1791 Full

Download as pdf or txt
Download as pdf or txt
You are on page 1of 14

Copyright  2002 by the Genetics Society of America

Persistence of Mhc Heterozygosity in Homozygous Clonal Killifish, Rivulus


marmoratus: Implications for the Origin of Hermaphroditism

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.

T HE mangrove rivulus (killifish), Rivulus marmoratus


Poey1880, is a small fish inhabiting temporal pools
and land-crab burrows in the coastal mangrove swamps
the ovarian component of the ovotestis (Cole and Noakes
1997), there is no evidence that these “females” are
reproductively active (Harrington 1975; Soto et al.
of northeastern Brazil, the Gulf of Mexico, the Carib- 1992).
bean, and the southern part of Florida (Huber 1992). Male R. marmoratus can arise in one of two principal
Its main distinction is its hermaphroditism—the ability ways. The so-called “primary males” can be produced
of single individuals to develop both ovaries and testis at high frequency in the laboratory by incubating devel-
(ovotestis) and produce both eggs and sperm (Har- oping embryos at temperatures between 18⬚ and 20⬚
rington 1961; Kallman and Harrington 1964; Har- (Harrington 1967; Harrington and Kallman 1968;
rington and Kallman 1968). The fish reproduce by re- Lin and Dunson 1995). They have no female reproduc-
leasing synchronously ovulated eggs and sperm, fertilizing tive organs and so produce only sperm (Harrington
the eggs internally, and laying the embryos after a short 1968). “Secondary males” or “false male gonochorists,”
period of development in the body (Harrington 1961, on the other hand, arise from either immature or ma-
1963; Atz 1965). R. marmoratus is the only vertebrate ture functional hermaphrodites by the loss of the female
known to reproduce by obligatory self-fertilization or function in response to shortened photoperiods (Har-
selfing (Vrijenhoek et al. 1989). Although laboratory- rington 1968). Males are either absent or exceedingly
reared individuals may pass through an early transient rare in most natural populations (Harrington and
phase of their development in which they possess only Kallman 1968; Davis et al. 1990). The only two known
exceptions to this rule are localities in the Netherlands
Antilles (Kristensen 1970) and in some of the Belize
cays, most notably the Twin Caye, in which the frequency
Sequence data from this article have been deposited with the
EMBL/GenBank Data Libraries under accession nos. AF550048– of males approaches 25% (Davis et al. 1990; Turner et
AF550092. al. 1992a).
1
Corresponding author: Max-Planck-Institut für Biologie, Abteilung Populations of R. marmoratus are assemblages of clones
Immungenetik Corrensstrasse 42, D-72076 Tübingen, Germany.
E-mail: akie.sato@tuebingen.mpg.de
distinguishable by an exchange of tissue grafts (Kall-
2
Present address: Osaka University Graduate School of Dentistry, De- man and Harrington 1964; Harrington and Kall-
partment of Oral Pathology, Suita, Osaka 565-0871, Japan. man 1968), electrophoretic enzyme surveys (Massaro
Genetics 162: 1791–1803 (December 2002)
1792 A. Sato et al.

et al. 1975; Vrijenhoek 1985), and DNA fingerprinting


(Turner et al. 1990; Laughlin et al. 1995). Individuals
comprising a clonal lineage are indistinguishable by
any of these techniques and highly homozygous. The
composition of the clonal lineages changes rapidly, of-
ten from year to year, presumably because of frequent
migrations of individuals between populations (Turner
et al. 1992b).
In populations in which males occur at high frequen-
cies, progeny testing combined with DNA fingerprinting
has revealed the individuals to be heterozygous at multi-
ple mini- and microsatellite loci (Lubinski et al. 1995).
The heterozygosity presumably stems from episodic out-
crossing between males and hermaphrodites releasing
unfertilized eggs. The circumstances under which the
outcrossing occurs are not known, but require a change
in behavior since hermaphrodites normally spawn alone
and are aggressive to other individuals of the same spe-
cies (Turner et al. 1992a).
The restriction of hermaphroditism to a single spe-
cies, or at most to a single clade of closely related species,
offers an opportunity to inquire into the circumstances Figure 1.—Map of Central America. Localities at which
under which this mode of reproduction arose. One of the samples were collected are indicated.
few genetic systems suitable for such an inquiry is the
major histocompatibility complex (Mhc), which encodes
proteins capable of presenting pathogen-derived pep- with the help of the Gigapack cloning kit (Stratagene), and
tides to receptors on thymus-derived lymphocytes and used to transform competent Escherichia coli NM514 bacteria.
so initiating an adaptive form of immune response Polymerase chain reaction (PCR) amplification: Standard
(Klein and Horejsı́ 1997). Some of the Mhc loci are PCR amplifications were performed in the PTC-200 Program-
mable Thermal Controller (MJR, Biozym, Hess. Oldendorf,
highly polymorphic (Klein and Figueroa 1986) with Germany) or in the GeneAmp PCR System 9700 (AB Applied
a large number of highly divergent alleles (allelic lin- Biosystems, Weiterstadt, Germany). One hundred nanograms
eages) maintained by balancing selection at sites speci- of genomic DNA was added to a reaction mixture consisting
fying the peptide-binding residues (PBRs) of the Mhc pro- of 1⫻ PCR buffer (50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-
teins (Hughes and Nei 1988). Heterozygosity at the Mhc HCl, pH 8.3, 0.0001% gelatin), 0.2 mm of each of the four
deoxynucleoside triphosphates (Amersham Biosciences), 1
loci is apparently advantageous because it broadens the mm of each of the sense and antisense primers, 2.5 units of
array of peptides that an individual can bind by its Mhc Taq DNA polymerase (Amersham Biosciences), and 0.4 units
molecules (Zinkernagel and Doherty 1974). Pfu DNA polymerase (Stratagene). The amplifications con-
In this study we used the polymorphism of the Mhc sisted of DNA denaturation for 1 min at 94⬚, followed by 35
loci in R. marmoratus to make inferences about the popu- cycles, each cycle consisting of 15 sec denaturation at 94⬚, 15
sec annealing at the required temperature, depending on the
lation structure of these fish and about the origin of primer combination, and 2 min extension at 72⬚. The reaction
their hermaphroditism. was completed by a final primer extension for 7 min at 72⬚.
Amplification of the mitochondrial control region: For the
initial PCR the published primers L15995 (Meyer et al. 1994)
MATERIALS AND METHODS and H00651 (Kocher et al. 1989) were used under standard
PCR conditions. On the basis of the sequence thus obtained,
Fish and DNA preparation: Forty-three fish collected at the killifish-specific primer pair KR1 (5⬘-CTCACCCCTAGCT
seven different localities were used (Figure 1, Table 1): 23 from CCCAAAGCTAG-3⬘) and KR2 (5⬘-TTTAAGCTACACGAGCC
Twin Caye, Belize (three different populations designated as CTAAGTTC-3⬘) was designed to amplify a 900-bp fragment
Bel, PG, and BEL2K); 2 from Dangriga, Belize (Dan); 2 from of the control region.
Tobacco Range (91-125); 4 from Brevard County, Florida; 5 DNA sequencing and analysis: Selected PCR products were
from Vero Beach, Florida (DS, CCHA); 1 from Marco Island, isolated from low-melting-point agarose (Life Technologies,
Florida; and 6 from Norman’s Pond Cay, Bahamas (BH). Total Eggenstein, Germany). Bands were stained with ethidium bro-
genomic DNA was prepared from fresh or ethanol-preserved mide, excised, and eluted with the aid of the QIAEX II gel
adult specimens as previously described (Sato et al. 1995). extraction kit (QIAGEN, Hilden, Germany). The eluted DNA
Production of cDNA library: Poly(A⫹) RNA was isolated was blunt ended, phosphorylated, ligated to SmaI-digested
from hepatopancreases of fish using an mRNA purification pUC18 plasmid vector with the help of the SureClone ligation
kit and cDNA was synthesized with the help of the TimeSaver kit (Amersham Biosciences), and used to transform E. coli
cDNA synthesis kit (Amersham Biosciences, Freiburg, Ger- XL-1 Blue competent bacteria (Stratagene). Double-stranded
many). The cDNA was then inserted into EcoRI-digested ␭gt10 DNA prepared with the aid of the QIAGEN plasmid kit was
vector (Stratagene, Heidelberg, Germany), in vitro packaged resuspended at a concentration of 1 ␮g/␮l and sequenced by
Mhc Heterozygosity in Rivulus 1793

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

Combination Name Sequence Location


1 KFF1 5⬘-GGGTTGGTTGATGATGTTCAGAT-3⬘ E2, codons 26–33 S
KFR1 5⬘-CCTCCCATAGTTCACATACTTCTT-3⬘ E3, codons 170–177 AS
2 Rima2F1 5⬘-GTTGATGATGTTCAGATTGATT-3⬘ E2, codons 28–34 S
KFR1 5⬘-CCTCCCATAGTTCACATACTTCTT-3⬘ E3, codons 170–177 AS
3 Rima134F1 5⬘-GTTGATGATGTTCAGATGTTTC-3⬘ E2, codons 28–34 S
KFR1 5⬘-CCTCCCATAGTTCACATACTTCTT-3⬘ E3, codons 170–177 AS
4 Rima5F1 5⬘-GTTGATGATGTTCAGATTGATGGC-3⬘ E2, codons 28–35 S
KFR1 5⬘-CCTCCCATAGTTCACATACTTCTT-3⬘ E3, codons 170–177 AS
5 KM1 5⬘-CCGATCAGCCAGAGTTTCAGAGGT-3⬘ I2, positions 1345–1370 S
KM2 5⬘-GAAGGCAGCATCAGTCCTGTCC-3⬘ E3, codons 148–156 AS
6 KM1 5⬘-CCGATCAGCCAGAGTTTCAGAGGT-3⬘ I2, positions 1345–1370 S
KM4 5⬘-GAAGGCAGCATTAGCCCCGTCC-3⬘ E3, codons 148–156 AS
7 KFF1 5⬘-GGGTTGGTTGATGATGTTCAGAT-3⬘ E2, codons 26–33 S
KM14 5⬘-GCTGTAGATTTATTCATGGATGGAGAA-3⬘ I2, positions 544–570 AS
8 KE31 5⬘-GTATGGCTGTGAATGGAATGATGAG-3⬘ I3, positions 1600–1624 S
KE32 5⬘-AGGGAGCTCCTCCCATAATTCAC-3⬘ E4, codons 174–181 AS
AS, antisense; E, exon; I, intron; S, sense.

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.

them. Distinct, well-separated, and statistically strongly

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.

otherwise identical or nearly identical sequences of a


given gene (not shown). Clade E1 contains a (GATA)n
repeat in this part of the gene.
Haplotype polymorphism: PCR-amplification experi-
ments suggested that not all the loci and all groups of
loci were represented in all the killifish populations.
The exhaustive typing of the four fish from Florida (nos.
32, 33, 35, and 36) was particularly informative in this
regard (Figure 6A, Table 3). It revealed the presence
of the A1, B1, C1, D1, D2, and D3 loci in the Florida
populations, but no evidence for the presence of any
of the other class I loci found in other populations.
Furthermore, differences were also apparent among
populations from the same area and even among indi-
viduals from the same population (Table 1). Thus, for
example, individuals 33 (Brevard County) and 35 (Vero
Beach) showed no signs of the presence of any loci
other than A1, D1, and D3. Similarly, no evidence of
the B1 locus could be obtained for the Belize samples,
which, by contrast, possessed the E, F, G, and H loci
apparently absent in the Florida samples.
Additional evidence for the variation in the number
of loci among individuals was obtained by Southern blot
analysis of DNA from two fish, no. 32 from Brevard
County, Florida and no. 39 from Tobacco Range, Belize
(Figure 6B). The DNA was digested with five enzymes
(EcoRI, HindIII, BamHI, MspI, and TaqI) and the blot
was hybridized with an exon 3 probe. The differences
in the number of hybridizing bands between the two
Mhc Heterozygosity in Rivulus 1797

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

Sequences in individual (number of clones)


Band 32 33 35 36
I A1*01 (1) A1*01 (1) A1*01 (2) A1*02 (1)
A1*02 (6) C1*01 (1)
B1*01 (1)
II A1*01 (5) A1*01 (3) A1*02 (2)
C1*01 (1)
III A1*02 (4) A1*02 (4)
B1*01 (2) B1*01 (2)
C1*01 (1)
IV A1*02 (2) A1*01 (2) A1*01 (2) A1*02 (2)
Figure 6.—Southern blot hybridization of killifish genomic B1*01 (4) B1*01 (4)
DNA with a 300-bp-long fragment of a class I Mhc gene (exon D1*02 (1) C1*01 (1)
3). (A) DNA from the four indicated individuals was digested D1*01 (1)
with HindIII. (B) DNA from two individuals was digested with Genomic
the indicated restriction endonucleases. DNAa A1*02 (1) A1*01 (1) A1*01 (1) B1*01 (3)
B1*01 (3) D1*01 (1) D1*01 (2) R1 (1)
R1 (1) D2*02 (1) R4 (2) C1*01 (2)
as 56% in some populations. Presumably the difference D1*02 (1) D3*01 (1) D2*01 (2)
between the Mhc- and non-Mhc-based estimates is due D2*01 (4) D2*02 (1)
to selection, which is absent at the neutral loci of the D3*01 (1) R5 (2)
D3*02 (1)
earlier studies but demonstrable in the case of the Mhc
loci (Hughes and Nei 1988). To determine under what a
Undigested, unseparated DNA.
conditions a selfing population could become nearly
fully homozygous at neutral loci while retaining up to
44% heterozygotes at loci under balancing selection, from 2 to 40 and the Ns becomes of the order of 100.
we carried out a computer simulation in which we varied The Ns ⫽ 100 value corresponds to N ⫽ 5 ⫻ 104 and
the selfing rate r, while keeping other population param- s ⫽ 0.002, but since simulation with N ⫽ 5 ⫻ 104 would
eters (population size N, mutation rate u, and selection have taken a long time, the actual parameters used were
intensity s) constant. The N was estimated from the N ⫽ 200 and s ⫽ 0.5.
average nucleotide diversity (␲) at the control region The results of the simulation are summarized in Fig-
of the mtDNA. Using the relationship ␲ ⫽ 2Nf v (see ures 8–10. They reveal that under conditions of neutral-
Nei and Kumar 2000) and values ␲ ⫽ 0.0047 ⫾ 0.0023, ity and low Nu value (0.02), the frequency of homozy-
v ⫽ 5.6 ⫻ 10⫺8 substitutions per site per generation, we gotes, φ, is close to 100%, irrespective of the variation
obtained Nf ⫽ 42,000 ⫾ 21,000 or ⵑ5 ⫻ 104. Since in in the selfing rate (Figure 8). It is only at Nu values
a selfing population most individuals can transmit their ⬎0.02 that the influence of selfing becomes apparent.
mtDNA to the next generation, here N is approximately When Nu ⫽ 0.1 and r ⫽ 0.90, the simulated value of φ
equal to Nf. The mutation rate ␮ at nonsynonymous becomes 96.6 ⫾ 4.2%, which is close to the observed
sites in the segment of Mhc loci controlling the PBR, value of φ (Taylor et al. 2001). When balancing selec-
the region under balancing selection, has been esti- tion is introduced in the simulation process under these
mated for cichlid fish to be 3 ⫻ 10⫺9 per site per genera- conditions (i.e., r ⫽ 0.90 and Nu ⫽ 0.02), the frequency
tion (or per year since in these fish, one generation of homozygotes is determined by the value of Ns: as Ns
equals ⵑ1 year; see Figueroa et al. 2000). Using this increases, φ decreases (Figure 9). At Ns ⫽ 100, φ ⫽ 58%,
estimate and taking the length of the PBR as L ⫽ 139 which is close to the value observed for the Mhc loci. We
sites (assuming correspondence between fish and hu- conclude therefore that the observed difference in the
man PBR positions), we obtain the estimate of Nu ⫽ frequency of homozygotes between neutral loci and loci
0.02, where u is the mutation rate of the PBR. The under balancing selection can be accounted for by assum-
selection intensity parameter s was estimated from the ing a selfing rate of 0.90, Nu of 0.02, and Ns of 100.
observed average nucleotide diversity at Mhc introns The difference between the heterozygosity of neutral
using the relationship ␲ ⫽ 4N␮fs, where fs, the scaling vs. Mhc loci was one observation in want of an explana-
factor of balancing selection, is a function of Ns and tion. Another was the large genetic distance (up to 0.026)
Nu (Takahata 1990, 1993). The observed nucleotide between some of the Mhc alleles. Such alleles differ by
diversity of Mhc loci ranged from 0.001 to 0.026. Using multiple substitutions whose accumulation requires
4N␮ ⫽ 4 ⫻ 5 ⫻ 104 ⫻ 3 ⫻ 10⫺9 ⫽ 0.0006, fs ranges long persistence of an allelic lineage in a population.
1800 A. Sato et al.

Figure 7.—Maximum-parsimony tree of R. marmoratus Mhc


class I exon 3 nucleotide sequences based on the heuristic Figure 8.—Parameters affecting the frequency of homozy-
search algorithm. The majority consensus tree using the mid- gotes (φ) at different selfing rates (r ) as determined by com-
point rooting option is shown. Major clades are indicated. puter simulation. For a description of the simulation, see text.
N, effective population size; u, mutation rate; s, selection inten-
sity.
However, in a population consisting of selfing individu-
als the odds are stacked heavily against the persistence
of allelic lineages and it is therefore necessary to define r, the intensity appears to be approximated by s(1 ⫺ r)
the conditions that could explain the presence of multi- (N. Takahata, personal communication). Thus when
ple allelic lineages in the killifish populations. Here, too, r is as large as 90%, selection operates with the inten-
we resorted to computer simulation. To generate allelic sity of s/10 in a selfing population. Therefore, by taking
lineages at an Mhc locus (defining a lineage as a collec- Ns ⫽ 100 in a random mating population, the effective
tion of genes sharing substitutions at the sites under Ns value in a selfing population becomes 10.
balancing selection), we started the simulation with a The killifish Mhc data reveal that their Ns value is of
homozygous gene pool of size 2N and let the pool evolve the order of 100, suggesting that the Ns in a random
under random mating with a mutation rate u and selec- mating population is much larger, perhaps of the order
tion intensity s, until it reached an equilibrium in which of 1000. We therefore ran two replications of simula-
the loss of allelic lineages was balanced out by the emer- tions to examine the effect of a larger Ns on φ and Td
gence of new lineages. (This took 100N to 4000N gen- under the conditions of Ns ⫽ 1000, Nu ⫽ 0.02, and r ⫽
erations, depending on the mutation rate.) At that point 0.90. The result shows that Ns ⫽ 1000 does not affect
we introduced selfing at a rate r, keeping track of individ- φ much: The simulated value of 56.4% ⫾ 0.3 is similar
ual lineages and recording the time of their disappear- to the observed φ-value (and to the simulated value
ance from the pool. The time from the onset of selfing when Ns ⫽ 100). However, Ns ⫽ 1000 affects Td strongly.
until the moment when only one of the original (“ances- While under the conditions Ns ⫽ 100, Nu ⫽ 0.02, and
tral”) lineages present at the outset remained for the r ⫽ 0.90, the estimated Td value is 23 ⫾ 14; when Ns ⫽
first time was designated Td. To obtain the average Td, 1000, Td becomes 51 ⫾ 19 in units of N generations. By
the simulation was repeated 1000 times for each of the assuming a generation time of 1 year for killifish and
different selfing rates. N ⫽ 50,000, the persistence time of the allelic lineages
As expected, the Td depended on the selfing rate r: is 2.5 ⫾ 0.9 million years (MY). The implications of
It decreased as the rate increased. At the rate r ⬎ 0.90, these results for the interpretation of the genetic com-
the Td dropped rapidly to values obtained for neutral position of the killifish populations and for the origin
loci (Figure 10). At r ⫽ 0.90, the Td depended on the of the species hermaphroditism are described in the
parameters Nu and Ns: The lower the former and the discussion.
larger the latter, the longer the persistence time Td. To
simulate conditions corresponding to the observations,
DISCUSSION
we chose one value of the Nu parameter (Nu ⫽ 0.02)
and one value of the Ns parameter (Ns ⫽ 100) to obtain In the population samples analyzed in this study, only
Td ⫽ 23 ⫾ 14 in units of N generations. However, the four pairs of individuals and one quintet were found
rapid reduction of Td to the neutral level (Figure 10) to be identical in their class I genes (Table 1). The
indicates that selfing influences selection intensity: Het- differences between the individuals were in the pres-
erozygotes tend not to be produced in a selfing popula- ence or absence of loci (haplotype polymorphism) and
tion and this weakens the intensity. With the selfing rate in the alleles found at shared loci (allelic polymor-
Mhc Heterozygosity in Rivulus 1801

Figure 9.—The effects of balancing selection on the fre-


quency of homozygotes (φ) at Nu ⫽ 0.1 and selfing rate of
r ⫽ 0.95, as determined by computer simulation. For explana-
tion, see text. N, effective population size; u, mutation rate; s, Figure 10.—Persistence of allelic lineages in a population
selection intensity. Standard errors are indicated by segments. of selfing individuals under different selfing rates (r ) and
chosen Ns and Nu parameters, as determined by computer
simulation. N, effective population size; u, mutation rate; s,
phism). Genetic distances obtained by pairwise compari- selection intensity. Persistence time, expressed as 2N genera-
tions, is measured from the onset of selfing until only one of
sons of Mhc alleles revealed that the Mhc composition the ancestral lineages is left. For details, see text.
of the killifish population resembled that of other fish
species, for example, that of the Lake Victoria haplo-
chromine flock (Figueroa et al. 2000). Hence, at this of heterozygosity at microsatellite or other loci has been
level of analysis, the Mhc class I heterogeneity of the reported for other populations in which males are rare.
killifish populations is comparable to that of the haplo- The Mhc analysis of R. marmoratus, by contrast, reveals
chromines, even though the former are considered to that only 4–44% of the individuals are heterozygous at
be a single species (albeit a species to which the defini- these loci (Table 1). The computer simulation results
tion based on breeding behavior does not apply), while explain this reduction in the level of Mhc heterozygosity
the latter are a collection of several hundred closely by the high degree of selfing in the R. marmoratus popu-
related species. These observations are consistent with lations. That Mhc heterozygotes have not been entirely
the interpretation of the killifish populations as agglom- eliminated is explained by the assumption of a selfing
erates of highly differentiated clones. The clonal hy- rate of ⬍1.0. Apparently, not only in the Twin Caye
pothesis was originally formulated on the basis of tissue population, in which male R. marmoratus have been
grafting experiments (Harrington and Kallman found at a relatively high frequency (Davis et al. 1990;
1968) and was later strengthened by microsatellite typing Turner et al. 1992a), but also in other populations,
data (Turner et al. 1990, 1992b). The Mhc analysis de- males occasionally occur and are responsible for the
scribed in the present study provides an explanation genetic mixing of the otherwise selfing clones.
for the grafting results since differences at class I Mhc The wide range of the genetic distances between the
loci are known to be the main source of histoincompati- Mhc alleles found in pairwise comparisons indicates that
bility between individuals (Klein and Horejsı́ 1997). the allelic differences stem from two sources: the ances-
In one important aspect, however, the results of the tral allelic lineages that were present in the population
Mhc analysis are at variance with the results of both the at the time it switched from outbreeding to obligatory
Mhc typing of other fish species and the microsatellite selfing and that have not been entirely eliminated as
typing data—the heterozygosity of the loci. Mhc typings yet by the selfing process and mutations that continue
of other fish species, indeed of most other jawed verte- to arise and are promoted by balancing selection. The
brates, generally indicate very high frequency of hetero- current state of Mhc diversity in the R. marmoratus popu-
zygotes at some of the class I and class II loci, usually lations is therefore apparently the result of a complex
close to 100%. On the other hand, microsatellite typing interplay of several factors among which the rate of
of killifish populations reveals high heterozygosity in selfing, the intensity of balancing selection, and the
only one population, that of Twin Caye in Belize, in persistence of ancestral allelic lineages are the most
which males occur in abundance in addition to the important ones.
hermaphrodites (Lubinski et al. 1995). The heterozy- The mangrove killifish R. marmoratus and its close
gosity of this population is explained by the assumption relative R. occellatus are the only two hermaphrodites
of hermaphrodites outcrossing with males. No evidence among the ⬎100 defined species of Rivulus, and of
1802 A. Sato et al.

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
eages suggest that obligatory selfing in R. marmoratus Atz, J. W., 1965 Hermaphroditic fish. Science 150: 789–797.
must be ⬍80,000 years old. Clark, J. A., W. E. Farell and W. R. Peltier, 1978 Global changes
The Mhc analysis provides a glimpse into the possible in postglacial sea level: a numerical calculation. Quat. Res. 9:
265–287.
mechanism by which hermaphroditism may have arisen Cole, K. S., and D. L. G. Noakes, 1997 Gonadal development and
in R. marmoratus. The genetic distances between the sexual allocation in mangrove killifish, Rivulus marmoratus (Pisces:
killifish class I genes, including those between alleles Atherinomorpha). Copeia 1997: 596–600.
Davis, W. P., D. S. Taylor and B. J. Turner, 1990 Field observations
found in different putative clones, are too great to have of the ecology and habits of mangrove rivulus (Rivulus marmora-
been attained in the last 2.5 MY (Klein et al. 1993). tus) in Belize and Florida. Ichthyol. Explor. Freshwaters 1: 123–
The divergence of the genes must have begun in the 134.
Figueroa, F., W. E. Mayer, H. Sültmann, C. O’hUigin, H. Tichy
nonhermaphroditic ancestors and some of the variabil- et al., 2000 Mhc class II B gene evolution in East African cichlid
ity must have then been bequeathed to R. marmoratus. fishes. Immunogenetics 51: 556–575.
This observation implies that hermaphroditism could Gilbert, D. G., 1996 SeqPup Version 0.6f: A Biosequence Editor and
Analysis Application (http://iubio.bio.indiana.edu/soft/molbio).
not have arisen in a single individual, but rather that Harrington, R. W., 1961 Oviparous hermaphroditic fish with inter-
the switch from the separate-sexed condition to her- nal fertilization. Science 134: 1749–1750.
maphroditism and to selfing involved a whole popula- Harrington, R. W., 1963 Twenty-four hour rhythms of internal
self-fertilization and oviposition by hermaphrodites of Rivulus
tion of individuals and most likely occurred gradually. marmoratus. Physiol. Zool. 36: 325–341.
At least two stages must be postulated in the evolution Harrington, R. W., 1967 Environmentally controlled induction
of hermaphroditism. In the first stage, a mutation (or of primary male gonochorists from eggs of the self-fertilizing
hermaphroditic fish Rivulus marmoratus. Biol. Bull. 132: 174–199.
mutations) produced protogynous hermaphrodites that Harrington, R. W., 1968 Delimitation of the thermolabile pheno-
functioned first as females and then transformed into critical period of sex determination and differentiation in the
males. The mutation then spread through the popula- ontogeny of the normally hermaphroditic fish Rivulus marmoratus.
Physiol. Zool. 41: 447–460.
tion among individuals bearing highly diverged Mhc Harrington, R. W., 1975 Sex determination and differentiation
class I genes. At this stage, hermaphroditism may have among uniparental homozygotes of the hermaphroditic fish Rivu-
been facultative so that the fish were reproducing by lus marmoratus (Cyprinodontidae Atheriniformes), pp. 249–262
in Intersexuality in the Animal Kingdom, edited by R. Reinboth.
selfing and outcrossing. In the second stage, another Springer, New York.
mutation (or mutations) transformed successive her- Harrington, R. W., and K. D. Kallman, 1968 The homozygosity
Mhc Heterozygosity in Rivulus 1803
of clones of the self-fertilizing hermaphroditic fish Rivulus marm- Nei, M., and T. Gojobori, 1986 Simple methods for estimating the
oratus (Cyprinodontidae Atheriniformes). Am. Nat. 102: 337–343. numbers of synonymous and nonsynonymous nucleotide substi-
Haug, G. H., and R. Tiedemann, 1998 Effect of the formation of the tutions. Mol. Biol. Evol. 3: 418–426.
Isthmus of Panama on Atlantic Ocean thermohaline circulation. Nei, M., and S. Kumar, 2000 Molecular Evolution and Phylogenetics.
Nature 393: 673–676. Oxford University Press, Oxford.
Huber, J. H., 1992 Review of Rivulus—Ecobiogeography, Relationships. Saitou, N., and M. Nei, 1987 The neighbor-joining method: a new
Société Française d’Ichtyologie, Paris. method for reconstructing phylogenetic trees. Mol. Biol. Evol.
Hughes, A. L., and M. Nei, 1988 Pattern of nucleotide substitution 4: 406–425.
at major histocompatibility complex class I loci reveals overdomi- Sanger, F., S. Nicklen and A. R. Coulson, 1977 DNA sequencing
nant selection. Nature 355: 167–170. with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:
Kallman, K. D., and R. W. Harrington, 1964 Evidence for the 5463–5467.
existence of homozygous clones in the self-fertilizing hermaphro- Sato, A., F. Figueroa, C. O’hUigin, D. N. Reznick and J. Klein,
ditic teleost Rivulus marmoratus Poey. Biol. Bull. 126: 101–114. 1995 Identification of major histocompatibility complex genes
Kimura, M., 1980 A simple method for estimating evolutionary rates in the guppy, Poecilia reticulata. Immunogenetics 43: 38–49.
of base substitutions through comparative studies of nucleotide Sato, A., D. Klein, H. Sültmann, F. Figueroa, C. O’hUigin et al.,
sequences. J. Mol. Evol. 16: 111–120. 1997 Class I Mhc genes of cichlid fishes: identification, expres-
Klein, J., 1986 Natural History of the Major Histocompatibility Complex. sion, and polymorphism. Immunogenetics 46: 63–72.
John Wiley, New York. Sato, A., F. Figueroa, B. W. Murray, E. Málaga-Trillo, Z. Zaleska-
Klein, J., and F. Figueroa, 1986 Evolution of the major histocom- Rutczynska et al., 2000 Nonlinkage of the major histocompati-
patibility complex. CRC Crit. Rev. Immunol. 6: 295–386. bility complex of class I and class II loci in bony fishes. Immunoge-
Klein, J., and V. Horejsı́, 1997 Immunology. Blackwell Scientific Pub- netics 51: 108–116.
lishers, Oxford. Soto, C. G., J. F. Leatherland and D. L. G. Noakes, 1992 Gonadal
Klein, J., R. E. Bontrop, R. L. Dawkins, H. A. Erlich, U. B. Gyllen- histology in the self-fertilizing hermaphroditic fish Rivulus marm-
sten et al., 1990 Nomenclature for the major histocompatibility oratus. Can. J. Zool. 70: 2338–2347.
complexes of different species: a proposal. Immunogenetics 31: Swofford, D. L., 2001 PAUP: Phylogenetic Analysis Using Parsimony
217–219. (and Other Methods), Version 4.0b8a. Sinauer, Sunderland, MA.
Klein, J., Y. Satta, C. O’hUigin and N. Takahata, 1993 The molec- Takahata, N., 1990 A simple genealogical structure of strongly
ular descent of the major histocompatibility complex. Annu. Rev. balanced allelic lines and trans-species evolution of polymor-
Immunol. 11: 269–295. phism. Proc. Natl. Acad. Sci. USA 87: 2419–2423.
Kocher, T. D., W. K. Thomas, A. Meyer, S. V. Edwards, S. Paabo et Takahata, N., 1993 Allelic genealogy and human evolution. Mol.
al., 1989 Dynamics of mitochondrial DNA evolution in animals: Biol. Evol. 10: 2–22.
amplification and sequencing with conserved primers. Proc. Natl. Taylor, D. S., M. T. Fisher and B. J. Turner, 2001 Homozygosity
Acad. Sci. USA 86: 6196–6200. and heterozygosity in three populations of Rivulus marmoratus.
Kristensen, I., 1970 Competition in three cyprinodont fish species Environ. Biol. Fishes 61: 455–459.
in the Netherlands Antilles. Stud. Fauna Curacao Carib. Isl. 119: Trowsdale, J., 1995 “Both man & bird & beast”: comparative organi-
82–101. zation of MHC genes. Immunogenetics 41: 1–17.
Kumar, S., K. Tamura, I. B. Jakobsen and M. Nei, 2001 MEGA Turner, B. J., J. F. Elder, T. F. Laughlin and W. P. Davis, 1990
2: Molecular Evolutionary Genetic Analysis Software. Arizona State Genetic variation in clonal vertebrates detected by simple-
University, Tempe, AZ. sequence DNA fingerprinting. Proc. Natl. Acad. Sci. USA 87:
Laughlin, T. F., B. A. Lubinski, E.-H. Park, D. S. Taylor and B. J. 5653–5657.
Turner, 1995 Clonal stability and mutation in the self-fertilizing Turner, B. J., W. P. Davis and D. S. Taylor, 1992a Abundant
hermaphroditic fish, Rivulus marmoratus. J. Hered. 86: 399–402. males in populations of a selfing hermaphroditic fish, Rivulus
Lin, H.-C., and W. A. Dunson, 1995 An explanation of the high marmoratus, from some Belize cays. J. Fish. Biol. 40: 307–310.
strain diversity of a self-fertilizing hermaphroditic fish. Ecology Turner, B. J., J. F. Elder, T. F. Laughlin, W. P. Davis and D. S.
76: 593–605. Taylor, 1992b Extreme clonal diversity and divergence in pop-
Lubinski, B. A., W. P. Davis, D. S. Taylor and B. J. Turner, 1995 ulations of a selfing hermaphroditic fish. Proc. Natl. Acad. Sci.
Outcrossing in a natural population of a self-fertilizing hermaph- USA 89: 10643–10647.
roditic fish. J. Hered. 86: 469–473. Vrijenhoek, R. C., 1985 Homozygosity and interstrain variation in
Massaro, E. J., J. C. Massaro and R. W. Harrington, 1975 Bio- the self-fertilizing hermaphroditic fish, Rivulus marmoratus. J.
Hered. 76: 82–84.
chemical comparison of genetically different homozygous clones
Vrijenhoek, R. C., R. M. Dawley, C. J. Cole and J. P. Bogart, 1989
(isogenic, uniparental lines) of the self-fertilizing fish Rivulus
A list of the known unisexual vertebrates, pp. 19–23 in Evolution
marmoratus Poey, pp. 439–453 in Isozymes, Vol. 3, edited by C. L.
and Ecology of Unisexual Vertebrates. Museum Bulletin No. 466, edited
Markert. Academic Press, New York.
by R. M. Dawley and J. P. Bogart. The State University of New
Meyer, A., M. Morrissey and M. Schartl, 1994 Recurrent origin York, Albany, NY.
of a sexually selected trait in Xiphophorus fishes inferred from a Weibel, A. C., T. E. Dowling and B. J. Turner, 1999 Evidence that
molecular phylogeny. Nature 368: 539–542. an outcrossing population is a derived lineage in a hermaphro-
Murphy, W. J., J. E. Thomerson and G. E. Collier, 1999 Phylogeny ditic fish (Rivulus marmoratus). Evolution 53: 1217–1225.
of the neotropical killifish family Rivulidae (Cyprinodontiformes, Wells, H., 1979 Self-fertilization: Advantageous or deleterious? Evo-
Aplocheiloidei) inferred from mitochondrial DNA sequences. lution 33: 252–255.
Mol. Phylogenet. Evol. 13: 289–301. Zinkernagel, R. M., and P. C. Doherty, 1974 Restriction of in-
Nagl, S., H. Tichy, W. E. Mayer, N. Takezaki, N. Takahata et al., vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis
2000 The origin and age of the haplochromine species flock within a syngeneic or semiallogeneic system. Nature 248: 701–702.
in Lake Victoria, East Africa. Proc. R. Soc. Lond. Ser. B 267:
1049–1061. Communicating editor: N. Takahata

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy