Transcript Speciation and Selection without Sex: the Bdelloid Rotifers

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
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.
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.
Darwin’s 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 didn’t…
Fast-forward to 2011…
The good news:
1. 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).
2. DNA sequences provide powerful tools for systematics.
The bad news:
1. 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)
2. Some biologists believe that species aren’t real.
3. 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…
Darwin: the Gap’s the Thing
sort
Gap in
Phenotypes
Cluster of similar phenotypes
Cluster of similar phenotypes
The Gap’s the Thing…But 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.
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
Species Clusters in Phylogenetic Trees of DNA
Sequences Reflect Phenotypic Clusters That We
See…But Also Detect Clusters That We Can’t See
What we see
What we infer
from sequences
But does this sequence gap separate species, or just varieties
within a species?
A Population/Evolutionary Genetic
Perspective on Species
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 > 2Ne generations.
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.
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.
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.
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.
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.
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?
?
?
?
?
Gaps: How Deep is Deep Enough
to 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 > 0.99
Gaps: How Deep is Deep Enough
to 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)
= average
q = /(1-4/3)
K = average
Express t in units of Ne
generations:
+
t
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!
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 don’t 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
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.
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 57:1465)
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:
1. Make a bootstrapped distance tree of DNA sequences from
the specimens to identify robust clades.
2. Get the pairwise sequence differences between the specimens.
3. Starting at the tip of the tree, find pairs of well-supported
sister clades and for each pair calculate K/q.
4. Use Noah Rosenberg’s 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.
5. Going toward the root of the tree, repeat until species are
found.
First Applied K/q to Delimit Species in
Asexual Organisms
Birky et al. 2010 PLoS One 5(5):1-11
QuickTime™ and a
decompressor
are needed to see this picture.
Bdelloid rotifers
Birky et al. 2005
Birky and Barraclough 2010
Fungus Penicillium
Oribatid mites
Nothrus, Platynothrus
Birky and Barraclough 2010
Green alga
Ostreococcus
Oligochaete
Lumbriculus variegatus
Heterotrophic marine flagellates
Some of Mike Robeson’s Soil Bdelloids
3 Cases Involving Singlets
K/q = 7.3
n1, n2 = 8,1
P > 0.98
K/q = 3.97
n1, n2 = 3,1
P = 0.94
K/q = 2.6
n1, n2 = 21,1
P ≈ 0.84
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 wasn’t sure that species are
real.
I just realized that this might have some advantages…
If species aren’t real, then they can’t go extinct.
We don’t need the Endangered Species Act.
Another Ancient
Asexual Organism
First Application to
Delimit Species in
Sexual Organisms
QuickTime™ and a
decompressor
are needed to see this picture.
Darwinulid ostracods
Schön, Pinto, Halse, Martens, Birky
(in preparation)
Copepod Hemidiaptomis
Federico Marrone et al. 2010
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.
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 5:e9835.)
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.
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.
1
2
3
4
q
K/q =
1 GQ8618302 GQ861828 0.0135 3 GQ861827 0.0136
0
4 GQ861826 0.0136
0
6
7
8
9
10
38
0 -
0.0038
6 GQ861832
0 .3 5 0 .3 6 0 .3 6 0 .3 6 0 .3 4 -
7 GQ861831
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 3
0.0017 -
8 GQ861825
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 3
0.0035
0.0017 -
9 GQ861824
0 .3 5 0 .3 6 0 .3 6 0 .3 6 0 .3 4
0.0073
0.0055
10 GU732830
0 .3 4 0 .3 5 0 .3 5 0 .3 5 0 .3 2
0.0052
0.0035
0.0048
0.0101 -
11 AY227378
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 2
0.007
0.0052
0.0078
0.0065
0 .3 5
11
0.0092
5 AY227379
K =
0.0115
5
0.0116
0.0117 -
q
0.008 -
0.0097 -
0.0074
Implementation of K/q Ratio Test (cont.)
4. In Noah Rosenberg’s 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 > 0.991675
Part of the table: rA
5
5
6
6
6
6
6
6
7
7
rB
4
5
1
2
3
4
5
6
1
2
TA
5
5
5
5
5
5
5
5
5
5
TB
5
5
5
5
5
5
5
5
5
5
Probability
0.990141
0.991036
0.982726
0.9872
0.989437
0.99078
0.991675
0.992314
0.983201
0.987677
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 doesn’t change the
conclusions.
Example 2: Ravens
QuickTime™ and a
decompressor
are needed to see this picture.
Common Raven
Corvus corax
QuickTime™ and a
decompressor
are needed to see this picture.
Chihuahuan Raven
Corvus cryptoleucos
Ravens (cont.)
Omland et al. (2000 Proc. R. Soc. Lond. B 267:2475; 2006 Molec.
Ecol. 15:795): 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
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.
Ravens
(cont.)
RavenCobAlignEditshort, NJ Uncorrected Tree
NJ
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 > 0.995
Common Raven-California vs.
Common Raven-Holarctic
K/q = 32.6 n1, n2 = 75, 17 P > 0.995
0.001 substitutions/site
Cynopterus horsfieldi C0399
Corvus coronoides
Corvus brachyrhynchos
Corvus albicolis MBM10981
Corvus albus LSUMZB
Corvus cryptoleucus LSUMZ
Corvus cryptoleucus AMNH
Corvus cryptoleucus NM528
Corvus cryptoleucus T X549
Corvus cryptoleucus NM602
Corvus cryptoleucus NM589
Corvus cryptoleucus NM523
Corvus cryptoleucus NM522
Corvus corax UCSB#26346
Corvus corax CA176
Corvus corax CA175
Corvus corax CA170
Corvus corax CA169
Corvus corax ID10
Corvus corax ID8
Corvus corax ID2
Corvus corax ID1
Corvus corax UCSB#26360
Corvus corax WA899
Corvus corax ID12
Corvus corax ID11
Corvus corax ID7
Corvus corax CA171
Corvus corax CA168
Corvus corax WASOA
Corvus corax UWBM#61493
Corvus corax UAM8803
Corvus corax AK955
Corvus corax Russia493
Corvus corax WACLE
Corvus corax WAF AI
Corvus corax UAM11373
Corvus corax UAM13489
Corvus corax UAM11374
Corvus corax UAM12982
Corvus corax UAM13315
Corvus corax UAM8175
Corvus corax UAM13312
Corvus corax UAM13313
Corvus corax UAM13316
Corvus corax UAM13317
Corvus corax UAM13318
Corvus corax UAM10748
Corvus corax UAM10887
Corvus corax UAM10891
Corvus corax UAM10888
Corvus corax UAM10889
Corvus corax UAM10890
Corvus corax UAM10754
Corvus corax UAM10752
Corvus corax UAM10749
Corvus corax UAM10753
Corvus corax UAM10750
Corvus corax UAM10886
Corvus corax UAM10803
Corvus corax UWBM61493
Corvus corax UWBM#53955
Corvus corax ME1
Corvus corax MA2
Corvus corax MBM#9018
Corvus corax MN353
Corvus corax UWBM57899
Corvus corax UAM10751
Corvus corax UAM10673
Corvus corax UAM8802
Corvus corax UAM13314
Corvus corax UAM13485
Corvus corax WA567
Corvus corax Siberia566
Corvus corax WI214
Corvus corax WA566
Corvus corax Mongolia899
Corvus corax Mongolia909
Corvus corax MN402
Corvus corax MN442
Corvus corax MN371
Corvus corax MN573
Corvus corax ID9
Corvus corax ID6
Corvus corax ID5
Corvus corax ID4
Corvus corax ID3
Corvus corax AK954
Corvus corax CCU86031
Corvus corax MA3
Corvus corax WAHAN
Corvus corax Siberia544
Corvus corax NM523
Corvus corax NM522
Corvus corax UAM13320
Corvus corax UAM10017
Corvus corax UAM8603
Corvus corax UAM13319
Corvus corax UAM10021
Corvus corax UAM7272
Corvus corax UAM7271
Corvus corax UAM10111
Corvus corax UAM9313
Corvus corax UAM7400
Corvus corax Siberia861
Chihuahuan
Raven
Common
RavenCalifornia
Common
RavenHolarctic
Example 3: Liverwort
Frullania 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. 56:1105-1114.
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.
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.
Example 4: Clouded Leopard
Kitchener et al., 2006
Four subspecies are actually two species (grey and black) based on phenotypes.
Clouded Leopard (cont.)
QuickTime™ and a
decompressor
are needed to see this picture.
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.
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. 48:285
Grania (cont.)
De Wit & Erséus 2010 J.
Zool Syst. Evol. Res. 48:285
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
didn’t consider these for species status
because the nuclear ITS sequence didn’t
separate them from sister clade, but it’s not
surprising that nuclear genes would
segregate later.
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.
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
doesn’t 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.
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:
1
2
3
1 GQ8618302 GQ861828 0.0135 3 GQ861827 0.0136
0
4 GQ861826 0.0136
0
No.
pairs10
5
6
7
8
9
10
11
0.0092
38
0 -
5 AY227379
0.0038
6 GQ861832
0 .3 5 0 .3 6 0 .3 6 0 .3 6 0 .3 4 -
0.0115
7 GQ861831
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 3
0.0017 -
8 GQ861825
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 3
0.0035
0.0017 -
9 GQ861824
0 .3 5 0 .3 6 0 .3 6 0 .3 6 0 .3 4
0.0073
0.0055
10 GU732830
0 .3 4 0 .3 5 0 .3 5 0 .3 5 0 .3 2
0.0052
0.0035
0.0048
0.0101 -
11 AY227378
0 .3 4 0 .3 5 0 .3 6 0 .3 6 0 .3 2
0.007
0.0052
0.0078
0.0065
K =
20
4
q
K/q =
0.0116
0.0117 -
q
0.0074
0.008 -
0.0097 -
0 .3 5
18
16
Barcode gap
14
12
10
#
pairs
8
6
4
2
0
0
0-1 1.1-2 2.1-3 ……………………….. .32 33 34
Percent sequence difference
1
2
3
4
5
6
7
8
9
10
11
35
12
36
13
14
Pairwise
differences
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:
1.Assumes species defined by systematists are real species. So systematists are
the only people who never make misteaks? Sets barcoding up for failure.
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.
+
+
=
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.
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 exists…statistical 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.
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 didn’t 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!