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 θST 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.