Figure 5 Genetic barriers obtained from BARRIER analysis based
on cpDNA in the years of (a ) 2016 and (b ) 2022 ofPrimulina heterotricha populations. Delaunay triangulation (black
lines) and inferred barriers (red, blue, and purple lines) separating
the different original regions which show the geographic location of the
genetic barrier.
The Mantel test showed a significant positive correlation between
pairwise genetic distance and geographic distance for both nrDNA and
cpDNA for the two study periods (Figure S2). The results of TajimaʼsD test and Fuʼs F s test are presented in Table 2 with the
associated simulated P -values. The values for D andF s were positive for 2016 nrDNA sequences (D = 0.80474,P > 0.10; F s = 1.811), 2016 cpDNA sequences
(D = 1.38922, P > 0.10; F s = 12.439),
and 2022 cpDNA sequences (D = 2.2589, P < 0.05;F s = 11.3), but were negative for 2022 nrDNA sequences (D= -1.18345, P > 0.10; F s = -3.937) (Table 2).
These results indicate that our sequences (with the only exception of
2022 cpDNA) are in agreement with the null hypothesis of constant
population size and neutral evolution. The hierarchical mismatch
analysis showed the distributions of differences for all populations
(Figure S3), from which the hypothesis of demographic population
expansion can be rejected.
| Discussion4.1 | Very quick effects of anthropogenic barriers on
phylogeographical patternsThe evaluation of the effects of habitat fragmentation on animal and
plant species has been traditionally addressed by comparing gene flow,
genetic differentiation, and genetic structure of populations on
fragmented and non-fragmented habitats simultaneously (Chung et al.,
2014a; Gao et al., 2015; Schlaepfer et al., 2018). For example, Su et
al. (2003) compared the genetic differentiation of populations
separated by the Great Wall of China with those not subjected to this
huge physical separation for six plants, and found attributable
differences to the presence of the wall during 600 years. On a second
example, the levels of genetic divergence in two crayfishes
(Faxonius validus and Faxonius erichsonianus ) were
significantly higher in streams impounded for 36–104 years than in
non-impounded streams in Alabama, United States (Barnett et al.,
2020). Direct comparisons (i.e. comparing before and after the
fragmentation event the populations that have actually been subjected
to such disturbance) are probably not available in the literature as
it is assumed that the time elapsed from fragmentation should be very
long, as enough generations should have passed to observe genetic
changes. In their meta-analysis, Schlaepfer et al. (2018) found that
effects are generally only observable after 50 years, although some
exceptions could apply, as cases in which only 1–5 generations have
passed.
Herein we have measured the fragmentation effects of anthropogenic
construction by using the same populations of a plant (Primulina
heterotricha ) two times within six years (i.e. two generations of the
study plant would have passed). We have detected a significant
geographic structure in eight populations (Figures 2, 3), and AMOVA
shows a high level of genetic variation between regions (Table 4),
indicating significant genetic differentiation and limited gene flow
among populations and regions. Both the phylogenetic tree and the
STRUCTURE results indicated that there are three distinct genetic
lineages, corresponding to three clades, i.e., northwest clade (NW
region), southwest clade (SW region), and southeast clade (SE region)
(Figures 2, 3). As one may expect for a species inhabiting a rugged
mountain area (and where some rivers were dammed since the late
20th century), such a significant geographic
structure was already detected before the expressway construction
(2016). Although the effects are not still considerable (probably due
to the insufficient time elapsed), we have been able to detect some
changes in the genetic structure of P. heterotricha just a few
years after the two expressways were completed.
The three clades detected for P. heterotricha would likely
predate the formation of the anthropogenic barriers of the Daguangba
Reservoir and Expressways 1 and 2, as the range of this species, which
occurs in mountains at relatively high elevations, is naturally
fragmented by river valleys. As it can be observed in Fig. 1 (and
Figures 4, 5), Expressway 1 has been constructed along the Changhua
River (whose valley is, at some parts, up to 3 km wide) that crosses
Hainan’s south-central mountain system in a SW–NE direction, and then
the river turns into the NW, where it is dammed by the Daguangba
Reservoir. Expressway 2 has been
constructed taking advantage of the Tongshi River valley in spite of
being much narrower. However, as our results show, the anthropogenic
constructions would have intensified the isolation effects of the
Changhua River and its associated valley, which has been cultivated
since a long time ago (Xiao et al., 2012). In addition, conventional
roads were built in these river valleys much earlier; for example,
road G224, which was completed in 1954, runs nearly the same route as
the two expressways.
The changes in the significance of barriers before and after road
construction in the BARRIER analysis (Figures 4, 5) suggest that
geographic isolation caused by human constructions is key for
understanding the present phylogeographical patterns of P.
heterotricha. Most of the first barrier for both the nrDNA and cpDNA
for the year 2016 corresponds to the separation between QX and WZ
populations, which does not match with any of the anthropogenic
constructions. In contrast, for the year 2022 the first barrier
coincides with the dam and the expressways for both sequences. Another
undisputable signal showing the important role of the anthropogenic
barriers on the genetic structure of P. heterotricha is the
sharp changes in the genetic affinities of population XA, both for
nrDNA and cpDNA (Figure 3), which may be caused by the isolation of
Expressway 1 but specially Expressway 2. Although it is hard to
discern the relative contribution of valleys and rivers on one hand,
and expressways on the other hand, the above-described changes in the
generic partners from 2016 to 2022 suggest that the two expressways
might contribute to shaping the phylogeographical patterns of the
island-endemic P. heterotricha within six years, corresponding
to two generations of the plant. Geographical discontinuity including
anthropogenic disturbance is an important factor in population
differentiation by weakening or blocking gene flow in many plant
species (Slatkin, 1985; Su et al., 2003; Kartzinel et al., 2013; Chung
et al., 2014b). These effects are even greater for small herbaceous
plants like P. heterotricha , with a poor dispersal potential
associated with small seeds dispersed largely by water.4.2 | Effects of anthropogenic barriers on genetic
structure
Although the different clades of P. heterotricha keep stable
population dynamics, as revealed by mismatch distribution analysis
(Figure S3), the plant would have suffered some detectable effects on
its genetic structure, caused by the reduced exchange of genes. Notably,
the sharing of ribotypes among the three groups of populations separated
by the expressway network almost disappeared, and population genetic
differentiation based on nrDNA increased both at the population level
(F ST[2016]= 0.0045, F ST[2022] = 0.0054, P< 0.01) and at the region level (Table S3) from 2016 to 2022.
In contrast, F ST remained invariable along time
with cpDNA at the population level (0.0098 in 2016 vs. 0.0097 in 2022;
Table S2) and even increased at the group level
(F ST only decreased between NW and SE regions,
which could be attributable to the Expressway 1 construction; Table S4).
This incongruence between nrDNA and cpDNA could stem both from the
different modes of inheritance of these two markers (nrDNA is
biparentally inherited, cpDNA is only maternally inherited) and from the
fact that pollen migration rates are often much higher than seed
migration rates (Ennos, 1994; Petit et al., 2005). Thus, one could
expect that the maternally-inherited cpDNA (which is only transmitted by
seeds) would be much less sensitive to the fragmentation effects,
particularly if we take into account that the anthropogenic barriers
would have affected pollen flow in a much higher extent than seed flow
(see below).
Genetic differentiation was higher between populations separated by the
dam than populations separated by the expressways (Tables S3, S4), i.e.
the Daguangba Reservoir showed a more obvious and significant barrier
effect on gene flow. Two factors could explain such an observed pattern.
Firstly, the reservoir was built in 1994 and nearly a period of 30 years
of isolation would have accumulated much more barrier effects on plant
dispersal than the newly completed expressways, with a six-year period
of effects or two generations of P. heterotricha . Secondly, the
dam has a water reservoir of about 6 km long and a water surface of 100
km2. Such physical impediment probably brings much
more barrier effects than the expressways, which are normally only
20–30 m wide. For P. heterotricha , its seeds are small and may
be largely dispersed by raindrops or water courses and its pollination
needs small-sized insects such as Amegilla leptocoma and A.
yunnanensis (Ling et al., 2017a). Such dispersal mechanisms would have
been affected by the Daguangba Reservoir, as dams could restrict
hydrochory (Andersson et al., 2000). Effects, however, would be much
more pervasive regarding pollen dispersal, as the flying distances of
these small insects would be hardly enough to connect the two banks of
the reservoir (separated by up to 4 km).
Despite having less impact than the reservoir, the effects of
expressways are still notable forP. heterotricha . In
addition to the physical barrier posed by the two high-capacity roads,
the rapid growth of traffic (especially after the end of the COVID-19
pandemic) with much more noise and accumulation of pollutants will
increase the levels of disturbance. The fast-moving traffic and changes
in wind conditions associated with expressways probably affected
pollinator movements of P. heterotricha thus decreasing pollen
flow. Negative effects of roads on the movement (and even survival) of
pollinators have been often detected
(Stephens et al., 2000;
Bhattacharya et al., 2003;
Baxter-Gilbert
et al., 2015; Fitch & Vaidya, 2021; Dániel-Ferreira et al., 2022).
Although road construction might change hydrological conditions (by
affecting natural flow pathways and water quality, e.g. Buchanan et al,
2013), we have not been able to find examples in the literature of roads
affecting hydrochory.
In conclusion, we provide evidence of the increased barrier effects of
anthropogenic constructions such
as reservoirs and expressways on genetic structure and phylogeographical
patterns within just two generations of this plant. These disruptive
effects involving habitat fragmentation may pose primary threats to
population regeneration, genetic diversity, and change the evolutionary
processes of plants, especially in endemic, threatened short-lived
plants. To alleviate such
negative pressure, we suggest establishing ecological corridors to
enhance gene flow between populations separated by these anthropogenic
barriers. These could include road tunnels and overpass woodlands, which
enhance the dispersal of seeds, including the movement of insects and
reptiles (such as lizards and frogs). Such ecological corridors might
also increase the vegetation integrity and habitat continuity between
the two sides of these barriers and thus be helpful for the long-term
persistence of the island-endemic P. heterotricha and other rare
and endangered species.