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DNAyou receive a blood Fossils: Could

Dr. Ross Barnett (UK)


that the mind craves.

transfusion from a Neanderthal?


The question, as strange as it sounds, gets to the crux of our yearning to truly understand extinct species of the past. What did they look like? How did they live? What blood flowed through their veins? The fossil record can tell us so much about the wonderful variety of life that this planet has sustained, but it is usually silent on the exquisite details

Over the past quarter of a century, a new way to study the recent past has emerged that really does have the power to answer questions about physiology, ecology, lifeappearance and diet. The method involves the study of DNA (deoxyribonucleic acid) from ancient material and the field is often simply known as Ancient DNA.

n 1984, a paper was published in the prestigious journal Nature (Higuchi R et al., 1984), looking at the genetic relationships of equids (horses). Using the new techniques of bacterial cloning and Sanger sequencing, the paper described attempts to identify where on the family tree the enigmatic quagga (Fig. 1) belonged. What made this paper stand out from the glut of phylogenetic studies then being published was that the last known quagga had died in captivity in Amsterdam a century before. The authors of the paper, Russ Higuchi and colleagues, had shown for the first time that DNA survived for an appreciable period after death. From this modest beginning, the field has grown almost exponentially, with constant press attention. It is thanks to the pioneering work of Higuchi that we currently now know infinitely more about whole extinct animal groups (for example, the Dinornithidae; or New Zealand Moa (Bunce M et al., 2003, 2009)) than was possible from fossils alone. In this short review, I will focus just on mammoths and hominids as these two groups serve as a good example of how the field has progressed. There is a glossary of the more technical terms (the first appearance of which in the text appears in bold italics) at the end of this article.

Mammoths
It is estimated that the permafrost of ancient Beringia (modern-day Siberia, Alaska and the Yukon) contains hundreds of mammoth mummies,

thousands of complete skeletons and millions of mammoth bones (Shapiro B et al., 2003). Locked in perennially frozen tombs, the bones, tusks and tissue retain an unparalleled biological record of the past 100,000 years (Fig. 2). As DNA degradation is subject to the same thermodynamic rules as any other chemical reaction, it has long been realised that frozen remains from polar regions should retain intact DNA longer than temperate and tropical regions. Capitalising on this in 1994, two research groups independently developed techniques for extracting DNA from mammoth bones (Hss M et al., 1994; Hagelberg E et al., 1994). At the time, this was the oldest DNA ever sequenced and served mostly as a proof of principle, showing that extinct Pleistocene megafauna were amenable to robust genetic analyses using the new technique of Polymerase Chain Reaction (see box, PCR). Since those pioneering experiments, the mammoth has been the go-to mammal for ancient DNA studies, combining as it does almost unlimited supplies, good preservation and the presence of extant close relatives (Elephas maximus, Loxodonta cyclotis and Loxodonta africana). As to which of the three surviving proboscideans the mammoth is most closely related to, the answer has proved surprisingly intractable. In all phylogenetic studies, the presence of suitable outgroups is necessary for the resolution of interrelationships, and the elephants and mammoths lack a suitable candidate. The nearest living outgroups are

Fig. 2. A mammoth tusk eroding out of a riverbank in the Russian tundra permafrost. Photo by L Dalen.

Fig. 1. The last captive quagga (Equus quagga quagga), photographed in London Zoo.

probably the Hyracoidea (hyraxes) or Sirenia (dugongs and manatees), which separated from the elephants possibly as far back as the Palaeocene. The Mammutidae (mastodons), Gomphotheriidae (gomphotheres) and Stegodontidae (stegodons), which made up the proboscidean family, are all now extinct. However, some survived into the late Pleistocene. The American mastodon, Mammut americanum, survived until the beginning of the Holocene and has been investigated through ancient DNA methods for use as a reliable outgroup. Despite early studies being plagued by contamination (see box, Problems), newer, more advanced techniques have succeeded in not only generating the complete mitochondrial genome of the mastodon (Rohland N et al., 2007), but also of a total of 18 woolly mammoths (Rogaev EI et al., 2006; Gilbert MTP et al., 2008; Gilbert MTP et al., 2007; Krause J et al., 2006; Poinar HN et al., 2006). The data has revolutionised our understanding of proboscidean evolution. We now know that the woolly mammoth and Asian elephant are sister species (Fig. 3) and actually diverged from each other roughly 2.5 to 5.6mya (Rohland N et al., 2007). Recent research has also produced the complete mitochondrial genome of the Columbian mammoth (Mammuthus columbi), long thought to be a morphologically and geographically distinct entity (Fig. 4) with a 1 to 2myr divergence from M. primigenius (Enk JM et al., 2011). Surprisingly, the Columbian mammoth mitochondrial genome appears to fall within the woolly mammoth diversity, suggesting hybridisation between the species. The massive genetic dataset of woolly mammoths has also allowed

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Woolly Mammoth Mammuthus primigenius

Asian Elephant Elephas maximus Savannah Elephant Loxodonta africana

Forest Elephant Loxodonta cyclotis

American Mastodon Mammut americanum

To Hyraxes amd Sirenians

Fig. 3. Phylogeny of elephants and mammoth with mastodon as an outgroup. Dashed line shows the distance to Hyracoidae and Sirenia.
4000BP 6500BP

Mammuthus columbi Mammuthus primigenius St. Paul Island Wrangel Island

Fig. 4. Distribution of late Pleistocene mammoth species and Holocene populations. Map outline by G Larson.

inferences to be made about their population structure and demographics. Widespread sampling coupled with radiocarbon dating has shown that mammoths exhibited population subdivision, including clades restricted to Asia and to North America. However, substantial dynamism was also found, with population replacement in Siberia by mammoths migrating from Alaska and the Yukon over the Bering land bridge (Debruyne R et al., 2008; Barnes I et al., 2007). Additionally, demographic reconstructions based on models incorporating DNA and radiocarbon data show that, unlike some Pleistocene mammals (for example, bison (Shapiro B et al. 2004) and musk-ox (Campos PF et al., 2010)), woolly mammoth did not undergo massive population declines at the same time as the deteriorating climate around the late glacial maximum (Debruyne R et al., 2008). In fact, the mainland woolly mammoth population appeared pretty healthy,

genetically - right up until their extinction. Perhaps this is evidence that their demise was due to factors so fast-acting that extermination was swift and decisive. Possibly, the best evidence as to the cause of the extinction of the mammoth has resulted from genetic studies of the last surviving island mammoth populations. Wrangel Island in the Arctic Ocean (Fig. 4) retained a viable mammoth population for more than 8,000 years after they went extinct on mainland Eurasia. Radiocarbon dates show Wrangel mammoths survived until at least 4,000 years ago in a slightly smaller (but not dwarfed) form (Vartanyan SL et al., 1993; Vartanyan SL et al., 2008). St Paul, one of the Pribilof islands in the Bering Sea (Fig. 4), also retained a Holocene mammoth population until at least 6,550 years ago (Enk JM et al., 2009; Guthrie RD, 2004). Both these populations have had ancient DNA sequenced. For the Pribilof mammoth, only one specimen was successful

for DNA work (dating from 6,550 years ago) and presented a highly unusual mitochondrial signature, different from any known mammoth population. Jacob Enk and colleagues have posited that this signature is that of the native Beringian land bridge population, whose last enclave as the sea-level rose was on the tiny (90km2) St Paul island, surviving on a remnant of mammoth steppe (Enk JM et al., 2009). It appears that humans never reached the Pribilofs before the modern era and, in the case of the St Paul mammoth, it was almost certainly a classical extinction vortex that doomed the population. The case of Wrangel Island is totally different. This large (8,000km2) island has an abundance of mammoth fossils and 42 of these have been both radiocarbon dated and sequenced for ancient DNA. The spread of dates ranging from the late Pleistocene to the mid Holocene allows some pretty interesting findings to emerge. Firstly, the Wrangel mammoths are clearly present before and after the change in sea level that actually isolated the island, showing that this was prime habitat, even before the Holocene. Secondly, although the level of genetic diversity is different in the Pleistocene and Holocene, the final Wrangel population was still diverse - indicative of a healthy, thriving population of a few hundred individuals. There is no gradual erosion of diversity as would be expected of a population that is on the brink of extinction due to natural causes. Instead, the date of last mammoths coincides very closely with the first archaeological evidence for humans on Wrangel. If it is indeed the case, as seems likely, that Homo sapiens was responsible for the extinction of the mammoth (Surovell T et al., 2005), then the onus is on us to learn as much as possible about the living animal. Towards this end, the mammoth was the second extinct animal (after the cave bear, Ursus spelaeus (Noonan JP et al., 2005)) to have its complete nuclear genome sequenced (Poinar HN et al., 2006; Miller W et al. 2008). Analysis of the mammoth genome has thrown up some unusual results. A gene for coat colour (mc1r) has been investigated and found to come in two different forms - one dark and one blonde (Workman C et al., 2010; Rmpler H et al., 2006). A

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small percentage of mammoths may have had a light coloured coat as an adaptation allowing them to keep warm in the high Arctic sun. Similarly, the genes for - and /- globin chains of mammoth haemoglobin have been sequenced and shown to vary from African and Asian elephants at key positions. Mammoth haemoglobin has even been reconstructed in the lab and shown to have a unique biochemistry. Specifically, whereas the job of haemoglobin is to deliver oxygen to cells around the body, this reaction is less efficient at low temperatures. While this confers no disadvantage to tropical elephants, an inability to oxygenate tissues at low temperatures would be fatal for arctic-dwelling mammoths. In fact, mutations in the haemoglobin protein show that, while the oxygen transfer reaction is equally efficient for mammoths and elephants at core body temperature, the mammoth haemoglobin outperforms the elephant haemoglobin at the low temperatures likely to be found in mammoth bodily extremities.

Neanderthals
Ever since the recognition of strange human-like bones from the Feldhofer Grotte in the Neander valley of Germany (Fig. 5), the evolution of our own species has been a topic of vigorous discussion. Perhaps the greatest potential the field of ancient DNA holds is in its ability to illuminate our origins and go some way to answering the question where do we come from? Unlike the permafrostpreserved mammoths, most of the remains of our putative ancestors come from tropical Africa and are either too old or too degraded to contain any viable DNA. Therefore, it is the Eurasian Neanderthals (Fig. 6) who have been the focus of productive study. In particular, the salacious idea that Neanderthals might have interbred with early modern humans has been explored through genetic means. Towards the end of the twentieth century, two schools of thought on the Neanderthal contribution to our genomes had come to an impasse. Multi-regionalism claimed that there was morphological continuity between archaic hominids and modern humans, and one merged into the other - Neanderthal to European, Homo erectus to Asian. An alternative

viewpoint suggested that as modern humans moved out of Africa and they completely replaced the archaic human species they found in Europe and Asia (Aiello LC, 1993). The first ancient DNA study on Neanderthals appeared in 1997 and painstakingly recounted the efforts of Krings and associates to retrieve DNA from the type material, a Feldhofer humerus (Krings M et al., 1997). The special problems that present themselves when working on ancient human or near-human remains were well addressed in this study. As expected, contamination with the DNA of modern humans almost completely swamped the endogenous Neanderthal DNA, thanks to over a century of intensive handling and study of these iconic fossils. Assiduous laboratory technique teased apart the contaminant sequences and true Neanderthal DNA in a series of reproducible, overlapping fragments. This tour-de-force study revealed the first authentic hominid DNA and forever changed our understanding of our relationship to the Neanderthals. In an apparent boost to the out-ofAfrica paradigm, the mitochondrial DNA sequences were clearly separate from anything found in modern human diversity, suggesting that the Neanderthals had not contributed to the modern human gene-pool. Over the next decade, ancient DNA from a total of 15 Neanderthal individuals (Fig. 6) was sequenced, including from sites outside the previously accepted Neanderthal homeland,

proving that they had ranged as far east as Siberia (Krause J et al., 2007). Each additional sequence grouped with the Feldhofer individual, to the exclusion of all modern humans (Fig. 7). It seemed that the controversy was settled - the Neanderthals had been completely replaced by modern humans and even their genes were extinct. Then, in 2006, the first million basepairs of nuclear genome were published (Green RE et al., 2006) from a Croatian Neanderthal from Vindija cave. With advances in sequencing technology, a draft genome (~3 x 109bp) was assembled

Fig. 5. The Neanderthal calvarium from the Feldhofer Cave, part of the type material for Homo neanderthalensis.

Sc En

Ok Fe
Ml

Dv

Rv Si
Ch

Vi

Mz

Tt 2000 km 1000 mi

Fig. 6. Neanderthal distribution and sites for which DNA sequences have been obtained - Si, El Sidrn, Spain; Rv, Rochers de Villeneuve, France; Ch, La-Chapelle-aux-Saints, France; Sc, Scladina Cave, Belgium; En, Engis Cave, Belgium; Fe, Feldhofer Cave, Germany; Ml, Monte Lessini, Italy; Vi, Vindija Cave, Croatia; Mz, Mezmaiskaya Cave, Russia; Tt, Teshik Tash Cave, Uzbekistan; Ok, Okladnikov Cave, Russia. Dashed line indicates the accepted extent of Neanderthal range prior to the recovery of Neanderthal DNA from the Okladnikov Cave remains. Denisova Cave (Dv) is identified in blue. Map outline by D Dalet.

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Europeans

Asians Melanesians Africans

Neanderthals

Denisovans

Time 1.04Myr 466kyr

Fig. 7. Phylogenetic relationships amongst Denisovans, Neanderthals, and modern humans inferred from the mitochondrial genome.

Polymerase Chain Reaction (PCR)


The Polymerase Chain Reaction (PCR) was developed by Kary Mullis in the early 1980s. The technique is so important to the modern eld of biology that Mullis was awarded the Nobel prize for chemistry in 1993 as a result of his work. Before PCR, there were no straightforward methods for generating DNA sequences from targeted genomic areas of interest. PCR works by co-opting the enzymatic processes that cells use in vivo to replicate their DNA and funnels it towards regions of interest to researchers. The keystone molecule to the process is the DNA polymerase from the extremophile bacterium Thermus aquaticus. Whereas, for example, DNA replication in human cells occurs at a temperature of 37C, T. aquaticus (Taq for short), which lives in Yellowstone hot springs, can replicate DNA at temperatures of up to 80C. This stability, at a temperature that would denature most other DNA polymerases, allows it to be used in a process of thermal cycling (illustration below). Basically, PCR uses short fragments of DNA (primers) that ank a region of interest, in combination with Taq polymerase and free bases, with a template extract (that is, DNA extract for bone/tissue/blood and so on) and then subjects the reaction mix to a series of di erent temperature steps. At 95C, the double stranded DNA helix unzips to produce single stranded product. At 45 to 60C, the primers can bind to the single stranded DNA anking the region of interest. At 72C, the Taq polymerase can use the free bases to replicate the complementary DNA strand adjacent to the primer. Cycling through these three temperature steps allows the production of DNA at an exponential rate. Starting with just one template DNA molecule, 45 thermal cycles could produce 2^45 or 1 x 10^13 molecules of product. It is this sensitivity and ability to generate trillions of molecules that makes the eld of ancient DNA possible.
DNA extract xxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxx 45-60c DNA Primers DNA Bases C A A C T T C G A G T G C G 72c vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv ACGTGTCAAATCGGGAA AATGCACAGTTTAG ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ C G vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

95c

vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv ^^^^^^^^^^^^^^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^

vvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvvv ^^^^^^^^^^^^^^^^^^^ VVVVVVVVVVVVVVVVVV ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

Taq Polymerase

1st cycle = 21x 2nd cycle = 22x 3rd cycle = 23x nth cycle = 2nx

Cartoon of the PCR process.

from the remains of three female Vindija Neanderthals (Green RE et al., 2010), with additional coverage of Neanderthals from El Sidrn, Feldhofer and Mezmaiskaya. The results were astounding. In complete contrast to the mitochondrial data, the nuclear data showed clear evidence of admixture between Neanderthals and modern humans (Fig. 8). As much as 4% of the genome in modern non-African people could be inherited from Neanderthals. Surprisingly, this introgression is not confined to Europeans but seems instead to be spread evenly throughout populations from Europe, Asia and Austronesia. The most parsimonious explanation for this would be hybridisation between modern humans and Neanderthals at the southernmost extent of their range in the middle-east, shortly after the sapient diaspora from Africa. Data-mining of the published genome for gene differences between Neanderthals and modern humans has provided sites of interest for future study. Of potential signicance is the fact that of six genes shown to have multiple novel xed differences in the modern human genome, three are genes expressed in the epidermis, suggesting that human skin has been subject to varying selection pressures. Related to this, a targeted investigation of mc1r in Neanderthal individuals from Spain (El Sidrn) and Italy (Monte Lessini) found a loss of function mutation not previously identied in any of 3,700 modern humans (Lalueza-Fox C et al., 2008). Present-day Europeans, with loss of function mutations in mc1r, have pale skin and/or red hair. It seems that, in a convergent fashion, Neanderthals had also evolved a pale complexion to cope with the low levels of sunlight in Europe. Even our understanding of Neanderthal behaviour is amenable to ancient DNA research. The Spanish cave of El Sidrn is unique in containing multiple Neanderthal remains from a single deposition event - likely a roof collapse that killed members of a family or group. Among the remains were identied six adults, three adolescents, two juveniles and an infant. A combined battery of nuclear and mitochondrial DNA amplications resolved the sex and mitochondrial types of the

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Neanderthals. What was fascinating was the discrepancy in the patterns presented by the adults. Whereas all the males belonged to a single mitochondrial group, three distinct matrilines were found in the females. Taken together, these findings indicate a preference for patrilocality in Neanderthals. That is, males stayed close to their region of origin and females moved between groups from a wide range (Lalueza-Fox C et al., 2011).

Problems
The sensitivity of PCR to even a single template molecule makes it invaluable for ancient DNA research and prone to contamination from exogenous sources. In the early days of PCR research, the fatal combination of a lack of comparative DNA data and a lax attitude to meaningful experimental controls resulted in a rash of highly improbable publications claiming DNA from dinosaur bones and amber insects - work that inspired Michael Crichton to write Jurassic Park. For many of these results, the sequence was simply an unrecognised region of the human genome. DNA simply cannot survive for millions of years and it is difficult to find even late Pleistocene remains with authentic DNA. To survive the criticisms directed at the field, sets of guidelines were put in place for researchers to follow with the aim of reducing spurious results. The trichotomy of sterile technique, multiple controls and reproducibility brought the field back into line and still forms the basis of lab technique today. Ancient DNA labs can resemble crime scenes as researchers are obliged to wear protective overalls, face masks and gloves to prevent their own DNA from contaminating their samples (Illustration below). All pipettes, lab surfaces and equipment must be sterile before use and all plasticware is single-use only. Controls are done in parallel with sample extractions and PCRs to pick up contamination at different stages. PCRs are attempted multiple times from multiple extracts to ensure that the final sequences are reproducible. Finally, it is good practice to have a subset of samples independently processed from extraction to PCR to sequencing at an external collaborators lab. This external replication allows researchers to put some faith in their results not being due to contaminants endemic within their own lab.

Denisova
Having advanced our understanding of the human-Neanderthal relationship, the pioneers of ancient DNA had clearly set themselves the task of deciphering the total genome of Homo neanderthalensis. However, in the search for more Neanderthal material, something unexpected happened. At a cave known as Denisova (Fig. 6), at the farthest eastern range of the Neanderthal, some archaeologists uncovered a partial human phalanx of between 48 and 30,000 years old - potentially either modern human or Neanderthal. Eager for wellpreserved remains, the ancient DNA group led by Svante Pbo at Leipzig had attempted whole mitochondrial genome sequencing, reasoning that, at best, they would recover a new Neanderthal sequence, at worst, an early modern human. Instead, they uncovered a DNA signature neither sapient nor Neanderthal (Krause J et al., 2010), but totally outside the diversity known for all (Fig. 7). There was no precedent for a discovery like this. No other species of hominid was thought to have survived so late in Eurasia. This was molecular palaeontology. The Leipzig group used all the tricks in their biochemical toolkit to attempt sequencing of the nuclear genome of Homo denisova (Reich D et al., 2010). Further surprises awaited them. The nuclear genome gave a different picture again of the relationship between Denisovans, Neanderthals and moderns. It seemed that the Denisovans were the sister group to Neanderthals, a finding at odds with the mitochondrial data (Fig. 8). Incredibly, the Denisovans may have inherited their incongruent mitochondria from an even earlier hominid, in a further case of multiregional continuity.

Working in a clean room, researchers at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, took extensive precautions to avoid contaminating Neanderthal DNA samples. Image from Wikimedia commons.

Europeans

Asians Melanesians Africans

Neanderthals

Denisovans

Time 804kyr 640kyr

Fig. 8. Phylogenetic relationships among Denisovans, Neanderthals and modern humans inferred from the nuclear genome. Bidirectional arrows indicate admixture. Green arrows, Neanderthals and the ancestors of Europeans, Asians and Melanesians. Blue arrows, Denisovans and the ancestors of Melanesians. Red arrows, an earlier, unknown hominid and the ancestors of the Denisovans, to explain the difference in topology between nuclear phylogeny (Neanderthal and Denisovan clade) and mitochondrial phylogeny (Separate Denisovan branch).

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When the Denisovan nuclear genes were compared to modern humans, this pattern continued in an unexpected way. Melanesians - the group that now occupy islands east of Wallaces line, including Papuans, Aborigines and Polynesians - may have as much as 5% of their genome derived from Denisovans. This, coupled with their additional inheritance of Neanderthal genes as non-Africans, means that around 9% of the Melanesian genome could result from admixture with archaic hominid groups (Reich D et al., 2011). How had the populations of Oceania ended up with this genetic legacy from a species only known to have definitely occupied central Asia? Perhaps the Denisovans originally had a wide range over Asia that has hitherto remained undetected in the fossil record. And perhaps the original admixed inhabitants of mainland Asia have been replaced by later waves of Homo sapiens. Whatever the answer, we are now living in the golden age of palaeogenetic research. Wellpreserved Pleistocene fossils have changed our understanding of human evolution and the evolution of other mammal groups. Future research will almost certainly take us by surprise again.

Coda
In 2008, Carles Lalueza-Fox and colleagues (Lalueza-Fox C et al., 2009) published a study on the gene responsible for the ABO generation of blood types from the bones of two Spanish Neanderthals. They found both had a sequence deletion identical to that causing the O blood group in modern humans. As O is known as the universal donor, due to a lack of reactive antigens on the red blood cells, it seems likely that you could receive a blood transfusion from at least some Neanderthals.

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Glossary
Bacterial cloning: An early method of producing large quantities of DNA. Used plasmids (autonomous circular strands of DNA) that had the ability to incorporate free DNA randomly in solution. Under certain conditions, the plasmids could be taken up by bacterial cells. These could then be grown in large quantities on agar and the plasmids+DNA sequenced. Basepairs: In DNA, there are only four chemical bases that code the genetic information - Adenine, Guanine, Cytosine and Thymine. In the double helix, each of these bases in a single strand binds to a base on a complementary strand Adenine binds to Thymine and Cytosine to Guanine. Therefore, the DNA code has the ability to replicate any double strand from a single strand, following the rules of base pairing. Since this simple rule allows the complementary base of any single strand to be easily worked out, DNA is reported as a single sequence, representing just one of the helical strands. This sequence length is reported in number of basepairs or bp, with standard units of kilobasepairs (kbp) and megabasepairs (Mbp). Clades: In molecular phylogenetics, a clade is a group of sequences (representing individuals, populations or species) that group together due to their sharing a recent common ancestor, exclusive of other sequences in the analysis. Extinction vortex: The classic, preextinction scenario, where a reduction in population size causes an increase in inbreeding, which causes a dangerous accumulation of genetic defects, which reduces the population size and so on and so forth. A well-known example is the case of the Florida panther (Puma concolor coryi), which suffered from inbreeding defects such as undescended testes. The situation was remedied by introducing puma from western populations to improve the genetic fitness of the population. Genome: The complete set of DNA present in a cell or organelle. The genome is divided into genes, the majority of which code for a protein product. The sum of the proteins coded for in the genome and the effects of the environment are what we recognise as the phenotype (that is, outward appearance) of the organism. Mammoth steppe: The productive, arid grass ecosystem that predominated in Asia and Beringia supporting megafauna like woolly rhino (Coelodonta antiquitatis) and mammoth, very different from the resource poor taiga and tundra that are found there today. Mitochondria: Within animal cells, there are self-contained structures, or organelles, that deal with the various biochemical processes of life. The mitochondrion is involved with energy production and uses carbohydrate to produce energy-rich molecules through complicated electron transfer reactions. Mitochondria are thought to have evolved from free-living bacteria that were engulfed by the earliest animal cells to act as energy-producers. A legacy of this origin is that mitochondria still retain their own independent genome (usually about 16,000bp). Mitochondria, found in the cytoplasm of the cell in great numbers, are only inherited maternally. Nuclear genome: The cytoplasmic genomes present in the mitochondria only code for a few gene products, mainly involved with energy production and protein synthesis. The nuclear genome is much larger (for example, 3 x 109bp in humans) and codes for the vast majority of protein products that determine the appearance of the living animal. Whereas the mitochondrial genome is circular, the nuclear genome is composed of chromosomes (for instance, 46 in humans) that are shuffled together randomly during sexual reproduction to produce a complicated gene mix of maternal and paternal origin for each generation. Phylogenetics: The science of elucidating evolutionary relationships between species either through study of morphological or genetic characters. The results are often visually represented as a branching tree. Molecular phylogenetic analyses can use DNA, RNA or protein sequences and computer algorithms to produce statistically robust estimates of species relationships. These trees can be calibrated through knowledge of ancestral fossil species, and then used to test hypotheses about species relationships and evolutionary timings. Polymerase: An enzyme that can produce a complimentary copy of a single strand of DNA using individual DNA bases (Adenine, Guanine, Cytosine and Thymine). Sanger sequencing: A method of sequencing DNA that uses modified bases known as labelled chain terminators. The method works by taking advantage of the probabilistic incorporation of these labelled chain terminating bases into replicating DNA strands. A DNA polymerase replicates a template molecule using normal free bases and, every so often, a labelled chain terminator is introduced. When this happens, no further replication is possible. If four parallel reactions are performed with each using a different labelled chain terminating base (that is, A, C, T or G), then a mix of products will be produced that can be separated by size on a gel, with each labelled fragment representing the occurrence of that base.

Diary of events
October 2012 to December 2012
OCTOBER 2012 m 29th l 15th 6th & 7th - Rock n Gem fair
York Racecourse.

13th - Beachy Head, Sussex

Public fossil trip with fossil hunter Roy Shepherd. Adults 12, Children (under 16) 6. Unsuitable for children under 4.

13th & 14th - Rock n Gem fair


Newton Abbot Racecourse. Kempton Park Racecourse.

27th & 28th - Rock n Gem fair


NOVEMBER 2012 m 28th l 13th 3th & 4th - Rock n Gem fair
Cheltenham Racecourse.

10th - Beachy Head, Sussex

Public fossil trip with fossil hunter Roy Shepherd. Adults 12, Children (under 16) 6. Unsuitable for children under 4.

10th, 11th - Festival of Geology

Join us for a full weekend of talks, fieldtrips, exhibits, photography competition and plenty of fun activities. Based at the University College London.

17th - Sidcup Lapidary & Mineral fair


10am-4pm at The Emmanuel Church Hall, Hadlow Road, Sidcup, DA14 4AA. Tel: 020 8300 4770. www.sidcuplapminsoc.org.uk.

17th & 18th - Rock n Gem fair


Brighton Racecours.

18th - Folkestone with the UKAFH 24th & 25th - Rock n Gem fair
Farnham Maltings.

FREE Fossil hunt to Folkestone - Kent, where we will be collecting from the famous gault clay. Contact us to book your place.

25th - Oxford Fossil and Mineral show

The shows are free to visitors after 10.30am with a charge of 2 for an earlier preview. Held at the Exeter Hall, Kidlington.

DECEMBER 2012 m 28th l 13th 8th - Beachy Head, Sussex

Public fossil trip with fossil hunter Roy Shepherd. Adults 12, Children (under 16) 6. Unsuitable for children under four.

9th - Mortimer Forest, Shropshire

FREE Fossil hunt to Mortimer Forest. We have 14 locations to visit, and we will hope to find number of superb fossils Contact us to book your place.

JANUARY 2013

m 27th l 11th

FEBRUARY 2013 m 25th l 10th 23rd - Canterbury Fossil Roadshow

This years theme is Rocks to Riches. Join us with various local Kent geology groups attending with fossils and minerals on display and childrens activities.

KEY Rock n Gem fairs - For further details:


www.rockngem.co.uk. Rockwatch - For further details, www.rockwatch.org.uk. UKAFH - Free UK fossil hunting trips. www.ukafh.co.uk. Discovering Fossils - Public fossil trips www.discoveringfossils.co.uk

Other events m Full Moon (UK) 36.

l New Moon (UK) 37.

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