Article

Time and time again: Unisexual salamanders (genus Ambystoma) are the oldest unisexual vertebrates

Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.
BMC Evolutionary Biology (Impact Factor: 3.37). 08/2010; 10(1):238. DOI: 10.1186/1471-2148-10-238
Source: PubMed

ABSTRACT

The age of unisexual salamanders of the genus Ambystoma is contentious. Recent and ancient evolutionary histories of unisexual Ambystoma were proposed by a few separate studies that constructed phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other studies showing that unisexual Ambystoma represent the most ancient unisexual vertebrates, a recent study by Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago.
We re-examined the phylogenetic relationship of the unisexuals to A. barbouri from various populations using both mitochondrial markers as well as the complete mitochondrial genomes of A. barbouri and a unisexual individual from Kentucky. Lineage dating was conducted using BEAST and MultiDivTime on a complete mitochondrial genome phylogeny. Our results support a monophyletic lineage for unisexual Ambystoma that shares its most recent common ancestor with an A. barbouri lineage from western Kentucky. In contrast to the Robertson et al.'s study, no A. barbouri individual shared an identical or almost identical cytochrome b haplotype with any unisexual. Molecular dating supports an early Pliocene origin for the unisexual linage (approximately 5 million years ago). We propose that a unisexual-like cytochrome b numt (or pseudogene) exists in the controversial A. barbouri individuals from Kentucky, which was likely the cause of an erroneous phylogeny and time estimate in Robertson et al.'s study.
We reject a recent origin of unisexual Ambystoma and provide strong evidence that unisexual Ambystoma are the most ancient unisexual vertebrates known to exist. The likely presence of an ancient cytochrome b numt in some Kentucky A. barbouri represents a molecular "fossil" reinforcing the hypothesis that these individuals are some of the closest extant relatives to unisexual Ambystoma.

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RESEARC H ARTIC LE Open Access
Time and time again: unisexual salamanders
(genus Ambystoma) are the oldest unisexual
vertebrates
Ke Bi
1,2
, James P Bogart
1*
Abstract
Background: The age of unisexual salam anders of the genus Ambystoma is contentious. Recent and ancient
evolutionary histories of unisexual Ambystoma were proposed by a few separate studies that constructed
phylogenies using mitochondrial DNA markers (cytochrome b gene vs. non-coding region). In contrast to other
studies showing that unisexual Ambystoma represent the most ancient unisexual vertebrates, a recent study by
Robertson et al. suggests that this lineage has a very recent origin of less than 25,000 years ago.
Results: We re-examined the phylogenetic relationship of the unisexuals to A. barbouri from various populations
using both mitochondrial markers as well as the complete mitochondrial genomes of A. barbouri and a unisexual
individual from Kentucky. Lineage dating was conducted using BEAST and MultiDivTime on a complete
mitochondrial genome phylogeny. Our results support a monophyletic lineage for unisexual Ambystoma that
shares its most recent common ancestor with an A. barbouri lineage from western Kentucky. In contrast to the
Robertson et al.s study, no A. barbouri individu al shared an identical or almost identical cytochrome b haplotype
with any unisexual. Molecular dating supports an early Pliocene origin for the unisexual linage (~5 million years
ago). We propose that a unisexual-like cytochrome b numt (or pseudogene) exists in the controversial A. barbouri
individuals from Kentucky, which was likely the cause of an erroneous phylogeny and time estimate in Robertson
et al.s study.
Conclusion: We reject a recent origin of unisexual Ambystoma and provide strong evidence that unisexual
Ambystoma are the most ancient unisexual vertebrates known to exist. The likely presence of an ancient
cytochrome b numt in some Kentucky A. barbouri represents a molecular fossil reinforcing the hypothesis that
these in dividuals are some of the closest extant relatives to unisexual Ambystoma.
Background
Because of the absence of sex, a brief evolutionary life-
span is generally expected for unisexual and asexual
organisms [1]. Nevertheless, ancient unisexuals and
asexuals that pe rsist millions of years have been di scov-
ered in various taxa among plants, fungi and animals
[2]. With recent advances in molecular genetics and
phylogenetics, our knowledge of reproductive systems
and evolutionary histories of many unisexual and asex-
ual lineages has been quickly improved. Recent evidence
reveals that many unisexuals are capable of utilizing
modified reproduct ive modes and/or mitotic or meiotic
mechanisms to incorporate abitofsex [3-7], or they
can incorporate additional genetic material to c ompen-
sate for the suspected lethal effects caused by the accu-
mulation of deleterious mutations [7-10]. Thus,
although populations may be all female, unisexual are
not necessarily equivalent to asexual populations [5].
The discovery of ancient unisexual and asexual lineages
not only poses a dilemma for evol utionary theoreticians
but also p rovides an opportunity to address questions
that relat e to the prevalence and maintenance of sexual
reproduction.
Unisexuality in vertebrates has been discovered in
about 90 lineages of fresh water fish, amphibians and
reptiles [11], most of which are recently spun off from
* Correspondence: jbogart@uoguelph.ca
1
Department of Integrative Biology, University of Guelph, Guelph, Ontario,
N1G 2W1 Canada
Full list of author information is available at the end of the article
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Page 1
sexual relatives via interspecific hybridization [7,12].
North American unisexual mole salamanders of the
genus Ambystoma co-evolve with five distinct sexual
ambystomatids (A. laterale, A. jeffersonianum, A. tigri-
num, A. texanum,andA. barbouri) across the entire
unisexual d istribution [9,13-15]. Unisexual Ambystoma
persist as a parasitic entity by steal ing and incorporat-
ing sperm from sympatric sexual species via a complex
reproductive mode, kleptogenesis, to generate nearly 30
genomic combinations or biotypes, with ploidy levels
ranging from diploid to pentaploid [5,15]. Genomes in
unisexuals may not be transmitted unaltered. Recent
studies using genomic in situ hybridization (GISH)
demonstrate complex intergenomic exchanges in unisex-
ual populations [4,16-18].
Despite the comp lexity of their nuclear genomes, all
unisexuals contain a highly conserved mitochondrial
genome which is derived from A. barbouri, suggesting
that A. barbouri may b e the maternal ancestor of the
unisexual lineage [5,9,19]. Although the ancestry of uni-
sexual Ambystoma is not contested by these studies, the
age of the unisexual lineage is controversial. Unisexual
Ambystoma have been proposed as the most ancient
unisexual vertebrates known to exist [5,20,21]. Based on
a phylogeny constructed by the mitochondrial intergeni c
spacer and control region (mitochondrial non-coding
region or NCR), Bogart et al. [5] estimated that the uni-
sexual lineage and the closest relatives, an A. barbouri
lineage from Kentucky, are descended from the most
recent commo n ances tor 2.4-3.9 million years ago (Ma).
Their results are based on the observati on of a 3.91%
pairwise difference in the control region between uni-
sex uals and a few Kentucky A. barbouri individuals. On
the contrary, Robertson et al. [19] constructed a phylo-
geny using a different mitochondrial marker cytochrome
b (cyt-b) gen e and suggested that unisexual Ambystoma
have a very recent origin which could be less than
25,000 years ago. Strikingly, they found a few A. bar-
bouri specimensthatwerealsousedbyBogartetal.[5]
to have an identical or almost identical cyt-b haplotype
to unisexual individuals. Such a large discrepancy is dif-
ficult to understand because the two different mitochon-
drial DNA markers (NCR vs. cyt-b) that came from the
same A. barbouri individuals demonstrated distinctly
different evolutionary relationships to the unisexual line-
age (~3.91% vs. ~0%). Therefore, the question: are uni-
sexual Ambystoma ancient or recent? is unresolved.
To provide a clearer answer to this question, we r e-
examined the phylogenetic relationship of unisexuals
and A. barbouri from various populations using both
mitochondrial cyt-b gene and NCR as markers. We
especially focused on A. barbouri samples (JPB34337,
JPB34342, JPB34343, JPB34356) that demonstrated dis-
tinctly different sequence divergences to the unisexuals
in the two studies [5,19]. Given that the rate of substitu-
tion likely varies for different genes/regions in the mito-
chondrial genome, we compared the substitution rates
of complete mitochondrial genomes (mtgenomes) of
unisexual and A. barbouri. We sequenced the mtge-
nome of a Kentucky A. barbouri specimen (JPB34342)
that was used in both studies and provided conflicting
results. We chose to sequence the mtgenome of one tet-
raploid unisexual A. laterale -3jeffersonianum (or LJJJ)
from a northern Kentucky population as a representative
unisexual. This individual was chosen because it was
found to h ave the most common mitochondrial NCR
haplotype among unisexuals (haplotype B in [5]), it was
collected in a state (Kentucky) where unisexuals were
previously unknown, and it was geographically close to
the A. barbouri indiv iduals that were deem ed to be the
closest r elatives to unisexual Ambystoma. For compari-
son, we also sequenced mtgenomes of o ne A. barbo uri
and one A. texanum from Ohio to examine their phylo-
genetic relationships to the Kentucky unisexua l and
A. barbouri individuals. The time to the most recent
common ancestor (TMRCA) for unisexual lineage and
its closest rel ative was re-calculated based on the mtge-
nome phylogeny.
Results
Mitochondrial cyt-b and NCR trees
Primers Glu14100L, MLM651 and M2R (abbreviated as
GMM) were used to amplify and sequence a ~ 2200
base pairs (bp) mitochondrial fragment that included
cyt-b and N CR. A total of 46 cyt-b sequences, including
one from an outgroup species (Ambystoma laterale)
downloaded from GenBank, were compared (Table 1).
Of the 1141 bp resolved, 228 sites were variable and 143
sites were phylogenetically informative. A total of 42053
most parsimonious trees resulted from the parsimony
analysis with 335 steps, a consistency index (CI) of
0.758 a nd a retention index (RI) of 0.939. For NCR, 46
sequences including one A. laterale from GenBank were
recovered (Table 1). Of the 1065 bp resolved, 215 sites
were variable and 111 sites were phylogenetically infor-
mative. A to tal of 90 most parsim onious trees were
obtained from the parsimony analysis with 310 steps, a
CI of 0.768 and a RI of 0.944. For both markers, a GTR
+I+G model was selected as the best-fit model. The cyt-
b strict consensus tree (not shown) was nearly identical
to the Bayesian tree, but NCR strict consensus (not
shown) and Baye sian trees yielded slightly different
topologies. The Bayesian trees with the BPP and BSP
from the parsimony analyses are presented in Figure 1.
Both cyt-b and NCR tre es clustered all specimens into
four clades (Figure 1). All the unisexuals, irrespective of
their b iotypes and ploidy levels, formed a monophyletic
clade A. Clade A was sister to clade B that contained
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Table 1 Specimens used for phylogenetic construction by mitochondrial cyt-b and NCR in this study with GenBank
accession numbers
Clade Vouchers (JPB) Species or biotype* Locality GenBank No.
cyt-b, NCR
A 11312 LTT Pelee, ONT Essex Co. GU078473, GU078514
30066 LLJ NJ Sussex Co. GU078475, GU078516
31283 LLJ PA McKean Co. GU078476, GU078517
32232 LLJ NY Niagara Co. GU078477, GU078518
37103 LLJ MI Cass Co. GU078507, GU078548
37107 LLLJ MI Cass Co. GU078508, GU078549
37128 LLJ MI Cass Co. GU078509, GU078550
37816 LLJ QUE Mirabel Co. GU078510, GU078551
39932 LJJJ KY Kenton Co. GU078472
B 32518 A. barbouri KY Anderson Co. GU078478, GU078519
32519 A. barbouri KY Anderson Co. GU078479, GU078520
32521 A. barbouri KY Anderson Co. GU078480, GU078521
34326 A. barbouri KY Oldham Co. GU078481, GU078522
34337 A. barbouri KY Jessamine Co. GU078488, GU078529
34339 A. barbouri KY Jessamine Co. GU078489, GU078530
34341 A. barbouri KY Oldham Co. GU078490, GU078531
34342 A. barbouri KY Oldham Co. GU078469
34343 A. barbouri KY Oldham Co. GU078491, GU078532
34344 A. barbouri KY Oldham Co. GU078492, GU078533
34355 A. barbouri KY Mercer Co. GU078496, GU078537
34356 A. barbouri KY Mercer Co. GU078497, GU078538
34357 A. barbouri KY Mercer Co. GU078498, GU078539
34365 A. barbouri KY Jessamine Co. GU078502, GU078543
C 22765 A. barbouri OH Montgomery Co. GU078474, GU078515
34327 A. barbouri KY Franklin Co. GU078482, GU078523
34328 A. barbouri KY Franklin Co. GU078483, GU078524
34331 A. barbouri KY Fayette Co. GU078484, GU078525
34332 A. barbouri KY Fayette Co. GU078485, GU078526
34333 A. barbouri KY Fayette Co. GU078486, GU078527
34334 A. barbouri KY Jessamine Co. GU078487, GU078528
39346 A. barbouri OH Warren Co. GU078512, GU078553
34348 A. barbouri OH Warren Co. GU078493, GU078534
39349 A. barbouri OH Warren Co. GU078513, GU078554
34350 A. barbouri OH Warren Co. GU078494, GU078535
34359 A. barbouri KY Franklin Co. GU078499, GU078540
34360 A. barbouri KY Franklin Co. GU078500, GU078541
34364 A. barbouri KY Jessamine Co. GU078501, GU078542
34366 A. barbouri KY Jessamine Co. GU078503, GU078544
34368 A. barbouri KY Livingstone Co. GU078504, GU078545
34369 A. barbouri KY Livingstone Co. GU078505, GU078546
37710 A. barbouri OH Hamilton Co. GU078470
38876 A. barbouri OH Butler Co. GU078511, GU078552
D 34353 A. barbouri TN Rutherford Co. GU078495, GU078536
34553 A. texanum OH Clarke Co. GU078506, GU078547
37892 A. texanum OH Montgomery Co. GU078471
Out-group - A. laterale - NC_006330
Voucher numbers in bold represent specimens whose mtgenomes were sequenced.
* All unisexuals have at least one A. laterale [L] nuclear genome. The unisexuals used in the present study also have A. jeffersonianum [J] or A. texa num [T]
nuclear genomes.
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A. barbouri individuals from southern Ohio and west of
the Kentucky Rivers in central Kentucky. This clade cor-
responded to the A. barbouri western clade in Niedz-
wieckis study [22]. Clade C contained A. barbouri from
north or east of the Kentucky River and north of the
Ohio River, as well as the disjunct populations in wes-
tern Kentucky. This clade corresponded to the north-
ern clade that was identified by Niedzwiecki [22]. Clade
DincludedA. texanum and A. barbouri from Tennes-
see. In the NCR tree, the relationship of clade D in
respect to three other clades was, however, not well
resolved but the monophyly of clade A+ clade B was
strongly supported (BSP = 99; BPP = 100).
In Robertson et al. s st udy [19], four specimens
(JPB34337, JPB34342, JPB34343, JPB34356) of A. bar-
bouri were found to be clustered in the unisexual clade
and three of them (JPB34337, JPB34342, JPB34343)
shared the same cyt-b haplotype with some of the uni-
sex uals (Figure 1A). We sequenced the cyt-b gene from
the very same individuals but we found that none of
them shared an identical or almost identical haplotype
to any unisexua l. We examined the sequence chromato-
gram of cyt-b in these individua ls and only single signal
peaks were observed at every site. All four samples were
grouped in clade B with other A. barbouri where the
average cyt-b sequence divergence between A. barbouri
and unisexuals was 5.16% (Figure 1B). Likewise, no
A. barbouri shared the same or even a very simila r NCR
sequence with unisexuals (Figure 1C), and the ingroup
relationship in the NCR tree was concordant wi th that
obtained by Bogart et al. [5].
Amplification of cyt-b gene by MVZ15 and MVZ16
Bogart et al. [5] used primers MLMTHR and MLM651 to
obtain a ~1100 bp intergenic spacer and control region
from unisexual and A. barbouri specimens. We com-
pared our sequencing results using the same primers and
confirmed that MLMTHR and MLM651 produced the
identical sequences to those by using pri mers GMM. Pri-
mers MVZ15 and MVZ16 were used by Robertson et al.
[19] to amplify an ~800 bp cyt-b fragment from ambysto-
matids. In their study, A. barbouri individuals JPB34337,
JPB34342, JPB34343 and JPB34356 were found to contain
an identical or almost identical haplotype to unisexuals
which placed them in the unisexual clade. Using the
same primers, we failed to duplicate the same sequencing
results in JPB34342 and JPB34343 as discovered by
Robertson et al. [19]. Cyt-b failed to amplify in JPB34337
when using MVZ15 and MVZ16 but primers GMM con-
fir med that JPB34337 did not contain a unisex ual hapl o-
type. The results showed t hat these three individu als
shared A. barbouri cyt-b haplotypes that were grouped
with other A. barbouri in clade B ( Figure 1B). We found,
however, that JPB34356 did have a unisexual-like cyt-b
haplotype a s was found by Robertson et al. [19] using
MVZ15 and MVZ16 as PCR and sequencing primers.
This contradictory result was unexpected. We further
examined the sequenc e chromatogram of JPB34356 and
Figure 1 Bayesian gene trees of mitochondrial cytochrome b (cyt-b) gene from Robertson et al. [19][A] and the present study [B], and
Bayesian gene tree of non-coding region (NCR) by the present study [C]. The tree arrangement from Robertson et al. [19] has been slightly
modified. Left numbers along branches represent the Bayesian posterior probabilities (BPP) and right numbers represent bootstrap proportions
(BSP) derived from the parsimony analysis. The detailed information of specimens that were sequenced is listed in Table 1. *A unisexual-like cyt-
b numt likely exists in A. barbouri specimens JPB34337, JPB34342, JPB34343 and JPB34356 [A] so they were grouped within the unisexual clade.
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found that its sequence contained many heterozygous
sites (multiple signal peaks) in positions where the vari-
able sites between unisexuals and clade B A. barbouri
were located (Figure 2). At th ese heterozygous sites,
A. barbouri s sequence signals were generally weaker
than unisexual-like sequence signals so the latter would
be read by the program by default (Figur e 2). We ruled
out the possibility of DNA cross contamination and
mitochondrial heteroplasmy in JPB34356 because, using
primers GMM which targeted a much longer fragment
for PCR amplificatio n and sequencing, we consistently
found a pure A. barbouri haplotype from JP B34356. In
the sequence chromatogram, unisexual-like sequence sig-
nals were no longer detectable (Figure 2A). We used var-
ious primer combinations to amplify mitochondrial genes
of JPB34342 using a new DNA extraction in the present
study as well as an old DNA extraction used by Robert-
son et al. [19]. None of the amplicons showed any signs
of DNA contamination or mitochondrial heteroplasmy.
Additionally, t hrough sequencing the mtgenome of
A. barbouri JPB34342, unisexual J PB39932 and other
spe cimens we showed that cyt-b gene did not have any
duplicated component residing in the mitochondrial gen-
ome. A plausible explanation was that JPB34356 contained
a nuclear copy (or copies) of a unisexual-like mitochon-
drial cyt-b gene fragment (numt or pseudogene). When
using whole genomic DNA as template, both the actual
mitochondrial cyt-b gene and its nuclear numt could have
been co-amplified by MVZ15 and MVZ16. To further test
this prediction we used the mitochondrial DNA fragment
as a template and conducted sequencing PCR using
MVZ15 and MVZ16 as pri mers. The results showed that
no heterozyg ous sites existed in the sequence chromato-
gram (Figure 2A) and JPB34356 did not contain a unisex-
ual-like cyt-b haplotype in its mitochondrial genome.
Another primer combination using MVZ15 and MLM651
(targeted a ~2200 bp fragment too) was also used to
amplify cyt-b using JPB34356 whole genomic DNA as
template and no unisexual-like signals were detected in
that sequence chromatogram.
Mtgenome phylogeny
A total of 17 mtgenome sequences, including 13 down-
loaded f rom GenBank, were used for p hylogenetic con-
struction(Table2).ThecombinedDNAdataset
contained 9647 bp with 3464 variable sites and 2361
were phylogenetically informative. Parsimony analysis
generated a sing le tree 8803 steps in length (CI = 0.569,
RI = 0 .614). For Bayesian analysis, a GTR+I+G model
was selected as the best-fit model. The tree topologies
derived from parsimony and Bayesian analyses were
similar, with slight differences in the r elationships of
Figure 2 Cyt-b fragment chromatograms showing the presence of unisexual-like cyt-b numt in Ambystoma bar bouri individual
JPB34356. [A] Amplification and sequencing of cyt-b from JPB34356 using primers GMM, or using MVZ15/16 with mitochondrial DNA as
template. [B] Amplification and sequencing of cyt-b from JPB34356 using primers MVZ15/16 with whole genomic DNA as template. [C]
Amplification and sequencing of cyt-b in unisexual individual JPB30066, either using primers GMM or MVZ15/16 with whole genomic DNA as
template. Arrows point to four sites that differ between A. barbouri and unisexual Ambystoma.
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some outgroup species, and we present the Bayesian
tree in Figure 3. The monophy ly of Kentucky A. bar-
bouri JPB34342 and unisexual JPB39932 was strongly
supported by both analyses (BSP = 100; BPP = 100).
The overall sequence divergence of mtgenome
between the unisexual sample JPB39932 and A. barbouri
JPB34342 was 4.42% (Table 3). We divided the mtge-
nome into 18 partitions and the pairwise difference of
each partition ranged from 2.27% (concaten ated tRNAs)
to 7.03% (intergenic spacer). All 13 protein-coding genes
were rather variable between genomes of JPB39932 and
JPB34342. Except for ND4L (3.03%) and ATP8 (3.57%),
the p airwise differences of all other genes were no less
than 4.50%. JPB34342 was found to have an identical
cyt-b haplotype to unisexuals by Robertson et al. [19].
Our study cle arly demonstrated that neither the cyt-b
gene nor any other genes, tRNAs or NCR throughout
the mitochondrial genome were the same between
A. barbouri JPB34342 and the unisexual.
Molecular dating
The r elaxed lognormal clock analysis of the mtgenome
sequences by BEAST produced the s ame topology as
the Bayesian analysis. The divergence times estimated
by BEAST and MultiDivTime are listed in Table 4. In
general, the time estimates by BEAST and MultiDiv-
Time were largely congruent. We present a time-cali-
brated tree from BEAST in Figure 4. T he split between
unisexual Ambystoma and Kentucky A. barbouri (Node
A) took place about 5.3 Ma (CI 2. 4, 8.7) by BEAST, and
5.1 Ma (CI 2.7, 9.0) by MultiDivTime. Both analyses
agreed that the o rigin of the unisexual linage may date
back to early Pliocene.
Discussion
No Ambystoma barbouri shares an identical mitochondrial
cyt-b gene with unisexuals
The phylogenies generated using cyt-b, NCR as well as
mtgenome all support the hypothesis that unisexual
Ambystoma form a monophyletic g roup and share the
most recent common ancestor with an A. b arbouri line-
age from Kentucky [5,9,19]. The age of unisexual
Ambystoma wa s in high di sagreement b ecause four
A. barbouri individuals (JPB34337, JPB3434 2, JPB34343,
JPB34356) examined by two previous studies demon-
strated distinctly different phylogenetic relationships to
the unisexual lineage. Robertson et al. [19] found that
these four A. barbouri were grouped with the unisexual
cyt-b clade that suggested a very recent origin of unisex-
ual lineage which was less than 25,000 years ago. On the
other hand, Bogart et al. [5] found a 3.91% sequence
pairwise distance in the control region between unisex-
uals and a few Kentucky A. barbouri individuals (includ-
ing JPB34342, JPB34343, JPB34356) and claimed an
ancient evolutionary history of unisexual Ambystoma,
approximately 2.4-3.9 Ma. When using primers targeting
a long mitochondrial fragment (~2200 bp), w e found
that no A. barbouri, including the four controversial
A. barbouri individuals used by Robertson et al. [19],
shared the same cyt-b gene sequence with unisexuals.
The average cyt-b sequence pairwise distance between
unisexuals (clade A) and their closest A. barbouri
Table 2 Species and unisexual biotype LJJJ used for mtgenome phylogeny and molecular dating analyses in this study
with their GenBank accession numbers
Species Vouchers GenBank Accession No. References
Ambystoma barbouri JPB34342 GU078469 This study
Ambystoma barbouri JPB37710 GU078470 This study
Ambystoma texanum JPB37892 GU078471 This study
Unisexual LJJJ JPB39932 GU078472 This study
Ambystoma californiense - NC_006890 [43]
Ambystoma laterale - NC_006330 [44]
Ambystoma tigrinum - NC_006887 [43]
Cryptobranchus alleganiensis - GQ368662 [45]
Cynops cyanurus - EU880309 [46]
Dicamptodon atterimus - GQ368657 [45]
Euproctus platycephalus - EU880317 [46]
Hynobius amjiensis - NC_008076 [47]
Notophthalmus viridescens - EU880323 [46]
Paramesotriton laoensis - EU880328 [46]
Taricha rivularis - EU880334 [46]
Triturus cristatus - EU880336 [46]
Xenopus tropicalis - NC_006839 JGI direct submission
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relatives (clade B) is as high as 5.16%. The examination
of mtgenome of the representative specimens further
demonstrated that neither t he cyt-b gene, nor other
genes/regions in the mitochondrial genome were identi-
cal or almost identical between unisexuals and
A. barbouri. The overall substitution rates throughout
the mitochondrial genome, especially in protein coding
genes, were found to be rather consistent. Through a
thorough sampling of A. barbouri across its distribution,
Niedzwiecki (personal communication) did not find any
Figure 3 A close phylogenetic relations hip of unisexual Ambystoma (JPB399 32) and Kentucky A. barbouri (JPB34342) inferred from
mtgenome phylogeny. Other species are incorporated to introduce calibration points for molecular dating analyses, and to serve as outgroups.
Left numbers along branches represent the Bayesian posterior probabilities (BPP) and right numbers represent bootstrap proportions (BSP)
derived from the parsimony analysis. BPP and BSP below 50 are not shown. Branch lengths are estimated by the Bayesian inference.
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haplotype that was more similar to the un isexua ls than
western clade Kentucky A. barbouri including specimens
JPB34337, JPB34342, JPB34343 and JPB34356. There-
fore, all our evidence clearly demonstrates that there is
no known A. barbouri that shares an identical cyt-b
gene with unisexuals.
Molecular dating demonstrates an ancient origin of
unisexual Ambystoma
In our BEAST analysis, the rate covariance among adja-
cent branches was 0.13 which was close to zero and
their 95% confident intervals spanned zero (CI -0.20,
0.46). This result suggests that rate aut ocorrelation is
insignificant [23] and BEAST (without rate-autocorrela-
tion assumptio n), rather than MultiDivTime (with rate-
autocorrelation assumption), would be more appropriate
for the data and calibration choices in our study.
Nevertheless, our results show that the time estimations
between BEAST and MultiDivTime are largely congru-
ent, especially within the ambystomatid salamanders
(Table 4). Both analyses show that unisexual Ambystoma
and Kentucky A. barbouri descended from their most
recent common ancestor about 5 Ma so this dichotomy
likely occurred in early Pliocene. More conservatively, if
we take the lowest value at 95% CI inferred by both
analyses, the unisexual lineage originated at least 2 Ma
(early Pleistocene). Generally, our estimate was consis-
tent with previo us time estimates of the o rigin of uni-
sexual Ambystoma [5,20,21] which validates them as the
most ancient unisexual vertebrate lineage known to
exist [2].
Niedzwiecki [22] suggested that the Kentucky and
Ohio Rivers likely act as barriers between western and
northern A. barbouri mtDNA clades (corresponding
respectively, to clades B and C in our study, Figures 1B
&1C). The Kentucky River might have been respons ible
for the divergence between A. barbouri from A. texa-
num as well as for the diver gence of geographically dis-
tinct A. barbouri mtDNA lineages. Using a rough
calibration approach (1.5% per million years for the A.
tigrinum complex) [24], Niedzwiecki [22] estimated that
various A. barbouri mitochondrial lineages were sepa-
ratedaround3.5-5Maandasimilartimewouldhave
elapsed since the split between the main A. barbouri
clades and A. texanum. Our study suggests that both
divergence times are possibly more ancient than these
estimates. In the BEAST analysis, for example, the
separation between K entucky A. barbouri (repr esented
by clade B, Figures 1B &1C) and Ohio A. ba rbouri
(represented by clade C, Figures 1B &1C) likely took
place 8.1 Ma (CI 4.4, 12.4). The split of western and
northern A. barbouri from A. texanum likely occurred
10.4 Ma (CI 6.0, 15.4). Our results indicate that both
events may have taken place in la te to middle Miocene
or at least in early Pliocene, suggesting a longer evolu-
tionary history for A. barbouri than expected.
Is a unisexual-like cyt-b numt present in A. barbouri?
Nuclear copies of mitochondrial genes or numts,which
evolve independently as paralogous copies of the origi-
nal mitochondrial DNA segment, are relatively common
and have been reported in various ta xa [25-30]. Failure
to discriminate real mi tochondrial sequences and numts
often confuses the genetic diversity of species and
produces erroneous phylogenies [31]. Because the
nuclear genome has an overa ll slower mutation rate
than the mitochondrial genome, estimates on divergence
times can be problematic [28,32]. Numts can originate
from all parts of the mitochondrial genome including
the cyt-b gene [33] and the majority of numts are fairly
short sequences [34].
Table 3 Sequence pairwise divergence of 18 genes/
regions of mtgenome between the unisexual sample
JPB39932 and A. barbouri JPB34342
Partitions Sequence pairwise distances
Overall 4.42%
12s 2.60%
16s 2.70%
ND1 5.68%
ND2 5.56%
COI 5.43%
COII 5.10%
ATP8 3.57%
ATP6 4.53%
COIII 4.85%
ND3 5.98%
ND4L 3.03%
ND4 5.26%
ND5 4.62%
ND6 5.61%
CYT-B 5.17%
Intergenic spacer 7.03%
Control region 3.64%
tRNAs 2.27%
Table 4 Divergence time means and 95% confidence
intervals calculated by BEAST and MultiDivTime*
Nodes BEAST MultiDivTime
A: 5.3 (2.4, 8.7) 5.1 (2.7, 9.0)
B: 8.1 (4.4, 12.4) 7.5 (3.9, 13.1)
C: 10.4 (6.0, 15.4) 9.2 (4.9, 15.9)
D: 20.2 (12.4, 28.8) 21.0 (12.0, 32.8)
E: 8.6 (4.7, 12.8) 12.5 (6.5, 21.5)
F: 23.6 (15.1, 33.1) 23.5 (13.7, 36.0)
*Time uni t is Ma (million years ago). The detailed description for each node
can be found in Figure 4.
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When using whole genomic DNA as template and pri-
mers MVZ15 and MVZ16 for PCR amplification and
sequencing [19], A. barbouri individual JPB34356 pos-
sessed two different PCR products. One of the two was
not detectable in the sequence chromatograms when
using mitochondrial DNA as template with MVZ15/
MVZ16 as sequencing primers, or using total genomic
DNA as template with o ther various primer combina-
tions. DNA cross contamination can yield sequence het-
erozygosity but contamination should have affected each
gene/region across the entire mtgenome. We tested the
old DNA extractions of A. barbouri individuals
(JPB34337, JPB34342 , JPB34343, JPB34356) u sed by
Robertson et al. [19], and we did not find any signs of
DNA contamination in any other genes/regions. Mito-
chondrial heteroplasmy may also be responsible for
mixed m itochondrial amplicons [35,36]. The possibility
that these A. barbouri individuals embrace A. barbouri
and complete unisexual-like mtgenomes is minimal as
proven by the experiments we conducted above. Mito-
chondrial heteroplasmy could also be caused by regional
mutations or duplications of some mitoch ondri al copies
[37,38]. If these A. barbouri individuals have two types
of mtgenomes which are heteroplasmic only in the cyt-b
gene, we should have d etected the heterozygous signals
in the cyt-b gene when using old/new total genomic
DNA extractions or mitochondrial DNA as templates
with any primer combinations for PCR and sequencing.
The most reasonable explanation is that A. barbouri
individual JPB34356 contains a unisexual-like cyt-b
numt. This unexpected discovery in JPB34356 has
resolved the mystery and provides a reasonable explana-
tion for the data that were obtained by Robertson et al.
[19]. We b elieve that a unisexual-like cyt-b numt was
present in some A. barbouri individuals, including
JPB34337, JPB34342, JPB34343, and JPB34356. It is ver y
possible, in their study, that the cyt-b sequences recov-
ered from these four A. barbouri were in fact unisexual-
like numts. Therefore, the subsequent ph ylogenet ic con-
struc tion and divergence time estimates that were based
on these putative numts were erroneous. Although we
did not detect stop codons in these numts,itisknown
Figure 4 A time-calibrated ph ylogeny inferring the origin of unisexual Ambystoma, fitted to a geological tim escale. Times for nodes
[A-F] are estimated by BEAST. The horizontal gray bars through these nodes indicate 95% credibility intervals from the BEAST analyses. Non-
ambystomatid specimens are not shown in the tree. Detailed time estimates can be found in Table 4. An early Pliocene origin for unisexual
Ambystoma is proposed.
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that numts do not always include stop codons or indels
and sometimes are indistinguishable to their mitochon-
drial orthologs [35]. It is possible, however, that the stop
codons were situated up/downstream beyond the region
amplified by the MVZ15/MVZ16 primers that we used.
Whether numts can be amplified may also depend on
the quality and quantity of nuclear DNA template in the
PCR reaction. In our study we failed to find or amplify
thesamesequencesinthreeother controversial speci-
mens JPB34337, JPB34342 a nd JPB34343, which likely
resultedfromthemorehighlydegradednucleargeno-
mic DNA from these samples.
Implications of having an ancestral cyt-b numt in
A. barbouri
Dating the origin of unisexual/asexual lineages is usually
based on the extent of genetic divergence from their
nearest sexual relatives [7,39]. In general, time estimates
are more robust if both mitochondrial and nuclear
genomic markers are used as inferences. Uncoupling
evolutionary trajectories between their mitochondrial
and nuclear genomes, however, make molecular dating
of unisexual Ambystoma depend solely on the mito-
chondrial genome. All unisexual Ambystoma share one
mitochondrial origin but the evo lutionary histories of
their nuclear genomes are much more complicated and
dynamic. It is clear that no re current hybridization
between a ny of the sexual sperm donors produces new
unisexual lineages [5,9] otherwise unisexual individuals
would contain a mitochondrial genome derived from
one or more of the extant sexual, sperm - donating spe-
cies of Ambystoma.Bogartetal.[15]showedthatA.
barbouri can serve as a sperm donor for unisexuals in a
single pond from southern Ohio. The cyt-b and NCR
haplotypes recovered from those A. barbouri were
found to be grouped in clade C (Figures 1B &1C), and
are distantly related to the unisexuals. Without recur-
rent hybridization, unisexual individuals have evolved an
extremely flexible reproductive mode by which they rely
on sexual sperm donors to perpetuate. Unisexuals
replace their nuclear genomes with those from sexual
sperm donors, historically and contempo rarily, through-
out their entire distribution [40]. Consequently, it is
impossible to use nuclear genome sequences for mole-
cular dating of unisexual Ambystoma.
Little and Hebert [41] have criticized the traditional
dating strategy used for unisexual and asexual organisms
[39] because dating, not only requires a thorough search
of extant taxa, but also related species that may have
gone extinct since unisexuality/asexuality originated.
Neiman et al. [42] reviewed asexual lineage longevity
among invertebrate and vertebrate taxa and criticized
the notion that there existed young and ancient asex-
ual lineages. They found that the distribution of asexual
lineages followed a re gular and linear age distributi on.
The tabulated ages of asexual vertebrate lineages (fish,
amphibians, and reptiles) were all less than 300,000
years old and could even be considered young compared
with the estimated age of a sexual species. The unisex-
ual Ambystoma lineage was included in their table of
asex ual lineag es with an age <25,000 years based on the
incorrect time estimate provided by Ro bertson et al.
[19]. Our present study demonstrates that unisexual
Ambystom a have persisted for about 5 million years and
we attribute this longevity to the fact that they are uni-
sexual, not asexual, and have a unique reproductive
mode (kleptogenesis)[5].
Because A. barbouri may have an ancient evolutionary
history [[22], this study], it is reasonable to assume that
there may have existed a closer common ancestor to the
unisexuals which became extinct at some unknown tim e
[5]. Finding a unisexual-like cyt-b numt in some A. bar-
bouri individuals suggests that its origination must have
taken place at about the same time as the hybridization
event that gave rise to unisexuals. Once a numt is estab-
lished in its descendants nuclear DNA, it would be
much more conserved than the mitochondrial haplotype
because the mitochondrial cyt-b mutates f aster than its
numt. The pre sence of an ancestral cyt-b numt in the
western Kentucky clade of A. barbouri would also sug-
gest that no other population of A. barbouri,including
any potential extinct lineages, needs to be considered
more closely related to the unisexuals.
Conclusions
Using multiple sets of empirical evidence and rigorous
statistical methodologies, we reject the conclusion of a
recent origin of unisexual Ambystoma and support the
hypothesis that unisexual Ambystoma is the most
ancient lineage in unisexual vertebrates known to exist.
The amplification of unisexual-like cyt-b sequence in a
few Kentucky A. barbouri individualsisthepossible
cause of the erroneous phylogeny and time estimate
determined by Robertson et al. [19]. The unisexual-like
cyt-b sequence, presumably a numt, in a few Kentucky
A. barbouri could represent a u seful molecular fossil
showing that the A. ba rbouri lineage from Kentucky
represents the closest relatives to the unisexuals. Inter-
estingly, our study shows that the unisexuals mitochon-
drial genomes, which are descended from ancestral A.
barbouri, seem to have changed little over time because
their cyt-b gene s are very similar to the ~800 bp ances-
tral cyt-b numt amplified sequence identified in some
extant A. barbouri individuals. Low mitoc hondrial DNA
variation among present unisexual popula tions have
been described before [5,20] and it might be attributed
to population bottlenecking of unisexuals prior to their
rapid expansion after the Last Glacial Maximum. The
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length, number of copies and distribution of the cyt-b
numt in A. barbouri is not yet known and there may b e
other A. barbouri individuals that have similar cyt-b
numts and/or other ancient signatures shared with uni-
sexuals. The confirmation of an ancient ancestry of uni-
sexual Ambystoma validates them as an excellent model
system for studying the evolution and maintenance of
unisexuality in vertebrates.
Methods
Samples
Ambystoma barbouri from various populations from
Kentucky, Ohio and Tennessee were examined. Because
unisexuals were found to share a highly conserved mito-
chondrial genome in previous studies [5,9,19,20], we
only sequenced a few unisexual individuals from several
geographically distant populations to represent the uni-
sexual lineage. The four specimens that were used for
sequencing of their mtgenomes were: A. barbouri from
Kentucky (JPB34342), unisexual LJJJ from Kentucky
(JPB39932), A. barbouri from Ohio (JPB37710), and
A. texanum from Ohio (JPB37892). To introduce cali-
bration points into the molecular dating analyses,
mtgenome sequences of 12 salamander species were
retrieved from GenBank [43-47]: A. californiense,
A. laterale, A. tigrinum, Cryptobranchus alleganiensis,
Cynops cyanurus, Dicamptodon atterimus, Euproctus
platycephalus, Hynobius amjiensis, Notophthalmus viri-
descens, Paramesotriton laoensis, Taricha rivularis,and
Triturus cristatus.Afrog,Xenopus tropicalis, was used
as an outgroup. Its mtgenome was also downloaded
from GenBank. Detailed sampling and sequen ce infor-
mation is listed in Tables 1 and 2.
Laboratory protocols
Total genomic DNA was extracted f rom frozen larvae,
adult muscle, heart or liver tissues using a standard phe-
nol-chloroform extraction method. We also tested some
DNA extractions used by previous studies [5,19]. A
combination of 22 primers [45] was used to amplify
contiguous a nd overlapping fragments that cov ered the
entire mitochondrial genome. PCR was conducted in a
25 μl mix including 30-50 ng of template DNA, 1 U
Taq DNA polymerase (TaKaRa), PCR buffer,
1.5 mM MgCl
2
, 0.2 mM of each dNTP, and 10 pmo l of
each primer. PCR cycling parameters were 95°C for
5 min as initial denaturation, followed by 30 cycles of
95°C for 30 sec, 45-50°C [45] for 45 sec, 72°C for 30 sec
to 2 min depending on the expected size of fragments
(approx. 1 min/kb), and a final step at 72°C for 5 min.
PCR products were verified on 1% agarose gels and pur-
ified using QIAquick PCR purification kits (Qiagen).
Sequencing was performed with the corresponding PCR
primers using BigDye 3.1 terminator sequencing
chemistry with an ABI 3730 (Applied Biosystems).
Sequences were assa yed using Sequenche r (version 4.5;
Gene Codes). For some large PCR fragments that were
more than 1600 bp, specific internal primers were
designed to obtain their complete sequences.
Primers Glu141 00L [45] and MLM651 [24] were used
to amplify a ~2200 bp mi tochondrial fragment that
included cyt-b, intergen ic spacer and control region. An
internal primer M2R (reverse primer, 5 -GTTGGTGG
TTTCTCGCCCTAAG-3) was de signed for sequencing
across the complete fragment. As a comparison, we also
used the same primers MVZ15 and MVZ16 [48] that
were used to amplify cyt-b genes by Robertson et al.
[19] to examine some of the A. barbouri individuals,
especi ally those that had an identical or almost identical
cyt-b haplotype to unisexuals. These two primers tar-
geted an ~800 bp partial cyt-b gene fragment [19]. Like-
wise, we used the same primers MLMTHR and
MLM651 [24] that were used by Bogart et al. [5] to
amplify NCRs from some specimens and the expected
size of PCR products was approximately 1100 bp [5].
The PCR annealing t emperature was set to 50°C for
MVZ15 and MVZ16, and was set to 46°C for MLMTHR
and ML651. The protocols of standard PCR reaction,
purification, sequencing and sequence alignment were
the same (above).
Phylogenetic methods
Three phylogenies were constructed using mtgenome,
cyt-b, and NCR, respectively. When using mtgenomes
for phylogen etic construction, w e excluded all the
tRNAs and non-coding regions and only included two
rRNAs and 13 protein-coding genes. This was necessar y
because of the absence of several tRNAs and the lack of
the intergenic spacer in mtgenomes of some outgroup
species as well as the difficulty in sequence alignment of
control regions. With the 13 protein-coding genes, the
third codon positions were eliminated because of high
substitution rates. Multiple su bsti tutions likely produce
noise in phylogeneti c and dating analyses [45]. Ambigu-
ousalignmentsinthetworRNAregionswerealso
excluded. Additionally, all the gaps in the alignments
were eliminated manually. Finally, a DNA dataset con-
taining all 17 DNA alignments (each with two rRNAs
and 13 protein-coding genes without third codon posi-
tions) was generated.
A maximum parsimony analy sis was conducted using
PAUP* (version 4.01b10) [49]. Each sequence w as trea-
ted as a taxon and each nucleotide was treated as a
character. All characters were weighted equally and
unordered. A heuristic search method via tree-bisection-
reconnection (TBR) branch swapping was used. Boot-
strap proportions (BSP) [50] with 1 ,000 replicates were
used to evaluate the nodal support. A Bayesian analysis
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was conducted using MrBayes (version 3.1) [51]. Model
selection was based on the Akaike information criterion
(AIC) as implemented in MrModeltest (version 2.2)
[52]. The best-fit model was used in subsequent Baye-
sian phylogenetic analysis. Four Markov chains were
used and the dataset was run for 10, 000,000 generations
to allow adequate time for convergence. Trees were
sampled e very 500 generations, and the last 5,000 trees
were used to estimate the consensus tree and the Baye-
sian posterior probabilities (BPP). The overall sequence
pairwise diverge nce between mtgenom e of Kentucky
A. barbouri and the unisexual was calculated using
MEGA (version 4.0) [53]. The mtgenomes were then
divided into 18 partit ions according to genes and
regions (one concatenated tRNAs, two rRNAs, two non-
coding regions and 13 protein-coding genes) and
sequence pairwise difference of each partition between
Kentucky A. barbouri and u nisexual respectively, was
calculated. Pairwise differences of cyt-b and NCR
between the major lineages of inter est were also calcu-
lated by MEGA.
Molecular dating
To infer the TMRCA for the unisexual lineage and its
closest relative, Kentucky A. barbouri,weincorporated
13 previously published mtgenome sequences to allow
the calibration points to be introduced to our analysis.
A com bined DNA dataset including two rRNAs and 13
protein-coding genes without thir d codon positions
were used for dating analyses. Gaps in the DNA align-
ment were manually excluded. The ingroup root of the
tree (Salamandroidea and Cryptobranchoidea) was con-
strained to be 151 to 170 Ma. This constraint was based
on the oldest salamander fossil known to exist, the sa la-
mandroid-like Iridotriton hechti (151 Ma) [54] and a
proposed maximal bound for the origin o f Caudata (170
Ma) [55]. The Taricha-Notophthalmus split was con-
strained to be at least 23 Ma (Taricha oligocenica from
the upper Oligocene) [56]. The Ambystoma-Dicampto-
don split was set to at least 55.8 Ma (Dicamptodon anti-
quus fromthelatePaleocene)[57];TheEuproctus
-Triturus split was set to 20-30 Ma (disjunction of the
Corsica-Sardinia microplate from the Iberian Peninsula)
[46]; The Cynops-Paramesotriton split was constrained
to be greater than 15 Ma (Procynops miocenicus from
the upper Miocene) [56]. The split of A. tigrinum-
A. californiense was s et to be around 5 Ma (the begin-
ning of Sierran uplift) [24]. Because most of the fossil
record we used in this study only provided a minimum
age for the origin, when setting the priors we used the
fossil ages as the lower bounds.
Molecular dating was conducted by a Bayesian
MCMC approach in the program BEAST (version 1.5.1)
[58] and MultiDivTime [59]. Using BEAST, the topology
and divergence t imes can be estimated simultaneously
from the data. BEAST input files we re generated with
BEAUTi (version 1.5.1) . A GTR+I+G wa s used to
describe the substitution model, a Yule process was
used to describe speciation, and an uncorrelated lognor-
mal (UCLN) model was used to describe the relaxed
clock [23]. We used a lognormal distribution for fossil
calibrations, and a normal distribution for the biogeo-
graphic calibrations. BEAST was run for 80,000,000 gen-
erations with samples taken every 1,000 genera tions.
Five independent MCMC runs were conducted and the
log and time tree files were combined using LogCombi-
ner (version 1.5.1). The results were examined by Tracer
(version 1.4.1) to confirm stationary distribution and
adequate effective sample sizes (ESS) that had been
obtained for all parameters. TreeAnnotator (version
1.5.1) was then used to summarize a best-supported tree
and annotate the tree with the mean age and posterior
probabilities of the nodes under investigation. FigTree
(version 1.2.3) was used to display the estimated tree
with node ages and the 95% confidence intervals. Pro-
grams BEAST, BEAUTi , LogCombiner, Tracer, TreeAn-
notator and FigTree were downloaded from http://beast.
bio.ed.ac.uk. In the MultiDivTime ana lysis, parameters
of the substitution model were first estimated by pro-
gram Baseml in the PAML package (version 4.3) [60].
The output from Baseml was then used in the Multidis-
tribute package to estimate the maximum likelihood of
the branch lengths and a variance-covariance matrix,
and to perform a MCMC Bayesian analysis for estimat-
ing the posterior distributions of substitution rates and
divergence dates. The tree presented in Figure 3 was
used as the reference topology for molecular dating ana-
lysis. The priors f or the ingroup root age mean (rttm)
and standard deviation (rttmsd) (Salamandroidea -
Cryptobranchoidea split, 151-170 Ma) were set to 1.60
and 0.1, respectively. The mean and standard deviation
of the prior distribution for the rate of molecular evolu-
tion at the ingroup root node (rtrate and rtratesd) were
both set to 0.12. The prior mean and standard deviation
for the Gamma distribution of the parameter controlling
rate variation over time (i.e. brownmean and brownsd)
were both set to 0.5. The Markov chain was run for
1,000,000 generations and sampled every 100 genera-
tions with an initial burn-in of 200,000 generations.
Three independent runs were performed to ensure the
convergence. The PAML package was downloaded from
http ://ab acus.gene.ucl.ac.uk/software/paml.html, and the
Multidistrib ute package from http://statgen.ncsu.edu/
thorne/multidivtime.html.
Acknowledgements
We thank J Bartoszek, J Ferner, MW Klemens, J Niedzwiecki, and J Davis for
providing samples, SB Hedges for pointing out the possibility of a numt,A
Bi and Bogart BMC Evolutionary Biology 2010, 10:238
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Hollis and J Gross for managing the sequencing facility at Guelph, J Fu and
D Noble for helpful discussions. This work was supported by an NSERC
(Canada) grant to J Bogart.
Author details
1
Department of Integrative Biology, University of Guelph, Guelph, Ontario,
N1G 2W1 Canada.
2
Museum of Vertebrate Zoology, University of California,
Berkeley, California 94720 USA.
Authors contributions
KB detailed the experimental design and performed most of the lab work,
data analyses and manuscript preparation. JPB conceived and directed the
study. Both authors contributed equally to this work in discussing research
strategy and development. Both authors read and approved the final
manuscript.
Received: 23 December 2009 Accepted: 3 August 2010
Published: 3 August 2010
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doi:10.1186/1471-2148-10-238
Cite this article as: Bi and Bogart: Time and time again: unisexual
salamanders (genus Ambystoma) are the oldes t unisexual vertebrates.
BMC Evolutionary Biology 2010 10:238.
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Bi and Bogart BMC Evolutionary Biology 2010, 10:238
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  • Source
    • "However, Bi and Bogart[25]described intergenomic recombination blocks on chromosomes of Ambystoma hybrids composed of A. laterale and A. jeffersonianum parental species. Despite authors did not provide divergence time of these species, Robertson et al.[68]showed that their divergence in cyt b (~ 10%) is more than twice as large than between A. laterale and A. texanum (~ 4%), which diversified about 10 MYA[69]. Recombination can therefore occur between genomes of species that are even more diverged than Cobitis under study (~ 7 MYA). "
    [Show abstract] [Hide abstract] ABSTRACT: Interspecific hybridization, polyploidization and transitions from sexuality to asexuality considerably affect organismal genomes. Especially the last mentioned process has been assumed to play a significant role in the initiation of chromosomal rearrangements, causing increased rates of karyotype evolution. We used cytogenetic analysis and molecular dating of cladogenetic events to compare the rate of changes of chromosome morphology and karyotype in asexually and sexually reproducing counterparts in European spined loach fish (Cobitis). We studied metaphases of three sexually reproducing species and their diploid and polyploid hybrid clones of different age of origin. The material includes artificial F1 hybrid strains, representatives of lineage originated in Holocene epoch, and also individuals of an oldest known age to date (roughly 0.37 MYA). Thereafter we applied GISH technique as a marker to differentiate parental chromosomal sets in hybrids. Although the sexual species accumulated remarkable chromosomal rearrangements after their speciation, we observed no differences in chromosome numbers and/or morphology among karyotypes of asexual hybrids. These hybrids possess chromosome sets originating from respective parental species with no cytogenetically detectable recombinations, suggesting their integrity even in a long term. The switch to asexual reproduction thus did not provoke any significant acceleration of the rate of chromosomal evolution in Cobitis. Asexual animals described in other case studies reproduce ameiotically, while Cobitis hybrids described here produce eggs likely through modified meiosis. Therefore, our findings indicate that the effect of asexuality on the rate of chromosomal change may be context-dependent rather than universal and related to particular type of asexual reproduction.
    Full-text · Article · Jan 2016 · PLoS ONE
  • Source
    • "For example, LJJ would signify a triploid unisexual salamander that possesses 1 A. laterale and 2 A. jeffersonianum nuclear genomes and would be one 'genomotype' [Lowcock, 1994] of more than 20 [Bogart, 2003; Bogart et al., 2009] diploid, triploid (3n), tetraploid (4n), or even pentaploid (5n) nuclear genomic combinations that have so far been identified. All unisexual genomotypes have at least one L nuclear genome and very similar mitochondrial genomes that distinctly differ from mitochondrial sequences in all 5 species [Hedges et al., 1992; Bogart, 2003; Bi and Bogart, 2010a]. Unisexual salamanders normally outnumber individuals of the sympatric, bisexual species in this complex [Bogart and Klemens, 1997; 2008]. "
    [Show abstract] [Hide abstract] ABSTRACT: Polyploid animals have independently evolved from diploids in diverse taxa across the tree of life. We review a few polyploid animal species or biotypes where recently developed molecular and cytogenetic methods have significantly improved our understanding of their genetics, reproduction and evolution. Mitochondrial sequences that target the maternal ancestor of a polyploid show that polyploids may have single (e.g. unisexual salamanders in the genus Ambystoma) or multiple (e.g. parthenogenetic polyploid lizards in the genus Aspidoscelis) origins. Microsatellites are nuclear markers that can be used to analyze genetic recombinations, reproductive modes (e.g. Ambystoma) and recombination events (e.g. polyploid frogs such as Pelophylax esculentus). Hom(e)ologous chromosomes and rare intergenomic exchanges in allopolyploids have been distinguished by applying genome-specific fluorescent probes to chromosome spreads. Polyploids arise, and are maintained, through perturbations of the 'normal' meiotic program that would include pre-meiotic chromosome replication and genomic integrity of homologs. When possible, asexual, unisexual and bisexual polyploid species or biotypes interact with diploid relatives, and genes are passed from diploid to polyploid gene pools, which increase genetic diversity and ultimately evolutionary flexibility in the polyploid. When diploid relatives do not exist, polyploids can interact with another polyploid (e.g. species of African Clawed Frogs in the genus Xenopus). Some polyploid fish (e.g. salmonids) and frogs (Xenopus) represent independent lineages whose ancestors experienced whole genome duplication events. Some tetraploid frogs (P. esculentus) and fish (Squaliusalburnoides) may be in the process of becoming independent species, but diploid and triploid forms of these 'species' continue to genetically interact with the comparatively few tetraploid populations. Genetic and genomic interaction between polyploids and diploids is a complex and dynamic process that likely plays a crucial role for the evolution and persistence of polyploid animals. See also other articles in this themed issue.
    Full-text · Article · Jun 2013 · Cytogenetic and Genome Research
    • "divergent lineages in the mtDNA gene tree (Shaffer and McKnight, 1996), and all diploid sexual species outside this clade. The Ambystomatidae also contains a complex of unisexual populations with a complicated evolutionary history (Bi and Bogart, 2010), and representatives from this group were not included in this study. To root the Ambystoma tree, 1–2 samples were included from all four extant Dicamptodon species (seven total individuals). "
    [Show abstract] [Hide abstract] ABSTRACT: The analysis of diverse data sets can yield different phylogenetic estimates that challenge systematists to explain the source of discordance. The mole salamanders (family Ambystomatidae) are a classic example of this phylogenetic conflict. Previous attempts to resolve the ambystomatid species tree using allozymic, morphological, and mitochondrial sequence data have yielded different estimates, making it unclear which data source best approximates ambystomatid phylogeny and which ones yield phylogenetically inaccurate reconstructions. To shed light on this conflict, we present the first multi-locus DNA sequence-based phylogenetic study of the Ambystomatidae. We utilized a range of analyses, including coalescent-based methods of species-tree estimation that account for incomplete lineage sorting within a locus and concordance-based methods that estimate the number of sampled loci that support a particular clade. We repeated these analyses with the removal of individual loci to determine if any a locus has a disproportionate effect on our phylogenetic results. Collectively, these results robustly resolved many deep and relatively shallow clades within Ambystoma, including the placement of A. gracile and A. talpoideum as the sister clade to a clade containing all remaining ambystomatids, and the placement of A. maculatum as the sister lineage to all remaining ambystomatids excluding A. gracile and A. talpoideum. Both Bayesian coalescent and concordance methods produced similar results, highlighting strongly supported branches in the species tree. Furthermore, coalescent-based analyses that excluded loci produced overlapping species-tree posterior distributions, suggesting that no particular locus - including mtDNA - disproportionately contributed to our species-tree estimates. Overall, our phylogenetic estimates have greater similarity with previous allozyme and mitochondrial sequence-based phylogenetic estimates. However, intermediate depths of divergence in the ambystomatid species tree remain unresolved, potentially highlighting a region of rapid species radiation or a hard polytomy, and which limits our ability to comment on previous morphologically-based taxonomic groups.
    No preview · Article · Apr 2013 · Molecular Phylogenetics and Evolution
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