cytochrome c (Danne
et al., 2012) in mitochondria. However, protein sequences are
preferred because they are more conserved than DNA ones,
and the concatenated genes provide more comprehensive information than a single one. Phylogenetic trees are constructed in several software programs (for example, MEGA, PAML,
Mrbayes, Tree-Puzzle, etc.) based on different models of evolution (Green, 2011).
The available whole-genome data has been a spotlight in algal research, which enables us to perform phylogenomic analysis. Phylogenomics refer to large-scale data mining and analyses of massive amounts of genome sequences. It is noted that
expressed-sequence-tags (ESTs) and genomes from mitochondria and plastids can be used to construct phylogenetic trees
(Hallstrom and Janke, 2009). The same set of genomic sequence
data can potentially result into different taxonomic relationships based on the unique orthologous genes of mitochondria
and chloroplasts. The differences reflect the distinct endosymbiotic events of these two organelles. We had suggested that in
phylogenomic trees built with algal ESTs and genomes of mitochondria and chloroplasts, Ulva prolifera was placed in a sister
position to Ulva linza but shared a similar chloroplast origin
with Pseudendoclonium akinetum (Jia et al., 2011).
In this study, as a part of the 1 000 Plants (1KP) Project
(http://www.onekp.com/) which covers more than 1 000 different species of plants by generating large scale gene sequence information, we focused on the phylogenomic analysis of Chrom-
ista algae, especially for brown algae.
2 Materials and methods
2.1 Sequence datasets from 1KP project
The assembled transcriptomic sequencing datasets were
downloaded from the website of 1KP Project, including 19 brown
algae species (Phaeophyceae) (Table 1). Totally, 786 783 495
base pairs are used to search for orthologs. The 18 complete
mitochondrial and 13 chloroplastic genomes of Chromista
algae, belonging to Ochrophyta, Cryptophyta and Haptophyta,
respectively, were downloaded from NCBI (Table 2). Their gene
number ranges from 19 to 51 for mitochondria and 81 to 147
for plastids.
2.2 Constructing phylogenetic trees
We performed several procedures to construct maximum
likelihood trees which are described as follow. First, all gene
DNA/protein sequences of 18 mitochondrial and 13 chloroplastic genomes collected from NCBI website were used as
references (including the outgroup Cyanophora paradoxa,
NC_001675.1, Phylum Glaucophyta). Second, considering possible horizontal gene transfer or gene exchanges between nucleus and organelles, we identified 17/41 typical genes shared
by the complete mitochondrion/chloroplast genomes, aiming
at excluding potential nucleus-originated genes. Third, the protein sequences of typical reference genes were used to perform
local TBLASTN searches against the assembled transcript datasets of our 19 brown algae from 1KP Project to obtain putative
algal orthologs. The best hits were selected and their alignment
information was also stored to do pairing of references and
samples. We compared the filtering power of different cutoff
E-values at 10−5, 10−10, 10−20, 10−30, 10−40 and 10−50, to achieve
reliable orthologs. Fourth, phylogenetic trees were constructed
by MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with
bootstrap method 1 000.
Table 1. Brown algal (Class Phaeophyceae, Order Ochrophyta) species information with assembled contigs from 1KP Project
Order
Family
Species
Contig#
Tot/Mb
Avg/bp
Lon/bp
Desmarestiales
Desmarestiaceae
Desmarestia viridis
53 140
24.8
467
11 018
Dictyotales
Dictyotaceae
Dictyopteris undulata
100 199
49.6
495
14 604
Ishigeales
Ishigeaceae
Ishige okamurai
78 583
48.2
614
14 784
Ectocarpales
Scytosiphonaceae
Colpomenia sinuosa
80 884
43.5
538
1 509
Petalonia fascia
89 229
52.6
590
12 350
Laminariales
Fucales
Laminariaceae
Scytosiphon dotyo
69 680
40.9
587
10 507
Scytosiphon lomentaria
75 666
47.0
621
13 047
Kjellmaniella crassifolia
88 084
41.9
476
8 676
Laminaria japonica
76 104
31.7
417
10 968
Laminaria japonica-2
75 714
33.4
441
12 165
Alariaceae
Undaria pinnatifida
72 256
34.9
483
8 628
Sargassaceae
Hizikia fusifrome
116 790
48.0
411
8 731
Sargassum hemiphyllum
87 021
44.9
516
12 861
Sargassum henslowianum
77 474
38.0
491
10 826
Sargassum horneri
75 127
39.0
519
11 559
Sargassum integerrimum
82 503
37.5
455
8 371
Sargassum muticum
81 102
43.3
534
9 887
Sargassum thunbergii
106 596
49.0
459
10 151
Sargassum vachellianum
69 871
38.5
550
12 856
Notes: Tot, Avg and Lon indicate total length/Mb, average length/bp and longest length/bp of contigs, respectively. # indicates the
numbers of contigs.
Phaeophyceae
Cryptophyceae
Coccolithophyceae
Ochrophyta
Cryptophyta
Haptophyta
EF508371.1
Pavlovaceae
Phaeocystaceae
Phaeocystales
Pavlovales
Noelaerhabdaceae
Isochrysidales
Chroomonadaceae
NC_009573.1
NC_020371.1
JN117275.1
Phaeocystis antarctica-2
Pavlova lutheri
NC_016703.1
JN022705.1
Emiliania huxleyi-2
Phaeocystis antarctica
NC_007288.1
NA
Hemiselmis andersenii-2
Emiliania huxleyi
NA
Hemiselmis andersenii
NC_000926.1
Rhodomonas salina
NA
Saccharina religiosa
Rhodomonas salina-2
NA
Saccharina ochotensis
Pyrenomonadaceae
NA
GQ358203.1
NA
Saccharina japonica-2
Saccharina longipedalis
Cryptomonas paramecium-2
NC_018523.1
Saccharina japonica
NC_013703.1
NA
NA
Saccharina diabolica
Cryptomonas paramecium
138
NA
Saccharina coriacea
110
99
99
109
118
NA
NA
145
145
146
81
81
NA
NA
NA
NA
NA
NA
NA
Saccharina angustata
NA
NA
138
NA
147
NA
NA
Gene#
Laminaria digitata
NC_016735.1
Guillardia theta
Laminariaceae
Laminariales
Fucus vesiculosus
NA
Pylaiella littoralis
Geminigeraceae
Fucaceae
Fucales
NC_013498.1
Ectocarpus siliculosus
NA
Pyrennomonadales
Ectocarpaceae
Ectocarpales
Dictyota dichotoma
NA
NCBI Accession
Chloroplast
Cryptomonadaceae
Dictyotaceae
Dictyotales
Desmarestia viridis
Species
Cryptomonadales
Desmarestiaceae
Family
Desmarestiales
Order
Notes: # and size indicate the gene numbers of the mitochondrial and chloroplastic genomes, respectively.
Pavlovophyceae
Class
Phylum
Table 2. The complete genome information of mitochondria and chloroplasts of reference algae in NCBI
95 281
105 651
105 651
105 297
105 309
NA
NA
135 854
135 854
121 524
77 717
77 717
NA
NA
NA
NA
130 584
NA
NA
NA
NA
124 986
NA
139 954
NA
NA
Size
NA
NA
NA
JN022704.1
NC_005332.1
EU651892.1
NC_010637.1
NA
NC_002572.1
NA
NA
NA
NC_013477.1
NC_013478.1
NC_013484.1
NC_015669.1
NC_013476.1
NC_013482.1
NC_013475.1
NC_013473.1
NC_004024.1
NC_007683.1
NC_003055.1
NA
NC_007685.1
NC_007684.1
NCBI Accession
NA
NA
NA
19
20
43
43
NA
43
NA
NA
NA
37
37
37
37
37
37
37
37
38
37
51
NA
37
38
Gene#
Mitochondrion
NA
NA
NA
28 660
29 013
60 553
60 553
NA
48 063
NA
NA
NA
37 657
37 656
37 657
37 638
37 657
37 657
37 500
37 605
38 007
36 392
58 507
NA
31 617
39 049
Size
96
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
97
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
2.3 Ka/Ks calculation
Ka/Ks calculation was performed by using MA method
and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006)
against the reference Fucus vesiculosus. We used similar methods to search for DNA orthologs with TBLASTX, rather than
TBLASTN.
rpl2 and rps3) with the total length of 4 292 aa and 1 210 variation sites, it revealed two lineages: Phylum Ochrophyta group,
and the others including Order Cryptomonadales, Pyrennomonadales, Isochrysidales, Phaeocystales and Pavlovales in
Phylum Cryptophyta, and Haptophyta. All species in Phylum
Ochrophyta (brown algae) were clustered in a single clade,
and were further divided into two sub-clades. One encompasses Order Laminariales and Ectocarpales, and the other includes Order Fucales (Family Sargassaceae) (Fig. 1). The result
is consistent with previous results from Silberfeld et al. (2010).
In addition, Order Desmarestiales and Asterocladales are also
included in Laminariales–Ectocarpales clade, along with Order
Ascoseiriales, Scytothamnales and Sporochnales (Charrier et
al., 2012), while some brown algae which are not covered in this
study were found in Fucales clade, for example, Order Nemodermatales, Tilopteridales and Ralfsiales (Silberfeld et al., 2010).
Brown algal ancestor was estimated to date back to more
than 200 million years ago, with a high diversity (Silberfeld et al.,
2010). There are many differences in the morphology of brown
algae, such as pneumatocysts, receptacles and conceptacles in
3 Results and discussion
To achieve the most reliable results and keep all sample
species, we have evaluated the powers of different filtering Evalues of 10−5, 10−10, 10−20, 10−30 and 10−50, and achieved fewer
genes with higher E-values. Finally, we chose E-values of 10−5
and 10−30 for mitochondrial and chloroplastic analysis, respectively. After alignment, we got the concatenated gene orthologs
of 4 292 aa and 4 053 aa for chloroplast and mitochondrion, respectively, which were used to build phylogenetic trees.
3.1 Brown algal taxonomy based on the chloroplastic tree
In the maximum likelihood tree of 13 chloroplast genes
(rpoB, rpl14, rpl3, rpl6, tufA, rps12, atpB, rps5, rpl5, rpl16, atpA,
98
78
97
Sargassum integerrimum
Sargassum vachellianum
Sargassum henslowianum
Sargassum hemiphyllum
Hizikia fusiformis
Sargassum horneri
100
99
Fucales
Sargassum muticum
Sargassum thunbergii
Ishige okamurai Ishigeales
100
100
Laminaria japonica
Laminaria japonica2
Laminariales
Ochrophyta
Petalonia fascia
100
Scytosiphon lomentaria
Colpomenia sinuosa
78
100
0.05
92
100
77
Saccharina japonica_r
Kjellmaniella crassifolia
Fucus vesiculosus_r
Dictyopteris undulata
100
100
Desmarestiales
Undaria pinnatifida
Ectocarpus siliculosus_r
100
Laminariales
Ectocarpales
Fucales
Dictyotales
Rhodomonas salina2_r
Pyrennomonadales
Rhodomonas salina_r
Cryptophyta
Guillardia theta_r
90
(Phaeophyceae)
Scytosiphon dotyo
Desmarestia viridis
99
96
Ectocarpales
Cryptomonas paramecium2_r
100
Cryptomonas paramecium_r
Pavlova lutheri_r
100
64
Emiliania huxleyi2_r
Emiliania huxleyi_r
Phaeocystis antarctica2_r
100
100
Cryptomonadales
Pavlovophyceae
Coccolithophyceae
Haptophyta
Phaeocystis antarctica_r
Cyanophora paradoxa_r
Fig.1. Maximum likelihood tree based on 13 concatenated chloroplastic protein sequences. The reference species are marked with
“_r” following the species name. The tree was built in MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with bootstrap method 1000. Bootstrap values lower than 60% are not shown.
98
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
Order Fucales, and stipe or young holdfast in Order Laminariales. A two-step cell division process (polystichous construction)
with a terminal growth is synapomorphic for Order Syringodermatales, Sphacelariales, Dictyotales and Onslowiales (SSDO)
(Charrier et al., 2012).
3.2 Brown algal taxonomy based on the mitochondrial tree
In the maximum likelihood tree of 5 mitochondrial genes
(rpl16, cob, rps12, rps3, and nad1) with the total length 4 053 aa
and 200 variation sites, the algal relationships (Fig. 2) are similar
to that in the tree built with nucleus genes (data not shown).
Phylum Ochrophyta is obviously separated with Phylum Cryptophyta and Haptophyta. We identified a distinct division in
Class Phaeophyceae: Laminariales-Ectocarpales clade vs Fucales clade, indicating that Fucales evolved independently from
Laminariales-Ectocarpales for a long period of time. This is in
agreement with that proposed by Wynne and Loiseaux (1976)
based on their studies on algal life cycles. Furthermore, it was
suggested that the earliest diverging phaeophycean orders
were Discosporangiales, Ishigeales and Dictyotales (Kawai et
al., 2007), and that Ishige okamurai and Dictyopteris undulata
seemed to be apart from most of the brown algae in our results.
The phylogenetic relationships were confirmed further by
the tree based on available complete cox1 sequences in NCBI
(Fig. 3). And we found that algae of Phylum Ochrophyta were
divided into two subgroups, Class Phaeophyceae and the other
9 species belonging to Class Chrysophyceae, Synurophyceae,
Bacillariophyceae and Raphidophyceae.
3.3 Other discoveries of brown algae
Kjellmaniella crassifolia was always described based on appearance (Patron et al., 2007). Here, we confirmed the previous
Saccharina ochotensis_r
Saccharina religiosa_r
Saccharina longipedalis_r
85 Saccharina japonica_r
Saccharina japonica2_r
82
Saccharina diabolica_r
Laminaria japonica
Laminariales
Laminaria japonica2
Saccharina angustata_r
Saccharina coriacea_r
Undaria pinnatifida
94
Kjellmaniella crassifolia
Laminaria digitata_r
78
Pylaiella littoralis_r
82
Petalonia fascia
61
Scytosiphon lomentaria
80
Colpomenia sinuosa
78
98
Ectocarpales
Scytosiphon dotyo
Desmarestia viridis_r
100 Desmarestia viridis
Ochrophyta
(Phaeophyceae)
Desmarestiales
Fucus vesiculosus_r
0.1
Sargassum hemiphyllum
69
99
Sargassum thunbergii
Hizikia fusiformis
90
97
Sargassum henslowianum
Sargassum vachellianum
Sargassum muticum
83
100
Sargassum integerrimum
65
64
Sargassum horneri
Dictyota dichotoma_r
98
Dictyopteris undulata
Ishige okamurai
100
Emiliania huxleyi2_r
Emiliania huxleyi_r
Rhodomonas salina_r
87
Fucales
Hemiselmis andersenii2_r
100 Hemiselmis andersenii
Dictyotales
Ishigeales
Haptophyta
Cryptophyta
Fig.2. Maximum likelihood tree based on 5 concatenated mitochondrial protein sequences. The reference species are marked with
“_r” following the species name. The tree was built in MEGA 5.1 based on Jones-Taylor-Thornton (JTT) model with bootstrap method 1000. Bootstrap values lower than 60% are not shown.
0.66
0.93
1
1
1
1
0.88
1
1
1
1
1
1
1
NC_015117.1
NC_016739.1
Ectocarpales
Fucales
Desmarestiales
Dictyotales
Phaeodactylum tricornutum
Synedra acus NC_013710.1
Heterosigma akashiwo NC_016738.1
Heterosigma akashiwo2 GQ222227.1
Chattonellales
Heterosigma akashiwo3 GQ222228.1
Chattonella marina NC_013837.1
Rhodomonas salina NC_002572.1
Hemiselmis andersenii NC_010637.1
Chondrus crispus NC_001677.2
1
Plocamiocolax pulvinata NC_014773.1
Gracilariophila oryzoides NC_014771.1
0.71
1
Gracilariopsis lemaneiformis JQ071938.1
Gracilariopsis andersonii NC_014772.1
1 Porphyra purpurea NC_002007.1
1
Pyropia yezoensis NC_017837.1
Porphyra umbilicalis NC_018544.1
Pyropia haitanensis NC_017751.1
Cyanidioschyzon merolae NC_000887.3
1
Cyanophora paradoxa NC_017836.1
Glaucocystis nostochinearum
1
1
Laminariales
Glaucophyta
Rhodophyta
Cryptophyta
Ochrophyta
Haptophyta
(Phaeophyceae)
Ochrophyta
followed by their NCBI accession number. Topology shows that there are three groups (Ochrophyta & Haptophyta, Cryptophyta & Rhodophyta, and Glaucophyta). Phylum Ochrophyta is divided into the two subgroups, Phaeophyceae and the others.
Fig.3. MrBayes tree based on the cox1 protein sequences exacted from publicly available mitochondrial genomes of Kingdom Chromista and Phylum Rhodophyta. Species names are
0.83
0.06
1
1
Saccharina religiosa NC_013477.1
Saccharina ochotensis NC_013478.1
1 Saccharina longipedalis NC_013484.1
Saccharina japonica NC_013476.1
1
Saccharina japonica2 NC_015669.1
Saccharina diabolica NC_013482.1
1
Saccharina angustata NC_013473.1
1
Saccharina coriacea NC_013475.1
Laminaria digitata NC_004024.1
Pylaiella littoralis NC_003055.1
Fucus vesiculosus NC_007683.1
Desmarestia viridis NC_007684.1
Dictyota dichotoma NC_007685.1
Emiliania huxleyi2 JN022704.1
Emiliania huxleyi NC_005332.1
Ochromonas danica NC_002571.1
Chrysodidymus synuroideus NC_002174.1
Thalassiosira pseudonana NC_007405.1
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
99
100
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
finding that K. crassifolia (now as Saccharina crassifolia) should
be placed in the clade of Laminariales (Charrier et al., 2012). It
should be noted that Saccharina and Laminaria are in a single
clade in the mitochondrial tree, but we identified Saccharina japonica in a distinct subgroup together with Ectocarpales in the
chloroplastic tree. It suggests that Saccharina and Laminaria
are in the same Order Laminariales, but they still show some
differences in the evolution of mitochondrion and chloroplast.
Hizikia fusifrome (Harvey) Okamura, which is shown as a
taxon of Sargassaceae, Phaeophyta (Ochrophyta) in algaeBASE,
is a brown sea vegetable called hijiki. Even now, there is a dispute for its taxonomic position to be named as Sargassum fusiforme (Burki et al., 2012; Sheng et al., 2011). Our results recognize its close relationship to genus Sargassum (Figs 1 and 2).
3.4 Non-brown Chromista algal taxonomy
Notably, non-brown Chromista algae show the same scenario away from Phaeophyceae. However, it was interesting that
the Haptophyta were within the Ochrophyta clade when analyzed based on cox1 protein sequences (Fig. 3), while they were
placed in the clade together with the Cryptophyta in Figs 1 and 2
based on multiple genes of chloroplasts and mitochondria. The
Haptophyta is a phylum of chlorophyll a/c, which was ever considered as a single class (Prymnesiophyceae Hibberd, or Haptophyceae Christensen ex Silva) and later was believed to be
composed of two distinct classes (Prymnesiophyceae and Pavlovophyceae) (Edvardsen et al., 2000). In algaeBASE, there are
three classes for Haptophyta: Prymnesiophyceae, Haptophyta
incertae sedis and Pavlovophyceae. The Prymnesiophyceae's
plastids are surrounded by four membrane layers indicating a
history of successive endocytosis. The Class Prymnesiophyceae
can be divided into four orders: Phaeocystales ord. nov., Prymnesiales, Isochrysidales, and Coccolithales.
Furthermore, we found that Rhodomonas salina and
Hemiselmis andersenii (Cryptophyta) are sisters to the Rhodophyta clade based on cox1 protein sequences (Fig. 3), suggesting that their mitochondria have a close relationship to
red algae. It was shown that cryptomonads are sisters to the
haptophyte algae, which also have a red-algal secondary plastid (Patron et al., 2007). The earliest secondary endosymbiosis
in Chromista, in which a host captured a red algal plastid, occurred about 1 274 million years ago (MYA). The cryptophyte
(Cryptophyta) first split about 1 189 MYA, and then the stramenopiles (Ochrophyta) and haptophytes (Haptophyta) split
about 1 047 MYA (Yoon et al., 2004).
The plastid genome of cryptophyte alga Guillardia theta is
almost completely comprised of clusters of genes that are found
on the rhodophyte Porphyra purpurea, confirming its common
ancestry with red algae that was suggested by Douglas and Penny (1999). There is considerable evidence that cryptomonad (or
cryptophytes in Phylum Cryptophyta) chloroplasts are closely
related to those of the heterokonts and haptophytes, and that
the three groups are sometimes united as the Chromista.
3.5 Differences between mitochondrial and chloroplastic
trees
We noticed the interesting fact that among Chromista algae, chloroplastic and mitochondrial genes suggested different phylogenies. According to our results, mitochondrial tree is
more conserved than the tree based on chloroplast genes, as we
found some differences in the Ochrophyta clade. For example, I.
okamurai is very close to Fucales based on chloroplastic genes
in Fig. 1, while in Fig. 2, we identified the mostly accepted ancient phylogenetic position for I. okamurai and D. undulata.
Ectocarpales and Laminariales seem to be split. One of the reasons might be that more genes were involved in building trees
(13 vs 5 genes used for chloroplast and mitochondrion, respectively), and phylogenetic structures were complicated. Another
key reason might be that the taxonomies depend on extracted
origin-unknown genes, whose evolution rate can introduce
modifications of phylogenetic tree. The following points should
be addressed here. First, a single gene has relatively limited diversity information, and phylogenetic trees must be constructed on the consensus regions of concatenated gene sequences.
Second, more algae species result in short consensus sequences
and less variations for building trees. Third, coding genes are
under various evolutionary pressures with diverse evolutionary rates. Finally, “endosymbiotic gene transfer (EGT) translocated chloroplast/mitochondrion-originated encoded protein
into the nucleomorph, and our extraction methods could not
distinguish whether they were originally from chloroplast/mitochondrion, or pseudogene transcripts in the nuclear genome.
However, EGTs have been shown to leave a footprint of ancient
photosynthetic activity in the nuclear genome (Burki et al.,
2012).
To assess evolutionary rates for individual genes, we calculated nonsynonymous (Ka) substitution rate, synonymous
(Ks) substitution rate, and Ka/Ks values by using MA method
and standard code of KaKs_Calculator 1.2 (Zhang et al., 2006)
against the reference F. vesiculosus. The ratio (Ka/Ks) indicates
neutral mutation (Ka/Ks = 1), negative (purifying) selection
(Ka/Ks < 1), and positive (diversifying) selection (Ka/Ks > 1).
Our results showed that the average Ks is much more than the
average Ka (3.53 vs 0.13 for chloroplast, and 3.98 vs 0.20 for mitochondrion), suggesting the genes' conservation (average Ka/
Ks: 0.043 and 0.057 for chloroplast and mitochondrion, respectively), and it seems the variances of Ka and Ks are associated
with their average values.
References
Burger G, Nedelcu A M. 2012. Mitochondrial genomes of algae. Berlin:
Springer Science & Business Media B V, 127–157
Burki F, Flegontov P, Oborník M, et al. 2012. Re-evaluating the green
versus red signal in eukaryotes with secondary plastid of red algal origin. Genome Bio Evol, 4(6): 738–747
Charrier B, Le Bail A, de Reviers B. 2012. Plant Proteus: brown algal
morphological plasticity and underlying developmental mechanisms. Trends Plant Sci, 17(8): 468–477
Cho G Y, Lee S H, Boo S M. 2004. A new brown algal order, Ishigeales
(Phaeophyceae), established on the basis of plastid proteincoding rbcL, psaA, and psbA region comparisons. J Phycol, 40(5):
921–936
Danne J C, Gornik S G, Waller R F. 2012. An assessment of vertical inheritance versus endosymbiont transfer of nucleus-encoded genes
for mitochondrial proteins following tertiary endosymbiosis in
Karlodinium micrum. Protist, 163(1): 76–90
Douglas S E, Penny S L. 1999. The plastid genome of the cryptophyte
alga, Guillardia theta: complete sequence and conserved synteny groups confirm its common ancestry with red algae. J Mol
Evol, 48(2): 236–244
Edvardsen B, Eikrem W, Green J C, et al. 2000. Phylogenetic reconstructions of the Haptophyta inferred from 18S ribosomal DNA
sequences and available morphological data. Phycologia, 39(1):
19–35
JIA Shangang et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 2, P. 94–101
Gray M W, Burger G, Lang B F. 2001. The origin and early evolution of
mitochondria. Genome Biol, 2(6): 1018.1–1018.5
Green B R. 2011. After the primary endosymbiosis: an update on the
chromalveolate hypothesis and the origins of algae with Chl c.
Photosynth Res, 107(1): 103–115
Hallstrom B M, Janke A. 2009. Gnathostome phylogenomics utilizing
lungfish EST sequences. Mol Biol Evol, 26(2): 463–471
Jia Shangang, Wang Xumin, Liu Guiming, et al. 2011. Gene expression
analysis of “green tide alga Ulva prolifera (Chlorophyta) in China. Genes Genom, 33(2): 173–178
Katz L A. 2012. Origin and diversification of Eukaryotes. Annu Rev Microbiol, 66: 411–427
Kawai H, Hanyuda T, Draisma S G A, et al. 2007. Molecular phylogeny
of Discosporangium mesarthrocarpum (Phaeophyceae) with
a reinstatement of the order Discosporangials. J Phycol, 43(1):
186–194
McFadden G I. 2001. Primary and secondary endosymbiosis and the
origin of plastids. J Phycol, 37(6): 951–959
Medlin L K, Cooper A, Hill C, et al. 1995. Phylogenetic position of the
Chromista plastids based on small subunit rRNA coding regions.
Curr Genet, 28(6): 560–565
Patron N J, Inagaki Y, Keeling P J. 2007. Multiple gene phylogenies support the monophyly of Cryptomonad and Haptophyte host lineages. Curr Biol, 17(10): 887–891
101
Phillips N, Burrowes R, Rousseau F, et al. 2008. Resolving evolutionary relationships among the brown algae using chloroplast and
nuclear genes. J Phycol, 44(2): 394–405
Rodríguez-Ezpeleta N, Brinkmann H, Burey S C, et al. 2005. Monophyly
of primary photosynthetic eukaryotes: green plants, red algae,
and glaucophytes. Curr Biol, 15(14): 1325–1330
Sheng Yingwen, Zhang Wen, Zhao Dan, et al. 2011. A morphological and molecular assessment of the genus Sinotubimorpha
(Halymeniaceae, Rhodophyta). J Syst Evol, 50(2): 146–152
Silberfeld T, Leigh J W, Verbruggen H, et al. 2010. A multi-locus time-calibrated phylogeny of the brown algae (Heterokonta, Ochrophyta, Phaeophyceae): Investigating the evolutionary nature of the
“brown algal crown radiation. Mol Phylogen Evol, 56(2): 659–674
Tan I H, Druehl L D. 1994. A molecular analysis of Analipus and Ralfsia
(Phaeophyceae) suggests the order Ectocarpales is polyphyletic.
J Phycol, 30(4): 721–729
Wynne M J, Loiseaux S. 1976. Recent advances in life history studies of
the Phaeophyta. Phycologia, 15(3): 435–452
Yoon H S, Hackett J D, Ciniglia C, et al. 2004. A molecular timeline for
the origin of photosynthetic eukaryotes. Mol Biol Evol, 21(5):
809–818
Zhang Zhang, Li Jun, Zhao Xiaoqian, et al. 2006. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomic Proteomic Bioinform, 4(4): 259–263