26 Molecular Phylogeny of Living Primates

Dr. Vijeta Dr. Vijeta

Contents:

1:  Introduction

2:  Methods to Study Molecular Phylogeny

2.2: Ouchterlony Immunodiffusion

2.3: Molecular Clocks

2.4: Immunoelectrophoresis

2.5: Protein Radioimmunoassay

2.6: Micro complement Fixation

2.7: Major Histocompatibility Antigen Recognition

2.8: Direct Study of Genetic Material

2.8.1: DNA Hybridization and Polymerase Chain Reaction

2.8.2: Restriction Fragment Length Polymorphism Analysis

2.8.3: Southern Blotting

3:    Mitochondrial DNA

4.    Problems of Phylogenetic analysis using Molecular Data

5.  Conclusion

6.  Summary

Learning Objectives:

• To understand the molecular phylogeny of living primates
• To understand the indirect methods to study Molecular Phylogeny
• To understand the direct methods to study Molecular Phylogeny

1. Introduction

Evolution is a complex subject encompassing the history of life and its governing processes. Our understanding of evolution is based on interpretation of patterns of diversity, ecology, behavior, visible morphology, and invisible molecular structure that have changed and continue to change through time. The widely varying organic forms that surround us are a product of diversification over hundreds of millions of years of geological time. Evolutionary time, on a geological scale, is the domain of paleontology, and calibration of rates for numerous important processes depends on evidence in the fossil record. The fossil record is necessarily the ultimate test of many systematic and evolutionary hypotheses. It is less complete for some groups of organisms than one would hope, and hypotheses about their evolution are consequently untested and untestable. Primates and mammals in general, have a relatively dense and continuous fossil record permitting hypothesis of relationships and rates to be explored in more depth than would otherwise be possible. Different approaches to evolution and different scales of inquiry yield patterns appropriate for understanding different processes. Some approaches answer specific questions, while others are more general (Gingerich, 1984). The genetic relationships among the living hominoids have been the subject of numerous molecular studies over the past 30 years. Recent DNA sequencing studies have focused upon resolving the genetic relationships among the major lineages of Old World monkeys. However, a complete evolutionary tree, or phylogeny, includes both the branching order and times of divergence of the species. Estimation of divergence dates within molecular phylogenies requires external calibration of the rate of molecular evolution, typically through interpretation of a few key fossils from the group of interest. From the early catarrhine fossil record, the date of divergence of the Old World monkey and the hominoid lineages has often been estimated to be about 25 to 30 million years ago (Stewart and Disotell, 1988).

Figure-1: Primate Phylogeny Models

Source: www.wwdd2.net/evotree.html

During the past four decades the technology of determining molecular structure has advanced at a rapid pace and has become much more accessible. When the idea emerged that studying genetic material in biology is fundamental to the understanding of all animal life, it caused great excitement and enthusiasm. There can be no doubt that the subject of human molecular genetics provided deep insights into the way our body works. Scientists are now able to selectively isolate and study even single fragments of DNA thought to be genes. Within this framework was the recognition that genetic material also provides insights that makes it possible to reconstruct the evolutionary history of extant organisms, and application of this ability to the study of nonhuman primates began. Probably the first application to several primate species, long before the 1960s, was the serological research by Nuttal in 1904. Since, these beginnings molecular approaches to primatology have often been controversial. Researchers learned that it was not always possible to solve unequivocally many of the phylogenetic and taxonomic puzzles within the primate order with these new approaches; for example, the taxonomic and evolutionary position of the enigmatic tarsiers, genus Tarsius, and whether it is justifiable to group them together with the higher primates rather than with the prosimians (a view that is held by some on the basis of only a few morphological similarities that are shared between extant tarsiers and higher primates) is still undetermined. Yoder, (2003) discussed that there are numerous genetic data sets that support the contention that the Strepsirhine- tarsier-anthropoid (sic) is a virtual trichotomy, with tarsiers being so derived as to be almost irresolvable as primates (Ankel-Simons, 2000).

Figure-2: Molecular Phylogeny of Living Primates

Source: https://www.google.co.in/Perelman P, Johnson WE, Roos C, Seuánez HN, Horvath JE, Moreira MAM, et al. (2011) A Molecular Phylogeny of Living Primates. PLoS Genet 7(3): e1001342. https://doi.org/10.1371/journal.pgen.1001342

DNA is the crucial coding substance that is replicated every time a cell divides. Similar to morphological studies, molecular studies require that recognized characters be compared with similar characters that are declared to be primitive or primary. Many molecular studies necessarily remain phenetic, which means based on similarity of phenotypic characters. It is impossible to be anything but phenetic for immunology and DNA hybridization, where there are no characters present, only distances between pairs of taxa. Thus, each evaluation is firmly based in other previously established assumptions about the grade of evolutionary advance of the species that are studied. Usually such decisions about the assumed evolutionary advance of the out group species employed are based on either morphological or cytogenetic characteristics. It is easy to see that it is perplexing for the molecular primatologist to hypothesize which of his study objects-either proteins or DNA sequences-are truly primitive and which traits of the different amino acids studied are derived. Consequently, even though molecular primatology can potentially contribute interesting insights into the possible relatedness of closely associated species, it often, does not offer simple or obvious solutions to taxonomic and phylogenetic questions (Ankel-Simons, 2000).

2.    Methods to Study Molecular Phylogeny

2.1: Precipitin reaction assay

One of-the earliest indirect methods applied to primates and many other mammals in the attempt to decipher their evolutionary relationships is called the precipitin reaction assay. It makes use of the functions of the immune system and its reactions to foreign protein molecules. The preferred vector initially was blood serum. Basically this analysis measures the degree of in vitro clotting or precipitate formation. First, antigens from the organism being tested are injected into a different animal (usually a rabbit or chicken). The resulting antiserum that builds up is mixed with either the original donor blood or the blood of closely related species. Application of this precipitate elicits different degrees of coagulation in the tested blood. Thus, for example, human antiserum not only causes a strong reaction with the donor human blood, but also with the blood of chimpanzees, gorillas, macaques, and so on in decreasing intensity according to taxonomic distance (Ankel-Simons, 2000).

2.2: Ouchterlony Immunodiffusion

Another method of studying molecular relationships is to analyze antisera to blood plasma or serum, a method that was perfected by Ouchterlony in 1958 and bears his name: Ouchterlony immunodiffusion. Immunodiffusion uses two protein samples that are simultaneously diffused (spread) against an antiserum on an agar-gel test plate. Initially in investigations involving primates, antisera were made to plasma or serum. Later this method was further developed and elaborated by making, antisera, to single, purified proteins. This technique was used to test primate albumins (which are synthesized early during fetal development) by Goodman (1962), who simply confirmed primate relationships more or less as they had originally been established based- on morphological similarities and dissimilarities. For example, this immunological test corroborated that the African great apes are closer to humans than they are to the orangutan.

Immunodiffusion technology is still used in immunological comparisons of proteins for phylogenetic evaluation. Researchers continue to use immunodiffusion techniques to investigate the evolutionary relationships among primates. Unlike earlier studies, and because of deeper understanding about molecular structure and reactivity, the sera used in tests are now purified. The antigen-antibody reaction is proportional to the closeness of relationship between the primates tested (Ankel-Simons, 2000).

2.3: Molecular Clocks

Sarich and Wilson (1967) first presented the idea that immunological differences between primates should be useful for the determination of the evolutionary time frame by dating phylogenetic trees. Thus, they invented the immunological clock. However, as all clocks must be properly regulated into equivalent time components comparable to seconds, minutes, or hours, it soon became obvious that it is not possible to properly calibrate any biological event: Neither biology nor evolution ever follow a steady time frame. The characteristic of biology is that it is irregular and that it functions in a random pattern. Consequently it is not possible to properly calibrate biological events or to formulate strict, invariable biological laws.

Bauer (1974) studied individual, purified proteins rather than non purified proteins. He prepared antisera to a series of purified human serum proteins in rabbits and compared them with serum proteins of other primates. Bauer created up to fifteen antisera to different serum proteins and tested them against serum samples from three apes (Pan, Gorilla, and Pongo), four Old World monkeys (Macaca, Cercopithecus, Erythrocebus, and Cercocebus), one New World monkey (Gebus), and one prosimian primate, (Galago). Bauer then inferred the number of antigen determinant sites for each protein tested and found that this number varied from 1 to 9, with an average of 2.4. The number of determinant sites that the different genera had in common was interpreted as an indicator of evolutionary relationship. The resulting evolutionary tree was in total agreement with the evolutionary tree created with the help of morphological characters: the Galago on its own branch, as are the New World monkeys, the Old World monkeys on the third branch clustered with the three great apes (orangutan, gorilla, and chimpanzee), the gorilla and chimpanzee being closest to humans.

2.4: Immunoelectrophoresis

The next step forward in the investigation of proteins was the application of electrophoresis, which studies the differences that relate to size and electric charge of protein molecules. Electrophoresis separates charged protein or nucleic acid molecules according to their net electrical charge and mass by drawing them through a filter material (paper or gel) using an electrical field. The molecules migrate in narrow bands along “wicks” toward the electrical pole that is charged opposite to their own electrical charge (from one pole to the other) at a differential rate. They can be stained to make them more visible and subsequently compared between species. Two-dimensional resolution of electrophoresis was applied to primate molecules with the help of starch gel electrophoresis and agar-gel precipitin testing examining the reaction of a variety of antisera to proteins (Ankel-Simons, 2000).

2.5: Protein Radioimmunoassay

Today, radioimmunoassay is carried out when only minute amounts of protein are available for evaluation. This technique has also been applied to fossils. Evaluation requires the material to be radioactive (Lowenstein, 1985). Albumin is often used for this procedure because it is abundant among vertebrates, relatively stable, and easily purified and requires only small amounts of tissue.

2.6: Micro complement Fixation

Serum albumins are predominantly used in an expeditious procedure that allows comparison of differences between homologous proteins. Micro complement fixation uses reactions between soluble, antigens and antibodies that are in a dilute solution. In this medium, only high- affinity antibodies will react with their antigens. Basically, a serum is tested for its reaction with an antiserum as it is progressively diluted. At each step there is a point when maximal precipitation occurs. This method was applied to help determine the relationship between humans and chimpanzees by Sarich and Wilson (1966, 1967) as well as to the study of albumins and transferrins by Cronin and Sarich (1975) to decipher the taxonomic relationships among Old World monkeys.

2.7: Major Histocompatibility Antigen Recognition

Another procedure that measures variations among major histocompatibility antigens is used in molecular primatology studies. All body cells in mammals carry cell-surface glycogens. These glycogens are involved in antigen recognition when an immune response to some foreign substance occurs. Major histocompatibility antigens vary between individuals; two classes of these antigens are recognized:-1 MHC antigens and II MHC antigens:-

Class I MHC molecules are triggered by antigens that originate within the cell and are therefore called endogenous antigens. Fragments inside the cell that originate from a foreign protein, such as a protein encoded by the genes of a virus, become bound to the 1 MHC molecules that, unlike class II MHC cells, occur in almost all cells with a nucleus. The class I MHC molecules bind with the foreign proteins and transport them to the cell surface where they can stimulate an immune response.

Class II MHC molecules are found only on the surface of cells that are involved in immune reactions. They are therefore called exogenous antigens. Class II MHC molecules are located on macrophages that process foreign antigen fragments on the outside of the cell. These exogenous antigens (such as fragments of bacterial or viral cells) are engulfed by the cell and are subsequently fractionated within the cell and then bound to II MHC molecules. The II MHC molecules then transport, the foreign particles back to the cell’s surface, where they are exposed to and attacked by other Cells of the immune system.

Many researchers have studied hemoglobins (types of proteins or amino acid sequences) as part of their endeavor to solve questions in primate phylogeny. One result from hemoglobin research that was carried out in the 1970s was the obvious conclusion that the lorisid Nycticebus is not monophyletic with the lemur genera Eulemur and Propithecus but belongs to a branch between lemurs and anthropoid primates. It was also documented that the New and Old World monkeys are phyletically separate from each other, and that the callithrichid, genus Callithrix, is closely related to the cebid monkey, Cebus. It was furthermore concluded that the Old World monkey genus Presbytis is closely related to genera Cercopithecus and Macaca, while the lesser ape genus Hylobates stands far separated from Gorilla, Pan, and Homo, with the latter two genera more closely related to each other than to Gorilla. The most surprising result of this evaluation was the purported close relationship determined between the two New World genera Callithrix and Cebus, which are ranked in different families according to conventional classification. Somewhat later results with the help of the same, methodology concluded that the lorisid Mycticcbus together with bushbabies (genus Galago), are more closely aligned with anthropoid primates than they are with the lemuroid genera Eulemur and Propithecus. Evaluation of genus Tarsias indicated that there are several characteristics that tarsiers share with anthropoid primates but that twice as many tarsier characteristics appeared to be independently acquired. Additional proteins such as myoglobin, fibrino-peptides, or eye lens crystal (small, globular proteins that are the principal components of the lens in the mammal eye) have been studied and provided more sketchy information about alleged relationships among primates: This sketchiness is likely to have been caused by the randomly scattered availability of the diversified protein aggregates derived from only a few primates (Ankel-Simons, 2000).

2.8: Direct Study of Genetic Material

In contrast, techniques that allow the direct analysis of DNA have become more widely used during recent years, and they have been applied to problems of population genetics as well as systematics. Importantly, direct phenotype studies are now applied in the assessment of genetic diversity of endangered animal populations, which is crucial for planning species conservation in the future. Such analyses are also advantageous because small tissue samples are sufficient for analysis, and DNA can even be obtained from extinct taxa. The techniques are as follows:

2.8.1: DNA Hybridization and Polymerase Chain Reaction: One of the techniques that allow direct comparison of different DNAs is the DNA hybridization technique. This method makes it possible to estimate the degree of DNA sequence differences between genomes. DNA hybridization is a potential tool to study DNA sequence evolution that supplements phenotypical evaluation of morphological characters. Therefore, since these methods of deciphering evolutionary process are independent, when the results of both are combined and evaluated together they can potentially be of great importance for the understanding of evolutionary sequences. The techniques are as follows:-

2.8.2: Restriction Fragment Length Polymorphism Analysis: Another way to study DNA directly is DNA restriction-analysis and restriction fragment length polymorphism (RFLP), which identifies DNA pieces and their internal structure with the help of restriction enzymes. Genomic DNA can be fragmented, or cut into smaller pieces, by bacterial or yeast enzymes called Restriction endo-nucleases (REs). Most REs work by recognizing a specific six- base pair palindrome sequence (reading exactly the same from left to right and right to left) and cutting the DNA at these sequences. The results are fragments of DNA of varying length that can be separated and visualized with the help of electrophoresis.

Figure-3: Evolution of Micro RNA in Primates

Source: https://www.google.co.in/McCreight JC, Schneider SE, Wilburn DB, Swanson WJ (2017)

Evolution of microRNA in primates. PLoS ONE 12(6): e0176596.

https://doi.org/10.1371/journal.pone.0176596

2.8.3: Southern Blotting: One very useful technique that makes it possible to study single DNA genes was described by Southern in 1975 and became widely known as Southern hybridization or Southern blotting. Southern separated RE-treated DNA fragments with the help of electrophoresis on an Agarose gel strip. Identification and separation of single DNA sequences is thus made possible as the radioactively marked probe and the gene to be studied are made visible by autoradiography. For example, the taxonomic position of the enigmatic prosimian genus Tarsius in relation to other primates has been reevaluated by Koop et al. (1989). Koop and his colleagues concluded front their investigation of ỏ- and β-globin sequences (globins are the protein constituents of hemoglobin; each hemoglobin molecule consists of four globin subunits— for example, the adult human hemoglobin is a combination of two α-globins and two β-globins) that hominoids are more closely related to Cercopithecoidea, followed by Cebidae, Tarsiidae, Lemuridae, rabbits, and goats. They, then opine that it is justified to place Tarsius together with anthropoids in suborder Haplorhini. This placement, however, is solely based on the evaluation of findings regarding one globin gene cluster (β-globin cluster) and a single globin gene (ỏ-globin, a gene from the α-globin gene cluster (Ankel-Simons, 2000).

3.    Mitochondrial DNA

Mitochondrial DNA has been extensively studied in the genus Macacci, a geographically widely dispersed and species-rich group of Old World monkey. All these studies have been done under the assumption that mtDNA is totally lost from the paternal line and is only maternally inherited Human mt DNA has also been used to investigate migration patterns of human populations. For example, it has been shown that the peoples of Polynesian islands are genetically homogeneous and their mt DNA is very uniform. The Polynesian islands are therefore thought to have been only very recently colonized by a small human founder group. In contrast, the human population of the western Pacific islands shows higher levels of genetic diversity; therefore, presumably, they were settled much earlier in human history than Polynesia.

Figure-4: Molecular phylogeny and Evolution of prosimians based on complete sequences of mitochondrial DNAs

Source: https://www.google.co.in/Matsui, A., Rakotondraparany, F., Munechika, I., Hasegawa, M., & Horai, S. (2009). Molecular phylogeny and evolution of prosimians based on complete sequences of mitochondrial DNAs. Gene, 441(1), 53-66.

4.   Problems of Phylogenetic analysis using Molecular Data

In primatology, molecular data have no real meaning unless they are brought into a phylogenetic context. Although there have been many attempts to give, phylogenetic significance to findings about proteins, immunological differences, DNA sequences, or mtDNA correlations, these have often been contradictory and disappointing. The reason for this lies in the functional background of molecular data. Even if molecular differences can be documented, they usually cannot directly be correlated with any morphological features (Muller, 1994). Such undesignated, characters must then be analyzed. An additional problem is the fact that most of the genetic coding regions (called exons) are separated by extensive areas of non-coding regions (introns). This simply means that most of the DNA in eukaryotic cells is not made up of functional genes. More questions than answers are resulting from the multitude of new research. One serious problem remains the lack of communication between research-groups using different methodologies (Ankel-Simons, 2000).

5.    Conclusion

All the methods of molecular study that are described above permit only indirect conclusions about the fundamental genetic basis underlying the structure of the proteins studied. Hence they are procedures that understandably open up numerous inroads for inaccuracy. Molecular primatology is especially useful in long-term research of both population dynamics and population genetics among wild primate populations in the attempt to decipher how evolution proceeds. DNA molecules can be helpful in solving uncertain paternity relationships among individual primates within groups that are the subject of long-term social behavioral studies. In addition, genetic fingerprinting is an indispensable tool for preserving genetic diversity of captive primate populations as part of the attempt to conserve and eventually reintroduce back to the wild endangered primates. Evolutionary or taxonomic evaluation of molecular data is only practicable when it is rooted in conventional morphological assessments (Ankel-Simons, 2000).

A multitude of molecular studies address human biology in particular and primate biology in general. However, many of these studies use techniques that are not compatible with each other or are based-on molecules of unknown function and hence are not always directly comparable or meaningful.

6.   Summary

Evolution is a complex subject encompassing the history of life and its governing processes. Our understanding of evolution is based on interpretation of patterns of diversity, ecology, behavior, visible morphology, and invisible molecular structure that have changed and continue to change through time. The genetic relationships among the living hominoids have been the subject of numerous molecular studies over the past 30 years. Recent DNA sequencing studies have focused upon resolving the genetic relationships among the major lineages of Old World monkeys. However, a complete evolutionary tree, or phylogeny, includes both the branching order and times of divergence of the species. DNA is the crucial coding substance that is replicated every time a cell divides. Similar to morphological studies, molecular studies require that recognized characters be compared with similar characters that are declared to be primitive or primary. Many molecular studies necessarily remain phenetic, which means based on similarity of phenotypic characters. It is impossible to be anything but phenetic for immunology and DNA hybridization, where there are no characters present, only distances between pairs of taxa. Methods to Study Molecular Phylogeny been provided such as Ouchterlony Immunodiffusion, Molecular Clocks, Immunoelectrophoresis, Protein Radioimmunoassay, Micro complement Fixation, Major Histocompatibility Antigen Recognition and Mitochondrial DNA. Direct Study of Genetic Material can be done through DNA Hybridization and Polymerase Chain Reaction, Restriction Fragment Length Polymorphism Analysis and Southern Blotting.

References

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• Sarich, V. M., & Wilson, A. C. (1967). Immunological time scale for hominid evolution. Science, 158(3805), 1200-1203.
• Bauer, K. (1974). Cross-reactions between human and animal plasma proteins. Humangenetik, 21(2), 179-192.
• Lowenstein, J. M., Scheuenstuhl, G., Eglinton, G., Westbroek, P., & Muyzer, G. (1991). Immunological methods in molecular palaeontology. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 333(1268), 375-380.
• Cronin, J. E., & Sarich, V. M. (1975). Molecular systematics of the New World monkeys. Journal of Human Evolution, 4(5), 357-375.