Polymorphism of the rod visual pigment between allopatric populations of the sand goby (Pomatoschistus minutus): a microspectrophotometric study
1 Department of Biosciences, University of Helsinki, PO Box 65 (Viikinkaari 1),
FIN-00014, Finland
2 Institute of Biotechnology, University of Helsinki, PO Box 65 (Viikinkaari 1),
FIN-00014, Finland
* Author for correspondence (e-mail: mirka.jokela{at}helsinki.fi)
Accepted 30 April 2003
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Summary |
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Key words: microspectrophotometry, rod photoreceptor, retina sand, goby, Pomatoschistus minutus, polymorphism, vision
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Introduction |
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Aquatic environments offer a wide range of strongly profiled, yet
reasonably stable or at least regularly recurring, spectral environments,
making underwater vision a gratifying field for comparative studies. There is
a vast literature on spectral adaptations in fishes (for reviews of older
literature, see, for example, Lythgoe,
1972,
1988
;
Bridges, 1972
; for more recent
studies relating spectral absorbance to opsin structure, see, for example,
Bowmaker et al., 1994
;
Hope et al., 1997
; Hunt et
al., 1996
,
2001
;
Yokoyama and Tada, 2000
).
However, little work has been done on incipient evolutionary adaptation
between separated populations of a single species inhabiting spectrally
different waters.
The sand goby (Pomatoschistus minutus) is a small marine fish
(adult length 5-10 cm) that occurs in considerable abundance along the coasts
of Europe from northern Norway to the eastern Mediterranean, including the
bracken-water of the Baltic Sea. This geographical span encompasses a wide
range of different light environments. For example, the peak of the light
spectrum at 30 m depth in the Baltic Sea lies around 550-560 nm, which is
displaced by some 80 nm towards longer wavelengths compared with that of the
Mediterranean (Jerlov, 1976;
Lindström, 2000
). As the
gobies spend their off-breeding season at depths of several tens or even
hundreds of meters (Koli,
1995
), one would expect that the Baltic population would benefit
from shifting
max of the dim-light receptor towards longer
wavelengths compared with its truly marine conspecifics.
Studying four populations of sand gobies (Baltic Sea, Swedish west coast,
English Channel and Adriatic Sea), we find small but consistent shifts in
max, from 503.0±0.3 nm (mean ±
S.E.M.) in the Adriatic to 508.3±0.5
nm in the Baltic, with the two other populations falling in between. We show
that even such a small shift may be significant for vision. Since the
differences could not be explained by a chromophore change, they must indicate
polymorphism of the opsin.
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Materials and methods |
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Recording
Before microspectrophotometrical (MSP) measurements, the living fish were
dark-adapted overnight. The frozen Adriatic sand gobies were thawed
individually for approximately 30 min in darkness at room temperature before
dissection. All subsequent manipulations were performed under dim red light.
The fish were decapitated and pithed. The eyes were dissected in physiological
saline (teleost Ringer) containing: 110 mmol l-1 NaCl; 2.5 mmol
l-1 KCl; 1 mmol l-1 CaCl2; 1 mmol
l-1 MgSO4; 10 mmol l-1 NaHCO3 and
10 mmol l-1 glucose. The solution was buffered to pH 7.2-7.4 with
10 mmol l-1 Hepes. The lens was removed, and pieces of retina
separated from the pigment epithelium were transferred to a drop of Ringer on
a cover slip and teased apart. Dextran (10-15%, Mr=70 kDa)
was added to the Ringer to prevent excess cell movements during recordings.
The sample was covered with a second cover slip, sealed at the edges with
vaseline and placed on the MSP stage.
Absorbance spectra were recorded with a single-beam, computer-controlled,
fast-wavelength scanning microspectrophotometer built at the University of
Helsinki (Govardovskii et al.,
2000; Ala-Laurila et al.,
2002
). The basic design is described in Govardovskii and Zueva
(2000
). A halogen lamp served
as a light source, and spectral scanning was achieved through a diffraction
grating attached to the head-moving lever of a Seagate ST-225 computer hard
disk drive. The grating was moved by a stepper motor grid, whose position was
controlled by a computer-driven step motor. Recordings were made on isolated
rod outer segments (OSs) or the outer segments of rods still attached to small
pieces of retina. OS dimensions were approximately 3 µmx30-40 µm.
The size of the measuring beam was adjusted to match the sample, typically to
approximately 75% of the OS width and nearly the full OS length. The beam was
linearly polarized in the plane of the discs. A baseline measurement was
obtained by scanning a clear area adjacent to the cell. The OS was then
scanned, and the ratio between the two measurements gave the absorbance
spectrum. The wavelength calibration was checked regularly, at least at the
beginning and at the end of each experiment, against the spectrum of a `blue
glass' standard, the spectrum of which had been accurately determined in a
Hitachi spectrophotometer. The recordings were carried out at room
temperature. For further technical details, refer to Govardovskii et al.
(2000
) and Ala-Laurila et al.
(2002
).
Analysis
The data were stored on the computer hard disk for later analysis. The
details of the analysis can be found in Govardovskii et al.
(2000), and only a brief
description is given here. Raw spectra from single cells were averaged and
normalized within each individual, and the resulting individual spectra were
corrected for zero offset. The position of the zero-line was computed as a
straight line least-square fitted to the long-wave tail of the spectrum
between 650 nm and 750 nm, where the absorbance of the visual pigment is close
to zero. High-frequency noise components were removed by Fourier filtering,
retaining 25-35 harmonics. Finally, the mean, zero-line-corrected and filtered
spectrum from each individual was fitted with the A1 template of Govardovskii
et al. (2000
), giving the
max. Differences in
max between the
populations were statistically evaluated using the SPSS 10.0.7 program. The
pairwise comparisons were based on Scheffe's test after an initial analysis of
variance (ANOVA) had indicated the presence of significant between-population
differences.
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Results |
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The histogram in Fig. 1C
shows the distribution of max values obtained by fitting
all the individual sand goby spectra. The different populations are symbolized
by the colours that mark the respective locations in
Fig. 1A. The means (±
S.E.M.) of
max calculated
across individuals within each population were 503.0±0.3 nm (A),
505.4±0.2 nm (S), 506.2±0.3 nm (E) and 508.3±0.5 nm (B).
An initial ANOVA indicated that there are significant differences between
populations (P<0.001). Pairwise comparison based on Scheffe's test
indicated that the Adriatic population differs from each of the others (A
vs B, P<0.001; A vs S or E, P<0.01),
as does the Baltic population (B vs S or E: P<0.05). By
contrast, the difference between S and E is not significant
(P>0.8).
A quick way for fish to red-shift their spectral sensitivity is to switch
the visual-pigment chromophore from A1 to A2 (e.g.
Dartnall and Lythgoe, 1965;
Bridges, 1972
). We therefore
paid special attention to the question of whether the relative bathochromic
shift of the Baltic rod pigment could be explained by some degree of A2
admixture. Since A1 and A2 spectra are of different shape, this can be studied
by comparing the quality of fit of a pure A1 template with that achieved by
some linear combination of A1 and A2 templates. The
max of
the A1 pigment was always chosen so that the mixture should give the best
possible fit to the main part of the recorded spectrum [the
max of the A2 component is tied to its A1 pair through the
Hárosi (1994
)
relationship]. In no individual could a perceptibly improved fit be achieved
by adding A2, and when the assumed proportion of A2 was increased above
approximately 2%, the fits clearly started to deteriorate. This is exemplified
in Fig. 2A for the Baltic
spectrum shown in Fig. 1B. The
best-fitting template for a 95%:5% A1:A2 mixture is seen to run systematically
above the recorded spectrum at long wavelengths.
Fig. 2B illustrates the
unacceptability of a conceivable `null' hypothesis that Adriatic and Baltic
fish actually have the same opsin and that the
max shift
from 503.0 nm to 508.3 nm is achieved just by mixing in a certain percentage
of A2 chromophore. Moving the peak to 508.3 nm would require a 39% admixture
of A2, producing a curve shape that is quite incompatible with the recorded
spectrum.
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Other gobies
When collecting sand gobies, we happened to catch a few specimens of two
other species of Pomatoschistus. It is of some interest to compare
the max of these close relatives with the range covered by
the sand goby populations. Thus, rod absorbance spectra were also recorded
from 11 common gobies (P. microps) from the Baltic Sea and two
marbled gobies (P. marmoratus) from the Gulf of Venice (on average,
30 cells from each individual). The
max values obtained
were 515.7±0.4 nm (P. microps) and 506.7 nm (the two P.
marmoratus individuals studied both yielded the same
max). Fig.
3A shows individual spectra from these species together with the
Adriatic sand goby spectrum from Fig.
1B. Fig. 3B
summarizes the results as a histogram similar to
Fig. 1C. For comparison, the
max ranges found in the different sand goby populations
have been plotted as bars above the histogram.
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Light environment
The gobies go into deep waters after the breeding season, to some 40 m in
the Adriatic Sea, starting in March, and at least tens of meters in the Baltic
Sea, starting in AugustSeptember. It is in these conditions that there
is least light overall, and spectral filtering by the water is most
pronounced. Thus, these are the conditions where spectral adaptation of the
rod pigment (as opposed to cone pigments) would be most important for visual
sensitivity, and considerations of the ecological significance of rod
max are likely to be most relevant. As an example, we shall
compare the calculated quantum catches of an Adriatic and Baltic sand goby and
a Baltic common goby, all assumed to live in a Baltic light environment.
The grey symbols in Fig. 4
show the spectral distribution of light at 10 m depth in a narrow bay of the
Baltic Sea, Pojoviken, close to where the Baltic gobies were caught. The
spectrum is reproduced from Lindström
(2000; curve B in his
fig. 1). The three other curves
are quantum catch spectra, obtained by convolving the 503 nm, 508.3 nm and
515.7 nm A1 templates with the light spectrum. It is immediately evident that
the highest
max value gives the best quantum catch (violet
curve for the common goby), while the lowest value gives the worst (blue curve
for the Adriatic sand goby). The total quantum catch in each case is
proportional to the integral of the spectrum across wavelengths, i.e. the area
under each curve. Thus, we find that the Baltic sand goby would catch 19% more
quanta than the Adriatic sand goby in this environment. The Baltic common
goby, however, is still 23% better than the Baltic sand goby and 47% better
than the Adriatic sand goby.
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Discussion |
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The spectral absorbance of visual pigments (determining the spectral
sensitivity of the organism) is an interesting characteristic from an
evolutionary point of view. It provides a case where a phenotypic trait
subject to strong natural selection depends directly on changes in a single
gene. A considerable amount of knowledge has been accumulated about amino acid
changes that modify the absorption spectra of visual pigments (see, for
example, Hunt et al., 1996,
2001
; Yokoyama,
2000
,
2002
). A start has also been
made towards identifying changes that control a second important functional
property of visual pigments, thermal stability
(Fyhrquist et al., 1998
).
Sequencing the opsins of individual fish (characterized by MSP) from the
different populations may allow us to establish very specific correlations
between amino acid substitutions and the spectral properties, the published
amino acid sequence of the English Channel population
(Archer et al., 1992
) serving
as a reference. Moreover, the results can be related to within-species
phylogeny based on other markers, whereby the common goby and the marbled goby
serve as outgroups.
Functional significance of the differences in
max
The qualitative consistency of the results may seem surprising, as the
max differences are so small as to leave doubts about their
functional significance. This is true especially considering seasonal, tidal
and other variations in the spectral transmission of the water as well as the
very different conditions encountered in the shallow and the deep waters
inhabited at different times of the year.
With respect to the conditions relevant for rod pigment adaptation, the
situation is less complex. Only in deep waters will the rod system
consistently have to face the limits to visual sensitivity and quantum catch
(together with thermal stability) become all important. Vision in shallow
waters in brighter light has to solve other problems, which mainly concern the
cone pigments (cf. Lythgoe,
1979,
1988
).
In Fig. 4, we considered
adaptation to the Baltic Sea in order to estimate how much quantum catch may
vary with the observed differences in max. This is a
reasonable example, as the Baltic red-shift of
max may
probably be regarded as a recent adaptation from an original marine state. The
gobies probably colonized the Baltic Sea in its last truly marine (Litorina)
phase, starting some 7000 years ago and gradually developing into the
present-day bracken-water condition. As shown in
Fig. 4, moving
max from 503 nm to 508.3 nm would confer a 19% quantum
catch advantage on a fish living at 10 m depth in Pojoviken Bay. Similar
convolution with an open-sea Baltic spectrum measured at 20 m depth
(Lindström, 2000
;
Fig. 1A) gives a 13% advantage
for the Baltic sand gobies compared with the Adriatic ones. Although the
ecological value of this is not negligible, it is worth noting that the
closely related common goby (P. microps,
max=515.7
nm) fares much better in the same environments, having a 31% and 47% quantum
catch advantage over the Adriatic sand gobies in the Baltic open sea and
Pojoviken environments, respectively. The rather modest adaptation of the
Baltic sand goby compared with the common goby could be due to different
earlier histories but might also indicate functional constraints in the
molecule that oppose further red-shifts along the routes open to this
particular opsin. Opposing pressures could arise, for example, from a
connection between increased long-wavelength sensitivity and increased thermal
noise (Barlow, 1957
;
Donner et al., 1990
;
Milder, 1991
). A general
empirical correlation has been found between the thermal stability and
max of visual pigments, although no strict physical
relation exists (Firsov and Govardovskii,
1990
; Fyhrquist,
1999
; Koskelainen et al.,
2000
). The loose but still significant correlation observed could
be interpreted to mean that the cost in thermal noise incurred by shifting
max varies depending on the exact amino acid sequence of
the opsin, so that a red-shift that gives a net signal-to-noise profit when
caused by a certain mutation or set of mutations in one opsin (e.g. the common
goby) might give a net loss when implemented in the partly different molecular
setting of another opsin (e.g. the sand goby).
A similar argument can be advanced to explain why the sand gobies have not
taken recourse to the obvious possibility of changing the chromophore to A2,
as used by many fish and amphibian species. Switching from A1 to A2 red-shifts
max by more than 20 nm
(Dartnall and Lythgoe, 1965
;
Hárosi, 1994
) but is
also known to lower the minimum energy for photoactivation and increase the
rate of thermal pigment activations and thus thermal noise
(Donner et al., 1990
;
Milder, 1991
;
Koskelainen et al., 2000
;
Ala-Laurila et al., 2002
). The
signal-to-noise change could easily be negative. With respect to chromophore
changes, however, it should further be noted that all photoreceptors in one
retinal area apparently receive similar proportions of A1/A2 from the pigment
epithelium (Loew and Dartnall,
1976
; Makino-Tasaka and
Suzuki, 1984
), and optimisation of the A1/A2 ratio may equally
well be driven by cone vision as by rod vision. Obviously, these arguments
concern `ultimate' causes of lack of A2. The `proximate' cause might simply be
that the fish lack the necessary enzyme for A1/A2 conversion.
Polymorphism within the same region?
Our mean max value for the English Channel sand gobies
(506.2 nm) is significantly higher than the value of 500.8 nm reported by
Archer et al. (1992
) for
specimens caught in the same area near Plymouth. In the 10 Plymouth
individuals studied here,
max ranged from 504.6 nm to 507.8
nm. The differences we are concerned with in the present work are small
overall, so we have to ask whether the difference between our value and that
of Archer et al. must indicate that different populations (in a statistical
sense) have been sampled even in this local region. At this point there seem
to be no strong reasons to think so. Firstly, trivial differences in
max reported by different investigators arise just from
using different visual-pigment templates. To evaluate this possibility, we
fitted the spectral data of Archer et al.
(1992
, their
fig. 1) with the Govardovskii
et al. (2000
) template. The
best fit was obtained with
max=502 nm, but, admittedly,
their data were certainly not consistent with
max=506.2 nm.
Secondly, however, their data had been collected from a very small sample of
rods in questionable condition, eight rods from five fish, while the typical
dimensions of the rod OSs (or the measurement beam fitting within the OS?) are
reported as 2 µmx5 µm. In our material, the dimensions of the OSs
were typically approximately 3 µmx30-40 µm. In view of the small
sample size and apparent fragmentation of the OSs, we would not regard the
discrepancy between their and our
max values as
significant.
On the other hand, there is of course no reason to exclude the possibility
of polymorphism within populations. This is the material on which natural
selection operates. Bowmaker et al.
(1975) have previously
suggested that the wide variation they found in rod
max
between individuals of the frog Rana temporaria (from a single
supplier but of unidentified provenance) could indicate genetic heterogeneity.
It may be significant that the strongest suggestion of within-population
polymorphism in our material is found among the Baltic sand gobies, which
display a particularly broad
max distribution spanning 5.7
nm (Fig. 1C).
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Acknowledgments |
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