Opsin gene duplication and diversification in the guppy, a model for sexual selection

Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada
Proceedings of the Royal Society B: Biological Sciences (Impact Factor: 5.05). 02/2007; 274(1606):33-42. DOI: 10.1098/rspb.2006.3707
Source: PubMed
ABSTRACT
Identification of genes that control variation in adaptive characters is a prerequisite for understanding the processes that drive sexual and natural selection. Male coloration and female colour perception play important roles in mate choice in the guppy (Poecilia reticulata), a model organism for studies of natural and sexual selection. We examined a potential source for the known variation in colour perception, by analysing genomic and complementary DNA sequences of genes that code for visual pigment proteins. We find high sequence variability, both within and between populations, and expanded copy number for long-wave sensitive (LWS) opsin genes. Alleles with non-synonymous changes that suggest dissimilar spectral tuning properties occur in the same population and even in the same individual, and the high frequency of non-synonymous substitutions argues for diversifying selection acting on these proteins. Therefore, variability in tuning amino acids is partitioned within individuals and populations of the guppy, in contrast to variability for LWS at higher taxonomic levels in cichlids, a second model system for differentiation owing to sexual selection. Since opsin variability parallels the extreme male colour polymorphism within guppy populations, we suggest that mate choice has been a major factor driving the coevolution of opsins and male ornaments in this species.

Full-text

Available from: Detlef Weigel, Sep 16, 2014
Opsin gene duplication and diversification
in the guppy, a model for sexual selection
Margarete Hoffmann
1
, Namita Tripathi
1
, Stefan R. Henz
1
, Anna K. Lindholm
2
,
Detlef Weigel
1
, Felix Breden
3,†
and Christine Dreyer
1,
*
,†
1
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tu
¨
bingen D72076, Germany
2
School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney,
New South Wales 2052, Australia
3
Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 156, Canada
Identification of genes that control variation in adaptive characters is a prerequisite for understanding the
processes that drive sexual and natural selection. Male coloration and female colour perception play important
roles in mate choice in the guppy (Poecilia reticulata), a model organism for studies of natural and sexual
selection. We examined a potential source for the known variation in colour perception, by analysing genomic
and complementary DNA sequences of genes that code for visual pigment proteins. We find high sequence
variability, both within and between populations, and expanded copy number for long-wave sensitive (LWS)
opsin genes. Alleles with non-synonymous changes that suggest dissimilar spectral tuning properties occur in
the same population and even in the same individual, and the high frequency of non-synonymous substitutions
argues for diversifying selection acting on these proteins. Therefore, variability in tuning amino acids is
partitioned within individuals and populations of the guppy, in contrast to variability for LWS at higher
taxonomic levels in cichlids, a second model system for differentiation owing to sexual selection. Since opsin
variability parallels the extreme male colour polymorphism within guppy populations, we suggest that mate
choice has been a major factor driving the coevolution of opsins and male ornaments in this species.
Keywords: guppy; LWS opsin; gene duplication; gene diversification; visual pigment; sexual selection
Abbreviations: LWS; long-wave sensitive; SWS; short-wave sensitive; RH1; rhodopsin; RH2;
rhodopsin-like 2; cDNA; complementary DNA; TM; transmembrane (domain); ID; intradisc
(domain); SNP; single nucleotide polymorphism; EST; expressed sequence tag
1. INTRODUCTION
The guppy (Poecilia reticulata) is one of the premier model
organisms for the study of phenotypic variation owing to
divergent natural selection and sexual selection
(Magurran & Ramnarine 2005). Male guppy colour
patterns in particular are highly polymorphic, both within
and between populations. This variation is a consequence
of different evolutionary and environmental contexts,
including predators (Endler 1980), carotenoid availability
(Grether et al. 1999) and female preferences (Houde
1997). Although females overall prefer conspicuous males
(Endler & Houde 1995), female preference also varies
within (Brooks & Endler 2001) and between populations
(Breden & Stoner 1987; Houde & Endler 1990; Endler &
Houde 1995). Variation in sensitivity of cone opsins has
been suggested as a mechanism explaining this variation in
female preference (Endler 1992). Despite the importance
of visually mediated sexual selection in the evolution of the
mating system of the guppy, very little is known about the
diversification of opsin proteins, which help tune
absorbance of visual pigments in vertebrates and thus play
an essential role in colour discrimination (Yokoyama &
Yokoyama 1996).
Teleost shes express four different cone opsins,
which determine sensitivity in red and orange (LWS),
green (rhodopsin-like (RH2) or KFH-G), blue and violet
(short-wave sensitive 2 (SWS2)) and UV (short-wave
sensitive 1 (SWS1)) regions of the spectrum; these cone
types also correspond to phylogenetically distinct gene
families (Yokoyama 2000; Trezise & Collin 2005). The
five sites rule suggests that replacement at a few tuning
sites can explain major shifts in maximum absorption of
vertebrate cone opsins (Yokoyama & Radlwimmer 1998;
Kochendoerfer et al. 1999). Guppy retinal absorption
spectra indicate sharp maxima for absorption of UV at
359 nm, violet/blue at 408 nm and green light at
464 nm, with a broader peak of absorbed orange and
red light between 520 and 580 nm. This latter peak
could be resolved into three maxima at approximately
533, 548 and 572 nm, and interestingly, individuals
differ in the occurrence of these three absorption
maxima, suggesting allelic variation in LWS opsin
genes (Archer et al. 1987; Archer & Lythgoe 1990;
Endler et al. 2001).
In the zebrafish, Danio rerio (Chinen et al. 2003), and in
the medaka, Oryzias latipes (Matsumoto et al. 2006), LWS
opsins are present in two tandemly duplicated copies.
Two isoforms have also been reported in Lucania goodei that
Proc. R. Soc. B (2007) 274, 33–42
doi:10.1098/rspb.2006.3707
Published online 26 September 2006
Electronic supplementary material is available at http://dx.doi.org/10.
1098/rspb.2006.3707 or via http://www.journals.royalsoc.ac.uk.
* Author for correspondence (christine.dreyer@tuebingen.mpg.de).
F.B. and C.D. contributed equally to the design of the guppy opsin
project.
Received 10 July 2006
Accepted 10 August 2006
33 This journal is q 2006 The Royal Society
Page 1
most probably also represent two gene copies (Fuller et al.
2003). Phylogenetic analysis suggests that these dupli-
cations are independent (Chinen et al. 2003). In contrast,
Southern blot analysis indicated that there is only a single
LWS opsin copy in the cichlid Neochromis nigricans (Terai
et al. 2002). While a given cichlid species generally has only
one or two LWS isoforms, an amazing degree of
interspecific LWS sequence variation has been documen-
ted (Terai et al. 2002; Carleton et al. 2005; Parry et al. 2005;
Spady et al. 2005). Since cichlid species in African rift lakes
have only arisen very recently, variation must have evolved
within a comparatively short time frame, indicating
diversifying selection acting on visual perception.
The molecular genetic basis underlying sexually
selected characters can reveal the pattern of selection
that has been acting on these characters and help to
discriminate among competing models explaining diversi-
fication and speciation owing to sexual and natural
selection. Therefore, we compared LWS opsin sequences
from several individuals and populations across the
geographical range of the guppy. We focused on LWS
polymorphisms, because these may provide a molecular
basis for the broad and variable red sensitivity of the guppy
retina, and because the perception of red and orange
signals plays a major role in mate choice of guppies.
2. MATERIAL AND METHODS
(a) Guppy strains
We examined the laboratory strain Wild Istanbul, as well as
offspring from wild-caught guppies that had been kept in the
laboratory for multiple generations (table 2).
(b) cDNA library and primer design
(i) Oropuche-2 retina library
Retinae were dissected from five adult females from an
Oropuche River, Trinidad, stock population and frozen in
liquid nitrogen. Total RNA was extracted using a Qiagen
RNAeasy Miniprep kit. After reverse transcription, comp-
lementary DNA (cDNA) was cloned into pDNRlib vector
using a creatorSMART cDNA construction kit (Clonetech).
The cDNA was amplified by 18 cycles of long-distance PCR
using BD Advantage 2 PCR enzyme mixture, following the
manufacturer’s protocols.
(ii) Quare-6 and Tranquille embryo libraries
Late- and very-late-eyed embryos were dissected from
pregnant females of Quare-6 and Tranquille stock popu-
lations. Total RNA was extracted using a Qiagen RNAeasy
Midiprep kit and enriched for polyACRNA using Qiagen
Oligotex on a spin column. cDNA was cloned as described
above, except that the primer extension protocol included only
6–8 cycles of PCR amplification. As part of an expressed
sequence tag (EST) sequencing project, cloned cDNAs were
isolated using Qiagen MagAttrac minipreps and sequenced
from the 5
0
end or both ends using pDNRlib-specific vector
primers on a Hitachi ABI 3730XL DNA Analyzer. cDNA
sequences were identified by NCBI BLASTN and BLASTX
(C. Dreyer, M. Hoffmann, C. Lanz, M. Riester, S. R. Henz,
and D. Weigel, manuscript in preparation).
(c) PCR analysis of genomic sequences
Consensus primers LWSfw1A (5
0
-TCT TAT CAG TCT
TCA CCA ACG G-3
0
) and LWSrev5 (5
0
-CAT GAC TAC
AAC CAT CCT GG-3
0
) were designed from the alignment of
Poecilia reticulata LWS cDNA with LWS sequences from
medaka (O. latipes, AB001604), blue fin killifish (L. goodei,
AY296740) and three cichlids (Dimidiochromis compressiceps,
AF247131; Astatotilapia velifer, AB090427; and Gaurochromis
simpsoni, AB090425). This primer pair amplifies a region
spanning part of exon 2, the highly variable exons 3 and 4
(Terai et al. 2002) and part of exon 5. LWSfw1B (5
0
-TTT
TAT CAG TCT TCA CCA ACG GC-3
0
) is a guppy-specific
primer designed from the LWS cDNA sequences. Additional
LWS haplotype-specific sequencing primers were LWSE-
nInsrev (5
0
-GAA GAA ATG AGG ATG CAG CA-3
0
),
LWS675AG (5
0
-GTA GCA CAA GAT GAT GAT ACC T-
3
0
), LWS675GG (5
0
-GTA GCA CAA GAT GAT GAT ACC
C-3
0
) and LWS675GC (5
0
-CAG GTA GCA CAA GAT GAT
GAT AG-3
0
). Primers for other opsin loci included:
rhodopsin (RH1) (RH1fw2, 5
0
-AAG TCA GCC TCC
ATC TAC AAC-3
0
; RH1rev, 5
0
-GCC TTG ATT ACA
TGC CTC TAC-3
0
), RH2-1 (RH2-1fw, 5
0
-GGA CGA GAC
CTC AGT TTC AG-3
0
; RH2-1rev, 5
0
-CTC CAG TAA AAC
ATG TTC CCT C-3
0
), and SWS1 (SWS1fw, 5
0
- TCA CCT
GTA CGA GAA CAT CTC-3
0
; SWS1rev, 5
0
- AAG AAC
GCA GGG ATG GTG AC-3
0
).
Genomic DNA was extracted using a DNeasy Tissue kit
(Qiagen), and amplified using Taq polymerase from New
England Biolabs and following the protocol: denaturation at
948C for 5 min, followed by 35 cycles of (948C, 30 s; 588C,
30 s; 688C, 90 s), and a final elongation step of 688C for
6 min. Sizes of amplification products are given in table 1.
Direct sequencing of PCR products for LWS showed that
many sites were polymorphic, and therefore PCR products
were purified with Bioline Quick Clean and cloned into
pGEMTeasy (Promega) vector. T7 and SP6 promoter
Table 1. Variability in guppy opsin cDNAs and genomic fragments.
cDNAs genomic fragments
opsin
clones among
18 000 ESTs
length coding
sequence (bp)
synonymous
SNPs
non-synonymous
SNPs length (bp)
synonymous
and non-coding
SNPs
non-
synonymous
SNPs
RH1 148 1062 3 1 497 4 0
RH2-1 14 1056 0 0 450 1 0
RH2-2 9 1036 1 0 n.d.
a
SWS1 10 1008 0 (6)
b
0 912 5C1 indel 0
SWS2B 2 1056 0 (2)
b
0 n.d.
a
LWS 5 1074 0 9 920 19 7
a
Not determined.
b
SNPs that could not be confirmed by sequencing of PCR amplification products are given in parentheses.
34 M. Hoffmann et al. Opsin diversity in the guppy
Proc. R. Soc. B (2007)
Page 2
primers were used to sequence clones. One PCR product,
amplified with primers fw1A and rev5 from a Xiphophorus
helleri individual purchased from an aquarium store, was
cloned and sequenced.
(d) DNA blot analysis
Genomic DNA was isolated from tail muscles of at least six
fishes from each of Quare, Tranquille and Cumana
´
strains,
and pooled for each strain. DNA was extracted using
QIAGEN genomic-tip 500/G columns. Six micrograms of
digested DNA was concentrated by ethanol precipitation,
resolved on a 0.8% agarose gel and transferred to a positively
charged nylon membrane (Roche). DNA was UV cross-
linked (STRATALINKER 2400) and pre-hybridized in 5!
SSC, 0.02% (w/v) SDS, 0.1% (w/v) N-lauroylsarcosine and
1% blocking reagent overnight at 628C. Cloned RH1, RH2-
1, SWS1 and LWS fragments were labelled using the PCR
DIG-Probe Synthesis Kit (Roche). Membranes were hybri-
dized overnight at 608C. The membranes were washed three
times in 0.1% SDS, 2! SSC (30 min, 608C), twice in 0.1%
SDS, 2! SSC (20 min, 628C), and twice in 0.1% SDS, 0.5!
SSC (15 min, 628C). Membranes were treated with blocking
solution, followed by 1 : 10 000 (v/v) anti-DIG-antibodies
(Roche). Detection was performed using CSPD chemi-
luminescent substrate (Roche). Blots were exposed to
Lumi-Film Chemiluminescent Detection Film (Roche) for
10–60 min.
(e) Comparison with other vertebrate long-wave
sensitive sequences
GCG (Wisconsin Package v. 10.3, Accelrys, Inc., San Diego,
CA) or C
LUSTALW (v. 1.81, Thompson et al. 1997) was used
to align guppy cDNAs with LWS sequences from GenBank
from other species of fish, chicken and human. Our alignment
follows the numbering system of Yokoyama & Radlwimmer
(1998) to facilitate comparison to substitutions known to
affect maximum absorbance of other vertebrate visual
pigments.
Given the high variability in these sequences and the
possibility that some of this variation were owing to PCR
artefacts, a conservative approach was taken for reporting
single nucleotide polymorphisms (SNPs). Only SNPs that
occurred in at least two individuals were analysed. In
addition, four sequences with frame shifts were omitted from
the analyses. Sequences were aligned using M
USCLE (Edgar
2004)andC
LUSTALX(Thompson et al.1997), and the program
C
OLLAPSE (v. 1.2, http://darwin.uvigo.es/software/collapse.
html) was used to identify unique haplotypes in the genomic
sequences. A BIONJ tree (Gascuel 1997) was constructed using
the X. helleri sequence as outgroup and using S
PLITSTREE4
software (Huson & Bryant 2006). An alignment of the set
of unique haplotypes was submitted as a population study
to GenBank.
(f ) Molecular evolution analysis
The rate of non-synonymous nucleotide substitutions per
non-synonymous site, relative to the rate of synonymous
nucleotide substitutions per synonymous site (Ka/Ks),
across all amino acid sites was calculated for the partial
LWS genomic sequences and the corresponding part of the
two LWS cDNAs. Branch-specific ratios were estimated
using the sliding window method of D
NASP v. 4.10.4 (Rozas
et al. 2003).
3. RESULTS
(a) Diversity of opsin cDNAs
We isolated cDNAs of RH1 and five different cone opsins
from libraries of whole embryos and adult retinas of the
position 0
3
7
K
R
R
R
R
R
R
P
K
K
R
0
5
5
V
L
L
L
V
V
V
V
L
V
V
0
5
6
S
S
A
S
A
A
S
S
A
A
A
0
6
5
V
V
V
I
V
V
V
I
V
V
V
0
6
8
V
V
V
V
V
V
V
T
T
T
I
1
3
1
Y
F
Y
Y
Y
Y
F
Y
F
Y
Y
1
3
4
S
S
S
A
S
S
S
S
A
S
S
1
4
0
A
A
A
A
A
A
A
A
G
A
G
1
7
1
A
G
A
G
G
G
A
A
A
A
A
1
8
0
A
S
A
S
A
S
S
S
S
A
A
1
8
2
V
A
V
F
V
V
V
V
V
A
V
1
9
4
Y
F
Y
F
Y
Y
Y
Y
Y
Y
Y
2
1
9
Y
Y
Y
F
Y
Y
Y
Y
Y
Y
Y
2
3
0
I
I
I/L
F
I
I
I
L
I
I
L
2
3
3
A
G
T
A/S
A
A
A
A
G
A
A
2
4
7
R
H
R
R
R
R
R
R
R
H
H
2
7
5
I
I
V
M
V
V
V
L
M
F
L
2
7
7
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
F
2
7
8
C
C
C
I
C
C
C
C
C
C
C
2
8
5
T
T
T
A
T
T
T
T
T
T
T
3
2
0
V
V
I
V
V
I
V
V
V
V
I
OR6-3 (variant 1)
OR6-4 (variant 2)
cichlids I
cichlids II
T. rubripes
O. latipes A (561)
L. goodei A (573)
L. goodei B (573)
A. fasciatus (558)
D. rerio 1 (558)
D. rerio 2 (548)
ID1 TM1 TM3 TM4 ID3 TM5 TM6 TM7
domains
RLAVVY SAG S VYYIARV Y C T VO. latipes B (562)
RH2-1
F
TA
A A A Y
YF
FAR
L
K
LL
V
TCL
I
Figure 1. Amino acid substitutions in LWS opsin protein sequences. Transmembrane domains (TM 1–7) and intradisc domains
(ID 1–6) are shown above. Amino acid position follows the bovine RH1 reference. Amino acids previously described as critical
for green/red spectral tuning (Yokoyama 2000) are colour-coded in green and red, respectively. RH2 (green) and LWS (red)
variable sites represent characteristic differences between green- and red-sensitive vertebrate pigments (Kochendoerfer et al.
1999). In addition, amino acids that differ between guppy OR6-3 and OR6-4 are colour-coded yellow and blue across all LWS
sequences. Cichlids I and II represent the major allelic types from Lakes Victoria and Nabugabo (Terai et al. 2002). For
comparison, the last line shows residues of guppy green opsin RH2-1. Wavelength of maximal absorption of proteins
reconstituted with 11-cis-retinal in vitro (l
max
) is given in parenthesis, if published. Accession numbers are given in table 3.
Opsin diversity in the guppy M. Hoffmann et al.35
Proc. R. Soc. B (2007)
Page 3
guppy (table 1) in the course of an EST project, whose
details will be reported elsewhere. The abundance of the
different cDNAs in the EST libraries gives a first hint of
their relative expression levels. RH1, for which we found
relatively little sequence variability, was the most abun-
dant opsin (table 1). The RH1 cDNAs from the Quare
strain were polymorphic at three sites, of which one was
non-synonymous, and fell into two haplotypes. cDNAs
from the other strains were polymorphic at a single
synonymous site each.
Alignments of the five different cone opsins, SWS1
(UV-sensitive), SWS2B (violet-sensitive), RH2-1 and
RH2-2 (green-sensitive) and LWS (red-sensitive) are
shown in the electronic supplementary material, figure 1.
Two different groups of RH2 cDNAs were isolated, similar
to the two different RH2 forms in other species (Neafsey &
Hartl 2005; Parry et al. 2005; Matsumoto et al. 2006).
Guppy RH2-1 and RH2-2 are distinguished by at least
two previously described candidates for tuning amino acid
substitutions: E139Q and S312A, corresponding to
positions 122 and 292 in bovine RH1 (22). The overall
amino acid identity between RH2-1 and RH2-2 is
approximately 80%.
The five full-length LWS opsin cDNAs, all from the
Oropuche population, cluster into two types: LWS_OR6-3
and LWS_OR6-4. These differ at nine sites, all of which
are non-synonymous (figure 1 and electronic supple-
mentary material, figure 1). Of these, A180S in the fourth
transmembrane (TM) domain was labelled in red and
green because it is one of the key tuning amino acids
known to shift the absorption maximum to longer
wavelength (12, 13). Several of the other non-synonymous
changes, labelled in yellow and blue to distinguish the
LWS_OR6-3 and LWS_OR6-4 isoforms, are likely to affect
spectral properties of LWS proteins (see below).
(b) Long-wave sensitive opsin copy number
We determined the minimum number of LWS opsin gene
copies by genomic DNA blots. Using restriction enzymes
that do not cut within the cDNA sequences, single
fragments were detected for RH1, RH2-1 and SWS1
(figure 2a). In contrast, multiple LWS fragments were
seen (figure 2a,b), suggesting a minimum of two gene
copies, given that some of these different bands could be
owing to allelic variation.
The two LWS opsin cDNA sequences differ in the
presence of a PvuII restriction site, allowing for their
differential detection. The observed hybridization pattern
indicates that there are additional PvuII sites flanking the
gene loci, and it favours the presence of a minimum of two
copies in tandem in all three strains analysed (figure 2c).
The pattern was the same after double digests with PvuII
and either BamHI or HindIII (data not shown). Different
individuals from the Cumana
´
and Quare strains had the
same fragment pattern, in agreement with results on
pooled DNA from individuals of the same strains (data not
shown). However, restriction length polymorphisms were
observed between strains and also within the Tranquille
strain (figure 2c).
(c) Genomic diversity of long-wave sensitive
opsin genes
We further investigated variation in LWS opsin genes by
direct sequencing of PCR products from 16 individuals
representing nine populations (table 2 and electronic
supplementary material, figure 2). The examined 920 bp
fragment codes for most of the tuning amino acids
(Yokoyama & Radlwimmer 1998; Kochendoerfer et al.
1999; Yokoyama 2000). There were 26 polymorphic sites
in this region, with many individuals being heterozygous at
multiple sites (electronic supplementary material,
figure 2). The SNP density was at least three times higher
in LWS than in RH1 and at least five times higher than in
the other cone opsins (table 1).
The genomic LWS product appeared to be
homozygous at all sites in a single Quare individual,
LQu47 f. Two individuals from the Aripo and Tacarigua
populations shared the same exon sequence, correspond-
ing to the sequence of LWS_OR6-4 cDNA, but were
heterozygous in introns. That this set of SNPs in the exons
was observed in three out of 11 individuals and one
cDNA, despite high population heterozygosity at multiple
sites, suggests that these non-synonymous SNPs are in
strong linkage disequilibrium. PCR amplification with
allele-specific primers after digestion of DNA further
supported the linkage of these SNPs, as explained in
electronic supplementary material, figure 2.
All other individuals were heterozygous at non-
synonymous sites and thus had the potential to express
multiple LWS proteins. Three Quare, one Oropuche and
one Tunapuna individual were heterozygous in at least 10
positions, seven of which caused amino acid replacements
(electronic supplementary material, figure 2). Cumana
´
and PV6 individuals were polymorphic for an indel, with
additional SNPs at up to 17 positions, six of which caused
non-synonymous substitutions. Two Tranquille individ-
uals had 18 heterozygous positions; these comprise the
SNPs found in the eastern strains plus the ones found in
individuals from the Aripo and Tacarigua populations.
(d) Long-wave sensitive opsin haplotypes revealed
by cloned PCR products
To determine the haplotypes and their distribution
between populations, we sequenced 193 clones from the
16 individuals investigated above (table 2). The PCR
products from all individuals but one were variable, and all
SNPs identified by direct sequencing of genomic amplifi-
cation products were confirmed with cloned products.
The simultaneous amplification of several copies of a gene
will probably give rise to some recombinant products
owing to template switching (Odelberg et al.1995).
Therefore, we restricted our analysis to 76 clones that
were observed at least twice, resulting in 15 unique
haplotypes (figure 3). Of these 76 sequences, 20 and 50%
encoded protein variants 1 and 2, respectively, corre-
sponding to cDNA sequences OR6-3 and OR6-4 (figure 3)
table 3.
(e) Molecular evolution of long-wave
sensitive haplotypes
We used the BIONJ algorithm (Gascuel 1997) to generate
a neighbour-joining tree of the 15 unique haplotypes listed
in figure 3. The tree has three major branches (figure 4).
As an outgroup we used genomic sequences from a
swordtail (X. helleri ) individual, which was polymorphic at
only two synonymous positions. The position of the
outgroup suggests that the deepest split is between variant
1 and a group comprising two other variants. Variants 1
36 M. Hoffmann et al. Opsin diversity in the guppy
Proc. R. Soc. B (2007)
Page 4
FMFM
Eco RI Hind III
SWS1
FMFM FMFMFM
Hind III
(a)
(b)(c)
Eco RI Hind III Bam HI Eco RI
FMFM
Eco RI Bam HI
LWS
RH1 RH2-1
Tr Cu Qu F1 F2 M1 M2 F1 M1 M2 F1 F2 M1 M2
Tr Cu Qu
8.6
7.4
6.1
4.9
3.7
8.6
7.4
6.1
4.9
3.7
2.8
8.60
7.40
6.10
4.90
3.70
2.80
1.52
1.90
1.48
1.16
0.99
Figure 2. Genomic DNA blots. (a) Genomic DNA from females (F) and males (M) of the Wild Istanbul strain digested with the
indicated enzymes and hybridized with probes for opsins RH1, RH2-1, LWS and SWS1. A BamHI site in the RH2-1 cDNA
sequence results in two RH2-1 opsin bands. (b) Pooled DNA of guppies from the Tranquille (Tr), Cumana
´
(C) and Quare (Qu)
strains digested with HindIII and hybridized with a LWS probe. (c) DNA from individual females and males of indicated strains
digested with PvuII and hybridized with a LWS probe.
Table 2. Number of LWS proteins and haplotypes in 16 individuals.
individual strain population proteins haplotypes
LQu47 f Lower Quare Quare R., Trinidad 1 2
LQu49 m Lower Quare Quare R., Trinidad 2 3
Qu135 f Quare 206-5 Quare R., Trinidad 3 3
Qu196 f Quare 206-5 Quare R., Trinidad n.d.
a
n.d.
Or56 m Oro 201-5 Oropuche R., Trinidad 2 2
Tu128 m Tunapuna Tunapuna, Trinidad 3 4
Ta16 m Tacariqua Tacariqua, Trinidad 1 2
Tr130 m Tranquille Tranquille, Trinidad n.d. n.d.
Tr131 f Tranquille Tranquille, Trinidad n.d. n.d.
AR138 m ARDC Aripo R., Trinidad 2 2
Ari18 f Lower Aripo Aripo R., Trinidad 1 2
En25 f CCFR Cumana
´
, Venezuela
b
44
En28 m CCFR Cumana
´
, Venezuela 3 3
En157 f CCFAH Cumana
´
, Venezuela n.d. n.d.
En158 f CCFNX Cumana
´
, Venezuela n.d. n.d.
PV6107 m PV6FIC Rio San Miguel, Venezuela 2 2
Xiphophorus helleri pet store unknown 1 2
a
Not determined.
b
Known in the aquarium trade as Endler’s Livebearer (Alexander & Breden 2004).
Opsin diversity in the guppy M. Hoffmann et al.37
Proc. R. Soc. B (2007)
Page 5
and 2 have further diversified in a way that suggests the
existence of seven protein isoforms (figure 3) occurring in
three phylogenetically distinct clades (figure 4). Variant 1
was found across the range of the guppy, in both the
distinct eastern and western Trinidadian strains (Fajen &
Breden 1992) as well as in the Venezuelan populations. In
contrast, variant 2 was found only in Trinidad. Variant 3 in
the Venezuelan populations Cumana
´
and PV6 apparently
replaces it.
The rate of non-synonymous nucleotide substitutions
per non-synonymous site, relative to the rate of synon-
ymous nucleotide substitutions per synonymous site
(Ka/Ks) can reveal whether selection is likely to have
acted on a set of sequences. Low Ka/Ks ratios are
indicative of purifying selection, while high Ka/Ks ratios
are taken as an indication of diversifying selection. We
used D
NASP (v. 4.10.4; Rozas et al. 2003) to estimate
branch-specific Ka/Ks ratios on the LWS phylogeny
(figure 5). The highest Ka/Ks values were found to
coincide with polymorphisms occurring in the fourth
TM domain.
(f ) Functional implications of long-wave sensitive
diversity in the guppy
According to the three sites rule (Yokoyama & Yokoyama
1996; Yokoyama & Radlwimmer 1998) for tuning of LWS
opsins, polarity changes caused by the substitutions
S180A, Y277F and T285A are the most important for
tuning vertebrate LWS (red-sensitive) opsins to shorter,
green-shifted wavelengths. Of these three amino acids,
only S180A is polymorphic between the LWS variants
1 and 2. Two additional polymorphisms, F131Y and
F194Y, also cause amino acid polarity changes. All of the
intraspecific substitutions found coincide with poly-
morphisms in LWS opsins of other fishes (figure 1). In
particular, LWS opsins of different species of cichlids from
Lakes Victoria and Nabugabo are polymorphic for at least
five of the positions that are variable in guppies, and the
cichlid LWS opsins have similarly been grouped into two
classes based on these substitutions (Terai et al. 2002).
To contrast similarities owing to descent and those
owing to convergent positive selection, we applied the
BIONJ algorithm both to the entire guppy sequences,
and only to the amino acids shown in figure 1, several
of which have been implicated as having tuning
function. The tree based on complete sequences shows
clustering of the different variants from the same
species, including guppies, L. goodei,medakaand
zebrafish, as well as clustering of the sequences from
two cichlid species (figure 6a). As expected, LWS opsins
of cichlids and guppies are most closely related to those
of medaka and fugu and most distant from zebrafish.
This molecular phylogeny suggests that the LWS opsin
variants found in different taxa have arisen by
independent duplications in each lineage. In contrast
to the phylogenetically consistent tree of the entire
sequences, analysis of only the discriminating amino
acids shown in figure 1 suggests functional similarity of
guppy OR6-4 and cichlid variant II, while OR6-3
clusters with both L. goodei variants (figure 6b). This
131 171 180 182 194 233 247
1_T 1 Y A A V Y A R Or56(2), Qu135(5), Tu128(4)
2_C 1 Y A A V Y A R Cu25(1), Cu28(1)
3_P 1 Y A A V Y A R PV6107(2)
4_C 1b YAA VYAH Cu25(2), Cu28(1), Cu157(5)
5_T* 1c F A A V Y A R Qu135(1), Tu128(1), AR138(1)
6_T 2 FGSAFGH LQu47(9), Ta16(3), Ari18(2)
7_T 2 FGSAFGH LQu47(2)
8_T 2 FGSAFGH AR138(3)
9_T 2 FGSAFGH Ari18(3)
10_T 2 FGSAFGH LQu49(1), Tu128(2)
11_T 2FGSAFGHOr56(7), LQu49(1), Qu135(2), Ta16(2), Tu128(1)
12_T 2b Y GSAFGH
LQu49(3), Ta16(0)
§
13_C 3a F A SAFA H Cu25(2), Cu28(3)
14_C 3b FGSAFA H
Cu25(2), Cu28(0)
§
15_P 3b FGSAFA H PV6107(2)
X. helleri Xi Y A S VYAH (2)
OR6-3 1 Y A A V Y A R P. reticulata cDNA
OR6-4 2 FGSAFGH P. reticulata cDNA
haplotype variant individual (no.of clones)position
Figure 3. LWS amino acid sequences deduced from cloned genomic PCR fragments. Haplotypes are numbered from 1 to 15,
followed by a letter indicating their origin (T, Trinidad; C, Cumana
´
; P, Rio San Miguel; see table 2). The resulting proteins are
colour-coded yellow (variant 1), blue (variant 2) and green (mixed).
Table 3. Accession numbers of LWS sequences used for
phylogenetic comparisons.
species GenBank accession number
Takifugu rubripes Q6J5K2
Oryzias latipes A AB223051
Oryzias latipes B AB223052
Lucania goodei A AY296740
Lucania goodei B AY296741
Astyanax fasciatus P22332
Danio rerio 1 Q7SZY0
Danio rerio 2 AB087804
Dimidiochromis compressiceps Q9183 9CICH
Yssichromis pyrrhocephalus AB090415
38 M. Hoffmann et al. Opsin diversity in the guppy
Proc. R. Soc. B (2007)
Page 6
pattern suggests convergent evolution of functionally
distinct LWS opsins in different fish taxa.
4. DISCUSSION
We found that the cone opsins of guppies are highly
polymorphic, both within and between populations,
resulting in at least two different green-sensitive opsins
and two highly differentiated LWS opsin isoforms. While
RH1, RH2-1 and SWS1 appear to be single copy genes,
LWS is found in at least two copies per individual. The 15
different LWS opsin haplotypes identified from nine
strains can encode seven different proteins, which can
clearly be grouped into three distinct forms. Only variant 1
was found in populations from both Trinidad and
Venezuela, while variants 2 and 3 were restricted to
Trinidad and Venezuela, respectively (figures 3 and 4).
The prevalence of non-synonymous substitutions known
to change maximum absorbance of visual pigments, along
with the high ratio of non-synonymous to synonymous
substitutions, suggests strong diversifying selection of
these proteins, especially in the functionally important
TM domain 4 (figure 5).
Our finding of five different cone opsins is compatible
with microspectrophotometric (MSP) studies of the
guppy retina, which suggest the presence of UV, blue/
violet, green and variable red/orange-sensitive cone
opsins. These long wavelength-sensitive cones are by far
the most polymorphic class, in which three different
absorption peaks at 533, 548 and 572 nm can be
distinguished (Archer et al. 1987; Archer & Lythgoe
1990). Although we found two different RH2 isoforms,
MSP data did not indicate the presence of functionally
different green sensitive cones, suggesting that the RH2
differences are too subtle to be detected. Alternatively,
expression of some visual pigments could be develop-
mentally regulated, as has been found for green opsins in
cichlids (Spady et al. 2006).
Archer & Lythgoe (1990) suggested the existence of
two LWS opsins with distinct absorption maxima, with
co-expression of both causing a third absorption peak, to
account for individuals with a single, two or three
absorption peaks for long wavelength light. Our data
provide a possible alternative explanation, since at least
some individuals have three different LWS forms (figure 3
and table 2). The MSP data indicated that about half of
the individuals contain two different LWS cones, with
most of the other individuals having either one or another.
Our genomic data are in broad agreement with this
conclusion, as most individuals have a repertoire of LWS
genes that should allow for at least two different proteins.
While the maximum number of isoforms observed within
one individual was four, a quarter of individuals seemed to
have only one type of LWS protein (table 2). There was
only partial overlap between the populations studied by
5_T
1_T
2_C
3_P
4_C
12_T
10_T
9_T
7_T
6_T
8_T
14_C
15_P
13_C
outgroup:
X. helleri
variant 1
97
97
86
98
83
69
76
74
51
58
93
100
88
62
76
61
70
79
71
99
94
88
88
variant 2
variant 3
100
0.001
11_T
Figure 4. BIONJ tree of guppy genomic LWS sequences. A BIONJ tree (Gascuel 1997) was constructed using SPLITSTREE4
(Huson & Bryant 2006). Bootstrap values above 50 are shown.
Opsin diversity in the guppy M. Hoffmann et al.39
Proc. R. Soc. B (2007)
Page 7
Archer & Lythgoe (1990) and the strains included in our
survey, and the percentage of individuals with a single
LWS opsin could differ between populations.
Our estimates of LWS haplotype number are con-
servative, although a low frequency of artefacts owing to
template switching during PCR cannot be excluded.
Several factors may have resulted in an underestimate of
the number of haplotypes per individual. For example, we
could not directly select for LWS alleles represented by
cDNA OR6-3 (variant 1) with allele-specific primers. In
addition, point mutations in the regions covered by the
consensus primers might have prevented detection of
some isoforms.
Long-wave sensitive opsin genes have been compared
between cichlid species that have only recently evolved in
East African rift lakes (Terai et al. 2002; Carleton et al.
2005; Parry et al. 2005; Spady et al. 2005). Although DNA
blot experiments did not indicate multiple LWS copies,
genomic PCR analysis revealed 14 different LWS alleles in
cichlids from Lakes Victoria and Nabugabo, with two
alleles found in most species (Terai et al. 2002). In cichlids,
differentiation of colour patterns and of the visual system
1
3
Ka /Ks
2
500100 300
v
ariant 1
2
3
nt
A
G
G
AV
SA
SA
Y
F
F
Y
F
F
A
G
A
R
H
H
TM 4 TM 5TM 3TM 2
Figure 5. Comparison of cDNA and genomic guppy LWS sequences. Exons contained in the genomic sequences representing
the 15 guppy haplotypes listed in figure 3 were aligned to the corresponding parts of the cDNAs. Sequences encoding variants 2
and 3 were pooled and compared to those encoding variant 1 (see figure 4) using a sliding window of 50 nucleotides and step size
10 for analysis of Ka/Ks with the program D
NASP v. 4.10.4 (Rozas et al. 2003). The amino acids encoded by the three main
variants are indicated below the abscissa showing numbers of the 582 nt long coding parts of the genomic fragments and the
position of TM domains is indicated in the bar diagram. The corresponding coding sequences of medaka LWSA and B were
pooled and used as outgroup for all guppy sequences to perform the McDonald & Kreitman (1991) test. The neutrality index
(NI) was 7.714, Fisher’s exact test p-value (two-tailed) 0.0025; G-test G-value 9.681, p-value 0.0019.
OR6-4
OR6-3
OR6-3
OR6-4
Cichlid I
(a)(b)
Cichlid II
100
100
100
100
100
100
100
100
100
100
100
99
99
97
90
88
88
Af
Cichlid II
Cichlid I
Tr
OlA
OlB
100
100
Dr2
Dr1
Dr1
Dr2
Af
LgA
LgB
OlB
LgB
LgA
OlA
Tr
100
100 100
95
86
81
99
99
99
96
Figure 6. Phylogenetic analysis of LWS sequences. (a) BIONJ tree of fish LWS opsins using alignments of amino acids 38–326.
OR6-3 and OR6-4 are the two guppy variants represented by cDNAs. The other species are: Af, Astyanax fasciatus; Dr, Danio
rerio (zebrafish); Lg, Lucania goodie; Ol, Oryzias latipes (medaka); Tr, Takifugu rubripes (fugu). Cichlid class I is represented by
Dimidiochromis compressiceps, and class II by the partial sequence of Yssichromis pyrrhocephalus.(b) Application of BIONJ
algorithm to amino acids shown in figure 1. Guppy sequences are boxed. Only bootstrap values above 50 are shown.
40 M. Hoffmann et al. Opsin diversity in the guppy
Proc. R. Soc. B (2007)
Page 8
have been associated with adaptation to different habitats
(Parry et al. 2005), and both of these factors have been
proposed as essential components of speciation.
The situation in cichlids, in which allelic diversity is
found predominantly between species (Terai et al. 2002),
contrasts with the one in guppies, in which eight strains of
the same species contain at least 15 different haplotypes
encoding seven different proteins, with obvious geographi-
cal differentiation. Importantly, much of the variation in
LWS opsins occurred within populations and even within
individuals in guppies. Guppies can live in different photic
environments that are distinguished by degree of trans-
parency and amount of canopy (Endler 1991, 1995), but
the main forces driving differentiation of male colour
patterns are thought to be sexual selection (Houde 1997)
and predation (Olendorf et al. 2006). Further detailed
analysis of a greater number of specimens from upper and
lower ranges of several northern Trinidad river systems
might reveal whether a correlation exists between opsin
gene pools and male ornamentation. How this colour
variation, one of the highest in vertebrates (Haskins et al.
1961), is maintained continues to be an important
question in evolutionary biology (Olendorf et al. 2006).
The colour diversity is due to as many as 40 genes with
polymorphic alleles for male coloration, most of which are
linked to the sex chromosomes (reviewed in Lindholm &
Breden 2002).
In contrast to the highly variable patterns of guppies,
five distinct male colour morphs coexist in Poecilia parae
and female mate choice may counteract loss of the less
frequent morphs (Lindholm et al 2004). In this context, it
would be interesting to know to what extent opsin gene
diversification has occurred in closely related poeciliids
such as P. parae.
Guppies also stand out among species studied for
sexual selection in that female preference functions are
variable within and between populations (Houde & Endler
1990; Endler & Houde 1995; Brooks & Endler 2001).
Sensitivity to UV light and the detection of black and
iridescent ornaments play a role in female preferences in
guppies (Houde 1997; Smith et al. 2002). However,
sensitivity to red and orange wavelength is likely to be
particularly important for differentiation because red and
orange ornaments are the most consistent targets of female
choice (Endler & Houde 1995; Houde 1997; Brooks &
Endler 2001). Further, sensitivity to red wavelengths
shows heritable variation in guppies (Endler et al. 2001),
and changes in wavelength sensitivity are expected to alter
female preferences. Hence, differentiation in LWS opsins
could select for variation in male nuptial colour patterns
(Endler 1992, 1993; Endler et al. 2001). Alternatively, the
variation in red and orange ornaments themselves might
select for diversification of red colour perception. Either
way, the highly polymorphic nature of opsins, and the
partitioning of some of this variation between individuals
within populations, provides a potential molecular frame-
work for future studies of coevolution between visual
perception and male ornamentation.
5. DATA DEPOSITION
The sequences reported in this paper have been deposited
at GenBank (accession nos DQ075246, DQ168659–
DQ168660, DQ234858–DQ234861, DQ435005–
DQ435019, DQ912023–DQ912026).
We thank Christa Lanz and Heike Keller for expert assistance
with sequencing, Markus Riester for database construction,
Stephen Russell for critical reading of the manuscript and
Richard Clark for helpful suggestions. We also thank two
anonymous reviewers for their helpful comments. Bernie
Crespi, Werner Mayer, Arne Mooers and Patrik Nosil read an
earlier draft of this manuscript. Guppies were provided by
David Reznick, Anne Magurran, Axel Meyer and Manfred
Schartl, or collected by F.B. in Venezuela in collaboration
with Donald Taphorn of UNELLEZ (permit no. 0497) and
in Trinidad with the permission of the Ministry of
Agriculture, Land and Marine Resources. This work was
funded by grants from the Natural Sciences and Engineering
Research Council of Canada, the German Academic
Exchange Service (DAAD), which enabled a sabbatical visit
of F.B. at the Max Planck Institute, and the Max Planck
Society, of which D.W. is a director.
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42 M. Hoffmann et al. Opsin diversity in the guppy
Proc. R. Soc. B (2007)
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    • "Subsequently, Endler (1992) [5] suggested that variations in visual properties contribute to differences in female preferences for male color patterns. Recent molecular genetic studies also revealed that guppies carry nine opsin genes, including an ultraviolet-sensitive gene (SWS1), two subtypes of blue-sensitive genes (SWS2-A and SWS2-B), two subtypes of green-sensitive RH2 genes (RH2-1 and RH2-2) and, remarkably, four subtypes of redsensitive LWS genes (LWS-1, LWS-2, LWS-3, and LWS-4) [28][29][30][31]. For LWS genes, the nomenclature of the first three genes follows [32] and that of the last follows [31]. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The visual system is important for animals for mate choice, food acquisition, and predator avoidance. Animals possessing a visual system can sense particular wavelengths of light emanating from objects and their surroundings and perceive their environments by processing information contained in these visual perceptions of light. Visual perception in individuals varies with the absorption spectra of visual pigments and the expression levels of opsin genes, which may be altered according to the light environments. However, which light environments and the mechanism by which they change opsin expression profiles and whether these changes in opsin gene expression can affect light sensitivities are largely unknown. This study determined whether the light environment during growth induced plastic changes in opsin gene expression and behavioral sensitivity to particular wavelengths of light in guppies (Poecilia reticulata). Results Individuals grown under orange light exhibited a higher expression of long wavelength-sensitive (LWS) opsin genes and a higher sensitivity to 600-nm light than those grown under green light. In addition, we confirmed that variations in the expression levels of LWS opsin genes were related to the behavioral sensitivities to long wavelengths of light. Conclusions The light environment during the growth stage alters the expression levels of LWS opsin genes and behavioral sensitivities to long wavelengths of light in guppies. The plastically enhanced sensitivity to background light due to changes in opsin gene expression can enhance the detection and visibility of predators and foods, thereby affecting survival. Moreover, changes in sensitivities to orange light may lead to changes in the discrimination of orange/red colors of male guppies and might alter female preferences for male color patterns. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0679-z) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016 · BMC Evolutionary Biology
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    • "chii breeding males have a yellow-white stripe on the dorsal fin, and their irises are not red (Chen and Chang 2005). The perception of visual signals is determined by two components: the physical characteristics of a habitat and the sensory properties of a receiver (Endler 1990 ); therefore , a change in one of these two components tends to drive the development of substitute signals in response to variable habitats (Chunco et al. 2007; Endler 1980), which has been demonstrated by several studies (Dalton et al. 2010; Fuller et al. 2005; Gray et al. 2008; Hoffmann et al. 2007 ). Based on the observation that the two aforementioned and closely related bitterlings exhibit distinct nuptial colorations, it is hypothesized that the differences in their nuptial colorations may be related to variations in photic properties of its habitats and/or spectral sensitivities of the fish. "
    Full-text · Dataset · Sep 2015
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    • "chii breeding males have a yellow-white stripe on the dorsal fin, and their irises are not red (Chen and Chang 2005). The perception of visual signals is determined by two components: the physical characteristics of a habitat and the sensory properties of a receiver (Endler 1990 ); therefore , a change in one of these two components tends to drive the development of substitute signals in response to variable habitats (Chunco et al. 2007; Endler 1980), which has been demonstrated by several studies (Dalton et al. 2010; Fuller et al. 2005; Gray et al. 2008; Hoffmann et al. 2007 ). Based on the observation that the two aforementioned and closely related bitterlings exhibit distinct nuptial colorations, it is hypothesized that the differences in their nuptial colorations may be related to variations in photic properties of its habitats and/or spectral sensitivities of the fish. "
    [Show abstract] [Hide abstract] ABSTRACT: Vision, an important sensory modality of many animals, exhibits plasticity in that it adapts to environmental conditions to maintain its sensory efficiency. Nuptial coloration is used to attract mates and hence should be tightly coupled to vision. In Taiwan, two closely related bitterlings (Paratanakia himantegus himantegus and Paratanakia himantegus chii) with different male nuptial colorations reside in different habitats. We compared the visual spectral sensitivities of these subspecies with the ambient light spectra of their habitats to determine whether their visual abilities correspond with photic parameters and correlate with nuptial colorations. The electroretinogram (ERG) results revealed that the relative spectral sensitivity of P. h. himantegus was higher at 670 nm, but lower at 370 nm, than the sensitivity of P. h. chii. Both bitterlings could perceive and reflect UV light, but the UV reflection patterns differed between genders. Furthermore, the relative irradiance intensity of the light spectra in the habitat of P. h. himantegus was higher at long wavelengths (480–700 nm), but lower at short wavelengths (350–450 nm), than the light spectra in the habitats of P. h. chii. Two phylogenetically closely related bitterlings, P. h. himantegus and P. h. chii, dwell in different waters and exhibit different nuptial colorations and spectral sensitivities, which may be the results of speciation by sensory drive. Sensory ability and signal diversity accommodating photic environment may promote diversity of bitterling fishes. UV light was demonstrated to be a possible component of bitterling visual communication. The UV cue may assist bitterlings in gender identification.
    Full-text · Article · May 2015 · Zoological studies
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