Reduction in muscle fibre number during the adaptive radiation of notothenioid fishes: a phylogenetic perspective
1 Gatty Marine Laboratory, Division of Environmental and Evolutionary Biology,
School of Biology, University of St Andrews, St Andrews, Fife, KY16 8LB,
Scotland, UK
2 Centro Austral de Investigaciones Cientificas (CADIC), Consejo Nacional de
Investigaciones Cientificas y Tecnicas (CONICET) CC92, Ushuaia, 9410, Tierra
del Fuego, Argentina
3 British Antarctic Survey, High Cross, Madingley Road, Cambridge, CB3 OET,
UK
4 Department of Biology, University of California, Riverside, CA 92521,
USA
* Author for correspondence (e-mail: iaj{at}st-and.ac.uk)
Accepted 30 April 2003
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Summary |
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Key words: Antarctic teleosts, growth, muscle fibre recruitment, Notothenioid fishes, phylogeny, skeletal muscle
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Introduction |
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The fish fauna of the continental shelf of the Southern Ocean is dominated
by a single sub-order of Perciformes, the Notothenioidei, which comprises at
least 125 species divided into eight families
(Eastman and Eakin, 2000).
Several authors have reported that Antarctic notothenioids have unusually
large diameter fibres, which can reach 100 µm in slow muscle and 500 µm
in fast muscle (Smialovska and Kilarski, 1981;
Dunn et al., 1989
;
Battram and Johnston, 1991
).
Slow muscle fibres have relatively high densities of mitochondria
(Johnston, 1987
;
Archer and Johnston, 1991
;
O'Brien et al., 2003
),
reaching 50% of fibre volume in some Channichthyids (haemoglobin-less
icefishes) (Johnston, 1989
).
However, mitochondria are found in the central zone of even the
largest-diameter slow fibres (Johnston,
1987
; Archer and Johnston,
1991
), consistent with the maintenance of adequate tissue
oxygenation at the low body temperature of these species
(Egginton et al., 2002
). Fast
muscle fibres with diameters of 400 µm have also been reported in
sub-Antarctic notothenioids from the Beagle Channel, although a relatively
restricted size range of fish was studied
(Fernández et al.,
2000
).
The ancestral form of the notothenioids is generally considered to have
been a temperate bottom-living species without a swim bladder
(Eastman, 1993). The success
of the radiation of the Antarctic notothenioids has been attributed to the
evolution of glycopeptide and peptide antifreezes, which enabled them to
adjust to the climatic cooling that occurred following the opening of the
Drake passage and establishment of the Antarctic Polar Front (APF) some 20-25
million years ago (Cheng and DeVries,
1991
; Eastman,
1993
; Clarke and Johnston,
1996
). The Bovichtidae, Pseudaphritidae and Eleginopidae are the
most basal notothenioid families and, except for a single Antarctic species of
bovichtid, are represented by non-Antarctic species with the plesiomorphic
condition of lacking antifreezes (Eastman
and Eakin, 2000
).
Members of six families of notothenioids are found outside the Antarctic in
the Beagle Channel, Patagonian Shelf, along the Pacific Coast of South America
and in the sub-Antarctic waters of New Zealand
(Eastman, 1993). One
bathypelagic species, the Patagonian toothfish Dissostichus
eleginoides (Nototheniidae), grows to 2 m total length and occurs on both
sides of the Polar Front, from a latitude of 40°S off the coasts of S.
America to 60°S south in the Antarctic. The distribution of D.
eleginoides is circum-Antarctic and its range overlaps with a sister
species D. mawsoni that is found around the Antarctic continental
shelf (Fisher and Hureau,
1985
). Evidence from a molecular phylogeny estimated using
mitochondrial 12S and 16S rRNA DNA sequences suggests that many sub-Antarctic
species evolved subsequent to the main radiation of the group, as recently as
7-9 million years ago (Bargelloni et al.,
2000
; Stankovic et al.,
2001
).
It has been suggested that the radiation of the Antarctic notothenioids has
been associated with a loss of characters or evolutionary function
(disaptation) followed by subsequent adaptive recovery
(Montgomery and Clements,
2000). Within the notothenioids, the family Channichthyidae
(icefishes) is notable for the loss of haemoglobin, which is thought to have
resulted from a single mutational event that deleted the entire ß-globin
gene and the 5' end of the linked
-globin gene
(Cocca et al., 1995
). Loss of
respiratory pigments is associated with a suite of compensatory adaptations in
the heart and peripheral circulatory system
(Tota et al., 1997
). These
include a relatively large ventricular muscle mass
(Johnston et al., 1993
), a
high blood volume (Acierno et al.,
1995
), coupled to a high output cardiac pump operating at low
frequencies and pressures (Tota et al.,
1997
). Six of the 15 species of icefishes also lack myoglobin in
their heart muscle (Moylan and Sidell,
2000
), involving at least three independent mechanisms including a
5-nucleotide insertion leading to premature termination in Champsocephalus
gunnari, an aberrant polyadenylation signal in Pagetopsis
macropterus (Vayda et al.,
1997
), and a duplicated TATAAAA sequence that interferes with
transcription in Chaenocephalus aceratus
(Small et al., 2003
). Some
notothenioids have undergone an ecological diversification to feed in the
water column involving changes in body shape, colour, and the attainment of
near neutral buoyancy through decreased mineralisation of the skeleton and the
accumulation of lipids (Eastman,
1993
,
1997
;
Klingenberg and Ekau, 1996
).
In some cases secondary pelagicism is associated with the retention of larval
characteristics into adult stages
(Eastman, 1993
;
Montgomery and Clements,
2000
), The resulting detrimental changes to the lateral line
sensory system have been compensated for by changes in central processing
mechanisms and behaviour (Montgomery and
Clements, 2000
).
The first aim of the present study was to test the hypothesis that a high maximum fibre diameter in notothenioid fishes was related to a reduction in the number of muscle fibres at the end of the recruitment phase of growth (FNmax). We then used phylogenetically based statistical methods to test whether fibre number was negatively related to body size and whether either body size or size-corrected fibre number showed significant phylogenetic signal. Finally, we estimated ancestral values for body size and fibre number to explore where during the evolutionary radiation reductions in fibre number occurred. A total of 16 species of Notothenioidei from three geographical provinces were studied, including representatives with benthic and secondarily pelagic lifestyles, and an independent phylogeny was constructed using sequence information from mitochondrial 12S rRNA genes.
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Materials and methods |
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Histology
A 0.5 cm transverse steak of the trunk was prepared at 0.7 standard length
(SL) using a sharp knife. The steak was either photographed using a
digital camera and macro lens or traced onto an acetate sheet in triplicate.
The total area of fast muscle was digitised (TCA). The steak was divided into
a series of up to 12 blocks (25 mm2), depending on the size of the
fish, so as to representatively sample all areas of the fast muscle. The
number of blocks was adjusted to sample 25-50% of one half of the steak.
Blocks were frozen in isopentane (2-methyl butane) cooled to near its freezing
point in liquid nitrogen (-159°C). Frozen sections were cut on a cryostat
at 7 µm thickness. Preliminary immonohistochemical investigations confirmed
the identity of muscle fibre types on the sections. Briefly, sections were
stained using standard methods (Johnston
et al., 1999) with the S-58 and F-59 antibodies
(Crow and Stockdale, 1986
),
which have been shown to identify slow and fast muscle myosin, respectively,
in a wide range of fish species (Devoto et
al., 1996
; M. Abercromby, unpublished results) (see
Fig. 2A,B). For the routine
quantification of fibre number, sections were stained with
HaematoxylinEosin. The cross-sectional areas of 1000 muscle fibres were
measured per fish, sampled equally between the blocks and the equivalent fibre
diameter computed. The total number of muscle fibres per trunk cross-section
was estimated as previously described; reproducibility was approximately 3%
(Johnston et al., 1999
).
Smooth distributions were fitted to the measurements of fibre diameter using a
kernel function as previously described
(Johnston et al., 1999
).
Values for the smoothing coefficients showed no systematic variation between
species and were within the range 0.085-0.25. The occurrence of muscle fibre
recruitment was determined on the basis of the presence of fibres less than 10
µm diameter. The final number of fast muscle fibres
(FNmax) was the mean ±
S.E.M. of the fibre number estimate for all
the fish in which fibre recruitment was complete.
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Estimation of a molecular phylogeny
Sixteen species of notothenioids belonging to five families were analysed.
Partial 12S mitochondrial rRNA sequences of ten notothenioid species were
retrieved from GenBank (E. maclovinus, AF145426, 341 bp; D.
eleginoides, AF145425, 340 bp; P. borchgrevinki, PBU90411, 390
bp; T. newnesi, TNU27527, 374 bp; P. tessellata, AF145433,
343 bp; N. coriiceps, NCMT12SG, 373 bp; N. rosii, AF145432,
341 bp; C. aceratus, CAMT12SG1, 373 bp; C. gunnari,
AF145424, 330 bp; H. antarcticus, U37137, 373 bp). cDNAs from six
other species were cloned for this study: C. gobio, P. sima, P. longipes,
P. magellanica, C. esox and H. bispinis. Two of the sequences
(from C. gobio, CGU87414, 250 bp, and C. esox, CES307046,
309 bp) were already available in GenBank but longer sequences were required
to improve the quality of the alignment. The new sequences of 12S
mitochondrial rRNA cloned were submitted to GenBank [accession numbers AY22775
(C. gobio), AY227776 (C. esox), AY227777 (H.
bispinis), AY227778 (P. sima), AY227779 (P. longipes)
and AY227780 (P. magellanica)].
Reverse transcriptasepolymerase chain reaction
Total muscle RNA was isolated using Qiagen Rneasy Mini and Midi Kits.
First-strand cDNA synthesis was carried out using 3' rapid amplification
of cDNA ends system (RACE, Gibco BRL, Life Technologies, Gaitheisburg, USA).
Polymerase chain reaction (PCR) was performed using two sets of primers. The
first set was Forward 5'-AAAAAGCTTCAAACTGGGATTAGATACCCCACTAT-3'
and Reverse 5'-TGAGTCAGAGGGTGACGGGGCGGTGT-3'
(Ritchie et al., 1997). The
second set was Forward 5'-GCGTAAAGGGTGGTTAGG-3' and Reverse
5'-TCTTACTGCTAAATCCTCC-3' (Stankovik et al., 2001). The PCR cycles
used for the first primer set were 94°C for 2 min for denaturation, 35
cycles of 94°C for 30 s, 61°C for 30 s and 72°C for 1 min and
elongation at 72°C for 5 min. The PCR cycles used for the second primer
set were 94°C for 2 min for denaturation, 35 cycles of 94°C for 30 s,
49°C for 30 s and 72°C for 1 min and elongation at 72°C for 5 min.
In all PCRs a proof-reading TAQ polymerase was used (Pfu from Promega UK,
Southampton, UK). DNA sequencing was performed in triplicate by The Sequencing
Service (School of Life Sciences, University of Dundee, Scotland;
http://DNASEQ.biocehm.dundee.ac.uk)
using DYEnamic ET terminator chemistry (Amersham Biosciences, Chalfont St
Giles, Bucks, UK) on Applied Biosystems automated DNA sequencers.
Alignment
The DNA sequences were put together in a FASTA file using BioEdit
(Hall, 1999) and aligned using
ClustalW at EMBL
(http://www.ebi.ac.uk/clustalw/)
with default parameters. The alignment was checked by eye using BioEdit in
order to be sure the automatic process was correct, and finally reduced to the
part of the alignment where most of the specific sequences were represented.
The final alignment of the 16 species was 383 bp long. The alignment was used
as the input for the phylogenetic analysis. The database was bootstrapped 100
times using SEQBOOT (Phylip package version 3.6). Maximum likelihood (ML)
analysis with a molecular clock assumption to assess divergence times was
performed using DNAMLK (Phylip package version 3.6). The analysis was carried
out using the following parameters previously calculated in PUZZLE version
5.0: transition/transversion ratio: 2.41, gamma distribution parameter
alpha=0.24. The consensus tree was assembled using CONSENSE (Phylip package
version 3.6). The node heights and branch lengths for the final tree used are
given in the Appendix.
Statistical analyses
We used both conventional and phylogenetically based statistical analyses.
For the latter, the phylogenetic topology estimated as described above (and
shown in Fig. 8) was used, and
two different sets of branch lengths were considered (see next paragraph). We
tested whether log10SLmax and
log10FNmax exhibit significant phylogenetic
signal (a tendency for related species to resemble each other;
Blomberg and Garland, 2002)
using the randomization test and associated MatLab program (PHYSIG.M)
described in Blomberg et al.
(2003
) and 1000 permutations.
Because log10FNmax was strongly related to
log10SLmax, we also tested for phylogenetic
signal in a size-corrected value of FNmax. Following
Blomberg et al. (2003
), we
first determined the allometric scaling exponent for the loglog
relationship using phylogenetically independent contrasts
(Felsenstein, 1985
),
calculated with the PDTREE program of Garland et al.
(1999
). We then divided the
value for FNmax by SLmax raised to the
appropriate scaling exponent (b=0.722 for real branch lengths,
b=0.964 for constant branch lengths), and then took the log of this
value. We used the K statistic of Blomberg et al.
(2003
) as a descriptor of the
amount of phylogenetic signal present in traits. A value of unity indicates
that a trait has exactly the amount of signal expected under Brownian motion
evolution along the specified topology and branch lengths, whereas values less
than unity indicate less signal than expected, and values of K greater than
unity indicate more.
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As discussed elsewhere, the branch lengths used for phylogenetic analyses
need to be tested for statistical adequacy and can have a large effect on the
results of analyses (e.g. see Garland et al.,
1992,
1999
;
Diaz-Uriarte and Garland,
1998
; Freckleton et al.,
2002
). The phylogenetic tree estimated as described above includes
estimates of branch lengths. These branch lengths may be referred to as `real'
because they are derived from data. However, they are derived from data on DNA
sequence divergence, whereas phylogenetically based statistical methods
require branch lengths in units proportional to expected variance of character
evolution for the actual characters (e.g. body size, fibre number) under study
(e.g. see Felsenstein, 1985
;
Garland et al., 1992
;
Freckleton et al., 2002
;
Blomberg et al., 2003
).
Therefore, such `real' branch lengths may or may not perform well for
statistical analyses. Hence, as an alternative set of branch lengths, we set
all segments to be equal in length (i.e. each segment was set to equal a value
of unity). We then compared the statistical performance of these two alternate
sets of branch lengths by three criteria. First, we used the diagnostic check
suggested by Garland et al.
(1992
) (see also
Diaz-Uriarte and Garland,
1998
), which involves plotting the absolute values of standardized
phylogenetically independent contrasts
(Felsenstein, 1985
)
versus their standard deviations and calculating the correlation
coefficient (not through the origin). A correlation closer to zero implies a
better fit of the branch lengths to the data. Second, we calculated the mean
squared error (MSE) in a generalized least-squares analysis (equivalent to the
variance of the standardized contrasts) using PHYSIG.M of Blomberg et al.
(2003
). Again, a smaller MSE
implies a better fit. We considered the foregoing two statistics for
log10 maximum length (SLmax: data reported in
Table 1) because it is the
primary independent variable in the analysis and because fibre number was
expected to be strongly correlated with SLmax. Finally, we
examined the independent contrasts regression (through the origin) of
log10FNmax on
log10SLmax. Here, a higher
r2 implies a better fit of the branch lengths to the data
(assuming that the data appear to fit the line well, e.g. linearity,
homoscedasticity of residuals, lack of outliers or influential points).
We used PDTREE (Garland et al.,
1999) to estimate ancestral values and 95% confidence limits for
both log10SLmax and
log10size-corrected FNmax. A clear sister group
to the notothenioids has not been identified, although characters may be
polarised relative to the Bovichtidae, and this family has often been used as
a `functional outgroup' (Eastman,
1993
). We therefore compared node 2 in the phylogenetic tree (see
Fig. 8), which represents the
ancestor of the Eleginopinae, Nototheniidae, Channichthyidae and
Harpagiferidae, with node 6, which is the ancestor of the Channichthyidae.
Comparison of the 95% confidence limits (c.l.) allowed us to test whether
either trait showed a significant change between the two nodes.
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Results |
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Another species showing a prolonged period of mosaic hyperplasia was D. eleginoides. Muscle fibres <10 µm diameter were present in specimens 35.6 cm SL or less, but absent from fish over 52 cm SL. It was not possible to establish the precise length at which fibre recruitment ceased in this species. The complete range of juvenile and adult stages was studied for P. tessellata (3.3-27.6 cm SL). Fibres less than 10 µm in diameter were only present in the four smallest specimens 3.3-3.5 cm SL. This suggests that fibre recruitment ceased between 3.5 and 7.7 cm SL, which is equivalent to 12.6-27.9% of the maximum-recorded length. The fast muscle of the smallest specimen of P. magellanica studied (9.5 cm SL) showed the characteristic mosaic pattern of fibres (Fig. 2G), but there were no fibres less than 10 µm diameter (not shown), indicating fibre recruitment had already ceased at 22% SLmax. Only the smallest N. coriiceps studied had muscle fibres less than 10 µm diameter, suggesting recruitment had ceased at approximately 16.8% of SL.
For all the other species, muscle growth was by fibre hypertrophy alone for the length ranges studied. For Channichthyidae (Chaenocephalus aceratus, Champsocephalus esox and C. gunnari) and the two Harpagifer spp., the fibres in particular regions of the myotome were of relatively uniform diameter (illustrated for C. aceratus in Fig. 3A). Thus the pattern of fibre diameter characteristic of mosaic hyperplasia was absent, which suggests that postembryonic growth was largely or entirely dependent on stratified hyperplasia.
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In the largest individuals of N. coriiceps (Fig. 3B), P. tessellata (Fig. 3C) and P. longipes (not illustrated) the fibres appeared to be undergoing splitting. The connective tissue sheath surrounding each fibre appeared to infiltrate and subdivide the fibre into 2-6 smaller daughter fibres (Fig. 3B,C), and in some cases there were aggregations of nuclei (arrowheads in Fig. 3C). Intermediate stages in this process were also occasionally observed (not shown). The incidence of `split fibres' in the largest fish remained low (1-3%) and was not sufficient to make a material difference to the estimate of fibre number.
Distribution of muscle fibre diameters
Smooth distributions were fitted to measurements of 800-1000 muscle fibres
per fish using a kernel function (Fig.
4A,B). The distributions of fast muscle fibre diameters in the
juvenile E. maclovinus studied were unimodal with a peak density that
increased from 10-40 µm diameter over the length range 4.4 (red line in
Fig. 4A) to 18.6 cm
SL. Some evidence for a bimodal distribution of fibre size was
observed in fish of 23.4-28 cm SL
(Fig. 4A). In individuals
greater than 26 cm SL there were no small diameter fibres, and in the
biggest specimens there was a broad unimodal distribution of fibre sizes with
a peak ranging from 100 to 200 µm diameter (blue line in
Fig. 4A). In contrast, fast
fibre diameters in the smallest specimens (3.3-8.6 cm SL) of P.
tessellata were bimodal (red line in
Fig. 4B), with the distribution
becoming unimodal and broader with increasing SL (green and blue lines in
Fig. 4B). Fibres less than 10
µm diameter were not present in fish of 8.6 cm SL or larger. In an
individual, 27.6 cm SL, close to the maximum size reported for this
species (Table 1), there was a
broad peak to the distribution from 250 to 500 µm diameter (blue line in
Fig. 4B). There was also a
distinct right-hand peak of fibres 20-100 µm diameter, which was not
present in fish 20-23 cm SL, probably corresponding to fibres
produced by the splitting process illustrated in
Fig. 3B,C.
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An estimate of the maximum fibre diameter per fish was obtained from the 99th percentile of the distribution and these values were similar to the average value of the 10 largest fibres measured. In all cases the maximum fibre diameter was linearly related to fish standard length (Fig. 5A,B), although there was significant interspecific variation in the slopes and intercepts of the relationships (Table 2). The highest slopes were found in those species with the lowest number of fast fibres per trunk cross-section (Fig. 5A,B) (Table 2). For the species that were close to their maximum body length, FDmax was 550 µm in P. longipes sp. and 600 µm in P. tessellata (Table 2). For the nine species where it could be determined, there was no relationship evident between the geographical zone in which the fish were captured and FDmax (Table 2).
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|
Muscle fibre number
FNmax was estimated for all 16 species. The change in
the number of fast muscle fibres (FN) per trunk cross-section in
relation to the total cross-sectional area of muscle (TCA) at 0.7 SL
for three of the species is shown in Fig.
6AC. Among species,
log10FNmax was positively and apparently
linearly related to log10SLmax
(Fig. 7). Fig. 7 also indicates that
size-corrected FNmax was apparently unrelated to
geographic origin, and inspection of Fig.
8 indicates no obvious relation with locomotory habit.
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Log10SLmax, log10FNmax, and log10size-corrected FNmax all showed highly significant phylogenetic signal (all P<0.001), irrespective of whether the real (as in Fig. 8) or arbitrary branch lengths (each segment length equal to unity) were used. Thus, for all traits examined, closely related species tended to have phenotypes that were more similar than for species chosen at random from the tree.
All three criteria suggested that setting all branch segments equal to unity in length was preferable for statistical analyses as compared with use of the real branch lengths. For log10SLmax, the correlation between the absolute values of the standardised contrasts and their standard deviations was -0.478 for the real branch lengths versus 0.190 for constant branch lengths. The corresponding MSE values were 0.162 and 0.109. Finally, r2 in the independent contrasts regression of log10FNmax on log10SLmax was 0.763 versus 0.845. Thus, we used constant branch lengths in the following analyses.
The independent contrasts linear regression (constant branch lengths) was:
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Finally, we tested the hypothesis that size-corrected FNmax was significantly lower at node 6 (supported by 88 out of 100 bootstraps) than at node 2 (supported by 100 out of 100 bootstraps), which would indicate an overall reduction in fibre number in the lineages leading to node 2. Because FNmax is strongly related to SLmax (see above and Fig. 7), we also compared SLmax at node 6 and node 2. The calculated 95% c.l. assuming constant branch lengths are shown in Table 3. The 95% c.l. of log10SLmax at node 2 overlapped with that for node 6, indicating no significant difference in the estimated nodal values. However, the 95% c.l. at node 2 for size-corrected log10FNmax do not overlap those for node 6, and so values at the two nodes can be considered significantly different. Thus, there has been a trend for the reduction of size-corrected FNmax but not SLmax in the lineages leading to node 6.
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Discussion |
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The notothenioid fishes comprise 8 families, 48 genera and 139 species
(Balushkin, 2000) or, more
conservatively, 8 families, 43 genera and 122 species
(Eastman and Eakin, 2000
).
Cladistic analysis of osteological features has contributed to resolving
notothenioid familial relationships
(Iwami, 1985
). There have been
several phylogenies based on nucleotide sequencing of mitochondrial 12S and
16S rRNA (Bargelloni et al.,
1994
; Ritchie et al.,
1997
) and nuclear 28S rRNA
(Lecointre et al., 1997
).
These molecular phylogenies implied extensive paraphyly, especially in the
Bovichtidae and Nototheniidae. Bovichtid species were placed at the base of
the tree in the sub-Antarctic zone whilst the `core' notothenioids are largely
Antarctic (Bargelloni et al.,
2000
). Based on mtDNA sequences, the diversification of this clade
is estimated at 15-20 million years (my) ago and after the formation of the
Antarctic Polar Front and climatic cooling
(Bargelloni et al., 2000
). The
phylogeny reported in Fig. 8
was in broad agreement with previous studies and assumed that C.
gobio was the most basal of the species studied. The bootstrap support
values from the Phylip analysis are shown in
Fig. 8. The family
Nototheniidae is probably paraphyletic, whereas Hapagiferidae and
Channichthyidae are monophyletic.
The Antarctic continental shelf waters have been less than 5°C for
about 12 my and today approach -1.86°C all the year around
(Clarke and Crame, 1989).
Notothenioids comprise 55% of fish species from the continental shelf and
upper continental slope of Antarctica and often represent in excess of 90% of
the species collected (Eastman,
1993
; Eastman and Eakin,
2000
; Clarke and Johnston,
1996
; Montgomery and Clements,
2000
). It has been argued that the low competitive environment
under which the notothenioid radiation has occurred has allowed a higher
tolerance of disaptation (evolutionary loss of function) than in other species
flocks (Clarke and Johnston,
1996
; Montgomery and Clements,
2000
). Examples of disaptation include the loss of respiratory
pigments in channichthyids and the incomplete canal formation in the lateral
line associated with secondary pelagicism and paedomorphosis
(Montgomery and Clements,
2000
).
The present study has shown that the radiation of the group has also been
associated with a progressive loss in the body size-specific maximum number of
fast fibres in the myotomal muscles (FNmax) of the more
derived species, e.g. node 6 versus node 2 in
Fig. 8. The maximum standard
length (SLmax) was a good predictor of
FNmax, explaining about 70% of the variation observed
among the 16 species studied (Fig.
7). Both log10SLmax and the
size-corrected values of log10FNmax showed
highly significant phylogenetic signal (P<0.001), and the amount
of phylogenetic signal was unusually high for the latter trait. Thus,
considering all 16 species in the study, related species tended to resemble
each other with respect to both traits. However, the 95% c.l. for the
estimated ancestral value of log10SLmax at node
2, representing the ancestor at the base of the notothenioid radiation,
overlapped with node 6, representing the ancestor of the Channichthyidae, one
of the more derived families (Fig.
8). This suggests that there has been no general trend for a
reduction in body size during the radiation of the group. Indeed, the
ancestral condition is generally thought to have been a small benthic species
(Eastman, 1993). In contrast,
there was statistical evidence for a reduction in
log10size-corrected FNmax between node 2 and
node 6, consistent with a general trend for reduced fibre number in the more
derived species. Thus, one of the largest species studied, Chaenocephalus
aceratus, which reaches 85 cm SLmax, had only 12 700
fast muscle fibres per trunk cross-section, or 7.7% of the 164 000 fibres in
E. maclovinus, which reaches a similar size
(Fig. 8). For comparison,
Atlantic salmon (Salmo salar L.) (Salmonidae) of 50-70 cm SL
have 550 000-1200 000 fast muscle fibres per trunk cross-section
(Johnston et al., 2000
).
An important consequence of the reduction in fibre number in the more
derived lineages of notothenioid fish is an increase in their maximum
diameter, which can reach 600 µm in some species depending on their final
body size (Fig. 5). Aerobic
muscle fibre types (slow muscles) that depend on oxygen delivery to support
their contractile activity also have correspondingly high maximum diameters in
some Antarctic fish, and can reach 100 µm
(Archer and Johnston, 1991;
Smialovska and Kilarski, 1981). Whether the maximum numbers of slow and fast
muscle fibres show a parallel reduction across the group remains to be
determined. For Atlantic cod, fibres attain a maximum diameter of 50 µm in
slow and 130 µm in fast muscle at 83 cm SL
(Greer-Walker, 1970
).
FDmax is approx. 220 µm in Atlantic salmon and is
attained at around 50 cm SL and
(Johnston et al., 2000
). The
maximum muscle fibre diameter in the highly active tropical top predator, the
Pacific Blue Marlin (Makaira nigricans Lacopóde 1803), is 50
µm for slow and 120 µm for fast muscle, even in fish exceeding 100 kg
total mass (I. A. Johnston, unpublished observations). Although the
comparative data are limited, the large maximum diameter of muscle fibres in
notothenioid fish does appear to be exceptional.
Interestingly, the pattern of fibre diameters observed in the juvenile and
adult stages of two recently diversified families of notothenioids, the
Channichthyidae and the Harpagiferidae, was not consistent with the
involvement of the mosaic hyperplasia phase of muscle fibre recruitment. This
was confirmed using laboratory reared H. antarcticus. Fibre number
was found to increase only twofold between the yolksac larvae and adult
stages, and postembryonic muscle growth was entirely supported by fibre
recruitment from localised germinal zones (stratified hyperplasia)
(Johnston et al., 2002).
Several characteristics of notothenioids, including the long pelagic larval
stage of many Antarctic species, the attainment of sexual maturity at a large
proportion of maximum size, the lack of haemoglobin in channichthyids, the
lack of scales in channichyids and many bathydraconids (reviewed in
Montgomery and Clements, 2000
)
and the reliance of embryonic and stratified hyperplasia during for growth in
adult stages, indicate that paedomorphy has been important in notothenioid
evolution.
Basic geometry dictates that an evolutionary increase in muscle fibre
diameter will decrease the surface-to-volume ratio of muscle fibres. This can
be expected to decrease basal energy requirements because of the concomitant
reduction in membrane leak pathways, which would mean that fewer
energy-utilising pumps were required to maintain ionic equilibria
(Hochachka, 1986). It has been
estimated that up to 40% of basal energy requirements are required to maintain
ionic gradients (Jobling,
1994
). Clarke and Johnston
(1999
) summarised literature
data on the metabolic rate of different fish taxa and found a significant
curvilinear relationship with temperature. However, there was no evidence that
notothenioids departed from the general trend of metabolic rate with
temperature in Perciformes (Johnston et
al., 1991
; Clarke and
Johnston, 1999
; Steffensen,
2002
). Notothenioid fish have additional energy costs compared
with other Perciformes associated with the synthesis of glycopeptide
antifreezes, which are present at high concentrations in the plasma and other
body fluids (Cheng and DeVries,
1991
). It is possible that reductions in other aspects of basal
metabolism, e.g. in relation to the loss of fibre number, serve to compensate
for the additional energy costs associated with antifreeze production.
Several `core' notothenioid species, including the icefish C. esox
and species from the genus Patagonotothen, are found outside the
Antarctic, mainly in Patagonian waters
(DeWitt et al., 1976). The
theory of an Antarctic origin for sub-Antarctic notothenioids is supported by
the distribution of antifreeze genes among representative species
(Cheng, 2000
). It has been
suggested that the presence of closely related species on either side of the
APF represents `jump dispersal' associated with episodes of climatic change
rather than passive vicariance (Bargelloni
et al., 2000
). However, it is noteworthy that several species of
notothenioids, including C. gunnari, which occur in the Antarctic,
are also found at some islands immediately to the north of the APF
(Fisher and Hureau, 1985
). The
relatively recent origin of the derived sub-Antarctic notothenioids
(Notothenidae and Channichthyidae) would then explain why they have a
relatively low muscle fibre number and high maximum diameter, traits that
originated in a colder and more stenothermal environment. E.
maclovinus and C. gobio have a greater size-corrected
FNmax than the other notothenioids studied, indicating
that they are probably the least derived species. Thus E. maclovinus
and C. gobio probably diverged from the other notothenioids with the
separation of South America from Antarctica around 25-20 million years ago,
and did not subsequently invade from Antarctica. This is probably the general
case for Bovichtidae, Pseudaphritidae and Eleginopidae, since they are all
represented by non-Antarctic species that lack antifreezes, except for a
single Antarctic species of bovichtid
(Eastman and Eakin, 2000
).
The rate of oxygen delivery to aerobic muscle fibres is a function of the
fibre diameter and factors that affect diffusion rate
(Egginton et al., 2002). The
latter include temperature, the distribution of mitochondria and lipid
droplets within the fibre, and overall metabolic demand
(Desaulniers et al., 1996
;
Londraville and Sidell, 1990
;
Egginton et al., 2002
;
O'Brien et al., 2003
). The
temperature-dependence of the state 3 respiration rate of isolated
mitochondria with pyruvate as substrate was described by a single quadratic
relationship for all Perciformes studied, with no significant upregulation of
the maximum rate of oxygen uptake per mg mitochondrial protein in Antarctic
and sub-Antarctic species (Johnston et
al., 1998
). Adequate oxygen delivery to large-diameter muscle
fibres is probably only possible because of the very low metabolic demand in
polar fishes at low temperature (Johnston
et al., 1991
; Clarke and Johnston, 1997;
Steffensen, 2002
). Modelling
studies indicate that a low fibre number and high maximum fibre diameter does
not limit adequate oxygen flux at low body temperatures (-2 to +5°C) in
notothenioids (Egginton et al.,
2002
). There is evidence that heat death in Antarctic fish is
linked to an oxygen limitation, resulting from a mis-match in oxygen delivery
and consumption at the tissue level
(Pörtner, 2002
;
Mark et al., 2002
). Thus, a
high FDmax may well constrain the upper thermal limit of
notothenioids, particularly in the case of the icefish C. esox and
other Channichthyids that lack respiratory pigments
(Egginton et al., 2002
).
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Acknowledgments |
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Footnotes |
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References |
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