Discussion
Our analysis of more than 20 million SNPs shows that eastern and western
North American monarchs have extremely low genome-wide genetic
differentiation. We did not detect any fixed nucleotide differences
between eastern and western monarchs, and even the smallest window
(100bp) size analyses indicated low maximum FSTvalues of 0.06, indicating a lack of genomic islands of differentiation.
The windows with maximum genetic differentiation between eastern and
western monarchs were low compared to the genome-wide average genetic
differentiation between subpopulations in other butterflye species
(Nadeau et al. , 2013;
Talla et al. , 2019;
Martin et al. , 2020). We found an
almost perfect genome-wide correlation between nucleotide diversity in
eastern and western monarchs, and genome-wide phylogenetic analyses
indicated no clustering of eastern and western monarchs. Both ∂a∂i and
Tajima’s D results suggest that eastern and western monarchs have a
similar effective population size. Importantly, both methods contrast
with population census data, which show much smaller numbers of western
than eastern monarchs (Schultz, Brown,
Pelton & Crone, 2017; Malcolm, 2018;
Pelton, Schultz, Jepsen, Black & Crone,
2019), and support the notion of frequent genetic exchange between
these monarchs.
Our results are in line with previous studies based on more limited
genetic markers, including allozymes
(Shephard et al. , 2002) and
microsatellites (Lyons et al. ,
2012). These findings also support observational, geographical, and
tagging studies that have suggested regular interchange between eastern
and western monarchs (Brower & Pyle,
2004; Dingle, Zalucki, Rochester &
Armijo-Prewitt, 2005; Morris, Kline &
Morris, 2015). Because the Rocky Mountains form a dispersal barrier for
monarchs, the high levels of interchange between eastern and western
monarchs indicated by our genomic analyses most likely occur during both
the spring migration – when north-flying monarchs from Mexico could end
up west of the Rocky Mountains (Brower &
Pyle, 2004) – and autumn migration, when monarchs from western North
America can end up migrating south to Mexico
(Morris et al. , 2015;
Billings, 2019). Indeed, population
genetic analyses of microsatellites are consistent with a radial
dispersal of monarchs from Mexico, including north-ward dispersal to
western North America (Pierce, Altizer,
Chamberlain, Kronforst & de Roode, 2015). Further tagging studies will
be necessary to map the migration routes of western monarchs
(Dingle et al. , 2005;
James et al. , 2018) and to
determine where actual genetic exchanges between eastern and western
monarchs are occurring. It is interesting to note that in our study,
three samples from Big Sur, California, appeared to cluster in both the
ADMIXTURE and SAGUARO analyses. This suggests potential genetic
sub-structuring in western North America, consistent with the non-random
migratory pathways of western overwintering monarchs inferred by tagging
and stable isotope studies (Naganoet al. , 1993; Yang et al. ,
2016).
As with many migratory species, monarch migration has a genetic basis,
and genome comparisons between migratory and non-migratory populations
have revealed strong evidence for the existence of migration-related
genes (Zhan et al. , 2014). While
migration per se is genetically determined and associated with
large-effect alleles, the lack of genomic divergence observed here
suggests that differences in migration routes, distances and
destinations for migratory monarchs are not. Our flight trials clearly
demonstrated differences in flight performance between eastern and
western monarchs, and these phenotypic differences may be driven by
either a large number of small-effect alleles or by differential gene
expression (Liedvogel et al. ,
2011) induced by environmental triggers in eastern and western North
America. To conclusively discern between these two alternatives, one
would ideally carry out genetic crosses between eastern and western
monarchs, and then release both parental genotypes and cross-progeny
offspring on both sides of the Rocky Mountains to study migratory
behavior, for example through the use of radio-tracking tagged monarchs
. However, current United States Department of Agriculture regulations
prohibit the transfer and release of monarchs across the Rocky Mountains
– partly based on the assumption that eastern and western monarchs are
genetically distinct populations – preventing such a definitive study
design.
However, some progress could be made by comparing the transcriptomes of
eastern and western monarchs throughout the year. Our preliminary gene
expression studies showed a trend for differential gene expression, and
one gene related to non-muscular motor activity was significantly
differentially expressed. While these results show that eastern and
western monarchs may differentially express migration-related genes
during active flight, it is likely that many other genes are
differentially expressed both during active flight, and in the
developmental stages leading up to migration. Previous studies have
shown different transcriptome profiles between breeding and migratory
monarchs in eastern North America, including differences in expression
of genes related to juvenile hormone production
(Zhu, Gegear, Casselman, Kanginakudru &
Reppert, 2009). Other studies have shown that non-migratory monarchs in
Australia, which evolved from migratory monarchs in North America
(Zhan et al. , 2014), have
retained the ability to enter reproductive diapause
(Freedman et al. , 2017), and that
exposing eastern North American monarchs to artificial light and
temperature conditions disrupts migration orientation behavior
(Tenger-Trolander, Lu, Noyes & Kronforst,
2019). Our study further suggests that environmental variation on the
east and west of the Rocky Mountains triggers monarchs to follow
different pathways to develop into eastern and western migrants. Such
factors may include the different species of milkweeds that monarchs use
in eastern and western North America
(Woodson, 1954;
Dilts et al. , 2019), as recent
work shows that milkweeds can significantly affect wing morphology
(Davis & de Roode, 2018;
Freedman & Dingle, 2018;
Decker, Soule, de Roode & Hunter, 2019).
A transcriptomics study examining differences during development and
flight between eastern and western monarchs would be important in
uncovering gene regulatory networks involved in migration ability, and
further shine light on how these highly similar genomes can give rise to
divergent migratory behavior.
Ultimately, determining the genetic or epigenetic basis of differential
migration in eastern and western monarchs will not only advance our
understanding of migration genetics, but may also have relevance for
conservation biology. The population size of eastern migrating monarchs
has dwindled over the last three decades
(Vidal & Rendón-Salinas, 2014;
Malcolm, 2018;
Boyle, Dalgleish & Puzey, 2019), with
some estimates indicating a decline over 80% from a high in 1996
(Semmens et al. , 2016). While
studies disagree on the primary cause, an emerging picture is that
monarch population decline is due to a combination of illegal logging at
the Mexican overwintering sites, climate change, agriculture-induced
loss of milkweed host plants in North America, and reduced availability
of nectar sources along the fall migration flyways
(Pleasants & Oberhauser, 2013;
Vidal, López-García & Rendón-Salinas,
2014; Inamine, Ellner, Springer &
Agrawal, 2016; Thogmartin et al. ,
2017; Boyle et al. , 2019;
Saunders et al. , 2019;
Wilcox, Flockhart, Newman & Norris,
2019). Western monarch population size has also declined
(Espeset et al. , 2016;
Schultz et al. , 2017), reaching
critically low levels in the 2018-2019 migrating season
(Pelton, 2018;
Pelton et al. , 2019). Monarch
migration has been coined an endangered phenomenon
(Brower et al. , 2012), and the
population decline has led a group of organizations and scientists to
petition the US Fish and Wildlife Service to protect monarchs under the
Endangered Species Act (Center for
Biological Diversity, Center for Food Safety, Xerces Society & Brower,
2014). Following recent advances in merging evolutionary biology with
conservation biology (Hendry et
al. , 2011; Lankau, Jørgensen, Harris &
Sih, 2011; Sgro, Lowe & Hoffmann, 2011;
Smith, Kinnison, Strauss, Fuller &
Carroll, 2014), a crucial aspect of this process is to determine the
adaptive capacity of monarch butterflies. This includes asking how much
adaptive genetic variation monarch populations harbor, and which
populations must be preserved to allow the species to adapt to changing
conditions and to preserve the processes that allow evolution to occur.
If future studies reveal that differential eastern and western migration
is driven by gene expression rather than by genetic differentiation,
then this would suggest that preservation of eastern monarchs could
potentially rescue western migration and vice versa . Future
studies, ideally involving reciprocal translocation experiments, will be
needed to address this important question.