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\begin{document}

\title{Paths of introduction: Assessing global colonization history of the most
successful amphibian invader}



\author[1]{Gabriel Jorgewich-Cohen}%
\author[2]{Rachel Montesinos}%
\author[1]{Rafael Henrique}%
\author[3]{Felipe Toledo}%
\author[4]{Amaël Borzée}%
\author[5]{Yang Yi}%
\author[6]{Wang Fei}%
\author[7]{Takeshi Igawa}%
\author[8]{Juan Guayasamin}%
\author[9]{Gabriel Laufer}%
\author[10]{Wilmar Bolivar}%
\author[11]{Ariel Rodriguez}%
\author[1]{Taran Grant}%
\affil[1]{Universidade de São Paulo}%
\affil[2]{UFMG}%
\affil[3]{UNICAMP}%
\affil[4]{Nanjing Forestry University}%
\affil[5]{Xishuangbanna Tropical Botanical Garden}%
\affil[6]{ShanghaiTech University}%
\affil[7]{Hiroshima University}%
\affil[8]{Universidad San Francisco de Quito}%
\affil[9]{Museo Nacional de Historia Natural}%
\affil[10]{Universidad del Valle}%
\affil[11]{Tierarztliche Hochschule Hannover}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
International socioeconomic relationships form the background that
underlies the history of invasive species. Species with economic value,
such as the North American bullfrog (Lithobates catesbeianus), are more
likely to become internationally distributed and to be subsequently
introduced to non-native areas and, consequently, become more difficult
to control. Using population genetics methods, we investigated the
invasion pathways, the connectivity among clusters in different
countries and the native population of origin of globally introduced
bullfrog populations. Throughout the analysis of seven microsatellite
nuclear loci, one fragment of the mitochondrial cytochrome b locus, and
historical information, four main lineages were identified and analyzed
along with previous findings. This species' capability to colonize
several countries from few starting lineages highlights the necessity to
control new propagule pressure to ensure successful management programs,
as high inbreeding and bottleneck effect seem not to diminish the
invasive success of this species. There is a consensus between markers
that most areas of South America belong to the same genetic population
while populations in Asia have a more complex history of introduction.%
\end{abstract}%



\sloppy


Paths of introduction: Assessing global colonization history of the most
successful amphibian invader

Gabriel Jorgewich-Cohen\selectlanguage{ngerman}¹ \textbar{} Rachel Montesinos² \textbar{} Rafael
dos Santos Henrique¹ \textbar{}Luís Felipe Toledo³ \textbar{} Amaël
Borzée\selectlanguage{english}4 \textbar{} Yang Yi5 \selectlanguage{english}\textbar{} Wang Fei6 \selectlanguage{english}\textbar{} Takeshi
Igawa7 \selectlanguage{english}\textbar{} Juan M. Guayasamin8 \selectlanguage{english}\textbar{} Gabriel Laufer9
\selectlanguage{english}\textbar{} Wilmar Bol\selectlanguage{ngerman}ívar¹\selectlanguage{english}0 \textbar{} Ariel Rodr\selectlanguage{ngerman}íguez¹¹ \textbar{}
Taran Grant¹

\emph{1 Laboratório de Anfíbios, Biosciences Institute, University Of
São Paulo. São Paulo, SP, Brazil}

\emph{2 Laboratório de Herpetologia, Departamento de Zoologia, Instituto
de Ciências Biológicas, Universidade Federal de Minas Gerais. Belo
Horizonte, MG, Brazil}

\emph{3 Laboratório de História Natural de Anfíbios Brasileiros
(LaHNAB), Departamento de Biologia Animal, Universidade Estadual de
Campinas. Campinas, SP, Brazil}

\emph{4 Laboratory of Animal Behavior and Conservation, College of
Biology and the Environment, Nanjing Forestry University, China}

\emph{5 Center for Integrative Conservation, Xishuangbanna Tropical
Botanical Garden, Chinese Academy of Sciences. Yunnan, China}

\emph{6 School of Life Science and Technology, Shanghaitech University.
Shanghai, China}

\emph{7 Amphibian Biology Center, Hiroshima University,
Higashihiroshima. Hiroshima, Japan}

\emph{8 Laboratorio De Biología Evolutiva, Instituto BIÓSFERA-USFQ,
Colegio De Ciencias Biológicas Y Ambientales COCIBA, Campus Cumbayá,
Universidad San Francisco De Quito USFQ. Quito, Ecuador}

\emph{9 Area Biodiversidad y Conservacion, Museo Nacional de Historia
Natural. Montevideo, Uruguay}

\emph{10 Research Group in Animal Ecology, Department of Biology,
Universidad Del Valle. Cali, Colombia}

\emph{11} \emph{Institute of Zoology, University of Veterinary Medicine
Hannover (TiHo)}

\textbf{Correspondence}

Gabriel Jorgewich-Cohen

Bioscience Institute, University of São Paulo, Rua do Matão, 101, Cidade
Universitária, CEP 05508-090, São Paulo, SP, Brazil

Email: gabrieljcohen2@gmail.com

\textbf{Funding information}

Grants and fellowships were provided by Sa\selectlanguage{english}o Paulo Research Foundation
(FAPESP \#2012/10000-5; \#2016/25358-3; \#2018/15425-0; \#2019/18335-5),
and the National Council for Scientific and Technological Development
(CNPq \#134290/2015-1; \#300896/2016-6; \#306823/2017-9)

\textbf{Abstract}

International socioeconomic relationships form the background that
underlies the history of invasive species. Species with economic value,
such as the North American bullfrog (\emph{Lithobates catesbeianus} ),
are more likely to become internationally distributed and to be
subsequently introduced to non-native areas and, consequently, become
more difficult to control. Using population genetics methods, we
investigated the invasion pathways, the connectivity among clusters in
different countries and the native population of origin of globally
introduced bullfrog populations. Throughout the analysis of seven
microsatellite nuclear loci, one fragment of the mitochondrial
cytochrome b locus, and historical information, four main lineages were
identified and analyzed along with previous findings. This species'
capability to colonize several countries from few starting lineages
highlights the necessity to control new propagule pressure to ensure
successful management programs, as high inbreeding and bottleneck effect
seem not to diminish the invasive success of this species. There is a
consensus between markers that most areas of South America belong to the
same genetic population while populations in Asia have a more complex
history of introduction.

\textbf{KEYWORDS}

Amphibia, Anura, globalization, invasion biology, population
establishment

\textbf{Introduction}

Human action influence natural environments in many ways, often with
unintended consequences. One of the most significant phenomena that
substantially increased with globalization is the invasion of alien
species. Together with habitat loss, it is the main reason for native
species' populations decrease and extinction (Lowe et al., 2000; Scheele
et al., 2019). Invasive species control efforts are expensive and
commonly ineffective (Ficetola \& Scali, 2010; Hulme, 2006), resulting
in the necessity to understand invasion pathways to avoid the
colonization of new species or the release of new propagules that might
increase genetic variability and fitness of already established
populations (Kolbe et al., 2004).

Alien species are sometimes intentionally spread for economic reasons,
such as agriculture, biological control, and pet trade (Chiaverano et
al., 2014; Ng \& Lim, 2010). In these cases, human history can be a
useful tool to help identifying and explaining translocation events. It
is also common to find information in the lay literature that can help
elucidate the origin and pathways of introduction. However, gathering
and interpreting information from such literature around the globe can
be difficult due to access limitations, language differences,
contradicting and misleading information. In this case, population
genetics can be a useful tool for reconstructing and understanding the
history behind the events of invasions (Ficetola et al., 2008; Bai et
al., 2012; Kamath et al., 2016).

The world's most widespread invasive amphibian species is the North
American bullfrog (hereafter bullfrog for short), \emph{Lithobates
catesbeianus} , which is native from eastern North America but currently
can be found in many countries in the Americas, Europe, and Asia, where
the species was introduced for frog farming and subsequently became
established (Kraus, 2009). Despite recognized impacts on native
populations (Both \& Grant, 2012; Schloegel et al., 2010; Li et al.,
2011; Borz\selectlanguage{ngerman}ée et al., 2017; O'Hanlon et al., 2018), there is a small
number of eradication attempts (Adams \& Pearl, 2007; Snow \& Witmer,
2010, Groffen et al., 2019), possibly as a reflection of the high cost
and low rate of success of these attempts (Adams \& Pearl, 2007). The
lack of information on history and paths of invasion, connectivity and
dispersal among populations and their genetic structure, is one of the
main reasons why management strategies tend to fail (Rollins et al.,
2009).

The first country in the world to be colonized by the American bullfrog
was Japan. The first importation consisted of only a few breeding pairs
from New Orleans, under the initiative of the biologist Shozaburo Watase
in 1918 (Maeda \& Matsui, 1999; Yanai, 2003; Hirai, 2004). New events
occurred in the 1920s (Minowa et al., 2008), 1952 (Goris, 1967; Ota,
2002; Kraus, 2009), 1953, 1954 and late 1950s (Ota, 1983; Kraus, 2009)
from undocumented sources.

Japan exported bullfrogs to Taiwan in 1924 and 1951 (Kraus, 2009;
Santos-Barrera et al., 2009). During the same period, Japan also
exported bullfrogs to Korea, first in 1959 when all individuals died,
and once again in the 1970's when farming took hold, allegedly
supplemented by bullfrogs originating from the USA (Groffen et al.,
2019). Until recently, Taiwan province was one of the greatest bullfrog
producers and exporters of the world, being the source of many
introductions in Southeast Asia. Some of these introductions happened in
Singapore (from the 1980s to present) (Ng \& Lim, 2010); Indonesia
(1984, Indo Prima Bullfrogs' owner, personal communication); and
Malaysia (Hardouin, 1997), which was one of the sources for the
Cambodian population (Neang, 2010).

China reconnected with Japan in the late 1950s, after getting isolated
by the split with the USSR due to political disagreements. China
received bullfrogs from Japan, but these animals died without producing
offspring (Li \& Xie, 2002; Bai et al., 2012; Liu \& Li, 2009; Xuan et
al., 2010). Political links at that period resulted in Cuba exporting
bullfrogs to China during this period (Li \& Xie, 2002; Bai et al.,
2012; Liu \& Li, 2009; Xuan et al., 2010).

Cuba was one of the first countries to receive bullfrog specimens. The
first known introduction happened in 1915-1916 (Kraus, 2009;
Santos-barrera et al., 2009), with other two events of introduction in
1927 and 1946. All animals were exported from the USA (Borroto-Páez et
al., 2015) and adapted well to the island.

In South America, bullfrogs have been introduced into 10 countries
(Akmentins \& Cardozo, 2009; Kraus, 2009; Urbina-Cardona \& Nori, 2011),
although no evidence of persistent populations exists in Chile and
Paraguay (F. Bauer, personal communication). Brazil was the first
country to be invaded, with two legal importations reported. The first
happened in 1935, while Brazil was facing a turbulent political period
known as the ``1930s revolution''. There was a pressure from some
segments of society for the diversification of the economy (Fonseca,
2012). The government started a breeding program with 300 couples
introduced by a Canadian technician named Tom Cyrril Harrison (Ferreira
et al., 2002). Apart from speculation about the Canadian origin of these
animals, Rueda-Almonacid (1999) reported the United States as the native
source population, although he did not state the basis for that
assertion. As noted by other authors ((Measey et al., 2017), the study
by Rueda-Almonacid (1999) contains mistakes; thus, we prefer to
disregard it

The second importation to Brazil happened in mid-1980s (Kraus, 2009)
with 20 pairs from Michigan (Schloegel et al. 2010). This population is
widespread in the country, especially in the state of Minas Gerais
(Jorgewich-Cohen et al., in press). Later, all other South American
countries received founders exclusively from Brazil (G. Laufer, ump.
data; Gallardo, 2004; Kraus, 2009; Pereyra et al., 2006) during the
1980s and 1990s, as part of a general effort to improve the economy
after the ``lost decade'' of the 1980s (Ghirardi et al., 2011; Laufer et
al., 2008).

Using population genetics data from within the native range (Austin et
al., 2004), some efforts were made to determine the source of alien
populations and the genetic structure in Europe (Ficetola et al., 2008),
western USA (Funk et al., 2011; Kamath et al., 2016), China (Bai et al.,
2012), and Brazil (Prim et al., 2003; Jorgewich-Cohen et al., in press),
but none of these studies aimed to reconstruct global pathways of
introduction. In order to reveal the paths of invasion of the main
bullfrog alien populations around the globe, combining our findings with
information from previous works and historical data, we focused on the
following questions: How many different lineages are there among alien
clusters around the world? Through what paths did these lineages get to
different countries? What is the native source of these introduced
populations?

\textbf{Materials and Methods}

We sampled a total of 416 bullfrogs (\emph{Lithobates catesbeianus} )
from 11 different countries between June 2013 and December 2017
(Supporting Information 1). Samples were obtained from feral specimens,
museums and private collections; and include samples from both
populations in Brazil collected by Jorgewich-Cohen et al. (in press).
Tissues from liver, muscle or skin were removed and stored in 90\%
ethanol at -20°C. DNA extraction was performed with the DNeasy Blood and
Tissue extraction kit (Quiagen, Valencia, CA, USA), following the
manufacture's guidelines.

Seven microsatellite loci were amplified following Austin et al. (2003),
with PCR conducted on a Veriti\selectlanguage{english} thermal cycler (Applied Biosystems)
using the following thermal profile: 95degC for 7 min followed by 10
cycles of 95degC for 30 s, touchdown from 62--57degC for 45 s, and 72degC for
30 s, followed by 30 cycles of 95degC for 30 s, 50degC for 30 s, and 72degC
for 30s, and final extension at 72degC for 7 min. The reaction mix, with a
total volume of 10 \selectlanguage{greek}µ\selectlanguage{english}l, contained 1.0 \selectlanguage{greek}µ\selectlanguage{english}l of buffer, 0.5 \selectlanguage{greek}µ\selectlanguage{english}l 2 mM dNTPs,
0.5 \selectlanguage{greek}µ\selectlanguage{english}l fluorescent dye (VIC for RcatJ11 and RcatJ44b; NED for RcatJ21
and RcatJ41; PET for RcatJ54 and Rcat3-2b or 6-FAM for RcatJ8; applied
biosystems), 0.5 \selectlanguage{greek}µ\selectlanguage{english}l of mixed forward and reverse primers (5 \selectlanguage{greek}µ\selectlanguage{english}M), 0.125
\selectlanguage{greek}µ\selectlanguage{english}l Taq polymerase, 3.375 \selectlanguage{greek}µ\selectlanguage{english}l distilled deionized water, and 3.0 \selectlanguage{greek}µ\selectlanguage{english}l
template DNA. We diluted the PCR products to a proportion of 1:4 and
submitted them to sequencing by a third party.

We used Gene Marker ver. 2.6.3 (SoftGenetics) to score results and
Microchecker (Van Oosterhout et al., 2004) to test for null alleles,
allele dropout, and stuttering. Around 22\% (91/416) of the samples were
genotyped again in order to evaluate the percentage of homozygotes that
were actually null alleles. Control samples were included in every
procedure.

To assess current genetic structure, we performed a discriminant
analysis of principal components (DAPC; Jombart et al., 2010) using the
adegenet package (Jombart, 2008) on platform R (R Core Team, 2017).
Samples were grouped by country of origin in cases where we could not
sample in more than one site or either when we could not get enough
samples to run the analysis without facing bias by using too different
sample sizes between locations. Samples from Brazil were divided based
on the known genetic population structure in the country
(Jorgewich-Cohen et al., in press).

In order to quantify the pairwise genetic differentiation between
populations observed on the DAPC, we used Jost's D index (Jost, 2008).
Next, we tested the significance of the differentiation between pairs of
populations with 10000 permutations. We subjected the results to a
Benjamini and Hochberg procedure (Hochberg \& Benjamini, 1990) in order
to control false discovery rates (FDR) and avoid type I error (Hauser et
al., 2019). These analyzes were conducted using the DEMEtics R package
(Jueterbock et al., 2010). To calculate the diversity indices: expected
(He) and observed (Ho) heterozygosity, number of alleles (A) per locus,
effective number of alleles (Ae) per locus, and size corrected Wright's
inbreed coefficient (Fis), we used the gstudio package (Dyer, 2014),
also on platform R. We checked for Hardy-Weinberg equilibrium (HWE) and
linkage disequilibrium (LD) deviations through a permutation procedure
with 100 batches of 1000 iterations, by performing a probability test in
Genepop ver. 3.4 (Rousset, 2008). P values were corrected with the
Benjamini and Hochberg procedure.

Samples that showed graphic overlap on the DAPC were considered the same
genetic population for further analysis. Based on those results, we
selected the most variable samples from each genetic population
(Supporting Information 2) and amplified a 1047bp segment of the
mitochondrial cytochrome b gene (cytb) that includes the fragment used
by Austin et al. (2004). We used a combination of the primers MVZ15L
(Moritz et al., 1992) and cyt-bAR-H (Goebel et al., 1999) and a thermal
profile for polymerase chain reaction that consisted of 95\selectlanguage{ngerman}°C for 10 min,
followed by 45 cycles at 95°C for 30 s, 50°C for 40 s, and 72°C for 40
s, with a final extension step at 72°C for 5min. The reaction mix, with
a total volume of 25 \selectlanguage{greek}µ\selectlanguage{english}l, contained 0.15 \selectlanguage{greek}µ\selectlanguage{english}l Go Taq G2 Flexi DNA
Polymerase (Promega corporation), 2.5 \selectlanguage{greek}µ\selectlanguage{english}l of Go Taq flexi Buffer, 1.0 \selectlanguage{greek}µ\selectlanguage{english}l
2 mM dNTPs, 2.0 \selectlanguage{greek}µ\selectlanguage{english}l 25 mM MgCl2, 1.0 \selectlanguage{greek}µ\selectlanguage{english}l of each primer (10 pM), 15.35 \selectlanguage{greek}µ\selectlanguage{english}l
distilled deionized water, and 2.0 \selectlanguage{greek}µ\selectlanguage{english}l template DNA. PCR amplification
products were cleaned using Agencourt AMPure XP DNA Purification and
Cleanup kit (Beckman Coulter Genomics, Brea, CA, USA), and they were
sequenced by a third party using fluorescent-dye labelled terminators
(ABI Prism Big Dye Terminators v. 1.1 cycle sequencing kits; Applied
Biosystems, Foster City, CA, USA) with an ABI 3730XL (Applied
Biosystems, Foster City, CA, USA).

We used Arlequin 3.5 (Excoffier \& Lischer, 2010) for most genetic
analysis. Two diversity indices were calculated: Hd and \selectlanguage{greek}π, \selectlanguage{english}described by
Austin et al. (2004) respectively as ``the relative frequencies of
haplotypes in a population, without consideration of their
relationships'' and the ``weighted mean of pairwise divergence among
haplotypes''. We also calculated the pairwise differences between the
introduced populations using \selectlanguage{greek}θ\selectlanguage{english}ST index and evaluated its significance by
performing 10,100 permutations. P values were corrected under the
Benjamini and Hochberg procedure (Hochberg \& Benjamini, 1990).
Significant differentiation between populations were interpreted as
cases of different native populations descendants, as it has been done
before (Funk et al., 2011; Kamath et al., 2016). We used analysis of
molecular variance (AMOVA) to assess the native origin of introduced
populations. Information on native range is available at Austin et al.
(2004), where four groups were delimited through a nested clade
analysis. To perform AMOVA, we gathered populations that showed
non-significant differentiation on \selectlanguage{greek}θ\selectlanguage{english}ST analysis and compared them with
each of four source populations. We also performed a significance test
with 10,000 permutations. The smallest value found between the four
AMOVAs was considered the indication of origin for the tested population
(Bai et al., 2012; Ficetola et al., 2008; Funk et al., 2011; Kamath et
al., 2016).

We used Geneious ver. 10.2.3 (Kearse et al., 2012) for sequence edition
and contig formation of the cytb sequences based on the chromatograms
obtained from the automated sequencer. All samples were sequenced in
both directions to check for potential errors. The sequences were
aligned with the MAFFT (Katoh \& Standley, 2013) plugin in Geneious
10.2.3 (Biomatters) with the G-INS-I strategy. We trimmed the sequence
alignment to a length of 937bp.

For better understanding haplotype relationship, we performed a
phylogenetic analysis under a maximum-likelihood (ML) based approach in
RAxML ver. 8 (Stamatakis, 2014) with 100 runs, and ML branch support was
evaluated by 10,000 bootstrap replicates, following the same
phylogenetic method conducted by Kamath et al. (2016). We used unique
haplotypes from our data and added haplotypes from Austin et al. (2003,
2004), Ficetola et al. (2008), Bai et al. (2012), and Kamath et al.
(2016) to compare the relationships among haplotypes from introduced and
native populations, and between those haplotypes that have not been
sampled in the native range. Tree rooting was based on the outgroup
method (Farris, 1982; Nixon \& Carpenter, 1993), and outgroup selection
was based on a previous phylogenetic analysis (Pyron, 2014). We
used\emph{Lithobates septentrionalis} to root the tree and \emph{L.
clamitans} , \emph{L. okaloosae} , and \emph{L. heckscheri} as
additional outgroup species (respective GenBank accession numbers:
AY083273, AY083281, AY083286, AY083299).

\textbf{Results}

\emph{Microsatellite analyses}

Allele dropout and stuttering measures were not significant and the
presence of null alleles was indicated for all loci. Genotyping error
was estimated to be 1.55\%. Most pairwise comparisons indicated
significant deviation from Hardy-Weinberg equilibrium (Supporting
Information 3) and linkage disequilibrium (Supporting Information 4)
after FDR corrections.

The DAPC (Fig. 1) presented clear structure composed by four populations
(BRA+BP2+COL+ARG+URU; SIN+CUB; CHI+KOR+BEL; JAP). There is almost
complete overlap among all South American countries, hereafter referred
as Population 1 (Pop1), including the two populations described in
Brazil (Jorgewich-Cohen et al., in press). Population 1 has a marginal
link with Cuba, which is almost completely overlapped with samples from
Singapore, together forming Population 2 (Pop2). Samples from the
Republic of Korea, Belgium and China are also greatly overlapped and
were considered as one unit (Pop3). Japan is completely isolated and was
considered as a separated population (Pop4). Calculation of pairwise
Jost's D index values (Table 1) revealed greater differentiation between
Pop3 and all others. The smallest differentiation reveled is between
Pop2 and Pop4, followed by the differentiation between Pop1 and Pop2.
The differentiation between pairs of populations was always significant
(p=0.001). With exception for Pop2, the observed heterozygosity (Ho) did
not match expected heterozygosity (He) and both populations showed
positive inbreeding values (Supporting Information 3).

\emph{Cytochrome b analyses}

Mitochondrial sequences provided information about the haplotypes
present in each area and, together with \selectlanguage{greek}θ\selectlanguage{english}ST differentiation index, we
were able to indicate which areas have the same source population. We
identified six haplotypes among all sampled populations (GenBank
accession number: XXXXX). Three of them have been sampled on native
range by Austin et al. (2004), two by Kamath et al. (2016) and one by
Bai et al. (2012) on invasive areas of the USA and China. Another three
haplotypes were sampled for the first time in the present work. The
genetic diversity for each site is available on Table 2.

The population differentiation test was significant following the
Benjamini and Hochberg procedure for all comparisons except Minas Gerais
(MG)--Cuba, Brazil--Ecuador, and Singapore--Japan--China (Table 3).
These results suggest that each of these groups have the same native
origin and, for this reason, they were pooled together for the AMOVA.
Belgium and Singapore were also nearly significant (0.05307), but were
not pooled together as Belgium differentiates from the other Asian
countries. AMOVA results showed similar covariance results between
native range areas (Cuba + MG: 18.41 -- 20.18; Asian countries: 24.05 --
31.01; Brazil + Ecuador: 17.68 -- 26.26; Belgium: 0.00 -- 7.87. (Table
4)).

Our phylogenetic analysis recovered topologies congruent with those
reported by Kamath et al. (2016), with USA native west and east clades
as monophyletic groups. Our haplotypes H02, H03, H04 and H06 are
clustered in the western lineage; whereas haplotypes H01 and H05 are
clustered in the eastern lineage.

\textbf{Discussion}

Several invasive species are known to have populations with low genetic
diversity (Bai et al., 2012; Rollins et al., 2009; Santos et al., 2013).
Besides cases where individuals from multiple native sources are put
together (Kolbe et al., 2004), low genetic diversity and increased rates
of inbreeding are expected due to sequential bottleneck effect (Estoup
et al., 2011). Countries that have been colonized by bullfrogs not
always received them directly from native sources, resulting in
populations with low genetic diversity, which facilitates tracking their
introduction pathway by genetic similarity. The association between
genetic data and information from specific literature along with
historical events can be helpful on understanding introduction pathways.

In our analysis, microsatellite and mitochondrial markers have shown
partially consistent results. There is a consensus between markers that
most areas of South America belong to the same genetic population. Two
different haplotypes were found in this continent (Fig. 2). One of them
(H01) is represented by 75\% of the samples from Brazil and Ecuador. The
other 25\% is represented by H02, which is the same haplotype found
among samples from Minas Gerais and Cuba on the cytb analysis. This
result is consistent with the DAPC, in which Cuba has only a marginal
link with the South American cluster. It also suggests that Ecuador
received bullfrogs from Brazil after the secondary introduction from
Michigan that might have happened before 1985.

Although no information about the native origin of the founders of Cuban
population is available {[}besides brief and weak conjectures based on
morphotype variation by Hoffman \& Noble (1927){]}, it is expected that
multiple events of introduction would increase genetic variation. Only
one haplotype was found in Cuba, suggesting a single source of origin or
a great loss of variability due to genetic drift since the last
introduction event in 1946. In any case, it is reasonable to consider
that samples from Cuba and Minas Gerais share their native source, as
shown in the \selectlanguage{greek}θ\selectlanguage{english}ST differentiation analysis. Both regions likely received
animals from Michigan, since there are no records of a secondary
introduction between Cuba and Brazil.

Given the records of close ties between Cuban and Chinese bullfrog
lineages in the literature, one might expect them to exhibit same
haplotypes, which would differ from those in Japan, since the only
documented introduction from Japan to China failed. However, two
haplotypes can be found in China. One of them is the same from Cuba,
while the other is found in all sampled Asian countries.

Literature indicates Japan as the source population to all countries of
Asia, besides China, and genetic data partially corroborates this
information. The two haplotypes found in Japan are very similar to one
another, with only one polymorphic locus, being recovered as sister
groups in our phylogenetic analysis. This could either mean that they
came from close areas of the native range or a single haplotype suffered
a mutation after introduced to Japan. One could be understandably biased
in favor of the second hypothesis, due to the lack of one of these
haplotypes in the rest of Asia, meaning the mutation happened after
Japan exported specimens to other countries. Unfortunately this is a
tricky question with many possible answers, such as: 1. Japan received
this haplotype from native source after exported animals to Taiwan; 2.
Japan received this haplotype before exporting animals, but it was not
sampled among the exported specimens; 3. Other countries received this
haplotype from Japan, but it was lost due to genetic drift; 4. We did
not sample this haplotype in other countries by casualty. Sampling in
DPR Korea Ri (2018) may be a step towards answering these questions as
these specimens have been introduced from Japan possibly before 1950,
and thus before a potential mutation was exported to other countries.

In any case, the haplotype H03 is widespread in Southeast Asia,
including China. Several different situations are likely to have
happened to cause this genetic homogeneity in Asia. The most
parsimonious hypothesis is that other translocations happened from Japan
to China. There are few reports that it may have happened at different
periods. Although no strong evidence is available, it is likely to have
happened during the Japanese invasion of China at the second
Sino-Japanese war in between 1937 and 1945, and another in 1959 with
three other events of exportation from Japan to China (Yang Yi, ump.
data).

Another possibility is that H03 was also present in Cuba, being
introduced from the same native source as the Japanese population at one
of the many events of introduction. It may have colonized China, later
disappearing from Cuba. This hypothesis might seem less probable, but
the low number of divergent sites between these haplotypes and the
shared positioning on the Western clade of the phylogenetic three
supports a kinship hypothesis. The native source population of bullfrogs
introduced in Asian countries is probably close to the native source of
the Cuban population.

Inferring the native source of invasive populations is a hard task,
especially when haplotypes found in alien populations were not sampled
in the native range, as observed in the present study. AMOVA results
were quite inconclusive. Finer sampling of the native area with more
loci sequencing is needed to elucidate the native population structure
(Bai et al., 2012; Kamath et al., 2016). This way, it will be possible
to better understand the origin of introduced populations. However, it
is worth mentioning that our results contradict prior literature. The
haplotype found by Bai et al. (2012) in China is the same we found in
Asia (H03) but AMOVA indicated west USA as the native source population
in their analysis, while our analysis indicated the overlap zone. We
found two new haplotypes in Asia that were not sample in their work.
Ficetola et al. (2008) indicates the East of USA as the native source of
Belgium and Greek populations, while our results indicate the West of
USA as the native source of Belgium population, after adding the new
discovered haplotype.

On the other hand, we concluded that USA's west zone is the native
source of the population from Cuba and Minas Gerais, corroborating the
results found by Kamath et al. (2016), as H02 is the same haplotype
found by them in Grand Teton National Park (GenBank Accession number
KX344492). This result adds questions about the origin of the haplotype
H02, as literature indicates that founders of the population from Minas
Gerais were imported from Michigan, located in the overlap zone.

Based on these analyzes, it is possible to conclude that Cuban and
Southeast Asian haplotypes share native distribution, which is apart
from South American's haplotype in Eastern lineage. However, the native
origin of these haplotypes cannot be designated. Nevertheless, the
information gathered from the literature (Supporting Information 5) and
from genetic data in the present work helps to shed light in the history
of introduction of the American bullfrog (Fig. 3), being helpful for
future efforts of control that might succeed by regulating sources of
introduction that keep nurturing invasive populations.

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\textbf{Tables}

Tables are in a separated file.

\textbf{Figure legends}

Figure 1. Discriminant analysis of principal components of genetic
variation among microsatellite samples of Lithobates catesbeianus
invasive populations.

Figure 2. Parsimony consensus tree with haplotypes from native range
(Austin et al., 2004), Europe (Ficetola et al., 2008), China (Bai et
al., 2012), non-native areas of USA (Kamath et al., 2016), and samples
from the present work. Clades East and West refer to distribution in the
native range (Austin et al., 2004). Map contains the haplotypes'
distribution around the world. Color legend follows both map and
phylogeny. Terminal labels with GenBank accession number starting with
AY correspond to haplotypes found in the native distribution (Austin et
al., 2004) and labels starting with KX correspond to haplotypes found in
the invasive distribution in the USA (Kamath et al., 2016).

Figure 3. Bullfrog invasion pathways diagram based on literature
information. Arrows and dates indicate the direction and date of each
event of invasion, question marks indicate unknown source of
introduction, and crosses indicates supposedly non-successful
establishment of the introduced animals. The green arrow indicates
possible successful genetic flow in disagreement with previous
literature.

\textbf{Data Accessibility Statement}

DNA sequences: Genbank accessions XXXXXX-XXXXXX; XXXX XXX: XXXXXXXXXX

\textbf{Competing Interests Statement}

\textbf{Author Contribution}

Gabriel Jorgewich-Cohen: Conceptualization (equal); data curation
(lead); formal Analysis (equal); investigation (equal); methodology
(equal); project administration (equal); writing -- original draft
(lead); validation (lead); visualization (equal). Rachel Montesinos:
Formal Analysis (equal); investigation (equal); methodology
(supporting). Rafael dos Santos Henrique: Visualization (equal); writing
-- review \& editing (lead). Luis Felipe Toledo: Data curation
(supporting); resources (supporting). Amael Borzee: Data curation
(supporting); resources (supporting). Yang Yi: Data curation
(supporting); resources (supporting). Wang Fei: Data curation
(supporting); investigation (supporting). Takeshi Igawa: Data curation
(supporting); resources (supporting). Juan Manuel Guayasamin: Data
curation (supporting); resources (supporting). Gabriel Laufer: Data
curation (supporting); resources (supporting). Wilmar Bolivar: Data
curation (supporting); resources (supporting). Ariel Rodriguez: Data
curation (supporting); resources (supporting). Taran Grant:
Conceptualization (equal); funding acquisition (lead); methodology
(equal); project administration (equal); resources (lead); supervision
(lead); writing -- review \& editing (equal).

\textbf{Acknowledgements}

We thank C. A. Agostinho, C. F. B. Haddad, M. T. Rodrigues, R. Lingnau,
R. B. Paradero, E. Grou, V. Silva, R. Maneyro, J. Faivovich, G. Louette,
S. Devisscher, A. Martel and K. P. Lim for sample donations; and M.
Targino and C. Rossi for laboratory and analysis assistance. This
research was supported by the Sa\selectlanguage{english}o Paulo Research Foundation (FAPESP
\#2012/10000-5, \#2016/25358-3, \#2018/15425-0, \#2019/18335-5) and
Conselho Nacional de Desenvolvimento Cient\selectlanguage{ngerman}ífico e Tecnológico (CNPq
\#134290/2015-1, \#300896/2016-6, \#306823/2017-9).\selectlanguage{english}
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