This is a research focused article based on the research of A.T. McLain et al. in their paper 'A Alu-based Phylogeny of Lemurs (Infraorder Lemuriformes) The link can be found in the references at the end of the article.
The Phylogeny of Lemurs: The Problems and how these can be resolved.
“Don’t be alarmed, giant freaks! While you were asleep, we simply took you to
our little corner of heaven. Welcome to Madagascar.” (Madagascar,
2005)
King Julian
 |
| Figure 1 A Ring-Tailed Lemur, Lemur catta, a 'real life' King Julian (Author's own, 2013) |
King Julian, the loveable character from the family film,
Madagascar, springs to mind whenever you mention Lemurs, to be more precise: the
infraorder Lemuriformes. With his impressive black and white striped tail, he
is in fact a Ring-Tailed Lemur, Lemur
catta. Endemic to the island of Madagascar, this group is vastly diverse in
both the way they look (phenotypically) and where they live (ecologically). There
is strong genetic and morphological evidence that supports the theory that all
living and extinct species of Lemur descended from a single common ancestor,
known as monophyly, that colonised Madagascar between 50 and 60 million years
ago, known as the early Miocene period. (Szalay & Delson, 1979) This ancestor may
have come to inhabit the island of Madagascar by a rafting event in which it
travelled across the Mozambique Channel from African mainland. (Switek, 2010) Following this
event, Lemurs continued to diversify to inhabit a wide variety of niches found
in Madagascar, possibly as a result of mutations in the genotype. (Flegr, 2009)
Division of the
Infraorder
Currently there is approximately 101 species of Lemur (Mittermeier, et
al., 2010)
however this number differs between experts which can be split into five
families. Since the 1980s there has been an increase in the number of new
species discovered or raised from subspecies status with 41 new lemur species
discovered from 2000-2010. The five families of Lemur are as follows:
Cherogaleidae, Daubentoniidae, Indriidae, Lemuridae and Lepilemuridae.
Cherogaleidae consist of 31 species which can be categorised
into 5 genera; Allocebus, Cheirogaleus,
Microcebus, Mirza and Phaner and
are known as the mouse, dwarf and fork-marked Lemurs. The aye aye is the only
species in the family Daubentoniidae and belongs to the genus Daubentonia. Around 400mm in length,
excluding the length of the large bushy tail they are unlike other
strepsirrhine primates, a clade of wet-nosed species (Switek, 2010), as they lack a
toothcomb and do not adopt the vertical clinging and leaping style of
locomotion that is seen in other species of Lemur. (Myers, 2000)
19 species of the indris, sifakas and wooly Lemurs can be separated into 3
genera: Avahi, Indri and Propithecus and these collate to be the
family Indriidae. The fourth family is that of the Lemuridae, which consists of
24 species in 5 genera, Eulemur, Hapalemur,
Lemur, Prolemur and Varecia and
contains the ring-tailed, brown, ruffled and bamboo lemurs. Finally, the family
Lepilemuridae which is made up of 26 species all in one genus, Lepilemur, which
was once placed within the family Lemuridae. Further to these 5 families are 17
known species of extinct lemurs that can be classified into 8 genera and three
families, their extinction believed to be related to the arrival of humans on
Madagascar about 2000 years ago.
Why the
controversy?
Even though there is plenty of literature on the phylogeny
of lemurs that agree on the monophyly of the infraorder and the grouping of
lemurs next to the infraorder Lorisiformes as a sister taxa to all living
primates, there is still a lot of controversy with the phylogeny of lemurs at
all taxonomic levels. Moreover, it also granted that the family Daubentoniidae
was the first to diverge from a shared common ancestor meaning it is a basal
lineage. However the family Daubentoniidae is sometimes separated in its own infraorder,
Chiromyiformes, due to a variation in phenotype and extreme divergence over
time.
There is still hullabaloo over the relationships of the four
remaining families and two alternative family-level phylogenies have surfaced,
both still in agreement of the basal location of Daubentoniidae. A basal taxon
is a group that diverged first in the history of the taxon. The first phylogeny
placing Indriidae, Cheirogaleidae and Lepilemuridae together, all basal to
Lemuridae. The second places Indriidae and Lemuridae together, next to
Cherogaleidae and Lepilemuridae. Both these phylogenies are pivotal around the
position of the family Indriidae and whether it is a basal lineage or is a more
recent divergence.
How do we solve
this conundrum?
There have already been many studies that have been carried
out already such as karyotyping, mitochondrial DNA analysis and combinations of
molecular and morphological analysis (just to name a few) to try and unravel
this mystery. The technique discussed here was
carriede out by (McLain, et al.,
2012) has already been used previously in earlier studies examining
Lemur phylogeny by combining it with other techniques such as mitochondrial
markers. (Roos, et al., 2004) The method used involved Alu-elements which are a family of primate specific mobile elements
and are a SINE of about 300 base pairs (bp) in length found in many primate
genomes. Alu elements originate from 7SL RNA in a common ancestor of all living
primates and can be classified into sub-families; AluJ being the oldest and can thus be found within the genomes of
all living primates and the subfamily of AluL
which has been assigned to elements found withing Lemuriformes. There are also
younger subfamilies specific to other species of primate, some still presently
active and some no longer producing subfamilies or new copies.
What is a SINE?
SINE is short for, Short
Interspersed Elements, and they are a class of genetic elements, known as
non-autonomous retrotransposons, that can use RNA intermediaries to copy and
insert themselves anywhere in the host genome (amplify themselves).
What make SINEs ideal to use as genetic markers in the
establishment of evolutionary relationships?
There are several reasons as
to why SINEs are used as genetic markers, one being that they are
nearly-homoplasy-free markers. This means that there is a very small amount, if
any, elements of the DNA that arose independently through evolution. Where
there is an absence of the element, this is known as the ancestral state and
every new element that arises is a distinct evolutionary event in a lineage.
This means that, any individuals sharing the same SINE at an orthologous locus
(a gene along a length of chromosome that didn’t diverge from an ancestral
locus) are thought to be of common ancestry. Secondly, a SINE is rarely deleted
precisely once it has been inserted and finally, SINEs are fairly easy to
assess using a locus specific PCR (polymerase chain reaction) assay.
Method
So how do we use Alu-elements to determine the phylogeny
of lemurs? The process used by (McLain et al,2012) can be divided into three
stages: Computational Methodology, PCR and DNA Sequencing and Phylogenetic
Analysis
Computational Methodology
In short, this is where the
design of primers for the PCR (polymerase chain reaction) took place. They were
designed using sequence from the Microcebus
murinus (grey mouse lemur) genome, obtained from GenBank. Genomic sequence
was also obtained for the following additional species: Lemur catta (ring-tailed lemur), Eulemur macaco (black lemur), Eulemur
coronatus (crowned lemur), Propithecus
coquereli (Coquerel’s sifaka), Daubentonia
madagascariensis (aye-aye) and Cheirogaleus
medius (fat-tailed dwarf lemur). This is so they could later be compared to
the genomic data of Microcebus murinus to differentiate whether certain Alu subfamilies were distinct to
particular lemuriform lineages and so more effective location of subfamilies
specific to a particular genera could take place. The sequences for the above
species we searched for supposed lemur-specific Alu insertations based upon seven previously identified AluL concensus sequences using a
programme called RepeatMasker. A concensus sequence is a way of representing
the results of multiple sequence alignment where different sequences are
compared to each other and it is the number of most frequent residues, whether
they are nucleotides or amino acids found at each position along the alignment.
(Pierce, 2002) For easier
examination of the findings from RepeatMasker, in-house Practical Extraction
and Report Language (Perl) was used to analyse the components parts and
describe their syntactic roles, as it is optimised for data processing. Following
this, elements that were identified in sections of the genome of Microcebus murinus as members of the AluL subfamily and were more than 280
base pairs in length were compared to four non-lemuriform primate genomes;
human, chimpanzee, orangutan and rhesus macaque using the BLAST-like alignment
tool (BLAT) which, to summarise, uses an index to identify regions in the
genome which are likely to be homologous, performs an alignment between these
regions, stitches together these aligned regions into genes and then finally revisits
any small internal exons possibly missed and adjusts large gap boundaries. (Kent, 2002) Another software
suite was then used to align sequences and identify the regions that were
suitable for primer building and then the Primer3Plus program was used to
design the oligonucleotide primers to be used for the PCR assay in the regions
surrounding the element. Finally, the primers were tested using a computer
using the insilico PCR tool on the
UCSC Genome bioinformatics site against the available primate genomes.
PCR and DNA Sequencing
The process of the polymerase
chain reaction (PCR) can be split into three stages: i) Separation of the DNA
strands (denaturation), ii) Annealing of the primers and iii) extension. The
amplification of each locus was performed in 25 μl reactions each consisting of
the following: 15 ng of DNA, 200 nM of each primer, 200μM of dNTPs
(deoxynucleotriphosphates) in 50 mM of potassium chloride (KCl), 1.5 mM of
magnesium chloride (MgCl2), 10mM of Tris-hydrogen chloride with a pH
of 8.4 and 2 units of Taq polymerase.
Taq polymerase is heat stable so it
isn’t denatured along with the DNA during the initial stage.
 |
Figure 2 Overview of
the PCR process After: (Unknown, 2013)
|
The PCR reaction conditions
The initial denaturation of
the DNA is carried out at 95°C for 1 minute and is then followed by another 32
cycles of denaturation at the same temperature. This proceeded by the annealing
of the primers. The proper annealing temperature for the primers is determined
by testing them along a temperature gradient of between 48°C and 65°C. Then
finally, the samples undergo extension at 72°C for 30 seconds each and then for
another minute.
Analysis of the PCR products
The products were then
analysed using Gel Electrophoresis, a technique uses 2% agarose gels stained
with 0.25 ug ethidium bromide and visualised with UV fluorescence. The agarose
gels are submerged in a tank filled with salt solution that conducts electricity
and using a pipette, the DNA samples are placed at the end. They are originally
coloured but are stained to allow their movement to be tracked as they move.As
the phosphate groups are negatively charged, once electrified, they will be
attracted to the positive electrode of the tank. A photo of the gel can then be
taken for further analysis later on. (Cold Spring Harbour Laboratory, 2013)
Products from the individual PCRs were then cloned and sequenced using chain
termination sequencing (Figure 3). To determine whether the locus being
examined was the same element across multiple species or whether it had arisen
through a different means, the resulting sequence data was aligned and then
examined.
Phylogenetic Analysis
 |
Figure 3 Chain termination method for sequencing DNA (Urry, 2008)
|
The
sequence data was inserted into a dollo parsimony matrix to construct a dollo
parsimony phylogenetic tree (Figure 4). Dollo parsimony is a method based on the
assumption that a particular compex character has been lost during the
evolution of a lineage and that it cannot be regained. It is used when trying
to work out the evolution of the genes of different lineages as multiple gains
of the same gene are improbable. (Albert, 2007) If an Alu element was present in the PCR assay
it was coded as ‘1’ for the given locus in the matrix and likewise, if it was
absent, it was coded as ‘0’. Any loci of a given species that couldn’t be
resolved were coded with a ‘?’. To test the statistical significance of each
branch on the resulting tree, 10,000 bootstrap replicates and a statistical
test for evaluating SINE insertations based on a likelihood model were
performed. Bootstrapping is a method that derives the confidence levels for
groups within the tree, in other words, how likely there position is in the
tree. To then create a visualisation of the tree, the data was used in
conjunction with a programme called FigTree.
What does the phylogenetic tree produced show?
The tree that was produced is
shown below and every clade, bar two, were strongly supported by the maximum
likelihood test and high levels of support from the bootstrap values, displayed
as numbers above the branches of the tree. The structure of the tree supports
the previous theory of the monophyly of the infraorder,because 10 shared
insertations support this node (shown by the number under the arrow on the
phylogenetic tree).The basal lineage of Daubentoniidae is further supported by
six loci, which was anticipated based on earlier studies that placed the family
basal to the other four families. The remaining four families can be divided
into two clades, Lepilemuridae-Cheirogaleidae and an Indriidae-Lemuridae clade,
supported by the cloning and sequencing of a single insertion locus in each
(MmM97 and LI1) which confirmed that they are actually the same element.
Findings for the Lemuridae
family
10 insertations provided
support for the monophyly of the Lemuridae and following this, Lemur and Haplemur were supported by the position of 8 loci as a sister clade
to Eulemur, with Varencia determined as a sister clade to all 3 generas (Lemur, Haplemur and Varencia). Three loci were recovered that were unique to Lemur catta (Ring-tailed lemur)
supporting the convention that it is the sole species in the genus Lemur.
 |
Figure 4 The most parsimonious tree generated from analysis
of 138 Alu insertions in Lemuriformes
|
The
genus Eulemur (Simons & Rumpler, 1988) was formed to
classify the ‘true’ lemurs after they were removed from Lemur after Lemur catta was found to be the sole
species of that genus. Strong support was found for the agreement of the macaco group (Eulemur macaco and Eulemur flavifrons) however further
establishment of the relationships between the other members of the genus was
difficult, particularly
within the closely related fulvus
group, shown by polytomy in the tree. The difficulty in identifying species
specific Alu elements could be due to
the recent divergence times, about 8 million years ago. There has been debate
over whether all the 12 Eulemur species currently recognised should be awarded
full species status or should continue to be a sub-species. The nine taxa
available for this study were classified as full species however, it was not
possible to obtain lineage specific Alu
elements to support this theory for each individual species.The relationship
between the two species of the macaco group
and the four species of the fulvus is
confirmed by a total of six shared insertation loci which also excludes them
from the other three Eulemur species
tested.
Findings within the
Cheirogalidae family
As shown by the phylogenetic
tree, Cheirogaleus was found to be a
basal lineage (supported by 19 loci) to the strongly supported sister-group
relationship between Microcebus and Mirza, shown by the bootstrap values of
100.
Findings within the Indriidae
Family
Now, the family at the root
of the controversy. Altogether, 8 loci were recovered, 2 of which were taken
from nuclear DNA sequence and were present in all four Indriidae species and of
the other 6 loci, four were present in all Indriidae species also. Additionally
to this, one locus was found to be specific the Avahi laniger (Eastern Wooly Lemur)
Conclusions
The results of the
investigation provide support for the already existing morphological and
genetic research about the relationships at the species levels within the
infraorder Lemuriformes. They also resolve the problem of the unresolved
relationships between the four families of Lemuridae, Indriidae, Cheirogaleidae
and Lepilemuridae supported by a robust tree that shows the basal lineage of
Daubentoniidae and the position of Indriidae in correspondance to the other
families.
What does this mean for future work?
Further research would be
required to fully determine the branching patterns of the fulvus group within Lemuridae and of the species of Indriidae as
polytomy is still present in the phylogenetic tree, this could be determined
with more work with Alu elements or
by techniques mentioned previously in this article. However, it has been
suggested from other studies that are consensus based approaches, like the work
done by McLain et al, offer more
support and resolution than those that are coalescent based approaches, which
attempt to trace all the alleles of genes shared by all the members of a
species to a single ancestral copy. (Ting & Sterner,
2012)
In addition to this, consensus based approaches also have their weaknesses. This
questions whether the previous work on primate phylogenetics are an accurate
representation so therefore it is important to keep re-evaluating the primate
molecular relationships using a combination of approaches. (Ting & Sterner, 2012)
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