Amphibia-Reptilia 33 (2012): 373-385
Weak genetic divergence between the two South American
toad-headed turtles Mesoclemmys dahli and M. zuliae
(Testudines: Pleurodira: Chelidae)
Mario Vargas-Ramírez1,3,∗ , Jan Michels2 , Olga Victoria Castaño-Mora3 ,
Gladys Cárdenas-Arevalo3 , Natalia Gallego-García3 , Uwe Fritz1
Abstract. Mesoclemmys dahli and M. zuliae are two endangered, little-known toad-headed turtles with small distribution
ranges in Colombia and Venezuela, respectively. Using the mitochondrial cytochrome b gene as a marker, we investigate
their phylogeographic differentiation. Furthermore, based on 2341 bp of mtDNA and 2109 bp of nDNA of M. dahli, M.
zuliae and allied chelid turtles, we infer their divergence time using a fossil-calibrated relaxed molecular clock approach.
Mesoclemmys dahli and M. zuliae are closely related species, with an estimated mean divergence time of 10.6 million years.
This estimate correlates with the uplift of the Serranía de Perijá, an Andean mountain chain separating their distribution
ranges, suggesting that this event could have caused the evolution of the two species. Haplotype and nucleotide diversities
of M. dahli are markedly higher than in Podocnemis lewyana, another endemic turtle species of Colombia. This pronounced
dissimilarity may reflect differences in the phylogeographies and demographic histories of the two species, but also different
habitat preferences.
Keywords: Colombia, molecular clock, mtDNA, nDNA, phylogeography, Venezuela.
Introduction
While the phylogeographies of many Western
Palaearctic and Nearctic turtles and tortoises
are well-known (e.g. Walker et al., 1998; Lenk
et al., 1999; Weisrock and Janzen, 2000; Karl
and Wilson, 2001; Fritz et al., 2006, 2007,
2008, 2009; Rosenbaum, Robertson and Zamudio, 2007; Amato, Brooks and Fu, 2008;
McGaugh, Eckerman and Janzen, 2008; Butler
et al., 2011; Pedall et al., 2011; Ureña-Aranda
and Espinosa de los Monteros, 2012), relatively
few studies have focused on species from Central and South America so far (Souza et al.,
2003; Pearse et al., 2006; Vargas-Ramírez et
1 - Museum of Zoology (Museum für Tierkunde), Senckenberg Dresden, A. B. Meyer Building, D-01109 Dresden,
Germany
2 - Department of Functional Morphology and Biomechanics, Institute of Zoology, Christian-AlbrechtsUniversität zu Kiel, Am Botanischen Garten 1-9, D24118 Kiel, Germany
3 - Instituto de Ciencias Naturales, Universidad Nacional
de Colombia, Apartado 7495, Bogotá, Colombia
∗ Corresponding author; e-mail:
[email protected]
© Koninklijke Brill NV, Leiden, 2012.
al., 2007, 2010a, 2012; González-Porter et al.,
2011; Fritz et al., 2012a, 2012b). Among the
South American species, members of the family
Chelidae are the least studied ones, with just one
publication on the phylogeography of Hydromedusa maximiliani (Souza et al., 2003). Chelidae,
with 23 species, is the most species-rich of the
seven families of turtles and tortoises in continental South America, and the most speciose
group within this family are the ‘toad-headed
turtles’ of the genus Mesoclemmys. Currently,
ten Mesoclemmys species are recognized, most
of which occur in central South America (Bour
and Zaher, 2005; Fritz and Havaš, 2007; van
Dijk et al., 2011). Mesoclemmys dahli (Zangerl
and Medem, 1958) and M. zuliae (Pritchard and
Trebbau, 1984) represent little-known, isolated
northern species with relict character (Bour and
Zaher, 2005).
Dahl’s toad-headed turtle (M. dahli), endemic
to Colombia, is the only chelid species occurring west of the Andes mountain range (Medem, 1966). It is known from the western part
of the Caribbean region of Colombia, including the departments of Córdoba, Bolivar, SuDOI:10.1163/15685381-00002840
374
cre and Atlántico (Zangerl and Medem, 1958;
Medem, 1966; Rueda-Almonacid et al., 2007)
and from a newly discovered, isolated population in the department of Cesar (Medina-Rangel
and Forero-Medina, 2008; fig. 1). Due to its
small distribution range and extreme habitat deterioration (Rueda-Almonacid et al., 2007), M.
dahli is listed in the threat category “Critically
Endangered” by the IUCN Red List of Threatened Species (IUCN, 2011). The closely related Zulia toad-headed turtle (M. zuliae), endemic to Venezuela, is confined to the Zulia
river and its tributaries in the south-western part
of the Maracaibo basin (Pritchard and Trebbau,
1984; Rueda-Almonacid et al., 2007; fig. 1).
Acknowledging its small distribution range, M.
zuliae is listed in the category “Vulnerable”
by the IUCN Red List of Threatened Species
(IUCN, 2011).
To date, some studies on the ecology and natural history of the two species have been performed (M. dahli: Medem, 1966; de la OssaVelasquez, 1998; Castaño-Mora and Medem,
2002; Rueda-Almonacid et al., 2007; ForeroMedina, Cárdenas-Arevalo and Castaño-Mora,
2011; M. zuliae: Rueda-Almonacid et al., 2007;
Rojas-Runjaic, 2009). Furthermore, the two
species were included in a study that investigated the patterns of geographical distribution
of South American chelid turtles (Souza, 2005).
The two species have similar habitat preferences (Medem, 1966; Pritchard and Trebbau,
1984; Rueda-Almonacid et al., 2007) and are
morphologically very similar (see images and
descriptions in Pritchard and Trebbau, 1984;
Rueda-Almonacid et al., 2007). Yet, they have
never been studied and compared using molecular markers. The present study aims to fill this
gap by analyzing their kinship at a molecular
genetic level and by applying a relaxed molecular clock to elucidate their divergence and biogeography.
M. Vargas-Ramírez et al.
Materials and methods
Sampling and laboratory procedures
Twenty-five saliva samples of Mesoclemmys dahli and four
tissue samples of M. zuliae were studied (fig. 1; Appendix).
The samples from M. dahli were collected in both regions
where the species is known to occur; 18 samples came from
the department of Córdoba and seven samples from the department of Cesar. The samples from M. zuliae were collected at the type locality and two additional sites, covering
largely its distribution range in north-south direction. For
inferring phylogeography, sequences of the mitochondrial
cytochrome b gene (cyt b) were generated for all samples.
For molecular clock calculations, the mitochondrial 12S
rRNA and NADH dehydrogenase subunit 4 (ND4) genes
and three nuclear loci, the oocyte maturation factor Mos
gene (C-mos), the intron 1 of the RNA fingerprint protein 35 gene (R35) and the recombination-activating gene
2 (Rag 2), of one sample each of M. dahli and M. zuliae
were sequenced. Sequences of the same mitochondrial and
nuclear DNA fragments were also produced for M. gibba,
Phrynops geoffroanus and P. hilarii, as far as these were not
available from GenBank (see Appendix).
Genomic DNA was extracted using a Qiagen DNA blood
extraction kit (Qiagen Benelux B.V., Venlo, The Netherlands), following the manufacturer’s instructions. The 12S
rRNA gene was amplified and sequenced using the primers
L1091 and H1478 (Kocher et al., 1989), and for the partial ND4 gene plus adjacent DNA coding for tRNAs, the
primers L-ND4 and H-Leu (Stuart and Parham, 2004) were
applied. The cyt b gene was amplified and sequenced in
two fragments overlapping by approximately 300 bp using the primer pairs mt-a-neu3 + mt-E-Rev2 and mt-cFor2 + mt-f-na (Fritz et al., 2006; Praschag et al., 2007).
For the nuclear loci the following primer pairs were used Cmos: G136 + G137 (Georges et al., 1998), R35: R35Ex1 +
R35Ex2 (Fujita et al., 2004), and Rag 2: F2-1 + R2-1 (Le et
al., 2006). PCRs were carried out in a total volume of 50 μl
containing 1 unit Taq polymerase (Bioron, Ludwigshafen,
Germany), 1 × buffer (as recommended by the supplier),
0.5 μM of each primer, and 0.2 mM of each dNTP (Fermentas, St. Leon-Rot, Germany). PCR products were purified using the ExoSAP-IT enzymatic cleanup (USB Europe GmbH, Staufen, Germany; modified protocol: 30 min
at 37°C, 15 min at 80°C) and sequenced on an ABI 3130xl
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA) using the BigDye Terminator v3.1 Cycle Sequencing
Kit (Applied Biosystems).
Phylogeographic analyses
DNA sequences were edited and aligned using CHROMAS
1.51 (http://www.technelysium.com.au/chromas.html) and
BIOEDIT 7.0.5.2 (Hall, 1999). For the 1067-bp-long cyt b
sequences of Mesoclemmys dahli, M. gibba and M. zuliae,
uncorrected p distances were calculated in MEGA 5.05
(Tamura et al., 2011). Evolutionary relationships between
sequences of M. dahli and M. zuliae were inferred using
TCS 1.21 (Clement et al., 2000). For M. dahli, haplotype
Weak genetic divergence between turtles Mesoclemmys dahli and M. zuliae (Testudines: Pleurodira: Chelidae)
375
Figure 1. Distribution of Mesoclemmys dahli and M. zuliae (based on Rueda-Almonacid et al., 2007) and sampling sites
(colour-coded). For M. dahli, the Córdoba population is indicated in green and the Cesar population in orange; sites of M.
zuliae, blue. The Serranía de Perijá is highlighted in red. Inset: juvenile Mesoclemmys dahli from the Cesar population (photo:
Guido Medina).
diversity (h) and nucleotide diversity (π ) were calculated
using DNASP 5.0 (Librado and Rozas, 2009). The influence of geography on genetic divergence within M. dahli
and between M. dahli and M. zuliae was examined using
Mantel tests as implemented in the software IBD (Isolationby-Distance; Bohonak, 2002). A first Mantel test was based
on uncorrected p distances and geographical distances (km)
between the two populations of M. dahli and M. zuliae. In
another Mantel test, the influence of the Serranía de Perijá, an Andean mountain chain separating the distribution
ranges of M. dahli and M. zuliae, was examined by combining a categorical matrix with genetic distances. In this matrix, the distance between the two populations of M. dahli
was coded with 0 (corresponding to the absence of a mountain barrier), while the distance between each population of
M. dahli and M. zuliae was coded with 1 (acknowledging
the presence of the mountain barrier).
Relaxed molecular clock
The phylogeny of Mesoclemmys dahli, M. zuliae and other
chelid turtle species was inferred to obtain a backbone for
molecular clock calculations. For this purpose, the 12S
rRNA, cyt b, ND4, C-mos, R35 and Rag 2 sequences of
M. dahli and M. zuliae were aligned with homologous sequence data of M. gibba, Chelus fimbriatus, Chelodina ru-
gosa, Phrynops geoffroanus, P. hilarii, Pelomedusa subrufa, Podocnemis expansa, and Indotestudo elongata (for
accession numbers, see Appendix). Sequences for each
taxon were concatenated. The resulting supermatrix was of
4450 bp length (including gaps), corresponding to 2341 bp
of mtDNA and 2109 bp of nDNA. The partial 12S rRNA
gene contributed 493 bp; the nearly complete cyt b gene,
1067 bp; the partial ND4 gene plus adjacent DNA coding
for tRNAs, 781 bp; C-mos, 390 bp; R35, 1025 bp; and
Rag 2, 694 bp. Based on this supermatrix, phylogenetic
trees were calculated using Maximum Likelihood (ML) and
Bayesian Inference (BI) analyses, applying the following
partition schemes: (1) unpartitioned, (2) by gene, i.e., each
gene corresponds to a distinct partition, and (3) maximum
partitioning, i.e., using each codon of each protein-coding
gene as distinct partition plus each non-protein-coding gene
or DNA block as distinct partition. Indotestudo elongata
(family Testudinidae, suborder Cryptodira) served for treerooting. ML analyses were run with RAxML 7.2.6 (Stamatakis, 2006) using the graphical user interface raxmlGUI 0.93 (Silvestro and Michalak, 2011) and the GTR +
G model across every partition. To explore the robustness
of the branching patterns, five independent ML searches
were performed using the fast bootstrap algorithm. Subsequently, 1000 thorough bootstrap replicates were calculated and plotted against the tree with the highest likelihood value. The BI analyses were run in MrBAYES 3.1
376
(Ronquist and Huelsenbeck, 2003) using four incrementally
heated Markov chains; posterior probabilities were obtained
from the 50% majority rule consensus tree. For each independent run, the variation in likelihood scores was examined by plotting − ln L scores against the number of generations, and the burn-in was set to sample only the plateau of
the most likely trees. In a conservative approach, 40% of all
sampled trees were discarded, although the plateau of likelihood values had been reached before. The best-fit model
of nucleotide substitution was established for each partition
using the Akaike information criterion of MrMODELTEST
(Nylander, 2002) and incorporated into a single tree search
(mixed model partition approach; Nylander et al., 2004).
The following models were suggested for the partitioning
by gene: 12S rRNA – GTR + G, cyt b – TVM + I + G,
ND4 – GTR + G, t-RNAs – TVM + G, C-mos – K80 + G,
R35 – TVM + G, and Rag 2 – HKY; and for the codons of
protein-coding genes in the maximum partitioning scheme:
cyt b 1st codon position – GTR + G, cyt b 2nd codon position – GTR + I + G, cyt b 3rd codon position – TIM + G,
ND4 1st codon position – TVM + G, ND4 2nd codon position – TVM + G, ND4 3rd codon position – HKY + I +
G, C-mos 1st codon position – HKY + I, C-mos 2nd codon
position – TrNef + G, C-mos 3rd codon position – K81uf +
G, Rag 2 1st codon position – TrN + G, Rag 2 2nd codon
position – GTR + G, and Rag 2 3rd codon position – HKY.
The divergence time of M. dahli and M. zuliae was
estimated by a Bayesian relaxed molecular clock approach (MULTIDISTRIBUTE package; Thorne, Kishino
and Painter, 1998; Thorne and Kishino, 2002). Fossil evidence was used for constraining the minimum ages of two
nodes within the obtained phylogeny. The split between
Chelodina rugosa and the South American chelids was calibrated with the range of 125.0-99.6 million years (ma),
based on the Lower Cretaceous record of Prochelidella cerrobarcinae (Cerro Barcino formation, Aptian-Albian?), the
oldest South American chelid (de la Fuente et al., 2011;
node A in fig. 4). Furthermore, the split between Pelomedusidae (represented by Pelomedusa subrufa) and Podocnemididae (represented by Podocnemis expansa) was set to
the lower and upper boundaries of the Valanginian (140.4136.2 ma), the Early Cretaceous stage from which the earliest podocnemidoid turtle is known (Cadena, 2011; node B in
fig. 4). Chelodina rugosa is a representative of Australasian
chelids that constitute the sister group of South American
chelids (Georges et al., 1998), and Prochelidella is thought
to be allied to the extant genus Acanthochelys of South
America (de la Fuente et al., 2011). Pelomedusa subrufa
and Podocnemis expansa are representatives of the families
Pelomedusidae and Podocnemididae, respectively. Among
extant turtles, Pelomedusidae and Podocnemididae together
are the sister group of Chelidae (Gaffney et al., 2006).
In order to determine the appropriate nucleotide substitution model parameters, the data set was analyzed using
the program PAML 3.13 (Yang, 1997). Subsequently, the
branch lengths and their variance-covariance matrix were
estimated with the program ESTBRANCHES. Using the application MULTIDIVTIME, Markov chains were run three
times with the settings numsamps = 1 000 000, sampfreq =
M. Vargas-Ramírez et al.
100 and a burn-in of 10 000 and compared to test the stability of the results. According to Cadena (2011), the prior for
the mean of the ingroup root age (rttm) was set to a minimum of 161.2 ma (with 2 ma SD), corresponding to the
split between (Pelomedusidae + Podocnemididae) and Chelidae. Mean and standard deviation of the rate of molecular
evolution at the ingroup root node (rtrate and rtratesd) were
0.0004341 substitutions per site and million years with 1
time unit = 1 ma (calculated with the mean of the branch
lengths from ESTBRANCHES). Mean and standard deviation of the Brownian motion constant (brownmean and
brownsd) were set to 0.006203 and bigtime to 220 ma according to the age of the oldest known chelonian, Odontochelys semitestacea (Li et al., 2008).
Results
Phylogeography
The 25 cyt b sequences of Mesoclemmys dahli
and the four cyt b sequences of M. zuliae were
assigned to two clearly distinct haplotype clusters in the parsimony network (fig. 2), differing by a minimum of 18 mutational steps. Sequences of M. dahli corresponded to 15 haplotypes; sequences of M. zuliae, to two haplotypes. Among haplotypes of M. dahli, some
loops occurred. The maximum number of mutational steps within M. dahli resembles the minimum divergence between M. dahli and M. zuliae. The two haplotypes of M. zuliae differed
by only one mutation. For the two populations
of M. dahli, no shared haplotypes were observed. However, this could be a bias due to
small sample size because the haplotypes of
each population did not form a distinct cluster,
but were rather randomly distributed in the network (fig. 2).
The mean uncorrected p distance between sequences of M. dahli and M. zuliae was 2.22%;
mean divergences within each species were
0.58% and 0.04%, respectively. The two populations of M. dahli differed by 0.77%. Within
the Córdoba population of M. dahli a sequence
divergence of 0.41% occurred; within the Cesar
population the divergence was 0.33%. The sequence of M. gibba differed by 14.16% from
that of M. dahli and by 13.27% from that of
M. zuliae. The Mantel tests revealed no signifi-
Weak genetic divergence between turtles Mesoclemmys dahli and M. zuliae (Testudines: Pleurodira: Chelidae)
377
correlation between genetic distance and the Serranía de Perijá acting as a geographical barrier
(Z = 4.4200, r = 0.9982, p = 0.001).
Haplotype diversities (h) of the populations
of M. dahli from Córdoba (n = 18) and Cesar (n = 7) were 0.889 and 0.952, respectively, and their nucleotide diversities (π) were
0.00531 and 0.00420. When all 25 sequences
were lumped together, h was 0.940 and π was
0.00622.
Divergence time of Mesoclemmys dahli and M.
zuliae
The topologies of the ML and BI trees were
congruent among all partition schemes and support values were always maximal or very close
to the maximum (fig. 3). Mesoclemmys gibba
was the sister taxon of M. dahli + M. zuliae.
A clade comprising Phrynops geoffroanus +
P. hilarii was sister to Mesoclemmys, and the
South American species Chelus fimbriatus and
the Australian species Chelodina rugosa were
the successive sister taxa. This phylogeny, consistent with expectations (Georges et al., 1998;
Gaffney et al., 2006), served as a backbone for
relaxed molecular clock calculations (fig. 4).
Obtained node ages are presented in table 1. The
mean divergence time of M. dahli and M. zuliae
was dated to 10.6 ma, with a standard deviation
of 2.8 ma and a 95% confidence interval of 6.216.9 ma.
Discussion
Figure 2. Parsimony network of cyt b haplotypes of Mesoclemmys dahli and M. zuliae, based on an alignment of
1067 bp length. Circle size indicates haplotype frequency.
Missing haplotypes are shown as small black circles. Each
line connecting haplotypes corresponds to one mutational
step. Colour code as in fig. 1; haplotype codes refer to the
Appendix.
cant correlation between genetic and geographical distances when cyt b sequences of M. dahli
and M. zuliae were compared (Z = 1165.9680,
r = 0.3642, p = 0.5060), but a significant
According to our analyses, Mesoclemmys dahli
is closely related to M. zuliae, which is in line
with their morphological similarity (Pritchard
and Trebbau, 1984). However, a phylogenetic
analysis using morphological characters could
not resolve the relationships among the ten currently recognized Mesoclemmys species (Bour
and Zaher, 2005). We included in our phylogenetic analyses three of these species and found
a well-supported sister group relationship of M.
dahli and M. zuliae, with M. gibba being the
378
M. Vargas-Ramírez et al.
Figure 3. Bayesian tree based on 2341 bp of mitochondrial DNA (12 S rRNA, ND4, cyt b) and 2109 bp of nuclear DNA
sequences (C-mos, R35, Rag 2; partitioned by gene). Support values are Bayesian posterior probabilities (top) and ML
bootstrap values (bottom); asterisks indicate maximum support under both methods. This tree was used for the Relaxed
Molecular Clock calculations.
successive sister taxon (fig. 3). Compared to M.
gibba, M. dahli and M. zuliae seem to be weakly
differentiated. The number of mutational steps
among haplotypes of M. dahli resembles the observed divergence between M. dahli and M. zuliae (fig. 2), and this pattern is also reflected by
low between-species divergences with respect
to uncorrected p distances of the cyt b gene.
The sequence divergence between M. dahli and
M. zuliae amounts only to 2.22%, while these
two species differ from M. gibba by 14.16% and
13.27%, respectively. With respect to cyt b sequences, the value of 2.22% is close to the lower
divergence limit for any distinct congeneric turtle and tortoise species. Yet, for some emydid turtles (Emys, Graptemys, Trachemys) even
lower species divergence values were reported
(Lamb et al., 1994; Fritz et al., 2005, 2012b). In
analogy to the widely used barcoding approach
(Hebert, Ratnasingham and de Waard, 2003) relying on divergences of the COI gene as a yardstick, uncorrected p distances of the cyt b gene
have been frequently used as a tool for species
delineation of chelonians (e.g. Vargas-Ramírez
et al., 2010b; Praschag et al., 2011; Stuckas
and Fritz, 2011; Fritz et al., 2012a, 2012b;
Kindler et al., 2012). However, these studies
pointed out that no universal thresholds should
be used because the critical divergence value
differs among various chelonian groups. Therefore, additional Mesoclemmys species need to
be studied before the status of M. dahli and
M. zuliae as distinct species should be challenged.
379
Weak genetic divergence between turtles Mesoclemmys dahli and M. zuliae (Testudines: Pleurodira: Chelidae)
Figure 4. Divergence time estimates using the MULTIDISTRIBUTE package. Dark grey bars at nodes represent 95%
confidence intervals. The light grey column on the right shows the time of the main uplift of the Serranía de Perijá (Late
Miocene-Pliocene; Kellogg, 1984). Letters indicate nodes calibrated with fossil evidence (see table 1). PP, Plio-Pleistocene.
Table 1. Results of the relaxed molecular clock analysis using the MULTIDISTRIBUTE package. SD = Standard deviation,
CI = 95% confidence interval. All dates are given in million years (ma).
Node
Mean
SD
CI
Mesoclemmys dahli + M. zuliae
Phrynops geoffroanus + P. hilarii
(Mesoclemmys dahli + M. zuliae) + M. gibba
Mesoclemmys + Phrynops
(Mesoclemmys + Phrynops) + Chelus
((Mesoclemmys + Phrynops) + Chelus) + Chelodina∗
Pelomedusa + Podocnemis∗∗
Chelidae + (Pelomedusa + Podocnemis)
10.6
17.5
41.0
64.2
87.0
121.7
99.6
160.3
2.8
5.0
6.7
7.8
7.8
3.2
10.1
2.0
6.2-16.9
10.01-29.4
28.4-55.0
48.6-79.5
70.4-101.2
113.0-124.9
78.7-118.4
156.4-164.2
∗ Node
A (fig. 4): Fossil constraint: 125.0-99.6 ma (Prochelidella cerrobarcinae; de la Fuente et al., 2011).
B (fig. 4): Fossil constraint: 140.4-136.2 ma (earliest podocnemidoid turtle; Cadena, 2011).
∗∗ Node
Haplotype and nucleotide diversities of M.
dahli are much higher than in another endemic turtle species of Colombia, Podocnemis
lewyana. Based on the rapidly evolving D-loop,
haplotype diversity (h) and nucleotide diversity
(π) of 119 samples of P. lewyana were 0.292
and 0.00006, respectively (Vargas-Ramírez et
al., 2012), contrary to h = 0.940 and π =
0.00622 in the 25 samples of M. dahli analyzed
in the present study for the more slowly evolving cyt b gene. This pronounced dissimilarity
may reflect differences in the phylogeographies
and demographic histories of the two species,
but also different habitat preferences. While P.
lewyana is a true river turtle, M. dahli is typically found in shallow and quiet ponds or small
380
brooks in seasonally dry tropical forest (Medem, 1966; Rueda-Almonacid et al., 2007), suggesting that the patchy structures in such habitat
may favour more genetic diversity than the occurrence along a river course.
Souza (2005) proposed that the distribution
of South American chelids is mainly correlated with major river or drainage basins. According to this hypothesis, the distribution of
M. dahli and M. zuliae is associated with the
Magdalena and Orinoco basins, respectively.
Notwithstanding that M. dahli occurs close to
the Magdalena river, its typical habitats are neither directly linked to this river nor to its major tributaries. Mesoclemmys zuliae lives in similar habitats as M. dahli. Moreover, the range
of M. zuliae is far away from the Orinoco
(Rueda-Almonacid et al., 2007), and the rivers
and streams of the Maracaibo basin are not
connected to the Orinoco basin. Unlike true
river turtles, as Podocnemis species, M. dahli
and M. zuliae seem therefore to be not tied to
the ecosystems of the Magdalena and Orinoco
rivers. Consequently, it is unlikely that the origin of M. dahli and M. zuliae is directly related to the hydrographic history of these rivers.
However, the estimated mean divergence time
of M. dahli and M. zuliae correlates quite well
with the uplift of the Serranía de Perijá (see
above) in the Late Miocene and Pliocene (Kellogg, 1984; fig. 4), suggesting that this orogenetic process could be directly responsible for
the evolution of the two species. Also in another South American chelid species, Hydromedusa maximiliani from eastern Brazil, phylogeographic structure is shaped by mountain
chains, and the estimated divergence time of the
major phylogeographic groups within H. maximiliani fits the uplift of the respective mountains
during the Pliocene and Miocene (Souza et al.,
2003).
Acknowledgements. We thank the Grupo de Conservación
y Biodiversidad of the Instituto de Ciencias Naturales de
la Universidad Nacional de Colombia, Fundación Biodiversa Colombia and Instituto de Biología Tropical Roberto
M. Vargas-Ramírez et al.
Franco (IBTRF) de la Universidad Nacional de Colombia
for institutional and logistical support. Carl J. Franklin (Amphibian and Reptile Research Center, University of Texas
at Arlington) and Ingo Pauler provided samples of Mesoclemmys zuliae. Christian Kehlmaier, Edgar Lehr, Anke
Müller, and Anja Rauh assisted in the lab. Ylenia Chiari and
João Lourenço shared sequences of Phrynops hilarii with
us. Massimo Delfino, Marcelo Sánchez-Villagra, Markus
Wilmsen and Juliana Sterli helped with literature of fossil chelids. Thanks to Guido Medina for the photograph of
M. dahli. Mario Vargas-Ramírez’ research in Germany is
funded by the Humboldt Foundation (Georg Forster fellowship).
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383
Submitted: February 28, 2012. Final revision received: July
25, 2012. Accepted: July 30, 2012.
Associated Editor: Sylvain Ursenbacher.
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Arache, Chimá, Córdoba, Colombia
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Momíl, Córdoba, Colombia (9°14 53.37 N 75°40 26.33 W)
Serradero, Purisima, Córdoba,
Colombia (9°14 33.17 N 75°43 0.27 W)
La Confianza, Lorica, Córdoba,
Colombia (9°14 14.29 N 75°48 30.13 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Arache, Chimá, Córdoba, Colombia (9°4 32.80 N 75°38 37.25 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
75°38 37.25 W)
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
Mesoclemmys dahli
(9°4 32.80 N
Locality
Species
M
L
K
J
JX139061 –
JX139060 –
JX139059 –
JX139058 –
JX139086
–
–
–
–
–
–
–
–
–
–
JX139047
JX139048
JX139049
JX139050
JX139051
JX139052
JX139053
JX139054
JX139055
JX139056
JX139057
D
E
E
E
F
G
H
H
H
I
J
–
–
–
–
–
–
–
–
–
–
–
JX139071
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
JX139082
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
C-mos
–
–
–
–
JX139078
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
R35
GenBank accession numbers
12S rRNA ND4
JX139046 –
JX139039
JX139040
JX139041
JX139042
JX139043
JX139044
JX139045
cyt b
C
A
A
A
A
A
B
B
mtDNA
haplotype
–
–
–
–
JX139073
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Rag 2
9151
9150
9149
9148
4699
4700
4703
4710
4701
4705
4706
4709
4715
4707
9147
4697
4694
4695
4712
4714
4711
4696
4698
MTD T
Table A.1. Mesoclemmys samples and outgroups used in the present study. MTD T numbers refer to saliva or tissue samples in the collection of the Museum of Zoology, Senckenberg
Dresden.
Appendix
384
M. Vargas-Ramírez et al.
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
Chimichagua, Cienaga de Zapatoza, Cesar,
Colombia (9°17 18.60 N 73°47 35.80 W)
10 km W of border of Cienagas de Juan Manuel National Park,
Zulia, Venezuela (9°12 44.52 N 72°30 59.66 W)
50 km N of border of Cienagas de Juan Manuel National Park,
Zulia, Venezuela (9°41 8.83 N 72°24 48.41 W)
Caño Madre Vieja, Colon, Zulia,
Venezuela – type locality (8°53 N 72°30 W)
Caño Madre Vieja, Colon, Zulia,
Venezuela – type locality (8°53 N 72°30 W)
Bolognesi, Ucayali, Peru
Mesoclemmys dahli
Mesoclemmys gibba
Mesoclemmys gibba
Phrynops geoffroanus
Phrynops hilarii
Chelus fimbriatus
Chelodina rugosa
Pelomedusa subrufa
Podocnemis expansa
Indotestudo elongata
Mesoclemmys zuliae
Mesoclemmys zuliae
Mesoclemmys zuliae
Mesoclemmys zuliae
Mesoclemmys dahli
Locality
Species
Table A.1. (Continued.)
–
–
–
–
–
–
–
–
–
b
b
a
b
O
N
JX139068
–
JX139069
JN999705
HQ172156
HQ172157
AF039066
AM943830
DQ080043
JX139067
JX139066
JX139065
JX139064
JX139063
JX139062
mtDNA
haplotype cyt b
JX139088
–
JX139089
JN999705
HQ172156
HQ172157
AF039066
AM943820
DQ080043
–
–
JX139087
–
–
–
12S rRNA
–
–
–
C-mos
–
–
–
R35
–
EF535304
JX139072
JN999705
HQ172156
HQ172157
FN645326
FM165620
DQ080043
–
–
–
AF109206
JX139084
JX139085
AF109203
AF039486
AF109208
AF109209
AY447980
–
–
JX139080
–
JX139081
–
AY339640
AY339641
FR717085
AM943843
HQ260650
–
–
JX139070 JX139083 JX139079
–
–
–
ND4
GenBank accession numbers
JX139075
–
JX139076
JX139077
–
–
FN645376
AM943839
HQ260657
–
–
JX139074
–
–
–
Rag 2
100
–
645
–
–
–
–
–
–
9156
9155
9154
9153
9336
9152
MTD T
Weak genetic divergence between turtles Mesoclemmys dahli and M. zuliae (Testudines: Pleurodira: Chelidae)
385