Báo cáo khoa học: Subunit sequences of the 4 · 6-mer hemocyanin from the golden orb-web spider, Nephila inaurata Intramolecular evolution of the chelicerate hemocyanin subunits pot

Subunit sequences of the 4 · 6-mer hemocyanin from the golden
orb-web spider,
Nephila inaurata
Intramolecular evolution of the chelicerate hemocyanin subunits
Anne Averdam, Ju¨ rgen Markl and Thorsten Burmester
Institute of Zoology, Johannes Gutenberg University, Mainz, Germany
The transport of oxygen in the hemolymph of many arth-
ropod and mollusc species is mediated by large copper-
proteins that are referred to as hemocyanins. Arthropod
hemocyanins are composed of hexamers and oligomers of
hexamers. Arachnid hemocyanins usually form 4 · 6-mers
consisting of seven distinct subunit types (termed a–g),
although in some spider taxa deviations from this standard
scheme have been observed. Applying immunological and
electrophoretic methods, six distinct hemocyanin subunits
were identified in the red-legged golden orb-web spider
Nephila inaurata madagascariensis (Araneae: Tetragnathi-
dae). The complete cDNA sequences of six subunits were
obtained that corresponded to a-, b-, d-, e-, f-andg-type
subunits. No evidence for a c-type subunit was found in this
species. The inclusion of the N. inaurata hemocyanins in a
multiple alignment of the arthropod hemocyanins and the
application of the Bayesian method of phylogenetic inference
allow, for the first time, a solid reconstruction of the intra-
molecular evolution of the chelicerate hemocyanin subunits.
The branch leading to subunit a diverged first, followed by
the common branch of the dimer-forming b and c subunits,
while subunits d and f, as well as subunits e and g form
common branches. Assuming a clock-like evolution of the
chelicerate hemocyanins, a timescale for the evolution of the
Chelicerata was obtained that agrees with the fossil record.

Keywords: Arthropoda; Chelicerata; evolution; hemocyanin;
subunit diversity.
Hemocyanins are large, copper-containing respiratory pro-
teins that serve to transport oxygen in many arthropod
species [1,2]. Hemocyanins and their sequences have been
identified in all arthropod subphyla, including the Onycho-
phora, Chelicerata, Crustacea, Myriapoda and Hexapoda
[3,4]. These proteins belong to a large superfamily that also
includes functionally divergent proteins such as phenol-
oxidases, as well as the crustacean pseudo-hemocyanins
(cryptocyanins), insect hexamerins and hexamerin receptors
[3,5–8].
Arthropod hemocyanins form hexamers or oligo-hexa-
mers composed of distinct or related subunits in the
75 kDa-range. In each subunit, the binding of oxygen is
mediated by a pair of Cu
+
ions that are coordinated by six
conserved histidine residues [1,2]. Based on immunological
differences, seven distinct hemocyanin subunit types have
been identified in the chelicerates that are termed a–g [9–11].
Depending on the taxon, chelicerate hemocyanins assemble
to quaternary structures of up to 8 · 6 subunits [1,11,12].
The 4 · 6-mer hemocyanin of the orthognath spider,
Eurypelma californicum (tarantula) is the best studied
hemocyanin in terms of structure, function and evolution
[13–17]. The formation of the 4 · 6-mer hemocyanin
requires the stoichiometric association of all seven subunit
types (4 · a,2· b,2· c,4· d,4· e,4· f,4· g), with
each subunit occupying a distinct position within the native

oligomer [18]. The complete amino acid and cDNA
sequences of all seven tarantula hemocyanin subunits have
been determined [19].
By a combination of electron microscopic and immuno-
logical methods, Markl and colleagues investigated the
structure and subunit composition of 40 different spider
species from 25 spider families [9,11,12]. These studies have
demonstrated that all investigated mygalomorph spiders
(Orthognatha; such as E. californicum) possess 4 · 6-mer
hemocyanins, but in some Araneomorpha, deviations from
that standard scheme have been observed. While the
ÔclassicalÕ 4 · 6-mer hemocyanins are also present in many
species of this taxon, many haplogyne and entelegyne
spiders contain 1 · 6or2· 6-mer hemocyanins. In these
hemocyanin-oligomers, some subunit types are absent. For
example, the hemolymph of the entelegyne hunting spider,
Cupiennius salei, contains a mixture of 1 · 6and2· 6-mer
hemocyanins [20]. The C. salei hemocyanin consists of six
distinct g-type subunits, while the subunits types a–f have
been lost in evolution more than 200 MYA [21].
For further understanding of chelicerate hemocyanin
structure and evolution, we have characterized the 4 · 6-
mer hemocyanin of an araneomorph spider. We show that
the hemocyanin of red-legged golden orb-web spider,
Nephila inaurata madagascariensis, consists of six distinct
polypeptides that can be assigned to the subunit-types a, b,
d, e, f and g. These additional sequences, as well as the
Correspondence to Ju
¨
rgen Markl, Institute of Zoology, University of

Mainz, Mu
¨
llerweg 6, D-55099 Mainz, Germany.
Fax: + 49 6131 3924652, Tel.: + 49 6131 392 2314,
E-mail:
Abbreviations: Hc, hemocyanin; MYA, million years ago.
(Received 13 May 2003, revised 18 June 2003,
accepted 26 June 2003)
Eur. J. Biochem. 270, 3432–3439 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03730.x
application of Bayesian methods for phylogenetic inference
also allow the first time a reliable reconstruction of the
intramolecular evolution of chelicerate hemocyanins.
Materials and methods
Animals
Six specimens of the red-legged golden orb-web spider,
Nephila inaurata madagascariensis (Chelicerata; Araneae;
Tetragnathidae; Fig. 1), were kindly provided by the
Zoological Garden ÔWilhelmaÕ in Stuttgart. The spiders
were kept at 28 °C with a 12-h light : 12-h dark cycle and
fed on insects. Specimens used in this study had a body
length of 4–5 cm and leg span of 12–16 cm.
Protein biochemistry
After immobilization of the spiders for 1 h at 4 °C, the
hemolymph was withdrawn by puncturing the heart with a
syringe at the median-dorsal region of the opisthosoma. The
hemolymph was collected in 20 lL50m
M
Tris/HCl, 5 m
M
CaCl

2
,5m
M
MgCl
2
, 150 m
M
NaCl,pH7.4.Hemocytes
and clotted material were removed by 10-min centrifugation
at 10 000 g. In some analyses, the hemocyanin was
dissociated by dialysis of the hemolymph over night at
4 °C in 130 m
M
glycine/NaOH, pH 9.6. SDS/PAGE ana-
lyses were carried on a 7.5% gel under reducing conditions
[22]. Native PAGE was performed on 7.5% polyacrylamide
gels without SDS and b-mercaptoethanol. For Western
blotting, the proteins were transferred to nitrocellulose at
0.8 mAÆcm
)2
. Nonspecific binding sites were blocked by 5%
nonfat dry milk in TBST (10 m
M
Tris/HCl, pH 7.4,
140 m
M
NaCl, 0.25% Tween-20) and the membranes were
incubated overnight at 4 °C with various anti-hemocyanin
Igs, diluted 1 : 5000 in 5% nonfat dry milk/TBST. The
filters were washed three times for 10 min in TBST and

subsequently incubated for 1 h with goat anti-(rabbit) Fab
fragments conjugated with alkaline phosphatase (Dianova)
diluted 1 : 10.000 in 5% nonfat dry milk/TBST. The
membranes were washed as above and the detection was
carried out using nitro blue tetrazolium and 5-bromo-4-
chloro-3-indolyl phosphate. Antisera against crude hemo-
cyanin preparations from Argiope aurantia and Araneus
diadematus were raised in rabbits [9,11,12]. Crossed immu-
noelectrophoresis of dissociated N. inaurata hemolymph
proteins was performed as described by Weeke [23],
applying anti-A. aurantia-hemocyanin antiserum.
Cloning and sequencing of hemocyanin cDNAs
Hematopoiesis was induced by bleeding about 1 week
before RNA preparation. The spider was shock-frozen in
liquid nitrogen and ground to a fine powder under
continuous addition of nitrogen. Total RNA was extracted
using the guanidine thiocyanate method [24]. Poly(A)
+
RNA was purified by the aid of the PolyATract kit
(Promega). A directionally cloned cDNA expression library
was established using the Lambda ZAP-cDNA synthesis
kit from Stratagene. The library was amplified once and
screened with a mixture of anti-A. diadematus- and anti-
A. aurantia-hemocyanin Igs. Positive phage clones were
converted to pBK-CMV plasmid vectors with the material
provided by Stratagene according to the manufacturer’s
instructions and sequenced on both strands by the com-
mercial GENterprise (Mainz, Germany) sequencing service.
Complete hemocyanin cDNA sequences were obtained by
primer walking using specific oligonucleotides.

Sequence analyses and molecular
phylogenetic studies
The web-based tools provided by the ExPASy Molecular
Biology Server of the Swiss Institute of Bioinformatics
() and the program
GENEDOC
2.6
[25] were used for the analyses of DNA and amino acid
sequences. The amino acid sequences of the N. inaurata
hemocyanin subunits were added by hand to the previ-
ously published alignments of chelicerate hemocyanin
sequences [19,21]. Nine selected arthropod phenoloxidases
[phenoloxidases from Penaeus monodon (accession
number AF099741), Pacifastacus leniusculus (X83494),
Marsupenaeus japonicus (AB065371), Tenebrio molitor
(AB020738), Bombyx mori (D49370, D49371 and E12578),
and Sarcophaga bullata (AF161260 and AF161261)) and
four crustacean hemocyanins (Panulirus interruptus hemo-
cyanin subunits a (P04254) and c (S21221), Homarus
americanus hemocyanin A (AJ272095) and Pacifastacus
leniusculus hemocyanin (AF522504)] were included in the
alignment and used as outgroups for tree reconstruction.
The final alignment is available from the authors upon
request. Distances between pairs of sequences were cal-
culated using the PAM [26] or the JTT [27] matrices
implemented in the
PHYLIP
3.6a2 package [28]. Tree
constructions were performed by the neighbor-joining
method and the reliability of the trees was tested by the

bootstrap procedure with 100 replications [29]. Bayesian
phylogenetic analyses were performed with
MRBAYES
3.01
[30]. The PAM, JTT or WAG [31] amino acid substitution
models with gamma distribution of rates was applied.
Metropolis-coupled Markov chain Monte Carlo sampling
was performed with four chains that were run for 100 000
generations. Prior probabilities for all trees were equal,
starting trees were random, trees were sampled every 10th
generation. Posterior probability densities were estimated
on 5000 trees (burnin ¼ 5000). Molecular clock calcula-
tions based PAM distances of orthologous subunits as
described [19,21], assuming that the Xiphosura and
Arachnida diverged about 450 MYA [7,32]. The confid-
ence limits were calculated using the observed standard
deviation of the protein distances.
Fig. 1. The red-legged golden orb-web spider, Nephila inaurata.
Ó FEBS 2003 Spider hemocyanin (Eur. J. Biochem. 270) 3433
Results
Characterization of
N. inaurata
hemocyanin
Hemolymph was collected from six specimens of N. inau-
rata. Electron microscopic images show the presence of
large particles that are indistinguishable in terms of size and
shape from the 4 · 6-mer hemocyanin of E. californicum,
indicating that N. inaurata contains a hemocyanin of similar
structure (data not shown). Denaturating SDS/PAGE
shows various main bands in the 65-kDa range, as expected

for a typical chelicerate hemocyanin subunit (Fig. 2). As
estimated from the Coomassie-stained gels, these polypep-
tides represent more than 90% of the total hemolymph
proteins, while other proteins form only a minor fraction.
Thus, the hemolymph was considered a crude hemocyanin
preparation and used as such in the following experiment.
The putative hemocyanin bands were recognized by four
polyclonal antibodies raised against different spider hemo-
cyanins (Fig. 2). Minor cross-reactions with other proteins
were observed with the anti- C. salei-, A. diadematus- and
A. aurantia hemocyanin Igs, probably due to come con-
tamination in the crude hemocyanin preparations used for
immunization. The anti-E. californicum hemocyanin Igs
were specific and only stained the hemocyanin bands. After
dissociation of the hemocyanin subunits in alkaline buffer,
the hemolymph samples were subjected to nondenaturing
PAGE (Fig. 3). In the low molecular mass region, six
distinct bands were identified that most likely correspond to
the hemocyanin subunits. At least two slowly migrating
bands were visible that probably represent nondissociated
or partially dissociated 4 · 6-mer hemocyanin, or nonres-
piratory proteins [33]. Two-dimensional, crossed immuno-
electrophoresis of dissociated N. inaurata hemolymph
proteins with anti-Argiope aurantia hemocyanin antiserum
shows the presence of six immunologically distinct compo-
nents that most likely correspond to distinct hemocyanin
subunits (Fig. 4).
N. inaurata
hemocyanin subunit sequences
A cDNA library that contains about 1.6 · 10

6
independent
clones was constructed from a single adult specimen of
N. inaurata. As no specific Igs against the hemocyanin of
this species are available, we used a mixture of antisera
Fig. 2. SDS/PAGE and Western blot analyses of Nephila inaurata
hemolymph proteins. Hemolymph proteins (2–3 lg) of N. inaurata
were applied per lane and stained with Coomassie Brilliant Blue
(lane 1). Immunodetection was carried out using Igs raised against the
hemocyanins of C. salei, A. diadematus, A. aurantia and E. californi-
cum as indicated (lanes 2–5). Igs were diluted 1 : 5000. On the left side,
the positions of the molecular mass marker proteins are given. Hc,
hemocyanin subunits.
Fig. 3. Native PAGE of N. inaurata hemolymph proteins. Total
hemolymph proteins (10 lg) of N. inaurata (left lane) and 10 lgof
purified hemocyanin of E. californicum (right lane) were applied. The
hemocyanin subunits were dissociated into subunits against alkaline
glycine/NaOH buffer before PAGE. The positions of the E. californ-
icum hemocyanin subunits a to g, as well as the nondissociated 24mer
and partially dissociated oligomers are indicated [33].
Fig. 4. Crossed immunoelectrophoresis of N. inaurata hemolymph pro-
teins. Four micrograms of dissociated N. inaurata hemolymph proteins
as antigen and anti-A. aurantia hemocyanin serum were used.
3434 A. Averdam et al. (Eur. J. Biochem. 270) Ó FEBS 2003
raised against the hemocyanins of the related web spiders,
Araneus diadematus and A. aurantia. Two hundred and
nineteen positive clones were identified, of which 124 clones
containing inserts between 1.5 kb and 2.5 kb were partially
sequenced at their 5¢-ends. Database comparisons show that
56 of these clones encode hemocyanin subunits. Based on

the similarities with the E. californicum sequences, 17 clones
were assigned to hemocyanin subunit a, 2 represent subunit
b, 13 subunit d, 11 subunit e, 7 subunit f and 6 subunit g.
No c-type subunit sequence was obtained. The complete
sequences of each subunit were obtained by primer walking
and have been deposited in the EMBL/GenBank databases
(Table 1).
Conceptual translation and comparisons with known
chelicerate hemocyanin sequences show that all six cDNA
sequences were complete and cover the whole coding
regions. The cDNA sequences comprise of 2078–2350 bp,
which include 22–73 bp of the 5¢-untranslated regions and
open reading frames of 1878–1893 bp. The 3¢-untranslated
regions comprise 137–419 bp, include the standard poly-
adenylation signals (AATAAA) and are followed by
poly(A)-tails of 19–58 bp. Multiple clones show the pres-
ence of allelic sequences for the subunits a, d, e and g that
are > 99.7% identical with the main cDNA sequence.
Most of the nucleotide substitutions are silent, only in each
a, d and g has a single amino acid substitution been
observed (data not shown).
The open reading frames of the hemocyanin subunits
translate into distinct polypeptides of 625–630 amino acids,
with calculated molecular masses in the range of 71–73 kDa
(Table 1). As in the E. californicum and C. salei hemocya-
nins [19,21], no signal peptides required for transmembrane
transport have been found in the N. inaurata subunit
sequences. Thus, the nascent proteins do not pass through
the Golgi-apparatus and the putative N-glycosylation sites
(NXT/S) in the primary structures are probably not used.

There are four strictly conserved cysteine residues in domain
3oftheN. inaurata hemocyanins (Fig. 5) that form two
disulfide bridges that stabilize the three-dimensional protein
structure [34,35].
Sequence comparison and chelicerate hemocyanin
evolution
A multiple alignment of the chelicerate hemocyanin amino
acid sequences was constructed and used for sequence
comparisons and phylogenetic inference (Fig. 5). Compar-
ison of the N. inaurata hemocyanin sequences with those of
E. californicum allows the unambiguous assignment to
distinct subunit types. The orthologous subunits of these
species share 69.1–76.2% of their amino acids, with the a
subunits being the most conserved and the b subunits the
least conserved proteins (Table 2). The similarity scores of
nonorthologous subunits is in the range of 55–64%. There
are only few amino acid insertions/deletion among the
chelicerate hemocyanin subunits that are located mainly in
the loop regions between alpha-helices 1.1 and 1.3, alpha-
helix 1.7 and beta-sheet 1B, beta-sheets 3B and 3C, and
alpha-helices 3.3 and 3.4.
Like with other web spiders belonging to the family of the
Tetragnathidae, the hemolymph of the golden orb-web
spider, N. inaurata, contains a 4 · 6-mer hemocyanin
[11,12]. Using electrophoretic and immunological methods,
we identified six distinct hemocyanin polypeptides. This
result is in line with the identification of six distinct cDNA
sequences, although we must consider that b-andc-type
subunits usually occur as stable heterodimer and therefore
may yield a single peak in crossed immuno-electrophoresis

[11]. Sequence comparison and phylogenetic analyses show
that the cDNAs correspond to subunits a, b, d, e, f and g.
Despite the identification of 56 independent hemocyanin
clones in the cDNA library, we found no evidence for the
presence of a cDNA clone corresponding to a c-type
subunit. We therefore designed various pairs of primers
based on the known sequence of subunit c of E. californicum
[19] (5¢fi3¢: bp 220–239, 591–608, 925–947, 3¢fi5¢: 947–
925, 1286–1265, 1474–1453) and used them for various PCR
experiments with the cDNA library as template. However,
we only obtained PCR products that correspond to the
known subunit b.
For reconstruction of chelicerate subunit evolution, either
three selected crustacean hemocyanin sequences or 11
arthropod phenoloxidases were included in the alignment.
Phylogenetic trees were calculated by the neighbor-joining
method based on protein distances estimated by the PAM or
JTT model of amino acid substitutions. While the grouping
of orthologous subunits from different species is strongly
supported, the relationships among the subunit types could
not be resolved with sufficient confidence (data not shown).
We therefore conducted Bayesian tree building methods.
Applying three different evolutionary models of amino acid
substitution, identical and solid reconstructions of the
diversification scheme of chelicerate subunit types was
obtained (Fig. 6). In all analyses, the clade leading to the
a-type subunits diverged first. This clade includes subunit II
of the horseshoe crab, Limulus polyphemus (LpoHc2). The
next branch is formed by the b-andc-type subunits of
N. inaurata and E. californicum, while there are common

Table 1. Molecular properties of the N. inaurata hemocyanin subunits. Accession number, EMBL/GenBank DNA data.
Subunit Accession number cDNA [bp]
a
Protein [Amino acids]
c
Molecular mass [kDa]
b
pI
c
NinHc-a AJ547807 2067 630 72.09 5.39
NinHc-b AJ547808 2060 628 72.59 6.06
NinHc-d AJ547809 2327 627 71.94 6.36
NinHc-e AJ547810 2106 625 71.00 5.49
NinHc-f AJ547811 2152 626 71.73 6.09
NinHc-g AJ547812 2059 626 71.19 5.88
a
Without poly(A) tail.
b
Including the initiator methionine.
c
Without initiator methionine.
Ó FEBS 2003 Spider hemocyanin (Eur. J. Biochem. 270) 3435
Fig. 5. Alignment of the amino acid sequences of N. inaurata and E. californicum hemocyanin subunits. Strictly conserved amino acids are shaded in
grey, the secondary structure elements as deduced from L. polyphemus subunit II [34] are indicated at the bottom. Other features are given in the
upper row: (*) copper binding histidine; (c) disulfide bridges. The abbreviations used are: NinHc-a to g, hemocyanin subunits a–g of N. inaurata (see
Table 1 for accession numbers); EcaHc-a to g, E. californicum hemocyanin subunits a–g (acc. nos: X16893, AJ290429, AJ277489, AJ290430,
X16894, AJ277491 and AJ277492).
3436 A. Averdam et al. (Eur. J. Biochem. 270) Ó FEBS 2003
branches of subunit types d and f,ande and g.TheC. salei
hemocyanins group with the g-subunit of N. inaurata, while

the hemocyanin subunit 6 of the scorpion Androctonus
australis (AauHc6) and subunit A of the horseshoe crab,
Tachypleus tridentatus (TtrHcA), (that form a common but
not significantly supported branch) are basal to all spider
g-subunits.
A timescale of chelicerate hemocyanin evolution was
inferred assuming that the LpoHc2 and the a-subunits of
N. inaurata and E. californicum on the one hand, and
TtrHcA and the arachnid g-subunits on the other hand are
orthologous proteins (see above). The fossil record suggests
that the Arachnida (A. australis, E. californicum, N. inau-
rata and C. salei) and the Xiphosura (L. polyphemus and
T. tridentatus) separated about 450 MYA [32]. Using the
PAM evolution model, a mean replacement rate of
0.66 ± 0.03 · 10
)9
amino acid substitutions per site per
year was calculated for the a-type subunits, and
0.60 ± 0.03 · 10
)9
for the g-type subunits. Based on these
predictions, we calculated that the hemocyanins of the
N. inaurata and E. californicum diverged about 279 ± 28
MYA (Fig. 7). The C. salei hemocyanins and N. inaurata
subunit g separated 222 ± 9 MYA. Assuming that scor-
pion hemocyanin AauHc6 is associated with the hemocy-
anins of the Araneae rather than with TtrHcA, we estimated
that AauHc6 split from the spider hemocyanins about
381 ± 32 MYA. The earliest divergence of chelicerate
hemocyanin subunits (i.e., a-type subunits vs. all others)

took place about 550 ± 45 MYA, the clade leading to the
b/c subunits emerged 545 ± 24 MYA, while the other
subunit types emerged about 450–470 MYA.
Discussion
Structure and subunit composition of
N. inaurata
hemocyanin
The absence of subunit c in N. inaurata is surprising, as
seven distinct subunits are required to build a functional
active 4 · 6-mer hemocyanin in E. californicum [16,18].
While we cannot rule out the possibility that we missed
subunit c in all of our analyses, it is, however, also possible
that the 4 · 6-mer hemocyanin of N. inaurata is in fact built
by only six subunit types. We must also consider the
findings of Kuwada and Sugita [36], which suggests that
subunit loss and duplication may also occur in some
mygalomorph spiders that are generally assumed to contain
4 · 6-mer hemocyanins [11,12].
In E. californicum, subunit c builds a stable dimer with
subunit b that is centred in the core of the 4 · 6-mer.
Table 2. Sequence comparison of N. inaurata and E. californicum
subunits.
N. inaurata E. californicum
DNA
identity [%]
Protein
identity [%]
NinHc-a EcaHc-a 72.0 76.2
NinHc-b EcaHc-b 66.4 69.1
NinHc-d EcaHc-d 70.1 74.9

NinHc-e EcaHc-e 69.6 71.3
NinHc-f EcaHc-f 70.1 76.2
NinHc-g EcaHc-g 69.6 72.8
Fig. 6. Phylogenetic tree of the chelicerate hemocyanin subunits. The
numbers at the nodes represent Bayesian posterior probabilities esti-
mated with the PAM model of amino acid substitution. Abbreviations:
AauHc6, A. australis hemocyanin subunit 6 (acc. no. P80476);
LpoHcII, subunit II of L. polyphemus (P04253); TtrHcA, T. trident-
atus hemocyanin a [42]; CsaHc1–6, C. salei hemocyanin subunits 1
through 6 (AJ307903 – AJ307907, AJ307909). For other abbrevia-
tions, see Fig. 5.
Fig. 7. Timescale of the evolution in the chelicerate hemocyanin sub-
units. The grey bars are the standard errors. Abbreviation: MYA,
million years ago; for abbreviations, see Figs 5 and 6.
Ó FEBS 2003 Spider hemocyanin (Eur. J. Biochem. 270) 3437
Assuming that the formation of hemocyanin quaternary
structure is similar in E. californicum and N. inaurata,
another subunit must have taken over the role and the
position of the missing c-subunit in the N. inaurata hemo-
cyanin that might be subunit b. Although we have no
experimental evidence for such an assumption, it is therefore
possible that the N. inaurata hemocyanin contains four
copies of subunit b.
Emergence of hemocyanin subunit diversity
in the Chelicerata
Our previous molecular phylogenetic analyses of the
evolution of the arthropod hemocyanin superfamily agree
that the chelicerate hemocyanin subunits are monophyletic
[7,19,21], but failed to provide a solid reconstruction for the
intramolecular evolution of the chelicerate hemocyanins.

On one hand, this was due to the limited number of
available sequences and on the other hand to the restrictions
of the phylogenetic methods. With the inclusion of the
N. inaurata hemocyanin sequences, the resolution increased
slightly, although the grouping of the different subunit types
is still inconsistent and the bootstrap values are low. With
the application of a Bayesian method for phylogenetic
inference [37], the resolution of the tree significantly
increased and resulted in high posterior probabilities
(Fig. 6). Notably, we obtained identical trees using three
evolutionary models of amino acid substitutions (PAM,
JTT or WAG).
The phylogeny presented here shows for the first time a
solid reconstruction of the diversification scheme of the
chelicerate hemocyanin subunits and the pattern of intra-
molecular evolution of this protein. The first gene duplica-
tion probably took place already in the chelicerate stemline,
close to 550 MYA, and gave rise to the a-subunits and the
ancestor of all other subunit types. Further differentiation of
subunit types occurred about 450–470 MYA, with the
exception of the b/c-subunits that diverged around
420 MYA. These calculations agree with the observation
of immunologically related subunits in L. polyphemus and
E. californicum [10–12]. The Bayesian reconstructions show
a common clade of the d and f subunit types on one hand,
and the e and g subunit types on the other. A close
relationship of subunits d and f is also supported by a
conserved common deletion of two amino acids between
b-sheets 3B and 3C (Fig. 5). Interestingly, the topology
presented here was essentially proposed already, on the

basis of antigenic determinants, by Markl and coworkers
[10–12]. Nevertheless, it should be noted that in previous
studies based on structural and immunological similarities,
AauHc6 was homologised with EcaHc-e [38,39], while our
phylogenetic inference strongly suggest an association of
AauHc6 with the g-type subunits (Fig. 5). Latter assump-
tion is, however, also supported by the higher similarity
scores of AauHc6 with the g-type than with the e-type
subunits.
A hemocyanin-based timescale for the evolution
of the Chelicerata
The orthologous hemocyanin subunits of E. californicum
and N. inaurata differ in 24–31% of their amino acids
(Table 2). Under the assumption of a clock-like evolution
and an arachnid–xiphosuran divergence of 450 MYA, these
differences translate into a divergence time of the species of
about 279 ± 28 MYA (Fig. 7). This estimate represents the
time of separation of the mygalomorph and araneomorph
spiders, and is in good agreement with previous calculations
(285 MYA [21]); and the fossil data [40]. The ancestors of
Nephila and Cupiennius separated about 222 ± 9 MYA
that reflect the time of divergence of the Orbiculariae and the
ÔRTA-cladeÕ within the entelegyne spiders [41]. The correc-
ted replacement rate for the g-type subunits led to slightly
earlier times ( 5%) of the differentiation of the C. salei
hemocyanins than estimated before [21] that are, however,
within the range of the expected standard deviations and are
still in line with fossil data [40]. According to our calcula-
tions, the differentiation of the Scorpiones from the other
Arachnids took place 381 ± 32 MYA that agrees again

with the fossils [32,40]. These accurate time estimates suggest
that the hemocyanin subunits are excellent tools to investi-
gate the evolution of the Chelicerata.
Acknowledgements
We thank J. Schultess for his help with the cDNA library, K. Kusche
for her advice and W. Gebauer for the EM pictures. This work was
supported by grants of the Deutsche Forschungsgemeinschaft (Bu956/
5; Ma843/4) and by the Feldbauschstiftung Mainz.
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