Identification of the wild genetic stock of lager-brewing yeast

I haven't had time to read the whole paper yet but I figured I wouldn't be the only one to find it interesting.

http://www.pnas.org/content/early/2011/08/17/1105430108

Caught the article on my BBC feed this morning.


Edit: Oops, may have gotten ahead of myself. It's just the abstract and not access to the whole paper.

Good to know a scientist with research paper access.

Microbe domestication and the identification of the
wild genetic stock of lager-brewing yeast
Diego Libkinda,1, Chris Todd Hittingerb,c,1,2, Elisabete Valériod, Carla Gonçalvesd, Jim Doverb,c, Mark Johnstonb,c,
Paula Gonçalvesd, and José Paulo Sampaiod,3
aLaboratorio de Microbiología Aplicada y Biotecnología, Instituto de Investigaciones en Biodiversidad y Medio-ambiente, Consejo Nacional de Investigaciones

Científicas y Técnicas (CONICET)-Universidad Nacional del Comahue, 8400 Bariloche, Argentina; bDepartment of Biochemistry and Molecular Genetics,
University of Colorado School of Medicine, Aurora, CO 80045; cDepartment of Genetics, Center for Genome Sciences, Washington University in St. Louis
School of Medicine, St. Louis, MO 63108; and dCentro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

Edited by John Doebley, University of Wisconsin, Madison, WI, and approved July 20, 2011 (received for review April 5, 2011)

Domestication of plants and animals promoted humanity’s transition
from nomadic to sedentary lifestyles, demographic expansion,
and the emergence of civilizations. In contrast to the well-documented
successes of crop and livestock breeding, processes of microbe
domestication remain obscure, despite the importance of
microbes to the production of food, beverages, and biofuels. Lagerbeer,
first brewed in the 15th century, employs an allotetraploid
hybrid yeast, Saccharomyces pastorianus (syn. Saccharomyces
carlsbergensis), a domesticated species created by the fusion of
a Saccharomyces cerevisiae ale-yeast with an unknown cryotolerant
Saccharomyces species. We report the isolation of that species
and designate it Saccharomyces eubayanus sp. nov. because of its
resemblance to Saccharomyces bayanus (a complex hybrid of S.
eubayanus, Saccharomyces uvarum, and S. cerevisiae found only
in the brewing environment). Individuals from populations of S.
eubayanus and its sister species, S. uvarum, exist in apparent sympatry
in Nothofagus (Southern beech) forests in Patagonia, but are
isolated genetically through intrinsic postzygotic barriers, and ecologically
through host-preference. The draft genome sequence of
S. eubayanus is 99.5% identical to the non-S. cerevisiae portion of
the S. pastorianus genome sequence and suggests specific changes
in sugar and sulfite metabolism that were crucial for domestication
in the lager-brewing environment. This study shows that combining
microbial ecology with comparative genomics facilitates
the discovery and preservation of wild genetic stocks of domesticated
microbes to trace their history, identify genetic changes, and
suggest paths to further industrial improvement.
beer yeast | next-generation sequencing | yeast ecology | yeast taxonomy
The beginning of agriculture and the domestication of plants
and animals are among the most decisive events in human
history because they triggered the rise of civilizations and the
attendant demographic, technological, and cultural developments
(1). The domestication of barley in the Fertile Crescent
(2) led to the emergence of the forebear of modern beer in
Sumeria 6,000 y ago (3). Beer and other alcoholic beverages
may have played a pivotal role in cementing human societies
through the social act and rituals of drinking (4) and by providing
a source of nutrition, medicine, and uncontaminated
water (5). Since the emergence of fermented beverages roughly
matches the domestication of plants and animals, it is likely that
some yeast lineages with favored traits were also unwittingly
domesticated.

In Europe, brewing gradually evolved during the Middle Ages
to produce ale-type beer, a process conducted by Saccharomyces
cerevisiae, the same species involved in producing wine and
leavened bread. Lager-brewing arose in 15th century Bavaria,
gained broad acceptance by the late 19th century (6), and has
since become the most popular technique for producing alcoholic
beverages, with over 250 billion dollars of global sales in
2008 (7). Unlike most ales and wines, lagers require slow, lowtemperature
fermentations that are carried out by cryotolerant
Saccharomyces pastorianus (syn. Saccharomyces carlsbergensis)
strains (8); two other cryotolerant Saccharomyces spp. have been
associated with beer as contaminants (Saccharomyces bayanus)
and with cider or wine fermented at low temperatures (Saccharomyces
uvarum) (9). S. pastorianus has never been isolated from
the wild, depends on humans for its propagation, and appears to
be an allotetraploid hybrid species of S. cerevisiae and an unidentified
species (10, 11). Several hypotheses have been advanced
for the source of the non-S. cerevisiae genome present in
S. pastorianus, including the taxonomically and genetically complex
species S. bayanus (12–14) and an unknown “lager” lineage
distinct both from S. bayanus and S. uvarum (11, 15). Identifying
the wild genetic stock of the cryotolerant subgenome of S. pastorianus
is necessary for resolving the taxonomy and systematics
of this important species complex, and for understanding the key
events that led to the domestication of lager yeast.
In contrast to extensive investigation into domestication of
crops and livestock (2, 16–19), studies of domestication of
eukaryotic microbes have been limited (20–24), perhaps because
of the inability to conduct direct field studies. Identifying the
genetic basis of traits under selection during domestication may
clarify the emergence of new traits and show the way toward
further improvement. Because domesticated lineages derive
from a subset of the original populations, a genetic bottleneck is
likely to have caused the disappearance of some alleles (17),
especially in microbes, which are often propagated clonally. In an
age of accelerated habitat destruction and diminishing biodiversity,
discovery of wild genetic stocks of domesticated
microbes will facilitate preservation of their genetic resources for
strain improvement.

Results and Discussion
Discovery of Wild Populations of Cryotolerant Saccharomyces. Saccharomyces
spp. are associated with oak trees (Fagaceae) in the
Northern Hemisphere (25, 26). Because species of the genus
Nothofagus (Southern beeches, also members of the Fagales)
occupy the oak niche in temperate regions of the Southern
Hemisphere (27), our survey in Northwestern Patagonia for Saccharomyces focused on woodlands containing populations of
Nothofagus antarctica, Nothofagus dombeyi, and Nothofagus pumilio,
within and near Lanin and Nahuel Huapi National Parks
(Argentina) (Fig. S1). We also surveyed stromata of Cyttaria hariotii
(an obligate ascomycete parasite of Nothofagus spp.) because
these fruiting structures are rich in simple sugars and provide
a favorable yeast habitat (28).Atotal of 133 samples of Nothofagus
bark, soil from underneath the trees, and Cyttaria stromata, collected
from 2006 to 2008, yielded 123 isolates of cryotolerant
Saccharomyces and two isolates of S. cerevisiae (Table S1).
For a preliminary identification of the cryotolerant Saccharomyces
isolates, we determined the DNA sequence of individual
genes, performed PCR-fingerprinting, and examined restriction
fragment length polymorphisms (RFLPs), using S. bayanus CBS
380T, S. uvarum CBS 395T, and S. uvarum CBS 7001 (referred to
as S. bayanus in genomics literature) as references (Fig. S2). The
isolates discretely fall into two groups: group A appears related
to S. bayanus (78 isolates); group B is closely related to S. uvarum
(45 isolates). The almost complete occupancy of the Nothofagus
niche by cryotolerant species contrasts with our ongoing survey
of Saccharomyces biogeography in the Northern Hemisphere oak
niche (North America, Mediterranean, Central Europe, and
Japan), where we have isolated ∼240 Saccharomyces strains from
more than 500 oak samples and observed that sympatric species
tend to have different growth temperature preferences. For
example, S. cerevisiae (thermotolerant) and Saccharomyces
kudriavzevii (cryotolerant) co-occur in Mediterranean regions,
but Saccharomyces paradoxus (thermotolerant) and S. uvarum
(cryotolerant) co-occur in temperate Europe and North
America (26). Therefore, the detection of a pair of cryotolerant
species in Patagonia and the near absence of thermotolerant
species suggest the Patagonian ecosystem supporting Saccharomyces
spp. may be unusual. One potential explanation is that
the relatively low annual average temperatures in our Patagonian
isolation sites [6/8 °C with mean low temperatures of −1/
−2 °C and mean high temperatures of 22/23 °C (29)] may favor
cryotolerant over thermotolerant species.

Ecological and Genetic Isolation of Two Cryotolerant Species. The
unanticipated detection of two closely related sympatric cryotolerant
populations prompted us to investigate the degree of
genetic isolation between them by measuring the meiotic sterility
of hybrids of the two populations. Spore viability within populations
was 89% to 91%, but hybrids produced only 7.3% viable
spores. Thus, the two populations exhibit considerable intrinsic
postzygotic isolation and can be considered to be two different
biological species, although they are phenotypically indistinguishable
(Fig. S3). Moreover, population A was found in association
with N. antarctica and N. pumilio, whereas population B was
associated with N. dombeyi (P < 10&#8722;7; Fisher’s exact test) (Table
1). The evergreen N. dombeyi prevails at mesic midelevation
sites, but N. antarctica and N. pumilio are deciduous and tend to
replace N. dombeyi at high-elevation and xeric sites (27), suggesting
that local niche-partitioning at least partially explains the
apparent coexistence of these two species. Detailed field and
laboratory studies into the causes of isolation are called for.
Genome Sequences Resolve Saccharomyces Taxonomy and Systematics.
The identification and taxonomy of S. bayanus, S. pastorianus,
and S. uvarum is problematic and controversial because
S. bayanus and S. pastorianus have only been isolated fromhumanassociated
fermentations. Indeed, all known representatives of
these two species have been suspected (S. bayanus) (13) or confirmed
(S. pastorianus) (10) to be interspecies hybrids. One potential
exception is the brewing contaminant NBRC 1948, which
was asserted to be a pure strain of S. bayanus based primarily on
RFLP evidence from all 16 chromosomes (15). A broad survey of
several strains previously assigned to S. bayanus, S. pastorianus,
and S. uvarum led to the conclusion that an additional “lager”
lineage or species exists that contributed its genome to S. pastorianus
(15). In contrast, the genome sequence of S. uvarum CBS
7001 lacks any signs of hybridization, introgression, or horizontal
gene transfer events from other Saccharomyces spp. (30).
To resolve these taxonomic and systematic issues and conclusively
identify our Patagonian strains, we generated draft genome
sequences of a representative from each Patagonian species and
several key brewing isolates by assembling millions of 36-bp
sequences, using the S. uvarum genome sequence as a reference.
Comparison of these genome sequences allowed us to conclusively
test: (i) whether our wild Patagonian populations A and B are
comprised of pure lineages; (ii) whether NBRC 1948 is a pure line
or a hybrid; (iii) the composition of the type strains of S. uvarum
(CBS 395T) and S. bayanus (CBS 380T); and (iv) the identity of the
non-S. cerevisiae moiety of the S. pastorianus genome.
S. eubayanus sp. nov. Is the Missing Wild Genetic Stock of S. pastorianus.

Comparison of the draft genome sequences revealed that the
two Patagonian species differ at &#8764;6% to 8% of nucleotides across
the genomes of surveyed strains [Fig. 1 (explained in detail in the
legend), and Datasets S1 and S2] (average divergence 6.89%). The
uniformity of sequence divergence suggests that there has been
little, if any, recent introgression between the Patagonian species,
consistent with the low spore viability of the hybrid cross. (Introgression
of regions that are small, subtelomeric, difficult to assemble,
or missing from some strains cannot be excluded.) The
species B strain is indeed closely related to the reference strain of S.
uvarum (average divergence 0.52%) and to its type strain CBS
395T, but the species A strain is closely related to the non-S. cerevisiae
moiety of the S. pastorianus lager yeast genome (average
divergence 0.44%). In contrast to these essentially pure strains, the
main component of the genome of the type strain of S. bayanus
(CBS 380T) is S. uvarum (67%), although there are substantial
contributions from species A (33%), including several large heterozygous
regions (19%of the genome). Similarly, large portions of
the genome of NBRC 1948 are derived from S. uvarum (37%), but
the majority of these portions are derived from species A (63%).
We also screened the short sequence reads for evidence of introgression,
hybridization, or horizontal gene transfer using two
different methods and the available Saccharomyces spp. reference
genomes (see SI Materials and Methods). We found no evidence of
foreign genes in either Patagonian strain or in the S. uvarum type
strain. Although these analyses cannot exclude foreign contributions
among genes missing from the Saccharomyces spp.
ortholog set, they are sensitive enough to readily detect multiple
subtelomeric contributions from S. cerevisiae in the two strains of
S. bayanus (CBS 380T and NBRC 1948) (Dataset S3).
Given the clear differences in ecological background and in
genomic constitution between the hybrid species S. bayanus and
the essentially pure species A, we propose regarding the wild
Patagonian lineage as a distinct species: S. eubayanus sp. nov.
Moreover, these genome sequence analyses firmly establish S.
eubayanus as the donor of the non-S. cerevisiae subgenome of S.
pastorianus, and exclude the contribution by an unknown “lager”
species substantially divergent from S. cerevisiae and S. eubaya-
nus (Dataset S4). Instead, a broad survey of strains (Fig. S2) and
the genome sequences of both of our representative strains of S.
bayanus (Fig. 1 and Datasets S1 and S3) suggest that the diversity
of this hybrid species can be explained by the contribution of
mixtures of alleles from S. uvarum and S. eubayanus, along with
some genes contributed by S. cerevisiae.

Evidence of Domestication. Domestication of crops and livestock
selects for desirable characteristics through directional breeding
so that domesticated lineages become genetically distinct from
their wild ancestors in ways that make them more useful to
humans (16, 17). To determine which genetic changes might
have been favored in brewing, we searched for differences between
the genome of S. eubayanus and three domesticated
strains associated with brewing (S. pastorianus and the triple
hybrids CBS 380T and NBRC 1948).

The disaccharide maltose is one of the most abundant sugars in
wort, so its utilization is a strongly selected trait in the brewing environment
(11, 31). Like S. pastorianus, the triple hybrid strains associated
with brewing contain subtelomeric maltose (MAL) gene
clusters horizontally transferred from S. cerevisiae, as well as the
S. cerevisiae SUC4 gene for processing the disaccharide sucrose
(Dataset S3). Surprisingly, we discovered that these three strains
share an identical chromosome translocation breakpoint within
ZUO1 (YGR285C) that fuses the right arm of S. eubayanus chromosome
VII to subtelomeric sequences on the right arm of S. cerevisiae
chromosome VII (Fig. 2 A and B). The transferred fragment
carries the S. cerevisiae IMA1 (YGR287C) gene that encodes isomaltase,
which catalyzes cleavage of the disaccharide isomaltose
(32). The identical breakpoints indicate a common origin for this
S. cerevisiae sugar-processing gene in all three hybrid strains and
suggest strong selection for optimal sugar utilization during brewing.
Sulfite formation is also important in lager-brewing because
sulfite is an antioxidant and flavor stabilizer (31). S. pastorianus
Weihenstephan 34/70 was previously noted to carry inactive copies
of both the S.cerevisiae and S. eubayanus SUL1 genes, while
retaining functional versions of both SUL2 genes (11), which encode
the high affinity transporters of sulfate, the metabolic precursor
of sulfite (33). Surprisingly, we found that both triple hybrid
strains contain the same frame-shift mutation as S. pastorianus in
their copies of S. eubayanus SUL1 (Fig. 2C). Interestingly, selective
expression of SUL2, especially the S. eubayanus allele, has been
shown to improve sulfite production (34). Because we found that
the SUL1 gene is intact in S. eubayanus, it is likely that the inactivation
of SUL1 is a consequence of artificial selection in the
brewing environment. The losses of SUL1 and some MAL transporters
suggest a tendency to selectively discard less-efficient nutrient
transport systems and retain or acquire others that are more
efficient under brewing conditions, a trade-off possibly resulting
from the 2D space constraints of the plasma membrane.

Evolution of S. pastorianus and S. bayanus Under Domestication.
Based on our ecological and comparative genomic analyses, S.
bayanus encompasses a set of hybrid strains known only from the
industrial brewing environment, whereas S. eubayanus exists as
an essentially pure lineage in natural conditions in Patagonia.
According to our model (Fig. 3), in the lager-brewery environment
of the 15th century (6) wild S. eubayanus genomes began
fusing with ale-type S. cerevisiae genomes to give rise to allotetraploid
hybrids, rare events that seem to have happened at least
twice (10). In these ancestors of modern-day S. pastorianus, mitotic
crossovers and other DNA repair mechanisms fused some
S. cerevisiae chromosomes to S. eubayanus chromosomes, making

some portions of the genome homozygous and other regions
aneuploid (10, 11). The S. eubayanus portions of these chromosomal
fusions that occurred in S. pastorianus would have
provided ample nearly identical sequence to seed the recombination
needed to introduce S. cerevisiae DNA into other S.
eubayanus and S. uvarum strains arriving in the brewing environment,
thus gradually giving rise to the complex S. bayanus
genome. Alternatively, the tremendous population sizes achieved
in the brewing environment may have allowed for the recovery
of extremely rare viable hybrid spores or for strains to
return to euploidy via a parasexual cycle. Because all S. cerevisiae
genes detected in the triple hybrids are subtelomeric in the S.
uvarum or S. cerevisiae reference genomes, we believe transformation
is the more likely mechanism because it requires only
one crossover proximal to the subtelomeric gene under selection,
and it explains the absence of widespread transfer of S. cerevisiae
proximal genes that would be expected under the other models.
Regardless of the mechanism of gene transfer, the identical
frame-shift mutations (SUL1), chromosome breakpoints (IMA1),
and identical hitchhiking sequences suggest that strong positive
selection was imposed by the brewers choosing the best strains or
by the competitive brewing environment itself to spread these
alleles to S. pastorianus and both S. bayanus triple hybrids.
It is surprising that European isolates of S. eubayanus have
never been found, despite records of yeast isolation since the late
19th century, including a recent emphasis on sampling more
natural arboreal environments (26). The facile recovery of this
species from Patagonia suggests that S. eubayanus may have been
absent in Europe until it was imported from overseas after the
advent of trans-Atlantic trade. In sharp contrast, its sister species,
S. uvarum, has been repeatedly isolated in Europe from
both artificial (e.g., brewing) and natural substrates (e.g., insects,
bark). Although additional environmental sampling is called for
worldwide, it seems likely that any natural European niches
suitable for S. eubayanus are occupied by other species, but that
it found great success through hybridization in the new artificial
environment of lager breweries.

The &#8764;7% genome-wide sequence divergence between S.
eubayanus and S. uvarum is the lowest level observed within the
Saccharomyces genus to result in genetic and ecological isolation
(Table S2). The geographic distribution and apparent niche
differentiation of these sister species make them a genetically
tractable system to study fungal speciation in allopatry with
secondary contact, or in sympatry because of ecological factors.

Taxonomy of S. eubayanus, S. bayanus, and S. uvarum. The nomenclature
of S. bayanus and S. uvarum has been confusing and
controversial for decades. High DNA-DNA reassociation values
between the type strains of these two species (35) led Naumov to
defend their merger, with S. uvarum initially considered a synonym
of S. bayanus (36), and later a variety (12). Other studies
employing molecular methods documented two well-separated
groups (37, 38), leading to proposals for the reinstatement of S.
uvarum as a separate species (13, 14). Unfortunately, the absence
of a clear resolution of these taxonomic issues caused CBS
7001, a strain selected for genome-sequencing (30), to be referred
to as S. bayanus (instead of S. uvarum) in most of the ensuing
genomics literature and databases. However, all known strains of
S. bayanus (including the type strain CBS 380T) appear to be
hybrids of S. eubayanus and S. uvarum that contain contributions
from S. cerevisiae in at least some cases (Fig. S2). Therefore, like
S. pastorianus, S. bayanus is not a “species” in the ecological and
evolutionary sense and is best viewed as a product of the artificial
brewing environment with no occurrence in nature. Because it
is now evident that the varietal and other designations adopted
by some researchers lack scientific support, we propose that
“S. uvarum” and “S. eubayanus” be used as descriptors of biologically
meaningful species, whereas “S. bayanus” and “S. pastorianus”
should be restricted to the domesticated and hybrid lineages.
Standard description. Standard description of Saccharomyces
eubayanus Sampaio, Libkind, Hittinger, P. Gonçalves, Valério,
C. Gonçalves, Dover et Johnston sp. nov.

Etymol.: The epithet is chosen to refer to the pure and natural
lineage that is related to the hybrid species Saccharomyces
bayanus Saccardo.

Cells globose to ovoidal (2.5–5 × 5–7 &#956;m). Pseudomycelium
absent. Asci oval and persistent containing two to four round
ascospores. The main diagnosis characteristic is the genome sequence
deposited in NCBI database. Salient physiological
properties are listed in Dataset S5. Strain CBS 12357 T (CRUB
1568T, PYCC 6148T) is designated as the type strain.
Latin description. Latin description of Saccharomyces eubayanus
Sampaio, Libkind, Hittinger, P. Gonçalves, Valério, C. Gonçalves,
Dover et Johnston sp. nov.

Species generis Saccharomycetis. Cellulae globosae ad ovoideae.
Pseudomycelium nullum. Asci ovales, persistentes, 2–4 ascosporis
globosis. Sequentia genomae totae in collectione sequentiarum
acidi nucleici NCBI numero SRP006155 in SRA030851 deposita.
Cultura typica CBS 12357T.

Domestication Processes Across Taxonomic Kingdoms. Domestication
is an inherently evolutionary process, the understanding of
which requires the inference of ancestral states, either by comparison
with wild progenitor populations or by archaeological
investigations. The characteristics of the previously undescribed
species S. eubayanus explain the instantaneous formation of
S. pastorianus by hybridization with S. cerevisiae. Selection imposed
by the brewing environment further refined lager yeast, and
we pinpointed several genetic changes attendant to lager production.
Given the diverse mechanistic natures of genetic changes
that occurred during crop and livestock domestication (2, 17, 18),
we anticipate that additional genetic changes that are not obvious
from sequence comparisons alone will be uncovered in the future,
such as changes in gene regulation and mechanisms to integrate
two genomes separated by millions of years of evolution. Identification
of these evolutionary changes and access to the previously
unknown wild stock promise to illuminate the role that fermented
beverages have played in human civilization and provide new
strategies for improving yeasts for brewing and biofuel production.

Materials and Methods
Yeast Isolation, Preliminary Characterization, and Crosses. The selective protocol
used for Saccharomyces isolations was previously described (26). Preliminary
identification was based on confirmation of Saccharomyces-type
ascospore production; DNA-sequencing of FSY1, FUN14, HIS3, MET2, RIP1
(Dataset S6), and the ITS region of rDNA; PCR-fingerprinting with primers
(GTG)5 and M13 (39); and PCR-RFLP using FUN14 digested with BanII, RIP1
digested with PstI, and HIS3 digested with RsaI (15) (Fig. S2). Isolated ascospores
of strains S. eubayanus CRUB 1568 and S. uvarum CRUB 1595 were
crossed to obtain interspecies hybrids. Hybridization was confirmed with
PCR-RFLP as above. Interspecific spore viability was determined by examining
362 ascospores produced by three independent hybrid strains. For the
assessment of intraspecific spore viability, &#8764;100 ascospores of each of the
two species (strains CRUB 1568, CRUB 1595) were studied.

Genome Sequencing and Analyses. The draft genomesequences ofmonosporicderivatives
of CRUB 1568 (FM1318), CRUB 1595 (FM1317), and NBRC 1948
(FM1309) were determined by assembling single-pass 36-bp Illumina sequence
reads to the S. uvarum CBS 7001 reference genome sequence (30), as described
previously (40) with modifications (SI Materials and Methods). Viable spores of
CBS 380T and CBS 395T were not recovered, so the genomes of these strains
were sequenced without further manipulation and shown to be aneuploid,
providing a likely explanation for their sterility. Genome sequence analyses
were performed using custom PERL scripts and standard software.

ACKNOWLEDGMENTS. We thank the Argentinean National Parks Administration
for collecting permits. We thank the NITE Biological Resource Center
of Japan for providing strain NBRC 1948, R. Ulloa for help during preliminary
field work, M. Weiss for preparing the Latin diagnosis, and R. A. Sclafani for
critical reading of the manuscript; and S. Fontenla and M. R. van Broock for
assistance and advice. This work was supported by Fundação para a Ciência e
a Tecnologia Grants PTDC/BIA-BDE/71734/2006 (to P.G. and J.P.S.), SFRH/BPD/
46471/2008 (to E.V.), UNComahue Grant CRUB B143 (to D.L.), National Institutes
of Health Grant GM032540 (to M.J.), and the James S. McDonnell
Foundation (C.T.H. and M.J.). C.T.H. is the Maclyn McCarty Fellow of the

And the figures.....

Appreciate it!