Genetica (2011) 139:199–207
DOI 10.1007/s10709-010-9537-x
Inactivation dates of the human and guinea pig vitamin C genes
Marc Y. Lachapelle • Guy Drouin
Received: 23 July 2010 / Accepted: 26 November 2010 / Published online: 8 December 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The capacity to biosynthesize ascorbic acid has
been lost in a number of species including primates, guinea
pigs, teleost fishes, bats, and birds. This inability results
from mutations in the GLO gene coding for L-gulono-
c-lactone oxidase, the enzyme responsible for catalyzing
the last step in the vitamin C biosynthetic pathway. We
analyzed available primate and rodent GLO gene sequen-
ces to determine their evolutionary history. We used a
method based on sequence comparisons of lineages with
and without functional GLO genes to calculate inactivation
dates of 61 and 14 MYA for the primate and guinea pig
genes, respectively. These estimates are consistent with
previous phylogeny-based estimates. An analysis of trans-
posable element distribution in the primate and rodent GLO
sequences did not reveal conclusive evidence that illegiti-
mate recombination between repeats has contributed to the
loss of exons in the primate and guinea pig genes.
Keywords Vitamin C  L-gulono-c-lactone oxidase 
GLO gene  Unitary pseudogene  Human  Guinea pig
Introduction
Ascorbic acid (vitamin C) plays an important role in a
diversity of physiological reactions, including collagen
Electronic supplementary material The online version of this
article (doi:10.1007/s10709-010-9537-x) contains supplementary
material, which is available to authorized users.
M. Y. Lachapelle  G. Drouin (&)
Département de biologie et Centre de recherche avancée en
génomique environnementale, Université d’Ottawa, 30 Marie
Curie, Ottawa, ON K1N 6N5, Canada
e-mail: gdrouin@science.uottawa.ca
synthesis, iron metabolism, and immunity (Padh 1990).
Given its importance, it is surprising that many species,
such as primates (Burns 1957; Pauling 1970), guinea pigs
(Zilva 1936), teleost fish (Dabrowski 1990), bats (Birney
et al. 1976), and passeriform birds (Chaudhuri and
Chatterjee 1969), have lost the capacity to synthesize it. In
these species, lack of adequate vitamin C supplementation
in their diet impairs many metabolic pathways and even-
tually leads to the development of scurvy. In humans and
guinea pigs, the inability to synthesize vitamin C is due to a
deficiency in L-gulono-c-lactone oxidase (GLO), the liver
enzyme responsible for catalyzing the last step of vitamin
C biosynthesis (Burns 1957). The molecular basis for this
deficiency was discovered in the early 1990s when it was
shown that these species possess highly mutated versions
of the GLO gene (Nishikimi et al. 1992, 1994). Having
undergone inactivating mutations sometime in their his-
tory, the human and guinea pig single copy GLO sequences
are therefore nonfunctional unitary pseudogenes (Graur
and Li 2000). The genetic basis for a lack of vitamin C in
other species is not yet known, but the observed cases of
GLO inactivity in primates and guinea pigs offer the pos-
sibility that this occurrence may be widespread.
Here we use data currently available from the various
genome sequencing projects to trace the molecular evolu-
tionary history of the GLO gene in species unable to bio-
synthesize vitamin C. To this end, we retrieved all
available GLO gene sequences and analyzed their substi-
tution record and transposable element distribution. Our
goals were to estimate dates of inactivation and to study
whether repetitive elements have had a role in inactivating
the GLO gene of these two species. Repetitive elements
have been shown to have a significant effect in mediating
chromosomal rearrangements (Szabó et al. 1999; Kolomi-
etz et al. 2002; Coghlan et al. 2005; Nakayama and Ishida
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200
2006; Uddin et al. 2006). Whether by illegitimate recom-
bination or through each element’s integration mechanism,
repeats are a potent source of genomic modification. Since
it was observed that the human and guinea pig GLO genes
lack some exons (Nishikimi et al. 1992, 1994), it could be
that illegitimate recombinations between repetitive ele-
ments were responsible for the inactivation of the human
and guinea pig GLO genes.
Materials and methods
Searches, sequences, and alignments
BLAST searches of the NCBI databases were done with
several different orthologues of the gene encoding L-gul-
ono-c-lactone oxidase. The sequences obtained and
accession numbers are provided in Table 1. A manual
alignment of the protein coding regions was done using
SequEdit version 0.912 (Drouin et al. 1999).
Inactivation date
Dating the time of inactivation of the GLO gene assumes a
molecular clock and is based on the observed pattern of
nucleotide substitution. Due to selective constraints, syn-
onymous nucleotide substitutions (S) usually accumulate at
a quicker rate than nonsynonymous (N) substitutions. This is
to be expected since nonsynonymous substitutions produce
changes in the translated amino acid sequence, which in
most cases is deleterious to the organism. The number of
synonymous substitutions per synonymous site (Ks) is thus
expected to be far greater than the number of nonsynony-
mous substitutions per nonsynonymous site (Ka), Ks  Ka.
A pseudogene, however, differs most prominently from its
functional counterpart at the level of its nonsynonymous
substitution rate. Whereas nonsynonymous substitutions are
Table 1 Accession numbers
Species (common name)
for sequences used in this study
Mus musculus (mouse)
Rattus norvegicus (rat)
Spermophilus tridecemlineatus
(squirrel)
Cavia porcellus (guinea pig)
Otolemur garnettii (galago)
Homo sapiens (human)
Pan troglodytes (chimpanzee)
Macaca mulatta (macaque)
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Genetica (2011) 139:199–207
negatively selected against within a functional sequence,
they are free to accumulate at a rate equal to the neutral
(synonymous) mutation rate (k) within a pseudogene (Graur
and Li 2000). Thus, for a pseudogene, we expect both kinds
of changes to be just as frequent, Ks = Ka. By comparing
the nucleotide substitutions between a pseudogene and its
functional orthologues, it is possible to approximate the time
when these rates became even (Echols et al. 2002).
To measure GLO genes inactivation dates, we used the
method described by Chou et al. (2002) to measure the
inactivation date of the human CMP-N-acetylneuraminic
acid hydroxylase gene. For a pseudogene, the total number
of nonsynonymous substitutions per site is equal to
kt1 ? fNk(t - t1). The first parameter is the number of
nonsynonymous substitutions per nonsynonymous site
since inactivation (t1) and the second is the number while
the gene was still functional (t - t1). Here t represents the
time when all species studied last shared a common
ancestor. The neutral mutation rate k is estimated as the
synonymous substitution rate, if we assume there are no
functional constraints against them. The fN value is the
fraction of neutral substitutions and is estimated as the ratio
Ka/Ks for the functional gene. Since Ks = Ka in a pseu-
dogene, fN becomes 1. This relationship is inversely pro-
portional to the gene’s level of functional constraint and
serves as an approximation of the selective pressure against
nonsynonymous substitutions.
The substitution record of the inactive GLO gene and its
orthologues is inferred from the observed nucleotide
changes by the maximum parsimony method. Individual
nucleotide substitutions are characterized as either synon-
ymous (S) or nonsynonymous (N) and preference is given
to possibilities requiring the least number of evolutionary
changes. When the order of substitution cannot be deter-
mined within a codon, the most likely change is calculated
from weighted averages, which assumes that all paths
leading to the observed mutation are equally likely.
Accession number(s) References
NM_178747 Li et al. 2006
NM_022220 Nishikimi et al.
1992
AAQQ01615738, AAQQ01184860, –
AAQQ01615740
D12762 Nishikimi et al.
1992
AAQR01573789, AAQR01573776, –
AAQR01573775,
AAQR01177479, AAQR01573774
AADC01074580, AADC01074579 –
AACZ02093370, AACZ02093372 –
AANU01121048, AANU01121047 –
Genetica (2011) 139:199–207 201

Synonymous substitution averages are represented by Sa

and nonsynonymous ones by Na. This analysis thus pro-
vides an inactivation date based entirely on the pattern of
nucleotide substitutions.

Repetitive element searches, alignments, and exon loss

The online program PlotRep (Toth et al. 2006) was used to

search for repetitive elements along the primate sequences.
The RepBase Update library of human and non-human
primate repetitive elements were used for these searches.
Areas with complete coverage from all four species
were aligned using ClustalW and analyzed for evidence of
illegitimate recombination (Thompson et al. 1994).

Results

Sequences

Blasting of NCBI databases with various orthologues of the

L-gulono-c-lactone oxidase gene resulted in a variety of
hits. This included numerous cartilaginous fish sequences
as well as at least one representative from most major
mammalian clades (results not shown). Of the species
unable to biosynthesize vitamin C, only the primate and
guinea pig sequences could be retrieved. More specific
searches aimed directly at teleost fish and bat genomic
databases were fruitless. A schematic representation of the
sequences used in this study is provided in Fig. 1. Only the
mouse, rat, human, chimp, and macaque sequences were
complete. Those of the squirrel and galago were obtained
from unfinished genome sequencing projects. The coding
regions of the guinea pig GLO gene were available from a
previous study (Nishikimi et al. 1992) and these were used
to infer the substitution record. Except for a few deletions,
including all of exon 5 and the 30 -end of exon 6, this
pseudogene sequence is complete. A full alignment of the
coding regions for all species is provided in Supplementary
Fig. 1.
A frameshift deletion was also observed at the 982nd bp
of the galago sequence. This deletion produces a premature
stop codon which would eliminate 53 amino acids from the
translated protein. However, previous work has demon-
strated that primates of the Lorisidae order, to which the
small-eared galago belongs, are able to biosynthesize
vitamin C (Pollock and Mullin 1987). Since the sequencing
project is still ongoing and we cannot be certain about the
actual state of the sequence, we simply inserted a gap to
complete the alignment and eliminated it from the analysis.
The squirrel sequence was assumed to be functional based
on the available body of literature (Drew et al. 1999).
Fig. 1 Schematic representations (not to scale) of the sequences used
in this study. Black boxes represent exons that are still present while
unfilled boxes with an X represent deleted exons or exon parts.
Dashed lines represent missing information
Inactivation date of the human GLO gene
A phylogenetic tree of the substitution record was con-
structed according to the maximum parsimony method
(Fig. 2). A total of 556 sites were analyzed, 435 of which
were nonsynonymous (lN) and 121 of which were synony-
mous (lS). Multiple substitutions within the same codon
were frequently observed, and when the order of these could
not be determined, weighted averages (half Na, half Sa)
were used to calculate the most probable change. Some sites
were ambiguous to classify (indicated by the symbol ‘‘?’’)
due to uncertainties in the ancestral sequence. At these
sites, the observed mutation could have resulted from two
equally parsimonious changes. Nucleotides from functional
sequences were preferentially considered as ancestral and,
when absolutely no informative support was available,
substitutions were evenly divided among the branches. The
inactivation date will therefore be calculated as a range, with
ambiguous sites either included or excluded from the
analysis.

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202 Genetica (2011) 139:199–207

234 C->T,S 378 C->A,N 1011 C->G,S ?301 G->C,N

235 A->G,N 387 G->A,S 1016 A->G,N ?330 C->T,S
?255 G->C,S 392 A->G,N 1047 G->A,S ?348 G->C,S
256 G->A,N *395 A->C,Na1 1081 A->C,N 406 G->T,N
279 T->C,S *396 C->T,Sa1 1091 T->A,N 858 G->A,S
280 G->A,N 401 T->C,N 1093 A->C,N 859 A->C,N
300 C->T,S 875 A->C,N *1208 G->C,Na1 865 A->G,N
323 A->G,N 889 T->C,N *1209 G->A,Sa1 887 C->G,N
*331 G->A,Na2 893 A->T,N 1213 G->A,N ?897 C->T,S
*332 C->T,Sa0 899 G->A,N 1228 T->C,N *970 G->A,Na2
338 C->T,N 919 G->C,N *1258 C->T,Na1.5 *971 C->G,Sa0
353 G->C,N 947 A->C,N *1260 G->A,Sa0.5 979 G->A,N
361 A->G,N 991 A->G,N 1269 G->A,S 987 C->G,N
374 C->T,N 1000 G->T,N 1295 C->T,N 1007 A->T,N
1027 A->G,N
1038 T->C,S
1039 G->A,N
1059 C->T,S
1095 G->C,N
1218 T->C,S
1288 C->T,S
1293 T->C,S
337 T->G,N 342 T->C,S ?1296 G->T,S
?348 G->T,S 375 A->G,N
383 C->T,N 414 G->C,N
404 T->C,N *872 G->A,Na1
410 C->G,N *873 C->T,Sa1
1010 C->T,N 892 G->A,N
1020 G->A,S ?897 C->G,N
1030 C->T,N 912 T->C,S
1034 G->A,N 916 C->T,N
1075 T->C,N *925 G->C,Na2
1207 C->T,N *926 C->T,Sa0
1272 C->A,N 948 G->A,S
1290 G->A,S 950 A->G,N
1300 C->T,S 964 C->A,N
990 C->T,S
1008 C->A,N
1031 G->A,N
1033 G->A,N
1045 C->A,N
1067 G->A,N
*1201 T->C,Na2
333 G->A,N 281 A->G,N *1202 G->T,Sa1
375 A->G,N 912 T->C,S *1203 T->C
973 A->G,N 1018 G->C,N 1212 G->C,N
990 C->T,S 1229 A->T,N 1217 T->C,N
1066 C->T,N 1314 C->T,S 1290 G->T,S
1297 T->C,N
1305 G->T,N

Human Chimp Macaque Galago

S = 16.5 S = 17.5
N = 45.5 N = 44.5
Fig. 2 Substitutions inferred in different primate lineages according
to the maximum parsimony method. A total of 556 sites common to
the human, chimpanzee, macaque, and galago were analyzed and the
corresponding rodent sequences were used as outgroups. The
observed substitution for each nucleotide is indicated along with an
S for a synonymous change or an N for a nonsynonymous change.
Asterisks indicate the presence of multiple substitutions within a
S = 17.5 S = 9
N = 52.5 N = 13
codon for which the order of substitution could not be determined
unambiguously and weighted averages were used to calculate the
most likely mutation type (Na, Sa). Question marks indicate sites for
which the ancestral sequence was not readily deducible and these
were evenly divided among the branches. The totals for each
substitution type are indicated for each species

The neutral mutation rate k was estimated from the and old calibration estimates) between the galago and other
average number of synonymous substitutions per site primates (Steiper and Young 2006). This gives a value
counted along each lineage and using a divergence date t of of k = (((16.5 ? 17.5 ?17.5 ? 9)/4)/121)/77.5 9 106) =
77.5 MYA (and a range of 67.1–97.7 MYA based on young 1.61 9 10-9 per site per year. This value is similar to the

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Genetica (2011) 139:199–207
neutral nucleotide substitution rate of 1.5 9 10-9 per site
per year calculated by Cooper (1999; cited in Winter et al.
2001) and by Chou et al. (2002). Calculating the fN value
from the observed mutations along the galago lineage gives
fN = (Ka/Ks) = (13/435)/(9/121) = 0.40. Combining these
values with the average number of nonsynonymous substi-
tutions per site incurred by the non-functional human,
chimpanzee, and macaque genes (Ka = ((45.5 ? 44.5
?52.5)/3)/435 = 0.109), allows us to calculate an inacti-
vation date of t1 = [(Ka/k) - fNt]/(1 - fN) = 61 MYA, and
a range of 48–68 MYA, when all sites are included. The
calculations with the six questionable sites excluded from
the analysis give k = 1.43 9 10-9, fN = 0.64, an inactiva-
tion date of 74 MYA and a range of 38–92 MYA.
Inactivation date of the guinea pig GLO gene
The phylogenetic tree of the inferred guinea pig substitution
record is provided in Fig. 3. Calculations were carried out in
the same manner as for the primate lineage with the
exception that the fN value was obtained from an average of
the functional mouse and rat genes. A total of 730 sites were
analyzed, 575 of which were nonsynonymous and 155 were
synonymous. We used a divergence date of 72 MYA, with a
confidence interval of 63.5–80 MYA, between the guinea
pig and the common ancestor of the mouse and rat as
t (Huchon et al. 2007). The estimates with all sites included
give k = 3.55 9 10-9, fN = 0.066 and Ka = 0.0626 for an
inactivation date of 13.8 MYA, with a confidence interval of
13.2–14.4 MYA. Excluding the five questionable sites from
the analysis gives k = 3.41 9 10-9, fN = 0.069 for a inac-
tivation date of 14.4 MYA, with a confidence interval of
13.8–15.0 MYA.
Exon loss and repetitive elements in the human
and guinea pig GLO genes
Analyses of the repetitive element distribution in the GLO
gene sequences were carried out to see if illegitimate
recombination between repeated sequences could have
contributed to exon loss. A variety of elements were found in
primate sequences, including the Alu and MIRb SINEs as
well as several types of LINEs (e.g., MLT1B; results not
shown). These were present at a frequency of about 1 per
1,000 bp for all lineages. Although much of the galago
sequence is still unavailable in GenBank (due to an unfin-
ished sequencing project), two regions of its GLO gene have
been sequenced and are suitable for comparison. These two
regions span exons 1 to 5 and exons 8 to 12. All sequences for
these areas were aligned and annotated for various repetitive
elements using PlotRep (Toth et al. 2006).
Figure 4a is a representation of the region upstream of
exon 4 of the galago and human sequences. The
203
chromosomal break that resulted in the deletion of exons 2
and 3 in the last common ancestor of humans, chimps, and
macaques can be traced to a region about 90 bp upstream
of a mammalian interspersed repetitive element b (MIRb)
where only the 30 end of the element is present, starting at
the 123rd bp of a full length MIRb. Since one would expect
that the full size element would straddle the observed
breakpoint, this suggests that MIRb elements were not
involved in the recombination event that led to the loss of
exons 2 and 3. The area between exons 8 and 12 was highly
fragmented and several lineage-specific insertions made it
difficult to propose any mechanism that may have led to the
loss of exons 8 and 11 (results not shown). Our analyses of
the repetitive elements found in the GLO genes of primates
therefore did not provide any conclusive evidence for their
involvement in exon loss for this gene.
Analyses of the repetitive elements found in the mouse,
rat and guinea pig GLO genes revealed that they contain
12, 11 and 1 repetitive elements, respectively (results not
shown). The single repetitive element found in the guinea
pig GLO gene is a PB1D10 repetitive element and is found
in its fourth intron. Since the single PB1D10 repetitive
element found in the rat GLO gene is in its second intron
and that the two PB1D10 repetitive elements found in the
mouse GLO gene are in its second and third intron (results
not shown), this element could not have been involved in
the loss of the fifth exon and part of the sixth exon of the
guinea pig GLO gene.
Discussion
With the ever increasing number of genomic sequencing
projects being undertaken, a total of eight sequences could
be retrieved for this study. At the time, fifteen other primate
sequencing projects have either been announced or initi-
ated (GOLD database v.2.0, http://www.genomesonline.
org/). Furthermore, of the other species unable to synthe-
size vitamin C, several teleost fish and three bat projects
are underway. As more data become available, we will
gain a clearer picture of the molecular evolution of the
L-gulono-c-lactone oxidase gene.
The inability of teleost fish, such as the rainbow trout,
the Japanese medaka, and the common carp, to biosyn-
thesize vitamin C has been well documented (Dabrowski
1990, 1994; Toyohara et al. 1996; Krasnov et al. 1998;
Moreau and Dabrowski 2000; Hwang and Lin 2002). Since
several ancestral actinopterygian fish species have been
observed to retain this ability and that all teleost fish tested
so far cannot synthesize vitamin C, the inactivating muta-
tion must have occurred shortly after the divergence of
these two lineages, some 200 to 210 MYA (Dabrowski
1994; Moreau and Dabrowski 2000). Given this long
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204 Genetica (2011) 139:199–207

?255 G->A,S 918 G->A,S 238 G->A,N 998 T->C,N

261 G->A,S 966 G->A,S 243 G->T,N 1011 C->T,S
264 T->C,S 981 A->G,S 252 A->G,S 1016 A->G,N
267 C->T,S 1032 G->A,S 262 G->A,N 1038 T->C,S
423 A->C,S 1092 C->T,S 273 C->G,S 1058 G->C,N
429 A->C,S 1140 A->G,S 282 C->G,N *1090 A->T,Na2
447 G->A,S 1167 C->T,S *289 C->T,Na1 *1091 T->G,Sa0
451 A->G,N 1170 G->A,S *291 A->G,Sa1 1095 G->A,N
465 C->T,S 1188 C->A,S 306 T->C,S 1107 T->C,S
471 C->A,S 1200 C->T,S 449 C->T,N 1116 T->C,S
477 C->T,S 1233 T->C,S 459 A->G,S 1124 G->A,N
498 G->A,S 1241 C->A,N 462 T->C,S 1128 G->T,S
?606 C->T,S 1251 C->T,S 475 A->C,N 1129 G->A,N
791 T->A,N 1253 C->A,N 478 G->C,N 1141 G->A,N
798 G->A,S 1263 A->G,S ?495 T->A,S 1145 A->G,N
819 T->C,S 1288 C->T,S 496 G->A,N *1168 G->C,Na2
860 A->G,N 1319 G->A,S 516 C->A,N *1169 G->A,Sa0
?897 C->T,S 805 G->A,N 1173 C->A,S
810 C->T,S 1176 G->A,S
820 C->T,N 1182 C->T,S
*862 A->T,Na1 1207 A->T,N
*864 C->T,Sa1 1231 C->T,N
865 A->G,N 1244 A->C,N
868 C->T,N 1248 C->T,S
887 C->G,N 1252 G->A,N
252 A->C,S 300 C->T,S 891 C->T,S 1270 G->A,N
277 G->A,N 301 A->G,N 912 T->C,S 1274 C->T,N
*437 C->A,Na1 *449 C->A,Na1.5 *970 G->A,Na2 1290 G->A,S
*438 A->G,Sa1 *450 C->A,Sa0.5 *971 C->G,Sa0 ?1296 G->C,S
*448 G->A,Na1 474 C->A,N 984 C->T,S 1308 G->A,S
*450 C->A,Sa1 513 C->T,S
474 C->T,S 786 C->G,N
499 C->A,S 835 C->A,N
799 T->C,N 873 C->T,S
886 A->T,N 915 C->A,S
1035 G->T,S 933 C->T,S
1056 C->G,S 957 G->A,S
1110 C->G,S 993 G->A,S
1126 C->T,S 1014 G->A,S
1131 C->T,S 1035 G->C,S
1260 T->C,S 1044 C->T,S
1305 G->A,S 1110 C->A,S
1311 G->T,S 1116 T->C,S
1128 G->A,S
1173 C->A,S
1176 G->A,S
1207 A->C,S
1222 A->G,N
1234 G->A,N
1308 G->A,S
1311 G->C,S

Mouse Rat Guinea Pig

S = 43 S = 48.5 S = 24
N = 10 N = 12.5 N = 36
Fig. 3 Substitutions inferred in different rodent lineages according to squirrel and primate sequences were used as outgroups. Symbols used
the maximum parsimony method. A total of 730 sites common to the are the same as for Fig. 2
mouse, rat, and guinea pig were analyzed and the corresponding

timescale, it is quite understandable that no GLO ortho-
logues could be retrieved from the available teleost gen-
omes. It is likely that the non-functional gene has acquired
so many mutations that it is now unrecognizable or that it
has been excised from the genome altogether. Furthermore,
gene transfer experiments attempting to restore vitamin C
biosynthesis in rainbow trout by adding the rat GLO gene
proved ineffective (Krasnov et al. 1998). While cDNA
transcripts were indeed observed in embryos, no GLO
protein or activity were observed in adults. This suggests
that the metabolic dysfunctions for vitamin C synthesis in
teleost fish may not solely depend on GLO activity.
Early estimates for the inactivation of the L-gulono-
c-lactone oxidase gene in primates placed the date between

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Genetica (2011) 139:199–207
Fig. 4 a Repetitive element A
distribution in the area upstream
of exon 4 of the galago and
Galago
human sequences. Diagonal
MLT1B
lines indicate the chromosomal
breakpoint area that led to the
loss of exons 2 and 3 in the last
common ancestor of human,
chimps, and macaques.
b Sequence alignment
highlighting the deleted region.
Homology with the MIRb Human
element begins only at the 123rd
bp of this element. Hu human,
Ch chimpanzee, Ma macaque,
Ga galago
B
35 and 55 MYA (Nishikimi et al. 1994; Challem and
Taylor 1998). These were based primarily on the postulated
date of divergence between Anthropoidea (Simiiformes;
new and old world monkeys, tarsiers) and prosimians
(Strepsirrhini; lemurs, galagos and lorises). Since the latter
retain the ability to endogenously synthesize vitamin C and
the former do not, it was thought that the inactivation must
have occurred sometime since their last common ancestor.
However, recent findings place this divergence a little
further up the evolutionary tree at about 77.5 MYA
(Murphy et al. 2004; Steiper and Young 2006). In light of
these new estimates, our calculated dates of between 61
and 74 MYA is consistent with this gene having been
inactivated soon after the divergence of Anthropoidea from
prosimians. While there is still some debate as to the
phylogenetic relationship of the Tarsiiformes lineage to
other primates, the fact that tarsiers also lack the ability to
synthesize vitamin C suggest that they are more closely
related to anthropoids than to prosimians (Pollock and
Mullin 1987). The recent study of Zhang et al. (2010), who
also used the Chou et al. (2002) method to calculate
inactivation dates, found the inactivation date of the
205
2 3 4
MIRb MIRb MIRb Alu
4
MLT1B MIRb MIRb
1000 bp
primate GLO gene to be between 30.5 and 42.9 MYA, rather
than between 61 and 74 MYA as we did. This is probably
due to the fact (as they point out themselves) that they used a
smaller number of sequences to calculate this date.
For the guinea pig, the calculated range of GLO inac-
tivation lies between 13 and 15 MYA, a date consistent
with the work of Nishikimi et al. (1992) who proposed a
date of less than 20 MYA. Limitations for both sets of
calculations reside predominantly in the amount of avail-
able data. In our case, only four primate and four rodent
sequences were used for the analysis. This limits the reli-
ability of our estimated neutral mutation rates and fN val-
ues, even if these seemed to be within acceptable ranges. A
larger number of sequences from related species would
have provided us with a more accurate picture. Further-
more, only between 42 and 55% of the total length of the
GLO gene protein coding regions are currently available
(Fig. 1). The completion of the numerous genome
sequencing projects should also allow further studies to use
complete sequences.
Another difficulty caused by this lack of sequences
concerns the sites which were difficult to characterize.
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206
Since an ancestral sequence could not be determined
unambiguously, the true direction of nucleotide change
could not be concluded. In some instances, one might
choose to simply eliminate these sites from the analysis.
However, because we were estimating substitution rates,
these could not simply be disregarded since a mutation had
evidently occurred. The bottom end of the inactivation
ranges were calculated with the ambiguous sites included.
Although ambiguous, the rates obtained when these are
considered do involve all observable substitutions.
Our results show that the inactivation dating method we
used gives the same results as a phylogenetic bracketing
approach when a particular gene has been lost in several
species and that the phylogeny (and divergence times) of
these species is known. On the other hand, this method is
useful when the phylogeny (and divergence times) of these
species is not known, such as in the case of the guinea pig
gene where a gene has been lost in a single lineage.
The analysis of repetitive element distribution in the
primate and rodent sequences did not reveal conclusive
evidence that illegitimate recombination between repeated
sequences were involved in the loss of exons in the
anthropoid and guinea pig GLO genes (Fig. 4 and results
not shown). However, since the exon/intron structure is
conserved between the human, chimp, and macaque, we
must infer that the exon deletions found in this gene were
already fixed in their last common ancestor some 30 MYA
(Steiper and Young 2006). The mechanisms which led to
the deletion of exons 1, 2, 3, 5, 6, 8 and 11 must therefore
have occurred prior to that. Such a long time span is suf-
ficient to have rendered any footprint or chromosomal
breakpoint unrecognizable. It is of interest, however, that
these deletions have occurred earlier rather than later.
Many types of repetitive elements are thought to have been
intensely active in the early part of the mammalian and
primate radiation (Pascale et al. 1990; Rowold and Herrera
2000; Zhang et al. 2003; Pace and Feschotte 2007). This
coincides with the fact that the common anthropoid GLO
gene has not sustained any gross modifications since at
least 30 MYA. A quick examination of the eight Alu
sequences present in the allele revealed that seven belon-
ged to the ancestral subfamilies AluJ and AluSx. These are
thought to have been active 60 and 44 MYA, respectively
(Price et al. 2004). Interestingly, it has been observed that
the bat genus Myotis has had a recent bout of DNA
transposon activity (Ray et al. 2007). As bat GLO gene
sequences become available, the nucleotide substitution
pattern will tell us whether vitamin C biosynthetic capacity
was lost during the same time frame.
The effects a nonfunctional GLO gene may have had on
anthropoid and guinea pig evolution is considerable. On
account of the various roles for vitamin C in metabolism, it
is surprising that the inability to synthesize it appears as
123
Genetica (2011) 139:199–207
often as it does. These species must consume a sufficient
amount only to survive, so how could there be any beneficial
aspects to this loss? It has been hypothesized that since
vitamin C acts primarily as an antioxidant, a GLO gene
inactivation should theoretically bring about elevated levels
of free radicals. Due to the mutagenic nature of these, the
likelihood of genetic mutations would increase and essen-
tially propel the evolution of these species (Challem 1997).
Another hypothesis suggests that formation of ascorbic acid
may have negative impacts on the liver. Synthesis would
involve the formation of deleterious hydrogen peroxide
(H2O2) and the depletion of glutathione stores. Therefore,
for species with an ample supply of vitamin C, the selective
pressure to preserve the ability of biosynthesis is lost
(Bánhegyi et al. 1996). Alternatively, as pointed out
repeatedly in the literature, loosing the capacity to synthe-
size vitamin C might simply not be disadvantageous to
species having a vitamin C-rich diet (e.g., Chatterjee 1973;
Birney et al. 1976; Nishikimi et al. 1992). The fact that the
inability to synthesize vitamin C in both humans and guinea
pigs is due to the loss of the same (GLO) gene is likely a
consequence of the fact that the enzymatic function of the
GLO protein is the last step in vitamin C production.
Whereas loosing this gene only affects the production of
vitamin C, loosing genes for the other enzymes of this
synthesis pathway would affect the production of many
other molecules (Linster and Van Schaftingen 2007).
As more sequence information becomes available,
molecular dating studies such as this one will become more
accurate. Postulating dates for the inactivation of genes can
provide important information in regards to when major
biochemical shifts occurred and their relation in the
broader evolutionary picture. These sequences will also
allow us to determine whether the inactivation of the same
(GLO) gene in humans and guinea pigs is due to chance or
is a feature of all species unable to synthesize vitamin C.
Acknowledgments We thank the two anonymous referees for their
useful and constructive comments on a previous version of this man-
uscript. This work was supported by a Discovery Grant from the Nat-
ural Science and Engineering Research Council of Canada to G. D.
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