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\begin{document}

\title{From nine to two: Ultra-barcoding develops a rigorous species
delimitation framework in Paris section Axiparis (Melanthiaceae)}



\author[1]{Yunheng Ji}%
\author[1]{Jin Yang}%
\author[2]{Jacob Landis}%
\author[3]{Shuying Wang}%
\author[1]{Zhenyan Yang}%
\author[1]{Lei  Jin}%
\author[3]{Li Li}%
\author[1]{Jun-Bo Yang}%
\author[4]{Ting Shuang Yi}%
\affil[1]{Kunming Institute of Botany Chinese Academy of Sciences}%
\affil[2]{Cornell University}%
\affil[3]{Kunming Institute of Botany Key Laboratory of Plant Diversity and Biogeography of East Asia}%
\affil[4]{Kunming Institute of Botany,Chinese Academy of  Sciences}%


\vspace{-1em}



  
  \date{June 16, 2024}


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\selectlanguage{english}
\begin{abstract}
Species delimitation is a difficult task in traditional morphology-based
taxonomy. The ultra-barcoding approach, which uses whole plastid genomes
(plastomes) and nuclear ribosomal DNA (nrDNA) regions as extended DNA
barcodes for species identification and delimitation, has been
recommended as one of the candidate techniques for plant barcoding 2.0.
Yet, the efficacy of this approach in delineating species boundaries
remains poorly understood. Here, we attempt to decipher species
delimitation in the taxonomically challenging clade, Paris L. section
Axiparis H. Li, using phylogenetic inference and multiple sequence-based
species delimitation methods (ABGD, SDP, and mPTP) utilizing complete
plastomes and nrDNA clusters from multiple accessions per described
species. The results suggest that only two species-level taxonomic units
that possess not only morphological uniqueness but also genetic
distinctiveness and evolutionary independence can be recognized in P.
sect. Axiparis. Therefore, previous taxonomic work overemphasized minor
intraspecific morphological differences to establish species, and thus
resulted in proliferation of as many as seven synonyms in this clade.
Inferred from ultra-barcoding analyses, we propose a taxonomic revision
of P. sect. Axiparis which will help inform future decisions regarding
species conservation in the commercially valuable and severely
threatened genus Paris. This case study indicates that the
ultra-barcoding approach has great promise for developing a rigorous
species delimitation framework that will facilitate credible taxonomic
revision especially in taxonomically difficult plant taxa.%
\end{abstract}%



\sloppy


\textbf{From nine to two: Ultra-barcoding develops a rigorous species
delimitation framework in}\emph{Paris} \textbf{section\emph{Axiparis}
(Melanthiaceae)}

\textbf{Running title: Species delimitation in}
\emph{Paris}\textbf{section \emph{Axiparis}}

\textbf{Yunheng Ji\textsuperscript{1,2*}, Jin Yang\textsuperscript{1,3},
Jacob B. Landis\textsuperscript{4}, Shuying Wang\textsuperscript{1,3},
Zhenyan Yang\textsuperscript{1}, Lei Jin\textsuperscript{1}, Li
Li\textsuperscript{1}, Junbo Yang\textsuperscript{5*}, Tingshuang
Yi\textsuperscript{5*}}

\textsuperscript{1}CAS Key Laboratory for Plant Diversity and
Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy
of Sciences, Kunming, Yunnan 650201, P. R. China

\textsuperscript{2}Yunnan Key Laboratory for Integrative Conservation of
Plant Species with Extremely Small Population, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, P. R. China

\textsuperscript{3}School of Life Science, Yunnan University, Kunming,
Yunnan 650009, P. R. China

\textsuperscript{4}School of Integrative Plant Science, Section of Plant
Biology and the L. H. Bailey Hortorium, Cornell University, Ithaca,
NY14850, USA

\textsuperscript{5}Germplasm Bank of Wild Species, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, P. R. China

\textbf{*Corresponding author email} : jiyh@mail.kib.ac.cn,
jbyang@mail.kib.ac.cn, tingshuangyi@mail.kib.ac.cn



\textbf{Abstract}

Species delimitation is a difficult task in traditional morphology-based
taxonomy. The ultra-barcoding approach, which uses whole plastid genomes
(plastomes) and nuclear ribosomal DNA (nrDNA) regions as extended DNA
barcodes for species identification and delimitation, has been
recommended as one of the candidate techniques for plant barcoding 2.0.
Yet, the efficacy of this approach in delineating species boundaries
remains poorly understood. Here, we attempt to decipher species
delimitation in the taxonomically challenging clade, \emph{Paris} L.
section \emph{Axiparis} H. Li, using phylogenetic inference and multiple
sequence-based species delimitation methods (ABGD, SDP, and mPTP)
utilizing complete plastomes and nrDNA clusters from multiple accessions
per described species. The results suggest that only two species-level
taxonomic units that possess not only morphological uniqueness but also
genetic distinctiveness and evolutionary independence can be recognized
in \emph{P} . sect.\emph{Axiparis} . Therefore, previous taxonomic work
overemphasized minor intraspecific morphological differences to
establish species, and thus resulted in proliferation of as many as
seven synonyms in this clade\emph{.} Inferred from ultra-barcoding
analyses, we propose a taxonomic revision of \emph{P} . sect.
\emph{Axiparis} which will help inform future decisions regarding
species conservation in the commercially valuable and severely
threatened genus \emph{Paris} . This case study indicates that the
ultra-barcoding approach has great promise for developing a rigorous
species delimitation framework that will facilitate credible taxonomic
revision especially in taxonomically difficult plant taxa.

\textbf{Key words}

DNA barcoding, plastome, ribosomal DNA, species boundary, taxonomic
revision, synonyms

\textbf{Introduction}

Species delimitation is the crucially important first step for designing
research in many fields of biology (Mace, 2004). Traditionally, species
boundaries are defined by taxonomists based on the analysis of
morphological variation (Lewin, 1981; Henderson et al., 2005), which has
resulted in massive disagreements over species identification and
delimitation due to either phenotypic plasticity or lack of
taxonomically robust morphological characters at the species level
(Hebert et al., 2003; de Queiroz, 2007). To compensate, additional data
types, such as molecular and ecological profiles, are needed to
explicitly decipher species boundaries (e.g., Sites and Crandall, 1997;
Sites and Marshall, 2003; Ellis et al., 2006; Bickford et al., 2007;
Eisenring et al., 2016) especially for taxonomically challenging taxa
(e.g., Duminil and Di Michele, 2009; Su et al., 2015; Lambert et al.,
2017; Cheng et al., 2020; Ma et al., 2020).

Analysis of DNA sequence variation can provide useful information for
identifying and delineating species (Brower et al., 1996; Hebert et al.,
2003; Pons et al., 2006; Duminil et al., 2012; Puillandre et al., 2012).
DNA barcoding, a technique that involves the standardized utilization of
one or a few sequence regions for species identification and
discrimination (Hebert et al., 2003; Kress et al., 2005; Hollingsworth,
2011; Hollingsworth et al., 2009, 2011, 2016), has been widely used as a
molecular tag for species diagnosis and delimitation (e.g., Hebert et
al., 2004; Liu et al., 2011; Kekkonen and Hebert, 2014; Mutanen et al.,
2015). Despite the mitochondrial \emph{COI} gene acting as the standard
DNA barcodes in animals (Hebert et al., 2003, 2004; Kekkonen and Hebert,
2014; Mutanen et al., 2015), reliable and accurate species
identification and delimitation based on standard DNA barcodes
(i.e.\emph{rbcL} , \emph{matK} , \emph{trnH-psbA} , and ITS) remains
problematic in plants, especially in lineages that have experienced
rapid radiations or complicated evolutionary histories (Hollingsworth et
al., 2009, 2011, 2016; Hollingsworth, 2011; Coissac et al., 2016).

With next-generation DNA sequencing (NGS) technologies, the concept of
DNA barcoding for plant species has been extended from a single or few
sequence regions to genome-wide sequence variations (Li et al., 2015;
Coissac et al., 2016; Hollingsworth et al., 2016). The development of
extended DNA barcoding approaches for plant species discrimination has
been referred to as `plant barcoding 2.0' (Hollingsworth et al., 2016).
The ultra-barcoding approach, which employs both whole plastid genomes
(plastomes) and nuclear ribosomal DNA (nrDNA) clusters as extended DNA
barcodes for resolution of species identification and delimitation (Nock
et al., 2011), is one of the recommended techniques for plant barcoding
2.0 (Hollingsworth et al., 2016). This approach yields a tremendous
amount of sequence variation, allowing far more sensitivity than
standard DNA-barcoding (Nock et al., 2011; Kane et al., 2012; Dodsworth,
2015; Li et al., 2015; Hollingsworth et al., 2016), and thus providing
far more genetic information than standard DNA barcodes to improve
resolution in species identification (Ruhsam et al., 2015; Firetti et
al., 2017; Fu et al., 2019; Ji et al., 2019a, 2020; Knope et al., 2020;
\selectlanguage{polish}Ś\selectlanguage{english}lipiko et al., 2020). Compared with Restriction-site Associated DNA
sequencing (Baird et al., 2008), another recommended approach for plant
barcoding 2.0 (Hollingsworth et al., 2016) that has been extensively
used to generate genomic data for species delimitation in recent years
(e.g., Wu et al., 2018; Donkpegan et al., 2020; Li et al., 2020; Ma et
al., 2020; Zhou et al., 2020), the above mentioned ultra-barcoding
approach is seldomly applied to investigate species boundaries. As a
result, its efficacy in delineating species is still poorly understood.

This study focuses on \emph{Paris} Linn. section \emph{Axiparis} H. Li
(Melanthiaceae), a well-supported monophyletic clade including nine
described species restricted to southwest China and the Himalayas (Li,
1998; Ji et al., 2006, 2019b; Huang et al., 2016). Discovery of the
first species (\emph{Paris vaniotii} ) of this group, dating back to
1906, was based on collections from Guizhou, southwestern China
(L\selectlanguage{ngerman}éveillé, 1906), with the remaining species have been described since
the 1980s (Takhtajan, 1983; Li, 1984, 1992; He, 1990; Ji et al., 2017;
Xu et al., 2019; Yang et al., 2019). The rapidly increasing species
count in the last 40 years led to considerable taxonomic confusion
in\emph{P. sect. Axiparis} . After critical examination of types and
specimens assigned to these species, it was found that, except for
slight differences in leaf shape and size (Figure 1), they exhibit high
levels of similarity in flower, fruit, and seed morphology.
Additionally, leaf shape and size of foliar displays have high levels of
intraspecific variations that sometimes far exceed the divergence used
for species diagnosis. High variability of key diagnostic morphological
characters makes species delimitation and correct species identification
a particularly difficult task in this clade.

Due to the above-mentioned aspects, \emph{P.} sect. \emph{Axiparis} is
in great need of taxonomic revision, and thus provides an ideal model to
evaluate the usefulness of ultra-barcoding approach for species
delimitation. In this study, we attempt to use the ultra-barcoding
approach to establish a basic understanding of species boundaries in
this taxonomically challenging lineage. Specifically, we employed a
genome skimming method (Straub et al., 2012) to recover complete
plastomes and nuclear rDNA sequences by sampling multiple accessions per
species within \emph{P.} sect. \emph{Axiparis} . Based on phylogenetic
inference and multiple sequence-based species delimitation methods, we
test whether the ultra-barcoding approach could develop a rigorous
species delimitation framework in \emph{P.} sect\emph{.
Axiparis.}Inferred from the current study, we discuss the prospects on
the ultra-barcoding approach as a tool to explore species boundaries in
taxonomically challenging plant groups.

\textbf{Materials and methods}

\textbf{Plant samples, shotgun sequencing, assembly and annotation}

In total, 51 individual plants from all nine described species
of\emph{P.} sect. \emph{Axiparis} were sampled, specifically \emph{Paris
axialis} (4 accessions), \emph{P. dulongensis} (8 accessions), \emph{P.
forrestii} (10 accessions),\emph{P. guizhouensis} (3 accessions),
\emph{P. lihengiana} (3 accessions), \emph{P. rugosa} (8 accessions),
\emph{P. tenchongensis} (7 accessions), \emph{P. vaniotii} (5
accessions), and \emph{P. variabilis}(3 accessions)(Table S1). Plant
material was collected from the field, and voucher specimens were
deposited at the herbarium of Kunming Institute of Botany, Chinese
Academy of Science (KUN). For each species, multiple individuals within
a species representing different localities were included. The sampling
strategy allows testing for species-level monophyly and to explore
species boundaries.

Total genomic DNA for each accession was isolated from
\textasciitilde{}10 mg silica gel dried leaf tissues collected in the
field using CTAB method (Doyle and Doyle, 1987). Approximately 5 \selectlanguage{greek}µ\selectlanguage{english}g of
purified genomic DNA was used to construct shotgun libraries with a
TruSeq DNA Sample Prep Kit (Illumina, Inc., USA) following the
manufacturer's instructions. Low coverage genome sequencing was
performed on the Illumina HiSeq 2500 system using paired end (2\selectlanguage{ngerman}×150)
reads. Raw reads were filtered to remove adaptors and low quality read
using the NGS QC Toolkit (Patel and Jain, 2012) with default parameters.

We used a reference-guided method for plastome and nrDNA assembly. The
published complete plastome (MN125565) and nrDNA sequence (MN174877)
of\emph{P. forrestii} was used as the reference to assembly newly
sequenced platomes and nrDNA sequences using GetOrganelle, a pipeline
developed by Jin et al. (2020). Briefly, the pipeline consists of a
customized python script, which conducts BLAST to search read mapping to
the reference plastome, was used to enrich plastid-like reads, and
assembled them into contigs with Bowtie v2.2.6 (Langmead et al., 2012),
with default parameters and preset options. Contigs were iteratively
extended to recover whole plastomes in SPAdes v3.10.1 (Bankevich et al.,
2012). For nrDNA assembly, we first excluded all plastid-like reads, and
used the steps described above to generate the nrDNA sequence (including
26S, 18S and 5.8S ribosomal RNA genes and the internal transcribed
spacers) of each accession.

The newly assembled plastomes were annotated with the Dual Organellar
Genome Annotator database (Wyman \emph{et al} ., 2004). The annotation
of protein-coding genes was further confirmed with a BLAST search
against the NCBI protein database. Genes putatively annotated as
transfer RNA (tRNA) were further verified by tRNAscan-SE 1.21 (Schattner
\emph{et al.} , 2005) with default parameters. The boundary of the
large-single copy (LSC), small-single copy (SSC), and inverted-repeat
(IR) regions for each plastome were visually examined and manually
adjusted with Geneious V10.2.3 (Kearse \emph{et al} ., 2012). For nrDNA
annotation, the ribosomal RNA genes and boundaries with the intergenic
transcribed spacer (ITS) regions were annotated and defined by
comparison with the reference in Geneious v10.2.3 (Kearse \emph{et al}
., 2012).

\textbf{Phylogenetic analyses}

Using inferred phylogenies, we tested whether the nine nominal species
within \emph{P.} sect. \emph{Axiparis} were monophyletic units. The
complete plastome and nrDNA sequences were aligned using MAFFT v7.450
(Katoh and Standley, 2013). For each dataset, phylogenetic relationships
were inferred using both maximum likelihood (ML) and Bayesian inference
(BI) methods. For each analysis, \emph{Paris thibetica}
representing\emph{P.} section \emph{Thibeticae} , the closest relative
of \emph{P.}sect. \emph{Axiparis} (Ji et al., 2019b), was selected as
the outgroup. The ML analysis was performed with RAxML-HPC BlackBox
v8.1.24 (Stamatakis, 2006), using the GTRCAT sequence substitution
model. The best-scoring ML tree for each dataset was produced with 1,000
bootstrap (BS) replicates to provide support values for each node. The
BI tree was inferred using MrBayes v3.2 (Ronquist and Huelsenbeck,
2003), with the sequence substitution model GTR + G (plastomes) and
GTR+I+G (nrDNA), which were recommended by Modeltest v3.7 (Posada and
Crandall, 1998) using the Akaike information criterion (Posada and
Buckley, 2004). Two independent Markov Chain Monte Carlo (MCMC) runs
were performed with one million generations, sampling every 100
generations, with the initial 25\% of the sampled trees as burn-in.
Posterior probability (PP) values were computed based on the remaining
trees.

\textbf{Sequence-based species delimitation}

Based on complete plastomes and nrDNA sequences, we used three different
approaches to explore putative species boundaries within \emph{P.}
sect.\emph{Axiparis} : the distance-based method automatic barcode gap
discovery (ABGD; Puillandre et al., 2012), the coalescence-based method
species delimitation plugin (SDP; Masters et al., 2011) in Geneious
v.10.2.3 (Kearse et al., 2012), and the tree-based method multi-rate
Poisson tree processes model (mPTP; Kapli et al., 2017). The ABGD
analysis was conducted with the online server
(http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html) with default
settings (Pmin=0.001, Pmax=0.1, Steps=10, X=1.5, Nb=20). All three
genetic distance models (JC69, K2P, and uncorrected\emph{P} -distances)
specified by the program were used. All assignments for intraspecific
divergence (\emph{P} ) values between 0.001 and 0.100 were recorded.
Next, we used the mPTP v0.2.3 algorithm (Kapli et al., 2017), an
improvement to PTP (Zhang et al., 2013), to estimate Bayesian support
for the putative species boundaries. The mPTP analysis was performed on
the web server (http://species.h-its.org/ptp/) with standard default
settings, using the inferred ML trees of plastome and nrDNA as inputs
since the branch lengths of ML tree represent number of sequence
mutations. For each analysis, the MCMC algorithm was conducted for 50
million generations, thinning every 100 generations and a 20\% burn-in.
Lastly, we performed SDP analyses to test whether species-like taxonomic
units proposed by ABGD and mPTP analyses are evolutionarily distinct
species. Based on ML and BI trees of plastome and nrDNA datasets, the
distinctiveness of these candidate species was estimated using
Rosenberg's \emph{P\textsubscript{(AB)}} : the probability of reciprocal
monophyly under a random coalescent model (Rosenberg, 2007), and
Rodrigo's \emph{P} \textsubscript{(\emph{R} D)}: the probability that a
clade has the observed degree of distinctiveness due to a random
coalescent process (Rodrigo et al., 2008).

\textbf{Results}

\textbf{Low coverage genome sequencing and assembly}

The summary of low coverage genome sequencing is shown in Table S2,
which produced 5.785 to 33.107 million paired-end clean reads per
accession. Of these, 70,765 to 1,044,267 and 3,875 to 66,322 were mapped
to the reference plastome and nrDNA, respectively. Based on these reads,
we assembled the complete plastomes and nrDNA for each sample, with an
average sequencing coverage ranging from 67.67× to 1,711.16× and 92.58×
to 1,710.43×, respectively. The reference-guided assembly generated the
complete plastomes of all samples, which possess a typical quadripartite
structure, with the sequence length varying from 156,061 to 157,653 bp.
The plastomes contain 114 genes, including 80 protein-coding genes, 30
tRNA genes, and four plastid rRNA genes. In addition, the assembly of
nrDNA entirely covered 18S, ITS1, 5.8S, ITS2 and 26S regions in all
accessions, with the sequence length being either 5,851 or 5,852 bp.

\textbf{Species-level monophyly}

Alignment of the plastome sequences yielded a matrix of 160,681
positions, in which we identified 1,724 variable sites (1.07\%) with
1,192 (0.74\%) being parsimoniously informative (Table S3). The ML and
BI analyses of complete plastomes produced highly similar tree
topologies (Figure 2). Two diverging clades were recovered in \emph{P.
sect. Axiparis} . Clade I (BS=100\%, PP=1.00) comprises accessions of
\emph{P. dulongensis} ,\emph{P. forrestii} , \emph{P. rugosa} , and
\emph{P. tengchongensis} . These species share the morphological
similarity of stamens numbering twice as many as the sepal number
(two-whorled stamens), and being geographically distributed from the
Hengduan Mountains (southwestern China) to the Himalayas (Figure 3).
Comparatively, Clade II (BS=100\%, PP=1.00) includes accessions of
\emph{P. axialis} , \emph{P. guizhouensis} , \emph{P. lihengiana} ,
\emph{P. vaniotii} , and \emph{P. variabilis} , whose stamen numbers are
three times as many as the sepal number (three-whorled stamen). These
species are distributed from central China to the Wumeng Mountains in
southwestern China (Figure 3). Strikingly, none of the nine nominal
species within \emph{P.} sect.\emph{Axiparis} were recovered as a
monophyletic unit in either ML or BI phylogeny.

The alignment of the nrDNA sequences produced 193 variable sites
(3.30\%) with 112 (1.91\%) being parsimoniously informative (Table S3).
ML and BI analyses of nrDNA sequences (Figure 4) recovered two major
clades within \emph{P.} sect. \emph{Axiparis} , corresponding to those
from the plastome reconstruction but with slightly lower support values
(clade I: BS=98\%, PP=1.00; clade II: BS=97\%, PP=1.00). Similar to the
plastome phylogeny, analyses of nrDNA sequences failed to resolve any of
the nine nominal species within \emph{P.} sect\emph{. Axiparis} as a
monophyletic unit.

\textbf{Species delimitation scheme}

Complete plasomes and nrDNA sequences of 51 individuals were used for
species delimitation analyses. Overall, ABGD, SDP, and mPTP analyses of
plastid and nuclear datasets produced highly congruent results that are
reflected in the phylogenetic trees (Figure 2 \& 4). The ABGD analyses
of complete plastomes (Table S4) and nrDNA sequences (Table S5) resulted
in a stable count (n = 2) of species division with a range of prior
intraspecific values (plastomes: \emph{P} = 0.001000--0.004642;
nrDNA:\emph{P} = 0.002000--0.007573) with JC69, K2P and \emph{P}
-distances initial and recursive partitions. One proposed species
includes individuals of \emph{P. dulongensis} , \emph{P. forrestii} ,
\emph{P. rugosa} , and \emph{P. tengchongensis} , while the other
comprises individuals of \emph{P. axialis} , \emph{P. guizhouensis} ,
\emph{P. lihengiana} , \emph{P. vaniotii} , and \emph{P. variabilis}
(Figure 2 \& 4). The mPTP analyses yielded the same delimitation scheme
as ABGD: all individuals were grouped into two species-like entities
that coincide with the two clades recovered by our phylogenetic analyses
(Figure 2 \& 4), and both received strong posterior support (Table S6).
Similarly, the SDA analyses showed that either plastome or nrDNA
dataset, excluding the outgroup, comprised two putative species (Figure
2 \& 4). For each dataset, both proposed species (corresponding to Clade
I and Clade II in the inferred phylogenetic tree) were distinct
with\emph{P\textsubscript{(RD)}} \textless{} 0.05, and significant
with\emph{P\textsubscript{(AB)}} \textless{} 10\textsuperscript{--5}
(Table S7).

\textbf{Discussion}

\textbf{Species delimitation scenario}

The alpha taxonomy of \emph{P.}sect\emph{. Axiparis} is still not well
understood, since nearly all nominal species show subtle morphological
differences. Both evolutionary and genetic profiles should be integrated
into species delimitation and taxonomic revision (de Queiroz, 1998;
Wiley and Mayden, 2000; Sites and Marshall, 2003). With that in mind,
this study aims to test whether the ultra-barcoding approach could
develop a rigorous species delimitation framework in \emph{P.}
sect\emph{. Axiparis} . To achieve this goal, we not only investigated
the species-level monophyly of each described species by sampling
multiple individuals per species, but also explore putative species
boundaries using multiple sequence-based species delimitation methods.

None of the nine described species within\emph{P.} sect.
\emph{Axiparis}were resolved as a monophyletic unit, suggesting that
genetic differentiation is low. Thus, their taxonomic status as separate
species needs to be reassessed. By contrast, both plastome and nrDNA
phylogenies recovered two clades that possess fairly distinct
morphological traits (Figure 2 \& 4) and distribution ranges (Figure 3).
Notably, the ABGD, SDP, and mPTP analyses consistently recognized the
two clades recovered by phylogenetic analyses as species-like taxonomic
units (Figure 2 \& 4). Briefly, the ABGD analyses partitioned all the
samples into two clusters comprised of individuals having two-whorled
and (Clade I) three-whorled stamens (Clade II). This implies that there
may be two distinct species with significant genetic gaps between them
(Puillandre et al., 2012). On the other hand, the mPTP analyses grouped
all accessions into two putative species with high posterior
delimitation probability, coinciding with the results found in the ABGD
analyses. The putative species boundaries proposed by ABGD and mPTP
analyses are further validated by the SDP analyses: with \emph{P}
\textsubscript{(\emph{R} D)} value \textless{} 0.05 and the
\emph{P\textsubscript{(AB)}} value \textless{} 10\textsuperscript{-5},
both of the two candidate species can be recognized as evolutionarily
distinctive entities (Rosenberg, 2007; Rodrigo et al., 2008). These
reinforcing results suggest that only two species-level taxonomic units
can be recognized in \emph{P} . sect.\emph{Axiparis} .

Notably, the two putative species possess not only distinct morphologies
but also allopatric distribution ranges, between which gene flow is
restricted. Therefore, it is reasonable to recognized them as distinct
species under the unified species concept (de Queiroz, 2007), given that
the species boundary proposed by our data reflects the unity of
morphological uniqueness, genetic distinctiveness, and evolutionary
independence. The findings suggest that previous morphology-based
taxonomic studies (Li, 1984; He, 1990; Li, 1992; Ji et al., 2017; Xu et
al., 2019; Yang et al., 2019) overemphasize intraspecific and minor
morphological differences to establish species, therefore leading to the
proliferation of synonyms. As a result, it is necessary to reduce
\emph{P. axialis} , \emph{P. guizhouensis} , \emph{P. lihengiana} , and
\emph{P. variabilis} as the synonyms of \emph{P. vaniotii} , and to
merge \emph{P. dulongensis} ,\emph{P. rugosa} and \emph{P.
tengchongensis} into \emph{P. forrestii} . The taxonomic revision
proposed here reduces the number of species in\emph{P} . sect.
\emph{Axiparis} from nine to two.

\textbf{Conservation Implications}

Taxonomy and species conservation are often assumed to be completely
interdependent practices (Mace, 2004). Estimating the number of species
under threat is an essential step in setting conservation priorities
(May, 1988, 1990; Margules and Pressey, 2000; Dirzo and Raven, 2003;
Mace et al., 2003). Prioritizing species for natural conservation
heavily depends on accurate delineation of species boundaries: well
delimited species are fundamental to allow adequate conservation and
biodiversity management (Hopkins and Freckleton, 2002; Sites and
Marshall, 2003; Mace, 2004). On the contrary, poorly delineated species
boundaries usually make it difficult to properly address conservation
issues (Rojas, 1992; Sites and Crandall, 1997; Prance et al., 2000;
Mace, 2004).

\emph{Paris} is a plant genus of great conservation concern with the
majority of species in the genus facing varying degrees of extinction
risk due to overharvesting of wild populations for medicinal purposes
(Ji, 2020). For instance, among the nine nominal species of
\emph{P.}sect. \emph{Axiparis} , \emph{P. dulongensis} and \emph{P.
lihengiana}are categorized as critically endangered (Qin et al., 2017;
Xu et al., 2019); \emph{P. forrestii} , \emph{P. guizhouensis} ,
\emph{P. rugosa} ,\emph{P. tengchongensis} and \emph{P. vaniotii} are
classified as endangered (Ji et al., 2017; Qin et al., 2017); \emph{P.
axialis} and\emph{P. variabilis} are identified as Vulnerable (Qin et
al., 2017; Yang et al., 2019). As suggested by our ultra-barcoding
analyses, prior taxonomic studies generally over-split species, and thus
proliferated as many as seven synonyms in \emph{P.} sect.
\emph{Axiparis} . In conservation practice, the poorly delineated
species boundaries within\emph{P.} sect. \emph{Axiparis} may lead to the
misallocation of limited conservation resource to widespread species
that are not at high risk of extinction (Agapow et al., 2004; Mace,
2004; Joppa et al., 2011). In this study, the ultra-barcoding approach
depicts a more accurate picture of the species diversity of \emph{P.}
sect. \emph{Axiparis} , which will help to better inform future
decisions regarding the species conservation in the genus \emph{Paris} .

On the other hand, commercial collection of rhizomes, seeds and
seedlings from wild populations pose the most serious threat to the
survival and future conservation status of \emph{Paris} species. With
the depletion of wild resources, some of the rare and endangered species
are getting collected as commercial products (Ji, 2020). Although rarity
alone does not mean a species is at a high risk of extinction, species
with small populations are undoubtedly more prone to extinct when
heavily disturbed by anthropogenic activities (Burgman, 2002). To
protect these species, it is necessary to take an effective way to end
entirely the collection of threatened \emph{Paris} species from the
wild. However, the rhizome, seed and seedling morphologies exhibit high
levels of interspecific similarity, and thus it is often difficult to
distinguish these products harvested from different \emph{Paris}species.
This makes the commercial collection and trade of \emph{Paris}products
difficult to monitor and manage.

As revealed by previous studies (e.g., Kane et al., 2012; Ji et al.,
2019a, 2020; Knope et al., 2020) and this study, the ultra-barcoding
approach performs well in discriminating species. More importantly, with
advances in NGS technologies, it has become much easier to generate
complete plastomes and nrDNA sequences even using trace and highly
degraded genomic DNA to construct sequencing libraries (Zeng et al.,
2018). Therefore, ultra-barcoding is likely an effective tool for the
identification of plant products to control the commercial collection
and trade of rare and endangered \emph{Paris} species.

\textbf{Prospects on ultra-barcoding approach for species delimitation}

The advancement of molecular-based approaches brought about great
progress in species identification and delimitation (Wiley and Mayden,
2000; Sites and Marshall, 2003; de Queiroz, 2007; Hollingsworth et al.,
2016). Concomitantly, a wide variety of species delimitation methods
have been developed, especially over the past 15 years (e.g., Rannala
and Yang, 2003; Yang and Rannala, 2010; Puillandre et al., 2012; Ence
and Carstens, 2011; Masters et al., 2011; Zhang et al., 2013). As a
result, it has been widely accepted that multiple methods should be
simultaneously employed to develop a robust species delimitation
framework, given that using different delimitation approaches allows
accommodations for the weaknesses of each approach (e.g., Hebert et al.,
2004; Aguilar et al., 2013; Kekkonen and Hebert, 2014; Mutanen et al.,
2015; Fujita et al., 2012). Unfortunately, previous studies using a
single or few sequence regions (e.g. the standard DNA barcodes)
indicated that employing multiple methods on the same data always
produced incongruent delimitation proposals (e.g., Camargo et al., 2012;
Mutanen et al., 2015; Jacob et al., 2018; Jirapatrasilp et al., 2019)
mainly due to the inadequacies of available genetic information to
properly delineate species boundaries (Carstens et al., 2013; Jacob et
al., 2018; Sukumaran and Knowles, 2017). Therefore, recent studies have
attempted to use complete plastomes and nrDNA sequences for species
delimitation (e.g. Ruhsam et al., 2015; Ji et al., 2020; Knope et al.,
2020). However, such studies did not employ multiple delimitation
approaches, but inferred tree topology alone and under the premise of
reciprocal monophyly to explore species boundaries. Factors such as
recent diversification, radiative speciation, and restricted
intraspecific gene flow may result in the absence of reciprocal
monophyly among closely related species, and thus not tracking species
boundaries (Ruhsam et al., 2015; Hollingsworth et al., 2016), which bias
delimitation schemes if only phylogenetic inference is consulted.
Therefore, these studies may provide little precise information to
evaluate the efficacy of either plastome or nrDNA barcoding approach for
species delimitation.

Using multiple sequence-based species delimitation methods (ABGD, SDP,
and mPTP), here we not only investigate the performance of the
ultra-barcoding approach in delineating species boundaries but also
examine to what extent these methods generate congruent delimitation
schemes. Although members of \emph{P.} sect. \emph{Axiparis} represent
recently diverged entities because their origins have been estimated to
no earlier than the late Miocene (Ji et al., 2019b), these delimitation
analyses generated highly congruent schemes in delineating species
boundaries (Figure 2 \& 4). Based on the results, the proposed taxonomic
revision establishes a clear-cut species boundary in \emph{P.}
sect.\emph{Axiparis} . The findings suggest that both complete plastomes
and nrDNA can provide more genetic information than standard DNA
barcodes to robustly delineate species boundaries. This study indicates
that the ultra-barcoding approach has great promise for developing a
credible species delimitation framework in taxonomically difficult
groups to provide guidance for taxonomic revision. In addition, the
approach offers great advantages in reliable species identification and
delimitation, which are important for formulating adequate conservation
and biodiversity management for those groups with great conservation
concerns. As discussed above, relying on reciprocal monophyly alone to
delineate species boundaries likely produces bias delimitation schemes.
The development of some coalescence-based species delimitation methods,
such as General Mixed Yule-Coalescent (GYMC; Fujisawa and Barraclough,
2013), species tree estimation using DNA sequences from multiple loci
(STACEY; Jones, 2017), Bayesian Phylogenetics and Phylogeography (BPP;
Rannala and Yang, 2013), and spedeSTEM (Ence and Carstens, 2011), may
provide the solution to this issue since these methods do not require
reciprocal monophyly of any species in focus within gene tree.
Unfortunately, we tried to use GMYC and STACEY to analyze our data, but
found that the BEAST runs using either complete plastomes or nrDNA
sequence to generate tree outputs ran for several weeks but failed to
produce results. In addition, both BPP and spedeSTEM are only suitable
for processing genomic segments no longer than 500 or 1,000 bp (Ence and
Carstens, 2011; Rannala and Yang, 2013), and thus cannot be used to
analyze ultra-barcoding data. Accordingly, there are still some
technical limitations to applying the ultra-barcoding approach to
species delimitation on a large scale. We look forward to the early
development of such coalescence-based method that enable to delineate
species boundaries with ultra-barcoding data in a time-saving manner.

\textbf{Acknowledgements}

We are grateful to Chengjin Yang, Guohua Zhou, Ren Zhao, Tingzhou Zhao,
Zhangming Wang, Fajian Yang, Juxiang Li, Changming Yang, Zhiping Zhang,
Yongxing Xiong, Guohai He, Yulong Li, Fengtian Lin and Qiang Zhao for
their helps in sample collection. This study is supported by the
National Natural Science Foundation of China (31872673), the NSFC-Joint
Foundation of Yunnan Province (U1802287), and a grant of the Large-scale
Scientific Facilities of the Chinese Academy of Sciences (No.
2017-LSF-GBOWS-02).



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\textbf{Data Accessibility Statement}

All sequences data generated in current study have been deposited into
NCBI GenBank database (accession number are presented in \textbf{Table
S1} ).

\textbf{Author Contributions}

YJ, JBY, and TSY designed the study. YJ, JBY, ZY, JY, and SW collected
data. YJ, JY, SW, ZY, LJ, and LL analyzed data. YJ wrote the manuscript.
JBL and TSY discussed the results and revised the manuscript.

\textbf{Figure legends}

\textbf{Figure 1.} Nine nominal species within \emph{Paris}
section\emph{Axiparis} , showing their leaf size and shape variations.
A,\emph{Paris axialis} ; B, \emph{P. dulongensis} (8 accessions);
C,\emph{P. forrestii} ; D, \emph{P. guizhouensis} ; E, \emph{P.
lihengiana} ; F, \emph{P. rugosa} ; G, \emph{P. tenchongensis} ; H,
\emph{P. vaniotii} ; I, \emph{P. variabilis} .

\textbf{Figure 2.} Phylogenetic tree of \emph{Paris}
section\emph{Axiparis} based on Maximum likelihood (ML) and Bayesian
inference (BI) analyses of complete plastomes. Well-supported nodes (BS
\textgreater{} 90\% and PP \textgreater{} 0.95) are indicated with a
star. Species delimitation schemes proposed by automatic barcode gap
discovery (ABGD), multi-rate Poisson tree processes model (mPTP), and
species delimitation plugin (SDP) of Geneious are reflected on the tree
topology.

\textbf{Figure 3.} Distribution of the nine nominal species
within\emph{Paris} section \emph{Axiparis.}

\textbf{Figure 4.} Phylogenetic tree of \emph{Paris}
section\emph{Axiparis} based on Maximum likelihood (ML) and Bayesian
inference (BI) analyses of nuclear ribosomal DNA (nrDNA). Well-supported
nodes (BS \textgreater{} 90\% and PP \textgreater{} 0.95) are indicated
with a star. Species delimitation schemes proposed by automatic barcode
gap discovery (ABGD), multi-rate Poisson tree processes model (mPTP),
and species delimitation plugin (SDP) of Geneious are reflected on the
tree topology.

\textbf{Supporting Information}

\textbf{Table S1.} Samples used in this study with locality, voucher and
GenBank accession numbers.

\textbf{Table S2.} Summary of Illumina sequencing.

\textbf{Table S3.} Characteristics of complete plastome and nuclear
ribosomal DNA datasets in \emph{Paris} section \emph{Axiparis} .

\textbf{Table S4.} The number of putative species recognized by
Automatic Barcode Gap Discovery (ABGD) analyses among 51 complete
plastomes using three distance metrics.

\textbf{Table S5.} The number of putative species recognized by
Automatic Barcode Gap Discovery (ABGD) analyses among 51 nuclear
ribosomal DNA (nrDNA) sequences using three distance metrics.

\textbf{Table S6.} Posterior delimitation probability of two putative
species proposed by mPTP analyses of complete plastomes and nuclear
ribosomal DNA (nrDNA) sequences.

\textbf{Table S7.} Species delimitation plugin (SDP) analyses show the
distinctiveness (Rodrigo's \emph{P} \textsubscript{(\emph{R} D)}) and
significance (Rosenberg's \emph{P\textsubscript{(AB)}} ) of the two
clades recovered by phylogenetic analyses of complete plastomes and
nuclear ribosomal DNA (nrDNA) as species-level taxonomic units.\selectlanguage{english}
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