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Multiple host transfers, but only one successful lineage in a

continent-spanning emergent pathogen
Wesley M. Hochachka, Andr A. Dhondt, Andrew Dobson, Dana M. Hawley, David H. Ley and Irby J.
Lovette
Proc. R. Soc. B 2013 280, 20131068, published 10 July 2013

Supplementary data

"Data Supplement"
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tml

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Multiple host transfers, but only one

successful lineage in a continent-spanning
emergent pathogen
rspb.royalsocietypublishing.org

Wesley M. Hochachka1, Andre A. Dhondt1, Andrew Dobson2,

Dana M. Hawley3, David H. Ley4 and Irby J. Lovette1
1

Lab of Ornithology, Cornell University, Ithaca, NY 14850, USA

Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544, USA
3
Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA
4
Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State
University, Raleigh, NC 27607, USA
2

Research
Cite this article: Hochachka WM, Dhondt AA,
Dobson A, Hawley DM, Ley DH, Lovette IJ.
2013 Multiple host transfers, but only one
successful lineage in a continent-spanning
emergent pathogen. Proc R Soc B 280:
20131068.
http://dx.doi.org/10.1098/rspb.2013.1068

Received: 26 April 2013

Accepted: 14 June 2013

Subject Areas:
health and disease and epidemiology,
genomics, evolution
Keywords:
Mycoplasma gallisepticum, phylogeny, house
finch, Haemorhous mexicanus, Carpodacus,
host transfer

Author for correspondence:

Wesley M. Hochachka
e-mail: wmh6@cornell.edu

Electronic supplementary material is available

at http://dx.doi.org/10.1098/rspb.2013.1068 or
via http://rspb.royalsocietypublishing.org.

Emergence of a new disease in a novel host is thought to be a rare outcome

following frequent pathogen transfers between host species. However, few
opportunities exist to examine whether disease emergence stems from a
single successful pathogen transfer, and whether this successful lineage
represents only one of several pathogen transfers between hosts. We examined the successful host transfer and subsequent evolution of the bacterial
pathogen Mycoplasma gallisepticum, an emergent pathogen of house finches
(Haemorhous (formerly Carpodacus) mexicanus). Our principal goals were to
assess whether host transfer has been a repeated event between the original
poultry hosts and house finches, whether only a single host transfer was ultimately responsible for the emergence of M. gallisepticum in these finches, and
whether the spread of the pathogen from east to west across North America
has resulted in spatial structuring in the pathogen. Using a phylogeny of
M. gallisepticum based on 107 isolates from domestic poultry, house finches
and other songbirds, we infer that the bacterium has repeatedly jumped
between these two groups of hosts but with only a single lineage of
M. gallisepticum persisting and evolving in house finches; bacterial evolution
has produced monophyletic eastern and western North American subclades.

1. Introduction
Transfer of a pathogen to a novel host is a common mechanism by which diseases emerge [14]. The first step in a host-transfer emergence of disease is the
transmission of the pathogen between its original host and the novel host. Prior
work suggests that following an initial jump to a new host a pathogen will often
not be well-adapted, and that in many cases, transmission will be restricted to a
few stuttering chains of infected individuals that ultimately lead to failure of the
pathogen to establish in the novel host species [3,5,6]. Before transfer to a novel
host can be followed by independent evolution, it is likely that repeated asymmetrical transfers of pathogens have happened in a sort of source sink
dynamics [5]; eventually, a lucky mutant is transferred that can sustain itself
in the new host. By contrast, anecdotal evidence exists for repeated introduction
of some pathogens into novel hosts [7,8], and repeated transfers are implicit in
our understanding of the major evolutionary shifts of human influenza [9].
Few large archives of samples exist for pathogens with non-human hosts
[10 12], and to the best of our knowledge none have been used to describe
the emergence of disease in a novel host. If archived specimens are available,
then phylogenetic reconstructions can be used to make inferences about disease
emergence and subsequent dynamics. Phylogenies make it possible to infer that
disease emergence resulted from a single successful host jump, when only a
single clade of pathogen is found in extensive sampling of the pathogens in a
novel host. Archived specimens further allow the examination of subsequent

& 2013 The Author(s) Published by the Royal Society. All rights reserved.

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M. gallisepticum circulating in domestic poultry? (iii) did western North American isolates derive from eastern ones,
consistent with a single host jump from poultry and later colonization of western North America? and (iv) when songbirds
other than house finches are infected by M. gallisepticum, is
the bacteria consistently from the same clade that infects
house finches? While the details of our results are specific to
this pathogenhost system, they provide important insights
that are relevant to patterns of evolution that will occur in
other pathogens, and more generally to invasive alien species
[28] that have successfully colonized a novel niche.

The bacterial isolates included in our study were derived from

one of three general sources: (i) field sampling of wild house
finches and other songbirds collected during the study of M. gallisepticum dynamics, (ii) isolates from diseased house finches or
other songbirds collected from wildlife rehabilitation facilities,
and (iii) isolates from poultry on farms collected in support of
disease control measures. Samples from all three sources were
collected non-systematically both temporarily and spatially,
and the sources vary in the precision with which collection
location was recorded or the accuracy with which locations
could be determined (detailed below). The non-systematic spatial
and temporal sampling does not allow these data to be used for
quantifying distributions of bacterial haplotypes or rates of evolution, but we have no expectation that this would bias
assessments related to the questions that we have outlined in 1.
When wild house finches and other songbirds were trapped
in the course of work (New York, Georgia, Wisconsin, Alabama)
to identify the prevalence of M. gallisepticum infections (source 1),
samples were collected following procedures as described in
Hawley et al. [26], for subsequent culture and identification
as described in the electronic supplementary material, section
Methods. We also worked with wildlife rehabilitation centres
in California, Virginia and North Carolina to obtain samples
from house finches and other songbirds that were brought into
these centres, presumably from nearby locations (source 2).
Locations of sample collection are shown in figure 1a, and
years of collection in figure 1b. Owing to the difficulty in successfully culturing field isolates, our collection protocol evolved over
the course of study. Ultimately, we had our highest success when
conjunctival swabs were placed into universal transport medium
(Becton Dickenson/Copan; Sparks, MD, USA) and shipped
overnight on cold packs to NC State University College of Veterinary Medicine (NCSU CVM; Raleigh, NC, USA). Owing to
improvements in our culture success with field-collected
samples, we have a better representation of songbird isolates
from the late 2000s and early 2010s than in prior years (figure
1b). Overall, the songbird isolates examined in this study represent approximately 52 per cent of those available to us, and
were chosen to be representative of the dates, locations and
hosts of isolates available. Six of eight of the M. gallisepticum
house finch isolates used in our experimental studies of virulence
[26,27] were examined in this study, and their identities are
noted in the electronic supplementary material, table S1. Samples
from all but one of the songbird isolates came from the archive
maintained by D.H.L. at NC State University (see the electronic
supplementary material, table S1). The remaining isolate, collected from a house finch in Ithaca, NY, was cultured in Ithaca
immediately prior to sequencing.
The sample isolates of M. gallisepticum from poultry (source 3)
were selected from a larger archive maintained by D.H.L. This
archive of poultry isolates has been built in the course of
D.H.L.s diagnostic work in support of disease control on poultry

Proc R Soc B 280: 20131068

2. Material and methods

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evolution of the pathogen in a novel host, with respect both

to longer-term spatial and temporal alterations in genomes
[10,11] and with finer-resolution changes in genetic diversity
at individual geographical locations [13,14].
One non-human system in which pathogen genotypes can
be examined throughout the process of emergence in a new
host is the bacterium Mycoplasma gallisepticum and its novel
host the house finch (Haemorhous (formerly Carpodacus) mexicanus). Mycoplasma gallisepticum is a worldwide pathogen of
domestic poultry and is relatively taxon-specific in the hosts
in which it commonly causes disease (Galliformes). The emergence of M. gallisepticum as a pathogen in house finches in
19931994 [15] was highly unusual and probably involved
substantial differentiation between house finch and poultry
lineages. Studies of a limited number of isolates suggest
M. gallisepticum emergence in house finches entailed rapid
differentiation of the bacterium during the colonization of
house finches [16,17]; this is consistent with a single (pointsource) origin. Nevertheless, anecdotal evidence [16,18]one
subsequent isolate each of apparent finch lineage and poultry
lineage M. gallisepticum crossing to poultry and house finches,
respectivelyalso suggested multiple transfers of M. gallisepticum between galliforms and finches. Following the emergence
of M. gallisepticum in house finches, other wild songbirds
(order Passeriformes) have also found to be infected with
M. gallisepticum; these appear to have originated from house
finches [19,20]. No large-scale comparisons have been made
of M. gallisepticum lineages in poultry, house finches and
other songbirds to validate the hypotheses of a point-origin
and single successful transfer of the pathogen in house finches
and subsequent transfer from house finches to other songbirds.
Multiple lines of evidence suggest that substantial spatial
and temporal genetic variation exists in M. gallisepticum that
has become established in house finches, and that this variation
may underlie systematic phenotypic changes in virulence
through space and time. The bacteria spread in house finches
from the point of emergence in the mid-Atlantic United
States (close to Baltimore) to the Pacific coast [21], apparently
across the northern Great Plains of the United States [22,23].
Work with a small number of isolates showed that some genetic differentiation has occurred [16,17,24] and suggests that
this genetic variation may be linked with systematic directional
phenotypic changes in virulence. Experimental studies suggest
that evolution of increasing virulence through time in lineages
of M. gallisepticum present in eastern North America, as well as
a drop in virulence with dispersal to western North America
[2527]. However, it is currently unclear whether eastern and
western isolates of M. gallisepticum represent distinct phylogenetic clades as a result of a single pathogen dispersal from
east to west. A clearer understanding of spatial and temporal
evolution in virulence of M. gallisepticum will require the examination of the evolutionary relationships among a larger
number of isolates of M. gallisepticum in house finches and
other songbirds.
In this paper, we take advantage of a large collection of
M. gallisepticum isolates from poultry, house finches and
other songbirds in order to examine the phylogenetic relationships among bacterial isolates collected from these hosts.
We ask the following questions: (i) is there evidence of repeated transfer of M. gallisepticum between domestic poultry
and house finches? (ii) irrespective of repeated host transfers, is there a single dominant clade of M. gallisepticum in
house finches and other songbirds that is distinct from the

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(a)

eastern songbird
western songbird
domestic poultry

12
no. isolates

10
8
6
4
2
0

1994

1998

2002
year

2006

2010

Figure 1. Locations and years of collection of isolates. (a) Approximate

locations from which songbird isolates of M. gallisepticum were collected.
Unfilled circles indicate locations from which at least some of the isolates
were of the same haplotype as the index isolate that was collected in Virginia; the single black-filled circle in Alabama indicates the only eastern
location from which the index haplotype was not identified. The total
number of isolates collected at these locations is proportional to the
square of the diameter of the plotted symbols. The Quebec location is plotted
as mid-way between the two potential collection sites, and the two central
California locations have been artificially offset to allow both to be seen.
(b) Calendar years from which isolates were collected. Unfilled bars indicate
numbers of isolates each year from eastern North American locations, yellowfilled bars indicate numbers of isolates from western (Californian) locations,
and black bars indicate years of collection of isolates from domestic poultry.
Mycoplasma gallisepticum was first confirmed in songbirds on the Pacific coast
of North America in 2004.
farms mainly in the southeastern United States. The chosen isolates
were largely drawn from the southeastern United States to be representative of the geographical distribution of many of the eastern
North American M. gallisepticum samples from house finches and
other songbirds used in our study. The precision of geographical
location for samples from poultry is lower than for samples from
wild birds for several reasons including the potential for interstate movement of poultry by large farm operations, and as a
result we have provided only location information to the level of
state in the electronic supplementary material, table S1, in order
to signify this imprecision. Our intent for including the large
number of isolates of M. gallisepticum from poultry was to allow
for a robust determination of whether the bacteria circulating in
house finches was genetically distinct from that in poultry or
whether regular host transfers were occurring, and not to make
interpretations of geographical patterns of variation within the M.
gallisepticum circulating in domestic poultry.

Proc R Soc B 280: 20131068

14

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(b)

Methods for laboratory culture and identification are described

in Ley et al. [15]. Mycoplasma gallisepticum is a nutritionally fastidious and labile bacterium, making the isolation and creation of
an archival library challenging. Each of the archived isolates was
cultured in Freys mycoplasma media (broth and agar) with
15 per cent swine serum medium [29], and colonies on agar identified as M. gallisepticum by direct immunofluorescence. Aliquots of
low passage broth cultures were frozen at 2708C. See electronic
supplementary material, section Methods for further information
about culturing M. gallisepticum.
All molecular genetic work was carried out at Cornell University, with samples from NC State University shipped to Cornell on
dry ice. We based our phylogeny of M. gallisepticum on partial
sequencing of 107 isolates (60 from house finches and other songbirds, and 47 from domestic poultry). We selected regions of the
genome to sequence without reference to functional genes (see
below for details), creating data that are appropriate for examining
patterns of genetic relatedness among isolates. This approach
allows a broad survey of isolates that complements the wholegenome sequencing of a far smaller set of isolates that is needed
to suggest candidate genes under selection during and subsequent
to the establishment of M. gallisepticum in house finches and related
species. Our research collaborations whole-genome sequencing
is reported on elsewhere [24], and has identified variation in multiple genes associated with levels of virulence [24], one of these
discussed in greater detail by Delaney et al. [17].
As the phylogenetic component of this survey was initiated
before full DNA sequences were available for any M. gallisepticum,
we initially used DNA from the index isolate of M. gallisepticum
in house finches [15] to construct a small-insert genomic library
from which we generated a panel of anonymous nuclear sequence
markers via direct Sanger sequencing of the Mycoplasma genomic inserts. We designed and optimized 13 PCR primer pairs from
within a corresponding set of M. gallisepticum insert sequences,
each targeting a region of 559869 nucleotides. See the electronic
supplementary material, section Methods for more information. Comparisons against subsequently published genomes of
M. gallisepticum confirmed that all of these regions aligned to, and
were distributed throughout, the M. gallisepticum genome. Location
in a reference M. gallisepticum genome of each amplified sequence,
gene identity and related information are included in the electronic
supplementary material, table S2; we have no reason to suspect
that any sequenced regions are under selection in a way that
would bias our phylogenetic results. All sequence data have been
archived in GenBank (accession numbers KC995288KC996678,
inclusive); electronic supplementary material, table S3 lists the isolate and primers used to obtain each GenBank sequence, with
isolate and primer names and further information provided in the
electronic supplementary material, tables S1 and S2.
Genomic DNA was purified using a DNAeasy kit (Qiagen,
Valencia, CA, USA), and all 13 regions were PCR amplified
and sequenced from all 107 isolates using standard methods.
Sequences were checked using SEQUENCHER v. 4.6 (GeneCodes,
Ann Arbor, MI, USA), and all sequences for each region were
aligned readily by eye. The combined length of all aligned regions
was 8399 nucleotides from a total genome slightly under one
million base pairs in length [30].
The phylogenetic relationships among all of the isolates
and associated posterior probability scores for branches within
this phylogeny were reconstructed using Bayesian multi-chain
MCMC analyses as implemented in MRBAYES v. 3.1.2 [31,32].
Analyses were conducted on the concatenated sequences from all
13 regions, as these have a shared phylogenetic history in a clonal
organism such as M. gallisepticum. MRBAYES analyses were run for
25 million generations under a globally applied GTR G I substitution model. Trees were sampled every 1000 generations, and
runs used two simultaneous Markov chains. Convergence was
assessed using the average standard deviation of split frequencies;

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(b)

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Finch_CA09.2
Finch_CA09.4

Finch_CA09.1

Finch_CA06.2

Finch_CA06.1

Finch_CA10.2
Finch_CA09.3
Finch_GA04.1
Finch_CA10.1
Finch_NC98.1
[AMGO]
Poultry
03.4

Finch_NC06.2
Finch_VA94.1
[AMGO (n = 2)]
[EVGR]

Finch_NC06.1
Finch_NC06.3
Finch_GA10.1

Finch
AL10.2

Finch
NY06.1

Finch
NY11.1

Finch_NY11.2
Finch_AL10.1

Figure 2. Phylogenetic relationships among haplotypes of M. gallisepticum isolated from (a) all songbird and poultry haplotypes used in our study, and (b) isolates
obtained from house finches and other songbirds. (a) All haplotypes recovered from house finches and other songbirds are indicated in red, and all haplotypes
recovered from poultry are indicated in black. Note the presence of one finch isolate recovered from domestic poultry and one poultry isolate recovered from a
house finch. The network diagram in (b) indicates a separation between haplotypes from California (in yellow) and those from eastern North America (open circles).
The black-filled circle indicates the house finch haplotype recovered from domestic turkeys in North Carolina, which clearly derived from the index isolate first
recovered from a house finch in 1994 and from which a second distinct haplotype (found in a house finch) was in turn derived. Grey text in (b) denotes isolates
from American goldfinch (AMGO) or evening grosbeak (Coccothraustes vespertinus; EVGR), with only isolate Finch_NC98.1 being unique to a songbird other than
house finches. Each dot on a connecting line and larger labelled circle represents a single nucleotide substitution. Circle areas monotonically increase with the
number of isolates belonging to each haplotype. In both (a) and (b), all haplotypes from house finches and other songbirds are labelled starting with finch,
followed by the two-letter code for the state in which the haplotype was first isolated, the last two digits of the calendar year in which the haplotype was
first isolated isolation, and a single arbitrary number unique to that haplotype. All haplotypes from domestic poultry are labelled with Poultry, the last two
digits of the calendar year of the first isolate identified with the haplotype and then an arbitrary number.

runs quickly reached convergence, and the burn-in was conservatively set to 500 000 generations. The resultant tree (figure 2a)
revealed that differences among the isolates from songbirds were
minor in comparison with the variation among M. gallisepticum
from poultry. Because of this similarity among songbird isolates, a
more appropriate method for describing the associations among
the isolates was to use statistical parsimony to create a haplotype
network describing the relationships within the recently diverged
finch clade of M. gallisepticum (figure 2b). We used TCS 1.21 [33]
to construct this haplotype network.

3. Results and discussion

When a pathogen transfers to a novel host, the expectation is
that the process involves: (i) repeated but typically short-lived

introductions of a pathogen into a novel host until one introduction is either preceded or followed by adaptations that
allow persistence, and then (ii) further adaptation of the
pathogen to its new host [5,6]; these adaptations potentially
restrict the pathogens ability to grow in its original host
species. The phylogeny produced from our collection of isolates is consistent with both aspects of this general model
for emergence of pathogens in novel hosts. Even with genotyping M. gallisepticum from a small proportion of all birds
infected since this disease emerged in house finches, we
found two haplotypes (Poultry_03.4 and Finch_NY01.1 in
figure 2) that imply the existence of continued exchanges of
M. gallisepticum both from poultry to songbirds as well as
from songbirds to poultry. Our phylogeny also shows a
highly supported divergence between the haplotypes of

Proc R Soc B 280: 20131068

Finch_CA09.1
Finch_CA09.2
Finch_CA09.3
Finch_CA09.4
Finch_CA10.1
Finch_CA06.1
Finch_CA10.2
100 Finch_CA06.2
Finch_NC98.1
Finch_NC06.1
Finch_NC06.2
Finch_NC06.3
Finch_AL10.1
100
Finch_AL10.2
Finch_NY06.1
Finch_NY11.2
Finch_NY11.1
Finch_GA10.1
Finch_VA94.1
Finch_GA04.1
Poultry_03.4
Poultry_01.1
100
Poultry_07.1
Poultry_07.2
95
Poultry_03.1
100
Finch_NY01.1
100
Poultry_07.3
100
Poultry_03.2
Poultry_04.1
Poultry_07.4
Poultry_03.3
Poultry_97.1
100
Poultry_01.6
99
100
Poultry_07.3
100
Poultry_99.3
94 100
Poultry_07.2
100
Poultry_01.5
Poultry_01.4
100
100 Poultry_00.2
100
Poultry_99.2
100
Poultry_01.3
100
100
Poultry_06.1
Poultry_05.1
Poultry_01.2
100
Poultry_99.1
Poultry_00.1
(a)

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Proc R Soc B 280: 20131068

house finches rather than the continued presence in poultry

of the lineage containing this haplotype. The viability of a
house finch M. gallisepticum in domestic poultry is not surprising, given that an isolate of M. gallisepticum from a house finch,
probably very similar to the index isolate from house finches,
has been experimentally introduced and found to be viable
in chickens [35]. Interestingly, the severity of disease that
resulted from this experimental infection was so mild that the
Finch_VA94.1 strain was suggested as a potential vaccine
strain of M. gallisepticum for domestic poultry [36]. Such low
virulence in its original host indicates that local adaptation to
house finches and concomitant lowered virulence in the original host taxa had occurred already within the first known
haplotype of house finch M. gallisepticum.
While we have documented only a very small number of
transfers of M. gallisepticum between hosts as disparate as
poultry and finches, we believe these observations show
that transfer rates are high enough that they did not limit
the emergence of M. gallisepticum as a pathogen of house
finches. Streicker et al. [12] reached a similar conclusion in a
study of movement of rabies lineages between bat species,
and the ranges of hosts that can be infected by more catholic
pathogens such as Salmonella [37] is also consistent with frequent opportunities for transfer of pathogens among host
species. While we have documented only a small number
of host transfersconfirming a second transfer from poultry
to house finches and identifying one new transfer of finchclade bacteria back to poultryin probabilistic terms, these
events are not rare: one new transfer to house finches from
60 isolates (1.7% of songbird isolates examined), and one
new transfer to poultry from 47 isolates (2.1% of poultry isolates). We suggest that these are minimum estimates of
transfer rates, because an unknown proportion of transfers
will not be detected either because of immediate failure of
the bacteria to establish or because the cross-host parasite
lineages will have gone extinct rapidly [3,5,6]. Such extinctions could be due either to poor adaptation to the novel
host per se, or to poor competitive ability relative to the established finch clade of M. gallisepticum. New poultry-clade
isolates that cause little or no obvious disease in songbirds
will also be under-sampled by our bias towards sampling
obviously diseased songbirds. Further, many transfers of
house finch M. gallisepticum back to poultry are likely to go
undetected, because the poultry most likely to be monitored
for infection come from large commercial farms that have biosecurity measures in place to minimize contact between
poultry and wild birds, whereas many exchanges may
occur with smaller non-commercial (backyard) flocks of
poultry with little or no biosecurity.
Our third objective was to determine whether
M. gallisepticum in house finches in eastern and western
North America represent independent lineages, and whether
the relationships among eastern and western M. gallisepticum
isolates are consistent with a single colonization of the
pathogen from eastern North America house finches [38].
While a previous study indicated genomic divergence of a
single western isolate (labelled Finch_CA06.1 in figure 2)
relative to other isolates [24], our phylogeny (figure 2b) indicates that the western isolates we have examined all have
a common origin and are derived from the haplotype of
M. gallisepticum originally isolated from finches in eastern
North America. Our phylogenetic reconstruction gave a
posterior probability score of 97% for this conclusion.

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M. gallisepticum typically isolated from house finches and other

songbirds, and those present in poultry in the southeastern
United States during a similar prolonged time period
(figure 2a). Note also the minimal variation among the finch
haplotypes relative to that found for M. gallisepticum from
poultry (figure 2a). The large number (figure 2b), large
geographical extent (figure 1) and long time period (see the
electronic supplementary material, table S1) from 1994 to
2011 over which the initial haplotype, Finch_VA94.1, was isolated, in concert with rapid diversification into multiple
lineages (figure 2b), are consistent with a recent colonization
and subsequent radiation of M. gallisepticum among house
finches [34] accompanied by occasional spillover into other
songbirds. The persistence of the initial finch haplotype
should not be taken to mean that the genetic make-up of
this haplotype has remained unchanged through time, but
merely that no changes were present in the marker genes
used in this study: full-genome sequences of five isolates
from this haplotype (VA94, NC95, NC96, NY01 and
WI01 in figure 4 of [24]) all differed slightly from each other.
Regarding the existence of repeated transfer of M. gallisepticum from poultry to house finches, we have confirmed the
suggestion [16] of a second poultry-clade isolate from house
finches. This poultry-type haplotype of M. gallisepticum (labelled
Finch_NY01.1 in figure 2a) that was isolated from an asymptomatic house finch in Ithaca, NY in 2001 falls clearly outside the
house finch clade of M. gallisepticum, as indicated by the high
posterior probability score. This second detected transfer to
house finches is also consistent with expectation that most host
transfers will not result in persistence of the pathogen in a
novel host, because this haplotype, isolated from a single house
finch displaying no outward clinical signs of disease, was not
subsequently detected in songbirds. Unfortunately, we have
very few isolates from Ithaca between 2001 and 2011 and examined only three for this study, so we have no effective way of
knowing how long the poultry-like haplotype (Finch_NY01.1)
persisted in house finches.
We have also identified a new instance in which
M. gallisepticum was transferred from songbirds to poultry.
This is the second such case, following the one described
by Ferguson et al. [18]. We found the Poultry_03.4 haplotype (black-filled circle in figure 2b) in four isolates from
domestic turkeys in North Carolina in 2003. Because these
turkeys were from four separate commercial farms belonging to the same company, we believe that they represent a
single back-transfer of M. gallisepticum to poultry with subsequent poultry-to-poultry transmission within the
companys facilities. The posterior probability score of
100% overwhelmingly supported placing these isolates
with the house finch clade of M. gallisepticum. This by
itself is not conclusive evidence for a host jump back to
poultry from house finches, because an alternative explanation is that a finch-like clade of M. gallisepticum
originated and continues to persist in poultry alongside what
we have termed the poultry clade. This latter explanation
would be consistent with two coalescence analyses that date
the divergence of M. gallisepticum in house finches from a
few years before the first isolation of the bacterium from finches
[17,24]. However, our isolate of finch-clade M. gallisepticum
from turkeys is intermediate between two isolates from
house finches in a network diagram of the finch-clade bacteria
(figure 2b). This intermediate position is more parsimoniously
explained by a back-transfer from the evolving bacteria in

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All capture and sampling work used to obtain isolates of M. gallisepticum was conducted in accordance with state and national
regulations, with required state and national permits, and with
approval of protocols by the Institutional Animal Care and Use Committees (IACUCs) of Cornell University ( protocol no. 2006094) and
Virginia Tech.

Acknowledgements. We thank L. Stenzler, A. Talaba, and C. Makarewich

for conducting the PCR and sequencing work. Staff at The Wildlife
Center of Virginia, California Wildlife Center, Lindsay Wildlife
Museum, Piedmont Wildlife Center, Triangle Wildlife Rehabilitation
Clinic and Cornell Lab of Ornithology were responsible for collecting
the majority the isolates of M. gallisepticum from wild birds used in
our paper. Other songbird sampling was coordinated by Barry
Hartup (Wisconsin) and Andy Davis (Georgia). Staff at many commercial poultry farms provided isolates of bacteria to D.H.L. in the
course of their work. Judith McLaren and Sile Huyan assisted with
M. gallisepticum culture, isolation, identification, and creation of the
isolate archive used in this study. We thank S. Geary and
E. Tulman for their insights during the conducting of the sequencing
work and additionally two anonymous reviewers during writing of
this paper.
Funding statement. This work was supported through NSF-EF grant no.
0622705 to A.A.D. under the NSF-NIH Ecology of Infectious Diseases
programme.

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