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Plastome-based backbone
phylogeny of East Asian
Phedimus (Subgenus Aizoon:
Crassulaceae), with special
emphasis on Korean endemics
Yongsung Kim
1
†
, Seon-Hee Kim
2
†
, JiYoung Yang
3
,
Myong-Suk Cho
4
, Marina Koldaeva
5
, Takuro Ito
6
,
Masayuki Maki
6
and Seung-Chul Kim
4
*
1
Department of Islands and Coast Biodiversity, Division of Botany, Honam National Institute of
Biological Resources, Mokpo, Republic of Korea,
2
Department of Botany, Graduate School of
Science, Kyoto University, Kyoto, Japan,
3
Research Institute for Dok-do and Ulleung-do Island,
Kyungpook National University, Daegu, Republic of Korea,
4
Department of Biological Sciences,
Sungkyunkwan University, Suwon, Republic of Korea,
5
Botanical Garden-Institute, Far Eastern Branch
of the Russian Academy of Sciences, Vladivostok, Russia,
6
Botanical Gardens, Tohoku University,
Sendai, Japan
Although the monophyly of Phedimus has been strongly demonstrated, the
species relationships among approximately 20 species of Phedimus have been
difficult to determine because of the uniformity of their floral characteristics and
extreme variation of their vegetative characters, often accompanied by high
polyploid and aneuploid series and diverse habitats. In this study, we assembled
15 complete chloroplast genomes of Phedimus species from East Asia and
generated a plastome-based backbone phylogeny of the subgenus Aizoon.As
a proxy for nuclear phylogeny, we reconstructed the nuclear ribosomal DNA
internal transcribed spacer (nrDNA ITS) phylogeny independently. The 15
plastomes of subg. Aizoon were highly conserved in structure and
organization; hence, the complete plastome phylogeny fully resolved the
species relationships with strong support. We found that P. aizo on and P.
kamtschaticus were polyphyletic and morphologically distinct or ambiguous
species, and they most likely evolved from the two species complex. The crown
age of subg. Aizoon was estimated to be 27 Ma, suggesting its origin to be in the
late Oligocene; however, the major lineages were diversified during the Miocene.
The two Korean endemics, P. takesimensis and P. zokuriensis, were inferred to
have originated recently during the Pleistocene, whereas the other endemic, P.
latiovalifolium, originated in the late Miocene. Several mutation hotspots and
seven positively selected chloroplast genes were identified in the subg. Aizoon.
KEYWORDS
Aizopsis, aneuploid series, Phedimus, polyploid, subgenus Aizoon, plastome, nrDNA ITS
Frontiers in Plant Science frontiersin.org01
OPEN ACCESS
EDITED BY
Mi-Jeong Yoo,
Clarkson University, United States
REVIEWED BY
Shih-Hui Liu,
National Sun Yat-sen University, Taiwan
Zelong Nie,
Jishou University, China
*CORRESPONDENCE
Seung-Chul Kim
sonchus96@skku.edu;
sonchus2009@gmail.com
†
These authors have contributed
equally to this work and share
first authorship
SPECIALTY SECTION
This article was submitted to
Plant Systematics and Evolution,
a section of the journal
Frontiers in Plant Science
RECEIVED 04 November 2022
ACCEPTED 23 February 2023
PUBLISHED 14 March 2023
CITATION
Kim Y, Kim S-H, Yang J, Cho M-S,
Koldaeva M, Ito T, Maki M and Kim S-C
(2023) Plastome-based backbone
phylogeny of East Asian Phedimus
(Subgenus Aizoon: Crassulaceae), with
special emphasis on Korean endemics.
Front. Plant Sci. 14:1089165.
doi: 10.3389/fpls.2023.1089165
COPYRIGHT
©2023Kim,Kim,Yang,Cho,Koldaeva,Ito,
Maki and Kim. This is an open-access article
distributed under the terms of the Creative
Commons Attribution License (CC BY). The
use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
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accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 14 March 2023
DOI 10.3389/fpls.2023.1089165
1 Introduction
The genus Phedimus Rafinesque comprises approximately 20
species worldwide and represents a lineage distinct from the more
broadly circumscribed “catch-all”genus Sedum (Ohba et al., 2000;
Mayuzumi and Ohba, 2004). Phedimus species are phenotypically
like Sedum species, but morphological characteristics (i.e., well-
developed rhizomes and flattened leaves with dentate margins) and
molecular phylogenetic studies strongly support the monophyly of
Phedimus and its segregation from Sedum sensu stricto (Hart, 1995;
Ohba et al., 2000;Mayuzumi and Ohba, 2004;Gontcharova et al.,
2006;Gontcharova and Gontcharov, 2009). Regarding the
phylogenetic position of Phedimus within the subfamily
Sedoideae, the nrDNA ITS phylogeny suggested that the clade
including Rhodiola L. and Pseudosedum A. Berger is sister to
Phedimus, while Sedum sensu stricto (Acre clade) is sister to the
Aeonium clade (Hart, 1995). In Phedimus, two major groups are
recognized at the subgeneric rank: subg. Aizoon (L.K.A. Koch ex
Schönland), Ohba & Turland, and subg. Phedimus. Approximately
five species of subg. Phedimus occur in regions from the Aegean to
South Persia and the North Caucasus and have purple or white
petals. In contrast, species in subg. Aizoon, which has between 12
and 15 species, occurs from East Europe in the South Urals to the
Far East and has yellow petals (Grulich, 1984;Gontcharova, 2000;
Hart and Bleij, 2003;Gontcharova et al., 2006). Based on the
nrDNA ITS phylogeny, two evolutionary lines within the genus
Phedimus seem to exist: the predominant European lineage
Phedimus and the Asian lineage Aizoon. These two evolutionary
lines were further recognized by Chao (2020), but interestingly, two
Chinese endemics in subg. Aizoon,P. yangshanicus (Guangdong
Province) and P. odontophyllus (W Hubei and SE Sichuan
Provinces; Fu et al., 2001), shared their most recent common
ancestor with European/Caucasian species rather than with con-
subgeneric species of Aizoon in East Asia. These results support the
idea that the two Chinese endemic species may represent a
phylogenetic link between Asian and European Phedimus
(Chao, 2020).
Of the approximately 15 species in subg. Aizoon in East Asia,
seven species of Phedimus are known to occur in Korea: P. aizoon,
P. ellacombeanus,P. kamtschaticus,P. middendorffianus,
P. takesimensis,P. zokuriensis, and P. latiovalifolium. Of the seven
species of Phedimus in Korea, four are narrowly restricted to
Ulleung Island (P. takesimensis), central Korea (P. zokuriensis and
P. latiovalifolium), and southern Korea (P. ellacombeanus), whereas
P. aizoon and P. kamtschaticus occur widely throughout the Korean
Peninsula. Recently, P. daeamensis, which is a narrow endemic to
Mt. Daeam, was described in Korea (Choi et al., 2022). Two species
that occur widely in Korea, P. aizoon and P. kamtschaticus, also
show a much broader geographical distribution in Russia, China,
Mongolia, and Japan. Phedimus middendorffianus, which occurs in
northern Korea, is also widely distributed in Russia and China. In
contrast, some species of the subg. Aizoon in East Asia is restricted
to certain countries, such as Russia (P. litoralis and P. sichotensis),
Japan (P. sikokianus), and China (P. odontophyllus,P. floriferus, and
P. yangshanicus). Owing to a lack of synapomorphic characters for
species, highly variable morphologies within species, and extensive
polyploidy and aneuploidy, the circumscription of species and
interspecific relationships within Phedimus in East Asia has been
problematic (Uhl and Moran, 1972;Chung and Kim, 1989;Amano,
1990;Amano and Ohba, 1992). Individual species within Phedimus
show diverse morphologies that often intergrade with other
recognized taxa, blurring species boundaries and resulting in the
recognition of species complexes and polymorphic species with
many varieties (Chung and Kim, 1989;Amano, 1990). Although the
extent of hybridization in nature is unclear, interspecific
relationships within Phedimus may be further complicated by
hybridization, with documentation of several natural hybrids in
the genus (Yoo and Park, 2016).
Nearly half of the species belong to subg. Aizoon occur in Korea,
three of which are narrow endemics, and phylogenetic relationships
among the seven species have been of particular interest (Chung
and Kim, 1989;Yoo and Park, 2016;Seo et al., 2020). Phedimus
aizoon is a polymorphic species with many varieties and is
distributed from eastern Siberia to Japan (Amano and Ohba,
1992). In Korea, it has been recognized as a highly variable
species, with infraspecific taxa ranging from one (Lee, 1980)to
four (Nakai, 1911), five (Lee, 1969), and seven (Oh, 1985). Chung
and Kim (1989) showed that P. aizoon intergraded with Phedimus
kamtschaticus, with the observation of several intermediate forms
between them. P. kamtschaticus is also a highly variable species in
Korea in terms of overall status, leaf size, and degree of leaf serration
and succulency (Chung and Kim, 1989). Three species present in
Korea, Phedimus ellacombeanus,P. takesimensis, and P. zokuriensis,
are closely related to P. kamtschaticus and are often treated as
conspecifics in various taxonomic treatments. P. ellacombeanus was
originally collected as P. kamtschaticus by Maximowicz in 1861
from Hakodate (Hokkaido, Japan) but later described as a new
species by Praeger (1917) based on the cultivated materials in
Hance’s Herbarium (Chung and Kim, 1989). Although its species
status has been controversial, P. ellacombeanus has been treated as a
distinct species from Korea (Chung and Kim, 1989). This species is
also known to occur in the type locality of Hakodate, Hokkaido in
Japan, but has been treated as a synonym of P. aizoon var.
floribundus (Nakai) H. Ohba (=P. kamtschaticus in Korea) (Ohba,
2002). Phedimus takesimensis, a morphologically variable species
endemic to Ulleung Island, was first described by Nakai (1919).
Phedimus zokuriensis, described by Nakai (1939), is endemic to Mt.
Sokri and neighboring mountains and is distinguished from
congeneric species by weak and creeping stems (Chung and Kim,
1989). It occurs on shaded, wet rocky surfaces in the forests of the
mountains of Sokri and Worak in central Korea. Lastly,
P. latiovalifolium was described recently from Geumdae-bong
Peak on Mt. Taebaek (Lee, 1992), and based on morphology, it
was suggested to be of hybrid origin between P. kamtschaticus and
P. aizoon or between P. aizoon and P. ellacombeanus (Lee, 2000;
Yoo and Park, 2016).
Although the monophyly of Phedimus is strongly supported by
molecular phylogenetic studies, interspecific relationships and
species entities within subg. Aizoon remain poorly understood
(Mayuzumi and Ohba, 2004;Gontcharova et al., 2006;Seo et al.,
2020). In the eastern Asian Sedoideae phylogeny, Mayuzumi and
Ohba (2004) revealed two major lineages within Phedimus, that is,
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org02
subg. Phedimus (P. spurius) and subg. Aizoon (P. kamtschaticus,
P. aizoon, and P. aizoon var. floribundus, and P. sikokianus) had
poor resolution in interspecific relationships. Gontcharova et al.
(2006) sampled Phedimus species primarily from various localities
in Primorsky Krai, Russia, and inferred their phylogenetic
relationships. In this study, based on nrDNA ITS sequences, they
identified two major lineages at the generic rank, Phedimus (=subg.
Phedimus) and Aizopsis (=subg. Aizoon), with basic chromosome
numbers of x= 14 and x= 16, respectively. In addition, interspecific
relationships among primarily Russian Far East species were
inferred with limited resolution and relatively low bootstrap
support (Gontcharova et al., 2006). To assess the anagenesis of
P. takesimensis on Ulleung Island, Seo et al. (2020) conducted a
phylogenetic analysis based on chloroplast noncoding regions and
nrDNA ITS sequences, providing limited support and relationships
among Korean populations of five taxa due to low resolution. In
addition, a molecular phylogenetic study to trace the cultivar
“Tottori Fujita”of Phedimus was conducted, finding its origin on
Ulleung Island P. takesimensis (Han et al., 2020). Lastly, the
phylogenetic position of the newly described species from China,
P. yangshanicus, was assessed based on nrDNA ITS sequences,
further confirming species relationships that have previously been
identified (Chao, 2020). Therefore, as of today, we have very limited
phylogenetic relationships among Phedimus species in East Asia,
and phylogenomic analysis based on the complete plastome using
broader sampling has never been conducted to gain insight into the
origin of Korean endemic species. Plastome-based phylogenomic
analysis has provided good resolutions and supports, demonstrating
its importance in various plant groups (e.g., Xie et al., 2019;Cho
et al., 2020;Xie et al., 2020;Yang et al., 2020;Yang et al., 2021).
Since the plastome represents the evolutionary history of maternal
lineages only, we employed nrDNA ITS sequences to complement
the phylogenetic inferences from the plastome sequences. Thus, the
aims of this study were to (1) characterize the chloroplast genomes
of Phedimus species in subg. Aizoon in East Asia; (2) generate
baseline plastome phylogenetic relationships; (3) infer species
relationships based on nrDNA ITS sequences as a proxy for
nuclear phylogeny and determine any incongruences between
plastome-based and nuclear phylogeny; and (4) gain insights into
the origin and evolution of endemic species of Phedimus in Korea.
2 Materials and methods
2.1 Plant materials
For each species, we tried to sample at least one accession from
its typical geographical range for complete plastome sequencing
(Table 1,Figure 1): P. sikokianus,P. kurilensis,P. selskianus, and P.
litoralis.Insomespecies(P. aizoon,P. middendorffianus,P.
ellacombeanus, and P. kamtschaticus), there was more than one
accession based on wild or cultivated origins. The plant materials
from the Botanical Garden Institute (Vladivostok, Russia) represent
transplanted ones from nature into cultivation with accurate species
identification. In the case of the Korean endemics, P. zokuriensis
and P. latiovalifolium, we sampled one accession from the type
locality, Mt. Sokri, and Geumdae-bong Peak, Mt. Taebaek,
respectively, in central Korea. Unfortunately, a newly described
Korean endemic, P. daeamensis, was not included in this study
owing to its recent description and lack of plant materials. Thus, we
sequenced the complete plastome of 15 accessions, representing 11
taxa. For sequencing of nrDNA ITS sequences, we sampled a total
of 89 accessions representing 10 taxa (Supplementary Table 1). All
these accessions were of wild origin, with accurate species
identification by Seung-Chul Kim, Masayuki Maki, Takuro Ito,
and Marina Koldaeva.
2.2 DNA isolation, nrDNA ITS Sanger
sequencing, NGS sequencing and
plastome assembly/annotation, divergent
hotspot identification, and selective
pressure analysis
Total genomic DNA was isolated using the DNeasy Plant Mini
Kit (Qiagen, Carlsbad, CA, USA), following the manufacturer’s
protocol. For nrDNA ITS sequencing, we followed the same
protocols of polymerase chain reaction (PCR) amplification and
subsequent sequencing as described by Seo et al. (2020).The
nrDNA ITS is a multi-copy nuclear gene, and different ribotypes
may exist within a single individual, complicating inferences about
species relationships. To minimize the potential complications of
ribotypes in species relationship inference, we used direct
sequencing of PCR products rather than cloning of ribotypes in
Phedimus species (Gontcharova et al., 2006;Chao, 2020;Seo et al.,
2020). In addition, we only included accessions with clean
sequencingresults.Thepairwisesequencedivergencewas
calculated based on the Kimura 2-parameter method (Kimura,
1980) using MEGA11 (Tamura et al., 2021). According to earlier
studies, we sequenced the entire plastome (e.g., Kim et al., 2019;
Cho et al., 2020;Yang et al., 2021;Yun and Kim, 2022). An Illumina
paired-end (PE) genomic library was constructed and sequenced
using the Illumina HiSeq platform (Illumina, Inc., San Diego, CA,
USA) at Macrogen Corporation (Seoul, Korea). Sequence reads of
the plastomes were assembled using the de novo genomic assembler
Velvet 1.2.10 (Zerbino and Birney, 2008) or NOVOPlasty version
4.3.1 (Dierckxsens et al., 2017). Annotation was performed using
Geneious R10 (Biomatters, Auckland, New Zealand) and
ARAGORN v1.2.36 (Laslett and Canback, 2004). The pairwise
sequence divergence was also calculated based on the Kimura 2-
parameter method (Kimura, 1980) using MEGA11 (Tamura et al.,
2021). The annotated plastome sequences were deposited in
GenBank under accession numbers (Table 1). The annotated
GenBank (NCBI, Bethesda, MD, USA) format sequence file was
used to draw a circular plastid genome map using the OGDRAW
software v1.2 (CHLOROBOX) (Lohse et al., 2007). We performed
DnaSP v6.10 (Rozas et al., 2017) sliding window analysis, with a
step size and window length of 200 bp and 800 bp, respectively, to
determine mutation hotspots (that is, the most divergent regions of
the plastome). To determine genes under positive selection, a site-
specific model was developed using EasyCodeML (Gao et al., 2019)
with CodeML algorithms (Yang, 1997). Seven codon substitution
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org03
TABLE 1 Characteristics of plastomes of the 15 Phedimus subg. Aizoon accessions used in this study.
Taxon/Locality
GenBank
Accession
Number
Total cpDNA
size (bp)/GC
content (%)
LSC size
(bp)/GC
content
(%)
IR size
(bp)/GC
content
(%)
SSC size
(bp)/GC
content
(%)
Number
of genes
Number
of
protein
coding
genes
Number
of tRNA
genes
Number
of rRNA
genes
Number of
duplicated
genes
1. Phedimus aizoon (two accessions)
Geumdae-bong Peak,
Jeongseon-gun, Taebaek-si,
Gangwon-do (Korea)
OP344959 151,732/37.7 83,089/
35.7
25,983/
42.8
16,677/
31.9 135 88 37 8 20
Khasansky District,
Primorsky Krai (Russia) OP344958 151,757/37.7 83,104/
35.7
25,976/
42.8
16,701/
31.9 135 88 37 8 20
2. Phedimus aizoon var. floribundus (one accession)
Kitakami-shi, Iwate Pref.
(Japan) OP344945 151,748/37.7 83,084/
35.7
25,988/
42.8
16,688/
31.9 135 88 37 8 20
3. Phedimus ellacombeanus (three accessions)
Hakodate-shi, Hokkaido
Pref. (Japan) OP344955 151,869/37.7 83,179/
35.7
25,984/
42.8
16,722/
31.9 135 88 37 8 20
Sochi Island, Namhae-gun,
Gyeongsangnam-do (Korea) OP344957 151,718/37.8 83,055/
35.7
25,983/
42.8
16,697/
32.0 135 88 37 8 20
Uje-bong Peak, Geoje Island,
Gyeongsangnam-do (Korea) OP344956 151,694/37.8 83,034/
35.7
25,982/
42.8
16,696/
31.9 135 88 37 8 20
4. Phedimus kamtschaticus (one accession)
Hanasaki cape,
Hanasakiminato, Nemuro-
shi, Hokkaido Pref. (Japan)
OP344954 151,849/37.7 83,174/
35.7
25,985/
42.8
16,705/
31.9 135 88 37 8 20
5. Phedimus kurilensis (one accession)
Kosmodemyanskaya Bay,
Kunashir Island, Sakhalin
Region (Russia)
OP344953 151,845/37.7 83,170/
35.7
25,985/
42.8
16,705/
31.9 135 88 37 8 20
6. Phedimus latiovalifolium (one accession)
Geumdae-bong Peak,
Jeongseon-gun, Taebaek-si,
Gangwon-do (Korea)
OP344952 151,787/37.7 83,124/
35.7
25,982/
42.8
16,699/
31.9 135 88 37 8 20
7. Phedimus litoralis (one accession)
Red Stones Bay, Reinecke
Island, Vladivostok Urban
Okrug, Primorsky Krai
(Russia)
OP344951 151,761/37.7 83,106/
35.7
25,977/
42.8
16,701/
31.9 135 88 37 8 20
8. Phedimus middendorffianus (two accessions)
Mt. Dosol, Yanggu-eup,
Yanggu-gun, Gangwon-do
(Korea)
OP344950 151,630/37.8 82,970/
35.8
25,982/
42.8
16,696/
31.9 135 88 37 8 20
Bikin, Pozharsky district,
Primorsky Krai (Russia) OP344949 151,557/37.8 82,890/
35.8
25,983/
42.8
16,701/
31.9 135 88 37 8 20
9. Phedimus selskianus (one accession)
Barabash, Khasansky
District, Primorsky Krai
(Russia)
OP344948 151,768/37.7 83,112/
35.7
25,977/
42.8
16,702/
31.9 135 88 37 8 20
10. Phedimus sikokianus (one accession)
Mt. Tsurugi, Tokushima
Pref. (Japan) OP344947 151,769/37.7 83,081/
35.7
25,985/
42.8
16,718/
31.7 135 88 37 8 20
11. Phedimus zokuriensis (one accession)
Mt. Sokri, Boeun-gun,
Chungcheongbuk-do (Korea) OP344946 151,746/37.7 83,087/
35.7
25,981/
42.8
16,697/
31.9 135 88 37 8 20
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org04
models (M0, M1a, M2a, M3, Mt, M8, and M8a) were constructed
and compared to detect positively selected sites using the likelihood
ratio test (LRT).
2.3 Phylogenetic analysis
For the ITS phylogeny, we included 106 accessions,
representing two species (three accessions) of subg. Phedimus
(S. obtusifolius was not available) and 15 species (103 accessions)
of subg. Aizoon, and based on previous studies, the genus Rhodiola
was used as an outgroup (Mayuzumi and Ohba, 2004;Gontcharova
et al., 2006;Chao, 2020;Messerschmid et al., 2020)(Supplementary
Table 1). Of the 106 accessions, newly sequenced ITS sequences (a
total of 89 accessions) included P. latiovalifolium (16 accessions),
P. zokuriensis (four accessions), P. takesimensis (14 accessions), P.
middendorffianus (six accessions), P. kurilensis (one accession), P.
ellacombeanus (nine accessions from Korea and Japan), P. litoralis
(two accessions), P. aizoon (18 accessions), P. selskianus (one
accession), and P. kamtschaticus (18 accessions). All the
sequences were edited and assembled using Sequencher v4.2.2
(Gene Codes, Ann Arbor, MI, USA) and Geneious R10
(Biomatters, Auckland, New Zealand). The ITS sequences for the
following species (P. stellatus,P. spurius,P. odontophyllus,P.
yangshanicus,P. hybridus,P. sichotensis, and P. sikokianus) were
also obtained from GenBank. We included these species (totaling 17
accessions) because some represent members of subg. Phedimus (P.
stellatus,P. spurius,P. odontophyllus, and P. yangshanicus), and
other species (P. hybridus,P. selskianus,P. sichotensis,andP.
sikokianus) have distinct species diagnostic features, minimizing
potential misidentifications and providing overall species
relationships in subg. Aizoon. The sequences were aligned using
Clustal X v1.83 (Thompson, 1997) with a final manual adjustment
using MacClade (Maddison and Maddison, 2002). Maximum
likelihood (ML) analysis was conducted using IQ-TREE v1.4.2
(Nguyen et al., 2015), with 1,000 replicate bootstrap (BS)
analyses, based on the best-fit model of TIM3e + G4 selected by
ModelFinder (Kalyaanamoorthy et al., 2017). For plastome
phylogeny, the complete plastome sequences were aligned using
MAFFT v7 (Katoh and Standley, 2013), and an ML phylogenetic
tree was constructed using IQ-TREE with 1,000 bootstrap replicates
(Nguyen et al., 2015). The best-fit evolutionary model for the
complete plastome sequences, TVM + F + I + G4, was selected
based on ModelFinder (Kalyaanamoorthy et al., 2017),
implemented in IQ-TREE v1.4.2. A representative species of
Rhodiola was used as the outgroup in ITS analysis (Messerschmid
et al., 2020). Given the lack of representative plastomes of subg.
Phedimus, we included Rhodiola species as part of the ingroup and
Umbilicus as the outgroup.
2.4 Molecular dating
Divergence times based on complete plastome sequences were
estimated using the Bayesian method (Drummond et al., 2006)
using BEAST version 1.10.4 (Suchard et al., 2018). The XML file for
analysis was prepared using the Bayesian evolutionary analysis
utility (BEAUTi). Owing to the lack of reliable fossils of Phedimus
and related genera of Crassulaceae, we considered two secondary
calibration points based on ITS phylogeny: an estimated Rhodiola
crown mean age of 7.17 Myr and a standard deviation of 4.87,
giving a range of 3.09–12.03 Myr, and the Phedimus and Rhodiola
clade stem mean age of 39.43 Myr, and a standard deviation of
14.31, giving a range of 24.91–53.74 Myr (Messerschmid et al.,
2020). We used the Yule process speciation prior, a lognormal
relaxed clock model, and the GTR-gsubstitution model, and then
the ucld.mean parameter was specified to be uniform with 0.333 as
the initial value, 0.00 as the lower limit, and 1 as the upper limit
(Drummond et al., 2006). Posterior distributions for each
parameter were estimated using an MCMC run for 400 million
FIGURE 1
Distribution map of Phedimus species sampled in this study.
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org05
generations with a sampling frequency of 50,000 generations. The
posterior distribution of all statistics was checked using Tracer
version 1.5 (Rambaut and Drummond, 2009) to assess convergence
and confirm whether the effective sample sizes (ESS) for all
parameters were larger than 200 (Drummond et al., 2012). In
addition, we used TreeAnnotator version 1.5 (http://
beast.bio.ed.ac.uk/TreeAnnotator) to produce a maximum
credibility tree of mean divergence time and 95% highest
posterior density (HPD) intervals with a posterior probability
(PP) limit (0.5) after removing the first 25% of trees as burn-in
(Drummond et al., 2012).
3 Results
3.1 Characterization of chloroplast
genomes, mutation hotspots, and
positively selected genes in Phedimus
species of subg. Aizoon
The complete plastome length of the subg. Aizoon ranged from
151,557 bp (P. middendorffianus; Primorsky Krai, Russia) to
151,869 bp (P. ellacombeanus;Hokkaido,Japan)(Table 1;
Figure 2). The large single copy (LSC) region, small single copy
(SSC) region, and two inverted repeat (IR) regions ranged from
82,890 bp (P. middendorffianus; Primorsky Krai, Russia) to 83,179
bp (P. ellacombeanus; Hokkaido, Japan), 16,677 bp (P. aizoon;
Geumdae-bong Peak, Korea) to 16,718 bp (P. sikokianus;
Tokushima Pref., Japan), and 25,976 bp (P. aizoon; Primorsky
Krai, Russia) to 25,988 bp (P. aizoon var. floribundus; Iwate Pref.
Japan), respectively (Table 1). All 15 accessions of the subg. Aizoon
contains 135 genes, including 88 protein-coding, eight ribosomal
RNA, and 37 transfer RNA genes. The overall guanine-cytosine
(GC) content ranged from 37.7% to 37.8%, and 20 duplicate genes
were found in the IR regions. Sliding window analysis using the
DnaSP program identified several highly variable genic and
intergenic (“–”) regions in 15 plastomes of subg. Aizoon:atpF–
atpH (Pi = 0.01223), ycf1 (Pi = 0.01015), trnH–psbA (Pi = 0.0076),
rbcL–accD (Pi = 0.00747), ndhF–rpl32 (Pi = 0.00622), rpoB–trnC (Pi
= 0.00601), psbZ–trnG (Pi = 0.00533), trnW–trnP (Pi = 0.0052), and
rps8–rpl14 (Pi = 0.00489) (Figure 3). The average nucleotide
diversity value (Pi) over the entire plastome was 0.00188. Among
the conserved genes, we identified seven genes with positively
selected sites (Table 2). These genes included the c-type
cytochrome synthesis gene (ccsA), chloroplast envelope
membrane protein (cemA), maturase K gene (matK), NADH
dehydrogenase subunit gene (ndhC), photosystem II protein gene
(psbJ), cytoplasmic ribosomal protein L22 gene (rpl22), and RNA
polymerase C2 gene (rpoC2).
3.2 Complete plastome
sequence phylogeny
For the first time, we obtained robust and well-resolved whole
plastome-based phylogenetic relationships among representative
species of subg. Aizoon in East Asia (Figure 4). The plastome tree
suggested that P. sikokianus, which is diploid and endemic to
southern Japan, diverged early within the subg. Aizoon and
further identified three major lineages, clades A–C. The first
lineage, clade A (100% bootstrap support, BS), included P.
ellacombeanus, sampled as P. aizoon var. floribundus from the
type locality of the species in Japan (Hokkaido, Japan), P.
kurilensis (Sakhalin, Russia), and P. kamtschaticus (Hokkaido,
Japan). All these taxa occur from northern Japan (Hokkaido) to
the southern Kuriles (Russia). In this clade, P. kurilensis, endemic to
the southern Kuril Islands and considered a synonym of P.
sikokianus, is sister to P. kamtschaticus from Hokkaido, Japan
FIGURE 3
Sliding window analysis of the whole plastomes of 15 accessions (11
taxa) of Phedimus subg. Aizoon.
FIGURE 2
Complete plastome map of Phedimus subg. Aizoon species in East
Asia. The genes inside and outside of the circle are transcribed in
the clockwise and counterclockwise directions, respectively. Genes
belonging to different functional groups are shown in different
colors. The thick lines indicate the extent of the inverted repeats (IR
A
and IR
B
) that separate the genomes into small single copy (SSC) and
large single copy (LSC) regions.
Kim et al. 10.3389/fpls.2023.1089165
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(100% BS). After the divergence of clade A, P. aizoon var.
floribundus (Iwate, Japan) is sister to the remaining clades of B
and C (100% BS). The second lineage, clade B (100% BS), included
P. middendorffianus (two accessions from Russia and Korea), P.
kamtschaticus (Korea), two accessions of P. ellacombeanus, and P.
zokuriensis (Korea). One Korean accession of P. middendorffianus
sampled from Gangwon-do Province is sister to P. kamtschaticus,
which was also sampled from Gangwon-do Province (moderate
FIGURE 4
Maximum likelihood phylogeny of subg. Aizoon, including 11 species of Phedimus, based on complete plastome sequences. Bootstrap support
values >50% are shown above and below branches. Asterisked 15 accessions are newly obtained in this study.
TABLE 2 Positively selected genes and sites detected in the plastomes of Phedimus subg. Aizoon species.
Gene
name Models np ln L Likelihood Ratio Test p-Value Positively Selected Sites
ccsA
M8 45 −1,393.266207
0.000000000 F 0.998**
M7 43 −1,465.917908
cemA
M8 45 −1,031.895178
0.000000000 105 H 0.950*
M7 43 −1,101.793428
matK
M8 45 −2,263.614460
0.000000000 199 L 0.971*
M7 43 −2,339.739355
ndhC
M8 45 −527.638862
0.000000000 26 L 0.976*
M7 43 −596.432768
psbJ
M8 45 −165.418110
0.000013861 27 I 0.974*; 28 G 0.952*; 29 L 1.000**;
30 G 0.995**; 32 S 0.955*; 33 L 0.993**
M7 43 −176.604574
rpl22
M8 45 −619.287439
0.000013861 18 S 0.972*,55 F 0.974*
M7 43 −675.381579
rpoC2
M8 45 −6,031.704371
0.014323525 1375 F 0.979*
M7 43 −6,035.950223
*p <0.05; **p <0.01. np represents the degrees of freedom.
Kim et al. 10.3389/fpls.2023.1089165
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support, 74% BS). The central South Korean Peninsula Endemic P.
zokuriensis is sister to the P. ellacombeanus sampled from Sochi
Island, which is in the southeastern part of the Korean Peninsula
(95% BS). Lastly, the third lineage, clade C (100% BS), included two
Korean endemic species (P. latiovalifolium and P. takesimensis), P.
aizoon (Korea), and species from Russia (P. selskianus,P. litoralis,
and P. aizoon). The monophyletic Ulleung Island endemic P.
takesimensis shares its most recent common ancestor with
Phedimus species from Russia (P. selskianus,P. litoralis, and P.
aizoon; 92% BS). The central South Korean peninsula endemic P.
latiovalifolium is sister to the clade containing species from Russia
and the Ulleung Island endemic P. takesimensis (84% BS). Overall,
plastome-based phylogeny of subg. Aizoon suggested that P.
takesimensis is monophyletic, while the more widely distributed
P. aizoon and P. kamtschaticus appear to be polyphyletic, which
requires further confirmation based on multiple accessions.
Pairwise sequence divergence based on Kimura 2-parameter
distances was shown in Supplementary Table 2.Theaverage
pairwise sequence divergence for all 15 accessions was 0.189%,
ranging from 0.008% (between P. kamtschaticus OP344954 and P.
kurilensis OP344953) to 0.425% (between P. takesimensis
NC026025 and P. sikokianus OP344947). The average pairwise
FIGURE 5
Maximum likelihood phylogeny of the genus Phedimus based on the nrDNA ITS sequences. Bootstrap support values >50% are shown below
branches. Accessions in circles are sequenced for their complete plastomes, and the rectangles represent two major clades of subg. Aizoon.
Kim et al. 10.3389/fpls.2023.1089165
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sequence divergence for four accessions of P. takesimensis on
Ulleung Island and three accessions of P. ellacombeanus was
0.053% and 0.215%, respectively. The type locality accession of P.
ellacombeanus (OP344955) in Japan showed quite divergent
pairwise sequence divergence from its conspecific populations
sampled in Korea: 0.307% between type locality accession
OP344955 and two Korean accessions versus 0.030% between two
Korean accessions of P. ellacombeanus.
3.3 nrDNA ITS phylogeny
The ITS ML tree revealed two major lineages within the genus
Phedimus (Figure 5; phylogram shown in Supplementary Figure 1).
Pairwise sequence divergence based on the Kimura 2-parameter
distances was shown in Supplementary Table 3. For all 106
accessions (including two subgenera Phedimus and Aizoon), the
average pairwise sequence divergence was 1.654%: 2.967% for
species of subg. Phedimus and 1.371% for species of subg. Aizoon.
Pairwise sequence divergence between two species of subg. Aizoon
(i.e., P. odontophyllus and P. yangshanicus) and the remaining
species was 7.064%, while between P. odontophyllus/P.
yangshanicus and subg. Phedimus was 1.254%. Accordingly, P.
odontophyllus and P. yangshanicus are more closely related to
species of subg. Phedimus than to consubgeneric species in subg.
Aizoon. The first lineage (99% BS) included two species of subg.
Phedimus (P. stellatus and P. spurius) and two species of
subg. Aizoon (P. odontophyllus and P. yangshanicus), confirming
non-monophyly of subg. Aizoon (Chao, 2020). According to ITS
sequence divergence, two species of subg. Aizoon,P. odontophyllus
and P. yangshanicus, are more closely related to members of subg.
Phedimus than those of subg. Aizoon (Supplementary Table 3). The
second lineage (100% BS) included all but two species of subg.
Aizoon (100% BS), and monophyletic P. hybridus (98% BS)
diverged first within this clade. Although bootstrap support values
within the subg. Aizoon restricted us from rigorously inferring
species monophyly and interspecific relationships, but some species
relationships could be postulated with caution. The ITS tree
identified one weakly supported clade 1 (59% BS), which included
P. middendorffianus,P. selskianus,P. kurilensis,P. sichotensis, and
P. takesimensis. The Ulleung Island endemic P. takesimensis is
monophyletic (96% BS) and embedded within the non-
monophyletic P. middendorffianus, sampled primarily from
northeastern (Jilin) China. One accession of P. middendorffianus
sampled from Primorsky Krai, Russia, was sister to the remaining
accessions within this clade. Phedimus sichotensis (AM039913 from
GenBank), which is often treated as a subspecies of P.
middendorffianus (P. middendorffianus subsp. sichotensis), is sister
to the clade containing primarily P. middendorffianus and P.
takesimensis (73% BS). The southern Kuriles (Russia) endemic P.
kurilensis accession sequenced in this study is sister to the clade
containing P. middendorffianus and P. takesimensis (80% BS).
In addition, the ITS tree also suggested that P. sikokianus
(AB08863 from GenBank), which is narrowly endemic to
southern Japan (Shikoku), is sister to the members of clade 2
(52% BS weak support; thus, it could be either sister to clade 1 or
2). Excluding P. sikokianus, the remaining clade 2 is strongly
supported (99% BS). Within clade 2, the central South Korean
Peninsula endemics P. zokuriensis and P. latiovalifolium are
monophyletic (84% and 94% BS, respectively), whereas the widely
FIGURE 6
Dated chronogram showing divergence time of 21 accessions of subg. Aizoon plastomes. Estimated mean ages are shown for each node, with 95%
high posterior density (HPD) in brackets. Internal node posterior probability (PP) is shown in red (PP ≥0.95), blue (0.95> PP >0.75), and green
(0.75 >PP). Two calibration points are shown as asterisked nodes based on a previous study (Messerschmid et al., 2020).
Kim et al. 10.3389/fpls.2023.1089165
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distributed P. aizoon and P. kamtschaticus are not monophyletic.
The ITS tree also showed that P. litoralis, endemic to Russia, was
deeply embedded within the P. aizoon lineage sampled primarily
from Primorsky Krai (Russia) and Heilongjiang (China) (56% BS).
Phedimus ellacombeanus sampled from the type locality of Japan
and Korea was polyphyletic. One accession sampled from
southeastern Korea, Igarigani Beach, Pohang (Gyeongsangbuk-do
Province), is closely related to P. kamtschaticus, sampled from
various parts of the Korean Peninsula, and P. aizoon, sampled
from Gangwon-do Province, Korea. Conversely, two accessions
sampled from southeastern Korea, that is, Uje-bong Peak and
Geoje Island (Gyeongsannam-do Province), are closely related to
P. aizoon and P. kamtschaticus collected from Korea, Russia, and
China. Furthermore, two accessions sampled from Hachuja Island
in the southern part of Korea are sister to the clade containing all
but P. sikokianus and one accession of P. kamtschaticus (Korea) in
clade 2. Two accessions of P. ellacombeanus from Sochi Island in the
southern part of Korea share their most recent common ancestor
with P. latiovalifolium in Gangwon-do Province. Lastly, one
accession collected from the type locality of P. ellacombeanus in
Hakodate (southern Hokkaido, Japan) is embedded within P.
aizoon from northeastern (Jilin) China (61% BS).
Several distinct lineages of highly polyphyletic P. aizoon were
found exclusively in clade 2 (Figure 5), including a lineage largely
from Gangwon-do Province (Korea)/Jilin (China) and a lineage
primarily from Primorsky Krai (Russia), Mongolia, and P. litoralis
(56% BS). Several lineages of P. kamtschaticus were also revealed in
Korea, including one lineage sampled from various parts of Korea
(<50% BS) and the other lineage, which is sister to the clade of P.
ellacombeanus (Sochi Island) and P. latiovalifolium. Both P. aizoon
and P. kamtschaticus were not only intermixed with each other but
also closely related to other congeneric species, such as P.
ellacombeanus and P. litoralis.
3.4 Molecular dating
Based on the complete plastome sequences, we estimated the
divergence times of the major lineages within the subg. Aizoon
(Figure 6). Without any representative species from subg.
Phedimus, the age of subg. Aizoon was estimated to be 27.09 Ma
(95% HPD, 13.02–44.34 Ma), suggesting its origin in the late
Oligocene. Within the subg. Aizoon,theearlydivergenceof
diploid P. sikokianus in southern Japan was immediately followed
by the divergence of the remaining lineages (26.79 Ma; 95% HPD,
13.12–43.86 Ma). The age of the estimated divergence of the
Ulleung Island endemic P. takesimensis from its continental sister
species was estimated to be 2.03 Ma (95% HPD, 1.15–3.70 Ma),
suggesting its early colonization soon after the formation of Ulleung
Island (ca. 1.8 Ma). The estimated divergence of the narrow
endemic P. zokuriensis in Korea from its sister P. ellacombianus
was inferred to be very recent at 0.15 Ma (95% HPD, 0.05–0.33 Ma)
during the Pleistocene (late Ionian), whereas that of the other
narrow Korean endemic P. latiovalifolium was inferred at 10.53
Ma (95% HPD, 4.02–34.35 Ma) during the late Miocene
(late Messinian).
4 Discussion
4.1 Plastome conservation, mutation
hotspots, and positively selected genes
in subg. Aizoon
Genome size, gene order, and number were highly conserved
within the subg. Aizoon (Table 1). In addition, the overall genome
size, gene numbers, and GC content were like those of the sister
lineage Rhodiola and other closely related genera (Hylotelephium,
Kalanchoe,Orostachys,Rosularia,Sedum,Sinocrassula,and
Umbilicus)(Kim and Kim, 2020;Zhao et al., 2020;Zhao et al.,
2022). This suggests that despite being morphologically and
cytologically extremely variable, major lineages of the subfamily
Sedoideae have highly conserved plastomes. In the case of the
hypervariable regions of plastomes (Figure 3), which can be used
as barcoding markers, we identified one ycf1 gene and three
intergenic regions (trnH–psbA,ndhF–rpl32,andtrnW–trnP),
which showed high sequence variation in the two sister lineages
Rhodiola and Phedimus (Zhao et al., 2022). These sets of mutation
hotspots in the two sister genera could provide useful phylogenetic
information for phylogeographic and population genetic studies at
the intraspecific level. Rhodiola species, which are mainly adapted to
alpine habitats in the Qinghai-Tibet Plateau and the Hengduan
Mountains, have been shown to contain three positively selected
genes (rpl16,ndhA, and ndhH) and one gene with a faster than
average rate of evolution (psaA)(Zhao et al., 2020). The products of
these genes may have been involved in the adaptive radiation of
Rhodiola to high altitudes, an environment with low CO
2
concentrations and high-intensity light. In the subg. Aizoon of the
Phedimus lineage, which was demonstrated to have significant
niche divergence from its sister Rhodiola (Zhao et al., 2020), we
identified different sets of positively selected genes (ccsA,cemA,
matK,ndhC,psbJ,rpl22, and rpoC2)(Table 2). Because Phedimus
species occur in various wider habitats (e.g., grassy slopes, shrub
thickets,meadows,rockystreamsides, sandy cliffs, mountain
steppes, stony and gravelly soils in forests, sandy shores,
deciduous fields and forests, subalpine meadows, limestone hills,
and rocks), these plastomic adaptations are assumed to contribute
to the diversification and range expansion of subg. Aizoon
throughout its geographical range. This also suggests that these
genes were most likely selected when the common ancestor of the
genus Phedimus diverged from its sister lineage, Rhodiola. However,
it is yet to be determined whether some species of subg. Phedimus,
which occur at high altitudes (e.g., P. stevenianus,P. spurius, and P.
obtusifolius), show any evidence of positive selection for the same
genes (ndhA,ndhH,rpl16, and psaA) detected in Rhodiola. It is also
necessary to investigate what drives radiating diversification and
adaptation to diverse ecological niches (e.g., Kapralov et al., 2013;
Zhang et al., 2021;Izuno et al., 2022).
Kim et al. 10.3389/fpls.2023.1089165
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4.2 Origin of Korean endemic
Phedimus species
One major objective of this study was to determine the origin
and evolution of the Phedimus species endemic to Korea. This
study, based on extensive sampling in eastern Asia, allowed us to
gain insights into the origin of P. takesimensis on Ulleung Island
for the first time. A previous study showed the monophyly of P.
takesimensis on Ulleung Island without concretely determining
the closest continental sister lineage(s) (Seo et al., 2020). The
current study further corroborates previous findings that the
morphological and genetic variation of P. takesimensis on
Ulleung Island were accumulated after a single colonization
event of a continental ancestral population on the island.
However, the ITS tree strongly suggests that monophyletic P.
takesimensis (96% BS) is embedded within paraphyletic P.
middendorffianus (Figure 5 and Supplementary Figure 1),
whereas the species of Phedimus from Russia (P. aizoon,P.
litoralis,andP. selskianus) are the closest continental relatives
based on the plastome tree (100% BS; Figure 4). All accessions of
P. middendorffianus sister to P. takesimensis, on Ulleung Island
were from northeastern (Jilin) China. It also commonly occurs
from eastern Siberia to the Russian Far East. Therefore, this
species is a possible continental progenitor species of P.
takesimensis on Ulleung Island, suggesting cautiously its origin
from northeastern Asia as a geographical source area, based on
ITS phylogeny (Figure 5). Although this geographical region
seems likely to be the origin of P. takesimensis,theplastome
phylogeny suggests that different sets of species, i.e., P. aizoon,P.
litoralis,andP. selskianus, are most likely progenitor species
(Figure 4). Of these three species, P. selskianus has a distinct
characteristic of densely grayish pubescent leaves. Phedimus
litoralis is a glabrous herb with an elongated, creeping, simple
rhizome, and strong stems. The last species in this clade was P.
aizoon from Primorsky Krai. Therefore, based on plastome
phylogeny, we cautiously suggest that P. aizoon-like species
from northeastern Asia (Jilin, China, or the Russian Far East)
may also be involved in the origin of P. takesimensis.The
estimated divergence time of P. takesimensis from its
continental progenitor species was estimated to be 2.03 Ma
(95% HPD, 1.15–3.70 Ma), suggesting its origin soon after the
formation of Ulleung Island (ca. 1.8 Ma) (Figure 6). Of the nearly
40 endemic species on Ulleung Island, P. takesimensis is an
example of its geographical origin from the Russian Far East
and northeastern China rather than from the Korean Peninsula
(Rubus takesimensis,Yang et al., 2019;Campanula takesimana,
Cheong et al., 2020;Prunus takesimensis,Cho et al., 2021),
southern Korean Peninsula/southern Japan (Rubus takesimensis,
Yang et al., 2019), and Japan/Sakhalin, Russia (Scrophularia
takesimensis,Gil et al., 2020).Thephylogeneticincongruence
between maternally inherited plastome sequences and the
multicopy nature of nrDNA ITS sequences further complicate
the origin of P. takesimensis. Additional studies based on genome-
wide SNPs (e.g., Cho et al., 2021) or reduced representation
sequencing approaches (e.g., Johnson et al., 2019)wouldbe
useful for future phylogenetic studies of subg. Aizoon in East Asia.
The taxonomic status of P. zokuriensis and its relationship
with other congeneric species is contentious. It can be
distinguished from congeneric species by its weak and creeping
stems. Such phenotypic variation from its congeneric species
could be caused by its forest habitats and is well within the
range of broadly distributed species, such as P. kamtschaticus or
P. aizoon. Because P. kamtschaticus tends to occur on rocky
surfaces in shaded forests and sunny forest edges instead of the
more open grassland habitats of P. aizoon, it is highly plausible
that P. zokuriensis is conspecifictoP. kamtschaticus or at least
closely related to it. Morphologically, P. zokuriensis is more closely
related to P. kamtschaticus than to P. aizoon. In addition, based on
extensive morphological analyses, Chung and Kim (1989) argued
that P. zokuriensis and P. ellacombeanus are not infraspecifictaxa
of P. kamtschaticus, and this could be difficult to determine based
on the ITS phylogeny owing to weak bootstrap support (<50% BS;
Figure 5). Plastome phylogeny suggests that P. zokuriensis is
embedded within the clade of P. ellacombeanus,whichwas
sampled from the southern Korean Peninsula (100% BS). This
plastome connection between accessions of forest habitat from the
central Korean Peninsula and coastal habitat from the southern
Korean Peninsula, including isolated islands, was unexpected,
owing to their geographical distance and morphological
differences. Nevertheless, based on a previous extensive
morphological investigation (Chung and Kim, 1989)andits
monophyly and close maternal relationship with P.
ellacombeanus from this study, the species status of P.
zokuriensis would be reasonable to maintain until different lines
of evidence suggest otherwise.
Phedimus latiovalifolium is also narrowly endemic to Gangwon-
do Province in the northern part of South Korea. It was originally
described by Lee (1992) but was later hypothesized based on
morphological intermediacy to have a hybrid origin between P.
aizoon and P. kamtschaticus or between P. aizoon and P.
ellacombeanus (Lee, 2000). Yoo and Park (2016), based on
morphological and allozyme studies, refuted its hybrid origin and
suggested a distinct taxonomic status for P. latiovalifolium. Our
current study, for the first time, demonstrated the monophyly of P.
latiovalifolium (94% BS, Figure 5) and suggested that it shared its
most recent common ancestor with broad-leaved maritime P.
ellacombeanus sampled from Hachuju and Sochi Island (Jeju and
Gyeongsangnam-do Province, respectively) and P. kamtschaticus
(71% BS). However, the complete plastome tree is less conclusive
but shows its relationship with close relatives, suggesting that it
shares the most recent common ancestor with species from the
Russian Far East (P. selskianus,P. litoralis, and P. aizoon) and
Ulleung Island endemic P. takesimensis in Korea (92% BS, Figure 4).
Like P. zokuriensis, we suggest that it would be reasonable to
maintain the species status of P. latiovalifolium because of its
morphological and allozyme distinctions (Yoo and Park, 2016)
and the strong monophyly demonstrated in this study. Unlike the
recent origin of two other Korean endemic species, P. takesimensis
and P. zokuriensis, it was suggested that the split of P.
latiovalifolium from its sister lineage might have occurred at
10.53 Ma (95% HPD, 4.02–34.35 Ma) during the late
Miocene (Figure 6).
Kim et al. 10.3389/fpls.2023.1089165
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4.3 Species boundary within subg. Aizoon:
Splitter versus lumper
This study allowed us to broadly assess species relationships
within Phedimus, thus giving us an opportunity to evaluate species
boundaries. Within the genus Phedimus, two major lineages are
recognized: the first clade includes two species of subg. Phedimus (P.
stellatus and P. spurius) and two species of subg. Aizoon (P.
yangshanicus and P. odontophyllus), while the second one
includes all but two species of subg. Aizoon (Figure 5). Two
species (P. stellatus and P. spurius) in this clade are diploids (P.
odontophyllus and P. yangshanicus of subg. Aizoon are unknown)
with a simple descending dysploidy series of x=7tox=6tox=5
(Hart et al., 1993;Hart and Bleij, 2003). These two species and other
members of subg. Phedimus are morphologically distinct and owing
to their largely contiguous geographical distribution in Eurasia, a
recent origin has been suggested (Hart and Bleij, 2003). Although
subg. Aizoon is a very distinct taxon based on morphological and
cytological characteristics (x= 8), its component species are much
less clear, except for the hirsute P. selskianus. Other taxa are very
difficult to separate and can be merged because of the uniformity of
their floral characters despite the extreme variation in vegetative
characters (Hart and Bleij, 2003). However, Hart and Bleij (2003)
followed a less rigorous and much more conservative approach,
recognizing approximately 14 species in subg. Aizoon;Fröderström
(1931) included all but one species (P. hybridus), either as a
subspecies or synonym in P. aizoon. We fully agree that large-
scale, comprehensive biosystematics studies of natural populations
are required to properly understand morphological and cytological
variation (Amano, 1990;Amano and Ohba, 1992;Hart and Bleij,
2003). However, our current study provides some insights into the
species boundaries in subg. Aizoon from East Asia. We found that
the monophyletic P. hybridus represents the earliest diverged
lineage within this subgenus in the ITS phylogeny. Despite the
poorly described nature of this species, P. hybridus appears to be a
distinct taxon within subg. Aizoon. The ITS phylogeny suggested
that P. selskianus, a very distinct hirsute species, is sister to the clade
containing P. sichotensis (Russia), P. kamtschaticus (Japan), P.
kurilensis (Russia), P. middendorffianus (China), and P.
takesimensis (Korea) (Figure 5). According to the plastome
phylogeny (Figure 4), it is sister to the clade of P. litoralis–P.
aizoon (Russia). Therefore, if P. selskianus can be recognized as a
distinct species, other species in this “clade 1,”such as P. sichotensis,
P. middendorffianus, and P. takesimensis (Hart and Bleij, 2003),
could also maintain cautiously distinct species status until different
lines of evidence suggest otherwise. We could also maintain distinct
species status for P. sikokianus (Japan), P. zokuriensis (Korea), and
P. latiovalifolium (Korea) in “clade 2.”Phedimus sikokianus has
been suggested to be a key species for understanding the evolution
of the whole subg. Aizoon because of the low diploid chromosome
number and the opposite leaves (Amano and Ohba, 1992;Hart and
Bleij, 2003). Its phylogenetic position is unresolved in the ITS tree
(Figure 5), but in the plastome tree, P. sikokianus first diverged
within the subg. Aizoon (Figure 4). The precise phylogenetic
position of P. sikokianus requires further study based on multiple
samples and a robust phylogenetic framework. We were unable to
include any members of subg. Phedimus, but as in the ITS tree, it is
still possible that P. sikokianus represents one of the early diverged
lineages in subg. Aizoon, as supported by cytological evidence
(Amano and Ohba, 1992).
Although Chung and Kim (1989) argued for the distinct
taxonomic status of P. ellacombeanus, it is uncertain whether it
should be recognized as a distinct taxon in Korea based on the
current study. One accession collected from the type locality in
Hakodate (Hokkaido, Japan) was embedded within the A. aizoon
clade in the ITS phylogeny (Figure 5). However, some accessions of
P. ellacombeanus sampled, including those previously reported by
Chung and Kim (1989), were positioned in various lineages within
“clade 2.”In the plastome phylogeny (Figure 4), the type locality P.
ellacombeanus was sister to the clade of P. kamtschaticus–P.
kurilensis from Russia, whereas two accessions sampled from
Korea were closely related to P. zokuriensis and P.
middendorffianus–P. kamtschaticus in Korea. The accessions from
Korea occurred on seashores or sunny rock surfaces in forests on
islands and tended to have broader spatulate leaves. However, we
noticed that the leaf characteristics described in previous reports
(Praeger, 1917;Praeger, 1921;Jacobsen, 1960;Clausen, 1975;Evans,
1985;Chung and Kim, 1989) were not observed in mature plants in
the current study. As we cultivated P. ellacombeanus sampled from
Geoje and Hachuja Islands at Sungkyunkwan University, we
observed that young emerging stems tend to have opposite to
subopposite very broad leaves with two to four crenate margins,
which fits the general description of P. ellacombeanus by Chung and
Kim (1989). In addition, two specimens cited by Chung and Kim
(1989) as P. ellacombeanus from Geoje Island (SNU, 66782) and
Chuja Island (SNU, 66785) are very similar to our collections,
especially the young Geoje Island accession. Therefore, this study
suggests that P. ellacombeanus, previously reported in Korea, may
not truly represent P. ellacombeanus originally described from the
type locality in Hakodate, Japan. Given their phylogenetic positions,
P. ellacombeanus accessions sampled from Korea could be
considered P. kamtschaticus sensu Korea (= P. aizoon var.
floribundus sensu Japan). Since P. ellacombeanus was described as
a new species based on cultivated materials and was originally
collected as P. kamtschaticus by Maximowicz, it is necessary to
investigate its taxonomic distinction from P. kamtschaticus based
on broad sampling, especially from Hokkaido, northern Japan, and
southern Kuriles, Russia.
Phedimus kurilensis was described by Voroshilov (1965) from
the island of Kunashir and is considered endemic to southern
Kuriles. Voroshilov later considered P. kurilensis to be a
subspecies of P. sikokianus based on several characteristics (Ohba,
2002). However, P. sikokianus is endemic to the southern part of
Japan, Shikoku, and occurs in the mountains (Ohba, 2002).
Therefore, the distribution and habitat may not support the
subspecies treatment of P. kurilensis as P. sikokianus. The same
species of P. kurilensis is thought to occur in the northern part of the
island of Hokkaido in Japan, perhaps under different names
(personal observation, Marina Koldaeva). The ITS phylogeny in
our study suggests that P. kurilensis is sister to the clade containing
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org12
P. middendorffianus and P. takesimensis (Figure 5). In addition, the
plastome phylogeny suggests that P. kurilensis is sister to P.
kamtschaticus, which was sampled from Hokkaido in our study
(100% BS). In the Flora of Japan (Ohba, 2002), P. kurilensis is
considered a synonym of P. kamtschaticus (Uhl and Moran, 1972).
This species is known to occur in Kamchatka, the Kuriles, and
Japan. The species description of P. kamtschaticus in the Flora of
Japan (2002) is quite different from that of Korea, China, and Russia
and from that by Hart and Bleij (2003). Although P. kurilensis and
P. kamtschaticus appear to be closely related, it is uncertain whether
P. kurilensis in the southern Kuriles and P. kamtschaticus in
Hokkaido are conspecific based on species description and
chromosome number. Based on the ITS phylogeny (Figure 5), it
seems that P. kurilensis is distinct from the rest of the P.
kamtschaticus lineages from Korea and China. Given its close
relationship with other species currently recognized (P. selskianus,
P. sichotensis,P. middendorffianus,andP. takesimensis), P.
kurilensis could maintain its distinct species status until other
lines of evidence indicate otherwise.
Phedimus litoralis is endemic to the Ussuriysk floristic region
(Hart and Bleij, 2003). We sequenced two additional accessions of
P. litoralis from Primorsky Krai, and these accessions were closely
related to accessions of P. aizoon, primarily sampled from
Primorsky Krai (Russia) and one accession from Heilongjiang
(China) (Figure 5). In addition, plastome phylogeny suggested
that P. litoralis is sister to P. aizoon sampled from Russia (100%
BS). Therefore, as recently suggested by Hart and Bleij (2003),P.
litoralis could be conspecifictoP. aizoon or at least closely related to
it. This P. aizoon lineage, including P. litoralis, appears to be distinct
from other lineages of P. aizoon.Phedimus sichotensis, another
Russian Far East endemic in southern Primorsky Krai, is almost
indistinguishable from many small forms of the variable P.
kamtschaticus and was considered a subspecies of P.
middendorffianus (Gontcharova, 2000). ITS phylogeny showed
that P. sichotensis was sister to the clade containing primarily P.
kurilensis–P. middendorffianus–P. takesimensis (73% BS).
Therefore, it is highly unlikely that P. sichotensis is related to P.
kamtschaticus, but it is closely related to P. middendorffianus, which
requires further confirmation based on a broader sampling.
Lastly, the taxonomic recognition of two widely occurring
species, P. aizoon and P. kamtschaticus, as separate species or
infraspecific levels could be problematic given their polyphyletic
nature and close relationships with other more distinct species.
Some distinct geographical lineages could be recognized for each
species, while two species could be intermixed in certain clades,
making the species boundaries of the two species difficult (Figures 4,
5). This could be due, in part, to different species descriptions
depending on the country. In the Flora of Japan (2001), two
infraspecific taxa were recognized for A. aizoon (var. aizoon and
var. floribundus) based on the number of flowering stems and leaves:
flowering stems fascicled and oblanceolate to lanceolate-ovate to
ovate leaves with apically serrated leaf margins for var. floribundus
versus flowering stems one or two and rhombic-elliptic to elliptic
leaves with regularly serrated leaf margins, except near the base for
var. aizoon. In Korea, these descriptions match those of P.
kamtschaticus in the former (var. floribundus)andP. aizoon in the
latter (var. aizoon). The species description of P. kamtschaticus by
Hart and Bleij (2003) fits that of P. kamtschaticus in Korea and China
and that of P. aizoon var. floribundus in Japan. Therefore, it is
possible that plants with taller, one- to two-pronged stems and more
uniform characters could be recognized as P. aizoon, whereas plants
with smaller and more variable characters could be recognized as P.
kamtschaticus or P. aizoon var. floribundus. The species description
of P. kamtschaticus (Fisch. et C.A. Mey) in Japan seems quite different
from that in Korea, China, Russia, and Hart and Bleij (2003).Itis
described as having numerous slender, fascicled flowering stems, 5–
10 cm tall; small oblanceolate leaves (1–2 cm long and 0.6–1cmwide)
with irregularly serrate leaf margins; and carpels patent in fruits. It is
known to occur in Hokkaido in Japan, southern Kuriles, and
Kamchatka (Russia); it has a chromosome number of n=16(2n=
4x= 32, tetraploid) (Uhl and Moran, 1972). P. aizoon subsp. aizoon
(= P. aizoon) is a highly polyploid species comprising an aneuploid
series with 37 different chromosome numbers, ranging from 2n=71
to 2n=124(2n=12x= 96, dodecaploid, most frequent). In contrast,
P. aizoon var. floribundus sensu Japan (=P. kamtschaticus sensu
Korea, China, and Russia) is tetraploid (2n= 32), hexaploid (2n=
48), and octoploid (2n= 64) (Ohba, 1982;Amano, 1990;Amano and
Ohba, 1992). Therefore, it is plausible that P. kamtschaticus sensu
Japan (n= 16) is conspecifictoP. aizoon var. floribundus sensu Japan.
Owing to the existence of different ribotypes, we were unable to
generate clean ITS sequences for accessions of P. kamtschaticus
sampled from Hokkaido, Japan. However, the complete plastome
sequence strongly suggests that P. kamtschaticus from Japan is closely
related to P. kurilensis (considered a synonym of P. kamtschaticus
sensu Korea, China, and Russia) and P. ellacombeanus (Hokkaido,
Japan) and distantly related to any accessions of P. aizoon var.
floribundus from Japan and P. aizoon accessions from Korea and
Russia (Figure 5). This suggests that P. kamtschaticus from Hokkaido
and southern Kuriles could represent a distinct taxon from P.
kamtschaticus, as currently recognized in Korea, China, and Japan.
In summary, the following relationships and diversification
processes have been proposed based on complete plastome and
nrDNA ITS sequences, pending independent confirmation based
on genome-wide nuclear data. After the divergence of P. sikokianus
and P. hybridus within the subgenus Aizoon during the mid-
Oligocene, the P. aizoon and P. kamtschaticus lineages diverged
further. It is hypothesized that certain distinct geographical lineages
of each species have become narrowly occurring local endemics: P.
takesimensis,P. zokuriensis, and P. latiovalifolium in Korea; P.
litoralis and P. sichotensis in the Russian Far East. It is also highly
plausible that species occurring in northern Japan and the Russian
Far East (P. middendorffianus,P. sichotensis,P. selskianus, and P.
kurilensis) shared their most recent common ancestor and
contributed to the origin of the insular endemic P. takesimensis
on Ulleung Island, Korea.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found below: https://www.ncbi.nlm.nih.gov/
Kim et al. 10.3389/fpls.2023.1089165
Frontiers in Plant Science frontiersin.org13
genbank/, OP344945-OP344959; https://www.ncbi.nlm.nih.gov/
genbank/, OP346879-OP346967.
Author contributions
YK, S-HK, and S-CK designed the experiments and YK, S-HK,
M-SC, MK, TI, MM, and S-CK collected the samples. YK, S-HK,
and JY performed the experiments and analyzed the data. YK and
S-HK drafted the manuscript and MK, TI, MM, and S-CK revised
the manuscript. All authors contributed to the article and approved
the submitted version.
Funding
This research was funded in part by the National Institute of
Biological Resources (NIBR, grant number 2020NIBR202005201),
under the program of “A Study on Plant Resources on DNA
Sequences Utilizing Next Generation Sequencing Technique (3rd year).”
Acknowledgments
We thank Dr. Hee-Young Gil for assistance with the field work.
We are also greatly indebted to Dr. Tadashi Yamashiro (Tokushima
University) for acquiring the collection permit and collecting
P. sikokianus.
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.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2023.1089165/
full#supplementary-material
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