B16-EvolveC.txt

Graham L. Kendall
All these files are available on
http://www.grahamkendall.net/
Modified 6/9/2008
I am found on IRC Efnet/Undernet as glk
Email grahamkendall74135@yahoo.com
All are free to use any of this material without limit.
*******************************************************************************
==
Weaknesses of evolution
Starting this summer, the state education board will determine the curriculum
for the next decade and decide whether the "strengths and weaknesses" of
evolution should be taught. The benign-sounding phrase, some argue, is a
reasonable effort at balance. But critics say it is a new strategy taking
shape across the nation to undermine the teaching of evolution, a way for
students to hear religious objections under the heading of scientific
discourse.
---
Already, legislators in a half-dozen states -- Alabama, Florida, Louisiana,
Michigan, Missouri and South Carolina -- have tried to require that classrooms
be open to "views about the scientific strengths and weaknesses of Darwinian
theory," according to a petition from the Discovery Institute, the
Seattle-based strategic center of the intelligent design movement.
---
The story mainly covers the local Texas aspects of the story, with quotes from
the state education board chairman ("I believe a lot of incredible things") and
some pro-evolution opponents.
I looked at the website where the Texans for Better Science Education lay out
examples of the "weaknesses" that should be taught. They're pretty weak, all
right. I think that most of these could be included in a science course as
"common myths about evolutionary theory."
Consider these:
---
The Cambrian explosion quickly produced all of the basically different body
structures, and some of these have since become extinct. This is very
different from the evolutionary tree of life, which suggests a slow and
gradual increase in body structures.
---
No, no it doesn't. Evolutionary theory provides no reason to think that body
structures should change at a slow constant rate. The synthetic theory
emphasizes why bursts of adaptive change should happen episodically.
---
Many life forms persist through large expanses of geologic time with
essentially no change. Evolution theory suggests that mutations occur randomly
over time and are selected to produce continuing change as the environment
continually changes.
---
No, no it doesn't. Some organisms may well have relatively constant
environments for millions of years.
---
Selective breeding has produced only very limited change with no new
structures occurring over thousands of years and multitudes of generations of
selection.
---
Umm... teosinte? I think that biology texts should devote a lot more attention
to selective breeding, as the best concrete examples of evolution in action.
So, that reflects on the basic problem with the idea of teaching evolution's
"weaknesses": A real weakness is not a matter of ignorance, but a matter of
evidence weighing in favor of some alternative hypothesis. We don't have that
here.


==
Cell manufacture
http://www.scienced aily.com/ releases/ 2008/06/08060414 0959.htm
[go to link for full article]

New Way To Think About Earth's First Cells

A team of researchers at Harvard University have modeled in the
laboratory a primitive cell, or protocell, that is capable of
building, copying and containing DNA.

Since there are no physical records of what the first primitive cells
on Earth looked like, or how they grew and divided, the research
team's protocell project offers a useful way to learn about how
Earth's earliest cells may have interacted with their environment
approximately 3.5 billion years ago.
The protocell's fatty acid membrane allows chemical compounds,
including the building blocks of DNA, to enter into the cell without
the assistance of the protein channels and pumps required by today's
highly developed cell membranes. Also unlike modern cells, the
protocell does not use enzymes for copying its DNA.
Led by Jack W. Szostak of the Harvard Medical School, the research
team published its findings in the June 4, 2008, edition of the
journal Nature's advance online publication.
"Szostak's group took a creative approach to this research challenge
and made a significant contribution to our understanding of small
molecule transport through membranes," said Luis Echegoyen, director
of the NSF Division of Chemistry.
When the team started its work, the researchers were not sure that the
building blocks required for copying the protocell's genetic material
would be able to enter the cell.
"By showing that this can happen, and indeed happen quite efficiently,
we have come a little closer to our goal of making a functional
protocell that, in the right environment, is able to grow and divide
on its own," said Szostak.
"We have found that membranes made from fatty acids and related
molecules -- the most likely components of primitive cell membranes --
have properties very different from those of the modern cell membrane,
which uses specialized pumps, channels or pores to control what gets
in and out," says Jack Szostak, PhD, of the MGH Department of
Molecular Biology and Center for Computational and Integrative
Biology, the report's senior author. "Our report shows that very
primitive cells may have absorbed nutrients from their environment,
rather than having to manufacture needed materials internally, which
supports one of two competing theories about fundamental properties of
these cells."
Szostak's team carefully analyzed vesicles comprised of different
fatty acid molecules and identified particular features that made
membranes more or less permeable to potential nutrient molecules. They
found that, while large molecules such as strands of DNA or RNA could
not pass through fatty acid membranes, the simple sugar molecules and
individual nucleotides that make up larger nucleic acids easily
crossed the membrane.
To further explore the function of a fatty acid cell membrane, the
researchers used activated nucleotides they developed for this study
that will copy a DNA template strand without needing the polymerase
enzyme usually required for DNA replication. After placing template
molecules inside fatty-acid vesicles and adding the activated
nucleotides to the external environment, they found that additional
DNA was formed within the vesicles, confirming that the nucleotide
molecules were passing through the fatty-acid membranes.

Related references:

Jack W. Szostak, Ph.D.
http://www.hhmi. org/research/ investigators/ szostak_bio. html

Mineral Surface Directed Membrane Assembly
(Origins of life and evolution of the biosphere, Feb. 2008; 37(1):67-82)
http://genetics. mgh.harvard. edu/szostakweb/ publications/ Szostak_pdfs/
Hanczyc_et_ al_2006_OLEB. pdf
[link may be line-wrapped]

Structure and Evolutionary Analysis of a Non-biological ATP-binding
Protein
(Journal of Molecular Biology, Aug. 10 2007; 371(2):501-13)
http://genetics. mgh.harvard. edu/szostakweb/ publications/ Szostak_pdfs/
mansy_etal_ JMB_2007. pdf


==
Parasite turns host into bodyguard
Many parasites simply eat away at their hosts from the inside. That may be bad
enough. But some go further: they manipulate their hosts behavior to benefit
themselves, producing some of natures most cruel and strange spectacles.
One notable example is that of the hairworm, which somehow induces its insect
hosts to commit suicide by jumping into water, where the hairworms go to
reproduce.
In this experimental setup, a caterpillar knocks a small parasitic bug off its
twig. The bug is seen rapidly dropping down at the lower right of the twig; the
caterpillar still hasn't finished its violent swing. Above the caterpillar are
the lightcolored wasp cocoons. (Courtesy A.H. Grosman et al.)
A new study describes yet another strange case of apparently parasiteinduced
behavioral changes: a creature that turns its host into its own, suicidally
devoted bodyguard.
After the parasitic wasp Glyptapanteles completes an early life stage as an
uninvited guest in the body of a caterpillar, the caterpillar exhibits
stunning changes, according to researchers. It stops eating and stays close by
the wasps, which by then are cocoons. It wraps them in a protective web of silk
and defends them against approaching predators with violent, relentless
headswings.
It continues this until the wasps emerge from their cocoons, then it dies,
according to the scientists, from the University of Amsterdam and University
of Vicosa in Brazil.
In experiments, when presented with a small predatory insect, 17 out of 19
parasitized caterpillars lashed out at the bug with repeated violent
headswings, the investigators wrote in a paper describing their work.
By contrast, only one of 20 unparasitized caterpillars showed this behaviour,
they wrote. The others hardly responded to the presence of the predator, even
when it was walking on the host. The paper is published June 4 in the online
research journal PLoS One.
The wasps bizarre life cycle begins when an adult lays eggs inside the
caterpillar. These develop into larvae that live on the animals body fluids.
They eventually crawl out of the caterpillar, to become cocoons shortly in
preparation for adulthood.This is when the caterpillar, known as Thyrinteina
leucocerae, acts as a bodyguard, the researchers said. The cocoons and
caterpillar live next to each other on a twig as the drama plays out.
Strictly speaking, the wasp is called a parasitoid, not a parasite, because it
only spends part of its life cycle as a parasite.
The researchers found that parasitoid cocoons guarded by caterpillars in the
wild suffered half as much predation as those without a bodyguard.
Its unclear how the wasp changes the behaviour of its host. But interestingly,
the investigators said, one or two parasitoid larvae normally remained behind
in the host after the others left. These larvae may be the ones that change
the caterpillars behaviour, thus sacrificing themselves for their brothers and
sisters, the researchers speculated.
==
 Hominids by Robert J. Sawyer, This book looks at the history of humankind on
this planet and all we have done to it.
==
Yeast use many of the same enzymes and cofactor vitamins and
minerals as humans and are an excellent model for human metabolism.

There are over 600 human enzymes that use vitamins or minerals as cofactors
==
A mutation permitting tooth development in chicken
embryos shows not only avian archosaur ancestry but
also process by which toothlessness arose in birds:
==
Darwinian: Our species homo sapiens arose, like all other species, from the
ordinary processes of evolution, which have continued to the present day.
Human nature is a collection of characteristics all susceptible to biological
explanation. These characteristics show variation in any one population. A
human population that breeds mostly within itself for many generations will
develop distinctive profiles of variation, as a result of ordinary biological
laws, causing it to diverge from other such populations. Neither individual
human beings nor human populations are equal. Some human-nature
characteristics can be shaped to some degree by "cultural" (i.e. social or
environmental) forces; some cannot. Biology rules!
==
Self-assembled Viruses Efficiently Carry Genes And Drug Molecules Into
Tumor Cells
http://www.scienced aily.com/ releases/ 2008/05/08053010 2627.htm

ScienceDaily (Jun. 2, 2008) (I12(B Viruses are true experts at importing
genetic material into the cells of an infected organism. This trait is
now being exploited for gene therapy, in which genes are brought into
the cells of a patient to treat genetic diseases or genetic defects.
Korean researchers have now made an artificial virus. As described in
the journal Angewandte Chemie, they have been able to use it to
transport both genes and drugs into the interior of cancer cells.

Natural viruses are extremely effective at transporting genes into
cells for gene therapy; their disadvantage is that they can initiate
an immune response or cause cancer. Artificial viruses do not have
these side effects, but are not especially effective because their
size and shape are very difficult to control(I12(Bbut crucial to their
effectiveness. A research team headed by Myongsoo Lee has now
developed a new strategy that allows the artificial viruses to
maintain a defined form and size.

The researchers started with a ribbonlike protein structure ((IA(B-sheet)
as their template. The protein ribbons organized themselves into a
defined threadlike double layer that sets the shape and size. Coupled
to the outside are "protein arms" that bind short RNA helices and
embed them. If this RNA is made complementary to a specific gene
sequence, it can very specifically block the reading of this gene.
Known as small interfering RNAs (siRNA), these sequences represent a
promising approach to gene therapy.

Glucose building blocks on the surfaces of the artificial viruses
should improve binding of the artificial virus to the glucose
transporters on the surfaces of the target cells. These transporters
are present in nearly all mammalian cells. Tumor cells have an
especially large number of these transporters.

Trials with a line of human cancer cells demonstrated that the
artificial viruses very effectively transport an siRNA and block the
target gene.

In addition, the researchers were able to attach hydrophobic (water
repellant) molecules(I12(Bfor demonstration purposes a dye(I12(Bto the
artificial viruses. The dye was transported into the nuclei of tumor
cells. This result is particularly interesting because the nucleus is
the target for many important antitumor agents.


==
Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness
Abstract
During the development of mammalian digits, clustered Hoxd genes are expressed
following a collinear regulatory strategy, leading to both the growth of digits
and their morphological identities. Because gene dosage is a key parameter in
this system, we used a quantitative approach, associated with a collection of
mutant stocks, to investigate the nature of the underlying regulatory
mechanism(s). In parallel, we elaborated a mathematical model of quantitative
collinearity, which was progressively challenged and validated by the
experimental approach. This combined effort suggested a two-step mechanism,
which involves initially the looping and recognition of the cluster by a
complex including two enhancer sequences, followed by a second step of
microscanning of genes located nearby. In this scenario, the respective rank
of the genes, with respect to the 5 extremity of the cluster, is primordial,
as well as different gene-specific affinities. This model accounts for the
quantitative variations observed in our many mutant strains, and reveals the
molecular constraint leading to thumbness; i.e., why a morphological
difference must occur between the most anterior digit and the others. We also
show that the same model applies to the collinear regulation of Hox genes
during the emergence of external genitalia, though with some differences
likely illustrating the distinct functionalities of these structures in adults.

In vertebrates, Hox genes belonging to the HoxA and HoxD clusters are
necessary for the development of the limbs (see Zakany and Duboule 2007).
Genes of both clusters are expressed in a complex manner, mostly through two
different phases, which depend on distinct regulatory controls (Nelson et al.
1996; Deschamps and van Nes 2005; Tarchini and Duboule 2006). During early
limb budding, Hoxd gene transcription is activated in a collinear fashion,
starting with Hoxd1 and progressing toward the 5 end of the cluster (Hoxd13).
This early phase of activation leads to the concomitant restriction in the
expression of the most 5-located genes (from Hoxd10 to Hoxd13) into the most
posterior-distal cells of the developing limb bud. Eventually, these posterior
Hox gene products will activate transcription of the gene sonic hedgehog
(Tarchini et al. 2006), a key determinant of limb growth and polarity (Riddle
et al. 1993), necessary for the second phase of limb development to occur.

This early phase of Hox genes activation corresponds to the organization and
patterning of both the stylopodium (the arm) and the zygopodium (the forearm).
This phase is soon followed by the morphogenesis of the most distal parts of
the limbs (the autopodium; hands and feet), which is accompanied by a second
wave of Hox genes activation, involving Hoxa13 as well as the four posterior
most genes of the HoxD cluster (from Hoxd10 to Hoxd13). It is during this late
phase of expression that the Shh signaling pathway further impacts on the shape
of the expression domain, thus imposing the anterior-to-posterior (AP) polarity
of our limb extremities (Drossopoulou et al. 2000). The existence of these two
separate modes of Hox gene expression has been associated with the distinction
between the most distal pieces of the limbs and the rest of the appendages in
term of evolutionary history. In this view, the autopodium is a neomorphic
structure that appeared in tetrapods, subsequent to the appearance of proximal
limbs (Sordino et al. 1995), in parallel to either the de novo emergence, or
the full recruitment of the appropriate regulatory sequences (see Spitz et al.
2003; Davis et al. 2007; Freitas et al. 2007; Gonzalez et al. 2007).

The nature of the genetic control underlying the early phase of Hoxd gene
expression in limb buds remains to be determined. However, molecular genetic
approaches in the mouse have revealed that transcriptional activation likely
depends on sequences located outside from the cluster itself, on the telomeric
side; i.e., 3 from Hoxd1 (Spitz et al. 2005). In contrast, the regulatory
elements that control the late phase of activation appear to locate
centromeric to the cluster (5 from Hoxd13), as judged by extensive genetic and
transgenic analyses. On the one hand, both spontaneous and engineered large
inversions have clearly positioned digit-specific enhancer sequences upstream
of (centromeric from) the HoxD cluster (Spitz et al. 2003, 2005). On the other
hand, transgenic analyses involving BAC clones as well as shorter DNA fragments
have pointed to two regions of critical importance for the expression of
5-located Hoxd genes in developing autopods.

The first of these DNA segments, located some 180 kb upstream of Hoxd13,
contains several global enhancer sequences, one of which active in developing
digit cells. This global control region (GCR) is conserved among all
vertebrates including teleostei, even though the fish counterpart was not able
to elicit a digit-specific expression when introduced into transgenic mice
(Spitz et al. 2003). The second sequence (Prox) was identified subsequently,
and lies between the GCR and the 5 end of the HoxD cluster. It is also found
in birds and amphibian, although not detected in teleostei genomes (Gonzalez
et al. 2007). In transgenic animals, this sequence can drive expression in
developing digits with a specificity slightly distinct from, and complementary
to, that of the GCR. Therefore, expression of Hoxd genes during the late phase
of limb development likely results from the combined action of these two
regulatory sequences (Gonzalez et al. 2007). In contrast, little is known yet
on the transcriptional control of Hoxa13 expression during digit morphogenesis.

In wild-type embryonic day 12.5 (E12.5) mouse embryos, the four contiguous
genes Hoxd10, Hoxd11, Hoxd12, and Hoxd13 are expressed under the control of
the GCR and Prox sequences. While their expression profiles in digits are
virtually identical to one another, their transcriptional outputs seems to
follow a collinear distribution, at least when considering steady-state levels
of mRNAs (Dolle et al. 1991). Hoxd13, the gene lying at the 5 extremity of the
cluster, is transcribed with maximal efficiency, whereas Hoxd10 is only weakly
expressed. Hoxd12 and Hoxd11 have intermediate amounts of transcripts, as
assessed by in situ hybridization. This quantitative difference makes Hoxd13
expression detectable in presumptive cells of future digit I (the most
anterior digit; the thumb in mammals), unlike the other three genes, whose
transcripts are not detected in these cells, most likely due to lower
expression levels. Consequently, the Hoxd13 transcript domain encompasses
those of the three other genes, an observation in contrast to the general
expression strategy of Hox genes and referred to as reverse collinearity
(Nelson et al. 1996).

This difference between Hoxd13 and other Hox genes is of critical importance
as it may help to individualize the morphology of digit I from those of the
other digits. There is indeed an exact correlation between digit morphology
and Hox gene expression, such that all reported cases where the thumb is
replaced by a more posterior (longer) type of digits, also show a gain of
posterior Hoxd genes expression in the presumptive thumb domain.
Alternatively, forced expression of posterior Hox genes (e.g., Hoxd11)
throughout the limb budi.e., including presumptive digit I cellsleads to the
disappearance of the thumb and transformation toward a more elongated digit
morphology, resembling posterior digits (e.g., Morgan et al. 1992). Therefore,
the globally weaker dose of Hox function in presumptive digit I cells, mostly
due to the nonexpression of Hoxd12, Hoxd11, and Hoxd10 there, is likely a key
factor in the morphological difference that exists between this future digit
and more posterior digits, which express these latter genes in addition to
Hoxa13 and Hoxd13.

The nature of the molecular mechanism underlying reverse collinearity is
unknown, yet it must rely on quantitative collinearity, since the Hoxd gene
expressed at the highest level (Hoxd13) is the only one transcribed in
presumptive digit I cells. By using a set of deletion and duplication alleles
produced by targeted meiotic recombination (TAMERE) (Herault et al. 1998), we
previously showed that the respective position of a gene was important, rather
than the specificity of its promoter. For example, when Hoxd12 was
experimentally positioned at the end of the cluster, where Hoxd13 normally
stands, it was expressed with the same efficiency as Hoxd13 (Kmita et al.
2002a), including in presumptive digit I cells, where it is normally silent.
Also, the introduction of supernumerary promoters at the proximity of the HoxD
cluster induced regulatory reallocations in digits (Monge et al. 2003),
suggesting that collinear expression of Hoxd genes in developing autopods was
the result of a global regulatory equilibrium, controlled by upstream
sequences and involving both the number and respective positions of target
transcription units.

In this study, we investigate this collinear process by using a quantitative
approach to precisely determine the impact of several mutated configurations
on the relative amounts of the various Hoxd mRNAs. We use both experimental
and theoretical approaches to elaborate a model of collinear regulation, after
several rounds of predictions and validations, a process made possible by the
large number of various mutant alleles available at this locus. We conclude
that the best-fit model involves both a strong topological component, but also
calls for some differences in promoter-specific responses. This model also
indicates that the collinear Hoxd genes regulations underlying the development
of both digits and external genitalia are very similar and likely implemented
by the same elements, even though differences exist in the local control of
some genes. Finally, this model accounts for the impossibility for all Hoxd
genes to be expressed equally strongly in presumptive digits cells (reverse
collinearity), and hence, it links a clustered genetic topography and the
associated regulatory constraints, to a particularly important morphological
output, the emergence and maintenance of thumbness.

In E12.5 mouse limb buds, the four most 5-located Hoxd genes are transcribed
in developing digits, with virtually identical expression profiles (Fig. 1A).
However, Hoxd13 is expressed at a much higher level and extends within the
most anterior presumptive digit one (Fig. 1A, arrowhead; Kmita et al. 2002a),
unlike the three other neighbor genes, whose mRNAs steady-state levels seem to
decrease following their respective rank on the cluster (Fig. 1A, quantitative
collinearity). This expression pattern in digits is controlled by two
regulatory sequences located in cis (GCR and Prox) (Spitz et al. 2003;
Gonzalez et al. 2007), which also regulate expression of the Evx2 and lunapark
(Lnp) genes in the same digit domain, as a result of their location within this
regulatory landscape (Fig. 1A; Spitz et al. 2003).

Figure 1.
Quantitative collinearity in developing digits. (A, top) Posterior Hoxd genes
are expressed in developing digits following a gradient of transcriptional
efficiency: Hoxd13 is expressed robustly, whereas more 3-located genes display
progressively (more ...)

Quantitative collinearity
In order to assess the various quantities of steady-state mRNAs, and hence to
establish a baseline for the relative expression levels of these genes in the
wild-type condition, the most distal parts of developing E13.5 digits were
dissected out, mRNA extracted and quantified by real-time RTPCR. At the same
time and for comparative purposes, the emergent genital bud was also dissected
out and treated similarly (Fig. 1B), since quantitative collinearity was
originally described in this developing bud (Dolle et al. 1991). The accuracy
of this quantitative approach was verified using a mouse carrying a deletion
of the Hoxd13 locus. In heterozygous mice, presumptive digits expressed almost
exactly half the amount of the corresponding mRNAs, whereas Hoxd13 transcripts
were expectedly not scored in the homozygous mutant background (Fig. 1C). This
result as well as several other lines of evidence (data not shown), indicated
that cross-regulatory interactions occurring between posterior Hox genes and
their products, if any, were not importantly involved in the final amounts of
transcripts. Consequently, this particular collinear regulation mostly, if not
entirely, derives from interactions in cis.

We next quantified the relative amounts of transcripts between the various
genes, such as to better visualize quantitative collinearity and to have a
starting point for both the evaluation of our various mutant configurations
and theoretical modeling (see below). By using comparisons with known amounts
of RNA, the results depicted in Figure 1D were obtained. While they globally
confirmed the existence of a collinear distribution, they revealed some
unexpected aspects. Because the amount of Hoxd13 mRNAs was arguably the
highest, it was arbitrarily fixed to 1, the other values being expressed in
percentages of this amount. In this scale, Hoxd8 transcripts were not detected
and Hoxd9 mRNAs represented <5%, which was at the limit of significance. This
may reflect some weak activity, not easily detected by in situ hybridization.
While Hoxd10 was clearly expressed, although at a low level, Hoxd11 and Hoxd12
had surprisingly almost identical levels of mRNAs, in the range of 35% of the
Hoxd13 amount. Altogether, quantitative collinearity was confirmed, yet with
no clear application to Hoxd12 and Hoxd11. Unlike what previous expression
studies had suggested, the steady-state levels of both Lnp and Evx2 mRNAs were
low, barely >10% of that of Hoxd13 (Fig. 1D).

Regulatory reallocations
Once these wild-type quantifications were established, we processed a set of
mutant strains where both the number and respective order of the genes had
been modified (Fig. 2). We first used three mutant strains where the most
posterior gene Hoxd13 had been deleted, either alone (Fig. 3A, red bars), in
combination with Hoxd12 (Fig. 3A, yellow bars), or together with both Hoxd12
and Hoxd11 (Fig. 3A, green bars). In all cases, quantitative collinearity was
maintained, but shifted along with the number of genes deleted. For example,
in the absence of the Hoxd13 locus, Hoxd12 was overexpressed by a factor of
two- to threefold, to reach 80% of wild-type Hoxd13 expression level. Both
Hoxd11 and Hoxd10 transcripts were also increased in amounts, although to a
lesser extent. A slight increase in Hoxd9 expression was also scored, yet
still below the amount of Hoxd10 mRNAs in the wild-type configuration (i.e.,
at the same respective positions).

Figure 2.
Stocks of mutant mice used in this study with, at the top, a drawing of the
wild-type HoxD cluster. Posterior Hoxd genes are depicted by using a color
code, from Hoxd13 in red, to Hoxd9 in blue. The position of conserved RXII is
shown by the light-blue (more ...)

Figure 3.
Regulatory reallocations. Real-time PCR comparisons of gene expression levels
in presumptive digits of either wild-type embryos (light-blue bars) or embryos
homozygous for deletions of Hoxd loci located in 5 of the cluster. The levels
are always (more ...)

When all three Hoxd13 to Hoxd11 loci were removed, Hoxd10 was increased, yet
not to the expected level for the leading gene; i.e., the gene positioned at
rank number 1. In this case, a very significant increase in the amount of
Hoxd9 mRNAs was detected (Fig. 3A, green bars), although also below the amount
expected for a gene located at the second position with respect to the 5 end of
the cluster. Also, in all deleted configurations, Lnp was not significantly
modified, except for a slight up-regulation in the del(13-11) mice. The same
holds true for the regulation of Evx2, even though in this case, the magnitude
of the variations was much higher. In particular, removing the Hoxd13 to Hoxd11
DNA interval induced an almost 300% increase in the transcription of Evx2,
whereas the shorter deletions had a less pronounced effect, though stronger
than that seen for Lnp.

From this set of experiments, we concluded that the collinear response in
transcript amounts was mostly, although not entirely, dependent on the rank of
the gene. A collinear response was obtained regardless of which gene was
positioned first, yet the shape of the response was not identical in all
cases, as clearly shown by the expression of Hoxd9 in the del(13-12) mutant
limbs, which was much below the level of Hoxd10 (Fig. 3A, yellow bars), unlike
the expression of Hoxd12 and Hoxd11 in the wild-type condition, where these
latter two genes were at the same respective positions and are expressed in
similar amounts (Fig. 3A, light-blue bars).

Conserved region XII (RXII)
RXII, a region of high DNA homology between various vertebrate species and
located between Hoxd13 and Evx2 (Kmita et al. 2002b) was previously proposed,
using in situ hybridization data, to help potential enhancer sequences to
contact between Hoxd13 and Evx2, thereby favoring expression of Hoxd13 (Kmita
et al. 2002a). By using this quantitative approach, we could not confirm this
original observation, despite RXII having a genuine effect on the
transcription of these target genes. Rather than equalizing transcript amounts
among the various target genes, the deletion of RXII increased the
transcriptional activities of Hoxd13 to Hoxd10 (Fig. 3B, cf. dark-blue and
light-blue bars). When combined with the deletions of either the Hoxd13 locus,
or of both the Hoxd13 and Hoxd12 loci, the same tendency was observed and
quantitative collinearity was maintained (Fig. 3B, orange and green bars)more
obviously, in fact, than upon the mere deletion of RXII. In this latter case,
expression of Hoxd11 was not significantly different from that of Hoxd12 when
using the Students t-test (Fig. 3B, dark-blue bars).

Interestingly, the major modification caused by deletion of RXII was scored on
Evx2 regulation, whose transcript level dropped down to 50%. As observed also
in the presence of RXII (Fig. 3A), the deletion of RXII in combination with
either Hoxd13 or Hoxd13 and Hoxd12 lead to an increase in the transcription of
Evx2. In this case, however, the increase was much less pronounced than in the
presence of RXII, and the level of Evx2 mRNAs eventually reached the wild-type
level (Fig. 3B, green bar), whereas a robust increase was seen with the same
deletion but in the presence of RXII (Fig. 3A, green bar). Altogether, the
gain of expression observed on Hox genes upon deletion of RXII was clearly
more important than the concomitant loss of Evx2 expression, indicating that
the increase was not due to a mere redistribution of regulations.

Modeling quantitative collinearity
As different mechanisms could underlie both the collinear regulation in the
wild-type locus and its observed variations in our mutant configurations, we
tried to elaborate a theoretical model that would best fit this initial set of
experimental data. We considered two major classes of mechanisms: those relying
on a scanning process, and those involving direct initial contacts between the
enhancers and the target DNA region(s), through the formation of loops (e.g.,
see Bulger and Groudine 1999; Tolhuis et al. 2002). Based on the first set of
experimental data mentioned above, two important general observations were
taken into account: First, while the rank of the gene with respect to the 5
end of the cluster is a key parameter for the expression level, it is not the
only element to consider. For example, regardless of which gene was positioned
at the place of Hoxd13 (rank I), this gene was highly expressed. The absolute
levels of expression were nevertheless significantly different from gene to
gene (Fig. 4A). Generally, genes located 3 of the breakpoint were not as
highly expressed as was predicted by their new position. For example, in
del(13), Hoxd12 reached only 80% of the Hoxd13 wild-type level, despite its
first position within the cluster (Fig. 4A). However, no simple rule could be
drawn, as shown by Hoxd11 expression in the same deletion, which in contrast
was 1.4-fold that of the Hoxd12 wild-type level (Fig. 4A). Also, Evx2
expression increased strongly together with the size of the deletions, even
though the respective position of this gene remained unchanged (Fig. 3A).
These observations suggested that slightly different, gene-specific affinities
were also involved.

Figure 4.
Modeling quantitative collinearity. (A) Expression levels of Hoxd genes as a
function of their relative position with respect to the 5 extemity of the
cluster (their rank), in the various genetic configurations. While gene rank
(more ...)

Second, the overall transcriptional activity of the locus, as measured by
cumulative amounts of transcripts, was function of the number of transcription
units present, such that the deletion of one or several genes led to a
corresponding overall decrease of mRNAs produced at the locus (with the
exception of the codeletion of RXII, as described above). This observation was
subsequently confirmed by increasing the number of genes, through various
duplications (see below), leading to a quasilinear relationship between gene
number and total mRNAs levels (Fig. 4B). This unexpected observation indicated
that the regulatory system is not saturated in the wild-type condition, and
that regulatory reallocations, following genomic modifications, do not simply
reflect the redistribution of the same general regulatory potential among a
reduced or increased number of target units.

After considering various model outlines, a particular class of models was
found to fit the above-mentioned constraints, while not requiring an undue
number of estimated parameters. These models assume a two-step process: In the
first phase, a complex involving regulatory sequences (GCR, Prox), together
with their bound factors, will interact with the Evx2-Hoxd13 intergenic region
(Fig. 4C). The probability that the GCR/Prox complex will bind the locus is
proportional to the number of promoters present at the locus.

Once this interaction has occurred, various probabilities exist to activate
either Lnp, Evx2, or Hoxd genes. These probabilities depend on the affinities
of the various promoters and the probability that the GCR/Prox complex did not
stop previously at upstream promoters. We thus assumed that the five Hox genes
promoters are not equivalent in their capacity to attract and fix the GCR/Prox
complex. This second step could occur via a microscanning process, over the
~40-kb large DNA interval containing from Hoxd13 to Hoxd10. Since all
promoters are at the vicinity of the enhancers complex, this latter can
discriminate and select in function of the respective affinities. In this
class of models, the maximum number of estimated parameters is nine (see
Materials and Methods). Two parameters define the regression between the total
number of transcripts and the number of promoters at the locus, and seven
parameters at most are required to account for the affinities of the seven
loci considered (Lnp, Evx2, and Hoxd13 to Hoxd9). Such models successfully
passed the test of the three observed features (Fig. 5): (1) the decrease in
total transcripts amount together with promoter number, (2) the modulation of
ranking by promoter efficiency to determine the expression levels, and (3) the
up-regulation of Evx2 whenever Hox genes are deleted. After definition of the
various parameters, this model, which includes components of both scanning and
looping mechanisms, fit the first set of data much better than any other model
relying exclusively on either a general scanning process or gene-specific
affinities (data not shown).

Figure 5.
Predicted versus observed expression levels. Comparison between expression
levels predicted by the model (yellow bars) and observed values in either the
wild-type (light-blue bars) or mutant configurations (red bars). The various
configurations are depicted (more ...)

The application of this model generated a set of values, which were then
compared with both the wild-type condition and the three deleted
configurations described above. In the wild-type condition, a very good match
was obtained between the predicted and the observed values (Fig. 5A). When
either the Hoxd13 locus, both Hoxd13 and Hoxd12 or the Hoxd13 to Hoxd11
interval were deleted from the model, the predicted values for reallocations
also matched the observed data set within error bars (Fig. 5BD, cf. red and
yellow bars), with the exception, perhaps, of the gain of Evx2 expression,
which was slightly but systematically underestimated by the model. Once
established, the model was used to make a number of predictions, which were
verified by using the appropriate mutant strains. In turn, the new results
were not only used to test the resilience of the model, but also to further
constrain the parameter values, progressively, through several rounds of
adjustment and experimental validation. The values (predictions) of the model
given in the figures below, for comparison with the experimental data sets,
are extracted from the final version of the model.

Duplication alleles
This model made clear predictions regarding the effects of either gene
duplications, or internal deletions. We experimentally challenged these
predictions, starting with three duplications obtained via our TAMERE system
(Herault et al. 1998). The first duplication was that of the Hoxd13 locus
alone (Fig. 6A), the second was a duplication of the entire Hoxd13 to Hoxd11
interval (Fig. 6B), and the third allele was an internal duplication of the
Hoxd12 to Hoxd11 locus (Fig. 6C). Generally, the predictions of the model
turned out to be rather precisely validated by the experiments. In particular,
the duplication of the Hoxd13 locus, leading to two copies of Hoxd13 positioned
at ranks 1 and 2 did not elicit a double amount of Hoxd13 mRNAs, emphasizing
again the prime importance of the promoters rank over the promoters affinity.
In this same duplication, however, the impact on Evx2 transcription was once
again not faithfully predicted, the observed down-regulation being more
important than anticipated (Fig. 6A).

Figure 6.
Duplication alleles. Observed (red) and predicted (yellow) gene expression
levels in three duplications. Wild-type levels (light blue) are shown on each
panel for comparison. (A) dup(13). (B) dup(13-11). (C) dup(12-11). In all
cases, the expression of (more ...)

Also, in the duplication of Hoxd13 to Hoxd11, the predicted level for Hoxd11
mRNAs at first appeared too high when compared with the experimental data
(Fig. 6B), yet the Students t-test indicated that this difference was not
significant. Beside this point, the observed matching was good, as was the
case for the internal duplication of both the Hoxd12 and Hoxd11 loci, where
the amount of Hoxd13 transcripts remained expectedly stable. In this latter
duplication, a robust increase in Hoxd11 mRNAs was scored, as predicted by the
model. This increase would not be expected based on a rank-only model, since
after duplication, one copy of Hoxd11 is now in the respective position of
Hoxd9; i.e., in a virtually silent position with respect to digit enhancers
(see above). This increase thus illustrates the affinity component of the
system (as predicted by the model), Hoxd11 performing better than Hoxd9 when
placed at the same genomic position. In all duplicated configurations, the
observed decrease in the amount of Hoxd10 mRNAs matched the predictions.

Interestingly, in contrast to what was observed with the various deletions,
the overall level of Hoxd gene transcripts increased together with the
importance of the duplications. For instance, a mild increase in total
transcription was detected with the duplication of Hoxd13, whereas a more
robust increase was scored upon duplication of the Hoxd13 to Hoxd11 interval
(addition of three transcription units). As mentioned earlier, this
relationship between the number of genes and the total amount of transcripts
was nearly linear (Fig. 4B).

Internal deletions
The predictions of the model concerning a set of five internal deletionsi.e.,
deletions that did not remove Hoxd13 from its first rankwere also challenged
experimentally (Fig. 7). In all these deletions, the observed levels of Hoxd13
mRNAs were stable, as predicted, except for the case of the del(12-11) where an
~20% increase was detected. Therefore, the general decrease in the amount of
Hoxd genes transcripts did not lead to an increase in Hoxd13 transcription.
Likewise, neither Evx2 nor Lnp showed significant variations in their
transcriptions, in agreement again with the model.

Figure 7.
Deletions excluding Hoxd13. Observed and predicted gene expression levels in
five internal deletions. Colors and schemes are as in Figure 6. (A) del(12).
(B) del(12-11). (C) del(12-10). (D) del(11). (E) del(11-10). All deletions
lead (more ...)

The effects of these deletions were thus scored mostly on the 3 side of the
breakpoints. Here again, the observed transcript levels were in good agreement
with the predictions. For example, Hoxd10 transcription was only moderately
increased in the absence of the Hoxd12 locus (hence, at respective rank 3)
(Fig. 7A), whereas a robust increase was scored for the same gene when located
at respective rank 2, after deletion of both Hoxd12 and Hoxd11 (Fig. 7B), thus
emphasizing the importance of the rank. Interestingly, when Hoxd12, Hoxd11 and
Hoxd10 were deleted (Fig. 7C), and hence, Hoxd9 was now neighboring Hoxd13 in
rank 2, this latter gene was not activated at a very high absolute level,
showing once again that the overall transcriptional activity distributed over
the cluster was not simply reallocated among the number of resident promoters.

While the deletion of the Hoxd11 locus generated data in good agreement with
the predictions (Fig. 7D), the internal deletion of both Hoxd11 and Hoxd10
generated the only truly aberrant measure of the whole experimental series.
This internal deletion indeed induced a very robust increase of Hoxd9
transcription, reaching the level of endogenous Hoxd12 (Fig. 7E).
Interestingly, the same 3 breakpoint was used in the del(12-10) configuration,
which did not lead to a similar increase (Fig. 7; cf. red bars in C and E).

Regulation in the genital eminence
During the emergence and further development of the external genital organs
(the future penis and clitoris), the same Hoxd genes are expressed with a
similar quantitative collinear distribution of transcripts (Dolle et al.
1991). This observation, as well as subsequent genetic data, have highlighted
the developmental similarities between distal limb buds and genital buds
(Kondo et al. 1997; Cobb and Duboule 2005), and suggested that Hoxd gene
expression in both structures could be controlled by the same regulatory
circuitry (Dolle et al. 1991; e.g., see Cohn 2004; Suzuki et al. 2004). We
thus investigated whether the model could also be applied to both wild-type
and mutant situations in the developing external genital organs. However, when
the parameters of the model were adapted after considering the entire series of
alleles, the global fit with the experimental data set was not optimal. This
could mean that either the structure of the model was different between
developing limbs and genitals, or alternatively, some classes of genetic
configurations (5 deletions, duplications, internal deletions) could not be
accounted for by the actual model; hence, their consideration to adjust the
parameters would decrease the general fit.

Consequently, we tried to exclude specific classes of genetic rearrangements
in the adjustment of parameters, by keeping the same model structure.
Interestingly, when we ignored the data set obtained with the various internal
deletions (not containing Hoxd13) to adjust the various parameters, we obtained
an excellent fit with the data sets derived from both 5 deletions (including
Hoxd13) and duplications (Fig. 8). As for the digits, the match with the
wild-type condition was very good (Fig. 8A) and the same (almost linear)
relationship was observed between the overall level of transcription and the
absolute number of transcription units present in this DNA interval (Fig. 8F).
The match between the model and either two 5 deletions [del(13) and del(13-12)]
(Fig. 8B,C), or two duplications [dup(13-11) and dup(12-11)] (Fig. 8D,E) was
very good, indicating that the regulatory mechanisms at work in both
developing digits and external genitalia were most likely the same.

Figure 8.
Hoxd gene regulation in the developing genital tubercle. The same model
structure was applied to investigate Hoxd gene regulation in the genital
eminence. The various parameters of the model were optimized using expression
values observed in the genital (more ...)

Because internal deletions had to be ignored to adjust the parameters, their
fit with the model was expectedly rather poor. For example, in the del(12-11)
configuration, Hoxd10 transcripts were much less abundant than anticipated by
the model (Fig. 8G, yellow bar). Also, the deletion of both Hoxd11 and Hoxd10
loci induced a decrease of Hoxd12 mRNAs, which were thus below the predicted
amount (Fig. 8I). Only the deletion of the Hoxd11 locus matched fairly well
the predictions of the model (Fig. 8H). Finally, and similar to what was
observed in digits, the deletion of the Hoxd11 and Hoxd10 loci induced a large
and unexpected gain in the transcription of Hoxd9 (Fig. 8I, red bar) that could
neither be explained nor accounted for by the model.

We used a quantitative approach to analyze the regulatory mechanism underlying
collinear Hoxd gene regulation during mammalian digit development. Such an
approach is necessary due to the sensitivity of various digit morphologies to
both the dose and quality of Hox gene functions delivered during fetal
development (Zakany et al. 1997; Zakany and Duboule 2007). Variations in this
quantitative regulation have been proposed to partially account for important
morphological differences between mammalian digits (e.g., Chen et al. 2005);
hence, the understanding of this regulatory control may help explain what
determines the various digital formulae of mammalian hands and feet.

The ontogeny of mammalian thumbness; a dosage effect linked to gene topology
In mammals, a clear difference is observed between the morphology of the most
anterior digit (the thumb) and the other digitsthe former generally displaying
a reduced size due to the presence of two phalanges only. At the molecular
level, very few genes are differentially regulated in various presumptive
digit domains, and hence could determine digit identities. Among these few
candidates, Hox genes are the most promising for at least three reasons.
First, the inactivation of HoxA and HoxD gene functions lead to either a
reduction or a complete agenesis of digits, including the thumb (Dolle et al.
1993; Fromental-Ramain et al. 1996; Kmita et al. 2005). Second, gain of
expression of posterior Hoxd genes (e.g., Hoxd12 and Hoxd11) in presumptive
thumb cells lead to a more elongated morphology, resembling posterior digits
(Morgan et al. 1992; Zakany et al. 2004). Finally, naturally occurring
mutations, which homogenize digital morphologies, are all associated with a
homogenization of Hox genes expression domains.

Among Hox genes expressed in digits during development, Hoxa13, the only
member of the HoxA cluster expressed there, is equally transcribed in all
digit primordia, including the thumb. Therefore, this critical difference in
shape ought to be determined by Hoxd genes and indeed both Hoxd10, Hoxd11, and
Hoxd12 are expressed exclusively in presumptive digits 25, whereas excluded
from digit I (the thumb). In contrast, Hoxd13, like Hoxa13, is expressed
throughout all presumptive digit cells. Accordingly, mice mutant for Hoxd13
have an abnormal thumb (Dolle et al. 1993). Interestingly, however, Hoxd13 is
expressed originally with the same posterior restriction than its three
immediate neighbors, with an apparent peak in posterior-distal cells and an
exclusion from digit I cells. Because Hoxd13 is expressed more robustly than
its neighbors, transcription is nevertheless subsequently detected in
presumptive thumb cells, thus encompassing all five digital primordia, leading
to what was referred to as reverse collinearity (Nelson et al. 1996). In this
view, reverse collinearity is the mere topological translation of quantitative
collinearity.

Therefore, the subtle transcriptional regulation of Hoxd genes in these cells
is likely a crucial parameter in the determination of thumbness, mostly by
reducing the global dose of HOX products present in digit I. This dosage
effect appears to be linked to gene topology, since mice deleted for the
Hoxd13 locus expressed Hoxd12 in developing digit I (Kmita et al. 2002a),
indicating that the gene positioned at the extremity of the cluster is always
expressed with maximal efficiency, regardless of its own specific regulatory
sequences. Accordingly, genes located at the second, third, fourth, and fifth
ranks respond to this regulation too, yet with lower efficiencies, leading to
their nontranscription in presumptive digit I cells. The resulting dose of
HOXA and HOXD proteins is thus higher in developing digits 25 than in digit I,
which will impact on the future morphologies, Hox genes being involved in the
control of cell proliferation and digit patterning. This difference between
digit I and the others suggests a regulatory explanation for the existence of
two developmental modules underlying the evolution of anthropoid distal
forelimbs (Reno et al. 2007).

In an evolutionary context, it is likely that the recruitment of this
regulation was an important step to accompany either the emergence, or the
expansion of digits in an ancestral tetrapod. Because of the built-in
asymmetry of the system, based on gene order, we can speculate that
quantitative collinearity was already at work in these ancestral autopods.
Consequently, a morphological distinction between the most anterior digit (the
thumb in mammals) and the others must have existed from the start. In this
view, while Hox gene clustering was certainly an efficient evolutionary
opportunity, it may have also importantly constrained the system to impose
some heterogeneity in digital morphologies. These morphological differences
may have been a basis for further adaptive solutions and selection thereof,
either by further elaborating on them, or by subsequent digital deletions or
reductions.

While many mammals indeed display little if any difference in the morphologies
of more posterior digits (e.g., the human digits 25), some vertebrate species
exhibit remarkably different, although neighboring, digits; for example,
associated with their flying behavior. In both bat and chicken wings, digits
can be variable in sizes and number of phalanges, without obvious changes in
Hox gene expression. It is nonetheless possible that slight quantitative
modifications be responsible for this effect (Chen et al. 2005).
Alternatively, the exact timing in the expression of these Hox genes in every
presumptive digit territory may subsequently affect digital morphologies.
While our various analytical tools reflect a frozen situation, a more dynamic
view of the system may reveal the nature of such differences. It is equally
possible that the Hox system be not used after the initial morphological
distinction has occurred between the most anterior digit and the others.

Modeling quantitative collinearity
Our quantitative approach, associated with the large collection of mutant
strains at this locus, allowed us to elaborate a model of large-scale gene
regulation that accounts for most of the observed results. In order to model
this regulation, we considered some facts as established, such as the
importance of gene position (the rank), the fact that two other genes were
also affected by this regulation (Evx2 and Lnp), and the known presence and
locationupstream of the gene clusterof regulatory sequences that control gene
expression in developing distal limb buds (the GCR and Prox elements) (Spitz
et al. 2003; Gonzalez et al. 2007). Other parameters were readily excluded,
due to their incapacity to explain the observed results. For example,
differences in various mRNAs half-lives, if at all involved, would have a very
minor impact. Also, models involving the scanning of the landscape by a protein
complex, after initial recognition of enhancer sequences (see Blackwood and
Kadonaga 1998) were rapidly discarded due to the difficulty of reconciling
them with any available data set. We thus focused on models based on an
initial looping, followed by the construction of a particular regulatory
microarchitecture. The predictions formulated from an early model were
subsequently tested by using the appropriate set of mutant strains. In turn,
the new data were used to adjust the various parameters such as to improve the
fit of the model. This bilateral process led to a final formulation built on
nine different parameters, which could account for the 69 quantitative
observations made by using 12 different mouse strains.

Surprisingly, the model helped to clarify two issues raised previously on the
basis of in situ hybridization patterns. First, the gene rank alone cannot
account for the observed transcriptional efficiencies. While of primary
importance, this parameter is not sufficient to explain the various data sets.
In fact, even wild-type absolute transcript levels indicate no real difference
between Hoxd12 and Hoxd11. Second, while largely increased, the transcript
levels of either Hoxd12 or Hoxd11, when placed at the first rank, never
reached the wild-type level of Hoxd13 transcripts. Also, the total amounts of
Hoxd13 transcripts in duplicated configurations containing two copies of this
locus were the same regardless of the rank of the second Hoxd13 copy; i.e.,
when placed either at rank number 2 or number 4. Therefore, the importance of
gene rank has to be somehow modulated by specific affinities between the
enhancer complex and the various gene promoters. In the model, the
introduction of parameters reflecting this previously unanticipated feature
(see Kmita and Duboule 2003) was necessary to reach the requested global fit.
Interestingly, the affinities derived from the model for Lnp, Evx2, and Hoxd9
are very similar to each other (see Materials and Methods), possibly
reflecting a generic, baseline promoter affinity, whereas Hoxd10 to Hoxd13
promoters would have evolved more robust affinities for the enhancer complex.

The second issue concerns the DNA segment initially contacted by the enhancer
complex. Our various data sets suggested a model whereby enhancers, in a first
step, contact the Evx2 to Hoxd13 intergenic region via a loop. In a second
step, the enhancer complex scans (senses) the immediate environment
preferentially from the 5 toward the 3 part of the cluster, regulated by
promoter-specific affinities. While anticipated, the importance of the
Evx2-Hoxd13 intergenic region does not seem to rely on the presence of the
highly conserved sequence RXII, unlike originally reported based on in situ
hybridization patterns (Kmita et al. 2002a). In this latter case, expression
of genes located at ranks number 2 and 3 were also detected in presumptive
cells of digit I, which was interpreted as a break in the collinear response.
Our quantification of transcripts derived from mice deleted for this sequence
XII indeed revealed that quantitative collinearity was maintained despite a
generally elevated level of Hoxd gene expression, which likely explains their
transcription in presumptive digit I cells.

In support of this explanation, the deletion used by Kmita et al. (2002a)
removed both the RXII and the Hoxd13 locus. Our analysis of this genetic
condition revealed a very robust transcriptional increase (>100%) of the
remaining three genes, explaining their expression in presumptive digit I
cells and, hence, the misleading impression that all patterns were alike in
these embryos. In the absence of RXII, Evx2 transcription was down-regulated,
which suggests that this sequence may be part of the Evx2-specific
transcriptional requirements (in the promoter region or in the 5 untranslated
region). However, the effects observed upon Hoxd gene transcription cannot be
explained solely on this basis, and call for a specific function for RXII. For
example, this conserved DNA sequence may act as a switch for the direction of
scanning, once enhancers have recognized the Hoxd13-Evx2 intergenic region.
The factors that favor the contact between the GCR/Prox enhancers and this
intergenic region remain to be characterized.

Finally, the model integrated an interesting phenomenon observed with both
deleted and duplicated configurations; i.e., the fact that the total amount of
transcripts produced in digit cells by all genes present in the landscape is a
function of the number of genes. This has several implications regarding the
transcriptional mechanism at work, as it means that the system is not
saturated, and adding more transcription units will increase the global output
of the system. This suggests that the wild-type situation likely corresponds to
a particular equilibrium, as imposed by the regulatory architecture of the
locus. Likewise, the deletion of several promoters will not merely reallocate
the regulatory potential over the remaining units, but instead, will produce a
novel equilibrium that will use less of the global transcriptional potential
available. One possibility is that such equilibrium imposes a number of
constraints to the systemfor example, in the spatial organizationpreventing
situations in which any number of genes would use their full regulatory
potential.

While in good agreement with the vast majority of the data sets obtained
experimentally, our model showed some minor and acceptable discrepancies with
the reality (for example, the underestimation of Evx2 down-regulation in
Hoxd13 duplication), as well as one major problem that remains unsolved; i.e.,
the behavior of Hoxd9 in the internal deletion del(10-11). In this case, an
almost 20-fold increase was scored in the expression of Hoxd9, whereas the
model predicted only a slight increase (Fig. 7E). This particular result is
the only one suggesting the manifestation of a new phenomenon, such as the
emergence of a novel, increased affinity for Hoxd9 following the fusion with
the 3 part of the Hoxd12 locus. The validation of this hypothesis would
require yet another set of genetic modifications, not readily available.

The digits versus genitals connection
Because of similar Hox expression dynamics during the development of both
digits and external genitalia, it was proposed that these structures share
both ontogenetic principles and a phylogenetic history (Dolle et al. 1991;
Kondo et al. 1997). Since then, the conservation of developmental principles
during the emergence and growth of these various buds has been largely
documented, notably through the presence and function of the same key
regulatory molecules and signaling pathways (for reviews, see Cohn 2004;
Suzuki et al. 2004). In fact, quantitative collinearity was originally
described in the growing genital eminence (Dolle et al. 1991), and we thus
looked at whether the model constructed for the digits could equally account
for the results obtained with developing external genitalia.

Clearly, the same type of model could be applied to the results obtained with
RNA extracted from developing genital buds. In particular, the model had a
very good fit for the duplicated configurations and the deletions including
Hoxd13 (5 deletions). Also, the quasilinear relationship between the total
transcriptional readout of the system and the number of transcription units
was also observed in genitalia. Altogether, these results suggested that the
regulatory strategy leading to quantitative collinearity was identical in both
digits and genitalia, and that these various structures not only use the same
genes, but also the same regulatory circuitry.

However, the consideration of all experimental data, as obtained from genital
bud RNAs, did not fit well, at first, with the model elaborated for digit
RNAs, even after adaptation of some parameter values. Interestingly, the set
of internal deletionsi.e., those deletions excluding Hoxd13was systematically
at odds with the model. Whenever these deletions were ignored and parameters
values adapted accordingly, a close to perfect match with the model was
obtained. This observation ought to be interpreted in two different contexts.
First, Hox gene function and regulation in these structures must be compared
at slightly different developmental time points (see Cobb and Duboule 2005),
as the distal part of the limb buds should be compared with the distal part of
the developing genitalia (the future penis, in males, or clitoris, in females)
and not with more proximal parts of the bud, the labioscrotal, and preputial
swellings, giving rise to either the scrotum, the labia, or the prepuce.
Because there was no clear morphological landmark to dissociate these parts at
our dissection time point, unlike in subsequent developmental stages, it is
possible that a contamination of more proximal cells induced a bias in the
results.

Another, nonexclusive explanation is that additional regulatory sequences
evolved within the gene cluster (e.g., see Gould et al. 1997) to fine-tune or
accompany the evolution of the external genital apparatus, on top of
pre-existing mechanisms. Internal deletions may have a particularly pronounced
impact on the function of these genital-specific sequences, thus leading to
results that cannot possibly be accounted for by the model. Such targeted
differences in the regulation of these genes may not be so surprising, given
the end result that is achieved in both cases. Indeed, in the external
genitals, the adaptive value may reside in the elongation of the future organ.
In digits, however, the function of these genes is not restricted to the mere
growth of the structures but also, as documented above, to their patterning.
These slightly different and sex-dependent contexts ultimately had their own
adaptive values, and it is perhaps not surprising if each structure evolved
some additional regulation, on top of a shared and potentially ancestral
regulatory circuitry.

Stocks of mice
The various mutant strains used in this study were described previously (Kmita
et al. 2002a, b; Tarchini and Duboule 2006; Di-Poi et al. 2007). Besides the
deletion of RXII, all mutant alleles were produced by TAMERE (Herault et al.
1998). Genotyping of mice and embryos was performed using Southern blot and
PCR analysis, according to standard procedures. For duplicated alleles, a
real-time PCR strategy was used to quantify copy numbers of the duplicated
segments, in order to discriminate between heterozygous and homozygous mutant
embryos (Supplemental Material).

RNA samples and real-time RTPCR
For each mutant line, heterozygous mice were crossed to obtain wild-type,
heterozygous, and homozygous mutant embryos. Presumptive digits and genital
tubercles were dissected from E13.5 embryos and stored in RNA later reagent
(Qiagen), until genotyped. RNA was isolated from individual embryo samples
using the RNeasy microkit (Qiagen) after disruption and homogenization with a
Polytron device (Kinematica). Single-stranded cDNA was synthesized using
SuperScript II RT (Invitrogen)

Real-time PCR primers and TaqMan probes were designed using Primer Express 2.0
software (Applied Biosystems), and PCR efficiencies were measured using serial
dilutions of cDNA. cDNAs were PCR-amplified in a 7900HT SDS system (Applied
Biosystems). Results were comparable when using either TaqMan or SYBR green
strategies. Specificity of the SYBR green reactions was determined by
examination of product melting curves. Relative quantities (RQ) were
calculated from the threshold cycle (Ct) values with the formula RQ = (1 +
E)-Ct, where E is the PCR efficiency calculated from standard curves. A mean
quantity was calculated from triplicate reactions for each sample and
normalized to two or three similarly measured quantities of housekeeping genes
(Rps9, Tbp, and Tubb). Sequences of primers and probes used in this assay are
listed in Supplemental Table S1.

For the determination of absolute transcripts levels, external calibration was
performed using a standard consisting of known amounts of the various mRNAs.
Sense RNA was produced by in vitro transcription of full-length or partial
cDNA clones (Supplemental Material) using T7 or SP6 RNA polymerases (Promega).
Standard curves were obtained by serial dilution of known amounts of these
RNAs, and reverse transcription with Drosophila L2 cell total RNA as a
carrier. Absolute quantification was performed by parallel real-time PCR
amplification of digit and genital bud total cDNA together with this standard
curve. Similar results were obtained using serial dilutions of a BAC clone
covering the locus as a standard (data not shown).



Looping and tracking model in digits
A preliminary analysis of the data revealed two striking features of this
system: First, the total amount of transcriptsi.e., the sum of absolute levels
of expression for all genes in a given configurationstrongly correlated with
the number of promoters present at the locus (Fig. 4B). The Pearson
coefficient is 0.93 in digits for the eleven configurations [excluding
del(10-11), see the text]. Second, the five Hoxd genes showed a constant
pattern where, in any given pair, the 5-located gene is more robustly
expressed than its 3-located neighbor. However, rank alone is not a good
predictor of absolute levels of expression (see the text). Therefore, we
designed a two-step model assuming the existence of seven different promoter
efficiencies (Elnp, Eevx, E13 to E9), which would determine a probability for
the GCR/Prox transcription complex to interact with any of these promoters. In
the first step, the GCR/Prox elements loop over and bind the locus somewhere
between Evx2 and Hoxd13 (Fig. 4C). The probability of this event is assumed to
be proportional to the number of promoters present at the locus. This will
determine the overall level of transcription; i.e., the total number of
transcripts from all the genes present. In a second step, the frequency f of
interaction between the GCR/Prox complex and either Lnp, Evx2, or Hoxd13, is
proportional to the efficiency E of their respective promoters. A potential
interaction with any downstream-located Hoxd gene is processive and will
depend on (1) the intrinsic efficiency of its promoter and (2) the cumulative
effect of previous interactions with all upstream-located Hoxd promoters.
Thus, the theoretical frequency fi of interaction of GCR/Prox with a Hoxd
promoter i is

where Qi is the probability that the GCR did not stop upstream of i in the
sweep, computable from the Ei; i.e., Q12 = (1-E13); Q11 = [1 - E13 - E12 * (1
- E13)] and so forth. Relative activities may then be obtained by

where Sfj is the sum over the seven genes. Absolute levels of activity (ai)
are computed by applying this ratio to the predicted total number of
transcripts for a given configuration (TTc), as derived from the linear
regression

These absolute levels of activity are to be compared with the observed
absolute levels of expression. The best fit between this model and the data
sets was looked for by using an optimum set of nine parameters: two parameters
for the linear regression to account for the total number of transcripts, and
seven parameters to account for the efficiencies of the promoters. The seven
latter parameters were derived from minimizing the sum of weighted least
squares:

where aip and aio are the predicted and observed absolute levels of
expression, respectively, Vi is the variance of measurements for the gene i,
and SS is the sum over all configurations and all genes. All computations were
done using Matlab 7.0.4 (The MathWorks) software.

Among the 69 data points, one observation, Hoxd9 in the del(10-11)
configuration, significantly deteriorated the goodness-of-fit of both the
linear regression and the least-square fit: The weighted sum of least squares
was almost halved when Hoxd9 in del(10-11) was excluded. Not taking this
observation into account in the fit (see the text), we obtained the following
optimum model efficiencies:



Efficiencies Ei in digits

Lnp 0.035
Evx2 0.031
Hoxd13 0.347
Hoxd12 0.198
Hoxd11 0.210
Hoxd10 0.107
Hoxd9 0.033
The total number of transcripts (TTc) was derived from the following
regression line, with R2 = 0.93 (Fig. 4B):


The corresponding predicted absolute levels for each gene in the 12
configurations are given in Supplemental Table S7 and compared with observed
values (Figs. 57).



Model in genitals
The same model was used to predict absolute transcripts levels in genitals. As
discussed in the text, internal deletions were ignored when minimizing the sum
of weighted least squares. The optimum model efficiencies, the regression
equation for the total number of transcripts, and the corresponding predicted
absolute levels are given in Supplemental Table S8 and compared with observed
values (Fig. 8).



Acknowledgments


We thank J. Zakany, M. Kmita, and B. Tarchini for sharing mice, and M.
Docquier and P. Descombes, from the NCCR genomic platform, for their help and
advice. This work was supported by funds from the canton de Geneve, the
Louis-Jeantet and Claraz foundations, the Swiss National Research Fund, the
National Center for Competence in Research (NCCR) Frontiers in Genetics, and
the EU programs Cells into Organs and Crescendo to D.D.



Footnotes


Supplemental material is available at http://www.genesdev.org.


Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1631708

Top
Abstract
Results
Discussion
Materials and methods
References
References


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==
Well, dinos & mammals are both amniotes but not
technically reptiles, since the clade Reptilia or
Sauropsida is reserved for diapsids. Its sister clade
is ours, the Synapsida, the mammal-like "reptiles" &
their mammalian descendants. However, if you regard
the anapsid ancestors of both diapsids & synapsids as
reptiles, then, yes we are also phylogenetically
"reptiles" or reptiliomorphs.

The problem with both common & scientific nomenclature
is that "reptile" can mean different things. The
evolutionary history of any lineage can be sliced up
in various ways. Cladistic taxonomy, based upon
shared, derived traits, allows great precision, but
leads to endless multiplication of categories,
compared to the Linnaean system.

By any cladistic analysis, however, birds are
reptiles, being diapsid archosaurs.

To review, the first amniotes, tetrapods capable of
reproducing away from water, & with skins that enabled
life in drier environments, had "anapsid" skulls, ie
lacking fenestrae, holes in their heads. These
creatures could be considered reptiles, in which case,
mammals are reptiles, as Dave says. But now customary
cladistic nomenclature is to reserve the technical
taxonomic term "Reptilia" (preferably Sauropsida) for
diapsids, those amniotes with two postorbital
fenestrae, opening in their skulls behind the eye to
allow for muscle attachment. The Synapsida, like us,
have a single postorbital fenestra, which in mammals
has become the opening through which our single-boned
jaw slides, guarded by our cheekbone.

I know it seems unnecessarily complicated, but
evolution produces gradients along which
categorization is somewhat arbitrary & semantic.

Dealing just with living forms, you can make a simple
cladogram. Draw a diagonal line across a sheet of
paper. Put "Crocodilians" at the upper right end (or
birds, or mammals, it doesn't matter, as long as you
get the branching right). Now a little farther back
down the main line, draw a line at an angle coming off
the base line to the left until it reaches the top of
the paper, at the same level as "Crocs". Write
"Birds" at its end. Repeat the process starting
farther down the main line, but write "Turtles"
instead (actually this position is still a bit
controversial, but genomic analysis seems to confirm
that turtles are more closely related to archosaurs
than lepidosaurs & are only secondarily anapsid).
Repeat it again & write "Lepidosaurs" (or squamates,
or lizards & snakes, & tuataras) at the end of this
branch, or show each of these groups branching off
along the side line. Now go way back down the main
line & draw a long branch at an angle up to the top &
write "Mammals" at the end of it. This is the Amniote
family tree. The tetrapod outgroup is Amphibians.

The mammal line branches off about 320 million years
ago, in the Carboniferous. Lepidosaurs (squamates &
tuataras) split from archosaurs (birds & crocs) in the
Permian, & birds diverged from crocs in the Triassic.
==
Genomic analysis shows that, while their skulls are
secondarily "anapsid", turtles aren't members of the
ancient clade Anapsida, but in fact diapsids more
closely related to Archosaurs than Lepidosaurs.
==
Leaving out a few clades, avian taxonomy goes like
this:

Birds are maniraptoran, coelurosaurian, tetanurian
theropod dinosaurs.

Birds & other dinosaurs are ornithodires.

Birds, other dinosaurs & other ornithodires (like
pterosaurs) are archosaurs.

Birds, other dinosaurs, other ornithodires & other
archosaurs (like crocs) are diapsids.

Birds, other dinosaurs, other ornithodires & other
archosaurs are diapsids (like lepidosaurs, ie lizards,
snakes & tuataras).

Birds, other dinosaurs, other ornithodires, other
archosaurs & other diapsids are members of the clade
Reptilia (also called Sauropsida), so all the above
taxa are equally reptilian in phylogeny.

The sister clade of Sauropsida within clade Amniota is
Synapsida, to which mammals such as ourselves & other
apes, other primates, other placentals, etc. belong.
==
Fossil reveals oldest live birth
A fossil fish uncovered in Australia is the oldest-known example of a mother
giving birth to live young, scientists have reported in the journal Nature.
The 380 million-year-old specimen has been preserved with an embryo still
attached by its umbilical cord.
The find, reported in Nature, pushes back the emergence of this reproductive
strategy by some 200 million years.
Until now, scientists thought creatures from these times were only able to
develop their young inside eggs.

When I looked at it, my jaw dropped. I said we are onto something big here

Before this find, the earliest evidence for this form of reproduction came
from reptile fossils dating to the Mesozoic Era (248 to 65 million years ago)
The team said the latest discovery had a remarkably advanced reproductive
biology, similar to modern sharks and rays.
The extremely well-preserved fossil represents a new species of "placoderm"
fish.
The placoderms were an incredibly diverse group and are thought to be the most
primitive known vertebrates with jaws.
These armoured fish dominated seas, rivers and lakes throughout the Devonian
Period (420-360 million years ago).
This latest placoderm specimen, which measures about 25cm (10in) in length,
was found in the Gogo area of Western Australia in 2005 by a team led by John
Long from Museum Victoria.

The fossil was found in Western Australia

Close examination revealed that the team had unearthed something unusual.
Professor Lane said: "When I looked at it, my jaw dropped. I said: 'we are
onto something big here'."
The team found an embryo and an umbilical cord, which had been exquisitely
preserved along with the female fish.
The scientists have named it Materpiscis attenboroughi, in honour of the
naturalist Sir David Attenborough, who first drew attention to the Gogo fish
fossil sites in the 1970s.
Sir David told the team that he was "very very flattered" to have had his name
given to such an "astonishing creature".
The discovery prompted the researchers to return to another fossil that they
had unearthed in 1986.
Close investigation revealed that this too contained evidence of live births -
it contained three embryos
Professor Lane said: "After we saw this, we realised we had totally nailed it,
everyone was convinced that this creature bore live young."
Until the latest fossil find, scientists thought life forms that existed
during these times had only evolved to reproduce using externally fertilised
eggs - a primitive version of the way fish spawn today.
Now, however, the team believes this ancient species bore live young through
internal fertilisation (viviparity).
Dr Long commented: "This is not only the first time ever that a fossil embryo
has been found with an umbilical cord, but it is also the oldest known example
of any creature giving birth to live young.
"The existence of the embryo and umbilical cord within the specimen provides
scientists with the first ever example of internal fertilisation - or sex -
confirming that some placoderms had remarkably advanced reproductive biology.
He added: "This is a world first fossil find, and it opens up a window into
the developmental biology of an entire extinct class of organisms."
Commenting on the paper, Zerina Johanson, a palaeontologist at London's
Natural History Museum, said: "It is extremely rare to find preservation like
this in the fossil record. This new discovery extends the record of viviparity
back almost 200 million years in the fossil record.
"Placoderms represent the most primitive group of jawed vertebrates, so this
work shows that the capacity for internal fertilisation and giving birth to
live young evolved very early during vertebrate history."
==
DNA rigins
Virtually the only gap that remains to be filled in is the matter of how DNA
arose in a prebiotic environment, and most of the answers are already in
place. Some example scientific papers covering the groundwork include:

A possible prebiotic synthesis of purine, adenine, cytosine, and
4(3H)-pyrimidinone from formamide implications for the origin of life by
Raffaelle Saladino, Claudia Crestini, Giovanna Costanzo, Rodolfo Negri and
Ernesto di Mauro, Bioorganic & Medicinal Chemistry, 9(5): 1249-1253 (May 2001)

A Self-Replicating Ligase Ribozyme by Natasha Paul & Gerald F. Joyce, Proc.
Natl. Acad. Sci. USA., 99(20): 12733-12740, 1st October 2002

A Self Replicating System by T. Tjiviuka, P.Ballester and J. Rebek jr.,
Journal of the American Chemical Society, 112: 1249-1250 (1990)

Carbonyl Sulphide Mediated Prebiotic Formation of Peptides by Luke Leman,
Leslie Orgel and M. Reza Ghadiri, Science, Vol 306, pp 283-286, 8 October 2004

Chemistry for the Synthesis of Nucleobase-Modified Peptide Nucleic Acid by R.
H. E. Hudson, R. D. Viirre, Y. H. Liu, F.Wojciechowski, and A. K. Dambenieks,
Pure Appl. Chem., 76: 1591-1598 (2004)

Nucelotide Synthetase Ribozymes May Have Emerged First In The RNA World by
Wentao Ma, Chunwu Yu, Wentao Zhang and Jiming Hu, The RNA Journal, 13:
2012-2019, 18th September 2007

Peptide Nucleic Acids Rather Than RNA May Have Been The First Genetic Molecule
by Kevin E. Nelson, Matthew Levy and Stanley L. Miller, Proc. Natl. Acad. Sci.
USA., 97: 3868-3871, 2000

Prebiotic Amino Acids As Asymmetric Catalysts by Sandra Pizzarello and Arthur
L. Weber, Science, 303: 1151, 20 February 2004

Racemic Amino Acids from the Ultraviolet Photolysis of Interstellar Ice
Analogues by Max P. Bernstein, Jason P. Dworkin, Scott A. Sandford, George W.
Cooper and Louis. J. Allamandola, Nature, Vol 416, pp 401-403, 28 March 2002

Ribozymes: Building The RNA World by Gerald F. Joyce, Current Biology, 6(8):
965-967, 1996

RNA-Catalysed RNA Polymerisation: Accurate And General RNA-Templated Primer
Extension by Wendy K. Johnston, Peter J. Unrau, Michael S. Lawrence, Margaret
E. Glasner and David P. Bartel, Science 292: 1319-1325 (18 May 2001)

RNA-Directed Amino Acid Homochirality by J. Martyn Bailey, J. Fed. Amer. Socs.
Exp. Biol., 12(6): 503-507

Self Replicating Systems by Volker Patzke and Gunter von Kiedrowski, ARKIVOC
5: 293-310, 2007

Self-Replication of Complementary Nucleotide-Based Oligomers by D. Sievers and
G. von Kiedrowski, Nature, 369: 221-224 (1994)

The Antiquity Of RNA-Based Evolution by Gerald F. Joyce, Nature, 418: 214-221,
11th July 2002
==
Speciation events observed in the laboratory, along with insights into
mechanisms for the origins of multicellularity:

Drosophila paulistorum: A Cluster of Species in Statu Nascendi by Theodosius
Dobzhansky & Boris Spassky, Proc. Natl. Acad. Sci. USA., 45(3): 419-428 (1959)

Evidence for rapid speciation following a founder event in the laboratory by
J.R. Weinberg V. R. Starczak and P. Jora, Evolution vol 46, pp 1214-1220, 1992

Experimentally Created Incipient Species of Drosophila by Theodosius
Dobzhansky & Olga Pavlovsky, Nature 230, pp 289 - 292 (02 April 1971)

Founder-flush speciation in Drosophila pseudoobscura: a large scale experiment
by A. Galiana, A. Moya and F. J. Alaya, Evolution vol 47, pp 432-444, 1993
(Speciation event in Drosophila melanogaster)

Phagotrophy by a flagellate selects for colonial prey: A possible origin of
multicellularity byM.E. Boraas, D.B. Seale and J.E. Boxhorn, Evolutionary
Ecology Vol. 12, no. 2, pp. 153-164. Feb 1998

The phagotrophic origin of eukaryotes and phylogenetic classification of
Protozoa by Tom Cavalier-Smith, International Journal of Systematic and
Evolutionary Microbiology vol 52, pp 297-354, 2002

Evolution of novel cooperative swarming in the bacterium Myxococcus xanthus by
Gregory J. Velicer and Yuen-tsu N. Yu, Nature vol 425, pp 75-78, 2003.

[2] Speciation events in the wild, including supporting evidence from
molecular phylogeny (and this is just in Cichlid fishes, by the way):

Adaptive Evolution And Explosive Speciation: The Cichlid Fish Model by Thomas
D. Kocher, Nature Reviews: Genetics, 5: 288-298 (April 2004)

Cichlid Species Flocks of the Past and Present by A. Meyer, Heredity vol 95,
419-420, 20 July 2005

Fractious Phylogenies by Thomas D Kocher, Nature, Vol 423, pp 489-490, 29 May
2003

Hybridisation and Contemporary Evolution in an introduced Cichlid Fish from
Lake Malawi National Park by J. Todd Streelman, S.L. Gymrek, M.R. Kidd, C.
Kidd, R.L. Robinson, E. Hert, A.J. Ambali and T.D. Kocher, Molecular Ecology,
vol 13, pp 2471-2479, 21 April 2004

Major Histocompatibility Complex Variation In Two Species Of Cichlid Fishes
From Lake Malawi by Hideki Ono, Colm O'hUigin, Herbert Tichy and Jan Klein,
Molecular and Evolutionary Biology, 10(5): 1060-1072 (1993)

Mitochondrial Phylogeny of the Endemic Mouthbrooding Lineages of Cichlid
Fishes from Lake Tanganyika in Eastern Africa by Christian Sturmbauer and Axel
Meyer, Journal of Molecular and Biological Evolution, Vol 10, No. 4, pp
751-768, 1993

Multilocus Phylogeny of Cichlid Fishes (Pisces: Perciformes) : Evolutionary
Comparison of Microsatellite and Single-Copy Nuclear Loci by J. Todd
Streelman, Rafael Zardoya, Axel Meyer and Stephen A Karl, Journal of Molecular
and Biological Evolution, Vol 15, No 7, pp 798-808, 1998

Origin of the Superflock of Cichlid Fishes from Lake Victoria, East Africa by
Erik Verheyen, Walter Salzburger, Jos Snoeks and Axel Meyer, Science, vol 300,
pp 325-329, 11 April 2003

Phylogeny of African Cichlid Fishes as Revealed By Molecular Markers by Werner
E. Mayer, Herbert Tichy and Jan Klein., Heredity, vol 80, pp 702-714, 1998

The Species Flocks of East African Cichlid Fishes: Recent Advances in
Molecular Phylogenetics and Population Genetics by Walter Salzburger and Axel
Mayer, Naturwissenschaft, vol 91, pp 277-290, 20 April 2004

Plus, the following paper contains an experimental test of selection
mechanisms applicable to the above:

Genetics of Natural Populations XII. Experimental Reproduction of Some of the
Changes Caused by Natural Selection by Sewall Wright & Theodosius Dobzkansky,
Genetics, 31(2): 125-156 (1946)
==
Evolution of a new functional gene observed: Apo-AI- Milano is a naturally
occurring genetic mutation that reduces the effects of cholesterol. It was
first observed in a single Italian family, and is currently spreading through
the Italian population. It is so effective drug companies are producing it
artificially as a cholesterol fighting agent.
http://en.wikipedia.org/wiki/Apolipoprotein_A1

Speciation examples: genetic studies show the Cichlid family of fishes has
diverged into dozens of new species in the last few thousand years in the
changing environments of Lake Victoria.
http://en.wikipedia.org/wiki/Cichlid_fish

abiogenesis  http://www.rockefeller.edu/evolution/
==
http://www.talkdesign.org/faqs/flagellum.html

http://www.millerandlevine.com/km/evol/design2/article.html

http://www.pandasthumb.org/archives/2007/07/current_biology.html

From The Origin Of Species To The Origin Of Bacterial Flagella by
Mark J. Pallen and Nick Matzke, Nature Reviews Microbiology, published
online 2006

Incidentally, Matzke published a paper in 2003, in which he predicted that
numerous homologies would be found between the proteins in the bacterial
flagellum and the Type 3 Secretory System, and lo and behold, those homologies
were later found to exist by other scientists working in the field. Score one
for an "imploding" branch of science there methinks.

Another paper on the subject is:

Stepwise Formation Of The Bacterial Flagellar System by Renyi Liu and Howard
Ochman, Proceedings of the National Academy of Sciences of the USA, 104(17):
7116-7121, 24th April 2007
==
Evolution is not something that happens overnight. It would take
generations for even minor changes to become apparent. It would take tens of
thousands of years for any significant changes and much longer for new species
to form. But there is evidence of evolution happening all around us today.
There are 8 different species of bears and many more sub species. There are
over 20,000 species of spiders. These are just 2 examples but nearly every
animal has many different varieties that have evolved differently. Recent
(remember recent in evolution terms is tens of thousands of years) evolution
is also easily seen in humans. Different skin color, facial features and body
structure that came about when pockets of the human population where isolated
for many generations. 
Darwin died in 1889. There has been a lot of research done
since then. I suggest that you dont use a single book written over 150 years
ago as your only source. But to address your comments, for the purpose of this
conversation it really doesnt matter where the original cell of life came from.
Intelligent design totally discredits the claim that species have evolved into
new species (although they do agree that minor changes have occurred). The
facts are that the oldest fossils are of very simple organisms. As time passed
the fossils became much more complex. NO fossils have been found for animals
that exist today. That in itself would lead us to believe that either species
evolved into new species or that new species have magically appeared over time. 
===
Note that phylogenetically, birds are members of
Reptilia.

http://www.pubmedce ntral.nih. gov/articlerende r.fcgi?artid= 1815256

Abstract

We report results of a megabase-scale phylogenomic
analysis of the Reptilia, the sister group of mammals.
Large-scale end-sequence scanning of genomic clones of
a turtle, alligator, and lizard reveals diverse,
mammal-like landscapes of retroelements and simple
sequence repeats (SSRs) not found in the chicken.
Several global genomic traits, including distinctive
phylogenetic lineages of CR1-like long interspersed
elements (LINEs) and a paucity of A-T rich SSRs,
characterize turtles and archosaur genomes, whereas
higher frequencies of tandem repeats and a lower
global GC content reveal mammal-like features in
Anolis. Nonavian reptile genomes also possess a high
frequency of diverse and novel 50-bp unit tandem
duplications not found in chicken or mammals. The
frequency distributions of &#8776;65,000 8-mer
oligonucleotides suggest that rates of DNA-word
frequency change are an order of magnitude slower in
reptiles than in mammals. These results suggest a
diverse array of interspersed and SSRs in the common
ancestor of amniotes and a genomic conservatism and
gradual loss of retroelements in reptiles that
culminated in the minimalist chicken genome.

Program

Comparative genomics is a central focus of modern
biology in part because it facilitates the
understanding of principles of genome evolution (13).
However, it is impractical to expect taxonomically
broad comparative studies to proceed rapidly for
nonmodel organisms on a whole-genome basis. A prime
example of our limited understanding from the present
handful of complete genomes is that we still do not
know the sequence of genomic events that led to the
structural diversity seen in mammalian genomes and
those of their sister group, the Reptilia, which
includes birds (4). The draft chicken genome (5)
substantially increases our understanding of amniote
comparative genomics, but evolutionary interpretation
relying solely on chickenmammal contrasts will remain
difficult without new data for phylogenetically
intermediate lineages. On the one hand, the common
amniote ancestor may have had a small genome as in
extant birds, with mammals and nonavian reptiles
independently acquiring transposable elements that
resulted in genome size increases in these two
lineages. On the other hand, the common amniote
ancestor may have had a large, repeat-rich genome as
in extant mammals, with multiple sequential reductions
in retroelement abundance occurring in the lineages
leading to the smaller genomes of nonavian reptiles
and birds (6). A third scenario might include a
combination of both independent gains and reductions
of specific genomic elements. Here we use a BAC- and
plasmid-end sequencing approach in exemplars of three
major nonavian reptile lineages, American Alligator
(Alligator mississippiensis) , Painted Turtle
(Chrysemys picta), and the Bahamian Green Anole
(Anolis smaragdinus) , to better characterize the
sequence of genomic changes underlying the
diversification of amniote genomes.
Little is known about the large-scale structure of
nonavian reptile genomes at the sequence level.
Alligator and turtle genome sizes are &#8776;30%
smaller than human, &#8776;50% larger than chicken,
and only &#8776;12% larger than Anolis, whose genome
size is close to the mean for nonavian reptiles.
Unlike alligator genomes, the anole, painted turtle,
and chicken contain a significant number of
microchromosomes (7), which we expect would be gene
rich as reported for chickens (8) and the soft-shelled
turtle (9). In general, it is unknown how the
macrochromosomes of reptiles differ from those of
mammals (10) and those of the nonavian reptiles
investigated here. The turtle and alligator species
investigated here have environmental as opposed to
genetic sex determination, and sex determination in
Anolis is inferred to be genetic based on some
karyological evidence (11). Several retroelement
lineages have been characterized in turtles and other
reptiles (1215). Projects in progress will produce
genome sequences for another bird, the Zebra Finch,
Taeniopygia guttata, and a lizard, Anolis
carolinensis. In the meantime, our goal in this
project was to quickly amass a moderate database of
primary sequence distributed throughout the genomes of
genomically understudied lineages, which can reveal
numerous genomewide trends that help characterize the
most fundamental aspects of genome structure. Although
the genomes we have investigated may not reflect
specific changes in subclades of diverse groups such
as squamates, any shortcomings of our limited
taxonomic sampling are overcome by our ability to
present a broad-brush window on genomic trends for
nonavian reptiles, thereby quickly placing the chicken
and mammal genomes in broader context.

(Snip Results & Discussion)

Conclusion

In summary, our analysis suggests that the ancestral
amniote genome featured a relatively low global GC
content as in mammals and a rich repetitive landscape
dominated by CR1 and MIR retroelements and an
abundance of AT-rich SSRs. Our finding of diverse CR1
lineages in nonavian reptiles qualifies a model in
which a chicken-like streamlined ancestral amniote
genome underwent expansion in mammals and nonavian
reptiles independently (6). Rather it implies a
complex scenario in which the diversity of CR1
elements in the ancestral amniote underwent a
wholesale replacement by L1 and related mobile
elements in mammals, and in which multiple sequential
reductions in diversity occurred in the lineages
leading to nonavian reptiles and birds. We expect that
further genomic scans in additional reptile species,
as well as further whole-genome sequencing projects,
will considerably refine the major features in reptile
genome evolution that we have outlined here.
==
http://palaeo.gly.bris.ac.uk/communication/boulton/evolution.html   dinos
==
A great deal is know about these immediate ancestors
to the dinosaurs.

http://en.wikipedia .org/wiki/ Archosaur

Clade Archosauria: Permian or Triassic to Present
(Birds & Crocodilians)

Archosaurs (Greek for 'ruling lizards') are a group of
diapsid reptiles represented by modern birds and
crocodilians. This group also includes extinct
non-avian dinosaurs, pterosaurs and relatives of
crocodiles.

There is some debate about when archosaurs first
appeared. Those who classify the Permian reptiles
Archosaurus rossicus and/or Protorosaurus speneri as
true archosaurs maintain that archosaurs first
appeared in the late Permian. Those who classify both
Archosaurus rossicus and Protorosaurus speneri as
archosauriformes (not true archosaurs but very closely
related) maintain that archosaurs first evolved from
Archosauriform ancestors during the Olenekian (early
Triassic Period).

The simplest and most widely-agreed synapomorphies of
archosaurs are:

* Teeth set in sockets, which makes them less
likely to be torn loose during feeding. This feature
is responsible for the name "thecodonts" ("socket
teeth"), which paleontologists used to apply to all or
most archosaurs.
* Antorbital fenestrae (openings in the skull in
front of the eyes but behind the nostrils), which
reduced the weight of the skull, a useful feature
since most early archosaurs had long, heavy skulls,
rather like those of modern crocodilians. The
preorbital fenestrae (sometimes called anteorbital
fenestrae) are often larger than the orbits (eye
sockets).
* Mandibular fenestrae (small openings in the jaw
bones), which may have reduced the weight of the jaw
slightly.
* A fourth trochanter (ridge for attaching
muscles) on the femur. This seemingly insignificant
detail may have made the evolution of dinosaurs
possible (all early dinosaurs and many later ones were
bipeds), and may also be connected with the ability of
the archosaurs or their immediate ancestors to survive
the catastrophic Permian-Triassic extinction event.

Archosaur takeover in the Triassic

The Synapsida (informally known as "mammal-like
reptiles") were the dominant land vertebrates
throughout the Permian, but most perished in the
Permian-Triassic extinction event. Lystrosaurus (a
herbivorous mammal-like reptile) was the only large
land animal to survive the event, becoming the most
populous land animal on the planet for a time.[1]

But archosaurs quickly became the dominant land
vertebrates in the early Triassic. The two most
commonly-suggested explanations[ citation needed] for
this are:

* Archosaurs made quicker progress than
mammal-like reptiles towards erect limbs, and this
gave them greater stamina by avoiding Carrier's
constraint. This is unconvincing since Archosaurs
became dominant while they still had sprawling or
semi-erect limbs, similar to those of Lystrosaurus and
other mammal-like reptiles.
* The early Triassic was predominantly arid,
because most of the earth's land was concentrated in
the supercontinent Pangaea. Archosaurs were probably
better at conserving water than mammal-like reptiles
because:

* Modern diapsids (lizards, snakes,
crocodilians, birds) excrete uric acid, which can be
excreted as a paste. It is reasonable to suppose that
archosaurs (diapsids and ancestors of crocodilians,
dinosaurs and birds) also excreted uric acid, and
therefore were good at conserving water. The
aglandular (glandless) skins of diapsids would also
have helped to conserve water.
* Modern mammals excrete urea, which requires
a lot of water to keep it dissolved. Their skins also
contain many glands, which also lose water. Assuming
that mammal-like reptiles had similar features, e.g.
as argued in Palaeos [1], they were at a disadvantage
in a mainly arid world. The same well-respected site
points out that "for much of Australia's
Plio-Pleistocene history, where conditions were
probably similar, the largest terrestrial predators
were not mammals but gigantic varanid lizards
(Megalania) and land crocs."

Main types of archosaurs (link has graphics of
anatomical differences)

Since the 1970s scientists have classified archosaurs
mainly on the basis of their ankles.[2] The earliest
archosaurs had "primitive mesotarsal" ankles: the
astragalus and calcaneum were fixed to the tibia and
fibula by sutures and the joint bent about the contact
between these bones and the foot.

The Crurotarsi appeared early in the Triassic. In
their ankles the astragalus was joined to the tibia by
a suture and the joint rotated round a peg on the
astragalus which fitted into a socket in the
calcaneum. Early "crurotarsans" still walked with
sprawling limbs, but some later "crurotarsans"
developed fully erect limbs (most notably the
Rauisuchia). And modern crocodilians are
"crurotarsans" which can walk with their limbs
sprawling or erect depending on how much of a hurry
they are in.

Euparkeria and the Ornithosuchidae had "reversed
crurotarsal" ankles, with a peg on the calcaneum and
socket on the astragalus.

The earliest fossils of Ornithodira ("bird necks")
appear in the Carnian age of the late Triassic, but it
is hard to see how they could have evolved from the
"crurotarsans" - possibly they actually evolved much
earlier, or perhaps they evolved from the last of the
"primitive mesotarsal" archosaurs. Ornithodires'
"advanced mesotarsal" ankle had a very large
astragalus and very small calcaneum, and could only
move in one plane, like a simple hinge. This
arrangement was only suitable for animals with erect
limbs, but provided more stability when the animals
were running. The ornothodires differed from other
archosaurs in other ways: they were lightly-built and
usually small, their necks were long and had an
S-shaped curve, their skulls were much more lightly
built, and many ornothodires were completely bipedal.
The archosaurian fourth trochanter on the femur may
have made it easier for ornothodires to become bipeds,
because it provided more leverage for the thigh
muscles. In the late Triassic the ornithodires
diversified to produce pterosaurs and dinosaurs.[3]

Phylogeny

(Diagram doesn't fit, but not that each of the two
main archosaur groups has living representatives; the
Crurotarsi have crocs & Ornithodira have birds, as the
name implies.)

`--Archosauria [Crown group Archosauria =
Avesuchia]
|--Crurotarsi
| |-?Ctenosauriscidae
| `--Crocodylotarsi
| |--Ornithosuchidae
| `--+--Phytosauria
| `--Suchia
| |--Prestosuchidae
| `--Rauisuchiformes
| |--Aetosauria
| `--Rauisuchia
|
|--Rauisuchidae
|
`--+--Paracrocodylo morpha
|
`--Crocodylomorpha (crocodiles and relatives)
`--Ornithodira
|--Pterosauromorpha
| |--Scleromochlus
| `--Pterosauria
`--Dinosauromorpha
`--Dinosauriformes
`--Dinosauria
|--Ornithischia
`--Saurischia
`--Aves (birds)
==
The modern taxonomic system of cladistics is based on
relatedness between groups of living things, relying
on shared characteristics derived from common
ancestors. It recognizes stem vs. more derived crown
groups, as well as out groups & sister taxons.

Here's a simplified cladistic summary of tyrannosaur
evolution, presented in narrative form, since detailed
cladograms can't be reproduced here, except as links.

Each node in a cladogram marks derived characteristics
distinguishing all the included groups sharing those
traits.

The outgroup to the archosaurs are all those diapsid
reptiles living & extinct more closely related to
lepidosaurs (snakes, lizards & tuataras) & (probably)
turtles than to crocs & birds. Their sister group is
either lepidosaurs or turtles. This node remains
controversial, with morphological & genetic evidence
inconclusive. (To simplify & shorten, I've left out
the extinct marine diapsids like ichthyosaurs,
plesiosaurs & mosasaurs.)

Crocs & their relatives form the sister taxon to the
group dinosaurs (including birds) & pterosaurs, which
are the sister taxon to the two orders of dinosaurs
(saurischians & ornithischians) .

The sister taxon to the saurischians (including birds)
is of course the ornithischians. The sister taxon to
the theropods (including birds) is the sauropods.

Among theropod dinosaurs, the sister taxon to the Late
Triassic Coelophysidae are less derived groups of
early meat-eaters. It now appears that this family
may be ancestral to all later theropods, in sister
groups Ceratosauria (Early Jurassic) & more derived
Tetanurae.

Among Tetanurae, the sister group to Avetheropoda, the
line leading to birds & tyrannosaurs, is now
considered the Megalosauroidea (which includes the
first dinosaur ever recorded, although no one knew in
1676 what the fossil was), alternately called
Spinosauroidea or Torvosauroidea.

Avetheropoda divided into the Carnosauria (including
Jurassic American predator Allosaurus), & the
Coelurosauria, a very large & diverse dinosaur group
that was especially common during the Cretaceous.

From Wiki:

"Thus, during the late Jurassic, there were no fewer
than four distinct lineages of theropods -
ceratosaurs, megalosaurs, carnosaurs, and coelurosaurs
- preying on the abundance of small and large
herbivorous dinosaurs. All four groups survived into
the Cretaceous, although only two - the abelisaurs and
the coelurosaurs - seem to have made it to end of the
period, where they were geographically separate, the
abelisaurs in Gondwana, and the coelurosaurs in
Asiamerica.

"Of all the theropod groups, the coelurosaurs were by
far the most diverse. Some coelurosaur clades that
flourished during the Cretaceous were the
tyrannosaurids (including Tyrannosaurus) the
dromaeosaurids (including Velociraptor and
Deinonychus, which are remarkably similar in form to
the oldest known bird, Archaeopteryx) , the bird-like
troodontids and oviraptorosaurs, the ornithomimosaurs
(or "ostrich dinosaurs"), the strange giant-clawed
herbivorous Therizinosauridae, and the birds, which
are the only dinosaur lineage to survive the end
Cretaceous mass-extinction.

"While the roots of these various groups must have
been in the Late or possibly even the Middle Jurassic,
they only became abundant during the Early Cretaceous.
A few paleontologists, such as Gregory S. Paul, have
suggested that some or all of these advanced theropods
were actually descended from flying dinosaurs or
proto-birds like Archaeopteryx that lost the ability
to fly and returned to a terrestrial habitat.

"Coelurosauria is a diverse group of theropod
dinosaurs that includes tyrannosaurs,
ornithomimosaurs, and maniraptors; Maniraptora
includes birds, the only coelurosaurs alive today. All
feathered dinosaurs discovered so far have been
coelurosaurs; in fact, some scientists believe that
most members of coelurosauria bore some kind of
feathers.

"Most coelurosaurs were bipedal predators. The group
includes some of the largest (Tyrannosaurus) and
smallest (Microraptor, Parvicursor) carnivorous
dinosaurs ever discovered. Characteristics that
distinguish coelurosaurs include:

* a sacrum (series of vertebrae that attach to the
hips) longer than in other dinosaurs
* a tail stiffened towards the tip
* a bowed ulna (lower arm bone).
* a tibia (lower leg bone) that is longer than the
femur (upper leg bone)"

Whatever the case may be, predecessor fossils abound
in the bird & tyrannosaur lineage.

Among Coelurosauria, the sister group to the more
birdy groups is probably the Compsognathidae, although
it may be the stem group or included within
Maniraptora.

http://en.wikipedia .org/wiki/ Compsognathidae

Tyrannosauria is the sister taxon to the group
containing Maniraptora (including birds) & the
Ornithomimosauria, members of which bear a superficial
resemblance to modern ostriches. Obviously, in this
case the sister taxon to Maniraptora (including birds)
is the Ornithomimosauria.

"Maniraptora ("hand snatchers") is a clade of
coelurosaurian dinosaurs which includes the birds and
the dinosaurs that were more closely related to them
than to Ornithomimus velox. It contains the major
subgroups Aves, Deinonychosauria, Oviraptorosauria and
Therizinosauria. Ornitholestes and the Alvarezsauridae
are also often included. Together with the next
closest sister group, the Ornithomimosauria,
Maniraptora comprises the more inclusive clade
Maniraptoriformes. Maniraptors first appear in the
fossil record during the Jurassic Period (see
Eshanosaurus) , and survive today as over 9,000 species
of living birds."

So, the tyrannosaurs would be cousins to the birds,
rather than their sisters. Looking at T. rex as a
big, flightless, carnivorous "bird" gives Thanksgiving
some new meaning!
==
In fact, his formal education was identical to
Newton's, but creationists don't disparage Sir Isaac
based upon his not having earned a PhD in a scientific
discipline. They might if they knew he were a secret
Unitarian.

Newton (BA, 1660) & Darwin (BA, 1830) were both
awarded Bachelor of Arts Degrees from Cambridge, later
upgraded to Master's, as was & is the practice at
Oxford & Cambridge. Newton earned no honors or
distinction, while Darwin finished fairly high overall
in his year. Due to decades of Puritanism, Cambridge
was in a slough of despond when Newton was up. The
education that Darwin received was superior, thanks in
large part to the influence of Newton's achievements
on the university's curriculum.

Darwin also benefited from his earlier medical studies
at Edinburgh. He learned a lot in Edinburgh,
including taxidermy from a black man, which stood him
in good stead later, & some marine biology, but was
ethically opposed to surgery without anesthesia. 
While there & later at Cambridge, he was dedicated to
sporting & nature study, already making scientific
contributions as an undergraduate.

He transferred to Cambridge, but concentrated as much
as possible on subjects that interested him rather
than requirements, taking advantage of all the
university offered. Through pursuits & interests like
collecting beetles, he became a protege of Prof.
Henslow.

Eventually, he graduated with honors, but with highest
marks in "natural theology", derived from the work of
Paley. His friendship & studies with Henslow were
comparable to a modern day college internship. 

After acing his exams, Darwin stayed on studying
geology, for the contemporary equivalent of a master's
degree. He spent a summer tramping Wales with the
Rev. Prof. Sedgwick, namer of the Cambrian Period &
arguably the greatest geologist of the time (certainly
one of the top two or three). Darwin worked in hard
core geology, where he demonstrated great ability, as
evinced in his early publications. 

Cambridge did not offer degrees in specialties; one
graduated in general studies oriented toward the
clergy but in the Anglican Church then, that included
lots of math & science. Darwin was as academically
qualified as anyone else in geology, or any of the
sciences. Doctorates were granted in only a few
professional specialities, unlike the extensive
specialization of today. 

His studies at Cambridge, while designed by the
university to prepare Anglican clergymen, enabled
Darwin to learn what he wanted to about nature, just
as had his ostensibly medical education. As part of
his required curriculum, he studied & enjoyed Paleyıs
"Natural Theology", but of course, it was essentially
a natural science text, or intended to be, when
published in 1802.

There were no undergrad degrees, let alone doctorates,
in scientific subjects in the 15th, 16th, 17th, 18th
or most of the 19th centuries, so Copernicus, Galileo,
Kepler & all the other giants upon whose shoulders
Newton stood should be equally disparaged by lying
creationist goons.

Both Newton & Darwin were both Fellows of the Royal
Society.

Darwin would be remembered today as the outstanding
naturalist of the 19th century even if he hadn't
discovered natural selection. "Naturalist" shouldn't
be considered derogatory, as of course lying
creationist swine try to suggest. It was the common
19th century term for a natural philosopher, ie
scientist, specializing in geology & biology rather
than chemistry, physics or astronomy. 

Many early modern scientific figures were of course
naturalists as well as chemists, physicist &
astronomers. Lavoisier comes to mind. While
primarily remembered today as a pioneering chemist, he
also made important contributions to geology & other
sciences.

As mentioned, putting his career into a 20th or 21st
century framework, his geological field studies with
Prof. Sedgwick in Wales in the summer after graduating
from Cambridge would have merited an MS degree at the
very least, & his biological studies with Henslow
deserved another MS. His first monograph, on the
creation of coral atolls, based upon his Beagle
experience, would easily qualify for a tenure-worthy
PhD. But at that point he was just getting started. 

One difference between Darwin & Newton is that Sir
Isaac remained single & stayed in academia, while
Darwin married his cousin & raised a large family. As
Wedgwoods, they didn't need an academic sinecure to
make ends meet. As a country gentleman, Darwin was
free to conduct research, experiment, collect & write,
which he did prodigiously.

Darwin is the greatest single contributor in history
to the science collections at the British Museum. 
Even before he returned from his five year voyage
around the world on HMS Beagle, he was nominated for
membership in the most prestigious science societies,
which marked professional scientists of his day, years
earlier than most contemporaries, on the basis of his
remarkable work.

But Darwinıs science recognition came from his voyage.
As noted, he made contributions to the collections at
the British Museum, astounding quantity & scope. His
specimens are still type specimens in ornithology,
mammals, botany & geology. 

This exceptional range of work got him nominated to
the Royal Society, which was then perhaps analogous to
the National Academy of Sciences here. His election
at such an early age is indication of his outstanding
scientific qualifications. 

Nor is it impossible even today for scientists without
PhDs to earn recognition for pioneering work, as Jane
Goodall demonstrates. Most of her great contributions
to ethology & primatology were made well before she
belatedly undertook a formal advanced graduate degree
program.

To quote from a 2007 science blogger: 

"In research, academic qualifications tend to fall by
the way. The question is whether you can devise
original ways to test important questions. People who
can do that tend to get promoted quickly ‹ like
Stanley Miller at the University of Chicago in 1953;
or like (paleontologist) Paul Serrano at Chicago
today...To make it in science, one needs a good,
testable hypothesis.. .

"Among other things, the serious papers propose tests
of the hypothesis that can be done, and they donıt
claim that a conspiracy keeps the ideas from getting
out ‹ instead explaining how it is that the best
scientists can continue to use as theory an idea that
may be wrong, usually in the form of ³hereıs how we
look at this problem today under the prevailing
theory, and hereıs how this new idea provides better
or more clear observations.² Darwinıs papers did that,
in coral atoll formation, in evolution theory as a
whole, in climbing plants, in insectivorous plants, in
human evolution, and in the importance of worms.

"Darwinıs legacy should be much more than just the
theory of evolution. Darwin revolutionized all of
science with his observation methods. He avoided
conjecture about what might happen whenever simple
observation could be devised to provide real data. So,
for example, rather that resort to the philosopherıs
ramblings on why ivy twines, he spent weeks in his lab
actually watching climbing vines grow, discovering the
methods of their twining. Itıs a stunningly simple
monograph (itıs available on the web), but no one had
thought, or bothered, to do that before. Darwinıs work
is still the standard in climbing vines, he covered it
so thoroughly and well."

I'd add his work on barnacles, still the standard, &
on orchids, to mention but a few of his astonishingly
varied contributions to life science. Wallace sent
Darwin his paper on natural selection because of
Darwin's reputation, after all, without knowing of
Darwin's own thoughts on the theory.
Darwin's election to prestigious, elite science
societies based upon even his early work shows
creationist lies about his credentials to be idiotic &
bearing false witness. There is no sin they aren't
willing to commit for their false cult. 
When you win the awards for having done
great work in science, you're a scientist.
In any case, the modern evolutionary synthesis doesn't
depend upon Darwin's work of 150 years ago.
==
Books by Charles Darwin
http://darwin- online.org. uk/contents. html#books

Some examples:

The Structure and Distribution of Coral Reefs [1842]

A monograph on the fossil Lepadidae, or, pedunculated cirripedes of
Great Britain [1851]

A monograph on the fossil Balanidae and Verrucidae of Great Britain [1852]

On the various contrivances by which British and foreign orchids are
fertilised by insects [1862]

On the movements and habits of climbing plants [1864]

Insectivorous plants [1875]

The different forms of flowers on plants of the same species [1877]

The power of movement in plants [1880]

The formation of vegetable mould, through the action of worms [1881]

Articles by Charles Darwin
http://darwin- online.org. uk/contents. html#periodicals
==
The Origins of Life: From the Birth of Life to the Origin of Language by
John Maynard Smith and Eors Szathmary (Paperback -
==
Dinosauria is presently
regarded as a Superorder containing the Orders
Saurischia & Ornithischia. The latter includes
families with "things on their backs", like
Stegosaurus & armored dinos; "things on their heads",
like Triceratops, & the "duck-billed" hadrosaurs.

T. rex' phylogeny:

Division: Archosauria
Subsection: Ornithodira
Superorder: Dinosauria
Order: Saurischia ("lizard hips"; outgroup
Ornithischia)
Taxon: Eusaurischia
Suborder: Theropoda ("beast foot"; outgroup Sauropoda)
Taxon: Neotherapoda
Infraorder: Tetanurae* ("stiff tails"; outgroup
Ceratosauria) 
Taxon: Avetherapoda (outgroup Spinosauroidia)
Taxon: Coelurosauria (feathered tetanurans; outgroup
Carnosauria)
Taxon: Tyrannoraptora (outgroup Compsognathidae)
Superfamily: Tyrannosauroidea (outgroup
Maniraptoriformes)
Family: Tyrannosauridae 
Subfamily: Tyrannosaurinae (outgroup Albertosaurinae)
Genus: Tyrannosaurus
Species: Rex

Other genera presently recognized in the subfamily
Tyrannosaurinae besides Tyrannosaurus are Tarbosaurus,
Daspletosaurus & Nanotyrannus.

*Tetanurae meaning "stiff tails", was named by Jacques
Gauthier on cladistic grounds in 1986 for a large
group of theropod dinosaurs. Gauthier's paper was the
first serious application of the science of cladistics
to vertebrate paleontology.

Tetanurae are defined as all theropods more closely
related to modern birds than to Ceratosaurus (e.g.
Padian et al., 1999). Gauthier considered it to
consist of Carnosauria and Coelurosauria, although
many of what he considered carnosaurs have been
regarded as coelurosaurs or basal tetanurans by
subsequent workers (but see Rauhut, 2003). Paul Sereno
(1999) named Neotetanurae for the node joining
Carnosauria (his Allosauroidea) and Coelurosauria,
excluding other tetanurans such as spinosauroids.
Padian et al. (1999) gave a synonymous definition for
Gregory Paul's (1988) Avetheropoda, but this
definition was published slightly later.

Phylogeny & anatomical details of Tetanurae:

http://www.users. qwest.net/ ~jstweet1/ tetanurae. htm

See the ancestry of other dinosaurs below. Click on
body shape for more different genera:

http://internt. nhm.ac.uk/ jdsml/nature- online/dino- directory/ body.dsml? disp=list& bodytype= 4&sort=Genus

For more on the more distant ancestors of dinosaurs,
please this on the Archosaur pelvic girdle & dinosaur
evolution:

http://www.bioone. org/perlserv/ ?request= get-document& doi=10.1666% 2F0094-8373(2001)027%3C0059% 3ATMOTAR% 3E2.0.CO% 3B2&ct=1

==
Neanderthals were separate species, says new human family tree
A new, simplified family tree of humanity, published on Sunday, has dealt a
blow to those who contend that the enigmatic hominids known as Neanderthals
intermingled with our forebears.
Neanderthals were a separate species to Homo sapiens, as anatomically modern
humans are known, rather than offshoots of the same species, the new
organigram published by the journal Nature declares.
The method, invented by evolutionary analysts in Argentina, marks a break with
the conventional technique by which anthropologists chart the twists and turns
of the human odyssey.
That technique typically divides the the genus Homo into various
classifications according to the shape of key facial features -- "flat-faced,"
"protruding-faced" and so on.
Reconciling these diverse classifications from a tiny number of specimens
spanning millions of years has led to lots of claims and counter-claims, as
well as much confusion in the general public, about how we came to be here.
Various species of Homo have been put up for the crown of being our direct
ancestor, only to find themselves dimissed by critics as failed branches of
the Homo tree.
The authors of the new study, led by Rolando Gonzalez-Jose at the Patagonian
National Centre at Puerto Madryn, Argentina, say the problem with the
conventional method is that, under evolution, facial traits do not appear out
of the blue but result from continuous change.
So the arrival of a specimen that has some relatively minor change of feature
as compared to others should not be automatically held up as representing a
new species, they argue.
The team goes back over the same well-known set of specimens, but uses a
different approach to analyse it, focussing in particular on a set of
fundamental yet long-term changes in skull shape.
They took digital 3D images of the casts of 17 hominid specimens as well as
from a gorilla, chimpanzee and H. sapiens.
The images were then crunched through a computer model to compare four
fundamental variables -- the skull's roundness and base, the protrusion of the
jaw, and facial retraction, which is the position of the face relative to the
cranial base.
When other phylotogenic techniques are used, the outcome is a family tree
whose main lines closely mirror existing ones but offers a clearer view as to
how the evolutionary path unfolded.
The paper suggests that, after evolving from the hominid Australopithecus
afarensis, the first member of Homo, H. habilis, arose between 1.5 and 2.1
million years ago.
We are direct linear descendants of H. habilis. H. sapiens started to show up
around 200,000 years ago.
None of the species currently assigned to Hom