Speciation and Selection without Sex: the Bdelloid Rotifers

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DNA Barcoding the Right Way A Theory-based
Method for Species Detection and Identification

• Bill Birky
• Department of Ecology and Evolutionary Biology
• The University of Arizona

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Biological Diversity is Discontinuous
My goal is to understand a remarkable and general
feature of nature that the diversity of
organisms does not present to us as a continuum
but as more or less distinct clusters of
individuals with different phenotypes that we
call species.
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Why We Should Care About Species

• Species are treated as fundamental units of
  biological diversity in areas of biology
  including
• systematics
• conservation
• population genetics
• evolutionary biology
• biogeography
• any research paper where we need to specify the
  experimental organism(s)
• How we define species and distinguish one from
  another really matters.

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Darwins Conflicted Views of Species 1868 The
Origin of Species 5th edition, p. 415
Hereafter we shall be compelled to acknowledge
that the only distinction between species and
well-marked varieties is, that the latter are
known, or believed to be connected at the present
day by intermediate gradations, whereas species
were formerly thus connected. Hence, without
rejecting the consideration of the present
existence of intermediate gradations between any
two forms, we shall be led to weigh more
carefully and to value higher the actual amount
of difference between them. At this point
Darwin had got it right. In this talk I will
follow his advice and weigh more carefully the
actual amount of difference between species,
relative to the differences within species. If
only Darwin had stopped here, but he didnt
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Fast-forward to 2011

• The good news
• We now have a proliferation of models of what
  species are (theoretical/conceptual definitions,
  often called species concepts) and analytic tools
  to assign individuals to species (operational
  definitions, often called species criteria).
• DNA sequences provide powerful tools for
  systematics.
• The bad news
• We have a proliferation of models and operational
  definitions. There is a state of warfare among
  adherents to different systematic doctrines...and
  astonishingly combative language and behavior of
  some partisans.. (Doug Futuyma)
• Some biologists believe that species arent real.
• Systematics is laissez faire when it comes to
  publishing actual species descriptions. Most such
  papers make no mention of species concepts or
  operational definitions.
• My approach to delimiting Eukaryotic species

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Darwin the Gaps the Thing
sort
Gap in Phenotypes
Cluster of similar phenotypes
Cluster of similar phenotypes
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The Gaps the ThingBut How Big a Gap?
Gap in Phenotypes
Gap in Phenotypes
Gap in Phenotypes
Can be addressed using very sophisticated
morphometric, physiological, or behavioral
analyses but this is much too time-consuming for
routine use and it is no help with environmental
sequences.
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Clades in Phylogenetic Trees of DNA Sequences
Often Reflect Phenotypic Clusters That We See
What we see phenotypic gap What we infer from
sequences genotypic gap
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Species Clusters in Phylogenetic Trees of DNA
Sequences Reflect Phenotypic Clusters That We
SeeBut Also Detect Clusters That We Cant See
What we see What we infer from sequences
But does this sequence gap separate species, or
just varieties within a species?
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A Population/Evolutionary Genetic Perspective on
Species
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Causes of Gaps and Clusters in DNA Sequence Trees
Accidental variation in the numbers of offspring
(random drift) produces transient, shallow gaps
and clusters of average depth 2Ne generations.
Physical isolation, reproductive isolation, or
adaptation to different niches produces deep gaps
and clusters of mean depth gt 2Ne generations.
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The Evolutionary Genetic Species Model

• This led me to the Evolutionary Genetic Species
  Model (EGSM) Evolutionary Genetic Species are
  inclusive populations that can be shown to be
  evolving independently from each other. They are
  independent arenas for mutation, selection, and
  random genetic drift. Their independence can be
  the result of adaptation to different niches, or
  physically isolation, or both. Erratum or
  reproductive isolation.
• This is a variant of the Evolutionary Species
  Concept that is (1) explicitly genetic so we can
  use it with DNA sequences (2) does not require
  the species be adapted to different niches and
  (3) does not require knowing that independence is
  permanent. Note that it is often difficult to
  tell whether two populations are evolving
  independently due to niche divergence or physical
  isolation.

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A Species Criterion or Operational Definition

• The Evolutionary Genetic Species Model is a
  conceptual definition it needs an operational
  definition or species criterion to say whether
  two or more individuals belong to one species or
  to two or more. This can be done in a number of
  ways. For example in sexual organisms, using the
  Biological Species definition and testing
  individuals for reproductive isolation by trial
  matings or indirect inference from population
  genetic data, morphology, etc. I am focusing on
  DNA sequence data.

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We Can Use Genes to Delimit EG Species, but Which
Gene(s) Should We Use?
Ideal gene responsible for reproductive
isolation or adaptation to different niches or
first gene to complete coalescence after physical
isolation. Gene responsible for isolation
completes lineage sorting when isolation is
complete. Usually a nuclear gene(s). Problem we
rarely know what this gene is. Never know with
environmental sequences.
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What Gene Should We Use?
Other nuclear genes in sexual organism,
different genes sort at different times by
chance, ranging from about the time of speciation
through average of 2Ne 4Nf and higher.
Second best organelle gene (mitochondrial or
chloroplast). Inherited uniparentally (usually
maternally in animals and plants), and
effectively haploid. Therefore effective
population size is Nf. This is 1/4 the
effective size for nuclear genes, so completes
lineage sorting 4 times faster. Organelle genes
detect speciation earlier.
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What Gene Should We Use?
Organelle genes have other practical
advantages All copies of a gene in a cell or
organism are identical at most sites.
Consequently one can PCR-amplify an organelle
gene and sequence the amplification products
directly without cloning. The mitochondrial
barcode gene (cox1 or CO1) can be amplified
from most animals with a universal pair of
primers and has an ideal amount of diversity for
identifying species.
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Gaps How Deep Is Deep Enough?
We have a gene that can detect early stages of
speciation. A tree of such a gene should show a
gap between species. But how deep must it be to
distinguish gaps between species from gaps
between clades within species?
?
?
?
?
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Gaps How Deep is Deep Enoughto Differentiate
Species?
This is the question I will answer by calculating
probabilities that the specimens came from
independently evolving populations.
P 0.5
P 0.81
My favorite cutoff is 95, so the probability of
single species is 5.
P 0.98
Note this is a purely hypothetical case,
probabilities are very rough approximations.
P gt 0.99
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Gaps How Deep is Deep Enoughto Differentiate
Species?
We need something to compare the between-clade
distances to. Solution compare to within-clade
distances. We can get the probabilities from the
ratio of sequence difference between two sister
clades (K) to the mean sequence difference within
the clades (q).

• K f(t,u)
• Express t in units of Ne generations
• K f(Ne,u) q f(Ne,u)
• Therefore K/q is dimensionless because Ne and u
  cancel.
• Good because Ne and u are usually unknown!

• average
• q p/(1-4p/3)

t
K average

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One More Problem Sampling
We do not see the tree I showed you earlier (A)
because most lineages are extinct. Tree B is
the phylogeny of the surviving individuals. But
we dont even see all of the survivors. We make
a tree (C) based on a very small sample of
individuals from an immense population.
A
B
C
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The problem is to use this to infer this and then
define species. We have to distinguish between
gaps and clusters formed by random drift, and
gaps and clusters formed by physical isolation or
adaptation to different niches or reproductive
isolation. And we must do this based on very
small samples of very large populations of the
few survivors of evolution.
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Fortunately, Noah Rosenberg showed how one can
calculate the probability that two populations
are reciprocally monophyletic (and therefore have
been evolving independently), given that the
samples are reciprocally monophyletic and we know
the ratio K/q. (Rosenberg 2003 Evolution 571465)
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Conceptual and Operational Definitions of Species

• Now we have a conceptual definition or model of
  species, the EGSM, and an operation definition or
  species criterion using K/q. In fact K/ q
  together with the sample sizes tells us the
  probability that a sample includes specimens from
  two species.
• Briefly
• Make a bootstrapped distance tree of DNA
  sequences from the specimens to identify robust
  clades.
• Get the pairwise sequence differences between the
  specimens.
• Starting at the tip of the tree, find pairs of
  well-supported sister clades and for each pair
  calculate K/q.
• Use Noah Rosenbergs table with K/q and the
  sample sizes to get the probability that that
  the samples came from independently evolving
  populations, i.e. from different species.
• Going toward the root of the tree, repeat until
  species are found.

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First Applied K/q to Delimit Species in Asexual
Organisms
Birky et al. 2010 PLoS One 5(5)1-11
Oribatid mites Nothrus, Platynothrus Birky and
Barraclough 2010
Bdelloid rotifers Birky et al. 2005 Birky and
Barraclough 2010
Oligochaete Lumbriculus variegatus
Fungus Penicillium
Heterotrophic marine flagellates
Green alga Ostreococcus
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Some of Mike Robesons Soil Bdelloids3 Cases
Involving Singlets
K/? 7.3 n1, n2 8,1 P gt 0.98
K/? 3.97 n1, n2 3,1 P 0.94
K/? 2.6 n1, n2 21,1 P 0.84
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I published paper with Tim Barraclough and Austin
Burt showing that asexual organisms can undergo
speciation, without using the word species.
Only later discovered that Austin wasnt sure
that species are real. I just realized that this
might have some advantages If species arent
real, then they cant go extinct. We dont need
the Endangered Species Act.
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Another Ancient Asexual Organism
First Application to Delimit Species in Sexual
Organisms
Darwinulid ostracods Schön, Pinto, Halse,
Martens, Birky (in preparation)
Copepod Hemidiaptomis Federico Marrone et al. 2010
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Applying K/q Method To Sexual Organisms
We require data in which cox1 or another
organelle gene has been amplified from a sample
of individuals, sequenced in both directions to
minimize sequencing errors, and sequences trimmed
to same length to avoid comparing apples and
oranges.
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Example 1 Pterapod (Sea Butterfly) Limacina
helicina
(Hunt et al. 2010 Poles apart the Bipolar
pterapod species Limacina helicina is genetically
distinct between the Arctic and Antarctic Oceans.
PLoS ONE 5e9835.)
Phylogenetic tree of cox1 sequences shows that
north and south circumpolar populations form
well-supported clades. Hunt et al. proposed that
these represented different species. I verified
this, using K/q to show that these are different
evolutionary genetic species.
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Implementation of K/q Ratio Test

1. Align and proofread sequences, trim to same
   length, remove gaps, etc.
2. Make Neighbor-joining (NJ) and bootstrapped NJ
   trees to identify pairs of sister clades with
   robust support which are candidates for EG
   species.
3. Make matrix of pairwise sequence differences and
   calculate K/q for candidates.
4. Or better, get some or all of this information
   from other people.

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Implementation of K/q Ratio Test (cont.)
4. In Noah Rosenbergs table, look up K/q (TA or
TB only goes as high as 5 in the table) and
sample sizes (rA, rB here, 6, 5) and read
probability that the populations from which the
samples came are reciprocally monophyletic and
evolving independently P gt 0.991675 Part of the
table
Important caveat The probability assumes that
the samples are representative of the entire
population. This can be tested, for example, by
showing that increasing the number and variety of
sample locations doesnt change the conclusions.
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Example 2 Ravens
Chihuahuan Raven Corvus cryptoleucos
Common Raven Corvus corax
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Ravens (cont.)
Omland et al. (2000 Proc. R. Soc. Lond. B
2672475 2006 Molec. Ecol. 15795)
mitochondrial and nuclear DNA sequences show
three clades Chihuahuan Ravens Common Ravens
from Europe, Asia, and most of the U.S. and most
Common Ravens from the Pacific Coast.
Pacific Coast ravens
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Ravens (cont.)

• I downloaded all 101 sequences of the raven
  mitochondrial cob
• gene from GenBank, plus outgroups.
• Same procedure as with Pterapods Sequences were
  aligned (one sequence was deleted because it
  could not be aligned). Trimmed sequences to 258
  bp consisting of 76 complete codons (except one
  was missing 1 bp at 5 end and one was missing 1
  bp at 3 end). Made Neighbor-joining trees with
  and without bootstrapping to identify sister
  clades. Calculated all pairwise sequence
  differences in PAUP. All ingroup sequence
  differences were 0.06, so I made no corrections
  for multiple hits.

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Ravens (cont.)
Chihuahuan Raven
Common Raven-California
Results verify three species Common
Raven-California vs. Chihuahuan Raven Using q
from Chihuahuan K/q 2.34 n1, n2 17, 7 P
0.93 (conservative) Using q from
Common-California K/q 15.0 n1, n2 17, 7 P gt
0.995 Common Raven-California vs. Common
Raven-Holarctic K/q 32.6 n1, n2 75, 17 P gt
0.995
Common Raven-Holarctic
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Example 3 LiverwortFrullania tamarisci
(Scalewort)
Jochen Heinrichs et al. 2010 One species or at
least eight? Delimitation and distribution of
Frullania tamarisci (L.) Dumort s. l.
(Jugermanniopsida, Porellales) inferred from
nuclear and chloroplast DNA markers. Mol.
Phylogenet. Evol. 561105-1114.

• I obtained the sequences from Jochen Heinrichs
  and edited them
• 1. Deleted taxa except for the clade identified
  as Frullania tanarisci sensu lato by Heinrichs et
  al.
• 2. Removed nuclear genes, leaving concatenated
  chloroplast genes
• trnL-F atpB-rbcL.
• 3. Trimmed these to ca. same length and removed
  most gaps.
• 4. Made Neighbor-joining tree and bootstrapped NJ
  tree to identify well-supported clades.

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Liverwort(cont.)
I used K/q to verify Heinrichs conclusion that
F. tamarisci is a complex of species, and to show
that two singlets and their sister clades are
probably samples from different species.
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Example 4 Clouded Leopard
Kitchener et al., 2006 Four subspecies are
actually two species (grey and black) based on
phenotypes.
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Clouded Leopard (cont.)
Buckley-Beason et al. 2006 NJ K2P tree of 771 bp
of mtDNA verifies species based on reciprocal
monophyly and deep divergences. By inspection,
K/q 4 and P(2 species) 0.95.
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Marine Enchytraeid Oligochaete Grania
In mitochondrial cox1 tree the established
species formed well-supported clades separated
from each other by deep gaps, judged by the
authors to show absence of gene flow in a long
time despite some of the species being
sympatric. E.g. one specimen was judged to be
well-separated its sister clade and, despite
being morphologically identical to G.
postclitellachaeta, was described as a new
species, G. occulta. Examination of the cox1 tree
showed that these clades have a sufficiently
large K/q ratio to easily qualify as EG species.
PDW15 vs. other G. postclitellochaeta may also be
distinct species (open circle). De Wit Erséus
2010 Genetic variation and phylogeny of
Scandinavian species of Grania (Annelida
Clitellata Enchytraeidae), with the discovery of
a cryptic species. J. Zool Syst. Evol. Res.
48285
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Grania (cont.)
De Wit Erséus 2010 J. Zool Syst. Evol. Res.
48285
Previously described species verified by K/q New
species, verified by K/q Other K/q species?
The K/q ratio should be used to determine the
probability that the yellow starred specimens
represent new species. Authors didnt consider
these for species status because the nuclear ITS
sequence didnt separate them from sister clade,
but its not surprising that nuclear genes would
segregate later.
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Potential Problems/Limitations of K/q Method
1. Problem of female philopatry, noted by
Weisrock et al. (2010) for lemurs The use of
mitochondrial or chloroplast genes will be
misleading if two populations have no female
migration, but male migration continues. Then the
two populations will be assigned to different
species by the K/q ratio of mito genes but males
will carry nuclear genes between the populations
and prevent independent evolution. When this is
suspected, it might be appropriate to use both an
organelle gene and a nuclear gene to track
males. 2. Because coalescence is a stochastic
process, a small proportion of nuclear genes are
expected to achieve reciprocal monophyly before
organelle genes. Unfortunately it is impossible
to identify those genes in advance, and it would
be very difficult to identify them after the fact.
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Potential Problems/Limitations of K/q Method
(cont.)
3. It bears repeating that the probability
assumes that the samples are representative of
the entire population. This can be tested as I
did for the bdelloid rotifers, by showing that
increasing the number of sample locations, the
number of samples per site, and the number of
individuals in the sample doesnt change the
conclusions. Increasing the sample coverage did
not split or lump species found with smaller
samples. But when K/q is large or q is in the
usual range for the group of organisms, it is
unlikely that additional sampling will increase q
enough to reduce the ratio significantly.
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Barcode Gap
As two populations diverge, a frequency
distribution of the pairwise differences among
their sequences becomes bimodal one peak for
differences within species, the other for
differences between species. The gap between the
peaks is sometimes called the barcode gap. Sea
butterfly example
20 10 0
Barcode gap
No. pairs
pairs
Pairwise differences
0-1 1.1-2 2.1-3 .. .32 33 34 35
36 Percent
sequence difference
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Barcode Gap (cont.)
No. pairs
Sequence difference

• Used by Consortium for the Barcode of Life (CBOL)
  and the International Barcode of Life project
  (iBOL) to identify gaps between sequences from
  already-described species. Critics of barcoding
  point to cases where gap fails to distinguish
  species, or splits a species, as failures of
  barcoding. But
• Assumes species defined by systematists are real
  species. So systematists are the only people who
  never make misteaks? Sets barcoding up for
  failure.

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Barcode Gap (cont.)

• 1. Problem assumes species defined by
  systematists are real species. So systematists
  are the only people who never make misteaks?
• 2. Critics of barcoding point to cases where gap
  fails to distinguish species, or splits a
  species, as failures of barcoding. But when data
  from more than two species are pooled, the gap
  can disappear if the different species pairs have
  different diversities. Testing barcoding by
  looking for a gap in data pooled from many
  species sets it up for failure.

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Barcode Gap (cont.)

• 1. Problem assumes species defined by
  systematists are real species. So systematists
  are the only people who never make misteaks?
• 2. Critics of barcoding point to cases where gap
  fails to distinguish species, or splits a
  species, as failures of barcoding. But when data
  from more than two species are pooled, the gap
  can disappear if the different species pairs have
  different diversities. Testing barcoding by
  looking for a gap in data pooled from many
  species sets it up for failure.
• 3. As practiced by CBOL/IBOL, barcoding has no
  theoretical rationale.
• Using the evolutionary species concept or my
  version of it and the K/q ratio to delimit
  species would solve these problems.

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The K/q Ratio Is Not Exclusive

• Use of the K/q ratio does not preclude the use of
  other methods to test whether a sample includes
  specimens from 2 evolutionary genetic species.
  For example
• If one could show that the specimens fell into
  groups that could mate only with members of the
  same group, this is evidence that the sample
  includes members of two different species even if
  they are sympatric.
• If individuals in a sample came from one or the
  other of two well-separated geographic locations
  and there was no migration between them, this is
  evidence that the populations in those regions
  would be evolving independently and so are
  different species.
• Note that the sampling problem still
  existsstatistical analysis is needed!
• Finding species by using DNA sequences is not the
  end of taxonomy! Whenever it is practical,
  species found in this way should be studied to
  find morphological traits that distinguish them
  reliably. Just as in traditional systematics, the
  behavior, ecology, and distribution of the
  species should be studied.

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I GRATEFULLY ACKNOWLEDGE

• Collaborators
• Bdelloid species
• Timothy Barraclough Silwood Park
• Diego Fontaneto Silwood Park
• Giulio Melone Claudia Ricci and Giulio Melone
  University of Milan
• Darwinulid species
• Isa Schön and Koen Martens Royal Belgian
  Institute of Natural Sciences, Brusssels, Belgium
• Ricardo Pinto University of Sao Paulo, Brazil
• Stuart Halse Bennelongia Pty Ltd, Wembley WA,
  Australia
• Many people for sharing their sequence files so I
  didnt have to download them from GenBank, and
  for invaluable discussions, comments, and
  suggestions.
• Rick Michod and all my colleagues for allowing me
  to keep my lab and office so that I might
  continue doing research after retirement.
• All of you for your kind attention!

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