Recap

 last  class  

q What  is  a  species  ?  

q Different  concepts  of  species  

   
 Summary  of  last  class  

§ All  the  naturalists  are  not  sa9sfied  with  any  one  of  the  concepts,    
but  every  naturalist  vaguely  knows  while  talks  about  species  

§ The  biological  concept  of  species  defines  as  the  members  of  the  
popula9on  that  freely  interbreed  and  produce  fer9le  progeny  

§ All  concepts  are  valid  to  date  but  s9ll  considered  as  flawed  
1. Typological  species  concept    
2. Nominalis9c  species  concept  
3. Biological  species  concept    
4. Evolu9onary  species  concept  
Molecular  Taxonomy  

Bama  Charan  Mondal  

Assistant  Professor  &    
DBT/Wellcome  Trust  India  Alliance  Fellow  
Department  of  Zoology,  BHU  
 Out  lines  of  today’s  class  

§ Importance  of  taxonomy  

§ Why  need  molecular  taxonomy  

§ How  it  works?    

§ Various  methods  used  in  molecular  taxonomy  

 
Importance  of  taxonomy  

Million  animal  species  are  iden9fied  and  described  by  taxonomists              

 −  this  is  ≤10%  of  the  organisms  in  our  world  
 
Several  thousand  years  will  take  to  iden9fy  and  describe  rest  of  the  species                  
 −  if  the  taxonomist  numbers  are  not  increased      
 
Why?  
 
To  know  which  species  are  endangered  or  threatened                  
                         −  we  must  know  what  they  are  and  what  we  have  to  conserve.    
Several  hundreds  of  species  may  become  ex9nct  before  we  discover  them.    

Narendran  2009  
 Taxonomy  

§ Science of classifying organisms Carl  Linnaeus  

(1707-­‐1778)  

§ Focused on morphological features and

physiological characteristics

§ Phenotypic approaches have proven unreliable,

insufficient, and challenging.
Taxonomic  Characters   Examples  
§ Bird  feather  
§ Morphological  Characters__   § Fish  scale  
§ Drosophila  sex  combs  

§ Behavioral  Characters      ____  § Courtship  pa_ern  

§ Ma9ng  song  
§ Bird  nest,  Spider  web  
§ Sweet/marine  water  fish  
§ Ecological  Characters        ____   § Fishes  in  the  pond  
§ Larvae  in  decaying  fruit  
§ Structural  protein  
§ Biochemical  Characters    ___   § Protein  hormone    
§ Neuropep9de    

§ Cytological  Characters    ____   § Karyotype  

Cytotaxonomy  
It  deals  with  the  categorizaJon  and  connecJons  of  organisms  by  applying  
comprehensive  chromosomal  research.    
 
§ Chromosome  number  is  oNen  used  and  referenced  feature  in  biology,  
§ Chromosomes’  qualiJes  (number,  structure,  and  behavior).    
§ Chromosome  numbers  are  mainly  determined  during  mitosis  (diploid).    
§ Centromere  locaJon  is  another  important  taxonomic  feature.    
§ MeioJc  acJvity  can  exhibit  heterozygosity  of  inversions  at  Jmes.    
Cytological  evidence  is  more  important  than  other  taxonomy  evidence.  
 
Cytotaxonomy  is  essenJal  because:    
o it  maintains  the  comparaJve  analysis  of  chromosomes,    
o and  tiny  differences  among  parJculars  are  frequently  idenJfied  using  this  approach.    
 
DNA  is  present  in  every  chromosome,  and  differences  in  each  DNA  are  
responsible  for  varia9on  across  people,  species,  genera,  and  everything  else.    
Taxonomic  Characters   Examples  
§ Bird  feather  
§ Morphological  Characters__   § Fish  scale  
§ Drosophila  sex  combs  

§ Behavioral  Characters      ____  § Courtship  pa_ern  

§ Ma9ng  song  
§ Bird  Nest,  Spider  web  
§ Sweet/marine  water  fish  
§ Ecological  Characters        ____   § Fishes  in  the  pond  
§ Larvae  in  decaying  fruit  
§ Structural  protein  
§ Biochemical  Characters    ___   § Protein  hormone    
§ Neuropep9de    
§ Cytological  Characters    ____   § Karyotype  
§ Molecular  Characters  
Molecular   Taxonomy  

§ Focus  on  data  derived  from  hereditary  material.    

§ Molecular  methods  enable  analysis  of  the  genotypic  features  that  

strengthen  the  similariJes  and  differences  among  all  organisms.    

§ Also,  explain  phenotypic  variaJon,  genotypic  variaJon  can  reveal  traces  

of  evoluJon  and  provide  insight  into  historical  relaJonships.  
Molecular  taxonomy:    
           all  methods  target  gene9c  features    
 
Ø  different    techniques  

Ø  used  to  assess  genus-­‐,  species-­‐  and  strain-­‐level  relaJonships  

In  1960s,  analysis  of  overall  DNA  base  composi9on  (molar  %  G+C)  varied  
among  unrelated  genera,    
 
But  these  values  were  consistent  for  species  of  the  same  genus.    
 
Give  the  potenJal  use  of  DNA-­‐based  classificaJon  for  bacteria.  
 
 
The  rise  of  molecular  taxonomy  is  credited  to  the  DNA-­‐DNA  hybridiza9on  
method  uses  the  complementarity  property  of  double-­‐stranded  DNA.  
 
Denatured  DNA  from  two  organisms  is  mixed  and    
homologous  regions  hybridize  to  duplexes.  
By  DNA-­‐DNA  hybridiza9on  method  

To  be  members  of  the  same  species,  if  >70%  of  their  genomes  hybridize    
 
It  can  dis9nguish  related  and  unrelated  species  
   
But  it  has  limita9on  to  use  for  assessing  intra-­‐species  rela9onships  
 
Labor-­‐intensive  technique    
§ require  large  amounts  of  input  DNA  
Polymerase  Chain  ReacJon  (PCR)  

1993
Gel  electrophoresis  

Stain  with  EtBr  

Under  UV  light  

Uses…  
1.  Allozyme  Polymorphisms  

Different  forms  of  enzyme  called  allozyme  

 
Aldehyde  oxidase  polymorphism  in  Drosophila  ananassae  

Ao10.98  
Ao11.00  
Ao20.98  
Ao21.00  

Kumar  2015  and  Singh  2020  

 Size  differen9al  PCR  
Employs  generic  PCR  primers  that  yields  different  length  amplicons.    
Mainly  targets  the  Intergenic  Transcribed  Spacer  regions  (ITS)  

Advantages  
§ Discriminate  a  range  of  species  simultaneously  
§ DifferenJaJon  by  electrophoresis,  no  need  sequencing  or  amplicons  processing  

Disadvantages  
§ Size  of  amplicon  needs  to  vary  substanJally  to  enable  discriminaJon  
§ ITS  regions  contain  repeJJve  regions  cause  PCR  products  with  mulJple  bands    
Summary  
Taxonomic  Characters   Examples  
§ Bird  feather  
§ Morphological  Characters__   § Fish  scale  
§ Drosophila  sex  combs  

§ Behavioral  Characters      ____  § Courtship  pa_ern  

§ Ma9ng  song  
§ Bird  Nest,  Spider  web  
§ Sweet/marine  water  fish  
§ Ecological  Characters        ____   § Fishes  in  the  pond  
§ Larvae  in  decaying  fruit  
§ Structural  protein  
§ Biochemical  Characters    ___   § Protein  hormone    
§ Neuropep9de    
§ Cytological  Characters    ____   § Karyotype  
§ Molecular  Characters  
 ConJnuaJon…  
Molecular  Taxonomy  

Bama  Charan  Mondal  

Assistant  Professor  &    
DBT/Wellcome  Trust  India  Alliance  Fellow  
Department  of  Zoology,  BHU  
bamacharan@bhu.ac.in  
scriptomes, and Proteomes
Nuclear  and  mitochondrial  components  of  the  human  genome  
l Human cell

Human family Nuclear genome Mitochondrial genome

16,500 base pairs

3,000,000,000 base pairs

Genomes | chapter 01 | figure 01
Terry The
Brown genome
| Fourth Editionis a store of biological information, but on its own it is unable
Brown  2018  
Arbitrary  Nuclear  DNA  markers  

§ RAPD  (Random  Amplified  Polymorphic  DNA)    

§ RFLP  (RestricJon  Fragment  Length  Polymorphisms)  

§ AFLP  (Amplified  Fragment  Length  Polymorphism)  

 RAPD  (Random  Amplified  Polymorphic  DNA)    

To  get  a  fingerprint  for  a  par9cular  species,    

uses  random  primer  to  get  mul9ple  PCR  products    

Only  one  primer  (8  to  15  bases)  

RAPD  analysis:  
RAPD  analysis  on  human  
RAPD  (Random  Amplified  Polymorphic  DNA)    

Advantages:  
§ Useful  for  discrimina9ng  closely  related  or  cryp9c  species  as  it  targets  
mul9ple  gene9c  loci  
§ DNA  fingerprint  generate  in  a  single  reac9on  
§ Data  used  for  phylogene9c  reconstruc9on  in  some  instances  

Disadvantages  
§ Reproducibility  issues  
§ Can’t  use  on  mixed  sample  
RFLP  (Restric9on  Fragment  Length  Polymorphisms)  
Involves  discrimina9on  of  species  based  on  restric9on  profile  of  amplicons  
RFLP  (Restric9on  Fragment  Length  Polymorphisms)  
 RFLP  (Restric9on  Fragment  Length  Polymorphisms)  
Involves  discrimina9on  of  species  based  on  restric9on  profile  of  amplicons  

Advantages  
§ Simultaneously  discriminate  a  range  of  species    
§ Used  on  a  range  of  geneJc  markers  (i.e.,  not  restricted  to  size  variable  markers)  
§ Provide  an  addiJonal  level  of  discriminaJon  if  differenJaJon  based  on  size  fails    
§ Able  to  detect  new  types  in  some  instances  

Disadvantages  
§ Requires  digesJon  of  PCR  product  
§ MutaJons  some  Jme  result  in  unidenJfied  RFLP  
 AFLP  (Amplified  Fragment  Length  Polymorphism)  
Involves  liga9on  of  adaptors  to  digested  DNA  followed  by  PCR  using  primers  
that  are  par9ally  adaptor  and  par9ally  gene-­‐specific  
AFLP  (Amplified  Fragment  Length  Polymorphism)  

Gel  picture  
AFLP  (Amplified  Fragment  Length  Polymorphism)  

Advantages  
§ Useful  for  discrimina9ng  closely  related  or  cryp9c  species  as  
it  targets  mul9ple  gene9c  loci  

§ Sensi9ve  and  robust  than  RAPD  

§ Used  for  phylogene9c  reconstruc9on  in  some  instances  

Disadvantages  
§ Requires  manipula9ons  in  addi9on  to  PCR  
§ Can’t  use  on  mixed  sample  
Specific  Nuclear  DNA  markers  
Variable  Number  of  Tandem  Repeat  (VNTR)  DNA  is  repeated  hundreds  of  
Jmes  in  human  genome.    
 
They  vary  in  number  in  different  loci  and  differently  in  individuals.    

Two  main  classes:    

1. Minisatellite:  DNA  referring  to  geneJc  loci  with  repeats  of  length  9-­‐65  bp  
2. Microsatellite:  DNA  with  repeats  of  2-­‐8  bp  long.    
 
Microsatellites  are  more  numerous  in  the  vertebrates  genome  than  
minisatellites.    
 Microsatellite  analysis  
PCR  amplifica9on  of  mul9ple  repeat-­‐containing  loci  that  are  hypervariable  
due  to  slipped-­‐strand  mispairing  muta9ons  

Advantages  
§ Targets  mulJple  geneJc  loci  that  useful  for  closely  related  or  crypJc  species.  
§ ParJcularly  useful  for  tracing  populaJons  
§ Automated  fragments  analysis  by  using  fluorescent  primers    
§ MulJplexed  PCR  for  detecJon  of  fragment  analysis  
§ Some  microsatellite  assays  apply  across  a  number  of  different  species  

Disadvantages  
§ Ini9al  assay  development  is  9me  consuming  
§ Can’t  use  on  mixed  sample  
 Figure 1. Development and use of microsatellite markers for species identification.
Microsatellite  analysis  
Microsatellite
CACACACA

Extract
Or genomic Transform
DNA Digest Ligate cut plasmids
DNA DNA into plasmid into bacteria
vector and grow
colonies

GTGTGTGT

Sequence Perform Transfer

positive hybridisation colonies onto
Design clones with microsatellite- membrane
forward and specific probes
reverse PCR
primers flanking
microsatellite
regions Fluorophore

Peaks indicate
GACTAAATCTGGTCTTATTT length of individual
CCGATTTCACACACAGATT amplicons
GTTGAGCAGTAAGGTATGA
Perform Perform
multiplex PCR fragment analysis
targeting multiple on fluorescent
microsatellites PCR products

Microsatellites occur at numerous loci within genomes and several of these markers
Jenkins  are usually
et  al.  Insects  2012  
Microsatellite  analysis  
With  advancing  technology,    

the  three  methods  to  iden9fy  microsatellites  have  exist:    

①  the  tradiJonal  method  of  screening  a  genomic  clone  library  

with  probes  complementary  to  commonly  occurring  repeat  
regions  as  previously  described;    

② an  automated  method  where  bioinforma9cs  somware  is  

used  to  search  publically  available  databases  for  
microsatellite  markers,  also  referred  to  as  data  mining;    

③  next  genera9on  sequencing  for  the  iden9fica9on  and  

screening  of  microsatellites    
scriptomes, and Proteomes
Nuclear  and  mitochondrial  components  of  the  human  genome  
l Human cell

Human family Nuclear genome Mitochondrial genome

16,500 base pairs

3,000,000,000 base pairs

Genomes | chapter 01 | figure 01
Terry The
Brown genome
| Fourth Editionis a store of biological information, but on its own it is unable
Brown  2018  
Arbitrary  Nuclear  DNA  markers  

① RAPD  (Random  Amplified  Polymorphic  DNA)    

② RFLP  (RestricJon  Fragment  Length  Polymorphisms)  

③ AFLP  (Amplified  Fragment  Length  Polymorphism)  

Specific  Nuclear  DNA  markers  
Variable  Number  of  Tandem  Repeat  (VNTR)  DNA  is  repeated  hundreds  of  
Jmes  in  human  genome.    
 
They  vary  in  number  in  different  loci  and  differently  in  individuals.    

Two  main  classes:    

1. Minisatellite:  DNA  referring  to  geneJc  loci  with  repeats  of  length  9-­‐65  bp  
2. Microsatellite:  DNA  with  repeats  of  2-­‐8  bp  long.    
 
Microsatellites  are  more  numerous  in  the  vertebrates  genome  than  
minisatellites.    
 Mitochondrial  DNA  markers  

RFLP  (RestricJon  Fragment  Length  Polymorphisms)  

Methods    
Mitochondrial  DNA  sequencing  

§ Conserved  Cytochrome  C  Oxidase  I  (COI)  gene  use  as  the  

universal  barcode  for  species  level  idenJficaJon.    
Cytochrome  c  oxidase  subunit  I  (COI  or  CO1  or  COX1  or  MT-­‐CO1)  is  
mitochondrial  DNA  encoded  subunits  of  respiratory  complex  IV.    
 
Complex  IV  is  the  third  and  final  enzyme  of  the  electron  transport  chain  of  
mitochondrial  oxidaJve  phosphorylaJon.  

LocaJon  of  the  COI  gene  in  the  human  mitochondrial  genome.    
COI  or  COX1  is  one  of  the  three  cytochrome  c  oxidase  subunit  mitochondrial  genes.  
 COI  is  a  gene  used  as  a  DNA  barcode  to  idenJfy  animal  species.    
 
The  COI  gene  sequence  is  suitable  for  this  role  because:  
§ its  muta9on  rate  is  fast  enough  to  dis9nguish  closely  related  species  
§ its  sequence  is  conserved  among  conspecifics.    
 
 
>2%  sequence  divergence  is  typically  detected  between  closely  related  animal  
species,    
 
sugges9ng  that  the  barcode  is  effec9ve  for  many  animals.    

DNA  barcodes  are  segments  of  approximately  600  base  pairs  of  
COI  gene  (5’  region)    which  help  in  cataloguing  the  biodiversity.  
DNA  barcoding  

600  base  pairs  of  COI  gene  (5’  region)    

DNA  fragments  used  for  barcoding  purpose:  

Singh  AK  2020  

DNA  barcoding  
Advantages  
§ Widely  used  in  arthropod  idenJficaJon  
§ Generic  primers  available  for  COI  barcode  region    
§ Useful  for  disJnguishing  closely  related  and  less  closely  related  taxa  
§ Alternate  markers  can  be  sequenced  if  COI  barcode  is  not  differenJal    

Disadvantages  
§ Need  database  of  sequences  for  comparison    
§ Prior  knowledge  of  the  barcoding  region  is  required    
§ Individual  sequences  not  provide  sufficient  data  when  studying  crypJc  species  
§ Mitochondrial  genes  are  maternally  inherited  that  decreased  barcode  diversity  
Jenkins  et  al.  Insects  2012  
Advantages  of  Molecular  data  

§ Heritable  
§ Unambiguous  
§ Quan9ta9ve  
§ Assessment  (homology)  is  easier  than  morphology  traits  
§ Abundant  and  robust  to    evolu9onary  distance  
§ Take  less  9me  
How  do  you  give  a  name  to  a  virus?  
Naming  the  coronavirus  disease  (COVID-­‐19)  and    
the  virus  that  causes  it  

Virus:    
Severe  Acute  Respiratory  Syndrome  Coronavirus  2  (SARS-­‐CoV-­‐2)  
Why  do  the  viruses  have  different  names?    
 
§ Different  processes  and  purposes  for  naming  viruses  and  diseases  

§ Viruses  are  named  based  on  their  gene9c  structure  to  facilitate  the  
development  of  diagnos9c  tests,  vaccines,  and  medicines    

§ Virologists  and  the  wider  scien9fic  community  do  this  work.    

§ Viruses  are  named  by  the  Interna9onal  Commi_ee  on  Taxonomy  of  
Viruses  (ICTV).    

§ ICTV  announced  “severe  acute  respiratory  syndrome  coronavirus  2  

(SARS-­‐CoV-­‐2)”  as  the  name  of  the  new  virus  on  11  February  2020.    

§ This  name  was  chosen  because  the  virus  is  gene9cally  related  to  the  
coronavirus  responsible  for  the  SARS  outbreak  of  2003.    
WHO    
2022  Nobel  Prize  in  Physiology  or  Medicine  
7 MAY 2010 VOL 328 SCIENCE www.sciencemag.org

RESEARCH ARTICLE
changed parts of their g
tors of these groups.

A Draft Sequence of the

Several features of DN
Pleistocene remains mak
The DNA is invariably d
Neandertal Genome age size of less than 200
it is chemically modified (
Richard E. Green,1*†‡ Johannes Krause,1†§ Adrian W. Briggs,1†§ Tomislav Maricic,1†§
almost always contain on
Udo Stenzel,1†§ Martin Kircher,1†§ Nick Patterson,2†§ Heng Li,2† Weiwei Zhai,3†||
dogenous DNA but large
Markus Hsi-Yang Fritz,4† Nancy F. Hansen,5† Eric Y. Durand,3† Anna-Sapfo Malaspinas,3†
microbial organisms that
Jeffrey D. Jensen,6† Tomas Marques-Bonet,7,13† Can Alkan,7† Kay Prüfer,1† Matthias Meyer,1†
after death. Over the pas
Hernán A. Burbano,1† Jeffrey M. Good,1,8† Rigo Schultz,1 Ayinuer Aximu-Petri,1 Anne Butthof,1
ancient DNA retrieval have
Barbara Höber,1 Barbara Höffner,1 Madlen Siegemund,1 Antje Weihmann,1 Chad Nusbaum,2
largely based on the pol
Eric S. Lander,2 Carsten Russ,2 Nathaniel Novod,2 Jason Affourtit,9 Michael Egholm,9
(PCR) (27). In the case o
Christine Verna,21 Pavao Rudan,10 Dejana Brajkovic,11 Željko Kucan,10 Ivan Gušic,10
Neandertals, four short ge
Vladimir B. Doronichev,12 Liubov V. Golovanova,12 Carles Lalueza-Fox,13 Marco de la Rasilla,14
determined by PCR: fragm
Javier Fortea,14 ¶ Antonio Rosas,15 Ralf W. Schmitz,16,17 Philip L. F. Johnson,18† Evan E. Eichler,7†
involved in skin pigment
Daniel Falush,19† Ewan Birney,4† James C. Mullikin,5† Montgomery Slatkin,3† Rasmus Nielsen,3†
the FOXP2 gene involved
Janet Kelso,1† Michael Lachmann,1† David Reich,2,20*† Svante Pääbo1*†
(29), parts of the ABO blo
a taste receptor gene (31).
Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe of ancient DNA can be m
and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal not allow the retrieval of
genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the genome of an organism.
Neandertal genome to the genomes of five present-day humans from different parts of the world The development of h
identify a number of genomic regions that may have been affected by positive selection in ancestral quencing technologies (33
modern humans, including genes involved in metabolism and in cognitive and skeletal development. genome-wide sequencing
We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with DNA extracted from anc
present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the and has recently made it
ancestors of non-Africans occurred before the divergence of Eurasian groups from each other. 1
 Vindija Cave into perspective with regard from Western Europe to 4- to 6-fold coverage on
rombones,
hese dertals

October 07, 2022

owder
other Neandertals, we generated a much smaller the Illumina GAII platform (SOM Text 9). These
using mans. H
amount
clean-room A sequence data from three BNe- sequences were aligned to the chimpanzee and
of DNA for the
andertal
or the pres- bones Vi33-16
from three additional
Vi33-25 Vi33-26sites (SOM human reference genomes and analyzed using a their m
, and8)three
Text that cover much of the geographical range similar approach to that used for the Neandertal wherea
ofs (Fig. 1A)
late Neandertals (Fig. 1B): El Sidron in Asturias, data. Autosomal DNA sequences of these indi- other p
Text 2]. The
Spain, dated to ~49,000 years B.P. (53); Feldhofer viduals diverged 8.2 to 10.3% back along the dertals,
usly Vi-80)
Cave in the Neander Valley, Germany, from which lineage leading to the human reference
3 by Malez genome,
Mezmaiskaya 20%, w
Neander Valley
ween sequenced
directly the type specimen found in 1856 considerably less than the 12.7% seen60-70,000
~ 40,000 in Nean- window
dated to ~42,000 years B.P. (54); and Mezmaiskaya dertals (SOM Text 10).
pectrometry We note that the diver-
Vindija Furt
> 38,000
Cave in the Caucasus, Russia, dated to 60,000 toEl Sidron
esent (B.P.) gence estimate for the Yoruba individual to the Papuan
sly used years
70,000 for B.P. (55). DNA divergences esti-~49,000 human genome sequence is ~14% greater than window
the deter-
mated for each of these specimens to the human previous estimates for an African American in- 2% div
uence (45).genome (table S26) show that none of dividual (56) and similarly greater than the
eference in the S
om layer I,
hem differ significantly from the Vindija individ- heterozygosity measured in another Yoruba in-
layer G. A Fig. 1. Samples and sites from which DNA was retrieved. (A) The three bones from Vindija from which
and 3.7
uals, although Neandertal
determined these estimates
DNA wasaresequenced.
relatively uncer- dividual
(B) Map showing the (33). This may besites
four archaeological duefrom
to differences
which bones in
were 2.2 to 2
ain due to theused
ain enough limited amount
and their of DNAdates
approximate sequence the alignment and filtering procedures between
(years B.P.). gence t
A ca
genome
www.sciencemag.org SCIENCE VOL 328 7 MAY 2010 711
low us
nC
humans
Of spe
human
(hg18) function
human- whole g
chimpanzee Neandertal genome
(panTro2) Neandertal nN nH divergence
panzee,
300000 1200000 30000
12.67% are like
6
 Neandertal genes and compared it with those from to detect positive selection in early mode

A Han- Han-
B
Neandertals French Chinese PNG Yoruba San Neandertals French Chinese PNG Yoruba San
-10

-8

-6

S
-4

-2

0
0 0

C
SNPs (ND)
SNPs
2
For  many  regions  of  the  genome,  the  variaJon  within  current  humans    is  old  enough  to  include  

(ND,s,e) / E(ND,s,e))
Fig. 4. Selective sweep screen. (A) Schematic illustration of
Neandertals  (leN).  Thus,  for  SNPs  in  present-­‐day  humans,  Neandertals  oNen  carry   1 the  derived  -­‐1  
the rationale for the selective sweep screen. For many
allele  (blue).    
regions of the genome, the variation within current humans 0
However,  in  isgenomic   r egions   w here   a n   a dvantageous  
old enough to include Neandertals (left). Thus, for SNPs m utaJon   a rises   ( right,   r ed   star)  and  sweeps  to  
high  frequency   or  fixaJon  ihumans,
in present-day n  present-­‐day  
Neandertals humans,  
oftenNeandertals  
carry thewderived
ill  be  devoid  of  -1derived  alleles.  
allele (blue). However, in genomic regions where an
 modern humans (81). The authors o
estimated the fraction of non-Africa
Han- Four  possible  scenarios  of  gene9c  mixture  involving  
French Chinese PNG Yoruba San Neandertals.    affected by “archaic” gene flow to
  almost an order of magnitude great
estimates,
Scenario  1  represents   gene   suggesting that theirfrom  
flow  into  Neandertal   observ
other  archaic  not
hominins,  
be entirely here  collecJvely  
explained referred  byto  gene
as  
Homo  erectus.  This  would  manifest  itself  as  segments  of  
Neandertals.
the  Neandertal  genome  with  unexpectedly  high  
divergence  from  pImplications
resent-­‐day  humans.   for   modern huma
One model
Scenario  2  represents   gene  for modern
flow   between   humanlate   origins s
Neandertals  aall nd  epresent-day
arly  modern  h umans  in  trace
humans Europe  
allatheir
nd/or  an
western  Asia.  
to a small African population that ex
Scenario  3  represents  gene  flow  between  Neandertals  
replaced
and  the  ancestors   of  all  narchaic forms
on-­‐Africans.   ofis  humans
This   the  most  with
Neandertals
ture.
par-­‐  simonious   Our analysis
explanaJon   of  our  oof the Neandertal
bservaJon.   Although  g
we  detect  gene  
notflow  
beonly   from  Neandertals  
compatible with this into  view
modern  
bec
humans,  gene  flow  in  the  reverse  direcJon  may  also  
dertals are on average closer to ind
have  occurred.    
Homo erectus Eurasia othan
Scenario  4  represents   to individuals
ld  substructure   in tAfrica.
in  Africa   hat   F
individuals
persisted  from   the  origin  of  in Eurasia today
Neandertals   carry regi
unJl  the  
ancestors  of  ngenome
on-­‐Africans   thatleN  are
Africa.  This  scenario  
closely relatedis  atolso  th
compaJble  with  the  current  data.    
andertals and distant from other pres
6. Four possible scenarios of genetic mixture mans. The data suggest that between 1
 Vol 464 | 8 April 2010 | doi:10.1038/nature08976

LETTERS
The complete mitochondrial DNA genome of an
unknown hominin from southern Siberia
Johannes Krause1, Qiaomei Fu1, Jeffrey M. Good2, Bence Viola1,3, Michael V. Shunkov4, Anatoli P. Derevianko4
& Svante Pääbo1

With  the  excepJon  of  Neanderthals,  from  which  DNA  sequences  of  numerous  
With the exception of Neanderthals, from which DNA sequences containing both Upper and Middle Palaeolithic elements has been
individuals  
of numerous individualshave  nhave ow  nowbeen  
been d etermined
determined 1 1 ,  the  number  
, the number reported (see and   geneJc  rInformation).
Supplementary elaJonships  of  
and genetic relationships of other hominin lineages are largely We extracted DNA from 30 mg of bone powder and converted it
other  hominin  
unknown. lineages  
Here we report a complete are  mitochondrial
largely  unknown.  
(mt) DNA Hinto ere  anwIllumina
e  report   a  complete  
sequencing library using DNA adaptors that carry
mitochondrial  
sequence retrieved from (mt)  
a bone DNA   sequence  
excavated in 2008 rin etrieved  
Denisova from   a  bone  
project-specific excavated  
barcodes. in  2a 008  
We next used recentlyin  
published protocol
Cave in the Altai Mountains in southern Siberia. It represents a for targeted sequence retrieval called primer extension capture (PEC)1
Denisova  
hitherto unknown Cave  
typeiof n  hominin
the  Altai  
mtDNAMountains  
that shares a commonin  southern  to isolate Siberia.   It  represents  
mtDNA fragments from the entirea  hmitochondrial
itherto   genome.
unknown  
ancestor with tanatomically
ype  of  hominin  
modern m human tDNA  
mtDNAs about 1.0 million years ago. This indicates that it derives
andthat   shares  aThe
Neanderthal  GAcommon   a
isolated fragments ncestor   w
were sequenced ith  a
from natomically  
both ends on the Illumina
II platform, using 76 cycles for each read. Fragments were included
modern  
from a hominin human  
migration and  outN ofeanderthal  
Africa distinct from mtDNAs  
that of the about  
in further1.0  analyses
million   years  
if their forwardago.   This  reads
and reverse indicates  
overlapped by at
that  it  ofderives   from   theabone
 hominin  
ancestors of Neanderthals and of modern humans. The stra-
tigraphy the cave where was found msuggests
igra9on   thato ut  oThis
the f  Africa  
removesdall is9nct  
fragments from   that  
bpo f  the  
least 11 base pairs (bp) and thus could be merged into single sequences.
over ,134 from analysis, but reduces
ancestors  of  Neanderthals  
Denisova hominin lived close in time
2–4
and and  of  modern  humans.  The  straJgraphy  of  the  cave  
space with Neanderthals errors at the 39 ends of Illumina reads where error rates are highest21,22.
as well as with modern humans . We generated 1,178,300 merged sequences, of which 93,349 (7.9%)
where  
The firstthominin
he  bone  
group was   found  
to leave Africaswasuggests  
Homo erectus that  about
the  Daligned
enisova   to thehrevised
ominin  
Cambridgelived   close  Sequence
Reference in  9me  (rCRS)23 using
and   space  
years w ith  N eanderthals  
. Archaeological asawell s  wasell  a s  w ith  manodern  
iterative h umans 2–4.   MIA . This alignment program is
5 1
1.9 million (Myr) ago genetic data mapping assembler,
indicate that at least two groups of hominins left Africa after this event: particularly suitable for aligning ancient DNA sequences because it
first, the ancestors of the Neanderthals between 500,000 and 300,000 takes into account the frequent sequence errors associated with base
years ago (500 and 300 kyr ago, respectively), presumably Homo damage in ancient DNA sequences24,25. Owing to the library amplifica-
PhylogeneJc  tree  of  complete  mtDNAs.    

J Krause et al. Nature 464, 894-897 (2010) doi:10.1038/nature08976

 Neanderthals differ from modern humans at an average of 202 nuc-
leotide positions, the Denisova individual differs at an average of rates
385 positions (Fig. 2), and the chimpanzee at 1,462 positions (Sup- site r
Distribu9on  
plementary of  pairwise  
Information). nucleo9de  
The Denisova hominin differences  
mtDNA thus selec
(dN/
prot
45
Human–human zees
40 nin
Number of comparisons

35 caus
30 prob
Neanderthal–human even
25
20 and
15
(see
Denisova–human A
10
the r
5
and
0 the
0 50 100 150 200 250 300 350 400
Pairwise nucleotide distance initi
perh
Pairwise  nucleoJde  differences  from  all  pairs  of  complete  mtDNAs  from  54  present-­‐  day  and  
2 | Distribution direc
one  Figure
Pleistocene   of pairwise
modern  human,   nucleotide
six  Neanderthals   and  tdifferences. Pairwise
he  Denisova  hominin  are  shown.  
nucleotide differences from all pairs of complete mtDNAs from 54 present- the
day and one Pleistocene modern human, six Neanderthals and the Denisova awai
Thank  you