Differential loss of embryonic globin genes during the radiation of placental mammals.
ABSTRACT The differential gain and loss of genes from homologous gene families represents an important source of functional variation among the genomes of different species. Differences in gene content between species are primarily attributable to lineage-specific gene gains via duplication and lineage-specific losses via deletion or inactivation. Here, we use a comparative genomic approach to investigate this process of gene turnover in the beta-globin gene family of placental mammals. By analyzing genomic sequence data from representatives of each of the main superordinal clades of placental mammals, we were able to reconstruct pathways of gene family evolution during the basal radiation of this physiologically and morphologically diverse vertebrate group. Our analysis revealed that an initial expansion of the nonadult portion of the beta-globin gene cluster in the ancestor of placental mammals was followed by the differential loss and retention of ancestral gene lineages, thereby generating variation in the complement of embryonic globin genes among contemporary species. The sorting of epsilon-, gamma-, and eta-globin gene lineages among the basal clades of placental mammals has produced species differences in the functional types of hemoglobin isoforms that can be synthesized during the course of embryonic development.
- [show abstract] [hide abstract]
ABSTRACT: Typescript. Thesis (Ph. D.)--University of Oregon, 2000. Includes vita and abstract. Includes bibliographical references (leaves 150-160).
- [show abstract] [hide abstract]
ABSTRACT: The globin family of genes and proteins has been a recurrent object of study for many decades. This interest has generated a vast amount of knowledge. How-ever it has also created an inconsistent and confusing nomenclature, due to the lack of a systematic approach to naming genes and to failure to reflect the phylo-genetic relationships among genes of the gene family. To alleviate the problems with the existing system, here we propose a standardized nomenclature for the a and b globin family of genes, based on a phylogenetic analysis of vertebrate a and b globins, and following the Guidelines for Human Gene Nomenclature.Genes & Genetic Systems - GENES GENET SYST. 01/2006; 81(5):367-371.
- Fems Microbiology Letters - FEMS MICROBIOL LETT. 01/1999; 174(2):247-250.
Differential loss of embryonic globin genes during
the radiation of placental mammals
Juan C. Opazo*, Federico G. Hoffmann†, and Jay F. Storz‡
School of Biological Sciences, University of Nebraska, Lincoln, NE 68588
Edited by Morris Goodman, Wayne State University School of Medicine, Detroit, MI, and approved July 8, 2008 (received for review May 8, 2008)
The differential gain and loss of genes from homologous gene
families represents an important source of functional variation
among the genomes of different species. Differences in gene
content between species are primarily attributable to lineage-
specific gene gains via duplication and lineage-specific losses via
deletion or inactivation. Here, we use a comparative genomic
approach to investigate this process of gene turnover in the
?-globin gene family of placental mammals. By analyzing genomic
sequence data from representatives of each of the main superor-
dinal clades of placental mammals, we were able to reconstruct
pathways of gene family evolution during the basal radiation of
this physiologically and morphologically diverse vertebrate group.
Our analysis revealed that an initial expansion of the nonadult
portion of the ?-globin gene cluster in the ancestor of placental
mammals was followed by the differential loss and retention of
ancestral gene lineages, thereby generating variation in the com-
plement of embryonic globin genes among contemporary species.
The sorting of ?-, ?-, and ?-globin gene lineages among the basal
clades of placental mammals has produced species differences in
the functional types of hemoglobin isoforms that can be synthe-
sized during the course of embryonic development.
?-globin gene family ? gene duplication ? gene family evolution ?
genome evolution ? hemoglobin
divergence between orthologous genes. The differential gain and
loss of genes from homologous gene families represents a less
widely appreciated source of functional variation among the
genomes of different species (1–6). Differences in the comple-
ment of genes between species are primarily attributable to
lineage-specific gene gains via duplication and lineage-specific
losses via deletion or inactivation. The ?-globin gene cluster of
mammals represents an especially good model for investigating
of the most intensively studied multigene families from the
standpoint of molecular genetics and phylogenetic history (7–9).
The ?-globin gene cluster of mammals contains a set of devel-
opmentally regulated genes that are arranged in their temporal
order of expression (10–12). The ?-, ?-, and ?-globin genes
(HBE, HBG, and HBH, respectively) are expressed in embry-
onic erythroid cells and are descended from an ancestral HBE
gene. The ?- and ?-globin genes (HBD and HBB, respectively)
from an ancestral HBB gene. There are some exceptions to these
general patterns of stage-specific expression, because duplicated
copies of HBG genes have been recruited for fetal expression in
anthropoid primates (8) and duplicated copies of HBB genes have
In contrast to the diverse repertoire of ?-like globin genes in
eutherian (placental) mammals that have been studied to date,
early-expressed 5? copy and an ontogenetically later-expressed 3?
copy (14–18). Remarkably, the early- and late-expressed ?-like
globin genes in monotremes and therian mammals (marsupials and
fforts to identify genetic changes that underlie phenotypic
differences among species traditionally focus on nucleotide
?-globin gene in each of these two lineages (19). Whereas the
?-globin gene cluster of marsupials has retained the ancestral
two-gene structure, the addition of new early- and late-expressed
genes to the ?-globin gene cluster of eutherian mammals is attrib-
utable to several successive rounds of duplication and divergence
after the eutherian/marsupial split, which is thought to have oc-
curred ?170 Mya (20). Because the HBD gene is either weakly
expressed or completely nonfunctional in the majority of eutherian
mammals, the increased functional diversity of the ?-globin gene
cluster in eutherian mammals is mainly attributable to the expan-
sion of the ‘‘nonadult’’ HBE-HBG-HBH portion of the gene cluster.
Thanks to recent advances in the molecular systematics of
eutherian mammals, we now have a solid phylogenetic framework
for reconstructing pathways of gene family evolution in this mor-
phologically and physiologically diverse vertebrate group. Euther-
ian mammals are classified into four superordinal groups: Afroth-
eria (which includes elephants, hyraxes, manatees, aardvarks,
tenrecs, and allies), Xenarthra (which includes sloths, armadillos,
pangolins, carnivores, perrisodactyls, and cetartiodactyls), and Eu-
archontoglires (which includes primates, tree shrews, colugos, rab-
that a clade (Atlantogenata) composed of Afrotheria and Xenar-
thra is the sister group of all remaining members of the eutherian
?-globin gene cluster in the common ancestor of Boreoeutheria is:
5?-?-?-?-?-?-3? (13, 14, 16, 23). Because the genomic structure of
the ?-globin gene cluster has not been previously characterized in
yet to be elucidated.
Here, we report the results of a comparative genomic analysis of
the four main superordinal clades of eutherian mammals. Results
of our analysis demonstrate that the initial expansion of the
nonadult portion of the gene cluster in the ancestor of eutherian
mammals was followed by differential retention of ancestral gene
lineages among different clades, thereby generating variation in the
complement of embryonic globin genes among contemporary
species. By using a phylogenetic approach to reconstruct pathways
of gene family evolution during the basal diversification of euther-
retained an HBE gene at the 5? end of the cluster; (ii) most
Author contributions: J.C.O. and J.F.S. designed research; J.C.O. and F.G.H. performed
research; J.C.O. and F.G.H. analyzed data; and J.C.O. and J.F.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
*Present address: Instituto de Ecologı ´a y Evolucio ´n, Facultad de Ciencias, Universidad Austral
de Chile, Casilla 567, Valdivia, Chile.
†Present address: Instituto Carlos Chagas Fiocruz, Rua Prof. Algacyr Munhoz Mader 3775-CIC,
81350-010, Curitiba, Brazil.
‡To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
September 2, 2008 ?
vol. 105 ?
representatives of Xenarthra, Afrotheria, and Euarchontoglires
have lost the HBH gene while retaining the HBG gene; and (iii)
most representatives of Laurasiatheria have lost the HBG gene
while retaining the HBH gene.
Genomic Structure of the Mammalian ?-Globin Gene Cluster. We
obtained genomic sequences that spanned the ?-globin gene clus-
ters of 21 eutherian and 3 metatherian species. Comparison of the
?-globin gene clusters among the eutherian species in our study
revealed considerable variation in the size and membership com-
position of the gene family (Fig. 1). The number of putatively
functional genes in the cluster ranged from 2 in the pig to 8 in the
(11, 12), the nonadult genes—HBE, HBH, and HBG—were lo-
cated upstream of the late-expressed HBD and HBB genes. The
only exceptions involved en bloc duplications in the goat and cow,
where HBE and HBH genes in the 3? duplication blocks were
located downstream of the HBB gene in the 5? blocks (13).
We found that all eutherian species possess one to two copies of
the HBE gene at the 5? end of the cluster, and the vast majority of
either HBG or HBH, but never both. The variation in gene family
size is mainly attributable to variation in the number of HBB genes
at the 3? end of the cluster. Whereas myomorph rodents (e.g., Mus
four HBB genes, most species possess a single copy. The two
eulipotyphlan species in our dataset, the African pygmy hedgehog
and the Eurasian shrew, have lost HBB altogether (Fig. 1). In these
two species it appears that paralogous copies of the HBD gene are
solely responsible for synthesizing the ?-chain subunits of adult
hemoglobin. The variation in membership composition of the
the embryonic HBH and HBG genes and the late-expressed HBD
gene. Below we assess this variation in the complement of ?-like
globin genes in a phylogenetic framework. Because the genomic
structure of the ?-globin gene family has been characterized
previously in primates, rodents, and rabbits (all members of the
superorder Euarchontoglires), here we focus on resolving ortholo-
gous relationships of ?-like globin genes among species in Afroth-
eria, Xenarthra, and Laurasiatheria. The genomic structure of the
?-globin gene cluster has not been previously characterized in any
species from the former two groups.
Genomic Structure and Orthologous Relationships in Atlantogenata.
Among atlantogenatan species, we obtained complete coverage of
the ?-globin gene cluster in one xenarthran species, the nine-
tenrec. In the nonadult portion of the cluster, both species possess
a single copy of HBE and a single copy of HBG, although HBG is
present only as a pseudogene in the armadillo (Fig. 1). We did not
find a functional HBH gene in either of the atlantogenatan species
examined. The only trace of HBH was a fragment (spanning intron
2, exon 3, and the 3? untranslated region) in the armadillo gene
cluster that appears to be orthologous to the human HBHps
pseudogene (???) [supporting information (SI) Fig. S1]. In the
adult portion of the cluster, the armadillo possesses single copies of
the HBD and HBB genes, whereas the gene cluster of the tenrec
contains no trace of HBD, but contains four copies of HBB (Fig. 1).
In the nonadult portion of the cluster, phylogeny reconstructions
of flanking and intronic sequences strongly suggest that the HBE
in humans (Fig. 2). Phylogeny reconstructions based on upstream
genes in the two atlantogenatan species are orthologous to the dupli-
cated pair of HBG genes in humans (G?- andA?-globin) (Fig. 2).
In the adult portion of the cluster, phylogeny reconstructions
genes of armadillo and human are 1:1 orthologs, as are the HBB
genes of these same two species (Fig. 2). By contrast, in the same
phylogenetic trees, monophyly of the four HBB paralogs of the
rounds of lineage-specific gene duplication (Fig. 2). Curiously, the
four HBB paralogs of the tenrec have downstream flanking se-
quences that exhibit strong affinities to HBD-like sequences of
other species (Fig. 2 and Fig. S1). One possible explanation for this
pattern is that the coding region and upstream flanking region of
an ancestral, single-copy HBD gene were completely converted by
an HBB donor gene that has since been deleted in the tenrec
lineage. Subsequent rounds of duplication then produced a total of
four HBB-like gene copies that have each retained an unconverted
HBD-like downstream flanking sequence.
50). Diagonal slashes indicate gaps in genomic cover-
scale. The orientation of the cluster is from 5? (on the
left) to 3? (on the right).
Genomic structure of the ?-globin gene clus-
Opazo et al.
September 2, 2008 ?
vol. 105 ?
no. 35 ?
Genomic Structure and Orthologous Relationships in Laurasiatheria.
Among laurasiatherian species, we obtained complete coverage of
the ?-globin gene cluster in two eulipotyphlans, two bats, two
carnivores, one perissodactyl, and one cetartiodactyl. In the non-
adult portion of the cluster, we found that the African pygmy
hedgehog (order Eulipotyphla) is the only mammalian species that
possesses two copies of the HBE gene (Fig. 1). The coding regions
of these two HBE paralogs were distinguished by a total of two
synonymous substitutions, which suggests two possibilities: (i) the
two genes are the products of a relatively recent, lineage-specific
duplication event, or (ii) the two genes have a more ancient origin,
but have undergone a relatively recent, lineage-specific gene con-
version event. Although the horse possesses an HBGps pseudo-
gene, no trace of the HBG gene was found in any of the other
laurasiatherian species. All bats and carnivores in our sample
possess a single HBH gene and the horse possesses an HBH gene
in addition to an HBHps pseudogene. The HBH gene has been
inactivated or lost in the remaining laurasiatherian species (Fig. 1).
For the nonadult portion of the cluster, phylogeny reconstruc-
tions of flanking and intronic sequence show that the HBE genes
in laurasiatherian species are 1:1 orthologs of the HBE gene in
humans (or coorthologs in the case of the HBE-T1 and HBE-T2
genes in the hedgehog) (Fig. 3 and Fig. S2). Phylogeny reconstruc-
tions also indicated that the HBGps pseudogene of horse is sister
to the two HBG paralogs of humans, and that the HBH genes of
all laurasiatherian species are 1:1 orthologs of the HBHps pseudo-
gene in humans (or coorthologs in the case of the HBH gene and
the HBHps pseudogene in the horse) (Fig. 3). In combination with
the results for the atlantogenatan taxa (see above) and marsupials
(16, 19), these results indicate that the HBE genes of therian
mammals originated via duplication of a proto-HBB gene after the
the products of two successive rounds of duplication that occurred
after the eutherian/metatherian split (?170 Mya). For the adult
portion of the cluster, phylogeny reconstructions of flanking and
intronic sequence demonstrated that the HBB genes of laur-
asiatherian species are 1:1 orthologs of the HBB gene in humans,
and likewise for the HBD genes (Fig. 3). However, we did
identify several cases in which recombinational exchanges be-
tween HBB and HBD affected upstream flanking sequence (the
three HBD paralogs of the African pygmy hedgehog, HBD-T2
of dog, and HBB of cat) and intron 2 sequence (the HBDps
pseudogene of pig) (Fig. 3).
Results of our comparative genomic analysis revealed that the
initial expansion of the nonadult portion of the ?-globin gene
cluster in the ancestor of eutherian mammals was followed by the
differential loss and retention of ancestral gene lineages, thereby
generating partially overlapping inventories of embryonic globin
genes among contemporary species. All eutherian species have
retained at least one copy of the HBE gene at the 5? end of the
gene cluster. However, as a result of the sorting of HBG and
HBH gene lineages among the four main clades of eutherian
mammals, contemporary species possess either HBE paired with
HBG (e.g., lesser hedgehog tenrec, and the majority of species
in Euarchontoglires) or HBE paired with HBH (the majority of
species in Laurasiatheria) (Fig. 1). There are also several euth-
erian species that have independently lost both HBG and HBH
(e.g., armadillo, African pygmy hedgehog, Eurasian shrew,
guinea pig, and pig). In fact, as a result of independent inacti-
vations and deletions, the ?-globin gene clusters of the pig and
the guinea pig have independently reverted to the ancestral
5?-HBE-HBB-3? structure seen in marsupials. The armadillo,
African pygmy hedgehog, and Eurasian shrew also approximate
this ancestral state except that they have two or more copies of each
early- and late-expressed paralog (Fig. 1).
We inferred the differential loss of the embryonic HBG and
HBH genes by using a phylogenetic approach to reconstruct
pathways of gene family evolution in the four main groups of
eutherian mammals: Afrotheria, Xenarthra, Laurasiatheria, and
Euarchontoglires. Our model for the evolution of the nonadult
portion of the mammalian ?-globin gene cluster is graphically
depicted in Fig. 4. According to this model, two successive dupli-
cations of a proto-HBE gene gave rise to the HBG and HBH genes
in the ancestor of eutherian mammals after divergence from
marsupials (see also refs. 9, 23, and 24). Consequently, the full
complement of embryonic globin genes—HBE-HBG-HBH—was
mammals, Boreoeutheria (comprising Euarchontoglires and Laur-
asiatheria) and Atlantogenata (comprising Xenarthra and Afroth-
eria). Subsequent to the Boreoeutheria–Atlantogenata split (?105
Mya), the HBH gene was lost in the common ancestor of xenar-
thrans and afrotherians, and subsequent to the divergence of the
The ancestral three-gene set was also present in the common
divergence of these two groups (?85 Mya), the HBG gene was lost
in Laurasiatheria and the HBH gene was lost in Euarchontoglires.
Although HBH was deleted altogether in the gene clusters of
rabbits and rodents, a HBHps pseudogene has been retained in
nearly all primates that have been examined (9, 25). As shown in
Fig. 1, the HBH gene has also been independently lost in several
relationships among ?-like globin genes in species of
the superorder Atlantogenata based on 1 kb of 5?
relevant nodes was evaluated by using 1,000 pseu-
Maximum likelihood phylograms depicting
www.pnas.org?cgi?doi?10.1073?pnas.0804392105Opazo et al.
laurasiatherian taxa (e.g., African pygmy hedgehog, Eurasian
shrew, and pig).
These species differences in the complement of ?-like globin
genes are associated with differences in the functional diversity of
prenatally expressed hemoglobin isoforms. In bats, cats, dogs, and
Euarchontoglires (rabbits, myomorph rodents, and prosimian pri-
mates), prenatal hemoglobins incorporate ?-chain products of
HBE and HBG. The functional significance of this hemoglobin
isoform diversity remains to be elucidated.
Mode of Gene Family Evolution. Results of our analysis demonstrate
that the genomic structure of the mammalian ?-globin gene family
has been shaped by a mixed process of concerted evolution and
birth-and-death evolution. However, concerted evolution appears
to have been largely restricted to tandemly duplicated copies of the
same paralogous type (e.g., between HBB-T1 and HBB-T2 of
mouse or between HBG-T1 and HBG-T2 of anthropoid primates;
refs. 26, 27). In the adult portion of the gene cluster, ectopic
recombination between HBB and HBD paralogs has created
chimeric ?/? fusion genes in multiple, independent lineages (9, 23,
28–30). In the nonadult portion of the gene cluster, ectopic
recombination between HBE and HBG has created a chimeric ?/?
fusion gene in myomorph rodents (24, 31). Aside from this one
exception in rodents, we found no evidence of recombinational
exchange among the HBE, HBG, and HBH paralogs in any other
placental mammals. There are no pronounced differences in levels
of interparalog divergence between species that possess an HBE–
HBG gene pair (tenrec and most representatives of Euarchonto-
glires) vs. those that possess an HBE-HBH gene pair (most
representatives of Laurasiatheria). In species that possessed HBE
and HBG in tandem, levels of amino acid sequence divergence
that possessed HBE and HBH in tandem, levels of interparalog
divergence ranged from 19 to 22%. In comparison with the HBB
Maximum likelihood phylograms depicting relationships among ?-like globin genes in species of the superorder Laurasiatheria based on 1 kb of 5? flanking
Opazo et al.
September 2, 2008 ?
vol. 105 ?
no. 35 ?
genes, the nonadult genes are characterized by higher levels of
sequence conservation, which presumably reflects a higher level of
functional constraint (16, 23).
Gene Duplication, Functional Redundancy, and Evolutionary Innova-
tion. The differential loss of HBG and HBH genes among different
lineages of eutherian mammals may have been a purely stochastic
process such that the particular complement of genes inherited by
a given species was simply a matter of chance. However, the
particular complement of genes inherited by a given species may
help steer the trajectory of physiological evolution. The possession
of multiple, functionally redundant gene copies may provide in-
creased scope for evolutionary innovation because it allows dupli-
cated genes to take on new functions or divide up ancestral
Whereas monotremes and marsupials possess a single pair of
early- and late-expressed ?-like globin genes, the majority of
eutherian mammals possess a more functionally diverse repertoire
of globin genes. The expanded gene complement of eutherian
been recruited for fetal expression. It has been argued that the
acquisition of fetally expressed hemoglobin played an important
role in the life history evolution of anthropoid primates because it
facilitated an extended duration of fetal development (8). In New
World monkeys, HBG-T1 is expressed in nucleated erythroid cells
derived from the embryonic yolk sac (the ancestral condition), but
fetal liver. In Old World monkeys and apes, both HBG-T1 and
HBG-T2 are fetally expressed (35). The cooption of HBG for fetal
expression in anthropoid primates was probably facilitated by the
fact that redundant or semiredundant copies of the HBE and HBG
genes continued to perform their ancestral functions during the
the acquisition of fetally expressed hemoglobin would not have
been accessible if the ancestor of anthropoid primates had pos-
sessed only a single embryonic gene, as in contemporary
monotremes, marsupials, and some eutherian species like the
guinea pig. In a similar fashion, the cooption of the embryonic
?D-globin gene for expression during postnatal life appears to have
played an important role in the evolution of hypoxia tolerance in
Old World vultures and other birds that fly at high altitude (36).
In conclusion, two successive rounds of gene duplication and
divergence produced a set of three embryonic ?-like globin genes
in the ancestor of eutherian mammals. The differential loss and
group generated variation in the complement of embryonic globin
genes among contemporary species and variation in the functional
types of hemoglobin isoforms that can be synthesized during the
course of prenatal development.
Materials and Methods
Nomenclature for ?-Like Globin Genes. FollowingthenomenclatureofAguileta
HBH, respectively, and we refer to the late-expressed ?- and ?-globin genes, as
multiple rounds of duplication that have resulted in tandemly repeated sets of
genes—HBE-HBG-HBH—was present in the common ancestor of the two main clades of eutherian mammals, Boreoeutheria (comprising Euarchontoglires and
Laurasiatheria) and Atlantogenata (comprising Xenarthra and Afrotheria). Subsequent to the Boreoeutheria–Atlantogenata split (?105 Mya), the HBH gene was
Mya), the HBG gene was lost in laurasiatherians and the HBH gene was lost in Euarchontoglires. Latin crosses denote lineage-specific gene losses, either via deletion
www.pnas.org?cgi?doi?10.1073?pnas.0804392105Opazo et al.
DNA Sequence Data and Gene Identification. The genomic structure of the
?-globin gene cluster has been characterized for cow (Bos taurus), goat (Capra
hircus), and several species in the superorder Euarchontoglires (e.g., primates,
rodents, and lagomorphs; refs. 12, 13, 30, 38–41). For this subset of taxa, we
the ?-globin gene cluster of one xenarthran species (nine-banded armadillo,
Dasypus novemcinctus), one afrotherian species (lesser hedgehog tenrec, Echi-
nops telfairi), and eight laurasiatherian species, including two eulipotyphlans
(African pygmy hedgehog, Atelerix albiventris, and Eurasian shrew, Sorex ara-
neus), two bats (little brown bat, Myotis lucifugus, and greater horseshoe bat,
Rhinolophus ferrumequinum), two carnivores (cat, Felis catus, and dog, Canis
(pig, Sus scrofa) (Table S1). For these species, we identified globin genes in
unannotated genomic sequences by using the program Genscan (42) and by
2, version 2.2 (43).
Structure of the ?-Globin Gene Cluster and Orthologous Relationships. The
genomic structure of the ?-globin gene cluster in afrotherian, xenarthran, and
laurasiatherian species was investigated by using pairwise analyses of sequence
5? gene copy and 5 kb of downstream sequence flanking the most 3? gene copy.
When comparing gene families among species, it is often difficult to assign
true history of gene duplication and species divergence. Because interparalog
gene conversion is typically restricted to the coding regions of globin genes (26,
31, 44–46), we used phylogeny reconstructions of noncoding sequences (flank-
laurasiatherian species and another containing atlantogenatan species. In both
cases the human sequence was included as an outgroup.
Sequence alignments were carried out by using the program MUSCLE (47) as
implemented in the Berkeley Phylogenomics Group web server (http://
phylogenomics.berkeley.edu). Phylogeny reconstructions were based on coding
sequence, 1 kb of upstream flanking sequence, 1 kb of downstream flanking
sequence, and intron 2 (1,451 bp in the atlantogenata sequence alignment and
2486 bp in the laurasiatheria sequence alignment). Phylogenetic relationships
were inferred in a maximum likelihood framework by using Treefinder, version
January 2008 (48) and support for the nodes was assessed with 1,000 bootstrap
laurasiatheria sequence alignment, we used the HKY ? ? model (upstream
for helpful comments and suggestions. This work was supported by National
dation Grant DEB-0614342 (to J.F.S.), the Nebraska Research Council (J.F.S.), and
a University of Nebraska postdoctoral fellowship (to F.G.H.).
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