A broad genomic survey reveals multiple origins and frequent...
Transcript of A broad genomic survey reveals multiple origins and frequent...
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A broad genomic survey reveals multiple origins and frequent losses in the
evolution of respiratory hemerythrins and hemocyanins
José M. Martín-Durán§1*, Alex de Mendoza§2,3, Arnau Sebé-Pedrós§2,3, Iñaki Ruiz-Trillo2,3,4,
Andreas Hejnol1
1Sars International Centre for Marine Molecular Biology
University of Bergen
Thormøhlensgate 55
5008 Bergen, Norway
2Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra)
Passeig Marítim de la Barceloneta, 37–49
08003 Barcelona, Spain
3Departament de Genètica, Universitat de Barcelona
Av. Diagonal 645
08028 Barcelona, Spain
4Institució Catalana de Recerca i Estudis Avançats (ICREA)
Passeig Lluís Companys, 23
08010 Barcelona, Spain
§These authors contributed equally to this work.
*Corresponding author:
José M. Martín-Durán. Sars International Centre for Marine Molecular Biology. University
of Bergen, Bergen, Norway. Phone +47-55584291. Fax +47-55584305. [email protected]
© The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]
Genome Biology and Evolution Advance Access published July 9, 2013 doi:10.1093/gbe/evt102 by guest on July 15, 2013
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Abstract
Hemerythrins and hemocyanins are respiratory proteins present in some of the most ecologically
diverse animal lineages; however the precise evolutionary history of their enzymatic domains
(hemerythrin, hemocyanin M and tyrosinase) is still not well understood. We survey a wide dataset
of prokaryote and eukaryote genomes and RNAseq data to reconstruct the phylogenetic origins of
these protiens. We identify new species with hemerythrin, hemocyanin M and tyrosinase domains in
their genomes, particularly within animals, and demonstrate that the current distribution of
respiratory proteins is due to several events of lateral gene transfer and/or massive gene loss. We
conclude that the last common metazoan ancestor had at least two hemerythrin domains, one
hemocyanin M domain and six tyrosinase domains. The patchy distribution of these proteins among
animal lineages can be partially explained by physiological adaptations, making these genes good
targets for investigations into the interplay between genomic evolution and physiological
constraints.
Keywords: comparative genomics, hemerythrin, hemocyanin, tyrosinase, respiration, lateral gene
transfer.
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Text
Hemoglobins, hemerythrins and hemocyanins are three different respiratory proteins present
in animals (Terwilliger 1998). Hemoglobins have a Fe–protoporphyrin ring to reversibly bind
oxygen, and are the most common molecules for oxygen transport and storage in the Bilateria
(Weber and Vinogradov 2001). Globin proteins are widespread in the tree of life and, in animals,
respiratory globins likely evolved from a membrane bound ancestor that acquired a respiratory
function independently in different lineages (Blank and Burmester 2012; Roesner, et al. 2005). In
contrast to the widespread hemoglobins, hemerythrins and hemocyanins have been detected in
fewer animal groups. Hemerythrins transport oxygen using two Fe2+ ions that bind directly to the
polypeptide chain, and have been described in a cnidarian (Nematostella vectensis), priapulids,
brachiopods, some annelids and sipunculans (Bailly, et al. 2008; Terwilliger 1998). Recently, it has
been shown that regulation of iron homeostasis in vertebrates involves an E3 ubiquitin ligase
(FBXL5 gene) with an iron-responsive hemerythrin domain in its structure (Salahudeen, et al. 2009;
Vashisht, et al. 2009), although it is not clear how this hemerythrin domain-containing protein is
related to invertebrate respiratory hemerythrins. Hemocyanins are large proteins that have copper
binding sites to transport oxygen in arthropods and mollusks (Bonaventura and Bonaventura 1980).
Despite their shared name, arthropod and molluscan respiratory hemocyanins are considered to
have evolved independently from a common ancestral copper protein based on protein similarities
(Burmester 2001; van Holde, et al. 2001). A deeper understanding of the evolutionary history of
hemerythrins and hemocyanins, and thus of the different respiratory strategies in animals, is limited
by the absence of data for many invertebrate groups and, in particular, for unicellular holozoans and
other eukaryotes. In this study, we survey a wide phylogenetic distribution set of genomes and
RNAseq data to identify new hemerythrin and hemocyanin proteins, and we then reconstruct their
evolution within the eukaryote tree of life, with particular focus on animal lineages.
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In addition to the respiratory hemerythrin sequences previously characterized in animals
(Bailly, et al. 2008; Meyer and Lieb 2010; Vanin, et al. 2006), we identified respiratory
hemerythrins in the priapulid Priapulus caudatus, the arthropod Calanus finmarchicus, and the
bryozoans Alcyonidium diaphanum and Membranipora membranacea (see Supplementary File S1).
In bryozoans, no respiratory proteins have been previously described, despite the presence of a
circulatory system in these animals (Schmidt-Rhaesa 2007). Differently from other animal
hemerythrins, the hemerythrin domain shows a Ca2+-binding EF-hand domain in its N-terminal
region in both bryozoan species. Additionally, we identified an E3 ubiquitin ligase containing an F-
box domain together with a hemerythrin domain, as in FBXL5, in cnidarians and across bilaterally
symmetrical animals (see Supplementary Table S1), suggesting an ancient origin of the iron-sensing
system described in vertebrates. Our phylogenetic analyses show two major clades of hemerythrin-
containing proteins (Figure 1), one comprising the metazoan FBXL5 gene and most of the eukaryote
hemerythrins (clade A), and the other comprising the metazoan respiratory hemerythrins (including
the newly identified sequences from this study) (clade B). As shown in a previous report (Bailly, et
al. 2008), respiratory hemerythrins are closely related to some Naegleria gruberi hemerythrins, and
a sequence from the amoebozoan Acanthamoeba castellanii. To eliminate possible bacterial
contamination, we checked the gene structure and confirmed that the Acanthamoeba gene has
introns within the hemerythrin domain. The two major clades (A, non-respiratory, and B,
respiratory) are separated with high nodal support, which is in agreement with observed structural
differences (Histidine 74 being only present in clade B) (Thompson, et al. 2012). Interestingly,
clade B hemerythrins seem to be more common and highly diversified in prokaryotes (French, et al.
2008) than in eukaryotes. Metazoans may have acquired them during an ancient event of lateral
gene transfer (LGT), but, according to our phylogeny, it is more likely that clade B hemerythrins are
ancient and have been lost in many eukaryotic lineages, so far only present in three extant distant
lineages: Amoebozoa, Excavata and Metazoa. Clade B prokaryote hemerythrins have been shown
to bind oxygen as metazoan respiratory hemerythrins (Xiong, et al. 2000), but have been mostly
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related to oxygen sensing and aerotaxis processes (Isaza, et al. 2006; Xiong, et al. 2000) and oxygen
supply to other metabolic enzymes (Karlsen, et al. 2005). This suggests that the oxygen storage and
transport function of hemerythrins may have evolved independently in metazoans, given the lack of
functional data for the Excavata and the Amoebozoa. Under this scenario, the role of respiratory
hemerythrins in iron storage, metal detoxification and immunity observed in some annelids (e.g. the
leeches Theromyzon tessulatum and Hirudo medicinalis and the polychaete Neanthes diversicolor)
(Baert, et al. 1992; Demuynck, et al. 1993; Vergote, et al. 2004) are secondary specializations of this
type of proteins. Finally, non-respiratory hemerythrins (clade A) are quite common in eukaryotes,
and have recruited several companion domains in different lineages.
Searches for the hemocyanin M domain (arthropod hemocyanins) identified this copper
binding protein in amoebozoans, the fungus Aspergilus niger, the sponge Amphimedon
queenslandica, the ctenophore Mnemiopsis leidyi and the hemichordate Saccoglossus kowalevski
(Figure 2). Therefore it is likely to be a unikont synapomorphy. Our phylogenetic analyses show the
monophyly of all metazoan sequences, as well as of the main arthropod protein families (see
Supplementary Fig. S1). The relationship of the sponge and fungal sequences may be due to an
ancient LGT, though the presence of hemocyanins in the more distant amoeobozoans does not
support that idea. Moreover the A. niger gene has a N-terminal intron and is located between two
other fungal genes, making it less likely to come from a recently incorporated segment of metazoan
DNA. Furthermore, a pseudo-gene with a hemocyanin M domain is present in the fungus
Neosartorya fischeri (a congeneric species despite the name), but absent from all 6 other
Aspergillus genomes and also from all the other fungi sequenced to date. The newly identified
sequences in this study demonstrate that the N-domain of arthropod hemocyanins and related
proteins is a specific molecular signature of the Panarthropoda (Onychophora+Arthropoda),
although there is some degree of similarity of these regions in non-arthropod sequences. The
presence of hemocyanin-like proteins in the tunicate Ciona intestinalis with putative phenoloxidase
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activity suggested that respiratory hemocyanins evolved from an ancestral prophenoloxidase
(Immesberger and Burmester 2004). Given the absence of functional data for the non-bilaterian
animals (i.e. the ctenophore M. leidyi and the sponge A. queenslandica) and our phylogeny (Figure
2), this is still the most parsimonious functional explanation for the evolution of the respiratory
properties of arthropod hemocyanins.
An extensive search for the tyrosinase domain (molluscan hemocyanin) demonstrated a
wide distribution of this copper-binding protein across metazoan lineages (see Supplemental Table
S1), with the remarkable exception of arthropods. The absence in arthropods could be associated
with the expansion and diversification of the hemocyanin M domain in this lineage, which can
exhibit similar activities to the tyrosinase domain, for example in melanin biosynthesis (Sugumaran
2002). In contrast to a previous analysis (Esposito, et al. 2012), our phylogenetic reconstruction of a
broader dataset shows that the animal tyrosinase domains group in 6 independent clades (clades A–
F) (Figure 3), which are further supported by the domain architecture of the proteins nested in each
clade (e.g. clade D and clade F). The tyrosinase domain that gave rise to the molluscan
hemocyanins is related to brachiopod and tunicate sequences (clade B), and the series of
duplications that lead to the typical arrangement of 8 tyrosinase domains in tandem (Bonaventura
and Bonaventura 1980) is specific to mollusks. With the exception of clade E, which is restricted to
non-bilateral animals (Figure 3), the other clades exhibit an extremely patchy distribution across
bilaterally symmetrical animals (see Supplementary Table S1), not only between major animal
groups, but also within the same group (e.g. in mollusks, Crassostrea gigas has only a clade D
tyrosinase, Lottia gigantea has clade A and D tyrosinases, and Sepia officinalis has both clade B and
D tyrosinases). Despite the poor resolution of deeper nodes, our phylogenetic scenario at least
strongly supports 3 independent origins of metazoan tyrosinases. Clades A, B and F are well
supported (PP>0.9) and nested with non-metazoan sequences. The other 3 clades (clades C, D and
E) are not robustly supported, but have unique domain architectures and do not significantly cluster
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with other metazoan groups, therefore they might also come from independent origins. Moreover,
our phylogenetic analysis demonstrates that the tyrosinase-containing proteins of plants likely
originated due to a LGT event from bacteria, corroborated by these proteins exhibiting the same
domain architecture (see Figure 3).
Altogether, our data clarify the origins and evolutionary history of the alternative respiratory
strategies observed in animals (Figure 4). Respiratory hemerythrins, arthropod hemocyanins and
molluscan respiratory tyrosinases originated independently from enzymatic domains that were most
likely already present in the last common metazoan ancestor. Although their function in early
branching lineages that do not possess circulatory systems needs to be elucidated (e.g. the function
of hemerythrins in the cnidarian N. vectensis or the hemocyanin M domain in sponges and
ctenophores), the co-option of these domains for respiratory purposes occurred independently, and
most likely took place at the base of the Protostomia (hemerythrin), the (Pan-)Arthropoda
(arthropod hemocyanins), and the Mollusca (molluscan tyrosinase “hemocyanins”). Accordingly,
the similarities observed between arthropod and molluscan hemocyanins (e.g. use of copper to
reversibly bind oxygen as a respiratory strategy, oligomerization and secretion to the hemolymph)
are the result of convergent evolution. The evolutionary history of hemerythrins and hemocyanins is
characterized by frequent losses, even after a respiratory function has been acquired when a higher
selective pressure against loss could be expected. For instance, the use of tyrosinase as an oxygen
transport molecule seems to be absent in some groups of mollusks, such as solenogasters and
pteriomorphids (e.g. C. gigantea, as also shown in this study) (Lieb and Todt 2008), in which it was
probably replaced by other respiratory proteins that have evolved independently in these lineages.
This is the case for gastropods in the group Planorbidae, which lack hemocyanin in their
hemolymph and which utilize an extracellular hemoglobin (evolved from an intracellular
myoglobin present in the gastropod radula muscle) as an alternative strategy for oxygen transport.
This high molecular mass hemoglobin has a higher affinity for oxygen than the ancestral
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hemocyanin (Lieb, et al. 2006). Similar adaptations are also observed within the Crustacea, such as
in branchiopods, ostracods, copepods, cirripeds and decapods, which lost hemocyanins and evolved
hemoglobins as respiratory proteins (Terwilliger and Ryan 2001). In the water flea Daphnia magna,
for instance, the tandemly duplicated gene cluster of hemoglobin genes shows multiple hypoxia
inducible factor (HIF) binding sites, which dramatically induce the expression of hemoglobins
when daphnids are exposed to hypoxia (Gorr, et al. 2004; Kimura, et al. 1999). The extremely
patchy distribution of these proteins across the animal phylogeny can be partially understood by the
different biochemical properties of their oxygen binding domains and the changing physiological
needs of each particular animal lineage, that make one or the other respiratory protein more
effective in their function as oxygen carriers. Recent studies show that many enzymatic genes have
complex evolutionary histories, with massive gene losses in most of the eukaryote genomes
sampled, but retention in certain tips of the tree of life (Allen, et al. 2011; Attenborough, et al. 2012;
de Mendoza and Ruiz-Trillo 2011; Stairs, et al. 2011). In contrast, transcription factors, signaling
pathways and adhesion molecules, for instance, can be traced back in a congruent phylogenetic
pattern (Pang, et al. 2010; Sebe-Pedros, et al. 2011; Sebe-Pedros, et al. 2010; Srivastava, et al.
2010). In some cases, the patchy phylogenetic distribution observed in enzymatic families could be
explained by multiple events of LGT, although the phylogenetic signal is often not strong enough.
Together with gene structure and synteny analysis we do not find strong evidences of LGT, with the
exception of plant tyrosinases (see above). Moreover, the study of the evolution of respiratory
proteins emerges as an ideal model to study the interplay between molecular evolution, biochemical
constraints and physiological-ecological needs.
Material and Methods
All potential hemerythrin, hemocyanin and tyrosinase sequences were identified by
HMMER searches against the Protein, Genome, and EST databases at the NCBI (National Center
for Biotechnology Information), and against completed genome/transcriptome projects databases
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publicly available or that are being conducted in our laboratories (sequences available in
Supplementary File S1) with the default parameters and an inclusive E-value of 0.05. The retrieved
sequences were aligned using MAFFT (Katoh, et al. 2002) L-INS-i algorithm, and then manually
inspected to remove those hits fulfilling one of the following conditions: 1) incomplete sequences
with more than 99% sequence identity to a complete sequence from the same taxa; 2) sequences
that showed extremely long branches in the preliminary maximum likelihood trees; and 3) incorrect
gene model predictions. The final alignment was carried out using the MAFFT G-INS-i algorithm
(for global homology). Maximum likelihood (ML) phylogenetic trees were estimated by RaxML
(Stamatakis 2006) and the best tree from 100 replicates was selected. Bootstrap support was
calculated from 1,000 replicates. Bayesian inference (BI) analyses were performed with
PhyloBayes (Lartillot and Philippe 2004), using two parallel runs for 500,000 generations and
sampling every 100. Bayesian posterior probabilities (BPP) were used for assessing the statistical
support of each bipartition. The domain architecture of all retrieved sequences was inferred by
performing a Pfam scan with the gathering threshold as cut-off value. The domain information was
used to assess the reliability of each sequence of the initial dataset, to help define protein families
according to their architectural coherence, and to assess the level of functional and structural
diversification of hemerythrins, hemocyanins and tyrosinases across the eukaryote lineages.
Acknowledgements
The authors thank J. Ryan for his advice and M. leidyi sequences, members of the S9 lab for
collecting material and discussion, G. Richards for reading and improving the manuscript, G.
Torruella and X. Grau-Boved for their help and J. Achatz for his support. This work was funded by
the Sars International Centre for Marine Molecular Biology to J.M.M.-D. and A.H., and an ICREA
contract, an European Research Council Starting Grant (ERC-2007-StG-206883), and a grant
(BFU2011-23434) from Ministerio de Economía y Competitividad (MINECO) to I.R.-T. A.S.-P.’s
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salary was supported by a pregraduate FPU grant and A.d.M.’s salary from a FPI grant, both from
MICINN.
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chemotaxis protein. Biochemistry 39: 5117-5125. by guest on July 15, 2013http://gbe.oxfordjournals.org/
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Figure Legends
Fig. 1— Maximum likelihood (ML) phylogenetic tree of the hemerythrin domain as obtained by
RAxML. The tree is rooted using the midpoint-rooted tree option. 1,000 replicate bootstrap values
(BV, in black) and Bayesian posterior probabilities (BPP, in red) are shown for each node. A black
dot in the node indicates BV>95% and BPP>0.95. Metazoan hemerythrins are highlighted by
colored rectangles. Domain architectures are shown for major lineages (abbreviations and accession
numbers of each domain are listed in Supplementary File S2).
Fig. 2— Maximum likelihood (ML) phylogenetic tree of the hemocyanin M domain as obtained by
RAxML. The tree is rooted using the amoebozoan hemocyanin genes as outgroup. 1,000 replicate
bootstrap values (BV, in black) and Bayesian posterior probabilities (BPP, in red) are shown for
each node. A black dot in the node indicates BV>95% and BPP>0.95. Metazoan and panarthropod
hemocyanins are highlighted by colored rectangles. The Aspergilus niger hemocyanin sequence is
enclosed by a red circle. Domain architectures are shown for major lineages (abbreviations and
accession numbers of each domain are listed in Supplementary File S2).
Fig. 3— Maximum likelihood (ML) phylogenetic tree of the tyrosinase domain as obtained by
RAxML. The tree is rooted using the midpoint-rooted tree option. 1,000 replicate bootstrap values
(BV, in black) and Bayesian posterior probabilities (BPP, in red) are shown for each node. A black
dot in the node indicates BV>95% and BPP>0.95. Metazoan tyrosinase clades are highlighted by
colored rectangles. Domain architectures are shown for major lineages (abbreviations and accession
numbers of each domain are listed in Supplementary File S2).
Fig. 4– Scenario for the evolution of hemerythrins and hemocyanins in metazoans. As shown in this
study, the metazoan ancestor likely had 2 different hemerythrin domains (oxygen subtype and iron-
related subtype), one hemocyanin M domain and 6 independent tyrosinases (A–F subtypes). In the
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last common ancestor to cnidarians and bilaterians, the iron-related hemerythrin subtype evolved
into FBXL5, an E3 ubiquitin ligase involved in iron homeostasis. The diversification of bilaterally
symmetrical animals was accompanied by extensive independent losses of these proteins in the
different lineages, which can be partially related to the colonization of new niches and
environments. The adaptation of the oxygen hemerythrin subtype to respiratory purposes occurred
at least in the last common ancestor of protostome animals. On the contrary, the adaptation of the
hemocyanin M domain and the clade B tyrosinases to oxygen transport and storage occurred
independently in panarthropods and mollusks, respectively. Results from groups indicated with a 1
superscript originated from RNAseq data.
Supplementary Material
Table S1– List of main groups analyzed, with special focus on metazoans, and presence (black dot)/
absence (white dot) of the different clades of hemerythrins, hemocyanins and tyrosinases.
1The tyrosinase domain was not found in other flatworm genomes.
2Other annelids, such as Helobdella robusta, and sipunculids contain respiratory hemerythrins in
their genomes.
Table S2– List of domains detected in the study, with the abbreviations used in the main figures and
their respective PFAM accession numbers.
Supplementary File S1– Sequences used in the phylogenetic analyses. For each sequence,
accession number (where possible), species name, and sequence information (gene, domain and/or
phylogenetic clade) is indicated.
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Phaeodactylium tricornutum
Thalassiosira pseudonana
Achantamoeba castellaniiAchantamoeba castellanii
Capsaspora owczarzakiAchantamoeba castellanii
Achantamoeba castellanii
Chlorella variabilis
Capsaspora owczarzaki
Bacteria
Ectocarpus siliculosus
Spizellomyces punctatusMicromonas pusilla
Ectocarpus siliculosusClamydomonas reinhardtii
Achantamoeba castellanii
Bacteria
2.0
MetazoanFBXL5
Haptophyta
Fungi
Bacteria
ChlorophytaStramenopile
Amoebozoa
unicellular Holozoa
green algae
Fungi
Fungi
Fungi
Embryophyta
Fungi
Trichomonas vaginalis
unicellular Holozoa
unicellular Holozoa
Archaeplastida
Embryophyta
unicellular Holozoa
Metazoanhemerythrins
Naegleria gruberi
Bacteria
Bacteria
Bacteria
Bacteria
Bacteria
Archaea
Archaea
Naegleria gruberi
Archaea
BacteriaBacteria
BacteriaBacteria
BacteriaBacteria
Archaea
Naegleria gruberi
Ministeria vibrans
Bacteria
Naegleria gruberi
Naegleria gruberiNaegleria gruberi
Ministeria vibrans
Volvox carteri
Volvox carteri
Cyanophora paradoxa
Bacteria
BacteriaMonosiga brevicollis
4810
10
0
518
3
47
23
9
8
00
1
1
1
49
92
88
62
0
96
0
1
2
11
2
85
14
8
47
19
94
6959
39
51
4046
86
3
8242
14
41
21
38
14
16
2089
20
2311
1135
24
31
11
1615
53
3632
1615
96
3
370
41
52
9123
17
2
93
010
0
0
1
2
18
6
30
30
Hry F-boxLRR
Hry
HryEF-hand (Bryozoa)
1
0.99
0.99
0.77
0.97
0.67
0.59
--
-
-
-
-
0.91
-
-
-
-
-
-
-
-
-
-
-
-
0.66
-
1
0.99
-
0.82
-
0.9
-
-
-
-
-
-
-
-
0.97-
-
0.99
0.980.63
1
0.99
0.9
1
0.79
-
-
-
0.970.86
1
-
-
-
-
-0
-
0.99
1
0.55
0.88
-
0.95
0.6
0.81
--
-
0.87
0.52
--
0.62
1
0.82-
-
0.82
0.59
0.95
-
-
Hry
Hry
Hry
Hry
Hry
Hry
Hry
Hry PolyCyc2
Hry
Hry
HryADK Bromo
Hry
Hry
Hry
HryScdA
Hry
Hry
HryMCPsign
Hry AMP bind
HryMCPsign
Hry
HryHry
HryAnk2 Ank
Hry
HryMCPsignHAMP
HryMCPsignLactB cNMPbcNMPb
HryMCPsign
HryMCPsignHAMP
Hry
HryAnk2RasGEFNADbFADbFlavo1
Hry
GST N3 GST C2 Hry
HryGST N3
Hry zfCHY zfRING2Hry Hry
Hry Hry zfRING2
zfCHY zfRING2Hry Hry
zfCHY zfRING2Hry
zfCHYHry Hry
Hry
Hry zfCHY zfRING2Hry Hry zfRING2
HryDUF4395 C4d ma tran
GlutRed HryDUF4395 C4d ma tran
GlutRed HryC4d ma tran
ECH GlutRed HryDUF4395 C4d ma tran
Hry
Iron metabolism
O2 transport
Clade A
Clade B
1
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Saccoglossus kowalevskii
Cheliceratehemocyanins
Myriapodhemocyanins
Crustaceancryptocyanins
Crustaceanhemocyanins
Insecthexamerins
InsectproPhenolOxidases
CrustaceanproPhenolOxidases
Tunicata
Ctenophora
Epiperipatus sp.
47
62
74
Aspergillus nigerAmphimedon queenslandica
58
66
28
38
Dictyostelium fasciatumPolysphondylium pallidum
Schistocerca americanaPacifastacus leniuscus
Homarus americanusPalinurus vulgaris – 3
Palinurus vulgaris – 2Palinurus vulgaris – 1
Palinurus elephas – 4
Penaeus vannameiCallinectes sapidusCancer magister
Insect hemocyanins
50
Onychophora
N M C
M C
0.5
Pan-Arthropoda
Amoebozoa
Fungi
0.78
0.97
1
0.78
1
0.56
0.53
0.95
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BacteriaBacteriaBacteria BacteriaBacteria
Tyr Tyr
Tyr
Tyr DWL
Tyr DWLTyr
PlantTyr DWL
Tyr
Tyr DWL KFDV
Tyr DWL KFDVTyr
TyrBacteria
Fungi Tyr
Bilateria Tyr
Tunicata
Brachiopoda TyrTyr
MolluscTyrosinases
(Hemocyanins)
Bilateria
Tyr
Bilateria Tyr
Non-bilaterianmetazoans Tyr
Tyr
TyrvW-A
TyrKtetra
SushiSushi Sushi
Cnidarians
Ctenophores
SpongesTyr
Bilateria Tyr vW-C vW-DvW-C vW-C
TyrBacteriaTyr
ProtostomiaTyr
Tyr ShK ShK
Tyr ShK ShK ShK ShK
Ichthyosporea Tyr
Ichthyosporea
Stramenopile Tyr
Ichthyosporea Tyr
Choanoflagelata Tyr Kazal-2 Kazal-2Kunitz
Tyr Tyr Kazal-2 Kazal-2Kunitz
Tyr Tyr
TyrKunitz
Fungi
Tyr
TyrDUF866
TyrNnf1
Tyr zf-MYND
TyrChitin bind 1 Chitin bind 1
Clade A
Clade B
Clade C
Clade E
Clade F
Clade D
57
59
84
2915
36
8
65
12
42
9339
18
58
25
47
35
Bacteria88
26
58
55
StramenopileTyr
Tyr Tyr
Bacteria
14
34
30
10
7
3010
5
4
2
53
12
49
6
6975
42
0.3
0.92
-
--
1
0.93
0.991
0.93
0.54
1
0.79
0.97
0.72
0.98
0.98
-
0.99
0.6
0.78
1
0.77
0.99
0.62
1
0.59
-
-
- 0.84
0.94
11
0.99
0.83
0.92
-
-
Tyr Tyr Tyr Tyr TyrTyr Tyr Tyr
Cubind
Itrans
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Cnidaria
Xenoturbellida
Acoelomorpha1
Chordata
Echinodermata
Hemichordata
Chaetognatha
Nematoda
Nematomorpha
Tardigrada
Onychophora1
Arthropoda
Priapulida1
Loricifera
Kinorhyncha
Bryozoa1
Entoprocta
Cycliophora
Annelida
Mollusca
Nemertea1
Brachiopoda1
Phoronida
Gastrotricha
Platyhelminthes
Gnathostomulida
Micrognathozoa
Rotifera
Ctenophora
Porifera
Trichoplax
Deu
t.
Bila
teria
Pro
tost
omia
1
Iron HryO2 Hry
Hemocyanin M
A B C D E F
Tyrosinase:
1 Metazoan ancestor:
FBXL5O2 Hry
Hemocyanin M
A B C D F
Tyrosinase:
2 Bilaterian ancestor:
Respiratory hemocyanin
3
FBXL5
Hemocyanin M
C F
Tyrosinase:
3 Deuterostomian ancestor:
FBXL5O2 Hry
Hemocyanin M
A B C D F
Tyrosinase:
4 Protostomian ancestor:
2
Iron Hry FBXL5
Tyr E
4
Respiratoryhemerythrin
Respiratory tyrosinase
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