Discussion
Analysing the multifarious factors potentially generating population
differentiation across six related Neotropical plant species differing
in pollination strategy, we here detected consistently stronger
isolating effects (particularly IBD) across localities of bee- than
vertebrate-pollinated species (Table 1, Fig. 4). The two bee-pollinated
species were the only ones where current climatic conditions contributed
substantially to explaining population differentiation. These results
are in line with the expectation that more mobile pollinators (i.e.
flying vertebrates) may connect populations more effectively than less
mobile (i.e. (small) bee) pollinators (Wessinger 2021). Even though
large bees (pollinators of M. maxima ) are generally considered as
relatively mobile (Gamba & Muchhala 2020), their flight activity is
strongly reduced under adverse weather conditions in tropical mountains,
likely limiting (large-distance) pollen dispersal (Dellinger et al.
2021). Within localities, we also detected IBD more frequently in bee-
than vertebrate-pollinated localities (Table S14). Against expectations,
however, we did not find consistent differences in nucleotide diversity,
heterozygosity or disparity according to pollination strategy (Fig. 2).
This suggests that, at least across small spatial scales (i.e. within
localities, between adjacent localities), bees may be equally effective
outcross pollinators as vertebrates
(Opedal et al. 2017,
Schmidt-Lebuhn et al. 2019). In the following, we discuss our findings
in the context of other factors potentially influencing present-day
population genetic structure such as mountain topography, habitat
suitability and Pleistocene climatic fluctuations.
Our study species share the same macroevolutionary background (same
tribe), but differ in distribution ranges and ecosystems colonized.
Bee-pollinated Ad. adscendens has a wide distribution in lowland
rainforests in tropical Latin America, while the other five species
inhabit montane cloud forests with relatively continuous (M.
phlomoides , M. tomentosa ) or patchy (A. costaricensis ,M. maxima , M. sanguinea ) distributions (Fig. S1). We may,
hence, expect partly idiosyncratic responses to isolating barriers and
(current and past) climatic habitat suitability. Following the
expectation that mountain terrain generates strong physical barriers, we
did indeed detect significant IBRTerrain in all species
but A. costaricensis (Table 1). It was, however, never recovered
as factor best explaining population differentiation (Tables 2). While
rugged mountain terrain may act as effective barrier even across small
spatial scales (i.e. populations three and six in M. phlomoides ,
Fig. 1), admixture was overall high among close (< 12km)
localities in all species, suggesting considerable connectivity by both
insect and vertebrate pollinators among localities (Fig. 3).
Ectothermic insect pollinators may be affected more strongly by harsh
abiotic climatic conditions (i.e. low temperatures, high precipitation,
strong winds) than vertebrate pollinators (Cruden 1972, Dellinger et al.
2021). Accordingly, we found evidence of isolating effects related to
current climatic conditions (Table 2, Fig. 4) only in the two
bee-pollinated species (IBE in Ad. adscendens and
IBRHabitat in M. maxima ). In Ad.
adscendens, the marked separation into two clusters (Fig. 1A, 3A)
clearly reflects the combination of IBD, isolating effects related to
mountain topography, and current climatic conditions. The south-western
localities are cut off from the north-eastern localities by the central
Costa Rican mountain range. These mountains feature moist and cool cloud
forests, which are generally unhostile to smaller bees, the pollinators
of Ad. adscendens (Dellinger et al. 2019). Along the western
coast, occasional dry habitats
(i.e. Nicoya peninsula), on the
other hand, represent unsuitable habitats for the moisture-adapted
plants (Pröhl et al. 2010). In accordance with this, our niche models
estimated the ‘least-cost’ path connecting the southern and northern
localities of Ad. adscendens through the south-eastern lowlands
along the Caribbean side of the high Central American mountains (Fig.
1A, Patten & Smith-Patten 2008). In M. maxima , the marked
differentiation among localities 3 and 4 may further indicate
disproportionately strong effects of climatic conditions on ectothermic
bee-pollinator activity. These two localities were significantly
differentiated from each other genetically, albeit only 20 km apart. Our
habitat-suitability models indicated that locality 4 lies in a
climatically less suitable area (Fig. 1). Our own experimental work in
montane Meriania species in the Andes has established a strong
link between bee pollinator activity and current weather conditions,
with high bee activity under sunny conditions, but almost no flower
visitation under cool and rainy conditions (Dellinger et al. 2021). It
is hence possible that the difference in habitat suitability strongly
reduced the probability of bee flight among these localities, hence
generating strong population differentiation. Interestingly, we also
found individuals of M. maxima in locality 4 to differ
morphologically (i.e. smaller flowers, non-revolute leaf basis,
Dellinger, pers. obsv.). Whether these observed phenotypic differences
are the result of random genetic drift, or a response to different
selection pressures, remains to be investigated.
Among the vertebrate-pollinated species, we did not find isolating
effects caused by habitat suitability (IBRHabitat) or
environment (IBE; Fig. 1, Table 2). Indeed, cloud forests form a
relatively continuous ecosystem particularly on the eastern slopes of
the Andes and Central American mountains
(Balslev 1988, Luebert & Weigend
2014), and possibly provide continuously suitable habitats for the
cold-adapted vertebrate pollinators. The marked population
differentiation observed between Northern and Southern Ecuadorian
populations of M. sanguinea and M. tomentosa , on the other
hand, follows the well-known biogeographic barrier of the dry
Girón-Paute valley (Jørgensen &
Ulloa Ulloa 1994, Escobar et al. 2020). This demarcation line, part of
the Amotape-Huancabamba zone, has
acted both as dispersal barrier for montane species as well as a
corridor for lowland species
(Weigend 2002, Trénel et al.
2008).
Understanding how tropical plants reacted to Pleistocene climatic
fluctuations, i.e. whether they retracted into refugia (‘dry-refugia’
hypothesis) or underwent down- and upslope migrations (‘moist-forest’
hypothesis), remains a major conundrum (Ramírez-Barahona & Eguiarte
2013). If species retracted into small refugia, climatic instability
through time should explain population genetic variation
(Helmstetter et al. 2020). In our
study, we found significant associations between genetic differentiation
and climatic instability in five species (Table 1), but IBI was
recovered as factor best explaining variation inFST only in M. tomentosa . Indeed,
modelling past climatic habitat suitability indicated that the cloud
forest species A. costaricensis, M. maxima, M. phlomoides andM. tomentosa retained relatively continuous suitable habitats
along the Central and Northern South American mountain ranges throughout
the Pleistocene (Fig. S3). These results support the ‘moist-forest’
hypothesis, with downslope and subsequent upslope migration during
Pleistocene climatic fluctuations (Fig. S14), and even range expansion
in M. phlomoides (Fig. S3c). Further, at the scale of our study
localities, there was some evidence of habitat contraction during the
LGM in A. costaricensis (continuously suitable around locality 4,
Fig. S6) and M. maxima (continuously suitable around localities
1, 2 and 5, Fig. S7). This suggests the possibility for in-situ
persistence of these cloud-forest species in part of the distribution
range (without necessarily contraction into isolated refugia), a pattern
documented for other montane Neotropical plant lineages (Ornelas et al.
2019). M. sanguinea , on the other hand, is the only species in
our sample occurring in the high-elevation cloud-forest-Páramo-ecotone.
This species may have undergone more prominent refugial retraction in
Southern Ecuador and Peru (Fig. S3, S14). Indeed, our models indicate
little connectivity among the southern Ecuadorian localities (south of
the biogeographic barrier of the Amotape-Huancabamba zone) and markedly
differentiated Northern Ecuadorian locality during the LGM (Fig. S7).
Finally, in lowland bee-pollinated Ad. adscendens , suitable
habitats were likely extensive in lowland Amazonia during the LGM, with
mostly continuously suitable habitats along the (eastern) coast of
Central America (Fig. S6, Pröhl et al. 2010).
Taken together, our results are highly valuable in adding to the limited
data available on the diverse processes shaping population genetic
differentiation of tropical plants, including differences in pollination
strategy. While we highlight that a wider sampling across bee-pollinated
Merianieae is required to firmly nail the role of pollinators in
promoting population differentiation, our result on stronger isolation
among bee- than vertebrate-pollinated populations suggests a critical
role of pollinator mobility in shaping population-level processes.
Extrapolating to a macroevolutionary perspective, pollinator shifts are
often invoked as “key innovations” spurring diversification (van der
Niet et al. 2014). Potentially, pollinator shifts may also alter a
population’s susceptibility to isolation and, consequently, its
potential for allopatric divergence. Interestingly, in various
Neotropical/Andean plant groups, shifts from bee to vertebrate
(particularly hummingbird) pollination go hand in hand with increases in
diversification rates (e.g.
Serrano-Serrano et al. 2017,
Lagomarsino et al. 2017). This is somewhat counter intuitive, however,
if vertebrates are expected to buffer isolating effects among
populations (see Serrano-Serrano et al. 2017 for discussion on
additional factors). Clearly, comparative studies, ideally focusing on
small monophyletic plant complexes, and documenting both pollination
ecology and population genetics of multiple populations across the
landscape (e.g. Opedal et al. 2017), will be essential for resolving the
relative contribution of pollinator shifts in spurring or limiting
speciation through gene flow (Abrahamczyk et al. 2014). To date, we
largely lack pollinator observations from multiple populations of the
same plant species in the (Neo)tropics, and hence know little about the
variability of pollinator composition across a species’ distribution
range (pollinator mosaic, Gowda &
Kress 2013). Within our own dataset, we have such information for a
subset of all populations only (Table S1). While we documented the same
functional groups acting as pollinators in most populations, we
documented effective hummingbird and rodent pollinators in Southern
Ecuadorian populations of M. sanguinea , and hummingbird and bat
pollinators in Northern Ecuador. Obtaining such natural history
information, in addition to population genomic data, will be key for
adding a more realistic understanding to the processes governing
speciation and hence contributing to the exceptional diversity observed
in the (Neo)tropics today.