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\begin{document}

\title{Phylogeographic patterns driven by river isolations in an island-endemic
montane plant}



\author[1]{Shaojun Ling}%
\author[2]{Juli Caujapé-Castells}%
\author[1]{Liang Tang}%
\author[1]{Mingxun Ren}%
\affil[1]{Hainan University}%
\affil[2]{Jardin Botanico Canario "Viera y Clavijo"}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Hainan Island in south China is a key part of the globally important
Indo-Burma biodiversity hotspot, while the origin and maintenance of its
species richness remains largely unexplored. In this study, we combined
nuclear ITS and cpDNA trnL-trnF and ycf1b sequences to evaluate the
genetic structure and phylogeographic patterns of the Hainan-endemic
Primulina heterotricha (Gesneriaceae). The results showed significant
phylogeographic patterns with low within-population genetic diversity
and significant genetic differentiation among populations (Fst = 0.708 \selectlanguage{ngerman}±
0.319 and 0.826 ± 0.209 for nrDNA and cpDNA). Three clades were
identified with little gene flow (Nm \textless{}\textless{} 1 for nrDNA
and cpDNA), which is supported by our STRUCTURE v.2.3.4 analyses. Our
analyses detected two vicariance events at c. 0.83 and c. 0.48 Myr, and
suggest that these three genetically-separated groups were isolated by
two big rivers (Changhua River and Wanglou River), that likely acted as
barriers to gene flow. The magnitude of isolation was positively
correlated to the size of the rivers, with the greater barrier effect
associated with the larger Changhua River. Our results highlight for the
first time the critical role of riverine isolation in the patterns of
intraspecific evolution of plant populations on Hainan Island.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Original Article}

Phylogeographic patterns driven by river isolations in an island-endemic
montane plant

Shaojun Ling\textsuperscript{1,2}, Juli
Caujap\selectlanguage{ngerman}é-Castells\textsuperscript{3}, Liang Tang\textsuperscript{1,2} and
Mingxun Ren\textsuperscript{1,2}*

\textsuperscript{1}Key Laboratory of Genetics and Germplasm Innovation
of Tropical Special Forest Trees and Ornamental Plants (Hainan
University), Ministry of Education, Haikou 570228, China

\textsuperscript{2}Center for Terrestrial Biodiversity of the South
China Sea, Hainan University, Haikou 570228, China

\textsuperscript{3}Departamento de Biodiversidad Molecular y Banco de
DNA, Jardín Botánico Canario \selectlanguage{english}'Viera y Clavijo' -- \selectlanguage{english}Unidad Asociada CSIC,
Cabildo de Gran Canaria, Camino del Palmeral 15, 35017 Las Palmas de
Gran Canaria, Spain

\textbf{Running title} : Phylogeography driven by rivers

*Corresponding author. E-mail: renmx@hainanu.edu.cn\textbf{Abstract}
Hainan Island in south China is a key part of the globally important
Indo-Burma biodiversity hotspot, while the origin and maintenance of its
species richness remains largely unexplored. In this study, we combined
nuclear ITS and cpDNA \emph{trn} L-\emph{trn} F and\emph{ycf} 1b
sequences to evaluate the genetic structure and phylogeographic patterns
of the Hainan-endemic \emph{Primulina heterotricha} (Gesneriaceae). The
results showed significant phylogeographic patterns with low
within-population genetic diversity and significant genetic
differentiation among populations (\emph{Fst} = 0.708 \selectlanguage{ngerman}± 0.319 and 0.826
± 0.209 for nrDNA and cpDNA). Three clades were identified with little
gene flow (\emph{Nm} \textless{}\textless{} 1 for nrDNA and cpDNA),
which is supported by our STRUCTURE v.2.3.4 analyses. Our analyses
detected two vicariance events at \emph{c.} 0.83 and\emph{c} . 0.48 Myr,
and suggest that these three genetically-separated groups were isolated
by two big rivers (Changhua River and Wanglou River), that likely acted
as barriers to gene flow. The magnitude of isolation was positively
correlated to the size of the rivers, with the greater barrier effect
associated with the larger Changhua River. Our results highlight for the
first time the critical role of riverine isolation in the patterns of
intraspecific evolution of plant populations on Hainan Island.

\textbf{KEYWORDS} Biogeography, Changhua River, Isolation,
Phylogeographic patterns, Hainan Island

\textbf{1 INTRODUCTION}

Geographical isolation refers to a situation where populations become
separated by physical barriers such as islands and mountains, and has
long been considered as a key driving force in speciation (MacArthur \&
Wilson, 1967; Li et al., 2011; Robin et al., 2015). Geographical
barriers can fragment distribution ranges and gene flow dynamics between
different sides of the barriers, thereby allowing the increase in
population differentiation. As a result, populations become fragmented
and genetically isolated and, in some cases, new species may evolve
(Crimmins et al., 2011; Li et al., 2011; Robin et al., 2015; Demenou et
al., 2016). The knowledge of such processes is crucial for gaining
insight into the origins and evolution of biodiversity, but also useful
for formulating biodiversity conservation strategies. Consequently, the
identification of geographic barriers using genetic data is increasingly
emphasized in modern biodiversity conservation studies (Li et al\emph{.}
, 2011; Robin et al\emph{.} , 2015; Tan et al\emph{.} , 2020).

Hainan Island in south China harbors \emph{c.} 4800 seed plant taxa in
total and about 500 endemics (Francisco-Ortega et al\emph{.} , 2010a, b;
Yang, 2013), and is a distinctive part of the globally important
Indo-Burma biodiversity hotspot (Myers et al\emph{.} , 2000). Notably,
the richest biodiversity and 80\% of the endemic species occur in the
south-central mountains of the island (Yu et al\emph{.} , 2001;
Francisco-Ortega et al\emph{.} , 2010a, b), which are largely located
within Hainan Tropical Rainforest National Park. Home to rich tropical
plant species, this Park is an important natural gene pool for the
entire globe. However, several large rivers, mountains and valleys cause
a pronounced geographical fragmentation in this region (Ling et al.,
2017a, b; Li et al., 2020), with potentially negative effects on
ecosystem integrity and the biodiversity conservation of the Park. Such
patterns offer opportunities to study the mechanisms underlying
evolutionary species diversification in the island, which involves both
isolation by the sea and by riverine and mountainous barriers.

The family Gesneriaceae in Hainan Island is notable for its extremely
high species endemism (Wei, 2010; Ling et al., 2017a, 2017b). Fourteen
genera and 25 species of Gesneriaceae are currently found on the island,
including two endemic genera and nine endemic species (Li \& Wang, 2005;
Yang, 2013; Ling et al., 2017a, 2020b). The species endemism proportion
of Hainan Gesneriaceae is 30\%, a great value similar to that of the
nearby Guangxi Province (Ling et al., 2017b), long believed to be the
species distribution and endemism centre of Gesneriaceae in SW China due
to its widespread limestone landscapes (Li \& Wang, 2005; Wei et al.,
2004; Wei, 2010). All these Hainan endemics are found on crown lineages
(Ling et al., 2017b), suggesting a recent origin (Ling et al., 2017b,
2020a). However, the mechanisms and processes underlying speciation
patterns of these narrow endemics have received little attention.

In this investigation, we examine the influence of rivers and mountains
on the genetic divergence and phylogeographic patterns in the
Hainan-endemic \emph{Primulina heterotricha} (Merr.) Y.Dong \& Yin
Z.Wang (Gesneriaceae) using both nuclear and chloroplast DNA sequences.
This study addresses three specific questions to test if the
geographical isolation of populations by rivers and valleys on the
island were determinant to the speciation, distribution and genetic
diversification of \emph{P. heterotricha} : (1) What is the genetic and
phylogeographic structure of \emph{P. heterotricha} populations? (2) Can
these patterns be associated with any geographic barriers? (3) Did these
barriers play a role in the speciation and adaptation history of this
montane plant?

\textbf{2 MATERIAL AND METHODS}

\textbf{2.1 Plant material}

\emph{Primulina heterotricha} is an endemic perennial shrub with thick
rhizome that inhabits limestone or granite streamsides in forested
valleys, widely distributed in south-central mountain systems of Hainan
Island (Li \& Wang, 2005; Wei, 2010; Yang, 2013). Its populations
display a limited range of morphological diversity including irregular
pale yellow or purple corollas. The plant has zygomorphic tubular
flowers which are pollinated by several insects,
especially\emph{Glossamegilla malaccensis} (Anthophoridae) Friese and
\emph{G. yunnanensis} Wu (Anthophoridae)(Ling, 2017). The capsule is
erect with small brown and fusiform shaped seeds, indicating a weak
dispersal capability with no wind- or animal-mediated dispersal
features.

\textbf{2.2 Sample collection and laboratory procedures}

From 2015 to 2017, we conducted a thorough sampling across the known
distribution range in the south-central mountain systems of Hainan
Island and we collected 217 leaf samples from 10 populations of \emph{P.
heterotricha} (Table 1, Figure 1). Each sampled leaf was dried in a
separate plastic bag containing 20 to 30 g of silica gel. We collected
two individuals with flowers and fruits as representative herbarium
vouchers, and these have been deposited in HUTB, under accession
numbers\emph{Ling \& Ren 2016091902} and \emph{Ling \& Ren 2016091501} .
Total genomic DNA for each sample was extracted using the CTAB method
(Doyle \& Doyle, 1987), and served as the template for the polymerase
chain reaction. DNA quality and quantity on 0.8\% agarose gels stained
with 2.5 \selectlanguage{greek}μ\selectlanguage{english}l Goldview (Aidlab Biotechnologies Co., Ltd) was detected by
AL2000 DNA maker (Aidlab Biotechnologies Co., Ltd) in DTU-48
spedtrophotometer (Hangzhou Miu Instruments Co., Ltd, China).

One nuclear ribosomal DNA (nrDNA) sequence, the ITS region comprising
spacer 1, the 5.8S gene and spacer 2 (White et al\emph{.} , 1990) and
two chloroplast DNA (cpDNA) intron-spacer region \emph{trn} L-\emph{trn}
F (Taberlet et al\emph{.} , 1991) and \emph{ycf} 1b (Dong et al\emph{.}
, 2015) were used in this study (Table 2). PCR reactions were set up in
a volume of 25 \selectlanguage{greek}μ\selectlanguage{english}l composed of 20 \selectlanguage{greek}μ\selectlanguage{english}l ddH\textsubscript{2}O, 2.5 \selectlanguage{greek}μ\selectlanguage{english}l
10\selectlanguage{ngerman}×Buffer, 0.5 \selectlanguage{greek}μ\selectlanguage{english}l 10 mM dNTPs, 0.5 \selectlanguage{greek}μ\selectlanguage{english}l each 5 \selectlanguage{greek}μ\selectlanguage{english}M primer, 0.5 \selectlanguage{greek}μ\selectlanguage{english}l DNA
template and 0.5 \selectlanguage{greek}μ\selectlanguage{english}l 5 U/\selectlanguage{greek}μ\selectlanguage{english}l Taq polymerase (Aidlab Biotechnologies Co.,
Ltd). The PCR reactions were carried out on the 2720 Thermal cycler
(Applied Biosystems by Life Technologies, made in Singapore) and Veriti
96-Well Thermal Cycler (Applied Biosystems by Life Technologies, made in
Singapore). The PCR program for ITS1/2 and \emph{trn} L-\emph{trn} F was
designed to an initial denaturation at 94 \selectlanguage{ngerman}ºC 5 min, followed by 35
cycles of 1 min at 94 ºC, 1 min at 55 ºC, 1 min at 72 ºC, and with a
final extension of 10 min at 72 ºC. Amplification of \emph{ycf} 1b used
the following protocol: 4 min at 94 ºC, 35 cycles of 30 s at 94 ºC, 40 s
at 58 ºC, and 1 min at 72 ºC, ending with 10 min at 72 ºC. All the PCR
products were verified by cataphoresis. The amplicons were sequenced by
an ABI 3730 DNA Analyzer based on the BigDye Terminator Cycle Sequencing
Ready Kit (Applied Biosystems, Foster City, CA) in BGI (Beijing Genomics
institution).

\textbf{2.3 Genetic diversity}

The chromatograms from both directions of the ITS1/2 and cpDNA sequences
were edited with the software BioEdit (Hall, 1999) for base confirmation
and contiguous sequences editing. The sequences were manually aligned
where necessary using MEGA v.6.5 (Kumar et al\emph{.} , 2008). All
sequences have been deposited in Genbank. The two non-coding cpDNA
sequences were assembled as a single locus by SequenceMatrix v.1.7.8
(Vaidya et al., 2011), and a Partition Homogeneity Test (PHT) vs the ITS
sequences was carried out with PAUP* v. 4.0a164 (Swofford, 2002). Since
the test resulted in the non-homogeneity of both matrices, we analysed
the geographic distribution of ribotypes/chlorotypes for nrITS and cpDNA
sequences separately.

DNASP v. 6.12.01 (Rozas et al\emph{.} , 2017) was used to compute the
number of ribotype/chlorotype (\emph{h} ), haplotype diversity (Hd)
within populations, polymorphic sites (S), nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}),
average number of nucleotide difference (K).

\textbf{2.4 Population structure analysis}

To explore the phylogeographic structure of ribotypes/chlorotypes, gene
diversity within populations (\emph{h\textsubscript{S}} ), gene
diversity (\emph{h\textsubscript{T}} ), index of gene diversity of total
populations (\emph{N\textsubscript{ST}} ) and index of genetic
differentiation between populations (\emph{G\textsubscript{ST}} ) were
calculated using the HAPLONST (Pons \& Petit, 1996). This software was
also used to compute the test statistic \emph{U} that compares the
values of \emph{N\textsubscript{ST}} and \emph{G\textsubscript{ST}} ,
which indicates the presence of phylogeographic structure
if\emph{N\textsubscript{ST}} is higher than
\emph{G\textsubscript{ST}}(Pons \& Petit, 1996).

Since the cpDNA is maternally inherited in \emph{Primulina} , there is
no recombination among loci and structure analysis makes no sense for
such dataset. Population structure of nrDNA sequences was inferred using
the Bayesian clustering procedure implemented in the software STRUCTURE
v.2.3.4 (Evanno et al\emph{.} , 2005), that identifies the most probable
number (\emph{K} ) of genetic clusters of origin of the sampled
individuals, and assigns individuals to clusters. We used Markov Chain
Monte Carlo (MCMC) iterations as implemented in the software to explore
the parameter space considering individual memberships to the
\emph{K}clusters, ranging from \emph{K} = 1 (null hypothesis of
panmixia) to\emph{K} = 10 (the total number of populations sampled).
Three independent runs were performed with an admixture model at
10\textsuperscript{5} MCMC iterations and a 10\textsuperscript{5}burn-in
period. The most likely number of population groups (\emph{K} ,
indicating the number of true clusters in the data) and the model values
(\emph{^K} ) according to the second-order rate of Change of
cluster\emph{K} that best fit the data was calculated in Structure
Harvester (Earl \& vonHoldt, 2012). The graphical representation of
results was accomplished in the CLUMPAK server
(http://clumpak.tau.ac.il/index. html).

The genetic differentiation (\emph{Fst} ) and gene flow (\emph{Nm} )
between populations and among different regions for nrDNA and cpDNA
sequences separately. An Analysis of Molecular Variance (AMOVA) was
conducted on nrDNA and cpDNA sequences separately to test genetic
differentiation within populations, among regions and among populations
within regions using GENALEX v. 6.503 (Peakall \& Smouse, 2012). To test
whether there was local genetic variation attributable to isolation by
distance (IBD), Nei'\selectlanguage{english}s genetic distances (Nei, 1973) calculated with by
MEGA v. 6.5 and geographic distances (in km) between all pair-wise
combinations of the ten populations sampled were subjected to a Mantel
test (Mantel, 1967) in GENALEX v. 6.503. The genealogical relationships
between ribotypes/chlorotypes were inferred from the Median-Joining
network (MJ) of NETWORK v4.6.1.0 (http://www.fluxus-Engineering.com/).
In order to identify and quantify potential genetic discontinuities and
biogeographic boundaries between populations from both nrITS and cpDNA
datasets, we calculated the Monmonier'\selectlanguage{english}s maximum-difference algorithm in
Barrier v.2.2 (Manni et al., 2004). The robustness of these barriers was
assessed by bootstrap, as in the Barrier v.2.2.

\textbf{2.5 Phylogenetic relationships of ribotypes}

Phylogenetic relationships for the identified ribotypes were
reconstructed with Maximum Likelihood (ML) and Bayesian Inference (BI)
using MEGA v.6.5 (Kumar et al\emph{.} , 2008) and MrBayes v.3.2.6
(Ronquist et al\emph{.} , 2012). \emph{Primulina pteropoda} (W.T.Wang)
Yan Liu was used as outgroup with sequences of nrDNA (Gao et al\emph{.}
, 2015, Genbank with accession number DQ872827).

We inferred the optimal model of nucleotide substitution using
MrModeltest 2.3 (Nylander, 2004), based on the AIC (Akaike Information
Criteria) (Akaike, 1981). The most suitable model (GTR+I+G) was used in
both Maximum Likelihood (ML) and Bayesian inference (BI) analysis. ML
analysis was conducted using MEGA v.6.5 with the optimal substitution
model, and 1000 bootstrap (BS) replicates were carried out to assess the
support of the resulting groups. BI analysis was conducted using MrBayes
v.3.2.6. The Markov Chain Monte Carlo (MCMC) was run for 10 million
generations, sampling every 10,000 generations across four independent
Bayesian runs. The first 2500 trees (25\% of total trees) were discarded
as burn-in, and the remaining trees were summarized in a 50\%
majority-rule consensus tree with the posterior probabilities (PP).
Chain convergence was assessed by checking the effective sample size
(ESS) that was bigger than 200 for each parameter in Tracer v.1.6
(Rambaut \&+' Drummond, 2007), and the length and PP of each branch were
visualized by FIGTREE v.1.4.2 (Rambaut, 2009).

\textbf{2.6 Molecular dating of chlorotypes and population historical
dynamics}

Divergence dating of the chlorotypes in a Bayesian framework was
performed in BEAST v.1.7.5 (Drummond et al\emph{.} , 2012). Owing to the
absence of fossil records of Gesneriaceae with known ages, we
used\emph{P. pteropoda} (with an estimated age of \emph{c.} 1.31 Myr;
Gao et al\emph{.} , 2015; Genbank accession number KF498219) to
calibrate a molecular clock, on the grounds that it has the closest
phylogenetic relationship with \emph{P. heterotricha} (Gao et al\emph{.}
, 2015). Four independent Markov Chain Monte Carlo (MCMC) runs were
analyzed for 80 million generations and sampled every 1000 steps with
the nucleotide substitution model GTR+I+G, as selected by MRMODELTEST
2.3 (Nylander, 2004) based on the Akaike Information Criteria (AIC). The
Yule speciation tree priors and the relaxed clock model with an
uncorrelated lognormal distribution were followed. The resulting trees
from the four runs were combined by LOGCOMBINER (Bouckaert et al\emph{.}
, 2014), discarding the first 25\% of generations as burn-in. We
specified a normal distribution with standard deviation of one million
years for all the time priors. TRACER 1.5 (Rambaut \& Drummond, 2007)
was used to access the chain convergence and ensuring ESS values was
well above 200. The maximum clade credibility (MCC) tree was summed up
using TREEANNPTATOR in the BEAST package (Bouckaert et al\emph{.} ,
2014). The averages and PP of age estimates were visualized by FIGTREE
v.1.4.2 (Rambaut, 2009).

The probable ancestral biogeographical scenarios in the phylogeny
of\emph{P. heterotricha} chlorotypes were reconstructed separately by a
Statistical Dispersal-Vicariance Analysis (S-DIVA) by using the S-DIVA
(Yu \emph{et al.} , 2010), which is parsimony-based and favours
vicariance events without any prior assumptions about area relation (Yu
et al\emph{.} , 2010). We chose to be cautious in our definition of
regions, and arranged three areas based on the geographic barriers and
contiguities: (1) NW-Northwest of Changhua River, including the
populations BW, YJ, EX, WX and YG; (2) SE-Southeast of Changhua River,
including the populations WZ, QX, XA and QL; (3) SW-Southwest of
Changhua River, consisting only of the population JF. The chlorotype
trees for S-DIVA analysis were generated in BEAST v.1.7.5, including the
outgroup \emph{P. pteropoda} (see above).

In order to detect possible recent range expansions, Tajima'\selectlanguage{english}s
\emph{D}(Tajima, 1989) and Fu'\selectlanguage{english}s \emph{F} s (Fu, 1997) were calculated
for testing the deviations from the null hypothesis of constant
population size and neutral evolution for each DNA fragment, pairwise
mismatch distribution and neutrality tests for all populations and
clades (NW, SE and SW) were conducted in DNASP v. 6.12.01 based on nrITS
and cpDNA separately. These tests show a unimodal shape in the mismatch
distribution of populations that experienced historical expansion.

\textbf{3 Results}

\textbf{3.1 nrITS and cpDNA dataset acquisition and assessment}

Twelve ITS ribotypes were identified based on 16 variable nucleotide
sites. The ITS1/2 length was 701 bp, nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}) was
0.00390, the average number of nucleotide difference (K) was 2.681, and
haplotype diversity (Hd) was 0.810 (Table 2). The geographical
distribution of ribotypes showed that only a few ribotypes were shared
across the species distribution range, whereas most of them were private
to a single population (Table 1, Figure 1).

The two cpDNA sequences used contained 50 polymorphic sites, and yielded
19 chlorotypes. Nucleotide diversity (\selectlanguage{greek}π\selectlanguage{english}) was 0.00860, average number of
nucleotide difference (K) was 11.634, haplotype diversity (Hd) was 0.919
(Table 2). All chlorotypes were exclusive to a single or a group of
neighbouring populations (Table 1, Figure 1). The total length of the
combined chloroplast alignments was 1404 bp (730 bp and 674 bp in size
for \emph{trn} L-\emph{trn} F and \emph{ycf} 1b, respectively), a
congruency test implied a high level of homogeneity of the two
chloroplast sequences (\emph{P} \textgreater{} 0.5) and a striking
non-homogeneity between nrDNA and cpDNA (\emph{P} \textless{} 0.5).
Ribotype and chlorotype diversities (Hd) of the 10 sampled populations
ranged from 0 to 0.586 and 0 to 0.711 respectively (Table 1).

\textbf{3.2 Phylogeographical patterns and population genetic structure}

Overall ribotype and chlorotype diversities (\emph{h\textsubscript{T}} )
were 0.908 and 0.970 respectively, and within-population ribotype and
chlorotype diversity (\emph{h\textsubscript{S}} ) were 0.181 and 0.228
(Table 2). Remarkable population differentiation was detected, with
a\emph{G\textsubscript{ST}} = 0.801 and 0.765
and\emph{N\textsubscript{ST}} = 0.855 and 0.833. The test
statistic\emph{U} resulted in significant divergence
between\emph{N\textsubscript{ST}} and
\emph{G\textsubscript{ST}}(\emph{N\textsubscript{ST}} \emph{>
G\textsubscript{ST}}\selectlanguage{english} , \emph{P} < \selectlanguage{english}0.05), showing a significant
phylogeographical patterns of \emph{P. heterotricha} in Hainan Island.
The 10 populations of \emph{P. heterotricha} clustered into three major
groups according to the STRUCTURE analysis (Figure 4), the likelihood of
the nrDNA dataset was also the highest when samples were clustered into
three lineages (K=3, Figure S2).

The nrDNA and the cpDNA sequences respectively estimated an
among-population genetic differentiation (\emph{Fst} ) of 0.708 \selectlanguage{ngerman}± 0.319
and 0.826 ± 0.209, gene flow level (\emph{Nm} ) of 1.44 ± 3.94 and 0.25
± 0.73, the majority \emph{Nm} values were less than one (Table 3).
Furthermore, the estimated \emph{Nm} of among-region was 0.40 between NW
and SE, 0.10 between SE and SW and 0.04 between NW and SW for nrDNA
sequences, and 0.41, 0.07, 0.07 for cpDNA sequences. According to the
AMOVA, 72\% and 55\% of genetic variation among the three regions was
respectively attributable to the nrITS and cpDNA differentiation
(respectively 17\% and 33\% among populations, and 11\% and 12\% within
populations, Table 4).

The Mantel tests showed a low but highly significant positive
correlation between genetic and geographical distance matrices
(respective R\textsuperscript{2} values of 0.2544 (\emph{P} \textless{}
0.001) and 0.1479 (\emph{P} \textless{} 0.001), Figure S1), indicating a
slight IBD. The BARRIER analyses suggested two strong barriers to gene
flow among the populations of \emph{P. heterotricha} (Figure 5); one of
them separated the NW and SW populations (red line, 93\% BS), and the
other separated the SE populations from the other (green lines, 90\%
BS). We detected a third, weaker barrier (blue line with BS below 80\%)
that separated of populations WZ and QX with XA and QL based on the
nrITS dataset.

\textbf{3.3 Phylogeny of ribotypes}

Bayesian inference (BI) and Maximun likelihood (ML) trees separated the
12 ribotypes into three different monophyletic lineages with high PP/BS
values. These correspond to populations in the NW, the SE and the SW
(Figure 2A). Consistently, the phylogenetic tree (Figure 2B) also showed
three independent and well-supported clades.

\textbf{3.4 Molecular dating of cpDNA and demographic history}

The BEAST-derived chronogram of 19 chlorotypes was supported by maximum
PP, and revealed three diverging genetic lineages (Figure 3A), which
correspond to the NW, SE and SW clades revealed by the chlorotype
Median-Joining network (Figure 3B). The time-calibrated phylogenetic
tree placed the split of \emph{P. heterotricha} at \emph{c.} 0.83 Myr
(Figure 3A). The splitting time of the remaining chlorotypes from clades
NW and SE was estimated at around 0.48 Myr, roughly at the end of early
Pleistocene, and most other chlorotypes were dated between
\emph{c.}0.26-0.05 Myr (Figure 3). And two vicariance events were
detected among three clades, first events gave rise to the genetic split
in SW and the rest clades, second events offered opportunity to the
genetic divergence in SE and NW clades (Figure S3).

The results of Tajima\selectlanguage{english}'s \emph{D} test and Fu'\selectlanguage{english}s \emph{Fs} test were
presented in Table 2 with the associated simulated \emph{P} -values. The
values for \emph{D} was negative and \emph{Fs} was positive for nrDNA
sequences (\emph{D} = -0.00585, \emph{P} \textgreater{} 0.10; \emph{Fs}=
0.538, \emph{P} = 0.132), but all were positive for cpDNA sequences
(\emph{D} = 0.98381, \emph{P} \textgreater{} 0.10; \emph{Fs} =
9.119,\emph{P} \textless{} 0.01) (Table 2), indicating an insignificant
less and excess of rare nucleotide site variants for nrDNA and cpDNA
sequences separately compared to the expectation under a neutral model
of evolution, and an significant excess of rare haplotypes for nrDNA and
cpDNA sequences over what would be excepted under neutrality. The
Hierarchical mismatch analysis showed the distributions of differences
for all populations, clade NW, SE and SW were ragged or multimodal
(Figure S4), which rejected the hypothesis of demographic population
expansion of these population clades.

\textbf{4 DISSCUSION}

\textbf{4.1 Phylogeographical patterns driven by river isolation}

Through the examination of the ribotypes and chlorotypes of 217
individuals from 10 populations of the Hainan-endemic herbaceous\emph{P.
heterotricha} in the south-central mountain system of Hainan Island, low
within-population genetic diversity and high genetic differentiation
(\emph{Fst} ) among populations were revealed (Figure 1). There was a
significant phylogeographical structure and AMOVA showed high genetic
variation mainly from inter-regions (Table 4). Three clades NW, SE and
SW have evolved in prolonged isolation, as intimated by bayesian
analysis (Figure 3), which is supported by results of STRUCTURE v.2.3.4.
(Figure 4), and low gene flow (\emph{Nm} ) among regions were detected.
Two main genetic barriers were identified based on Monmonier'\selectlanguage{english}s algorithm
(Figure 5), which spatially concordant with the Changhua River and
Wanglou River in south-central mountains system of Hainan Island (Figure
2, 3 and 4).

The average population genetic divergence detected in \emph{P.
heterotricha} is high overall (\emph{G\textsubscript{ST}} = 0.801 and
0.765 for nrITS and cpDNA gene respectively), considering that genetic
differentiation among populations turns to be stronger
where\emph{G\textsubscript{ST}} \textgreater{} 0.25 (Buso et al\emph{.}
, 1998). \emph{Primulina heterotricha} grows widely in rocky streamsides
of forest valleys and humid or arid limestone and granites habitat,
which are widespread in south-central Hainan Island. Yet, there was a
slight IBD, although pollen or seed dispersal is very limited among most
populations (\emph{Nm} \textless{}\textless{} 1, Table 3) owing to the
discussed isolation effect of Changhua River, and the lack of adaptation
to long-range seed dispersal. Furthermore, the distribution of effective
pollinators (genus \emph{Glossamegilla} ) is interrupted easily by
rivers and mountains, and it even shows altitudinal differences within
the same mountain system (Zhong et al\emph{.} , 2014).

Geographic discontinuities are indeed a significant factor for
population differentiation through weakening or blocking gene flow
(Slatkin, 1985), and their impacts apparently have driven regional
genetic differentiation within \emph{P. heterotricha} populations. These
results are in line with previous studies in other groups, showing that
the geographic barrier associated with the course of Changhua River may
have stimulated lineage diversification. Two examples are the multiple
intra-specific genetic lineages detected in the endemic
species\emph{Metapetrocosmea peltata} (Merr. et Chun) W. T. Wang
(Gesneriaceae) and genus \emph{Oreocharis} (Gesneriaceae), which also
presented a strong phylogeographical population structures (Li et
al\emph{.} , 2020; Ling et al\emph{.} , 2020a).

The major role of mountains and valleys in the origins and evolution of
plants has been widely reported in the Andes (Antonelli et al., 2009;
Pennington et al\emph{.} , 2010), Ghats (Robin et al\emph{.} , 2015) and
Hengduan Mountain (Liu et al\emph{.} , 2013). And similar results of
genetic structure associated with sharp geographical discontinuities
have been reported in many Asian plants and animals. Regarding other
families and phyla, \emph{Myricaria laxiflora} (Franch.) P.Y. Zhang et
Y.J. Zhang (Tamaricaceae) in the Three Gorges mountain region (Liu et
al\emph{.} , 2009), \emph{Taxus wallichiana} Zucc. (Taxaceae) in the
Himalaya-Hengduan Mountains region (Liu et al\emph{.} , 2013), and
several endemic montane birds from the Western Ghats (Robin et
al\emph{.} , 2015), all showed nested genetic patterns across complex
mountain system isolated by valleys or rivers. The ensuing gene flow
limitation, genetic drift enhancement and possibly natural selection,
can likely have induced the genetic structure detected, that may in the
long term lead to reproductive isolation and speciation.

Our genetic data pinpoint the lower Changhua River as the stronger
gene-flow barrier between NW and SW clades (93\% bootstrap) with
minimum\emph{Nm} = 0.04 and 0.07 for nrDNA and cpDNA sequences, followed
by a secondary influence of the Wanglou River, separating SE and SW
clades with \emph{Nm} = 0.10 and 0.07 for nrDNA and cpDNA sequences,
then the third influence of the upper Changhua River dividing NW and SE
clades with \emph{Nm} = 0.40 and 0.41 for nrDNA and cpDNA sequences
(Figure 5, 90\% bootstrap). Notably, the Nansheng River splits nrITS
clade SE into two subclades (BS below 80\%, Figure 5A). Besides,
according to the BARRIER analysis, the isolation magnitudes were
proportional to the size of the rivers; the lower Changhua River
features the greatest impact, possibly owing to its much larger width
(1660 m, vs only 300 m of the upper Changhua River and 160 m of the
Wanglou River), and bigger discharge volume.

\textbf{4.2 The role of rivers on evolutionary history of \emph{P.
heterotricha}}

Our phylogenetic trees and dating analyses convincingly indicate that
the closest phylogenetic relationship of \emph{P. heterotricha}
is\emph{P. pteropoda} (mainly distributed in the South of mainland
China), and the colonization event that gave rise to \emph{P.
heterotricha}occurred at \emph{c.} 0.83 Myr, approximately coinciding
with the formation of the Qiongzhou Strait (Zhao et al\emph{.} , 2007).

The analyses of the ribotypes and chlorotypes converge in suggesting a
widespread pristine distribution of \emph{P. heterotricha} in the
south-central mountain system of Hainan Island. At \emph{c} . 0.83 Myr
(Figure 3), two vicariance events gave rise to the genetic split of the
species first in two clades (SW and the rest), and later in three clades
(SW, NW and SE, Figure 2, 3, S3). Meanwhile, The barriers represented by
the Changhua and Wanglou rivers are very old, as their origins date back
to Hercynian-Indosinian ages (236-257 Myr) and Late Yanshanian ages
(\emph{c.} 99 Myr), respectively (Wang et al\emph{.} , 2015), long
before the estimated differentiation within \emph{P. heterotricha} and
in other Hainan endemic Gesneriaceae (Ling et al\emph{.} , 2017b). Thus,
gene flow restrictions induced by these rivers may have affected many
species of the biota of Hainan Island throughout long geological
periods.

Cogent with genetic isolation between clade SW and other two clades
(at\emph{c.} 0.83 Myr) and between clades NW and SW (starting at
\emph{c.}0.48 Myr), one possibility to explain the high differentiation
of the three current lineages of \emph{P. heterotricha} is limited gene
flow after the colonization of the south-central mountain (feasibly
triggered by the tropical monsoon, Ling et al\emph{.} , 2017a, b). The
small brown and fusiform shaped seeds intimate weak dispersal
capabilities either by anemochory (i.e. wind-dispersal) or zoochory
(i.e. animal-mediated dispersal), deep and wide rivers may enhance
barriers of seeds dispersal. Furthermore, at the estimated time of
isolation between clades the water discharge of Changhua River and
Wanglou River was probably greater, consistent with the increased
precipitation regime that prevailed in the area in the early Holocene
(Zheng et al\emph{.} , 2004). Both factors likely contributed to the
strengthening of the earlier genetic barriers and shaped the observed
inter-regional genetic differentiation.

This scenario is not incompatible, however, with the possibility of
multiple colonization events from the mainland to Hainan (the distance
from the mainland is quite low, some 20 km across the straits of
Qiongzhou), followed by secondary contact and further dispersal and
isolation to originate the three genetic lineages. This would also help
explain the high genetic variation levels and structuring detected
in\emph{P. heterotricha} . Similar processes related to low distances to
the mainland and the geological ontogeny of the islands have been
suggested to be frequent in other islands and archipelagos with high
levels of endemicity (Caujap\selectlanguage{ngerman}é-Castells et al., 2017; Stuessy et
al\emph{.} , 2014). Finally, the possibility of independent colonization
events to the three areas of current distribution of \emph{P.
heterotricha} is not supported by the mismatch distribution analysis.
However, three clades of \emph{P. heterotricha} had no observable
morphological differentiation within south-central mountain system,
which may indicate the species could be at an early diversification
stage.

\textbf{4.3 The drivers of biodiversification in Hainan Island}

As one of world-wide biodiversity hotspot, the richest biotic assembly
were shaped in south-central mountains of Hainan Island. Apart from
island isolation and monsoon climate factors, the phylogeographical
patterns of multiple clades of Gesneriaceae were driven by Changhua
River (Li et al., 2020; Ling et al., 2020a), which blocked gene flow
between populations, and even facilitated significant distinctive
climates on different sides (Xing et al., 2012). Thus, differ from
uplift-driven diversification in the Hengduan Mountains (Xing \& Ree,
2017), current plant diversity in South America under Andean orogeny
(Luebert \& Weigend, 2014), the high biodiversity and endemism may be
caused by the positive isolation effect of Changhua and Wanglou Rivers.

However, Changhua River has potentially negative effects on landscape
pattern and ecosystem integrity of Hainan Tropical Rainforest National
Park by declining connection degree and increasing landscape
fragmentation. The further habitat isolation and fragmentation caused by
the expressway G9811 probably possess negative ecological effects in
future. So, the ecological corridor should be constructed across
Changhua River and expressway to lower ecological risk.

\textbf{5 CONCLUSIONS}

Our investigation revealed three well supported genetic clades in the
Hainan island endemic plant \emph{P. heterotricha} , and proposed
hypotheses to construe the phylogeographic patterns detected (Figure 2
and 3). The genetic results highlight for the first time the critical
role of riverine isolation caused by the Changhua and Wanglou Rivers in
the patterns of intraspecific evolution of plant populations on Hainan
Island. However, such barrier effects for the speciation mechanism of
this island-endemic herb and other similar plants are still in need of
further illumination, especially by reproductive studies, and enhanced
mainland sampling. Both are important to construe the origins of the
species\selectlanguage{english}' high current genetic diversity.

Importantly, the three groups of populations detected are genetically
quite distinct, which calls for their independent management in any
conservation strategy, in order to avoid disrupting their current
genetic structure and their prospects of future evolution.

\textbf{ACKNOWLEDGMENTS}

This work was funded by the Innovative Team Program of Hainan Natural
Science Foundation (2018CXTD334), National Natural Science Foundation of
China (31670230 and 41871041). We thank Zhe Zhang, Ke Tan for field
sampling and data analyses, staffs from Mt. Bawang and Yingge National
Nature Reserve for providing various helps in the field.

\textbf{CONFLICT OF INTEREST}

We have no conflicts of interest to declare.

\textbf{AUTHOR CONTRIBUTION}

\textbf{Shaojun Ling:} Data curation (lead); Formal analysis (lead);
Investigation (equal); Writing-original draft (lead). \selectlanguage{ngerman}\textbf{Juli
Caujapé-Castells:} Writing-review \& editing (supporting). \textbf{Liang
Tang:} Conceptualization (equal); Writing-review \& editing
(supporting). \textbf{Mingxun Ren:} Investigation (equal);
Conceptualization (equal); Funding acquisition (lead); Project
administration (lead); Supervision (lead); Writing-review \& editing
(supporting).

\textbf{DATA AVAILABILITY STATEMENT}

All newly acquired sequences have been deposited in GenBank
(http://www.ncbi.nlm.nih.gov) under accession numbers MW165394-MW165405
(ITS1/2), MW196628-MW196646 (\emph{trn} L-\emph{trn} F), MZ391871,
MZ479364-MZ479381 (\emph{ycf} 1b).

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Taylor, J. (1990). Amplification and Direct Sequencing of Fungal
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\& Liu, D. M. (2012). I\emph{nventory of Plant Species Diversity of
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National Academy of Sciences of the United States of America}, 114(17),
E3444--E3451. https://doi.org/10.1073/pnas.1616063114 Yang, X. B.
(2013). \emph{Flora of Hainan}. Science Press. Yu, S. X., Zang, R. G.,
\& Jiang, Y. X. (2001). Spatial analysis of species diversity in the
tropical vegetations along the vertical belt at Bawangling Nature
Reserve, Hainan Island. \emph{Acta Ecologica Sinica}, 9, 1438--1443. Yu,
Y., Harris, A. J., \& He, X. (2010). S-DIVA (Statistical
Dispersal-Vicariance Analysis): a tool for inferring biogeographic
histories. \emph{Molecular Phylogenetics and Evolution}, 56(2),
848--850. https://doi.org/10.1016/j.ympev.2010.04.011 Zhao, H. T., Wang,
L. R., \& Yuan, J. Y. (2007). Origin and time of Qiongzhou Stait.
\emph{Marine Geology \& Quaternary Geology}, 27, 33--40. Zheng, Z.,
Deng, Y., Zhang, H., Yu, R. C., \& Chen, Z. X. (2004). Holocene
environmental changes in the tropical and subtropical areas of the South
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Zhong, Y. F., Zhang, Z., Song, X. Q., \& Zhou, Z. D. (2014). Pollination
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different altitudes. \emph{Biodiversity Science}, 22(4), 467--475.
https://doi.org/10.3724/SP.J.1003.2014.13243\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected individuals. &
\textbf{TABLE 1} Sampling information, haplotype information, haplotype
diversity within-population (Hd) of \emph{P. heterotricha}, private
haplotypes are showed in FontBlod. N, number of collected
individuals.\tabularnewline
Code & Sampling sites & N & Coordinates & Elevation (m) & nrITS & nrITS
& nrITS & cpDNA & cpDNA\tabularnewline
& & & & & Ribotype (No. of individuals) & Hd & & Chlorotype (No. of
individuals) & Hd\tabularnewline
BW & Mt. Bawang & 16 & N19\selectlanguage{ngerman}º07\selectlanguage{english}'13''/E109\selectlanguage{ngerman}º09\selectlanguage{english}'36'' & 1011 & R1(16) & 0 & 0
& C1(11), C2(5) & 0.458\tabularnewline
YJ & Mt. Yajia & 16 & N19\selectlanguage{ngerman}º04\selectlanguage{english}'34''/E109\selectlanguage{ngerman}º08\selectlanguage{english}'50'' & 1163 & R1(16) & 0 & 0 &
C1(9), C2(1), \textbf{C19(6)} & 0.575\tabularnewline
EX & Mt. Exian & 31 & N19\selectlanguage{ngerman}º00\selectlanguage{english}'48''/E109\selectlanguage{ngerman}º00\selectlanguage{english}'47'' & 920-1026 & R1(29),
R2(2) & 0.124 & 0.124 & C4(18), \textbf{C5(2)}, \textbf{C6(5)},
\textbf{C7(6)}, \textbf{C8(3)} & 0.711\tabularnewline
WX & Wangxia & 15 & N19\selectlanguage{ngerman}º01\selectlanguage{english}'26''/E109\selectlanguage{ngerman}º07\selectlanguage{english}'05'' & 450 & R1(11), R2(1),
\textbf{R11(2)}, \textbf{R12(1)} & 0.467 & 0.467 & C1(12), C4(3) &
0.342\tabularnewline
YG & Mt. Yingge & 9 & N19\selectlanguage{ngerman}º01\selectlanguage{english}'27''/E109\selectlanguage{ngerman}º34\selectlanguage{english}'11'' & 600 & \textbf{R10(8)},
R6(1) & 0.222 & 0.222 & \textbf{C3(9)} & 0\tabularnewline
WZ & Mt. Wuzhi & 25 & N18\selectlanguage{ngerman}º53\selectlanguage{english}'45''/E109\selectlanguage{ngerman}º40\selectlanguage{english}'43'' & 442-823 & R5(13),
R6(2), \textbf{R7(10)} & 0.586 & 0.586 & \textbf{C18(25)} &
0\tabularnewline
QX & Mt. Qixian & 30 & N18\selectlanguage{ngerman}º43\selectlanguage{english}'10''/E109\selectlanguage{ngerman}º42\selectlanguage{english}'00'' & 590-1000 & R1(2),
R5(1), \textbf{R9(27)} & 0.190 & 0.190 & \textbf{C14(27)},
\textbf{C15(3)} & 0.186\tabularnewline
XA & Mt. Xian'\selectlanguage{english}anshilin & 16 & N18\selectlanguage{ngerman}º53\selectlanguage{english}'39''/E109\selectlanguage{ngerman}º25\selectlanguage{english}'25'' & 783 & R6(1),
R8(15) & 0.125 & 0.125 & \textbf{C16(10)}, \textbf{C17(6)} &
0.500\tabularnewline
QL & Mt. Qianlongdong & 26 & N18\selectlanguage{ngerman}º35\selectlanguage{english}'13''/E109\selectlanguage{ngerman}º26\selectlanguage{english}'04'' & 611-679 & R1(1),
R8(25) & 0.076 & 0.076 & \textbf{C11(11)}, \textbf{C12(9)},
\textbf{C13(6)} & 0.673\tabularnewline
JF & Mt. Jianfeng & 33 & N18\selectlanguage{ngerman}º46\selectlanguage{english}'11''/E108\selectlanguage{ngerman}º51\selectlanguage{english}'57'' & 918-966 &
\textbf{R3(27)}, \textbf{R4(6)} & 0.306 & 0.306 & \textbf{C9(25)},
\textbf{C10(8)} & 0.378\tabularnewline
& Total & 217 & & & & 2.099 & 2.099 & & 3.826\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01. &
\textbf{TABLE 2} Sequences of the primers used for genetic studies of
\emph{P. heterotricha}, and main descriptors of the obtained fragments
S, polymorphic sites; h, number of haplotypes; Hd, haplotypes diversity;
\selectlanguage{greek}π, \selectlanguage{english}nucleotide diversity; K, average number of nucleotide difference; *,
\emph{P} \textless{} 0.05; **, \emph{P} \textless{} 0.01.\tabularnewline
DNA fragment & Primers sequences & S & h & Hd & \selectlanguage{greek}π & K & Fragment size &
Tajima'\selectlanguage{english}s D & Fu'\selectlanguage{english}s \emph{F}s & References\tabularnewline
ITS1/2 & ITS1: 5'\selectlanguage{english}TCCTCCGCTTATTGATATGC 3' & 16 & 12 & 0.810 & 0.00390 &
2.681 & 701 bp & -0.00585 & 0.383 & White \emph{et al}.,
1990\tabularnewline
& ITS2: 5'\selectlanguage{english}GGAAGGAGAAGTCGTAACAAGG 3' & & & & & & & & &\tabularnewline
\emph{trn}L\emph{-trn}F & c: 5'\selectlanguage{english}CGAAATCGGTAGACGCTACG 3' & 10 & 6 & 0.750
& 0.00359 & 2.544 & 730 bp & 1.35790 & 5.417** & Taberlet,
1991\tabularnewline
& f: 5'\selectlanguage{english}ATTTGAACTGGTGACACGAG 3' & & & & & & & & &\tabularnewline
\emph{ycf}1b & \emph{ycf}1bF: 5'\selectlanguage{english}ACATATG CCAAAGTGATGGAAAA 3' & 42 & 16 &
0.906 & 0.01363 & 8.637 & 674 bp & 8.63671 & 7.228** & Dong \emph{et
al}., 2015\tabularnewline
& \emph{ycf}1bR: 5'\selectlanguage{english}CCTCGCCGAAAATCTGATTGTTGTGAAT 3' & & & & & & & &
&\tabularnewline
\emph{trn}L\emph{-trn}F and \emph{ycf}1b & & 52 & 19 & 0.919 & 0.00860 &
11.634 & 1,404 bp & 0.98381 & 9.119** &\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
\textbf{TABLE 3} Pairwise comparisons of \emph{Fst} estimates (above
diagonal) and deduced \emph{Nm} values (below diagonal) between
populations of \emph{P. heterotricha} based on nrITS and cpDNA
separately. \emph{Fst} values presenting significant differentiation are
in bold, ``-'' indicates estimate unavailable due to the absence of
polymorphism within the corresponding populations. & \textbf{TABLE 3}
Pairwise comparisons of \emph{Fst} estimates (above diagonal) and
deduced \emph{Nm} values (below diagonal) between populations of
\emph{P. heterotricha} based on nrITS and cpDNA separately. \emph{Fst}
values presenting significant differentiation are in bold, ``-''
indicates estimate unavailable due to the absence of polymorphism within
the corresponding populations. & \textbf{TABLE 3} Pairwise comparisons
of \emph{Fst} estimates (above diagonal) and deduced \emph{Nm} values
(below diagonal) between populations of \emph{P. heterotricha} based on
nrITS and cpDNA separately. \emph{Fst} values presenting significant
differentiation are in bold, ``-'' indicates estimate unavailable due to
the absence of polymorphism within the corresponding populations. &
\textbf{TABLE 3} Pairwise comparisons of \emph{Fst} estimates (above
diagonal) and deduced \emph{Nm} values (below diagonal) between
populations of \emph{P. heterotricha} based on nrITS and cpDNA
separately. \emph{Fst} values presenting significant differentiation are
in bold, ``-'' indicates estimate unavailable due to the absence of
polymorphism within the corresponding populations. & \textbf{TABLE 3}
Pairwise comparisons of \emph{Fst} estimates (above diagonal) and
deduced \emph{Nm} values (below diagonal) between populations of
\emph{P. heterotricha} based on nrITS and cpDNA separately. \emph{Fst}
values presenting significant differentiation are in bold, ``-''
indicates estimate unavailable due to the absence of polymorphism within
the corresponding populations. & \textbf{TABLE 3} Pairwise comparisons
of \emph{Fst} estimates (above diagonal) and deduced \emph{Nm} values
(below diagonal) between populations of \emph{P. heterotricha} based on
nrITS and cpDNA separately. \emph{Fst} values presenting significant
differentiation are in bold, ``-'' indicates estimate unavailable due to
the absence of polymorphism within the corresponding populations. &
\textbf{TABLE 3} Pairwise comparisons of \emph{Fst} estimates (above
diagonal) and deduced \emph{Nm} values (below diagonal) between
populations of \emph{P. heterotricha} based on nrITS and cpDNA
separately. \emph{Fst} values presenting significant differentiation are
in bold, ``-'' indicates estimate unavailable due to the absence of
polymorphism within the corresponding populations. & \textbf{TABLE 3}
Pairwise comparisons of \emph{Fst} estimates (above diagonal) and
deduced \emph{Nm} values (below diagonal) between populations of
\emph{P. heterotricha} based on nrITS and cpDNA separately. \emph{Fst}
values presenting significant differentiation are in bold, ``-''
indicates estimate unavailable due to the absence of polymorphism within
the corresponding populations. & \textbf{TABLE 3} Pairwise comparisons
of \emph{Fst} estimates (above diagonal) and deduced \emph{Nm} values
(below diagonal) between populations of \emph{P. heterotricha} based on
nrITS and cpDNA separately. \emph{Fst} values presenting significant
differentiation are in bold, ``-'' indicates estimate unavailable due to
the absence of polymorphism within the corresponding populations. &
\textbf{TABLE 3} Pairwise comparisons of \emph{Fst} estimates (above
diagonal) and deduced \emph{Nm} values (below diagonal) between
populations of \emph{P. heterotricha} based on nrITS and cpDNA
separately. \emph{Fst} values presenting significant differentiation are
in bold, ``-'' indicates estimate unavailable due to the absence of
polymorphism within the corresponding populations. & \textbf{TABLE 3}
Pairwise comparisons of \emph{Fst} estimates (above diagonal) and
deduced \emph{Nm} values (below diagonal) between populations of
\emph{P. heterotricha} based on nrITS and cpDNA separately. \emph{Fst}
values presenting significant differentiation are in bold, ``-''
indicates estimate unavailable due to the absence of polymorphism within
the corresponding populations.\tabularnewline
& BW & YJ & EX & WX & YG & WZ & QX & XA & QL & JF\tabularnewline
nrITS & nrITS & nrITS & nrITS & nrITS & nrITS & nrITS & nrITS & nrITS &
nrITS & nrITS\tabularnewline
BW & & 0 & 0.033 & 0.127 & \textbf{0.941} & \textbf{0.686} &
\textbf{0.920} & \textbf{0.875} & \textbf{0.960} &
\textbf{0.970}\tabularnewline
YJ & - & & 0.033 & 0.127 & \textbf{0.941} & \textbf{0.686} &
\textbf{0.920} & \textbf{0.875} & \textbf{0.960} &
\textbf{0.970}\tabularnewline
EX & 14.50 & 14.50 & & 0.070 & \textbf{0.911} & \textbf{0.673} &
\textbf{0.900} & \textbf{0.824} & \textbf{0.902} &
\textbf{0.959}\tabularnewline
WX & 3.44 & 3.44 & 6.67 & & \textbf{0.877} & \textbf{0.596} &
\textbf{0.779} & \textbf{0.594} & \textbf{0.640} &
\textbf{0.883}\tabularnewline
YG & 0.03 & 0.03 & 0.05 & 0.17 & & \textbf{0.775} & \textbf{0.928} &
\textbf{0.915} & \textbf{0.948} & \textbf{0.963}\tabularnewline
WZ & 0.23 & 0.23 & 0.24 & 0.34 & 0.15 & & \textbf{0.306} &
\textbf{0.537} & \textbf{0.560} & \textbf{0.825}\tabularnewline
QX & 0.04 & 0.04 & 0.06 & 0.14 & 0.04 & 1.13 & & \textbf{0.828} &
\textbf{0.865} & \textbf{0.938}\tabularnewline
XA & 0.07 & 0.07 & 0.11 & 0.34 & 0.05 & 0.43 & 0.10 & & \textbf{-0.030}
& \textbf{0.935}\tabularnewline
QL & 0.02 & 0.02 & 0.05 & 0.28 & 0.03 & 0.39 & 0.08 & 17.00 & &
\textbf{0.955}\tabularnewline
JF & 0.02 & 0.02 & 0.02 & 0.07 & 0.02 & 0.11 & 0.03 & 0.03 & 0.02
&\tabularnewline
cpDNA & cpDNA & cpDNA & cpDNA & cpDNA & cpDNA & cpDNA & cpDNA & cpDNA &
cpDNA & cpDNA\tabularnewline
BW & & \textbf{0.271} & \textbf{0.680} & \textbf{0.094} & \textbf{0.901}
& \textbf{0.976} & \textbf{0.958} & \textbf{0.950} & \textbf{0.929} &
\textbf{0.983}\tabularnewline
YJ & 1.35 & & \textbf{0.674} & \textbf{0.199} & \textbf{0.800} &
\textbf{0.947} & \textbf{0.931} & \textbf{0.931} & \textbf{0.904} &
\textbf{0.970}\tabularnewline
EX & 0.24 & 0.24 & & \textbf{0.541} & \textbf{0.763} & \textbf{0.718} &
\textbf{0.700} & \textbf{0.783} & \textbf{0.657} &
\textbf{0.891}\tabularnewline
WX & 4.51 & 2.01 & 0.44 & & \textbf{0.679} & \textbf{0.888} &
\textbf{0.869} & \textbf{0.890} & \textbf{0.835} &
\textbf{0.949}\tabularnewline
YG & 0.05 & 0.13 & 0.15 & 0.27 & & \textbf{1.000} & \textbf{0.985} &
\textbf{0.970} & \textbf{0.964} & \textbf{0.993}\tabularnewline
WZ & 0.01 & 0.03 & 0.20 & 0.07 & 0 & & \textbf{0.915} & \textbf{0.953} &
\textbf{0.734} & \textbf{0.992}\tabularnewline
QX & 0.02 & 0.04 & 0.21 & 0.09 & 0.01 & 0.05 & & \textbf{0.937} &
\textbf{0.659} & \textbf{0.985}\tabularnewline
XA & 0.03 & 0.04 & 0.14 & 0.07 & 0.02 & 0.02 & 0.03 & & \textbf{0.911} &
\textbf{0.952}\tabularnewline
QL & 0.04 & 0.05 & 0.26 & 0.11 & 0.02 & 0.18 & 0.26 & 0.05 & &
\textbf{0.974}\tabularnewline
JF & 0.01 & 0.02 & 0.06 & 0.03 & 0 & 0 & 0.01 & 0.03 & 0.01
&\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{TABLE 4} AMOVA analysis of \emph{P. heterotricha} based on nrITS
and cpDNA sequence data. **, \emph{P} \textless{} 0.001. & \textbf{TABLE
4} AMOVA analysis of \emph{P. heterotricha} based on nrITS and cpDNA
sequence data. **, \emph{P} \textless{} 0.001. & \textbf{TABLE 4} AMOVA
analysis of \emph{P. heterotricha} based on nrITS and cpDNA sequence
data. **, \emph{P} \textless{} 0.001. & \textbf{TABLE 4} AMOVA analysis
of \emph{P. heterotricha} based on nrITS and cpDNA sequence data. **,
\emph{P} \textless{} 0.001. & \textbf{TABLE 4} AMOVA analysis of
\emph{P. heterotricha} based on nrITS and cpDNA sequence data. **,
\emph{P} \textless{} 0.001. & \textbf{TABLE 4} AMOVA analysis of
\emph{P. heterotricha} based on nrITS and cpDNA sequence data. **,
\emph{P} \textless{} 0.001.\tabularnewline
& Degree of freedom & Sum of squares & Variation components & Percentage
of variation & Fixation indices\tabularnewline
nrITS & & & & &\tabularnewline
Among regions & 3 & 407.352 & 2.336 & 72** &
\emph{F}\textsubscript{CT}=0.718**\tabularnewline
Among populations within regions & 6 & 65.788 & 0.560 & 17** &
\emph{F}\textsubscript{SC}=0.610**\tabularnewline
Within populations & 207 & 74.040 & 0.358 & 11** &
\emph{F}\textsubscript{ST}=0.890**\tabularnewline
Total & 216 & 547.180 & 3.254 & &\tabularnewline
cpDNA & & & & &\tabularnewline
Among regions & 3 & 1241.755 & 6.099 & 55** &
\emph{F}\textsubscript{CT}=0.550**\tabularnewline
Among populations within regions & 6 & 429.121 & 3.709 & 33** &
\emph{F}\textsubscript{SC}=0.744**\tabularnewline
Within populations & 207 & 264.175 & 1.276 & 12** &
\emph{F}\textsubscript{ST}=0.885**\tabularnewline
Total & 216 & 1935.051 & 11.084 & &\tabularnewline
\bottomrule
\end{longtable}

Figure legends:

\textbf{FIGURE 1} Sampling sites and geographic distribution of the 12
ribotypes (A) and 19 chlorotypes (B) found in 10 populations
of\emph{Primulina heterotricha} from Hainan Island.

\textbf{FIGURE 2} (A) Molecular phylogeny and (B) network of the 12
ribotypes from \emph{Primulina heterotricha} based on nrITS data,
numbers on the branches showed the Posterior probability (PP)/Bootstrap
value (BS). The relative sizes of the circles in the network are
proportional to the ribotype frequencies. ^, vicariance events detected
by S-DIVA. The dashed lines represent the isolation of two rivers in
Hainan Island, and the size of dashed lines was correspondent to the
magnitude of isolation. The red frames indicate different clades.

\textbf{FIGURE 3} (A) Chronogram for 19 chlorotypes from \emph{Primulina
heterotricha} and outgroup based on combined \emph{trn} L\emph{-trn} F
and\emph{ycf} 1b data, analysed using a relaxed molecular clock
approach, posterior probabilities (PP) are showed above branches. (B)
Network of 19 chlorotypes from \emph{Primulina heterotricha} based on
combined\emph{trn} L\emph{-trn} F and \emph{ycf} 1b data, the relative
sizes of the circles in the network are proportional to the chlorotypes
frequencies, and missing chlorotypes are represented by small black
spot. ^, vicariance events detected by S-DIVA. The dashed lines
represent the isolation of two rivers in Hainan Island, and the size of
dashed lines was correspondent to the magnitude of isolation. The red
frames indicate different clades.

\textbf{FIGURE 4} Histogram of the STRUCTURE for all individuals at K=3
based on ITS1/2, the size of arrow lines were correspondent to the
isolation level of rivers.

\textbf{FIGURE 5} Results of Barrier analysis based on the (A) ITS1/2
and (B) combined \emph{trn} L\emph{-trn} F and \emph{ycf} 1b, indicating
the spatial separation of \emph{Primulina heterotricha} populations,
Delaunay triangulation (black lines) and inferred barriers (red, green
and blue lines) separating the different original regions which showing
the geographic location of the genetic barrier, black spots represent
corresponding population location. NW, northwest of Changhua R.,
including populations BW, YJ, EX, WX and YG; SE, southeast of Changhua
R., including populations WZ, QX, XA and QL; SW, southwest of Changhua
R., including population JF.

\textbf{FIGURE S1} The results graph of the relationship between genetic
and geographic distance for 10 populations based on the (A) nrITS and
(B) cpDNA.

\textbf{FIGURE S2} Bayesian inference analysis of nrITS for the
determination of the most likely number of genetic clusters (K).

\textbf{FIGURE S3} Graphical output from S-DIVA. (A) Graphical results
of ancestral distributions at each node of the phylogeny of chlorotypes
from \emph{P. heterotricha} obtained by S-DIVA. Alternative ancestral
ranges of nodes I and II (with frequency of occurrence) are shown in pie
chart form. Posterior probabilities (PP) are indicated by the sides of
pie charts. (B) Colour blocks to possible ancestral ranges at different
nodes. NW, northwest of Changhua R., including populations BW, YJ, EX,
WX and YG; SE, southeast of Changhua R., including populations WZ, QX,
XA and QL; SW, southwest of Changhua R., including population JF. ^,
possible vicariance events detected by S-DIVA.

\textbf{FIGURE S4} Mismatch distribution analysis plots across all
populations of (A) nrITS and (E) cpDNA and clade NW region of (B) nrITS
and (F) cpDNA, clade SE region of (C) nrITS and (G) cpDNA, clade SW
region of (D) nrITS and (H) cpDNA.

\textbf{FIGURE 1}\selectlanguage{english}
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\textbf{FIGURE 3}\selectlanguage{english}
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\textbf{FIGURE S1}\selectlanguage{english}
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\textbf{FIGURE S2}\selectlanguage{english}
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\textbf{FIGURE S3}\selectlanguage{english}
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\textbf{FIGURE S4}\selectlanguage{english}
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