Genetic analysis of cavefish reveals molecular convergencein.docx

Genetic analysis of cavefish reveals molecular convergence
in the evolution of albinism
Meredith E Protas1, Candace Hersey2, Dawn Kochanek3, Yi
Zhou2, Horst Wilkens4, William R Jeffery5,
Leonard I Zon2, Richard Borowsky3 & Clifford J Tabin1
The genetic basis of vertebrate morphological evolution has
traditionally been very difficult to examine in naturally
occurring populations. Here we describe the generation of a
genome-wide linkage map to allow quantitative trait analysis of
evolutionarily derived morphologies in the Mexican cave tetra,
a species that has, in a series of independent caves, repeatedly
evolved specialized characteristics adapted to a unique and
well-studied ecological environment. We focused on the trait
of albinism and discovered that it is linked to Oca2, a known
pigmentation gene, in two cave populations. We found
different deletions in Oca2 in each population and, using
a cell-based assay, showed that both cause loss of function
of the corresponding protein, OCA2. Thus, the two cave
populations evolved albinism independently, through
similar mutational events.
The relatively closed, often nutrient-poor, and lightless
environment
of caves represents a marked change in ecological conditions to
which
several entrapped species have adapted. Obligate cave-dwelling
ani-
mals, called troglobites or troglodytes, are characterized by a
remark-
able convergence of eye and pigment loss across diverse species
such as

spiders, isopods, salamanders and fish1.
There are 86 known troglodytic species of fish2. The best
studied is
the Mexican tetra, identified by some authors as Astyanax
mexicanus
and others as Astyanax fasciatus; the two names should be
considered
synonymous in the present context and the species will be
referred to
herein as Astyanax. This species has 29 cave populations in the
karst
region of the Sierra de El Abra of northeast Mexico and one
additional
population in Guerrero (Fig. 1a)3,4. A surface, or river-
dwelling, sister
population of the cave morph lives in southern Texas and
northeastern
Mexico and can still interbreed with the cave morph.
Phenotypically,
the cave and surface morphs are very different; among other
char-
acteristics, the cave morph has a greater weight per unit length,
less
pigment, regressed eyes, larger nostrils, more maxillary teeth,
more
cranial neuromasts and more taste buds, as well as differences
in
feeding, schooling and aggressive behaviors (Fig. 1b–d)4,5.
Molecular
phylogenetic studies indicate that several cave populations
indepen-
dently evolved these characteristics6–8.
To provide a framework in which to study the genetics of this

species, we made a microsatellite linkage map. We have
isolated and
Rio Sabinas
Rio Frio
Molinoa b
c
d
Pachón
Pachón
Japonés
Ciudad Valles
kilometers
0 5 10 15
Ciudad Mante
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Surface
Molino
R
io

M
an
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M
EXICO
Figure 1 Phenotype and locations of albino cave populations of

Astyanax
mexicanus. (a) Map of the area in Mexico where the different
cave
populations are found. Dots represent cave populations. Caves
with red dots
are Molino, Pachón and Japonés, all of which contain a majority
of albino
individuals. Inset map at bottom shows the location of the
region within
Mexico. (b) A representative surface fish. (c) A representative
Molino
cavefish. (d) A representative Pachón cavefish.
Received 20 July; accepted 13 October; published online 11
December 2005; doi:10.1038/ng1700
1Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115, USA. 2Children’s Hospital Stem Cell
Program, Department of Hematology/Oncology,
Howard Hughes Medical Institute, Children’s Hospital Boston,
Boston, Massachusetts 02115, USA. 3Cave Biology Research
Group, Department of Biology, New York
University, 1009 Main, 100 Washington Square East, New
York, New York 10003, USA. 4Zoological Institute and
Zoological Museum, University of Hamburg, Martin-
Luther-King-Platz 3, 20146 Hamburg, Germany. 5Department of
Biology, University of Maryland, College Park, Maryland
20742, USA. Correspondence should be
addressed to C.J.T. ([email protected]).
NATURE GENETICS VOLUME 38 [ NUMBER 1 [ JANUARY

2006 107
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designed primers corresponding to over 600 microsatellites.
Using a
backcross from the Molino cave population with 111 progeny,
we
obtained 35 linkage groups composed of 267 markers, out of
300
markers genotyped, with a coverage of 1,916 cM (Fig. 2 and
Supplementary Table 1 online). Astyanax has 25 chromosomes,
sug-
gesting that with the addition of more markers, some of the
linkage
groups would collapse. We are also genotyping a larger F2 cross
from
another cave, Pachón, which should coalesce some of the
linkage
groups and allow for comparisons between the two cave
populations.
We identified a number of statistically significant, quantitative
loci
for different traits present in the cave form, most of which will
be
described elsewhere. Here we focus on one such trait, albinism.
Previous genetic studies have indicated that albinism in the
Pachón
cave is caused by a single recessive mutation9,10. In the Molino
backcross, albinism mapped to a single locus in linkage group
16
with a LOD score of 17.29 at microsatellite marker 218E,
accounting
for 49.4% of the variance in this trait (Fig. 3a). A similar
analysis of
the Pachón F2 cross mapped the locus for albinism to the same
location with a LOD score of 17.98 at marker 218E, accounting
for

42.6% of the variance in this trait (data not shown). This
coincidence
of loci responsible for albinism raises the following three
possibilities:
the two cave populations could have the same mutation in the
same
gene, different mutations in the same gene or mutations in
distinct
but closely linked genes. To address the latter possibility, we
performed
a complementation test between a Molino individual and a
Pachón
individual, which yielded only albino offspring (Fig. 3b). Thus,
albinism in these two cave populations is caused by mutations
in
the same gene.
To identify the gene responsible for albinism in Astyanax, we
genotyped individuals of the Molino backcross for a series of
candi-
date genes, based on known albinism loci in mouse and humans:
tyrosinase (Tyr), tyrosinase-related protein-1 (Tyrp1) and ocular
and
cutaneous albinism-2 (Oca2) (Fig. 2). One of these genes, Oca2,
mapped to the albino locus and increased the LOD score in the
Molino backcross to 68.6 (Fig. 3c), now accounting for 93.1%
of the
variance of this trait, and the LOD score in the Pachón F2 cross
to
60.66, now accounting for 71.6% of the variance of this trait
(data
not shown). Furthermore, there is a perfect association between
the genotype of the Oca2 marker and the phenotype of albinism
in
all successfully genotyped individuals of both the Molino

backcross
(105 individuals) and the Pachón F2 cross (215 individuals).
Although the function of Oca2 is unknown, it is the most
commonly
mutated gene in cases of human albinism11 and is also
responsible for
pigmentation phenotypes in mouse and medaka12,13. To test
whether
Oca2 mutations are responsible for albinism in cavefish, and,
more
importantly, to identify the specific genetic lesions in Oca2
responsible
for albinism, we compared the sequence of the Oca2 cDNA in
surface,
Pachón and Molino individuals (Supplementary Fig. 1 online).
We
found numerous differences in the Oca2 sequences present in
the two
cave populations as compared to their surface counterparts (Fig.
4a
and Supplementary Fig. 1). The Pachón cave population had
three
polymorphisms that could affect Oca2 function: two were amino
acid
changes in conserved residues, and the last was a deletion
extending
from within intron 23 through most of exon 24, such that the
cDNA
includes part of intron 23 fused to the last nine base pairs of
exon 24
and the 3¢ UTR (Fig. 4a and Supplementary Fig. 1). The Molino
cave
population had only one major difference: exon 21 was missing
(Fig. 4a and Supplementary Fig. 1).

The missing exons observed in the Molino and Pachón Oca2
sequence could, in principle, be explained by either alterations
in
splicing or deletions of genomic DNA. Amplifying from
genomic
DNA, we found that in both cases, the observed losses of exonic
sequence in the Molino and Pachón Oca2 cDNAs were
attributable to
genomic deletions (Supplementary Fig. 2 online).
24 206A
209E0
18
6
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103A
219A
207F
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15A
232A
231C
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0

129B
43A
32 33 34 35
113 33b
95
90
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237A
213F82
80
73
66 30C
26A
105B
43C54
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44
218E
132B
34
205E
210A
9

0
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220B
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224C
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37 208A
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47 109C
119A52
66 113C
139B23
12
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0
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228E227D
103C
203A
47 115C
15C35
30
27
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228D
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220E0
42
40
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21

0 219B
111B
109B
219E
226B
49 105A
223C
229B
21
16
7
0
235D
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58 3c
47 3B
127B35
16 8C
133C0
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211E
143A0 0
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0
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31302928272625242322212019181716
Oca2
Tyrp1
Tyr
0 237E
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0 0 0
0
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0 0 0 0 0 0 0 123C
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211B
235B
241B
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202A
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15
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182
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Figure 2 Microsatellite linkage map of Astyanax mexicanus.
The names of the microsatellite markers are to the right of each
linkage group and the positions
of the markers (in cM) are on the left. Markers in three genes
(Oca2, Tyr and Tyrp1) are labeled in red.
108 VOLUME 38 [ NUMBER 1 [ JANUARY 2006 NATURE
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To test whether the polymorphisms in the cave populations’
Oca2
sequences cause albinism through loss of function of Oca2, we
used
the melan-p cell line, which is a melanocyte cell line generated
from an
Oca2-deficient mouse14. We made constructs expressing wild-
type
surface-fish Oca2 and surface-fish Oca2 modified with each of
the
cavefish polymorphisms driven by the human ubiquitin
promoter. We
first transfected the surface-fish Oca2 construct to ascertain
whether
wild-type Astyanax Oca2 could complement Oca2 function in
mouse
cells. Indeed, melan-p cells transfected with the surface-fish
Oca2
showed high levels of pigmentation (Fig. 4b–d). We then
transfected
cells with constructs encoding the different amino acid
substitutions
seen in the Pachón cave. Both point mutations caused rescue of
pigmentation (Fig. 4e–j). However, neither the deletion found in
the
Molino cave nor the deletion found in the Pachón cave caused
rescue

of pigmentation (Fig. 4k–p). Therefore, the deletions cause loss
of
function of OCA2, whereas the two point mutations do not
drastically
affect the function of OCA2 in this cell line, strongly
suggesting that
the exon 21 deletion is the mutation that causes albinism in the
Molino cave population and the exon 24 deletion is the mutation
that
causes albinism in the Pachón population.
The identification of different inactivating mutations in Oca2 in
the
Molino and Pachón populations suggests that albinism evolved
independently in these two caves, by convergent evolution in
the
same gene. The independent evolution of Oca2 in the two caves
is
further supported by analysis of amino acid changes and neutral
base
changes; these polymorphisms do not group the Molino and
Pachón
populations together to the exclusion of the surface population
(Supplementary Table 2 online).
We also examined another cave popula-
tion, Japonés, that contains albino indivi-
duals (Fig. 1a). In a complementation test
between an albino Japonés individual and an
albino Pachón individual, only albino off-
spring resulted, suggesting that albinism in
the Japonés population also arose through
mutations in the Oca2 gene (data not
shown). Sequencing of Oca2 from one albino
Japonés individual showed that neither the
Molino nor the Pachón deletion was present

(data not shown), though the two point
mutations found in the Pachón cave were
found. Together with the complementation
data, this result suggests that a third, inde-
pendent mutation in the Oca2 gene, perhaps
in a regulatory sequence, is responsible for
the convergent albinism phenotype in the
Japonés cave.
The parallel evolution of loss of pigmenta-
tion and loss of eyes within the species
Astyanax is mirrored in many other cave
dwelling organisms. The most debated issue
about cave animals is why this loss of eyes
and pigmentation occurs. There are three
main theories1,8,9,15. First, the genes respon-
sible for structures or pathways that are not
advantageous in a dark environment, such as
eyes or pigmentation, might accumulate dele-
terious mutations and, over time, completely
degenerate. Second, it might be advantageous
to lose eyes and pigmentation to conserve the
energy or space that these structures con-
sume. Third, the genetic changes that cause loss of eye and
pigmenta-
tion might cause changes in other structures or pathways that
might
be advantageous in the cave environment.
The identification of the mutations responsible for loss of
pigmen-
tation in Astyanax allows us to consider the molecular nature of
the
variation facilitating this change in response to the cave
environment.

It is notable that Oca2 has repeatedly mutated in the cave
populations
we examined. It is possible that in the cave environment, loss of
Oca2
function is actually advantageous, for some as yet unknown
reason.
Alternatively, Oca2 might be mutated more often than other
pigmen-
tation pathway genes in Astyanax simply because Oca2
mutations do
not seem to have any deleterious effects aside from loss of
pigmenta-
tion and problems with vision. It is possible that some of the
other
pigmentation genes have more pleiotropic effects and that those
mutations are not as viable; for example, all of the mutations in
zebrafish Tyr that cause complete loss of pigmentation are only
semiviable16. A final explanation is that Oca2 is the most
frequently
targeted pigmentation gene in Astyanax for the same reasons
that it
seems to be in humans: first, Oca2 presents a very large target
size for
mutagenesis, being 345 kb in humans, with 24 exons11. Second,
human Oca2 maps to a region characterized by repetitive
sequences,
which are often associated with chromosomal rearrangements
and
deletions17. Although it is not yet known whether the Astyanax
Oca2
locus is similarly characterized by a large size and a large
number of
associated repeat sequences, the most parsimonious explanation
seems
to be a combination of a lack of deleterious pleiotropic effects
in

conjunction with the structure of Oca2 itself. These sorts of
features
may, in general, predispose certain loci to be targets for
evolutionary
80
Molino backcross Molino backcross
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B E
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20 40 60
cM
LG 16 LG 16
80 100 120 0 20 40 60
cM
80 100 120
O
c
a
2
Molino × Pachón
a

b
c
Figure 3 Oca2 is linked to albinism in both the Pachón and
Molino cave populations. (a) The albino
locus in the Molino backcross is in linkage group (LG) 16. The
LOD score is plotted against the
distance (in cM) across this linkage group. (b) An albino
Pachón � Molino hybrid, showing
noncomplementation. (c) The marker in Oca2 is located at the
albino locus in the Molino backcross
and its inclusion in the analysis greatly increases the LOD
score.
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forces effecting morphological change. The existence of at least
86
species of cavefish2, a subset of which show albinism, will
allow for
further examination of the parallel evolution of this trait in the
cave
environment.
We have seen that albinism has evolved in two different cave
populations through independent changes in the same gene.
Other
studies have also shown that one gene is responsible for the
same
morphological change in multiple populations or species:
ectodyspla-

sin (Eda) in body armor and Pitx1 in pelvic reduction in the
three-
spine stickleback18,19, ovo (shaven-baby) in trichome loss in
different
drosophilids20, and MC1R in pigmentation in pocket mice,
jaguars
and several avian species21,22. Thus, the same morphology
often
evolves by mutation in the same gene, possibly because it is the
most efficient or best way for a certain phenotype to evolve.
Another issue often discussed regarding the evolution of
morpho-
logical change is whether coding changes or regulatory changes
generate new traits. Regulatory mutations have an advantage as
agents
of morphological change in that they can alter a gene’s activity
in a
subset of the regions in which it is expressed. Regulatory
mutations
have been identified or implicated in morphological change in
plate
armor and pelvic reduction in sticklebacks18,19, wing-spot
pigmenta-
tion in Drosophila biarmipes23, muscle mass in pigs24 and
trichome
loss in drosophilids20. Often it is difficult to confirm that a
gene has a
specific regulatory mutation, as these mutations can span large
areas
of sequence. In contrast, coding differences are easier to find. It
is
notable that many of the examples of evolutionary change
involving
coding mutations that have previously been described, including
our

example of Oca2 in albinism in Astyanax, involve overall
changes in
pigmentation21,22. This could be because pleiotropic effects of
pig-
mentation genes are minimal and usually do not affect the
viability of
the organism.
We have identified specific genetic lesions responsible for the
parallel evolution of albinism in different cave populations of
Astya-
nax, and found that they represent convergent genetic events in
separate populations. The genetic tools we have developed will
facilitate further investigation into the molecular bases for the
evolu-
tion of specialized morphological characteristics in the unique
and
well-studied cave environment.
METHODS
Crosses. We used five crosses in this study: a Molino backcross,
a Pachón
F2 cross, a Pachón backcross, a Molino � Pachón
complementation cross
and a Pachón � Japonés complementation cross (see
Supplementary
Methods online).
Microsatellite identification. To make a genomic library, we
digested genomic
surface-fish DNA with Sau3AI and cloned fragments of 500–
700 bp into
BamH1-linearized pBluescriptSK+ (Stratagene). We then

electroporated the
library into SURE electroporation-competent cells (Stratagene).
A 32P- or
digoxigenin-end-labeled (CA)12 probe was used to hybridize
colony lifts and
colonies hybridizing to the probe were sequenced using T3 and
T7 primers.
Primers to detect microsatellites that had ten or more repeats
were
designed using Primer3. The following tag sequence was added
to the 5¢ end
of every forward primer: 5¢-CACGACGTTGTAAAACGAC-3¢
(Y.Z. and L.I.Z.,
unpublished data).
Genotyping and linkage map construction. We genotyped 111
individuals
for 300 microsatellite markers in the Molino backcross and 235
indi-
viduals with 254 microsatellites in the Pachón F2 cross. PCR
reactions
were 10 ml in volume and contained 0.1 mM MgCl2, 6 mM
Tris-HCl,
pH 8.3, 30 mM KCl, 0.006% glycerol, 0.25 mM dNTP mix
(Roche),
0.06% Tween, 0.06% Nonidet P-40, 0.25 units of Taq DNA
polymerase

(Roche), 5 nM forward primer, 200 nM reverse primer and 200
nM of the
fluorescent tag primer 5¢-CACGACGTTGTAAAACGAC-3¢
labeled with
one of two phosphoramidite conjugates, Hex and Fam, using a
PCR program
as described25. Each PCR reaction was primed with the specific
forward
primer. The majority of cycles used the forward primer in
excess—the
fluorescently labeled tag (Y.Z. and L.I.Z., unpublished data).
Three
Fam-labeled PCR products and three Hex-labeled PCR products
were pooled
and run on an ABI 3700 with GeneScan-500 ROX size standard
(Applied
Biosystems). Genotyper software (version 3.6) was used to
analyze the
genotyping results. The program JoinMap was used to make the
microsatellite
linkage map using the Kosambi mapping function, a jump
threshold of 5.0 and
Surface Oca2
Pachón Oca2

Molino Oca2
a
b c
f
i
l
o
e
h
k
n
d
g
j
m
p
Figure 4 Different Oca2 exon deletions in the Molino
population and

the Pachón population cause loss of function of the OCA2
protein.
(a) Schematics of the surface, Pachón and Molino Oca2 coding
regions.
Asterisks in the Pachón Oca2 represent changes in conserved
amino acid
residues: red asterisk, methionine to valine; blue asterisk,
proline to serine
(see Supplementary Figs. 1 and 2). In the Pachón coding
sequence, exon
24 is almost completely deleted. Following exon 23 are
additional sequence
(intron 23), the last few amino acids of exon 24, and the 3¢
UTR. The
Molino coding sequence is identical to that of the surface-fish
Oca2 except
that exon 21 is missing. (b–d) Transfection of the unmodified
surface-fish
Oca2 construct results in highly pigmented cells. (e–g)
Transfection of the
construct encoding the methionine-to-valine substitution, found
in the
Pachón cave, results in pigmented cells, although they are
slightly less
pigmented than the cells transfected with the surface-fish

sequence.
(h–j) Transfection of the construct encoding the proline-to-
serine
substitution results in highly pigmented cells. (k–p)
Transfection of the
construct with the extra intron 23 sequence and exon 24
deletion (k–m)
or the construct with the exon 21 deletion (n–p) does not rescue
pigmentation. b,e,h,k,n show bright-field pictures of the melan-
p cell line
transfected with different constructs; c,f,i,l,o show the
corresponding
transfected cells visualized by fluorescence (all constructs were
co-injected
with pUB-GFP26); and d,g,j,m,p provide schematics of the
constructs
transfected in each experiment.
110 VOLUME 38 [ NUMBER 1 [ JANUARY 2006 NATURE
GENETICS
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a ripple function. For the Molino backcross, 267 of 300 markers
were grouped
into 35 linkage groups using a LOD threshold of 4.0.
QTL mapping. We used the interval mapping function of
MapQTL26 to

determine the albino locus and determined significant LOD
scores using a
permutation test.
Cloning and mapping of albinism candidate genes. We obtained
fragments of
Astyanax Tyr, Trp1 and Oca2 by degenerate PCR. For primers
and specifics, see
Supplementary Methods. The fragment that we initially
amplified by degen-
erate PCR of Oca2 contained exons 11–23. We were able to
amplify exon 1 of
Oca2 from the DNA of surface fish by designing primers to the
area of exon 1
that is most conserved between zebrafish and fugu. Using a
forward primer in
exon 1, 5¢-GAGCCCAGGGTCATCAGG-3¢, we obtained the
sequence through
exon 11. To obtain the 5¢ and 3¢ ends, we used the SMART
Race cDNA
kit (Clontech).
Genomic analysis of missing Oca2 exons in Pachón and Molino
populations.
We carried out PCR to exons 21 and exons 14 for genomic DNA
from all
individuals of the Molino backcross using the following

primers: exon 21F,
exon 21R, exon 14F and exon 14R (see Supplementary Table 1
online for all
primer sequences). For the Pachón backcross, we amplified
intron 23 and exon
24 to the 3¢ untranslated region using the following primers:
intron 23F, intron
23R, exon 24F and 3¢ UTR.
Sequencing of full-length Oca2 coding region for Surface,
Molino and
Pachón fish. We extracted RNA from fin clips and whole fish
using Trizol
(Invitrogen), and we made cDNA using AMV reverse
transcriptase (Roche). To
amplify the full-length cDNA, we used primers from the 5¢ and
3¢ untranslated
regions of Oca2. In some cases, we amplified Oca2 in two
pieces for ease of
amplification, using a reverse primer in exon 12 and a forward
primer in exon
10. We compared Oca2 sequences from five surface fish, five
Molino fish and
two Pachón fish.
Making overexpression constructs. We cloned surface, Molino
and Pachón

full-length sequences into pGEM-T Easy (Promega). We cloned
the missing
exon 21 (Molino) and missing exon 24 plus extra sequence
(Pachón) cloned
into the surface-fish construct using MluI and SmaI. pUB-
GFP26, a vector that
encodes GFP under the control of the human ubiquitin promoter,
was used as
the backbone for the overexpression constructs. pUB-GFP was
blunted with
SmaI and MscI, removing the GFP, and pGEM-T Easy surface-
fish Oca2,
surface-fish Oca2 minus exon 21 and surface-fish Oca2 minus
exon 24 were
blunted with PvuII. The different Oca2 fragments and the pUB
fragment were
ligated together (Takara), putting surface-fish Oca2 under the
control of the
ubiquitin promoter. To make the point mutations found in the
Pachón cave, we
carried out site-directed mutagenesis using the QuikChange site
directed
mutagenesis kit (Stratagene) on the pUB-surface Oca2
construct.

Cell line assay. We obtained melan-p cells from D. Bennett’s
group and
cultured them as described14. We then transfected the cells on
3-cm plates
using Fugene (Roche) and 1 mg of each construct. The
transfections were as
follows: pUB-GFP alone26, pUB-GFP with pUB-surface Oca2,
pUB-GFP with
pUB-surface minus exon 21, pUB-GFP with pUB surface minus
exon 24 and
the proline-to-serine amino acid substitution, pUB-GFP with
pUB-surface
Oca2 with the methionine-to-valine substitution, and pUB-GFP
with pUB-
surface Oca2 with the proline-to-serine substitution. After
transfection, we
waited 4 d for pigmentation to develop.
Accession numbers. GenBank: A. mexicanus microsatellite
markers,
BV678703–BV678968; A. mexicanus Oca2, DQ232591.
Note: Supplementary information is available on the Nature
Genetics website.
ACKNOWLEDGMENTS
This work was supported by US National Science Foundation

grant IBN0217178
to R. Borowsky and C.J. Tabin. The authors thank L. Mekios for
phenotyping
and fish maintenance, the Bennett group for the use of the
melan-p cell line,
T. Matsuda for the pUB-GFP construct, R.C. Albertson for
critical reading of the
manuscript, and C. Peichel, R.V. Pearse II, J.L. Galloway and J.
Rivera-Feliciano
for help and advice.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial
interests.
Published online at http://www.nature.com/naturegenetics/
Reprints and permissions information is available online at
http://npg.nature.com/
reprintsandpermissions/
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Acknowledgments: We thank the anonymous reviewers for their
thoughtful and insightful critiques, which substantively
improved
this manuscript. Supported by the Singapore University of
Technology
and Design–Massachusetts Institute of Technology International
Design Center (IDG31300103) and by Natural Sciences and
Engineering Research Council (Discovery Grant 125517855).
Supplementary Materials
www.sciencemag.org/content/343/6177/1373/suppl/DC1
Materials and Methods

Figs. S1 to S4
Tables S1 and S2
References (26–70)
18 June 2013; accepted 31 January 2014
10.1126/science.1242063
Fossilized Nuclei and Chromosomes
Reveal 180 Million Years of
Genomic Stasis in Royal Ferns
Benjamin Bomfleur,1* Stephen McLoughlin,1* Vivi Vajda2
Rapidly permineralized fossils can provide exceptional insights
into the evolution of life over geological
time. Here, we present an exquisitely preserved, calcified stem
of a royal fern (Osmundaceae)
from Early Jurassic lahar deposits of Sweden in which
authigenic mineral precipitation from
hydrothermal brines occurred so rapidly that it preserved
cytoplasm, cytosol granules, nuclei, and even
chromosomes in various stages of cell division. Morphometric
parameters of interphase nuclei match
those of extant Osmundaceae, indicating that the genome size of
these reputed “living fossils” has
remained unchanged over at least 180 million years—a
paramount example of evolutionary stasis.
R
oyal ferns (Osmundaceae) are a basal
group of leptosporangiate ferns that have
undergone little morphological and an-
atomical change since Mesozoic times (1–6).
Well-preserved fossil plants from 220-million-
year-old rocks already exhibit the distinctive ar-

chitecture of the extant interrupted fern (Osmunda
claytoniana) (2), and many permineralized os-
mundaceous rhizomes from the Mesozoic are
practically indistinguishable from those of mod-
ern genera (3–5) or species (6). Furthermore, with
the exception of one natural polyploid hybrid
(7), all extant Osmundaceae have an invariant
and unusually low chromosome count (7, 8), sug-
gesting that the genome structure of these ferns
may have remained unchanged over long periods
of geologic time (8). To date, evidence for evo-
lutionary conservatism in fern genomes has been
exclusively based on studies of extant plants
(9, 10). Here, we present direct paleontological
evidence for long-term genomic stasis in this
family in the form of a calcified osmundaceous
rhizome from the Lower Jurassic of Sweden with
pristinely preserved cellular contents, including
nuclei and chromosomes.
The specimen was collected from mafic vol-
caniclastic rocks [informally named the “Djupadal
formation” (11)] at Korsaröd near Höör, Scania,
Sweden [fig. S1 of (12)]. Palynological analysis in-
dicates an Early Jurassic (Pliensbachian) age for
these deposits (11) (fig. S2), which agrees with
radiometric dates obtained from nearby volcanic
necks (13) from which the basaltic debris originated.
The fern rhizome was permineralized in vivo by
calcite from hydrothermal brines (11, 14) that per-
1Department of Palaeobiology, Swedish Museum of Natural
History, Post Office Box 50007, SE-104 05 Stockholm, Sweden.
2Department of Geology, Lund University, Sölvegatan 12,

SE-223 62 Lund, Sweden.
*Corresponding author. E-mail: [email protected]
nrm.se (B.B.); [email protected] (S.M.)
Fig. 1. Cytologicalfeaturespreservedintheapicalregion
of the Korsaröd fern fossil. (A) transverse section through
the rhizome; (B) detail of radial longitudinal section showing
typical pith-parenchyma cells with preserved cell membranes
(arrow), cytoplasm and cytosol particles, and interphase nuclei
with prominent nucleoli; (C) interphase nucleus with nucleolus
and intact nuclear membrane; (D) early prophase nucleus with
condensing chromatin and disintegrating nucleolus and
nuclear membrane; (E and F) late prophase cells with coiled
chromosomes and with nucleolus and nuclear membrane
completely disintegrated; (G and H) prometaphase cells
showing chromosomes aligning at the equator of the nucleus;
(I and J) possible anaphase cells showing chromosomes at-
tenuated toward opposite poles. (A), (C to E), (G), and (I)
are from NRM S069656. (B), (F), (H), and (J) are from NRM
S069658. Scale bars: (A) 500 mm; (B) 20 mm; (C to J) 5 mm.
21 MARCH 2014 VOL 343 SCIENCE www.sciencemag.org1376
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http://www.sciencemag.org/
colated through the coarse-grained sediments short-
ly after deposition (table S1). The fossil is 6 cm
long and 4 cm wide and consists of a small (~7 mm
diameter) central stem surrounded by a dense man-

tle ofpersistentfrondbaseswithinterspersed rootlets
(Fig. 1). Its complex reticulate vascular cylinder
(ectophloic dictyoxylic siphonostele), parenchym-
atous pith and inner cortex, and thick fibrous outer
cortex are characteristic features of Osmundaceae
(1, 3–5, 12) (fig. S3). Moreover, the frond bases
mantling the rhizome contain a heterogeneous scle-
renchyma ring that is typical of extant Osmunda
sensu lato (1, 3, 4, 12) (fig. S4). The presence of
a single root per leaf trace favors affinities with
(sub)genus Osmundastrum (1, 3, 6, 12).
The specimen is preserved in exquisite sub-
cellular detail (Fig. 1 and figs. S4 and S5). Pa-
renchyma cells in the pith and cortex show
preserved cell contents, including membrane-
bound cytoplasm, cytosol granules, and possible
amyloplasts (Fig. 1 and fig. S5). Most cells con-
tain interphase nuclei with conspicuous nucleoli
(Fig. 1, figs. S4 and S5, and movies S1 and S2).
Transverse and longitudinal sections through the
apical part of the stem also reveal sporadic dividing
parenchyma cells, mainly in the pith periphery
(Fig. 1). These are typically preserved in prophase
or telophase stages, in which the nucleolus and nu-
clear envelope are more or less unresolved and the
chromatin occurs in the form of diffuse, granular
material or as distinct chromatid strands. A few
cells contain chromosomes that are aligned at the
equator of the nucleus, indicative of early meta-
phase, and two cells were found to contain chromo-
somes that appear to be attenuated toward opposite
poles, representing possible anaphase stages.
Some tissue portions in the stem cortex and the
outer leaf bases show signs of necrosis and pro-

grammed cell death (fig. S6).
Such fine subcellular detail has rarely been
documented in fossils (15–17) because the chances
for fossilization of delicate organelles are small
(16) and their features are commonly ambiguous
(17). The consistent distribution and architec-
ture of the cellular contents in the Korsaröd fern
fossil resolved via light microscopy (Fig. 1 and
fig. S4), scanning electron microscopy (fig. S5),
and synchrotron radiation x-ray tomographic
microscopy (SRXTM) (fig. S5 and movies S1
and S2) provide unequivocal evidence for three-
dimensionally preserved organelles.
Positive scaling relationships rooted in DNA
content can be used to extrapolate relative ge-
nome sizes and ploidy levels of plants (18–21).
We measured minimum and maximum diame-
ters, perimeters, and maximum cross-sectional
areas of interphase nuclei in pith and cortical
parenchyma cells of the fossil and of its extant
relative Osmundastrum cinnamomeum. The mea-
surements match very closely (Fig. 2), with mean
nuclear perimeters of 32.2 versus 32.6 mm and
mean areas of 82.2 versus 84.9 mm2 in the fossil
and in extant Osmundastrum, respectively. The
equivalent nuclear sizes demonstrate that the
Korsaröd fern fossil and extant Osmundaceae
likely share the same chromosome count and DNA
content, and thus suggest that neither ploidization
events nor notable amounts of gene loss have
occurred in the genome of the royal ferns since
the Early Jurassic ~180 million years ago [(8),
see also discussion in (9, 10)]. These results, in

concert with morphological and anatomical evi-
dence (1–6), indicate that the Osmundaceae rep-
resents a notable example of evolutionary stasis
among plants.
References and Notes
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I. J. Leitch, J. Greilhuber, J. Doležel, J. F. Wendel, Eds.
(Springer-Verlag, Wien, 2013), vol. 2, pp. 307–322.
11. C. Augustsson, GFF 123, 23–28 (2001).
12. See supplementary materials available on Science Online.
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1383–1385 (1978).
16. K. J. Niklas, Am. J. Bot. 69, 325–334 (1982).
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18. A. E. DeMaggio, R. H. Wetmore, J. E. Hannaford,
D. E. Stetler, V. Raghavan, Bioscience 21, 313–316 (1971).
19. J. Masterson, Science 264, 421–424 (1994).
20. I. Símová, T. Herben, Proc. Biol. Sci. 279, 867–875 (2012).
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Acknowledgments: We thank E. M. Friis and S. Bengtson
(Stockholm) and F. Marone and M. Stampanoni (Villigen) for
assistance with SRXTM analyses at the Swiss Light Source,
Paul
Scherrer Institute (Villigen); G. Grimm (Stockholm) for
assistance
with statistical analyses; B. Bremer and G. Larsson (Stockholm)
for providing live material of Osmunda; M. A. Gandolfo Nixon
and J. L. Svitko (Ithaca, New York) for permission to use
images
from the Cornell University Plant Anatomy Collection (CUPAC;
http://cupac.bh.cornell.edu/); the members of Tjörnarps
Sockengille (Tjörnarp) for access to the fossil locality; A.-L.
Decombeix
(Montpellier), I. Bergelin (Lund), C. H. Haufler (Lawrence,
Kansas),
N. Tian (Shenyang), Y.-D. Wang (Nanjing), and T. E. Wood
(Flagstaff, Arizona) for discussion; and two anonymous referees
for
constructive comments. This research was jointly supported by
the
Swedish Research Council (VR), Lund University Carbon Cycle
Centre (LUCCI), and the Royal Swedish Academy of Sciences.
The
material is curated at the Swedish Museum of Natural History
(Stockholm, Sweden) under accession nos. S069649 to S069658

and S089687 to S089693.
Supplementary Materials
www.sciencemag.org/content/343/6177/1376/suppl/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S6
Table S1
References (22–36)
Movies S1 and S2
17 December 2013; accepted 21 February 2014
10.1126/science.1249884
Fig. 2. Morphometric
parameters of inter-
phase nuclei of extant
O. cinnamomeum com-
pared to those of the
Korsaröd fern fossil. Col-
ored box-and-whiskers plots
in upper graph indicate
interquartile ranges (box)
with mean (square), me-
dian (solid transverse bar),
and extrema (whiskers);
dashed colored lines in
lower graph indicate linear
fits (n = 76 versus n = 37
measured nuclei for extant
O. cinnamomeum versus
the fossil).
www.sciencemag.org SCIENCE VOL 343 21 MARCH 2014
1377

REPORTS
DOI: 10.1126/science.1249884
, 1376 (2014);343 Science
et al.Benjamin Bomfleur
Genomic Stasis in Royal Ferns
Fossilized Nuclei and Chromosomes Reveal 180 Million Years
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http://www.sciencemag.org/about/permissions.dtl
http://www.sciencemag.org/about/permissions.dtl
http://www.sciencemag.org/content/343/6177/1376.full.html
http://www.sciencemag.org/content/suppl/2014/03/19/343.6177.
1376.DC1.html
http://www.sciencemag.org/content/343/6177/1376.full.html#ref
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http://www.sciencemag.org/content/343/6177/1376.full.html#rel
ated-urls
http://www.sciencemag.org/cgi/collection/paleo

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