Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 December 2020 | 12(17): 17263–17275
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
doi: https://doi.org/10.11609/jott.6532.12.17.17263-17275
#6532 | Received 08 August 2020 | Finally
accepted 08 December 2020
Genetic and reproductive
characterization of distylous Primula reinii in the Hakone volcano, Japan: implications for
conservation of the rare and endangered plant
Masaya Yamamoto 1, Honami Sugawara 2, Kazuhiro Fukushima 3,
Hiroaki Setoguchi 4 & Kaoruko Kurata 5
1 Hyogo University of Teacher
Education, 942-1 Shimokume, Kato-city, Hyogo
673-1494, Japan.
2,3,5 Graduate School of Education,
Yokohama National University, 79-2 Tokiwadai,
Hodogaya-ku, Yokohama, Kanagawa 240-8501, Japan.
4 Graduate School of Human and
Environmental Studies, Kyoto University, Yoshida Nihonmatsu,
Sakyo-ku, Kyoto 606-8501, Japan.
1 myamamo@hyogo-u.ac.jp
(corresponding author), 2 xxhonami.s@gmail.com, 3 kazuhiro-kazuhiro-73@docomo.ne.jp,
4 setoguchi.hiroaki.2c@kyoto-u.ac.jp, 5 kaoruko@ynu.ac.jp
Editor: Mandar Paingankar,
Government Science College Gadchiroli, Gadchiroli, India. Date of
publication: 26 December 2020 (online & print)
Citation: Yamamoto, M., H. Sugawara, K.
Fukushima, H. Setoguchi & K. Kurata
(2020). Genetic and reproductive
characterization of distylous Primula reinii in the Hakone volcano, Japan: implications for
conservation of the rare and endangered plant. Journal of Threatened Taxa 12(17): 17263–17275. https://doi.org/10.11609/jott.6532.12.17.17263-17275
Copyright: © Yamamoto et al. 2020. Creative Commons Attribution
4.0 International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: This work was financially supported by ProNatura Foundation Japan,
Support Society of Faculty of Education
and Human Science of Yokohama
National University, and Grants-Aid
for Science Research from Japan Society for Promotion
of Science (#19K16219).
Competing interests: The authors
declare no competing interests.
Author details: Masaya Yamamoto is an assistant professor in
Hyogo University of Teacher Education. Honami Sugawara is a school teacher in Tokyo, Japan. Kazuhiro Fukushima is also a school
teacher in Shizuoka Pref., Japan. Hiroaki Setoguchi
is a professor in Kyoto University. Kaoruko Kurata is an associate professor in Yokohama
National University.
Author contribution: KK conceived the project; MY, HS
and KF collected the data; MY led the writing of the manuscript. HS and KK
contributed critically to the drafts and give final approval for publication.
Acknowledgements: Fieldworks are conducted under
permits from Kanto Regional Environment Office, Ministry of the
Environment. We are grateful to Misato Wakai and Saki Sugihara, undergraduate and graduate
students in the Kurata laboratory (Yokohama National
University) for help in field surveys.
Abstract: Genetic and ecological evaluation
are crucial in effective management of rare and endangered species, including
those exhibiting complex breeding systems such as distyly. We studied a threatened distylous
herb Primula reinii in the Hakone volcano,
central Japan, to obtain baseline information of reproductive and genetic
status towards conservation. In two
representative populations inhabiting a central cone and somma of the volcano, population size, floral morph ratio,
stigmatic pollen deposition, and fruit-set were measured. Using microsatellite markers, we evaluated
genetic diversity, structure and differentiation of populations. Population bottlenecks and historical changes
in population size were also estimated from genotype data. We found significant deviation from equal
morph ratios in the central cone population, which also exhibited skewed mating
success together with a high frequency of pollination within the same
morph. These trends were not detected in
the somma population.
From genetic insights, the central cone population showed slightly lower
genetic diversity, whereas no significant deviation from Hardy-Weinberg
equilibrium was found in either population.
The estimated moderate genetic differentiation and admixed genetic
structure suggest recent lineage divergence and/or gene flow between
populations. While robust evidence for a
recent bottleneck was not obtained in our analyses, a clear signature of
historical population contraction was detected in the central cone population.
Our findings suggest that the skewed morph ratio strongly influenced the
reproduction of small and isolated populations in the short-term, highlighting
the vulnerability of distylous plant populations
under ongoing anthropogenic pressure.
Keywords: Distyly,
morph bias, reproduction, genetic diversity, volcanism.
INTRODUCTION
Worldwide, numerous plants are
already threatened by human-caused stress (e.g., habitat destruction) and
climate changes (Jackson & Kennedy 2009).
Among these plants, species having a sophisticated entomophilous
breeding system such as distyly (heterostyly) are
likely to be the most vulnerable to the detrimental effects of isolation and
unreliable pollination service due to anthropogenic environmental alteration (Washitani et al. 2005).
Distyly is a floral polymorphism, where
populations have two floral morphs (a long- and short-styled morph; hereafter,
referred to as the L- and S-morphs) that differ reciprocally in the heights of
stigmas and anthers in flowers. Besides
the morphological differences, distylous plants
usually have a heteromorphic incompatibility system that prevents selfing and intra-morph mating (Barrett 2002); only
cross-pollination (i.e., legitimate pollination) between L- and S-morphs
results in seed setting. In theory, such
morphologically and physiologically disassortative mating between floral morphs
generally leads to an equilibrium with equal morph ratios, as a result of
negative frequency-dependent selection and simple inheritance of distyly (Heuch 1979; Barret &
Shore 2008). Accumulated evidence in distylous plants, however, has provided numerous examples
of variation in population morph ratios (e.g., Kéry
et al. 2003; Brys et al. 2008; Meeus
et al. 2012). It has been advocated that
floral morph bias can be governed by several factors, such as stochastic and
deterministic events (Matsumura & Washitani 2000;
Kery et al. 2003), maternal fitness differences
between morphs (Hodgins & Barret 2006), and a combination of weak
heteromorphic incompatibility and pollen limitation (Barret 1989; van Rossum et
al. 2006; Brys et al. 2008).
Skewed morph ratios have often
been found in small isolated distylous plant
populations (e.g., Matsumura & Washitani 2000; Kery et al. 2003).
Furthermore, it is well known that the deviation of morph frequencies
from a 1:1 ratio can have negative reproductive and genetic consequences for
populations. Indeed, skewed morph ratios
result in the limited availability of compatible mates, which can contribute to
reduced reproductive success (Kery et al. 2003; Wang
et al. 2005; Pedersen et al. 2016) and increase the effects of genetic drift
(Byers & Meagher 1992). Moreover, a
combination of the loss of effective pollinators and the absence of a strict heteromorphic
incompatibility system can increase inbreeding (selfing
and biparental inbreeding) in morph-biased populations (Barret 1989; Wilcock
& Neiland 2002; van Rossum & Triest 2006).
Another consequence of skewed morph ratios is an increase in genetic
drift and inbreeding, which can lead to a loss of genetic diversity (van Rossum
& Triest 2006; Meeus et
al. 2012) and may eventually result in the breakdown of distyly
(Washitani 1996).
Therefore, conservation studies on threatened distylous
species which integrate ecological and genetic approaches are indispensable for
assessing current status and predicting future extinction probability, as well
as for planning effective conservation and/or restoration strategies (Washitani et al. 2005).
The present paper investigated Primula
reinii Franch. et Sav.
(Primulaceae), an endemic primrose that inhabits
mountainous regions of Japan. As in most
primroses, the plant is a self-incompatible distylous
species (Richards 2003). Because of its
attractive and relatively large flowers with dwarf foliage, the primrose has
suffered from anthropogenic activity (e.g. horticultural exploitation) in the
wild. Based on the rarity and serious
reductions in numbers and populations, the species is listed in the ‘Vulnerable’
category of the latest Japanese Red List (Ministry of the Environment
2019). Despite required effective
management, especially for small populations exposed to ongoing human
activities, little is known about their population status, reproductive success,
or remnant genetic diversity. Simultaneously,
this might provide an opportunity to study the immediate responses of a distylous plant population to demographic changes. In this
study, we assessed the genetic and reproductive status of two neighborhood populations of P. reinii
to provide baseline information pertinent to the conservation and preservation
of this rare and endangered primrose. To
discuss factors affecting reproductive success in natural habitats, we measured
population size, morph frequency, stigmatic pollen deposition, and fruit-set
within a population, and evaluated the genetic diversity and structure within
and among populations.
MATERIALS
AND METHODS
Plant species and study site
Primula reinii is a diploid (2n = 24) perennial
herb occurring as a chasmophyte on wet shaded rocky
cliffs in the mountains (Image 1). The
natural populations are rare and usually small and isolated from each other
because of their narrow edaphic niche and low dispersal ability arising from
the nature of the species. A single ramet produces one to two pink flowers from mid-April to
early May. Although flower visitors are
not well-known in this species, its narrow corolla tube and recent pollinator
observations in their related species (Yamamoto et al. 2018) imply that
long-tongued flying insects, such as bumblebees and bee flies can be effective
pollinators for the species. Under
cultivation conditions, the generation time (between seed germination and first
flowering) of the species is estimated to be 2–3 years (Yamamoto et al. 2017).
Fieldwork was conducted from
April 2013 to October 2014. We selected P.
reinii populations from two sites in the
Hakone volcano within a special protected zone of the Fuji-Hakone National Park
in central Japan (Fig. 1a). These sites
were severely isolated by a volcanic landform, i.e., caldera (distance: ca. 7
km) (Fig. 1b). One was on Mt. Kintoki (KIN population, 35.289’N, 139.004’E, 1,212m) and
the other was on Mt. Komagatake (KOM population,
35.228’N, 139.021’E; 1,275m). In both
sites, the primrose occurs on rock outcrops near the mountain top in which
populations are composed of scattered patches within an area of approximately
20 × 20 m2. Although a few
smaller patches also located alongside the studied populations, these rim
populations were sparse and separated geographically from the studied center population (>100 m). Thus, each studied population was considered
to be one reproductive unit.
Formation and strong eruptive
activities of the Hakone volcano initiated 650–350 ka and continued until 3ka
(Nagai & Takahashi 2008). The mean
annual precipitation and average air temperature at this area are 2,132mm and
8.80C, respectively (http://en.climate-data.org/location/769594/,
accessed 7 February 2019). Currently,
the Hakone volcano is an attractive destination to tourists. There is more than 10,000 people living
within the caldera, and an approximate average of 60,000 tourists visit the
area every day. Including Primula reinii, approximately 80% of endangered plants found in
the volcano are presumed to have decreased due to habitat destruction and
horticultural exploitation (Osawa & Inohara 2008).
Measurements
of basic population traits, pollen deposition, and fruit-set
During the
flowering season, the number of all flowering individuals and flowers within each
population, along with flower morph (L- or S-morph), were recorded. Whether the two morphs were equally frequent
within a population (i.e., deviations from a 1:1 morph ratio) was investigated
with Chi-square goodness-of-fit tests.
To
elucidate the role of pollen limitation and morph-ratio variation on female
reproductive success, we measured the stigmatic pollen load. In the natural
fields, 20 fully-opened flowers were collected from each population in the
afternoon and carefully transported to the laboratory the same day. In the laboratory, flowers were dissected,
and stigma removed and mounted on a microscope slide in aniline blue staining
solution (0.1% aniline blue in 0.1 M K3PO4). Under a compound microscope (Olympus), we
directly counted the number of legitimate (pollen from the opposite morph) and
illegitimate (pollen from the same morph) pollen grains on stigmas of each
floral morph based on pollen size differences (L-morph: 18.0 ± 0.1 μm; S-morph: 28.6 ± 0.1 μm; Y. Ojima, unpublished data).
At the
beginning of the fruiting season (August), the population mean for fruit-set
per flower was measured with the exception of some flowers that were used for
the measurements of stigmatic pollen loads.
Even if the fruit was set, the reproductive success will strongly depend
on fruit predation (e.g., Matsumura & Washitani
2000; Yamamoto et al. 2013). Thus, we
continued observations until October that was immediately before avulsion of
the capsules.
All field
surveys described above were conducted in 2013 and 2014 for KOM and KIN
population, respectively.
Population
sampling, DNA extraction, and microsatellite genotyping
In each
population, 32 plants were sampled randomly (without distinction of floral
morph types) from the entire area occupied by each population for genetic
analysis. Leaf materials were collected
and dried in silica gel. Before DNA extraction, leaves were homogenized with a
disposable homogenizer (Biomasher 2; Nippi Co., Tokyo, Japan) to a fine powder. Total DNA was extracted from 40 to 80 mg
silica-dried leaf tissue using the grass-fiber filter
method (Takakura 2011). The extracted
DNA was dissolved in a TE solution and stored at 40C until use.
After the preliminary
marker screening, the genotypes of each individual were characterized following
seven microsatellite markers that were originally developed for Primula sieboldii E. Morren: ga0161,
ga0218, ga0580, ga0691, ga1140, Pri0141, and 2ca135 (Ueno et al. 2003, 2006,
2009; Kitamoto et al. 2005). PCR amplifications and
allele-size determination of fragment analysis were performed in accordance
with the methods described by Yamamoto et al. (2017).
Population
genetic analysis
For all
seven microsatellite loci, the absence of linkage disequilibrium (LD) and the
presence of null alleles were tested using Genepop
v4.2 (Raymond & Rousset 1995). The LD test was verified using a Markov chain
method with 1,000 dememorization steps, and 1,000 iterations per batch. Null allele frequencies were estimated by
maximum-likelihood estimator based on the expectation-maximization algorithm
(Dempster et al. 1977) with the default setting.
The
following measures were calculated for each population: number of alleles (A),
effective number of alleles (AE), number of private alleles (AP),
expected heterozygosity (HE), and inbreeding coefficient (FIS).
Deviations from the Hardy-Weinberg equilibrium were determined by the exact
test and permutations. All measurements
were calculated using GenoDive v2.0 (Meirmans & van Tienderen
2004). GenoDive
was also used to compute the population’s genetic differentiation pairwise FST
and G’ST indices (Hedrick 2005), and FST
was tested for significance using 10,000 permutations.
To estimate
genetic structure of P. reinii
populations in Hakone volcano, we used the model-based clustering method
STRUCTURE 2.3.4 (Pritchard et al. 2000) and non-model-based method principal
component analysis (PCA). STRUCTURE
analysis was conducted for all samples across the two populations. Under an
admixture model with correlated allele frequency, 20 independent simulations
were run for each K (K = 1–5) with 5 × 105 Markov
chain Monte Carlo (MCMC) steps and a burn-in period of 105
interactions. The most likely value of K
was determined by the ΔK method (Evanno et al.
2005) with STRUCTURE HARVESTER 0.6.94 (Earl & vonHoldt
2012). CLUMPAK (Kopelman et al. 2015)
was used to average the outputs from multiple STRUCTURE runs and produce the
graphical results. The F value,
the amount of genetic drift between each cluster and a common ancestral
population, was also calculated for each cluster. The PCA analysis was
performed using the package adegenet 2.0.1 (Jombart 2008) in R 3.5.2 (R core Team 2018).
To detect a
genetic imprint of past population bottlenecks, we first used the
heterozygosity excess method (Cornuet & Luikart 1996) implemented within the program BOTTLENECK
v1.2 (Piry et al. 1999). This method is suitable to detect very recent
and less severe bottlenecks, and has low false positive error rates
(Williamson-Natesan 2005). All simulations were done with mutation-drift
equilibrium conditions (2,000 replicates) under the stepwise mutation model
(SMM), infinite allele model (IAM), and two-phase mutation model (TPM: 70% SMM
and 30% IAM). A two-tailed Wilcoxon
signed-rank test was used to determine a significant excess of heterozygosity.
We also
calculated the M-ratio (Garza & Williamson 2001) for each
population using Arlequin (Excoffier et al.
2005). The M-ratio test is
considered to have a greater detection power for ancient and moderate-to-severe
population declines in comparison with the former method (Williamson-Natesan 2005). M-ratio
represents the number of alleles relative to the range in allele sizes. After a severe bottleneck, the number of
alleles should reduce faster than the allelic size range, which results in a
reduced M-ratio (Garza & Williamson 2001). Thus, the magnitude of the decrease reflects
the severity and duration of the reduction in population size, and generally an
M-ratio <0.68 is indicative of the presence of a bottleneck (Garza
& Williamson 2001).
Finally, we
conducted a Bayesian demographic analysis using the R package, Vareff (Nikolic & Chevalet
2014). In contrast to the first two
moment-based methods, this coalescent-based approach can examine temporal
changes in the effective population size (Ne).
The function Vareff simulates prior changes in
the effective population size from microsatellite data by resolving coalescent
theory and using an approximate likelihood MCMC (Nikolic & Chevalet 2014).
After a series of preliminary runs, we used the prior parameter settings
for each population (Table S1), following recommendations from Nikolic & Chevalet (2014). We
set the estimated mutation rate to 5 × 10–4 (Estoup
et al. 2002) for all loci, and ran each analysis under a two-phased mutation
model with a proportion of 0.22 for multiple mutations (Peery et al. 2012), for
105 MCMC steps (NumberBatch = 1,000,000, LengthBatch = 10), sampling every 10 steps (SpaceBatch = 10) with an acceptance ratio of 0.25 (AccRate = 0.25), after burning of 10,000 steps. Estimations of sizes were searched for from
sampling time to 5,000 and 500 generations ago.
RESULTS
Population
traits and morph ratio
Each
population trait is summarized in Table 1.
A total of 72 flowering individuals and 99 flowers were found in the KIN
population, whereas the KOM population had fewer (52 individuals and 69
flowers). The morph ratio in KIN did not
deviate significantly from a 1:1 ratio (L-morph ratio = 0.54), even in the
number of flowers (L-morph ratio = 0.52).
In contrast, the number of flowering individuals of the L-morph was
significantly higher than that of the S-morph in KOM (L-morph ratio = 0.65) and
even higher for the number of flowers (L-morph ratio = 0.70).
Pollen
deposition
Stigmas of
both floral morphs received pollen grains in each population, but the numbers
varied greatly between the individuals, ranging from zero to 321. In the KIN population, no differences in
stigmatic pollen loads were detected between morphs (Fig. 2a). In addition, the proportion of deposited
legitimate pollens was not significantly different between both morphs (Fig.
2c), while the proportions varied greatly among the L-morph stigmas in
comparison with the S-morph. This is
complemented by the result that the S-morph stigmas received significantly more
legitimate pollen grains than the L-morph stigmas (Fig. 2b), implying that
S-morph stigmas were pollinated more effectively than the opposite morph.
In contrast, in the KOM
population, L-morph stigmas received a significantly greater number of pollen
grains than the S-morph stigmas (Fig. 2a).
After classifying pollen grains, however, we found no legitimate pollen
grains loaded on the L-morph stigmas (Fig. 2b); that is, most L-morph stigmas
were covered with a large quantity of illegitimate pollen. Although several S-morph stigmas were
legitimately pollinated, similar to other populations (Fig. 2c), there was no
significant difference in the number of legitimate deposited pollen grains
between the two morphs (Fig. 2b).
Fruit-set
At the population level,
fruit-set ratio was much higher in the KIN population than in the KOM
population (32.3 % and 14.3 %, respectively) (Fig. 2c). Within a population, both L- and S-morph
scored comparable values in the KIN population (37.1 % and 26.7 %,
respectively). In contrast, fruit-set of
L-morph in the KOM population was less than half of the opposite morph (10.5 %
and 27.3 %, respectively). We continued
monitoring until October, but no evidence of fruit predation was found in
either population, namely fruit-set was almost unchanged throughout the
fruiting season.
Genetic diversity
LD between locus pairs was not
significant. Although the frequencies of
the majority of the null alleles were lower than 0.1, higher frequencies of
null alleles were detected on 2ca135 and ga1140 loci in the KOM population
(0.227 and 0.114, respectively). As the
presence of null alleles may affect the estimation of genetic diversity or
differentiation, we excluded the two loci and repeated several analyses to
compare results between seven and five microsatellites. This trial revealed no clear difference in
the results based on 5 vs. 7 loci (Table S2).
Thus, seven loci were used in all analyses described below.
Genetic diversity parameters for
the two populations are presented in Table 2.
In total, 63 alleles were amplified by seven microsatellite markers,
with an average 9.0 alleles per locus.
All diversity measurements were slightly higher in the KIN population (A
= 6.3, AE = 3.3, AP = 2.9 and HE
= 0.652) than in the KOM population (A = 6.1, AE =
2.6, AP = 1.9 and HE = 0.544). The inbreeding coefficient value (FIS)
was positive and comparable between the populations, but each value did not
deviate significantly from zero.
Genetic structure and evidence a
recent bottleneck
A moderate genetic
differentiation was detected between populations (FST =
0.115, P < 0.001; G’ST = 0.286). The STRUCTURE analysis based on the ΔK
method indicated that ΔK was 462.5 for K = 2 and ΔK were
<3 for other values of K.
Therefore, the optimal ΔK for K = 2 showed that the
best-fit model for the 64 sampled individuals of P. reinii
revealed two clusters (Fig. 3a).
Although several admixed individuals were found in each population, all
samples formed a clear genetic structure between the two populations. The F values of clusters produced by
STRUCTURE analysis were higher in the KOM population (F = 0.186) than in
the KIN population (F = 0.086), indicating that the KOM population had
undergone a larger genetic drift compared to that of the KIN population. In the PCA (Fig. 3b), the first two axes
explained 17.0% and 10.7% of the variances in the experimental data,
respectively. The results also
distinguished the two populations, suggesting the existence of two genetic
units corresponding to each population.
In BOTTLENECK analyses, the
two-tailed Wilcoxon signed-rank test provided statistical support (P
< 0.025) to the presence of a recent bottleneck in the KOM population under
the SMM and TPM, whereas no evidence was found in KIN (Table 2). On the contrary, the M-ratio test
indicated that both populations experienced a reduction in population
size. The M-ratio values were
0.312 and 0.338 for the KIN and KOM populations, respectively (Table 2). A clear signature of historical population
contraction was detected only in KOM via the third method, the Bayesian
population demographic analysis. The
bottleneck began approximately 1,000 generations ago (Fig. 4a), whereas a
gradual decline was settled at least 100 generations ago (Fig. 4b). In contrast, the KIN population seems to have
historically had a large constant population size (Fig. 4a); however, recent
changes were unclear due to the broad confidence levels (Fig. 4b).
DISCUSSION
Reproductive status and genetic
diversity
The observed low reproduction in
KOM is congruent with reports that morph-biased populations experience reduced
reproduction (Byers & Meagher 1992; Kery et al.
2003; Wang et al. 2005; Pedersen et al. 2016).
Given that almost all stigmas were covered with L-morph pollen grains
(Fig. 2), it is plausible that frequent self- or intra-morph (i.e.,
illegitimate) pollination had occurred among the KOM L-morphs. Therefore, our
ecological data indicate that the low fruiting success in KOM L-morphs was
caused by stigmatic clogging (Yeo 1975) as a consequence of the skewed morph
ratio. Because L-morph flowers generally
produce greater amounts of pollen grains than S-morph flowers (Richards 2003),
it is apparent that the total pollen pool within KOM was occupied by a large
amount of L-morph pollen. Similar to our
results, previous studies in distylous plants showed
higher female reproductive success in the relatively less abundant morph than
the dominant morph (e.g., Wyatt & Hellwig 1979;
Thompson et al. 2003; Wang et al. 2005; García-Robledo
& Mora 2007). Thus, these results
may demonstrate negative frequency-dependent patterns of reproductive success
in the distylous primrose.
The indices of genetic diversity
were relatively high and comparable between the two populations (Table 2),
despite the skewed morph ratio observed.
In addition, each population exhibited low FIS levels
with no significant deviation from the Hardy–Weinberg equilibrium. These results allow for the conclusion that Primula
reinii growing in the volcano had maintained
sufficient genetic diversity as a result of outbreeding.
Overall, this
study suggests the persistence of distylous
self-incompatibility system in the P. reinii
populations. Nevertheless, determination
of the exact causes of floral morph bias in KOM was not possible based on the
limited ecological and genetic data currently available. Because skewed morph ratios are often
explained by several biotic and abiotic factors as discussed in the
Introduction, there is a need for future studies investigating the ability of selfing and intra-morph mating, maternal fitness
differences between morphs, pollinator assemblage, and population demography.
Genetic differentiation and
structure
Our molecular analysis showed
that genetic differentiation was moderate between the two populations (FST
= 0.115). Additionally, signs of genetic
admixture between the populations were detected in PCA and STRUCTURE analyses (Fig.
3). There are at least two non-exclusive
explanations for this: recent lineage divergence and gene flow. According to accumulated geographical
surveys, Mt. Kintoki (locality of KIN pop.) and Mt. Komagatake (locality of KOM pop.) formed approximately ca
350–300 ka (Nagai & Takahashi 2008) and ca 27–20 ka (Kobayashi 1999; Nagai
& Takahashi 2008), respectively.
Formation of the central cone (i.e., Mt. Komagatake)
clearly corresponded to the period of the last glacial maximum (LGM; ca 25–15
ka), suggesting that the KOM population was established at least after the last
glacial period. The observed high F value
(STRUCTURE analysis) and low private alleles in KOM may support a migration
scenario that the population experienced a founder effect arising from a
post-glacial refugial isolation and subsequent migration from the lowland of
the caldera to the high-altitude areas of the central cone during the late
Pleistocene and Holocene. Hence, it is
plausible that the detected genetic admixture between populations suggests
incomplete lineage sorting (i.e., sharing ancestral polymorphism between
populations) due to recent lineage divergence.
Given the geographically close
relationship between the populations (Fig. 1b), the presence of contemporary
gene flow will also be taken into consideration. Because the two populations are severely
isolated by a volcanic landform, gene flow mediated by pollen would be a
plausible hypothesis. Moreover, in the
flowering season we found claw marks, a useful indicator for the pollination
services provided by bumblebees (Washitani et al.
1994), on the petals of each population.
This may suggest that the bumblebees have a key role in pollination
within the Primula reinii populations. Although bumblebees are known as strong-flying
insects (e.g., Rao & Strange 2012), previous observations in other Primula
species have demonstrated that pollen transfer by bumblebees generally occurs
within short distances (e.g., Ishihama et al.
2006). Therefore, we determined that the
pollen flow between the populations might occur contemporarily but on very rare
occasions. Nevertheless, deciding among
the possible explanations for the genetic composition of the primrose in the
Hakone volcano is difficult due to the weak evidence based on an insufficient
number of loci.
Recent and historical demography
The two tests for a recent
bottleneck yielded mixed results (Table 2).
Based on the BOTTLENECK analysis, only the KOM population exhibited
excess heterozygosity. In contrast, the M-ratio
test supported a recent population size reduction in both populations. As mentioned above, however, because these
inconsistent results might be attributed to the low statistical power of our
sample size (e.g., number of loci or individuals), our results should be
interpreted with caution. Nevertheless,
such conflicting results often indicate the severity or timing of the reduction
in population size (Williamson-Natesan 2005; Marshall
et al. 2009; Padilla et al. 2015; Tóth et al. 2019),
and were expected due to the differences in power detecting a bottleneck (Peery
et al. 2012).
Considering the robust results in
KOM, it is likely that the morph-biased population may have undergone more
recent and severe bottlenecks in comparison with another population. In theory, the BOTTLENECK analysis can
demonstrate population bottlenecks over a period of 0.2–4.0 Ne generations (Cornuet & Luikart 1996). Assuming for KOM population of Ne = 100 (Fig.
4) and a generation time of 2–3 years, it translates into approximately 50–1000
years before the present. On the other
hand, a clear sign of recent (within 100 generations) population bottleneck was
not found in the Bayesian demographic analysis (Fig. 4). Therefore, based on results from a series of
demographic analyses, it is difficult to draw a definitive conclusion on
whether recent bottlenecks occur or not, and thus, we defer a final conclusion
until more genetic data are available in the future.
Contrary to this, the Bayesian
demographic analysis provided strong evidence in support of a historical
population bottleneck in KOM inhabiting the central volcanic cone. The first signs of population decline would
have occurred 2–3 ka (assuming a generation time of 2–3 years). This timeframe post-dates a climatic warming,
known as the Jomon optimum transgression, that
occurred approximately 6ka, implying that historical population bottlenecks
were likely due to volcanic activities as opposed to climatic events. According
to geological records, the last major eruption of the volcano was from the
central cone in 2.7–2.9 ka (Kobayashi 1997; Kobayashi et al. 2006), and
intermittent phreatic eruptions continued until present-day. Although speculative, these evidences may
support the idea that the historical population declines experienced by the KOM
could have been associated with repeated eruptive activities in the central
cone. Perhaps, the detected recent
bottlenecks in KOM are caused by eruptive activities rather than human
activities.
On the other hand, the estimated
effective population size in the KIN population inhabiting the somma mountains was large and constant in the long term,
suggesting that the population has been maintained without suffering from
volcanic eruptions occurring in the central cone. Further studies for the lineage divergence
and demographic history of P. reinii in
this region, using more informative datasets (e.g., single nucleotide
polymorphisms), will be valuable because volcanism is one of the key abiotic
factors in the plant’s diversification and distribution in Japan (e.g., Yoichi
et al. 2017; Nagasawa et al. 2020), located in the Pacific Ring of Fire.
Implication for conservation
Our study
suggests that morph imbalances are striking effects on the reproduction of P.
reinii population in the short-term. Accordingly, a measure of morph ratio should
be given top priority in conservation management of the species, and
enhancement of habitat monitoring should be considered as in situ managements
to protect remnant individuals and to maintain optimum morph frequencies from
horticultural exploitation. Considering
the observed negative frequency-dependent patterns of reproductive success, if
heteromorphic self-incompatibility is totally strict in P. reinii, the skewed morph ratio in KOM may be
improved in the future when regeneration is successful. However, the exact
breeding system of the species remains poorly understood. Therefore, in addition to other examinations
(e.g., the germination requirements and the effect of storage time of seeds)
towards a future ex situ conservation strategy, the levels of within
morph fertility and selfing ability should be
resolved immediately to evaluate the medium- to long-term risk of extinction in
the remnant populations across species distribution ranges.
The two surveyed populations in
the Hakone volcano were distinguished by two genetic clusters, suggesting that
each population should be divided into a different management unit to maintain
evolutionary distinctiveness and ecological viability (Moritz 1994; Frankham et al. 2002).
The moderate genetic differentiation and the presence of large amounts
of private alleles between the populations highlight this suggestion; thus,
artificial inter-population crossing should be avoided in this case.
Nevertheless, the lack of samples from other parts of the volcano will
influence the estimated genetic structure.
Thus, an exhaustive population sampling, including other remnant small
population, is required to elucidate the genetic structure and demographic
history of P. reinii occurring in the
Hakone volcano as is also needed for planning conservation strategies.
To our knowledge, this is the
first conservation genetics study on threatened plants in the Hakone volcano,
which harbors approximately 1,800 plant species
(Tanaka 2008). Thus, the results
discussed here will be useful for designing both in situ and ex situ
conservation strategies for P. reinii
as well as other plants inhabiting the volcano and shed light on the
instability of plant populations due to the impacts of volcanism and human
activities. Our study highlights the
importance of studies in conservation, integrating ecological and genetic
approaches to accurately assess the population status of endangered species and
draw up effective conservation strategies.
Table 1. Number of blooming
plants during the flowering season in each population. Results of χ2 goodness-of-fit
tests for the similarity between the two morphs.
Number of |
L-morph |
S-morph |
Total |
χ2 |
P |
KIN pop. |
|
|
|
|
|
Flowering individuals |
39 |
33 |
72 |
0.50 |
0.48 |
Flowers |
51 |
48 |
99 |
0.09 |
0.76 |
KOM pop. |
|
|
|
|
|
Flowering individuals |
34 |
18 |
52 |
4.92 |
0.03 |
Flowers |
48 |
21 |
69 |
10.57 |
0.001 |
Table 2. Genetic diversity and
detection of a recent population bottleneck of the two Primula reinii populations.
Pop. code |
Genetic diversity measurements |
P values of Wilcoxon
test |
M-ratio |
|||||||
A |
AE |
AP |
HE |
FIS |
IAM |
SMM |
TPM |
Mean |
SD |
|
KIN |
6.3 |
3.3 |
2.9 |
0.652 |
0.068 |
0.109 |
0.297 |
0.813 |
0.312 |
0.221 |
KOM |
6.1 |
2.6 |
1.9 |
0.544 |
0.073 |
0.296 |
0.007 |
0.015 |
0.338 |
0.199 |
total |
9.0 |
2.8 |
- |
0.598 |
0.070 |
- |
- |
- |
- |
|
A, mean number of alleles; AE,
mean number of effective alleles; AP, mean number of private
alleles; HE, expected heterozygosity; FIS,
coefficient of inbreeding; IAM, infinite allele model; SMM, stepwise mutation
model; TPM, two-phase mutation model.
Table S1. Prior parameter
settings for each population using VarEff software.
Parameters |
KIN |
KOM |
Description |
JMAX |
4 |
4 |
Number of when the effective
size has changed |
DMAX |
10 |
7 |
the maximal distance between
alleles |
NBAR |
100 |
100 |
prior value for the effective
population size |
RHOCORN |
0 |
0 |
coefficient of correlation
between effective population size in successive intervals |
VARP1 |
3 |
3 |
variance of prior
log-distribution of effective population size |
VARP2 |
3 |
3 |
variance of prior
log-distribution of time intervals |
GBAR |
10000 |
5000 |
number of generations since the
assumed origin of the population |
Diagonale |
0.5 |
0.5 |
a smoothing parameter |
Table S2. Genetic diversity
measurements and population differentiation between the two populations based
on the selected five loci (ga0161, ga0218, ga0580, ga0691
and Pri0141).
Pop. code |
A |
AE |
HE |
FIS |
FST |
G’ST |
KIN |
6.0 |
3.3 |
0.637 |
0.020 |
0.111 |
0.249 |
KOM |
5.0 |
2.1 |
0.472 |
0.029 |
For
Image & Figures - - Cliick Here
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