A Brief Molecular Phylogeny focused on Twenty-Nine Species within the
Testudinidae Family using Mitochondrial Cytochrome b Gene
Christine Chessler
Tortoises within the family Testudinidae are some of the most familiar and widely
distributed groupings of turtles. However, many researchers feel that their phylogenetic
relationships are relatively controversial due to molecular studies being limited by
adequate taxonomic sampling, in addition to studies addressing only specific questions
aimed only at subsets within a family (ie: only the genus Gopherus). So, where exactly
does the gopher tortoise “fit” phylogenetically among the tortoise family (Testudinidae)?
Importantly, what are the distinguishing clades among this family? Also, are these clades
supported by shared synapomorphies and biogeography? Available cytochrome b
nucleotide sequences for the gopher tortoise and 29 other members in the Testudinidae
family, inlcuding an outgroup species from a sister family, were obtained from the NCBI
nucleotide database and aligned using Seaview + MAFFT. A Maximum Likelihood
(ML) phylogenetic tree was constructed using RAxML and modified with the aid of
FigTree software. Five distinct clades were found among the ML phylogenetic tree
constructed. Since some branches and clades had relatively low bootstrap values, possible
synapomorphies among these clade groupings were examined and found to validate
relationships among the Indotestudo + Testudo + Malacocherus and Geochelone sp. +
Pyxis + Dipsochelys clade groupings. Additionally, observing biogeographical locations
for clade Geochelone sp. + Pyxis + Dipsochelys validated the clade grouping based on a
common ancestor more than likely dispersing to these locations. The clade of Manouria
and Gopherus was shown to represent the divergence between the Asian and North
American lineages. Other potential relationships and dispersal mechanisms were also
examined within the other clades. Modifications may be made to obtain more sequences
and species in a future analysis to possibly garner stronger bootstrap support for those
relationships provided.
Keywords: Testudinidae, phylogenetics, cytochrome b gene, biogeography, tortoises
Introduction
Tortoises within the family Testudinidae are some of the most familiar and widely
distributed groupings of turtles. The family has 49 living species in 12 extant genera (Le
et al, 2006). The Testudinidae family is said to have evolved 200 million years ago from
the early Anapsid reptiles (Krenz et al, 2005). This family is the only known living reptile
classification with the Anapsid skull structure (Alderton, 1988). The earliest Testudine
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fossils have been reported to be found in Asia (Le et al, 2006). Most members in this
family are characterized by high-domed shells and stout scaly limbs that allow them to be
classified as terrestrial tortoises. The majority of these land tortoises occur in Africa and
Asia, but a few species are in America and Europe (The Reptile Database).
Many researchers feel that the phylogenetic relationships in this family are
relatively controversial due to molecular studies being limited by adequate taxonomic
sampling, in addition to studies addressing only specific questions aimed only at subsets
within a family (ie: only the genus Gopherus) (Le et al, 2006; Lamb and Lydeard, 1994).
To allow the reader to become familiar with the phylogenetic structure of the members of
the Testudinidae family, I will provide a very brief review of selected individual genera
and individual species, focused only on those most ecologically significant (and with
abundant taxonomic information available). Potential discussion on the synapomorphies
within clades will be saved for the Results & Discussion of this paper and will not be
discussed in this Introduction.
The genus Manouria is said to form the most basal testudinid lineage that
includes a novel sister relationship between the Asian Manouria and North American
Gopherus species (Le et al, 2006). Among the genus Gopherus are those tortoises
capable of using their forefeet for digging (Ashton and Ashton, 2008). The Gopherus
genus is the only grouping within the Testudinidae family to occur in North America
(The Reptile Database). G.polyphemus and G. aggassizi will be only two of the four
species in this genus to be featured in the phylogenetic construction in this paper.
Within the genus Geochelone are the commonly known giant land tortoises
(Alderton, 1988). The species that comprise this genus are some of the largest known
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tortoises in the world (Caccone et al, 2002). The taxonomic history of just the Galapagos
tortoise (G.nigra sp.) is said to be very complex and even fairly unresolved to this day; up
to 15 species or subspecies have been described and vary among the island chain of the
Galapagos (Caccone et al, 2002). With the individuals used in this phylogenetic
summary, only G.pardalis species are actually sub-species, while the others are
recognized as individual species within the genus Geochelone (Table 1.1). Le et al (2006)
found that the taxon is polyphyletic and may be found among atleast four independent
clades in some cases. However, previous research (Meylan and Sterrer, 2000), found
support for a monophyletic Geochelone (Le et al, 2006). Tortoises within this genus, and
the Dipsochelys genus, have been said to have occupied the Galapagos Islands by means
of rafting to these islands during the early Eocene (Le et al, 2006).
The Dipsochelys genus is believed now to contain only two true living species-the
Seychelles giant tortoise (Dipsochelys hololissa) and Arnold's Giant Tortoise
(Dipsochelys arnoldi) also known as the Seychelles saddle-backed tortoise (Palkovacs et
al, 2003). Although these species are still alive, they are categorized as being extinct in
the wild and can now only be found in zoos and in captive-breeding programs (Palkovacs
et al, 2003). The genus was found in Madagascar, the Seychelles Islands, Comoros and
Aldabra. By 1840, they appeared eliminated from all islands except Aldabra due mainly
to overexploitation by European settlers (Palkovacs et al, 2003).
The chosen outgroup to root the phylogenetic tree presented in this summary was
Deirochelys reticularia in the family Emydidae (Buhlmann, Gibbons, and Jackson,
2008). This species is a semi-aquatic turtle that utilizes freshwater habitat, in addition to
terrestrial areas (Buhlmann, Gibbons, and Jackson, 2008). This species was chosen as an
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outgroup due to its family (Emydidae) having a previously known sister relationship to
the Testudinidae family (Le et al, 2006). Le et al (2006), also chose this species as an
outgroup to construct a similar phylogeny of the Testudinidae family; so given this past
research I also chose to use a member of this family as the root of my phylogenetic tree.
Given my graduate research on gopher tortoises, I initially wanted to know more
about where exactly the gopher tortoise “fit” phylogenetically among the tortoise family
(Testudinidae). However, as I researched the existing phylogeny, I also wanted to
address where the specific clades were situated among Testudinidae and also which clade
the gopher tortoise fit within. Lastly, I wanted to group each clade based on their
geographical locations to see if the specific locales had anything to do with these clade
groupings. In this project summary, I plan to address these questions with the
construction of a basic phylogenetic tree focused on 29 members of the Testudinidae
family (with an outgroup in Deirochelys reticularia; a known sister relationship used as
an outgroup in previous studies (Le et al, 2006).
Materials and Methods
Initially, NCBI (http://www.ncbi.nlm.nih.gov/) was searched for keyword gopher
tortoise. The nucleotide database of NCBI was then utilized to find a specific gene to
construct a phylogeny of the family Testudinidae (my given objective). Within the
database search, gopher polyphemus cytochrome b gene (GenBank: DQ497307.1) was
chosen to construct my tree due to its common use in phylogenetic tree construction and
past studies, as previously discussed. A BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
was run on the cytochrome b gene with the parameters of Testudinidae specified under
organism search set so I could obtain only those genes within the tortoise family to
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analyze. Through this BLAST, all non-replicated species and sequences of cytochrome b
gene, partial cds that the NCBI database had for individuals in the Testudinidae were
utilized in constructing my final phylogenetic tree. The total sequences obtained for
analysis were 30, including an additional outgroup sequence chosen (Deirochelys
reticularia, GenBank: AF258877.1) to root the tree, due to its known sister taxa
relationship to the Testudinidae family, as previously discussed in the Introduction. All
sequence accession/GenBank numbers are listed in Table 1.1 for reference.
Multiple sequence alignment programs, Seaview v.4 (Gouy, Guindon, and
Gascuel, 2010) with MAFFT v.6.240-2 (Katoh et al, 2002) using einsi setting were used
to align the 30 sequences. DNA alignment, instead of proteins, was done given my
objective of constructing a phylogeny and also due to the simplicity of my dataset. To
create a maximum-likelihood (ML) based phylogenetic tree, I used RAxML v.7.0.4
(Stamatakis, 2006). Within this program, I set my parameters to run 100 bootstraps. The
output tree I used was the bipartitions tree file of which I then put into FigTree v.1.3.1
(Rambaut, 2009) to obtain a visual representation of my constructed tree. Additional
modifications, such as re-root tree modification, highlighting of clades, and various
alterations were done to my tree using FigTree v.1.3.1.
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Table 1.1. Total cytochrome b, partial cds, sequences (N = 30) used in the phylogenetic
tree construction. Each is listed with the accession numbers (NCBI database) and
scientific name.
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Results and Discussion
Based on my tree construction, I proposed to split my tree into 5 different clades
(Figure 1.1). These clades were decided on by the common ancestor nodes, but also by
some of the high bootstrap values highlighting the likelihood of these branches and
potential groupings. The most recent research (Le et al, 2006) has also used similar clade
groupings based on their phylogeny, which appeared similar to mine. Some of those
groupings with low bootstrap values were further confirmed as sharing a common
ancestor and being a clade through distinguishing possible synapomorphies shared in the
grouped taxa, in addition to adding biogeographical information to my phylogenetic tree
(Figure 1.2)
The 5 main clades represented in Figure 1.1 are as follows: Manouria +
Gopherus, Geochelone sp. + Homopus + Chersine, Geochelone sp + Kinixys,
Geochelone sp. + Pyxis + Dipsochelys, and Indotestudo + Testudo + Malacocherus.
Given some of the low bootstrap values, I turned to distinguishing any known
synapomorphies among these taxa to further strengthen their proposed clade relations.
However, if the Manouria and Gopherus clade have any apparent synapomorphies to link
these two together in a sister relationship is often debatable, with some researchers
reporting no know synapomorphies and some reporting mental glands in both (Le et al,
2006).
The Indotestudo + Testudo + Malacocherus clade is said to share a few common
characteristics. Le et al (2006) suggest possible shared characteristics to include: the
processus inferior parietalis meeting the quadrate and partially covering the prootic, a
ventral tip of the processus interfenestralis, in addition to the presence of sutures between
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this process and the surrounding bones. The 2 clades of Geochelone sp + Kinixys and
Geochelone sp. + Pyxis + Dipsochelys, both of the Indian Ocean/Madagascar Islands,
have been observed to have numerous synapomorphies among their members. G.radiata
and G. yniphora have been observed to both possess a ventral ridge on the maxillapremaxilla suture and keels on the supraocciptal crest. Additionally, the close
relationship that exists between Pyxis, G.yniphora and G.radiata has been supported by
the synapomorphy of an indistinct fenestra postotica (Le et al, 2006; Caccone et al,
2002). Within the Dipsochelys genus is still contention, as some researchers believe that
all three forms may actually represent a single species and population originating solely
from Aldabra (Le et al, 2006, Lamb and Lydeard, 1994).
The addition of geographical locations to the phylogenetic tree made for a further
confirmation of relationships among taxa and in some cases strengthened a relationship
when a low bootstrap value was present. All biogeographical information was obtained
from Le et al, 2006 & Caccone et al, 2002. As previously mentioned, the earliest
Testudine fossils were reported in Asia (Le et al, 2006; Krenz et al, 2005). Manouria and
Gopherus represent the divergence between the Asian and North American lines (Figure
1.2), which is more then likely a consequence of crossing the Bering Strait early in the
Eocene. As can be seen from Figure 1.2, two clades with low bootstrap support actually
are grouped together in the same locations. The clades: Geochelone sp. + Pyxis +
Dipsochelys and Geochelone sp. + Homopus + Chersine were the two which had the
most relationship based on geographical location, as all individuals within the clade
grouping were found on the Island of Madagascar or Africa, respectively. Geochelone
sp. + Homopus + Chersine are endemic to Africa, while there is good evidence that
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Geochelone sp. + Pyxis + Dipsochelys dispersed from Africa to the Indian Ocean area
by way of sea currents, due to tortoises well-known ability to float and survive in
freshwater for up to six months (Le et al, 2006; Caccone et al, 2002). In addition, those
tortoises located in South America (some species within the Geochelone sp + Kinixys
clade, Fig 1.2) may have also diverged from Africa by way of westward sea currents (Le
et al, 2006; Krenz et al, 2005; Caccone et al, 2002).
Given some of the low bootstrap values obtained in this analysis, some
modifications could be made if a future analysis were to be done. Additionally, since I
was only able to obtain 29 species of the 49 species, my analysis may not be as accurate
as one that contained the remaining 20 species. Since my gene options and species were
limited by the data available for the gopher tortoise and the Testudinidae family, I could
have potentially broadened my search to another nucleotide database (besides NCBI) or
another gene. Lastly, relationships among some of the proposed clades are simply known
throughout past research to be problematic and still quite unresolved. For instance, the
relationships and clade groupings of the Geochelone species are known to be very
muddled and skewed due to inadequate knowledge on which are sub-species, individual
species, or even possibly all the same species originating from one population (Caccone
et al, 2002). Until relationships like this are resolved, a complete and accurate analysis of
the relationships among the Testudinidae family will be hard to confirm and agree upon.
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Figure 1.1. Phylogenetic tree (ML) of 29 species in the Testudinidae family. Individual
clades are highlighted in individual colors.
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Figure 1.2. Phylogenetic tree (ML) of 29 species in the Testudinidae family with
emphasis on geographical locations within individual clades (colored).
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