1791 Full
1791 Full
1791 Full
Akie Sato,*,1 Yoko Satta,† Felipe Figueroa,* Werner E. Mayer,* Zofia Zaleska-Rutczynska,*
Satoru Toyosawa,*,2 Joseph Travis‡ and Jan Klein*
*Max-Planck-Institut für Biologie, Abteilung Immungenetik, 72076 Tübingen, Germany, †The Graduate University for Advanced Studies,
Department of Biosystems Science, Hayama, Kanagawa 240-0193, Japan and ‡Department of Biological Science,
Florida State University, Tallahassee, Florida 32306-4340
Manuscript received July 28, 2002
Accepted for publication September 30, 2002
ABSTRACT
The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the
only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population
structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of
the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced.
Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose
ⵑ20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of
R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations
and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci,
as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc
alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these
findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must
have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing
multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by
mutations, each of which spread through the entire population and was fixed independently in the
emerging clones.
TABLE 1
Origin and Mhc genotypes of killifish examined
Sample
no. Stock Location Genes found to be presenta
1 Bel49.3 Twin Caye, Belize C1*04, H1*03, H1*04
2 Bel20.1 Twin Caye, Belize A1*03, C1*04, H1*03
3 Bel12.23 Twin Caye, Belize R6
4 Bel44.29 Twin Caye, Belize E2*01, H1*01; mtDNA typing
5 Bel44.28 Twin Caye, Belize C1*05
6 Bel44.27 Twin Caye, Belize E2*01, H1*05
7 Bel51.31 Twin Caye, Belize A1*03, C1*04
8 Brevard10 Brevard County, Florida A1*02, B1*0203, B1*0201
9 Brevard16 Brevard County, Florida A1*03; mtDNA typing
10 PG3-3 Twin Caye, Belize A1*02, C1*05
11 PG9-5 Twin Caye, Belize C1*03, E1*01, E2*01, H1*01
12 PG9-4 Twin Caye, Belize E2*01, G1*02, H1*01
13 PG9-3 Twin Caye, Belize H1*02
14 PG11-1 Twin Caye, Belize H1*01; mtDNA typing
15 PG8-2 Twin Caye, Belize E2*01, H1*01
16 BH1-1 Norman’s Pond Cay, Bahamas H1*02
17 BH2-1 Norman’s Pond Cay, Bahamas E1*01, G1*03, H1*02; mtDNA typing
18 BH3-1 Norman’s Pond Cay, Bahamas H1*02
19 BH4-1 Norman’s Pond Cay, Bahamas E1*01, H1*02
20 BH5-1 Norman’s Pond Cay, Bahamas H1*02
21 BH6-1 Norman’s Pond Cay, Bahamas H1*02
22 DAN Dangriga, Belize C1*03; mtDNA typing
23 DS Vero Beach, Florida A1*02, B1*0201, B1*0202
24 CCHA Vero Beach, Florida A1*03; mtDNA typing
25 91-125 Tobacco Range, Belize A1*03, C3*01; mtDNA typing
31 Marco Island, Florida mtDNA typing
32 Brevard10 Brevard County, Florida A1*01, A1*02, B1*01, D1*02, D2*01, D3*01, D3*02, R1
33 Brevard16 Brevard County, Florida A1*01, D1*01, D2*02, D3*01
35 CCHA Vero Beach, Florida A1*01, D1*01, R4
36 DS Vero Beach, Florida A1*02, B1*01, C1*01, D1*01, D2*01 D2*02, R1, R5;
mtDNA typing
37 DAN Dangriga, Belize mtDNA typing
39 91-125 Tobacco Range, Belize Southern blot
48 DS Vero Beach, Florida B1*01, C1*01, D2*01
50 cDNA library Unknown C2*03, G1*01
61 BEL2K-86 Twin Caye, Belize C1*02
62 BEL2K-035 Twin Caye, Belize R3
63 BEL2K-028 Twin Caye, Belize F1*01
65 BEL-2K Twin Caye, Belize F1*03
66 BEL2K-028 Twin Caye, Belize F1*04
67 BEL2K-035 Twin Caye, Belize R2
69 BEL2K Twin Caye, Belize F1*03
70 BEL2K-2 Twin Caye, Belize D3*03
71 BEL2K-03 Twin Caye, Belize F1*02
72 BEL2K Twin Caye, Belize F1*01
a
Heterozygote gene combinations are underlined; R, putative recombinant.
the dideoxy chain-termination method (Sanger et al. 1977), to the site in the particular gene. They constituted 30% of
using the Thermo Sequenase fluorescent-labeled primer cycle the sequences.
sequencing kit (Amersham Biosciences). Sequencing reac- Southern DNA blotting and hybridization: Five micrograms
tions were processed by the LI-COR Long ReadIR 4200 DNA of genomic DNA was digested with restriction endonucleases
sequencer (MWG Biotech, Ebersberg, Germany). Scattered for 18 hr under the conditions recommended by the supplier
single-nucleotide changes differentiating a given clone from (Roche Diagnostics, Mannheim, Germany) and fragments
all other clones of a set obtained from the same DNA sample were separated by agarose gel electrophoresis and blotted
(individual) were taken for replication or sequencing errors. onto Hybond-N⫹ nylon filters (Amersham Biosciences). Pre-
As such, they were ignored in the final analysis and the nucleo- hybridization, hybridization, and probe labeling were carried
tides shared by all the other clones in the set were assigned out using the AlkPhos Direct kit (Amersham Biosciences).
1794 A. Sato et al.
TABLE 2
Primer combinations used in the present study
After the overnight hybridization, the filters were washed ac- RESULTS
cording to the AlkPhos Direct protocol. Following the applica-
tion of the chemiluminescent detection reagent CDP-Star of Cloning of killifish Mhc class I genes: The first R. mar-
the kit, Hyperfilm ECL (Amersham Biosciences) was exposed moratus (Rima) class I sequences were obtained by PCR
to the blot for 2 hr and developed. amplification of genomic killifish DNA using the primer
Computer simulation: The initial population consisted of pair KFF1 and KFR1 (Table 2). The sequence of the oligo-
N individuals, each individual carrying two identical alleles at
a given locus and each gene containing L sites. The effects nucleotides corresponded to the regions of exons 2 and
of mutation, drift, selection, and selfing were simulated in the 3 conserved between the guppy (Sato et al. 1995) and
process of sampling 2N genes for N individuals to create the haplochromine cichlid fish (Sato et al. 1997). The PCR
next generation. To simulate mutations, a random variable therefore amplified a region of the class I genes encom-
C1 (from 0 to 1) was chosen for each gene and if its value passing exon 2 from codon 36, the entire intron 2, and
was C1 ⱕ u (where u was the chosen mutation rate), a mutation
was introduced into that gene. Then another random variable exon 3 up to codon 169 (exons 2 and 3 being the most
C2 (from 0 to 1) was obtained and if its value was C2 ⱕ r variable parts of the class I genes). Subsequently, a cDNA
(where r was the chosen selfing rate), each second gene for library prepared from killifish RNA was used in anchor
an individual of the next generation was taken from the same PCR to obtain the 5⬘ and 3⬘ parts of the cDNA clones.
individual of the preceding generation as the first gene; other- The combined sequence of the clones covered the en-
wise it was sampled from a different individual. If the newly
generated individual was a homozygote, a third random vari-
tire coding region of the Rima class I genes (Figure
able C3 (from 0 to 1) was chosen. If C3 ⬎ s (where s was the 2). These initial sequences were then used to design
chosen selection intensity), the individual was discarded (to additional primers specific for the different groups and
simulate selection against homozygotes) and two new genes different regions of the class I genes present in 25 indi-
were sampled. Every 10/u generations, the values φ (frequency viduals representing seven different killifish populations
of homozygotes) and F (expected homozygosity) were re-
(individuals 1–25 in Table 1). Additional sequences
corded in 100 replicate experiments.
Phylogenetic analysis: Sequences were aligned using the were obtained by PCR amplification of fragments eluted
SeqPup 0.6f software for Macintosh (Gilbert 1996). Variabil- from agarose gels in a Southern blotting experiment
ity of the sequences was assessed with the help of the MEGA (see below), as well as of other samples. Altogether, 165
2.0 program (Kumar et al. 2001), using synonymous and non- sequences encompassing different lengths of Mhc class
synonymous substitutions estimated by the Nei and Gojobori I exon 2, intron 2, and exon 3 were obtained. Among
(1986) method for exons and Kimura’s two-parameter dis-
tances (Kimura 1980) for introns. Phylogenetic trees drawn these were 30 unique exon 2 sequences and 87 unique
by the neighbor-joining method (Saitou and Nei 1987) using exon 3 sequences (Figures 2–5).
the PAUP*4.0b8a program (Swofford 2001) were based on Southern blot analysis: To facilitate the assignment
p-distances for amino acid sequences and on Kimura’s two- of individual sequences to loci and alleles, four fish
parameter distances for nucleotide sequences. Trees were caught in Florida (two at a locality in Brevard County—
rooted at midpoint. Maximum-parsimony trees were drawn
by the same program using the heuristic search algorithm. nos. 32 and 33—and two in the Vero Beach area—nos.
Gaps were treated as missing data. The topological stability 35 and 36; see Figure 1) were chosen for exhaustive
of the trees was assessed by 500 bootstrap replications. analysis. The genomic DNA isolated from these fish was
Mhc Heterozygosity in Rivulus 1795
Figure 2.—R. marmoratus Mhc class I locus G1*01 cDNA sequence. The clones on which this sequence is based were obtained
from a cDNA library. The translated amino acid sequence in the IUPAC-IUB three-letter code is given underneath the coding
nucleotide sequence. The positions of exons as deduced in this study are indicated.
digested with the HindIII endonuclease, the digest was the amplification products were cloned, and the clones
separated by gel electrophoresis and blotted, and the sequenced. Altogether 21, 8, 7, and 20 clones were se-
filters were hybridized with a probe covering exon 3 of quenced from individuals 32, 33, 35, and 36, respectively
the killifish class I locus. Four different sizes of bands (Table 3). Disregarding differences apparently repre-
were revealed: 7.5 kb (band I), 5.5 kb (band II), 4.0 kb senting replication errors, 6 different sequences could
(band III), and 2.5 kb (band IV; Figure 6A). Bands I be distinguished in the collection of 56.
and IV were found to be present in all four individuals, Identification and characterization of class I loci and
band II in three individuals (nos. 33, 35, and 36), and alleles: The starting point of the identification was a
band III in two individuals (nos. 32 and 36). The DNA phylogenetic tree based on the collection of exon 3
restriction fragments from the areas of the gel corre- sequences. Both the maximum-parsimony (Figure 7) and
sponding to the position of the individual bands on the neighbor-joining (not shown) trees identified the same
filter were then eluted and amplified by PCR using the major clades; they differed, however, in the arrange-
exon 3-specific primer pair KE31 and KE32 (Table 2), ment of the clades and the branching patterns within
1796 A. Sato et al.
with the consensus sequence at the top and dots indicate missing information. The numbering starts with the first nucleotide of the G1*01 cDNA sequence (Figure 2) in an
Figure 3.—Nucleotide sequence alignment of the R. marmoratus Mhc class I exon 2. The alignment encompasses 54 codons of the 3⬘ part of exon 2. Dashes indicate identity
supported clades were taken to represent separate groups
of class I loci, provisionally designated by letters A–H.
Some of the clades consist of a single locus, and others
are composed of multiple loci designated by numbers.
Alleles at a given locus are also designated by numbers,
separated from the locus designation by an asterisk
(Klein et al. 1990). The assignments of loci and alleles
within clades were based primarily on the number of
distinct sequences obtained per individual. Other crite-
ria included the relationship among the sequences of
exon 2 and intron 2, distribution of diagnostic substitu-
tions (i.e., substitutions characterizing a given group or
locus), and the presence of other markers such as spe-
cific indels (data not shown). Intron 2 sequences (Fig-
ure 5) were particularly helpful in the assignment of
sequences to loci and alleles. The introns of the differ-
ent clades differ in their length, their sequence, and
the presence of indels at specific positions (data not
shown). The longest intron 2 sequence (that of clade
E) consists of ⬎521 sites; the other introns are shortened
by indels or truncations at their 3⬘ parts. The 5⬘ parts
of intron 2 of the different clades can be aligned unam-
biguously. The middle and the 3⬘ parts (where present)
are largely clade specific and alignable only partially or
not at all. The middle part contains a (CA)n repeat,
which varies in length even between individuals bearing
alignment of all available genomic and cDNA sequences. Only unique sequences are shown.
Figure 4.—Nucleotide sequence alignment of R. marmoratus Mhc class I exon 3. Asterisks indicate alignment indels. For other
explanations, see Figure 3.
individuals suggest that the two individuals differ in the crossing over in a chromosomal region populated with
organization of their class I regions. Assuming that all closely related loci.
or nearly all class I loci are located in the same chromo- Heterozygosity of class I loci: Heterozygotes at Mhc
somal region, as they are in other fish taxa (Sato et al. loci were found in two geographically separated regions
2000) and indeed in all vertebrates tested (Klein 1986; in the area of R. marmoratus distribution, Belize and
Trowsdale 1995), it follows that different class I haplo- Florida, at frequencies of 4 and 44%, respectively (Table
types exist in the killifish populations. The haplotypes 1). The one heterozygous individual in the Belize popu-
differ in the number and identity of loci they bear, some lation was found at Twin Caye, a locality at which the
of them consisting of only one locus and others bearing presence of males has been reported (Davis et al. 1990;
multiple genes. This situation is analogous to that well Turner et al. 1992a). The individual is therefore most
documented for the HLA class II (DR) haplotypes (Klein likely a product of a mating between a male and a
1986) and is presumably the consequence of unequal hermaphrodite belonging to a different clone. By con-
1798 A. Sato et al.
Figure 5.—Alignment of variable sites of representative R. marmoratus Mhc class I intron 2 sequences. Only variable sites of
the exon-flanking regions are shown; they are separated by a gap of unalignable sequence of ⵑ613–237 bp. For other explanations,
see Figures 3 and 4.
trast, the presence of males in the Florida populations are deposited in the GenBank database). One lineage
has not been observed and so the four heterozygotes appeared to be restricted to Belize populations, while
found in these populations might be the result of a the other was found to be present in Belize, Bahamas,
mutational divergence of clones that were originally and Florida populations. Pairwise comparisons of se-
homozygous at the Mhc loci. The smallness of the ge- quences revealed the identities of three sequences from
netic distance between the alleles in five of the six het- Belize individuals (nos. 14, 22, and 37) and two se-
erozygous combinations (0.006, 0.006, 0.012, 0.012, quences from Florida individuals (nos. 9 and 22). The
0.012 amounting to one or two substitutions) is in line largest difference of eight nucleotide substitutions was
with this proposition. Alternatively, males might also be observed between individuals 25 (Belize) and 36 (Flor-
present in the Floridian populations but at such low ida), giving a nucleotide diversity of 0.009 ⫾ 0.003.
frequencies that their existence has thus far gone unde- Using the haplochromine cichlid substitution rate of 5.6%
tected, or they may arise episodically. The overall fre- substitutions per site per 106 years (Nagl et al. 2000),
quency of Mhc heterozygotes in the sample of 40 individ- we estimate that the lineages diverged 80,000 ⫾ 30,000
uals tested is 12%. years ago. The identity or near identity of sequences
Genetic distance analysis: In pairwise comparisons, within each lineage suggests that R. marmoratus reached
the genetic distances between Mhc alleles found in the Central America and Florida relatively recently. Weibel
sampled R. marmoratus specimens range from 0.003 to et al. (1999) distinguished three mtDNA lineages in R.
0.076 at nonsynonymous sites, from 0.014 to 0.240 at marmoratus by the analysis of restriction fragment length
synonymous sites of exons 2 and 3, and from 0.001 to polymorphism: one restricted to South America, an-
0.026 for intron 2 sites (data not shown). The lower other to the Bahamas, and the third shared by the Flor-
parts of the ranges can be explained as being the result ida and Belize populations. From the estimated diver-
of divergences since the onset of selfing in R. marmora- gence times of these alleles, the authors concluded that
tus. The upper parts are characteristic of allelic lineages the dispersal to Belize, Bahamas, and Florida occurred
whose divergence predates the separation of the species ⵑ18,000 years ago, at the time of the last glacial, when
involved. Hence the genetic distance analysis reveals the the sea level in the Caribbean was ⵑ75 m lower than
presence of polymorphisms presumably generated both present (Clark et al. 1978).
before and after the onset of selfing (see discussion). Computer simulation: Earlier studies revealed a high
Dispersal time estimate based on mtDNA analysis: frequency (close to 100%) of homozygotes at non-Mhc
We sequenced ⵑ900 bp of mtDNA control region from loci in the R. marmoratus populations (Massaro et al.
10 R. marmoratus individuals (nos. 4, 9, 14, 17, 22, 24, 1975; Vrijenhoek 1985; Turner et al. 1990; Laughlin
25, 31, 36, and 37) and found two lineages distinguished et al. 1995). By contrast, this study provides evidence
by substitutions at four sites (not shown, but sequences that the frequency of homozygotes at Mhc loci is as low
Mhc Heterozygosity in Rivulus 1799
TABLE 3
Sequences found in gel blocks corresponding to bands I–IV
in the Southern blots of Figure 6A
the two, R. marmoratus is the only obligatorily selfing, maphrodites into synchronous ones, in which mature
synchronous hermaphrodite (Huber 1992). The two male and female gametes were produced at the same
species form a sister group to another species, R. caudo- time. If the mutation required homozygosity for its full
marginatus, which is nonhermaphroditic, and the whole phenotypic manifestation, it could have again spread
clade is well separated from all the other nonhermaph- through the population and thus assured the retention
roditic Rivulus species (Murphy et al. 1999). Hermaph- of much of the existing Mhc variability in the population.
roditism therefore presumably arose in the common If selfing were, for whatever reason (Wells 1979), ad-
ancestor of R. marmoratus and R. occellatus. Taking the vantageous, homozygotes for the mutation would have
rate of 0.25% substitutions per site per 106 years (cali- been selected, and obligatory hermaphroditism would
brated on the African rivulid “Roloffia” geryi and on the have been fixed independently in the different emerg-
split of the African and South American continents ing clones into which the population was being frag-
ⵑ100 MYA) and the mtDNA sequences of the cyto- mented. This scenario would account for the large ge-
chrome b, 12S ribosomal RNA, 16S ribosomal RNA, and netic distances between Mhc genes in a species that has
cytochrome oxidase I genes (Murphy et al. 1999), we presumably been reproducing by selfing for some 2.5
estimate that R. marmoratus and R. occellatus diverged MY. It would also, at the same time, account for the
from each other 5.1 MYA and that their ancestor di- small genetic distances between alleles of class I hetero-
verged from R. caudomarginatus ⵑ26 MYA. The her- zygotes. The former would have been founded long
maphroditism of the two sister species should therefore before the species shifted to obligatory, synchronous
be ⬎5 MY old. It apparently arose in South America, hermaphroditism; the latter would have arisen only in
perhaps in southeastern Brazil (Murphy et al. 1999), the last 2.5 MY.
after the closure of the Panama Isthmus, which was We thank Dr. Bruce J. Turner (Department of Biology, Virginia
completed ⵑ2.7 MYA (Haug and Tiedemann 1998). Polytechnic Institute and State University, Blacksburg, Virginia) for
Obligatory, simultaneous selfing in R. marmoratus, how- introducing J.K. and A.S. to this interesting model system and for
ever, must be of a much younger age. This conclusion generously providing killifish specimens for this study. We also thank
Prof. Naoyuki Takahata (Department of Biosystems Science, The
follows from the presence of ancestral allelic lineages Graduate University for Advanced Studies, Hayama, Kanagawa, Japan)
at the Mhc loci in this species. The simulation results for many useful suggestions regarding the interpretation of the data;
indicate that obligatory selfing must be ⬍2.5 MY old; as well as Sabine Rosner for outstanding technical assistance and Jane
otherwise the ancestral lineages would have been elimi- Kraushaar for no less indispensable editorial assistance.
nated. The mtDNA data lower this age estimate further.
The estimate of the divergence of the mtDNA control
region lineages and the low divergence within the lin- LITERATURE CITED
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