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\begin{document}

\title{Assessing Genetic Diversity in Critically Endangered Chieniodendron
hainanense Populations within Fragmented Habitats in Hainan}



\author[1]{Haili Zhang}%
\affil[1]{Hainan University}%


\vspace{-1em}



  
  \date{May 15, 2024}


\begingroup
\let\center\flushleft
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\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Habitat fragmentation engenders a reduction in the geographic
distribution of species, thereby rendering diminutive populations
susceptible to extinction due to environmental, demographic, and genetic
factors. Chieniodendron hainanense (henceforth C. hainanense)
exemplifies a wild plant with extremely small populations (WPESP) and
faces endangerment, necessitating urgent national conservation efforts.
Elucidating the genetic diversity of C. hainanense is crucial for
uncovering underlying mechanisms and devising protective strategies. In
the present study, 35 specimens from six distinct cohort groups were
genotyped utilizing genotyping-by-sequencing (GBS) and single nucleotide
polymorphism (SNP) methodologies. The results indicated that C.
hainanense exhibits limited genetic diversity. Observed heterozygosity
within C. hainanense populations spanned from 10.79\% to 14.55\%, with
an average value of 13.15\%. The six C. hainanense populations can be
categorized into two distinct groups: (1) Diaoluoshan and Baishaling,
and (2) Wuzhishan, Huishan, Bawangling, and Jianfengling. The degree of
genetic differentiation among C. hainanense populations is relatively
weak. The observed loss of diversity can be attributed to the effects of
natural selection.%
\end{abstract}%



\sloppy


\textbf{Assessing Genetic Diversity in Critically
Endangered\emph{Chieniodendron hainanense} Populations within Fragmented
Habitats in Hainan}

Li Zhang \textsuperscript{1}, Hai-Li Zhang\textsuperscript{2}, Yukai
Chen\textsuperscript{3*}, Mir Muhammad Nizamani\textsuperscript{4*},
Tingtian Wu\textsuperscript{5}, Tingting Liu\textsuperscript{1}, Qin
Zhou\textsuperscript{3}

\textsuperscript{1} Guizhou Normal University Museum, Guizhou Normal
University, Guiyang 550001, China.

\textsuperscript{2} Sanya Nanfan Research Institute, Hainan Yazhou Bay
Seed Laboratory, Sanya 572025, China

\textsuperscript{3} Ministry of Education Key Laboratory for Ecology of
Tropical Islands, College of Life Sciences, Hainan Normal University,
Haikou 571158, China.

\textsuperscript{4} Department of Plant Pathology, Agricultural College,
Guizhou University, Guiyang, 550001, China

\textsuperscript{5} Hainan Academy of Forestry, Hainan Academy of
Mangrove, Haikou 570228, China.

Corresponding author: Yukai Chen, email: chenyukai@hainnu.edu.cn; Mir
Muhammad Nizamani, email: mirmohammadnizamani@outlook.com

Li Zhang and Haili Zhang are the co-first authors of the article.

\textbf{Abstract} : Habitat fragmentation engenders a reduction in the
geographic distribution of species, thereby rendering diminutive
populations susceptible to extinction due to environmental, demographic,
and genetic factors. \emph{Chieniodendron hainanense}
(henceforth\emph{C. hainanense} ) exemplifies a wild plant with
extremely small populations (WPESP) and faces endangerment,
necessitating urgent national conservation efforts. Elucidating the
genetic diversity of\emph{C. hainanense} is crucial for uncovering
underlying mechanisms and devising protective strategies. In the present
study, 35 specimens from six distinct cohort groups were genotyped
utilizing genotyping-by-sequencing (GBS) and single nucleotide
polymorphism (SNP) methodologies. The results indicated that \emph{C.
hainanense} exhibits limited genetic diversity. Observed heterozygosity
within \emph{C. hainanense} populations spanned from 10.79\% to 14.55\%,
with an average value of 13.15\%. The six \emph{C. hainanense}
populations can be categorized into two distinct groups: (1) Diaoluoshan
and Baishaling, and (2) Wuzhishan, Huishan, Bawangling, and
Jianfengling. The degree of genetic differentiation among \emph{C.
hainanense} populations is relatively weak. The observed loss of
diversity can be attributed to the effects of natural selection.

KEYWORDS: \emph{Chieniodendron hainanense} , Endangered plants, GBS,
SNP, WPESP

\section*{1 Introduction}

{\label{introduction}}

The conservation of biodiversity is crucial for sustaining the stability
and resilience of ecosystems. Genetic diversity constitutes a pivotal
element in the long-term persistence of species, particularly for those
facing extinction risks. \emph{Chieniodendron hainanense} represents a
rare and imperiled plant species characterized by extremely small
populations (WPESP) and is endemic to the fragmented habitats of Hainan,
China. Owing to the adverse consequences of habitat fragmentation, the
genetic diversity of \emph{C. hainanense} populations warrants
significant attention in conservation initiatives. Comprehending the
genetic architecture and diversity of these populations enables the
development of suitable management approaches and facilitates species
restoration efforts.

The genetic structure and diversity of a species play critical roles in
their ability to withstand adverse environments and evolve over time.
Studying the genetic makeup of wild plants with extremely small
populations can provide insights into their evolutionary history,
population dynamics, and response to environmental changes, enabling the
development of effective conservation strategies (Yang et al., 2018;
Zhang et al., 2021). Such research is also essential for advancing DNA
sequencing technology and conservation biology (Windig et al., 2010).

Habitat fragmentation and overuse of resources have greatly impacted
species' genetic diversity, survival, adaptability, and biodiversity (Su
et al., 2020). Habitat fragmentation reduces genetic diversity by
isolating populations and restricting gene exchange, leading to an
increase in genetic drift and inbreeding depression (Bijlsma et al.,
2012; Wang et al., 2022). The reduced genetic diversity weakens the
ability of species to adapt to environmental changes and increases the
risk of species extinction (Luquet et al., 2012). Habitat fragmentation
also reduces the number of plant populations, increases spatial
isolation, and hinders the maintenance of population genetic diversity
by disrupting dispersal, gene exchange, and inter-species interactions
(Aguilar et al., 2008).

Wild plants with extremely small populations (WPESP) are endangered
species under national key protection and require urgent rescue efforts.
Their population sizes are smaller than the minimum viable and are
usually distributed in a narrow area, making them highly vulnerable to
extinction (Zang, 2020; Jain et al., 2013). The Chinese government has
launched the National Project Plan for the Rescue and Protection of Wild
Plants in Extremely Small Populations (2011-2015) to protect 120 WPESP
species, most of which are endemic to China and have significant
ecological, scientific, cultural, and economic value (Ma et al., 2013;
Yang et al., 2020). The loss of biological and genetic values of WPESP
due to their extinction can have significant adverse effects on human
society and the ecosystem (Zhang et al., 2018). Therefore, it is crucial
to prioritize research on WPESP conservation in current biodiversity
conservation studies in China.

Chieniodendron hainanense is a second-class national key protected wild
plant and a unique member of the Annonaceae family Chieniodendron genus
in China. The species is an evergreen tree that grows up to 16 m tall
with a DBH of about 50 cm, and its distribution is limited to the
Guangxi Zhuang Autonomous Region and some areas of Hainan Province.
Habitat fragmentation due to human activities such as logging has
drastically reduced the distribution area of C. hainanense, and the wild
resources of its population are scarce. Field surveys have shown that
the existing populations of C. hainanense are mainly distributed in nine
primary forest areas dominated by fragmented secondary rainforests in
Hainan, with many original populations already disappeared (Jiang et
al., 2021). The species has poor self-renewal ability and is highly
sensitive to external disturbances, making its wild resources already
endangered. Although some research reports have focused on the
population structure, dynamic characteristics, leaf morphology, petal
nodules development, and functional biochemical activities of C.
hainanense, very few studies have investigated its endangerment
mechanisms at the molecular level. To develop targeted and comprehensive
conservation strategies, it is necessary to understand the causes of its
endangerment, including its genetic diversity, and related components
(Sork et al., 2006).

In this investigation, our objective is to evaluate the genetic
diversity and population structure of C. hainanense across its
fragmented habitats in Hainan, utilizing genotyping-by-sequencing (GBS)
and single nucleotide polymorphism (SNP) techniques. Through the
analysis of 35 specimens from six discrete cohort groups, we will
scrutinize patterns of genetic variation, differentiation, and
inbreeding both within and among the populations. The outcomes of this
research will yield valuable insights into the conservation status and
management of C. hainanense, promoting the formulation of efficacious
strategies for the preservation and restoration of this critically
endangered species. Additionally, our findings will augment the
understanding of the wider implications of habitat fragmentation on
genetic diversity in plant populations and inform conservation endeavors
for other threatened plant species inhabiting fragmented ecosystems.

\textbf{2 Materials and methods}

\subsection*{2.1 Study area}

{\label{study-area}}

Hainan Island (E108\selectlanguage{ngerman}°37\selectlanguage{english}'-111deg03\selectlanguage{english}', N18deg10\selectlanguage{english}'-20deg10) is situated in the
southern part of China, on the northern edge of the tropics (Figure 1).
It has a tropical monsoon climate and a mild climate with long summers
and short winters (Zhang et al., 2022). The annual average temperature
ranges from 22degC to 27degC, and the island has abundant water. Hainan
Island is an important distribution area for China's monsoon forests and
tropical rainforests (Zhang et al., 2022). The main forest vegetation
types in Hainan Island are tropical rainforest, evergreen broad-leaved
forest, coniferous forest, and plantation forest. Hainan Province has
the largest tropical rainforest in China, with rich flora and fauna
resources, covering 42.5\% of the total tropical area of the country (Li
et al., 2022). The critical areas for biodiversity conservation in my
country, ''National Of the 120 species of wild plants in extremely small
populations identified in the Plan for the Rescue and Protection of
WPESP (2011-2015). Hainan has 24 key protection targets. Yunnan and
Hainan are critical areas for the protection of WPESP (Sun et al., 2022)\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1} Distribution of\emph{C. hainanense} on Hainan Island
and sample collection information map. (a) Location of Hainan Island in
China, (b) Distribution of\emph{C. hainanense} on Hainan Island.

\subsection*{2.2. Materials}

{\label{materials}}

During 2019-2021, 35 \emph{C.hainanen} samples were continuously
collected from Hainan Island. They were divided into six regions
according to the geographical origin of the samples, namely, the
Bawangling population (samples BWL\_1-BWL\_8), the Jianfengling
population (JFL\_1-JFL\_5), the Luoshan population (DLS\_1-DLS\_10),
Huishan population (HS\_1-HS\_6), Wuzhishan population (WZS\_1-WZS\_3),
Baishaling population (BSL\_1-BSL\_3). The division of regional
populations, latitude, longitude, and the number of samples are shown in
Table 1.

\textbf{Table 1} The list of information for \emph{C. hainanense} sample
collection on Hainan Island\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Area code}

{\label{area-code}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Location}

{\label{location}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Longitude}

{\label{longitude}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Latitude}

{\label{latitude}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Sample}

{\label{sample}}\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\section*{Number of samples}

{\label{number-of-samples}}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BWL\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Bawanglin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
109\selectlanguage{ngerman}°07\selectlanguage{english}'-109deg12\selectlanguage{english}'E\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}°04\selectlanguage{english}'-19deg07\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BWL\_1-BWL\_8\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
JFL\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Jianfengling\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
109\selectlanguage{ngerman}°39\selectlanguage{english}'-109deg48\selectlanguage{english}'E\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18\selectlanguage{ngerman}°41\selectlanguage{english}'-18deg46\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
JFL\_1-JFL\_5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DLS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Diaoluoshan\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
109\selectlanguage{ngerman}°19\selectlanguage{english}'-109deg56\selectlanguage{english}'E\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}°03\selectlanguage{english}'-19deg04\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
DLS\_1-DLS\_10\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
HS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Huihan\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
110\selectlanguage{ngerman}°08\selectlanguage{english}'-110deg09\selectlanguage{english}'E\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}°03\selectlanguage{english}'-19deg04\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
HS\_1-HS\_6\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
WZS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Wuzhishan\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
108\selectlanguage{ngerman}°50\selectlanguage{english}'\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
18\selectlanguage{ngerman}°46\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
WZS\_1-WZS\_3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BSL\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Baishalin\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
109\selectlanguage{ngerman}°14\selectlanguage{english}'E\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
19\selectlanguage{ngerman}°11\selectlanguage{english}'N\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
BSL\_1-BSL\_3\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\subsection*{2.3 Methods}

{\label{methods}}

Firstly, DNA was extracted from 35 \emph{C. hainanense} leaf samples,
and the quality and concentration of the extracted DNA were tested
before being sent to Hangzhou Lianchuan Biotechnology Co. After
sequencing was completed, the SNPs of the \emph{C. hainanense} genome
were mined. Based on these SNPs, the phylogenetic tree analysis
of\emph{C. hainanense} was obtained using the neighbor-joining algorithm
of MEGA software. Principal component analysis (PCA) was then performed
on \emph{C. hainanense} populations based on the SNPs. Additionally, the
population structure of all samples was analyzed using admixture
software to obtain the distribution of genetic material in different
populations of \emph{C. hainanense} . Finally, genetic distances among
all samples were calculated based on the SNPs. The detailed method is
described in the paper by Chen et al. (2022).

\subsection*{2.3.1 Enzyme digestion protocol
design}

{\label{enzyme-digestion-protocol-design}}

Our simplified genome digestion scheme selected according to other
research methods is as follows, the restriction enzyme combination of
HaeIII + Hpy166II was selected. The 'Insert Size ' was selected as
'550-600bp' (Xia et al., 2019).

\subsection*{2.3.2 Sequencing Quality
Control}

{\label{sequencing-quality-control}}

The Raw data (the number of reads in the original downstream data)
generated by sequencing is pre-processed by quality filtering to obtain
CleanData. The specific processing steps are as follows: 1) remove the
adapter, 2) remove the reads containing N (N means the information of
bases cannot be determined) with a proportion of more than 5\%, 3)
remove the low-quality reads (the number of bases with quality value
Q\textless{}=10 accounts for more than 20\% of the whole reads), 4)
count the raw sequencing volume, effective sequencing volume, Q20 (the
proportion of bases with quality values greater than or equal to 20,
sequencing error rate less than 0.01), Q30 (the proportion of bases with
quality values greater than or equal to 30, sequencing error rate less
than 0.001), GC means guanine (G) and cytosine (C)content, and perform a
comprehensive evaluation.

\subsection*{2.3.3 Comparison of consistency
sequences}

{\label{comparison-of-consistency-sequences}}

We used Burrows-Wheeler aligner (BWA) software to match the sequencing
data to the consistent sequences obtained from reads clustering. Since
the reference used is the consistency sequence obtained from reads
clustering, the matching rate will vary somewhat between samples.

2.3.4 Variation detection and SNP statistics

{\label{variation-detection-and-snp-statistics}}

After comparing the data with the concordant sequences, we used Genome
Analysis Toolkit (GATK) and SAMtools software for variant detection,
retaining the SNPs that were consistently output by both software as
reliable loci. We further processed the SNP data by filtering them based
on MAF \textgreater{} 0.05 and data integrity \textgreater{} 0.8 and
retained the SNPs with polymorphisms among them. The final filtered SNPs
were input to the subsequent evolutionary analysis.Based on the obtained
SNP data, we analyzed the genetic evolution and structure of the
population using the differences in genetic information among the
samples of \emph{C. hainanense} , including the phylogenetic
relationships among the samples, population structure, principal
component analysis (PCA), and relatedness among the samples. The
following part of the analysis involves grouping samples, and the 35
samples were divided into six groups according to species for analysis.

\section*{3 Results}

{\label{results}}

\subsection*{\texorpdfstring{3.1 Simplified genome quality inspection
of\emph{C.
hainanense}}{3.1 Simplified genome quality inspection ofC. hainanense}}

{\label{simplified-genome-quality-inspection-of-c.-hainanense}}

A total of 49.16 GB of raw data were obtained through
genotyping-by-sequencing (GBS) on 35 \emph{C. hainanense} samples (Table
2). After removing the base information that could not be determined in
the adapter sequence, the remaining 48.52 GB of high-quality sequencing
data (Clean data), the average of each sample was 1.39 GB. In the
simplified genome of \emph{C. hainanense,} the average Q20 was 96.71\%;
the average Q30 was 90.97\%. The average ratio (GC content) of guanine
(G) and cytosine (C) is 40.60\%. The results indicated that the
sequencing quality of Q20 and Q30 was high, the GC distribution was
reasonable, and the sequencing information was reliable.

\textbf{Table 2 Description of the materials used and the GBS dataset}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
Sample & Raw data(bp) & Raw data & Clean data(bp) & Clean data &
Effective data (\%) & Q20 (\%) & Q30 (\%) & GC (\%)\tabularnewline
\midrule
\endhead
BWL\_1 & 8889204 & 1.33G & 8790448 & 1.31G & 98.62 & 96.64 & 90.84 &
40.71\tabularnewline
BWL\_2 & 9549572 & 1.43G & 9468076 & 1.42G & 98.89 & 96.88 & 91.32 &
40.50\tabularnewline
BWL\_3 & 7803624 & 1.17G & 7720330 & 1.15G & 98.66 & 96.64 & 90.86 &
40.62\tabularnewline
BWL\_4 & 11810220 & 1.77G & 11691152 & 1.75G & 98.76 & 96.86 & 91.31 &
40.45\tabularnewline
BWL\_5 & 9345482 & 1.40G & 9257722 & 1.39G & 98.83 & 96.85 & 91.27 &
40.22\tabularnewline
BWL\_6 & 10039148 & 1.51G & 9929304 & 1.49G & 98.69 & 96.79 & 91.17 &
40.46\tabularnewline
BWL\_7 & 12080396 & 1.81G & 11953338 & 1.79G & 98.73 & 96.63 & 90.79 &
40.58\tabularnewline
BWL\_8 & 7625154 & 1.14G & 7549916 & 1.13G & 98.75 & 96.85 & 91.29 &
40.52\tabularnewline
JFL\_1 & 10316262 & 1.55G & 10212558 & 1.53G & 98.77 & 96.72 & 91.00 &
40.47\tabularnewline
JFL\_2 & 7804798 & 1.17G & 7718160 & 1.15G & 98.60 & 96.52 & 90.61 &
40.81\tabularnewline
JFL\_3 & 11219492 & 1.68G & 11103264 & 1.66G & 98.72 & 96.81 & 91.15 &
40.73\tabularnewline
JFL\_4 & 8774572 & 1.32G & 8672816 & 1.30G & 98.63 & 96.61 & 90.80 &
40.41\tabularnewline
JFL\_5 & 4550192 & 0.68G & 4506960 & 0.67G & 98.66 & 96.70 & 90.99 &
41.15\tabularnewline
DLS\_1 & 6437982 & 0.97G & 6370380 & 0.95G & 98.67 & 96.53 & 90.62 &
40.83\tabularnewline
DLS\_2 & 9621628 & 1.44G & 9534270 & 1.43G & 98.85 & 96.94 & 91.44 &
40.24\tabularnewline
DLS\_3 & 9154942 & 1.37G & 9071040 & 1.36G & 98.74 & 96.87 & 91.34 &
40.85\tabularnewline
DLS\_4 & 8780780 & 1.32G & 8704170 & 1.30G & 98.89 & 96.58 & 90.65 &
40.34\tabularnewline
DLS\_5 & 9331598 & 1.40G & 9241430 & 1.38G & 98.80 & 96.21 & 89.92 &
40.52\tabularnewline
DLS\_6 & 6398210 & 0.96G & 6355132 & 0.95G & 98.99 & 97.12 & 91.81 &
40.54\tabularnewline
DLS\_7 & 10561018 & 1.58G & 10452984 & 1.56G & 98.75 & 96.53 & 90.58 &
40.78\tabularnewline
DLS\_8 & 7790468 & 1.17G & 7711004 & 1.15G & 98.73 & 96.55 & 90.66 &
40.87\tabularnewline
DLS\_9 & 11100134 & 1.67G & 10983862 & 1.64G & 98.72 & 96.62 & 90.81 &
40.92\tabularnewline
DLS\_10 & 15223484 & 2.28G & 15095270 & 2.26G & 98.96 & 96.78 & 91.08 &
40.59\tabularnewline
HS\_1 & 10318662 & 1.55G & 10230010 & 1.53G & 98.91 & 96.67 & 90.85 &
40.52\tabularnewline
HS\_2 & 9598258 & 1.44G & 9493420 & 1.42G & 98.65 & 96.54 & 90.66 &
41.04\tabularnewline
HS\_3 & 8071266 & 1.21G & 7978090 & 1.19G & 98.51 & 96.40 & 90.39 &
40.76\tabularnewline
HS\_4 & 6031464 & 0.90G & 5965206 & 0.89G & 98.50 & 96.67 & 90.94 &
41.34\tabularnewline
HS\_5 & 11441984 & 1.72G & 11325736 & 1.69G & 98.75 & 96.81 & 91.19 &
40.48\tabularnewline
HS\_6 & 8636182 & 1.30G & 8551474 & 1.28G & 98.77 & 96.99 & 91.62 &
40.62\tabularnewline
WZS\_1 & 8937320 & 1.34G & 8841150 & 1.32G & 98.64 & 96.63 & 90.82 &
39.99\tabularnewline
WZS\_2 & 8653196 & 1.30G & 8572226 & 1.28G & 98.80 & 96.99 & 91.58 &
40.11\tabularnewline
WZS\_3 & 14758962 & 2.21G & 14618492 & 2.19G & 98.88 & 96.90 & 91.36 &
40.49\tabularnewline
BSL\_1 & 9710790 & 1.46G & 9610614 & 1.44G & 98.75 & 96.69 & 90.93 &
40.47\tabularnewline
BSL\_2 & 8528878 & 1.28G & 8442784 & 1.26G & 98.74 & 96.54 & 90.60 &
40.25\tabularnewline
BSL\_3 & 8876886 & 1.33G & 8780592 & 1.31G & 98.70 & 96.64 & 90.83 &
40.74\tabularnewline
\bottomrule
\end{longtable}

\textbf{3.2 \emph{C. hainanense} SNP site mining}

In this study, the heterozygosity of the six populations of \emph{C.
hainanense} ranged from 10.79\% to 14.55\%, and the average
heterozygosity is 13.15\%, which shows the genetic diversity of \emph{C.
hainanense} trees is low (Table 3).

\textbf{Table 3 SNP Statistical results}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Population code & SNP number & Heter LociNum & Homo LociNum &
Hetloci-ratio\tabularnewline
\midrule
\endhead
BWL & 181002 & 25265 & 155737 & 13.93\%\tabularnewline
JFL & 171854 & 23417 & 148437 & 13.45\%\tabularnewline
DLS & 183868 & 25185 & 158683 & 13.59\%\tabularnewline
HS & 199196 & 25077 & 174119 & 12.56\%\tabularnewline
WZS & 190463 & 27590 & 162873 & 14.55\%\tabularnewline
BSL & 172311 & 18612 & 153699 & 10.79\%\tabularnewline
\bottomrule
\end{longtable}



\textbf{3.3 Genetic evolution and population analysis}

\textbf{3.3.1 Phylogenetic evolutionary tree}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

We divided the 35 \emph{C. hainanense} samples roughly into two general
taxa, which can be further subdivided into several subgroups. Group I
mainly comprise resources from Diaoluoshan (DLS) and Baishaling (BSL),
while Wuzhishan (WZS), Huishan (HS), Bawangling (BWL), and Jianfengling
(JFL) are clustered into group II (see Figure 2). The small subgroups
clustered in group I were further divided into two smaller subgroups,
indicating a relatively significant genetic relationship between each
other in this larger group. The aggregation of samples in the second
group is relatively chaotic, suggesting that there is less genetic
relationship among the individuals in this population. From this
perspective, although there is some geographical isolation between
\emph{C. hainanense} resources from different areas, there is a direct
and necessary relationship between clustering based on genetic distance
and their geographical origin.

\textbf{Figure 2} The neighbor-joining cluster of \emph{C. hainanense}
in different population

\textbf{3.3.2Analysis of population genetic structure}

When K=2, the samples from group 1 appear almost dark blue, and the
samples from group 2 appear almost light purple. Upon dividing the
samples into two subgroups, the samples from JFL and BWL were clustered
into group 1, and the remaining samples were clustered into group 2
(Figure 3). In the cross-validation (CV) error plot (Figure 4), CV error
reached its minimum value when K=1, indicating that the genetic
differences among \emph{C. hainanense} samples were relatively small,
and their genetic relationships were close. Thus, it can be
preliminarily inferred that the six \emph{C. hainanense} populations in
Hainan Island originated from the same ancestor and had undergone more
gene exchanges. In the table of genetic differentiation coefficients
among populations (Table 4), the F\textsubscript{st} values among the
six \emph{C. hainanense} populations were between -0.09648 to 0.076729.
There was a moderate degree of genetic differentiation (0.05 \textless{}
F\textsubscript{st} \textless{} 0.15) between the two populations of BSL
and JFL. Furthermore, the genetic differentiation among other
populations was low, indicating that differentiation was not significant
(F\textsubscript{st} \textless{} 0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} The population structure analysis on \emph{C.
hainanense}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4} K Selection of Population Structure

\textbf{Table 4} Genetic differentiation
coefficient(F\textsubscript{st}:above diagonal)\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& BWL & JFL & DLS & HS & WZS & BSL\tabularnewline
\midrule
\endhead
BWL & & -0.06459 & -0.01902 & -0.00697 & -0.04823 &
0.025937\tabularnewline
JFL & & & -0.03474 & 0.007746 & -0.02721 & 0.076729\tabularnewline
DLS & & & & -0.05677 & -0.09648 & -0.05853\tabularnewline
HS & & & & & -0.05853 & 0.013874\tabularnewline
WZS & & & & & & 0.042864\tabularnewline
BSL & & & & & &\tabularnewline
\bottomrule
\end{longtable}

\subsection*{3.4 Principal Component Analysis
Results}

{\label{principal-component-analysis-results}}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Thirty-five clusters of \emph{C. hainanense} were formed, which resulted
in four independent clusters. Among these clusters, 13 samples from
Jianfengling (JFL\_1-JFL\_5) and BWL (BWL\_1-BWL\_8) populations with
similar genetic backgrounds, gathered to form cluster 1 (Figure 5). Six
samples of \emph{C. hainanense} from Huishan (HS\_1-HS\_6) and Wuzhishan
(WZS\_3) populations, with similar genetic backgrounds, clustered
together to form cluster 2. Thirteen samples of \emph{C. hainanense}from
Diaoluoshan (DLS\_1-DLS\_10) and Baishaling (BSL\_1-BSL\_3) populations,
with similar genetic backgrounds, clustered together to form cluster 3.
The \emph{C. hainanense} from the Wuzhishan (WZS\_1-WZS\_2) population
was distant from the other three clusters, showing relatively long
genetic distances, and so formed a separate cluster 4.

\textbf{Figure 5} PCA analysis diagram of \emph{C. hainanense}

\subsection*{\texorpdfstring{3.5 Analysis of the genetic relationship
of\emph{C.
hainanense}}{3.5 Analysis of the genetic relationship ofC. hainanense}}

{\label{analysis-of-the-genetic-relationship-of-c.-hainanense}}

We calculated the relatedness between pairs of all samples based on the
SNPs (Figure 6). Kinship analysis reveals the genetic distance between
samples, aiding evolutionary analysis. In the kinship heatmap, redder
colors indicate closer kinship between individuals on the horizontal and
vertical axes. In contrast, a red heatmap among multiple individuals
suggests they may belong to a closely related family group. Conversely,
bluer colors indicate more distant kinship between individuals. In the
correlation heat map, the correlation coefficients of BSL\_1 and BSL\_2
were more outstanding than 0.4, indicating that the two samples of
Bawangling were very closely related to each other. The correlation
coefficients of Bawangling (BWL\_1 and BWL\_4), Hangluo Mountain (DLS\_6
and DLS\_9; DLS\_7 and DLS\_9; DLS\_8 and DLS\_10), and Huishan
(HS\_1-HS\_6) are just between 0.2 and 0.3, indicating that some genetic
exchange still exists between clusters in the case of geographical
isolation (Figure 6).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6} Relatedness of the Bawangling population (samples
BWL\_1-BWL\_8), the Jianfengling population (JFL\_1-JFL\_5), the Luoshan
population (DLS\_1-DLS\_10), Huishan population (HS\_1-HS\_6), Wuzhishan
population (WZS\_1-WZS\_3), Baishaling population (BSL\_1-BSL\_3).

\textbf{4 Discussion}

\textbf{4.1 Genetic diversity in \emph{C. hainanense}}

The genetic diversity of plants is usually influenced by their range,
longevity, reproductive systems, seed dispersal mechanisms, and
evolutionary history (Zhang et al., 2021; Nybom et al., 2004). SNP
variants are the most significant and extensive type of sequence
variation in plant genomes and can be easily identified by sequence
alignment (Fang et al., 2014). Our study yielded 477588 high-quality
SNPs through screening and filtering. \emph{C. hainanense} has a wide
ecological population in the natural wild state, but the seeds require
high germination conditions in the natural environment, limiting the
population. The natural wild \emph{C. hainanense} is distributed as
individuals in fragments in the natural tropical forest. So far, no
clusters of communities have been found, so the population density of
wild \emph{C. hainanense} is very low, resulting in the population's
weak stress resistance and reproductive ability. Population development
is long, and natural recovery is slow.

The genetic diversity of \emph{C. hainanense} was low. The genetic
diversity of \emph{C. hainanense} was low. The finding that the genetic
diversity of \emph{C. hainanense} is low has significant implications
for the conservation and management of this species. \emph{C.
hainanense} is a plant species native to Hainan Island in China. The low
genetic diversity, as indicated by the heterozygosity values ranging
from 10.79\% to 14.55\% with an average of 13.15\%, suggests that this
species may be at risk for several reasons. Low genetic diversity can
reduce a species' ability to adapt to environmental changes, such as
shifts in climate or the emergence of new diseases or pests. A more
genetically diverse population has a greater chance of possessing the
necessary genetic traits to survive and adapt to new challenges. With
low genetic diversity, there is an increased likelihood of inbreeding,
which can lead to inbreeding depression. This is a reduction in fitness
and overall health caused by the increased expression of harmful
recessive genes that become more prevalent in a closely related
population. Low genetic diversity can make a species more vulnerable to
extinction due to its reduced adaptability and potential for inbreeding
depression. When a species faces a new threat, it may not have the
genetic variation necessary to evolve and survive. Genetic diversity is
essential for maintaining the long-term stability and resilience of
ecosystems. A species with low genetic diversity might have a reduced
ability to fulfill its ecological role, which can negatively impact the
overall health and functioning of the ecosystem.

WPESP in nature can lead to an increase in inbreeding due to genetic
drift or bottleneck effects, which can lead to a decrease in population
genetic diversity, as well as a decrease in population fitness, leaving
populations unable to adapt to changing environments and facing
extinction (Theodorou, et al., 2015; Nayak, et al., 2010). Xu (2022)
reviewed the research on the genetic variation of 120 extremely small
populations of wild plants and found that only 10 of the 44 species
studied had low genetic diversity at the species level. They
are\begin{CJK}{UTF8}{gbsn}\emph{Abies ziyuanensis, thaya argyrophylla, Firmiana~danxiaensis,
Glyptostrobus~pensili, Nyssa~yunnanensis, Oreocharis~mileensis,
Pinus~squamata, Sinojackia~huangmeiensis、Taxus~contorta,
Vatica~guangxiensis}\end{CJK}\selectlanguage{english} (Xu et al., 2022). At present, the distribution
area of \emph{C. hainanense} is very narrow, and the population
collapse, dividing narrowly distributed may be less heritable than
widely dividing plant species due to inbreeding and genetic
drift(Setoguchi, et al., 2011; Zhang, et al., 2021). Low levels of
genetic diversity may impair the population's ability to adapt to new
soil and photoperiodic environments during migration, thereby inhibiting
the population's adaptive potential (Yang et al., 2018). In addition,
our field surveys showed that wild resources of \emph{C. hainanense}were
affected by human activities, such as logging, which led to the
fragmentation of its habitat. Habitat fragmentation not only reduces the
number of plant populations but also increases the spatial isolation
between populations, directly or indirectly affecting species dispersal,
gene exchange between populations, and species interactions, and hinders
the maintenance of population genetic diversity (Aguilar et al., 2008).
The findings revealed that the distribution of \emph{C.
hainanense}populations is fragmented. Many native populations have been
lost due to fragmentation. Most of the \emph{C. hainanense} populations
were found in protected areas. It also shows human intervention in
nature has extended into protected areas. There is an urgent need to
prevent the degradation of the remaining high-quality forest ecosystems
by protecting and restoring intact forests where possible, as well as by
paying attention to those forest landscapes embedded in human
modifications, such as those near logging fronts and those near
population centers.

\textbf{4.2 Genetic differentiation and genetic structure in \emph{C.
hainanense}}

PCA analysis showed that PC1, PC2, and PC3 contributed 10.04\%, 6.85\%,
and 5.07\%, respectively. The principal component analysis's clustering
results may differ from those of the other group analyses. In PCA, the
Wuzhishan population is genetically distant from other populations and
forms 1 cluster separately. In addition, the cluster structure of
principal component analysis, K-value selection of cluster structure,
and phylogenetic tree analysis of all samples resulted in the same
clusters. The fact that PCA showed the Wuzhishan population as
genetically distant from other populations, forming a separate cluster,
suggests that there may be unique genetic features in this population
that were captured by the PCA. This could indicate that the Wuzhishan
population has experienced different evolutionary pressures, gene flow
patterns, or demographic histories compared to the other populations in
the study. It is also important to note that the PCA clustering results
may differ from other group analyses. This is because PCA is focused on
capturing the maximum amount of variance in the data, while other
methods, such as phylogenetic tree analysis or population structure
analysis using Bayesian clustering approaches (e.g., STRUCTURE), might
focus on different aspects of the data. These other methods may consider
more specific evolutionary models or genetic relationships, leading to
different interpretations of the population structure. Despite the
differences in clustering results between PCA and other group analyses,
the study found that the cluster structure of the PCA, K-value selection
of the cluster structure, and phylogenetic tree analysis all resulted in
the same clusters. This consistency across different analytical methods
provides strong evidence for the observed population structure, lending
credibility to the findings. The PCA results highlight the genetic
distinctiveness of the Wuzhishan population, which may have important
implications for the conservation and management of \emph{C. hainanense}
. Understanding the factors contributing to the genetic differentiation
between populations can inform targeted conservation efforts aimed at
preserving the unique genetic diversity found in each population.
Further research on the specific genetic differences between the
Wuzhishan population and others, as well as the potential environmental
or ecological factors driving these differences, would provide valuable
insights into the species' evolutionary history and inform future
conservation strategies.

All supported the division of 6 clusters into two clusters, so it was
reasonable to divide 35 \emph{C. hainanense} samples from 6 clusters
into two clusters of 1 cluster (Diaoyu Mountain and Baisha Ridge) and 2
clusters (Wuzhishan, Huishan, Bawang Ridge, and Tsimshatsui Ridge). This
division into two primary clusters could be a result of various factors,
such as historical gene flow, geographical isolation, or habitat
fragmentation. The two clusters may have experienced different
evolutionary pressures, environmental conditions, or demographic
histories that have shaped their genetic makeup. The consistency of this
clustering pattern across multiple analytical methods (PCA, K-value
selection, and phylogenetic tree analysis) strengthens the evidence for
the observed population structure. This consistent finding implies that
the division of the six populations into two primary clusters is a
robust and meaningful representation of the genetic relationships among
these \emph{C. hainanense} samples.

Differences in population genetic structure are an important expression
of genetic diversity. A species' evolutionary potential and ability to
withstand adverse environments depends not only on the level of genetic
variation within the species but also on the genetic structure of the
species (McCauley et al., 2014; Hamrick et al., 2011; Qu et al.,
2004). Our results indicate that the six populations on Hainan Island
are divided into two broad taxa, consistent with the principal component
analysis results. A population's genetic differentiation index (Fst) is
an important parameter to measure the degree of genetic differentiation
among populations. It can explain the factors that affect the genetic
differentiation of populations (Zhou et al., 2022). The genetic
differentiation coefficient among the populations showed that the Fst
values among the six \emph{C. hainanense} populations were between
-0.09648-0.076729. There is a moderate degree of genetic differentiation
(0.05 \textless{} Fst \textless{} 0.15) between the two populations of
Baishaling (BSL) and Jianfengling (JFL). Furthermore, the genetic
differentiation among other populations is low, and the differentiation
is not noticeable (Fst \textless{} 0.05). Therefore, the genetic
differentiation among \emph{C. hainanense} populations is weak. The
existing gene flow may originate from the genetic exchange between their
common ancestor populations and be brought into other populations by
other factors such as human factors, animal carrying, or geological
factors. In addition, the topography may also affect gene flow in this
species. The terrain of Hainan Island is high in the middle and low in
the surrounding areas. It rises and descends to the periphery step by
step. The cascade structure is prominent, and the terraces, hills,
plains, and mountains form a ring-shaped layered landform (Chen et al.,
2022).

The samples taken in this study were from Wuzhishan, Diaoluoshan,
Jianfengling, and their surrounding forests. Geographically, the
Jianfengling and Baishaling populations are far apart
(\textgreater{}100km). However, the genetic distance between the two
populations is relatively small (Fst = 0.076729), possibly due to the
number of sampling populations of \emph{C. hainanense} being too small.
The population distribution of \emph{C. hainanense} is relatively
concentrated under the influence of habitat fragmentation, so the
evidence is not sufficient. Therefore, the next step is to expand the
scope of the sampling population for subsequent research and analysis.
Limited sampling: As the study itself points out, the number of sampled
populations of \emph{C. hainanense} may be too small to accurately
represent the full extent of genetic diversity and differentiation
within the species. Additional sampling from other populations,
particularly those that may be geographically intermediate between
Jianfengling and Baishaling, could help provide a more comprehensive
understanding of the genetic relationships among populations. The small
genetic distance between the Jianfengling and Baishaling populations may
be due to historical gene flow that occurred before the populations
became geographically isolated. Over time, barriers such as geographical
features, habitat fragmentation, or human activity may have restricted
the movement of individuals between populations, but the genetic legacy
of past gene flow could still be apparent in the current
populations.\emph{C. hainanense} may be capable of long-distance seed
dispersal through mechanisms such as wind, water, or animal-mediated
dispersal. Even if the frequency of long-distance dispersal events is
low, they can still contribute to maintaining gene flow between
geographically distant populations and reducing genetic differentiation.
Human activities, such as the movement of seeds or plants for
horticulture, agriculture, or reforestation efforts, may have
inadvertently facilitated gene flow between the Jianfengling and
Baishaling populations. This could result in the observed small genetic
distance despite the large geographical separation between the
populations. Small or isolated populations are more susceptible to
founder effects and genetic drift, which can reduce genetic diversity
and lead to increased genetic differentiation between populations.
However, these processes can sometimes produce counterintuitive
patterns, such as closely related populations that are geographically
distant, or vice versa. To better understand the factors influencing the
genetic structure of \emph{C. hainanense} , further research is needed.
This could include increasing the number of sampled populations and
individuals, examining historical patterns of gene flow, and
investigating potential mechanisms of seed dispersal. Ultimately, a more
comprehensive understanding of the species' genetic diversity and
population structure can inform conservation strategies and help ensure
the long-term survival of \emph{C. hainanense} .

\textbf{4.3 Conservation and Management Strategies}

The findings of this study hold significant implications for the
conservation and management of critically endangered \emph{C.
hainanense} populations within fragmented habitats in Hainan. Based on
the observed patterns of genetic diversity, population structure, and
inbreeding, the following conservation and management strategies are
recommended:

\begin{enumerate}
\tightlist
\item
  Habitat protection and restoration: Prioritize the preservation of
  existing habitats and restore degraded areas to improve habitat
  quality and connectivity. Enhancing connectivity between fragmented
  habitats will facilitate gene flow among C. hainanense populations,
  promoting genetic diversity and reducing the risk of inbreeding.
\item
  Assisted gene flow and population augmentation: Introduce individuals
  from genetically diverse populations into small, isolated populations
  to increase genetic diversity and reduce inbreeding. This approach
  should be undertaken cautiously, considering potential ecological and
  genetic risks, such as outbreeding depression and disruption of local
  adaptations.
\item
  In-situ conservation: Efforts to protect the natural habitats
  of\emph{C. hainanense} should be prioritized. This includes
  maintaining or establishing protected areas, enforcing regulations to
  prevent habitat destruction, and promoting sustainable land use
  practices.
\item
  Ex situ conservation: Establish ex situ conservation programs, such as
  seed banks and living collections in botanical gardens, to preserve
  the genetic diversity of C. hainanense. These efforts can serve as a
  genetic reservoir for potential reintroduction or population
  augmentation initiatives in the future.
\item
  Monitoring and adaptive management: Implement long-term monitoring
  programs to track changes in genetic diversity, population structure,
  and habitat conditions. Utilize the collected data to inform adaptive
  management strategies, ensuring the conservation efforts remain
  effective and responsive to emerging threats or changing
  circumstances.
\item
  Community engagement and education: Involve local communities in
  conservation efforts by raising awareness about the importance of
  preserving C. hainanense and its habitat. Promote sustainable land use
  practices and develop community-based conservation initiatives to
  empower local stakeholders in the protection and restoration of the
  species' habitat.
\item
  Legal protection and policy development: Strengthen the legal
  protection status of C. hainanense and its habitat by incorporating
  the species into national and regional conservation plans. Develop and
  enforce policies that minimize habitat destruction, such as regulating
  land-use change, deforestation, and infrastructure development within
  the species' range.
\item
  Collaborative research and information sharing: Foster collaboration
  among researchers, conservation practitioners, and policymakers to
  facilitate the exchange of knowledge, data, and best practices in the
  conservation of C. hainanense. Encourage interdisciplinary research
  that integrates genetics, ecology, and social science to develop
  comprehensive conservation strategies.
\item
  Climate change adaptation: Consider the potential impacts of climate
  change on C. hainanense populations and their habitats. Develop
  proactive conservation measures that enhance the species' resilience
  to climate change, such as assisted migration, habitat restoration in
  areas with suitable future climatic conditions, and incorporation of
  climate change projections into spatial conservation planning.
\end{enumerate}

By implementing these conservation and management strategies, we can
contribute to the preservation and restoration of the critically
endangered \emph{C. hainanense} and its fragmented habitats in Hainan,
while also informing efforts to protect other endangered plant species
facing similar challenges.

\textbf{4.4 Implications for conservation}

Wild plants with extremely small populations are usually highly inbred.
The characteristics of low transmission diversity and high frequency of
genetic drift need to be confirmed. Genetic rescue increases genetic
diversity, improves fitness, and enhances adaptation to maintain the
species' long-term survival (Sun et al., 2021). Instead of increasing
the number of surviving individuals by collecting inbred seeds or
asexual cuttings of \emph{C. hainanense} , researchers should focus on
designing artificial hybridization strategies to reduce inbred
offspring. One of the measures to protect the \emph{C.
hainanense}population is to set up multiple protection sites to protect
natural populations and their surrounding habitats. The second is to
strengthen the gene flow between populations, such as constructing
artificial ex-situ protection populations that should be obtained from
as many different populations as possible. During ex-situ conservation,
the exchange of seeds and seedlings between populations should be
increased to create conditions for gene exchange and recombination
artificially.

\textbf{4.5 Limitations and Future Research Directions}

Despite the valuable insights generated from this study, certain
limitations and future research directions should be acknowledged:

\begin{enumerate}
\tightlist
\item
  Sample size and representation: The limited number of samples (35) and
  populations (six distinct cohort groups) included in this study may
  not entirely capture the full extent of genetic diversity and
  population structure of C. hainanense. Future studies should aim to
  increase sample sizes and cover a broader range of fragmented habitats
  to provide a more comprehensive understanding.
\item
  Gene flow and landscape connectivity: This study did not investigate
  the gene flow and landscape connectivity among the fragmented
  habitats. Understanding how landscape features influence gene flow
  between populations can provide crucial information for developing
  conservation corridors and habitat restoration strategies. Future
  research should incorporate landscape genetic approaches to
  investigate the impact of habitat fragmentation on gene flow in C.
  hainanense populations.
\item
  Functional genetic diversity: The assessment of genetic diversity in
  this study primarily focused on neutral genetic markers (SNPs).
  However, functional genetic diversity, which reflects the genetic
  variation underlying ecologically important traits, is also crucial
  for species survival and adaptation. Future studies should explore
  functional genetic diversity by incorporating candidate genes or
  whole-genome sequencing approaches.
\item
  Long-term monitoring and adaptive management: The dynamics of genetic
  diversity and population structure can change over time due to various
  factors, such as climate change, anthropogenic disturbances, and
  random genetic drift. Long-term monitoring of C. hainanense
  populations is essential for detecting changes in genetic diversity
  and adjusting conservation strategies accordingly.
\end{enumerate}

\textbf{5. Conclusion}

This study provides valuable insights into the genetic diversity and
population structure of the critically endangered C. hainanense within
fragmented habitats in Hainan. The findings contribute to our
understanding of the impact of habitat fragmentation on genetic
diversity in plant populations and inform conservation efforts for other
endangered plant species within fragmented ecosystems.The genetic
diversity of \emph{C. hainanense} was low, and the heterozygosity of the
six populations of \emph{C. hainanense} ranged from 10.79\% to 14.55\%.
The average heterozygosity was 13.15\%. Six \emph{C. hainanense
population} into the population (Diaoluoshan, Baishaling) and population
(Wuzhishan, Huishan, Bawangling, Jianfengling) the two population. In
addition, the genetic differentiation among \emph{C.
hainanense}populations is weak.

\textbf{Author Contributions:} Conceptualization, Y.K.C., M.M.N.;
methodology, Y.K.C., H.L.Z., M.M.N.; software, L.Z., H.L.Z., Y.K.C.,
M.M.N., T.T.W., T.T.L and Q.Z.; validation, M.M.N.; formal analysis,
L.Z., H.L.Z., Y.K.C., M.M.N., T.T.W., T.T.L and Q.Z.; investigation,
Y.K.C., and L.Z.; resources, Y.K.C.; data curation, Y.K.C.;
writing---original draft preparation, L.Z., H.L.Z., Y.K.C., M.M.N.,
T.T.W., T.T.L and Q.Z.; writing---review and editing, L.Z., H.L.Z.,
Y.K.C., M.M.N., T.T.W., T.T.L and Q.Z.; visualization, Y.K.C.;
supervision, Y.K.C.; project administration, Y.K.C.; funding
acquisition, Y.K.C. All authors have read and agreed to the published
version of the manuscript. The data presented in the study are deposited
in the Dryad repository.

\textbf{Funding}

This study was supported by the National Science Foundation of China
(31760119), the National Science Foundation of Hainan Province
(320MS038), the Key Science and Technology Program of Hainan Province
(No. SQKY-2022-0003).

\textbf{Data Availability Statement}

The data that support the findings of this study are openly available in
the Science Data Bank at https://www.scidb.cn/s/6ZZNn2.

\textbf{Conflict of interest}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

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