Subunit sequences of the 4 · 6-mer hemocyanin from the goldenIntramolecular evolution of the chelicerate hemocyanin subunits Anne Averdam, Ju¨rgen Markl and Thorsten Burmester Institute
Trang 1Subunit sequences of the 4 · 6-mer hemocyanin from the golden
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- and g-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
classical 4· 6-mer hemocyanins are also present in many species of this taxon, many haplogyne and entelegyne spiders contain 1· 6 or 2 · 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· 6 and 2 · 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: markl@mail.uni-mainz.de
Abbreviations: Hc, hemocyanin; MYA, million years ago.
(Received 13 May 2003, revised 18 June 2003,
accepted 26 June 2003)
Trang 2application 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 Wilhelma in Stuttgart The spiders
were kept at 28C 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 4C, 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 lL 50 mMTris/HCl, 5 mM
CaCl2, 5 mM MgCl2, 150 mM NaCl, pH 7.4 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
4C in 130 mMglycine/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 mM Tris/HCl, pH 7.4,
140 mMNaCl, 0.25% Tween-20) and the membranes were
incubated overnight at 4C 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
diadematuswere 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 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 (http://www.expasy.org) and the program GENEDOC2.6 [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 leniusculushemocyanin (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 withMRBAYES3.01 [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 (burnin¼ 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.
Trang 3Characterization ofN 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· 106independent 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 lg of 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.
Trang 4raised 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 sequpres-ences 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
3 of the N 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 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- and c-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¢ fi 3¢: bp 220–239, 591–608, 925–947, 3¢ fi 5¢: 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- and c-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
a
Without poly(A) tail.bIncluding the initiator methionine.cWithout initiator methionine.
Trang 5Fig 5 Alignmentof 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).
Trang 6branches of subunit types d and f, and e and g The C 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)9for the g-type subunits Based on these predictions, we calculated that the hemocyanins of the
N inaurataand 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 ofN 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.
Trang 7Assuming 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 inauratahemocyanin 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
RTA-clade 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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