The Phylogeny of Lemurs: The Problems and how these can be resolved.

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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