Marc W Allard ; George Washington Univ
Systematics of Lipotyphlan Insectivores, An Early Lineage of Placental Mammals

Participant Individuals:
Graduate student(s) : Ginny Emerson; Barbara McNiff
Undergraduate student(s) : Katherine Salmon; Christina Thirurathukal; Jennifer Saionz; Scott Baker; Fred Winninger

Partner Organizations:
Other collaborators:

Collaborators
C. W. Kilpatrick, Department of Zoology, University of Vermont,
Burlington VT.

J. Ottenwalder, UNDP-GEF/ONAPLAN Biodiversity Conservation and
Management Project, Programa de las Naciones Unidas para el
Desarrollo, Santo Domingo, Dominican Republic

J. S. Farris, Molekylarsystematiska laboratoriet, Naturhistoriska
riksmuseet, Box 50007, S-104 05 Stockholm, Sweden.

J. Carpenter. Department of Entomology, American Museum of Natural
History, New York, NY 10024 U.S.A.

Contacts
Rodney L. Honeycutt, 210 Nagle Hall, Genetics, Texas A&M University
College Station, TX 77843

Michael M. Miyamoto, 223 Bartram Hall, Department of Zoology,
University of Florida Gainesville, FL 32611

Michael J. Novacek, Department of Vertebrate Paleontology, American
Museum of Natural History, Central Park West at 79th Street, New
York, NY 10024

Robert Asher, Dept. of Anatomy, SUNY, Stony Brook NY.

Suzette K. Mouchaty, Dept. of Genetics, Lund University. Sweden

Link Olsen, Mammals, The Field Museum of Natural History, Chicago

Activities and findings:

Research Activities:

SUMMARY OF COMPLETED PROJECT

The Insectivora (Mammalia; Eutheria; Lipotyphla) originated during
the radiation of placental mammals dating back to at least the
beginning of the Cenozoic. Today this group (shrews, moles,
hedgehogs, and their relatives) is one of the largest mammalian
orders, yet little is known about the systematic placement of these
taxa. First, it is disputed whether the group, as currently defined,
is monophyletic or requires further subdivision. Second, the
relationship of the Insectivora to other eutherian orders is not
clear. The traditional view places them at the base of the
phylogenetic tree as a sister-group to the other eutherian mammals.
More recently, workers have suggested that insectivores may be part of
a later clade that includes Carnivora and Tubulidentata on the basis
of skull characters. Still other workers examining genetic evidence,
a and ß hemoglobin, myoglobin and lens a-crystallin sequences propose
that the Insectivora are more closely related to the Carnivora and
Pholidota. Furthermore, the relationships of the six families that
comprise the order Insectivora are not well understood either. There
are many competing classifications with character support for many of
the possible combinations of inter-familial relationships. Determining
the placement of the Insectivora has implications for understanding
all of eutherian mammal systematics because these taxa have been used
as a primitive morphotype for polarizing character transformations.

Resolving these questions have required a multi-disciplinary approach
including molecular, morphological, and paleontological information.
The major objectives of this project were to: 1) collect partial
mitochondrial 12S ribosomal RNA (rRNA) and Nicotinamide adenine
dinucleotide dehydrogenase subunit 2 (NADH2) sequences for
representatives of all insectivore families as well as five other
eutherian orders; and 2) conduct phylogenetic analyses of gene
sequences separately, combined, and combined with published
morphological information. The purpose of this project was to provide
fundamental information about patterns of molecular systematics for
the earliest extant lineages of the eutherian radiation, with emphasis
on the Insectivora. This knowledge is important for understanding
more about the molecular evolution and systematics of all eutherians
including humans.

Our results clearly show that the lipotyphlan insectivorans are
critical for understanding the eutherian radiation. What is less
clear is exactly how they are related to the other orders of mammals.
Current genetic analysis does not support lipotyphlan monophyly,
though the evidence is weak. Insectivorans spread themselves out
across the mammalian tree with few inter-familial and fewer
inter-ordinal relationships well supported. This is in direct
contrast to morphological data which supports the monophyly of the
group. Combined analysis of all of the available data does not
support lipotyphlan monophyly, nor does it support the super-orders
Afrotheria, and Afrosoricidae. Our results suggest a primitive grade
of insectivorans at the base of the mammalian tree. Eutherian
relationships continue to challenge our ability to build well
supported phylogenetic hypotheses. Only with further evidence will
these groups become better established. This project seems to have
raised more questions then answers. The unusual phylogenetic
placement of these insectivorans have inspired many other mammalian
systematists to incorporate lipotyphlan insectivorans into their
phylogenetic studies. Future work may uncover the genetic properties
which make insectivorans unstable on the eutherian phylogeny. More
detailed results are listed in the technical information section.

Sequencing 12S rRNA and Nadh2 genes for representative Insectivores
(see Table). Training Graduate and undergraduate researchers in
automated sequencing techniques (see project participants).
Publishing the results of the research (see major journal
publications) as well as participation in numerous national and
international meetings.

Table: A summary of sampling among families and subfamilies of
Insectivora. Where complete sequences are available a * is present.
Some of this sequence is being published now and is not yet in
GenBank.

FAMILY SUBFAMILY SPECIES 12S NADH2
Talpidae Talpinae Parascalops breweri * *
Condylura cristata * *

Erinaceidae Hylomyyinae Echinosorex gymnurus * *
Hylomys suillus * *
Podogymnura truei * *
Erinaceinae Atelerix albiventris * *
Erinaceus europaeus * *
Paraechinus * *

Soricidae Soricinae Blarina brevicauda * *
Sorex palustris * *
Crocidurinae Crocidura guildenstaedtii * *
Suncus murinus * *

Tenrecidae Tenrecinae Tenrec ecaudatus * *
Echinops telfairi * *
Oryzorictinae Oryzorictes talpoides * *
Microgale talazaci * *

Solenodontidae Solenodon paradoxus * *

Chrysochloridae Eremitalpa granti * *
Amblysomus hottentotus * *

Sequences were also collected for several outgroup samples and for
representatives of the Archonta. Control region sequences were
collected for several solenodons as well.

GENERAL METHODS (followed Emerson et al., 1999. Cladistics
15:221-230.
Materials and Methods
Sequences of the mitochondrial 12S rRNA gene were obtained for 106
taxa through Genbank, one from a separate website, three from
Tanhauser et al. (1985), and eight were generated in our lab. The
range of taxa includes at least one representative for each of the
eighteen orders of eutherian mammals and four members of Metatheria
which compose the outgroup. The sequences were compiled from published
data, and were brought together to provide a greater amount of
sampling and comparable variation than previous studies of the 12S
rRNA gene.

The 118 sequences were aligned by hand, with the exception of a
highly variable region located near the 3' end of the gene. This region
was aligned using the alignment program 1.6 (Thompson et al.
1994). The alignment DS38659 is available from EMBL upon request by
electronic mail to NetServ@EBI.AC.UK. A maximum parsimony analysis was
conducted by performing 100 heuristic searches utilizing the random
addition sequence option and equal weighting in PAUP 3.1.1 (Swofford
1993). This method was chosen due to the large number of taxa and in
the interest of minimizing computer time. Support for groups was then
assessed by the parsimony jackknifing program Jac (Farris et al.
1996), set for 10000 replicates and a cut of 50. The resulting tree
was then reconstructed using MacClade 3.0 (Maddison and Maddison
1992). The four taxa Didelphis virginiana, Macropus giganteus,
Macropus robustus, and Dromiciops gliroides, were used to root trees
in analyses conducted with both PAUP and Jac programs.

PRESENTATIONS (1996-2000)
Invited:
2000 M.W. Allard. 'Mammalian molecular systematics: Searching for
the closest relative to the Primates. Department of Anatomical
Sciences, State University of New York, Stony Brook.

2000 M.W. Allard. 'Molecular systematics of mammalia with emphasis
on the Archonta:
measuring support for determining the closest relative to the
Primates." Department of Biology, University of Massachusetts,
Boston.

2000 M.W. Allard. 'Mammalian molecular systematics: Searching for
the closest relative to the Primates. American Museum of Natural
History, New York City.

1999 M.W. Allard. Molecular systematics of Mammalia. Are
Insectivores monophyletic and where do they place on the mammalian
tree? Department of Biological Sciences, Howard University,
Washington DC. (Topics in Evolutionary Biology 201, guest lecturer).

1998 M.W. Allard. Comparisons of Maximum Likelihood and parsimony
methods of phylogenetic reconstruction. Finnish Museum of Natural
History, Helsinki Finland.

1998 M.W. Allard (lecturer). Cladistic analysis of molecular data,
postgraduate course. Uppsala University, Institutionen for
Systematisk Botanik, Uppsala, Sweden. Six lectures were given titled:
Introduction to parsimony, Molecular data bases for nucleic acids and
proteins, Alignment of molecular sequences (2 lectures), Molecular
evolution of rates and dates, and The Maximum likelihood Method.

1998 M.W. Allard. Measuring Congruence among mammalian mitochondrial
genes: Applications of phylogenetic analysis. United States
Department of Agriculture, Kearneyville West Virginia.

1996 M.W. Allard. On weighting and congruence. Presented to the
Molecular Systematics Laboratory, Smithsonian Institution, Washington
DC. Also presented at Duke University.

National and International Meetings (1996-1999).
1999 Emerson, G.E. and M.W. Allard (poster, published abstract).
Phylogenetic instability of the eutherian mammal phylogeny: sampling
effects and inclusion/exclusion of variable characters. Willi Hennig
Society XVIII, Zoological Museum (Institut fur
Erziehungswissenschaften), Gottingen Germany.

1999 G.E. Emerson* and M.W. Allard. (published abstract).
Molecular systematics of lipotyphlan insectivores. Willi Hennig
Society XVIII, Zoological Museum (Institut fur
Erziehungswissenschaften), Gottingen Germany.

1999 G.E. Emerson* and M.W. Allard. (poster). Phylogenetic
relationships of lipotyphlan insectivores based on sequences from
mitochondria. Poster presented to the Society of Systematic
Biologists, University of Wisconsin, Madison.

1999 M.W. Allard (published poster). Biodiversity and molecular
systematic databases. First International Conference on Biodiversity
and renewable Natural Resources preservation, Al Akhawayn University,
Ifrane, Morocco.

1999 "The Robert Weintraub Research Program in Biodiversity,
Systematics and Evolution" (Lipscomb, Clark, Burns, Allard*, Hormiga,
Herendeen, Smith, and Knowlton. Poster presented at the First
International Conference on Biodiversity and renewable Natural
Resources preservation, Al Akhawayn University, Ifrane, Morocco.

1998 M.W. Allard (invited, published abstract). Parsimony,
weighting and maximum likelihood in molecular systematics and
Evolution, Willi Hennig Society XVII, Universidade de São Paulo, São
Paulo Brazil.

1997 M.W. Allard and J. M. Carpenter. On weighting and congruence.
Poster presented to the Society of Systematic Biologists, University
of Colorado, Boulder.

1997 G.E. Emerson* and M.W. Allard. (poster). Phylogenetic
relationships of lipotyphlan insectivores based on complete
mitochondrial 12S rRNA sequences. Poster presented to the Society of
Systematic Biologists, University of Colorado, Boulder.

1997 M.W. Allard. Congruence among mammalian mitochondrial genes.
Presented in the symposium on Mammalian Systematics and Evolution,
Willi Hennig Society XVI, The George Washington University, Washington
DC. Symposium of Mammalian Systematics, Symposium coordinator, M.W.
Allard and R. L. Honeycutt.

1997 G.E. Emerson* and M.W. Allard. (poster). Phylogenetic
relationships of lipotyphlan insectivores based on complete
mitochondrial 12S rRNA sequences. Poster presented to the Willi
Hennig Society XVI, The George Washington University, Washington DC.
Symposium of Mammalian Systematics.

Activities and findings:

Research Findings:

TECHNICAL INFORMATION
The information requested under this section of the final progress
report will be organized according to the publications that resulted
from the findings.

1. Allard, M. W. and J. Carpenter. 1996. On weighting and
congruence. Cladistics. 12:183-198.
A priori differential weighting of molecular characters is a common
methodological practice in molecular phylogenetics. This has been a
largely subjective exercise with few criteria for deciding which
characters to down-weight and how much to do so. A priori
differential weighting is conducted to remove heterogeneity from the
data sets and to improve the congruence among the informative, and
usually more conservative characters. In this part of the research,
we tested whether congruence is improved with a priori differential
weighting by using the incongruence length difference test on a linked
genetic data set consisting of 14 mammalian taxa and the 13 protein
coding genes of the mitochondrial genome. Weighting by omitting the
third codon position did not improve congruence with respect to the
equally weighted data, while weighting transversions did improve the
congruence between the 13 protein coding genes. Nonetheless, the most
parsimonious tree found from transversion weighting did not differ
from one using all of the data equally weighted. These results
support the use of examining all of the data equally weighted.

2. McNiff, B. and M. W., Allard. 1998. A Test of Archontan
Monophyly and the phylogenetic utility of the mitochondrial gene 12S
rRNA. American journal of Physical Anthropology. 107:225-241.
The relationships within the superorder Archonta, which contains the
orders Dermoptera (flying lemurs), Scandentia (tree shrews),
Chiroptera (bats), and Primates are examined through the analysis of
five newly derived and complete mitochondrial 12S rRNA sequences. The
new data is combined with 83 additional known mammalian sequences to
provide a full phylogenetic sampling. Phylogenetic hypotheses were
generated using parsimony analyses of all characters equally weighted
and transversions only. We also examined the effect that alignment
gaps had on the phylogeny. The Parsimony Jackknifer was used to
assess the level of ambiguity present in the sequence data, and
therefore the strength of the tree topologies. The conclusions that
12S rRNA is a reliable marker to a time depth of 100 million years
(Springer and Douzery 1996) is unsupported by these analyses. The
usefulness of 12S rRNA to aid in solving Archonta relationships and
others of similar time depth is found to be suspect. This research
has important implications in determining the Primates' closest living
relative.

3. Emerson, G., C. W. Kilpatrick, B. McNiff, J. Ottenwalder, and M.
W., Allard. 1999. Phylogenetic relationships of the order
Insectivora based on complete 12S rRNA sequences from mitochondria.
Cladistics15:221-230. Special issue: Hennig XVI Symposium on Mammal
Phylogeny. Guest editors: Marc W. Allard and Rodney L. Honeycutt.
Despite numerous studies there is no single accepted hypothesis of
eutherian ordinal relationships. Among the least understood mammalian
orders is the group Insectivora. Currently molecular and
morphological data are in conflict over possible relationships of the
living members. One of the primary criticisms concerning molecular
analysis is the noticeable lack of data from a well sampled group of
lipotyphlan insectivores. The mitochondrial 12S rRNA gene has been
widely used to resolve interordinal and intraordinal relationships
across a variety of mammalian taxa. This study compares 118 complete
mammalian 12S rRNA sequences representing all of the eighteen
eutherian orders and three metatherian orders, and includes as well
taxa from each of the six families of lipotyphlan insectivores in an
effort to assess the relationships within and surrounding this diverse
order. Insectivoran lineages are thought to have diverged
concurrently with the general radiation of mammalian orders. This
study suggests that the 12S rRNA sequences lack the ability to resolve
relationships extending into this period. This would explain the
polyphyly, unusual affinities, and low support derived in this and
other studies which apply 12S rRNA sequences to ordinal level
questions. The results of these analyses suggest that even this large
sampling of taxa is insufficient to provide well supported groups
among eutherian orders by overcoming the ambiguity present in
sequences of the 12S rRNA gene. Additional genes and species sampling
will be necessary to answer questions of insectivore relationships and
our ongoing work is an attempt to rectify this problem.

4. Allard, M. W., J. S. Farris, and J. M. Carpenter. 1999.
Congruence among mammalian mitochondrial genes. Cladistics 15:75-84.

Mitochondrial protein coding genes were combined into a single matrix
that included the 13 protein coding genes for 22 mammals, resulting in
11,448 characters each, or more than a quarter of a million base pairs
of mitochondrial sequence. This matrix was examined for 3 separate a
priori weighting strategies including; equal weighting, transversion
weighting, and codon weighting. The incongruence length difference
test obtained a significant value for equally weighted data and for
one of our differentially weighted analyses. Thus, in one case,
transversion only, weighting did effect previously incongruent data
matrices, making them congruent. The tree produced for the various
weighting strategies was identical in all of the treatments. These
results support the conclusion that a priori weighting schemes should
not be used to remove incongruence (presumably one does not want to
introduce incongruence), instead a simultaneous analysis strategy
which includes treating all of the data equally weighted should be
considered.

5. Marc W. Allard, Scott D. Baker, Ginny L. Emerson, C. W. Kilpatrick
and J. A. Ottenwalder. 2000 Conservation genetics of the Solenodon
paradoxus and the implications for conservation management. submitted
to the Journal of Mammalogy.
The Solenodon is an endangered species of insectivoran found in the
Caribbean, with Solenodon cubanus in Cuba, and Solenodon paradoxus on
Hispaniola. To date no genetic markers are available for conservation
management. Thus, we provide variable mitochondrial markers in the 5'
end of the control region to be used in the conservation management of
this species. We sequenced this region for three individuals. All
clones (n=3) sequenced for each individual were identical, indicating
that no heteroplasmy was observed. Approximately 400 bp were
collected for each of the three individuals. Six variable positions
in the sequences defined three haplotypes. Nucleotide diversity (*)
was determined to be 0.953% for solenodons. The nucleotide diversity
is high relative to other endangered species or populations which are
protected. The level of nucleotide diversity observed may result from
the inclusion of material from specimens that are either
geographically or reproductively isolated. Finally, control region R1
repeat structure was discussed with Solenodon only showing one repeat
and a possible second imperfect repeat.

Three other papers are in various stages of preparation.
6. Emerson, G., and M. W., Allard. 2000. Phylogenetic relationships
of the order Insectivora based on combined analysis of 12S rRNA and
Nadh2 sequences from mitochondria. To be Submitted to Syst. Biol.

7. Asher, R., Emerson, G., and M. W., Allard. 2000. Total evidence
of molecules and morphology does not support the super order
Afrotheria, and Afrosoricidae. To be Submitted to Journal of
Mammalian Evolution.

8. Allard, M. W., T. Yates, and R. L. Honeycutt. 2000. Molecular
systematics of the Talpidae to determine relationships among moles and
their relatives. To be Submitted to American Journal of Mammalogy.

Research Training:

Two graduate and five undergraduate researchers have been trained in
molecular systematic and evolutionary techniques. 1) One graduate is
still matriculating and is expected to graduate in the Fall of 2000.
She is planning for postdoctoral study in forensic sciences at the
American Registry of Pathology, Armed Forces Institute of Pathology,
Armed Forces DNA Identification Laboratory, for the DoD DNA Registry,
Rockville MD. 2) The other graduate student has started her own
business. Of the five undergraduates 3) one is attending medical
school at George Washington University, 4) one is a medical genetics
research technician in Boston, 5) one is doing her Ph.D. in genetics
at the Massachusetts Institute of Technology, 6) one is currently
working for the US customs office, and 7) one is finishing his senior
undergraduate year and plans to go on in veterinary medicine.

Education and Outreach:

Numerous opportunities were taken by myself to improve the publics
understanding of science. Two educational courses were taught as
well.

COURSES
Uppsala University, Uppsala, Sweden
Department of Systematic Botany, Institutionen for Systematisk
Botanik, Guest lecturer. Cladistic Analysis of Molecular Data,
postgraduate course, 9-20 March 1998. Class with lecture and computer
laboratory. Six lectures were given.

American Registry of Pathology, Armed Forces Institute of Pathology,
Armed Forces DNA Identification Laboratory, Rockville MD, USA
Introduction to Forensic DNA Analysis. Accreditation course,
June-August 1999. Class with lecture and computer demonstration.
Five lectures were given.

PUBLIC DISCUSSIONS OF SCIENCE

1997 Scientific expert on Discovery online (www.) for "The
week: Rats". This was coordinated with the Discovery channel
television show "Rats among us" on wild Discovery 8 p.m. ET/PT.

1997 Consultant for CBC, Calgary Canada and DC office in National
Press building, regarding recent advances in cloning technology. Kary
Schultz and Sarah Wolf

1998 Consultant for VH1, pop up video, Cinta Vega NYC, regarding
animal facts in pop up video of Gloria Estefan, In the Rhythms of the
night (to be aired on cable channel VH1).

1998 Ecuador, Galapagos Islands tour guide with Kathy Brown-Wing, for
Friends of the Museum of Comparative Zoology, Harvard University.

1998 Tanzania Safari tour guide with Kathy Brown-Wing, for Friends of
the Museum of Comparative Zoology, Harvard University. Also guided
this safari in 1989 and planning to go again in 1999.

1998 Consultant for Vogue Magazine, Heather Caldwell NYC, regarding
rat reproduction (to be published in an article on bizarre animal
sexual behavior).

1998 Consultant for Science magazine, Eliot Marshall Washington DC,
regarding information for his article "Elephantine gift stirs museum
debate" Science 280:1186-1187.

1999 Consultant for Washington Post, Eric Lipton Washington DC,
regarding information for his article "District Battles Legions of
Rats" 4/17/99 column A1.

1999 Interview with Radio Morocco (daily broadcast in english).
Morocco and the Environment: Biodiversity in Morocco. (contact
Zouhour Himmich).

1999 Consultant for National Park Service, Virginia District (contact
Ranger Lisa Smith), regarding information on genetic mutations in
white tailed deer.

2000 Interview for Radio Free Europe, Radio liberty (www.rferl.org),
host Andrew Tully Washington DC, regarding "U.S.: Consumers Resist
Genetically Altered Food". Program to air in Eastern Europe and
Published: 07/02/2000 20:18:04
(http://www.rferl.org/nca/features/2000/02/f.ru.000207134821.html).

2000 Tanzania Safari tour guide with Kathy Brown-Wing for Harvard
Museum of Natural History, Travel Program, Harvard University
(Dec.-Jan.).

2000 Kenya Safari tour guide with Kathy Brown-Wing for Harvard Museum
of Natural History, Travel Program, Harvard University (Plans pending
August).

2000 Costa Rica tour guide with Kathy Brown-Wing for Harvard Museum of
Natural History, Travel Program, Harvard University (Plans pending
July).

Journal Publications:
Allard, M. W., B. E. McNiff, and M. M. Miyamoto., "Support for interordinal relationships with emphasis on Primates and their archontan relatives.", Molecular Phylogenetics and Evolution, vol. 5, (1996), p. 78. Published

Allard, M. W. and J. Carpenter., " On weighting and congruence.", Cladistics, vol. 12, (1996), p. 183. Published

McNiff, B. and M. W., Allard, "A Test of Archontan Monophyly and the phylogenetic utility of the mitochondrial gene 12S rRNA.", American journal of Physical Anthropology, vol. 107, (1998), p. 225. Published

Emerson, G., C. W. Kilpatrick, B. McNiff, J. Ottenwalder, and M. W., Allard, "Phylogenetic relationships of the order Insectivora based on complete 12S rRNA sequences from mitochondria.", Cladistics, vol. 15, (1999), p. 221-230. Published

Allard, M. W., J. S. Farris, and J. Carpenter, "Congruence among mammalian mitochondrial genes.", Cladistics, vol. 15, (1999), p. 75.
Published

Allard, M. W., R. L. Honeycutt, and M. J. Novacek, "Introduction: Advances in higher level mammalian relationships.", Cladistics Special issue: Hennig XVI Symposium on Mammal Phylogeny. Guest editors: Marc W. Allard and Rodney L. Honeycutt. , vol. 15, (1999), p.
213. Published

Marc W. Allard, Scott D. Baker, Ginny L. Emerson, C. W. Kilpatrick and J. A. Ottenwalder., "Conservation genetics of the Solenodon paradoxus and the implications for conservation management", Journal of Mammalogy, vol. , (2000), p. . Submitted

Emerson, G., and M. W., Allard, "Phylogenetic relationships of the order Insectivora based on combined analysis of 12S rRNA and Nadh2 sequences from mitochondria.", Syst. Biol., vol. , (2000), p. . in preparation

Asher, R., Emerson, G., and M. W., Allard., " Total evidence of molecules and morphology does not support the super order Afrotheria, and Afrosoricidae.", Journal of Mammalian Evolution, vol. , (2000), p. . in preparation

Allard, M. W., T. Yates, and R. L. Honeycutt., "Molecular systematics of the Talpidae to determine relationships among moles and their relatives", American Journal of Mammalogy., vol. , (2000), p. . in preparation

Book(s) of other one-time publications(s):
Allard, M. W., R. L. Honeycutt, and M. J. Novacek., "Advances in higher level mammalian relationships." , bibl. One volume of the journal will be dedicated to this published symposium on mammalian systematics, (1999). Cladistics special volume of journal Published
of Collection: Allard, M. W. and R. L. Honeycutt, "Cladistics, Special issue of Mammalian Systematics."

Internet Dissemination:

http://www.gwu.edu/~clade/faculty/allard/ http://www.gwu.edu/~clade/faculty/allard/data.html

The project is discussed in detail on my web page.
In addition, all of the data collected from this project as well as my
laboratory is available through web links at this site.

Representative sequences are linked to Entrez, and all alignments of
our sequences are available at this web site
(http://www.gwu.edu/~clade/faculty/allard/data.html) or through links
to EBI which houses most of our alignments.

The web site has additional links to other relevant web sites
concerning molecular systematics, the evolution of mammals, and
information on insectivorans.

Other Specific Products:

Physical collection (samples, etc.)

Clones are available for several Solenodon control region fragments

The TA Cloning method was employed using the pBluescript® II sk (-)
vector. This vector was blunt end digested with EcoRV, ethanol
precipitated, and then underwent the addition of 3' deoxythymidine
residues by T4 DNA ligase during a 2 hour incubation at 72°C. The
vector was then phenol/chloroform extracted and ethanol precipitated
to remove reagents. DNAs amplified for insertion into the vector were
purified through Centricon-100 centrifugal filter devices (Amicon,
Millipore) prior to ligation. Ligations and transformations followed
standard protocols (Sambrook et al., 1989). Transformed colonies were
picked and grown overnight in 4 ml of 2x Luria Bertani medium plus
ampicillin. Two µl of the overnight culture were used as the template
in a subsequent PCR that included the same primers under conditions
originally used to produce the amplicon for ligation. This PCR
amplification indicated whether or not the insert was present for a
particular clone.

Investigators with the proper permits will be mailed frozen samples of
the clones. DNA may also be made available to potential
collaborators.

Data or databases

Numerous nucleotide sequences have been collected for a large number
of insectivorans as well as other related mammals. All of the
sequences and the aligned matrices used in our published results are
available on my web site http://www.gwu.edu/~clade/faculty/allard/.
These sequence data are also submitted to published databases such as
GenBank, Entrez, and EBI. Most of these public databases are cross
listed on my home web site.

Most of these sequences are mitochondrial genes of mammals.

Besides housing the published data from these studies. I have made
links to many relevant web sites concerning mammalian evolution,
molecular systematics and the lipotyphlan insectivorans.

My web site http://www.gwu.edu/~clade/faculty/allard/ is publicly
available on the George Washington University web site. Any
investigator who wishes to use the data is free to down load these
sequences and alignments.

Contributions:

Contributions within Discipline:

The disciplines which this project will help develop the most include
both molecular systematics and mammalian systematics. This project
has advanced our understanding of the role of congruence in molecular
systematics studies by providing examples of data showing when
congruence is both present and absent among data sets. In 1996 very
few studies had examined congruence in phylogenetic studies but now
many investigators measure congruence using the tests promoted in our
manuscript (Allard and Carpenter 1996 and later in Allard et al.
1999).
This project has also advanced our understanding of the relationships
among the lipotyphlan insectivores and among interordinal comparisons
of eutherian mammals (McNiff and Allard 1998; Emerson et al. 1999,
Allard et. al. 1999). This project has demonstrated the importance of
insectivores in understanding the early mammalian radiations.
Similarly, this project and our results has encouraged many other
investigators to explore the relationships among the lipotyphlan
insectivores and to date both morphological and molecular studies have
been started to further examine this group. It has come to the point
that any study of higher level mammalian systematics must include a
diversity of insectivorans.
Furthermore by understanding Archonta relationships we have gain a
better understanding of who primates closest relative is. I have
interacted with several people in the biomedical industry who would
like to be able to address this question (e.g. Diagnon and
Pharmacopeia).

Contributions to Other Disciplines:

This project has contributed to several other fields of study. I
have interacted with investigators at the USDA in plant disease
sciences to understand the systematics of Erwinia amylovora, an
economically important plant pathogen (Brown, E., M. W. Allard, and T.
van der Zwet. 1998. Phylogenetic characterization of the eubacterial
lcrD gene family: Molecular evolutionary aspects of pathogen-induced
hypersensitivity in plants. Cladistics 14:45-62; and Brown, E., T. M.
O'Brien, R. M. Davis, M. W. Allard, and T. van der Zwet. 2000.
Metabolic fingerprints reveal a convergent evolutionary history
reflective of pathological partitions in the genus Erwinia. Submitted
to Antonie van Leeuwenhook). The interest generated from my funded
project of mammals encouraged a plant scientists to examine their
problem in a similar way. This same research colleague (Dr. E. Brown)
now is conducting research for the food and drug administration where
understanding the evolution of important pathogens will be critical
for the US economy, particularly where this affects plant pathogens of
crops.

Genetic Diversity: My interests in evolutionary questions extend
beyond those for the Mammalia. Through various collaborations, I have
advised students and faculty on research for a number of other taxa as
well. With researchers at the USDA and FDA, we have been using the
lcrD gene sequences and metabolic typing (i.e. to determine nutrient
utilization patterns of bacteria) to characterize an economically
important plant pathogen, the Eubacterial genus Erwinia (see above).
Other faculty (e.g. Dr. D. Lipscomb) and their students have been
trained to use the equipment in my laboratory for molecular
systematics studies. Currently, these researchers are collecting
sequence data for ciliated protists (Ciliophora), sea anemones
(Cnidaria), and green algae (Chlorophyta). They consult with me on
both methods and interpretation of results. Future collaborations
might include studies of fishes, nemertean worms, molluscs, and
bacteria. By expanding the taxonomic breadth examined in my
laboratory, I hope to attract additional funding, students, faculty,
and off campus collaborators.

Contributions to Education and Human Resources:

Two graduate and five undergraduate researchers have been trained in
molecular systematic techniques directly related to this grant. Five
additional faculty and students have been trained in the laboratory
examining other taxa. These techniques are widely shared in
biomedicine and in the biotechnology industry. These skills are
extremely valuable for many disparate fields of science and
engineering. Molecular biology has become the tool for the 21st
century with many different questions being addressed using the same
over-riding methodologies. Learning analytical skills help students
to become critical thinkers and improves their computer proficiency.
Personnel trained in my laboratory are able to transfer these skills
to a wide variety of disciplines thus improving the pool of scientists
available for activities in engineering and science.

Contributions to Resources for Science and Technology:

This grant has added to my own training in computerization and
information resources. With this training I acted as liaison with our
campus telecommunications office to establish 10T internet connections
for all of the laboratories and offices in the department of
biological sciences. Also, I have advised the University in
developing an MS degree program in the Computational Sciences.
Furthermore, I have collaborated with the Physics department
(collaborations with a research associate, one postdoctoral student
and an undergraduate) in developing a parallel virtual machine for the
George Washington University. This increased computational strength
is needed for the analysis of the large genetic data matrices
generated from this project as well as analyses in particle physics.
The direct collaboration between these disparate fields of Biology and
Physics has already provided new insight into ways to improve computer
parallel programs and the installation of equipment.

Contributions Beyond Science and Engineering:

Probably the most important benefit to the public welfare comes in
the education and development of human resources. The students that I
trained in my laboratory and through the auspices of this project are
trained to think critically. Through this project I have trained
researchers who can operate independently and who are problem solvers.
In the past I have trained students in avian population genetics who
have gone on to study the genetic causes of blindness. Thus, the
training they receive in my laboratory is of a general nature allowing
students the time to grow and develop into their careers. The
students who are trained in these programs have long productive lives
ahead of them and the training they receive will benefit them with the
ability for wise decision making. We need to train more leaders and
critical thinkers to advance the public welfare.

Several examples of more direct contributions to regulatory policy
and business includes one trainee (Ph.D. student) who has gone on to a
job in the Food and Drug Administration, where now he is using
molecular systematic methods to improve FDA policy.

In regards to business. I have several ongoing collaborations with
other investigators. One project involves using the comparative
method in studies of drug development (Dr. Horlick, Pharmacopeia, and
now with Millennium, Boston). I also am collaborating on a molecular
systematic project to assess genes potentially involved with Malaria
resistance (Dr. McKenzie Harvard University). All of these
interactions have been facilitated with the funding provided from this
NSF grant.