Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis, and polyphyletic nature of S. lutescens var. intermedia

Abstract

Both Salvia akiensis and S. lutescens (Lamiaceae) are endemic to Japan. Salvia akiensis was recently described in 2014 in the Chugoku (= SW Honshu) region, and each four varieties of S.lutescens distributed allopatrically. Among varieties in S.lutescens, var. intermedia show a disjunctive distribution in the Kanto (=E Honshu) and Kinki (= W Honshu) regions. Recent field studies of S.lutescens var. intermedia revealed several morphological differences between the Kanto and Kinki populations. Here, I evaluated these differences among Salvia lutescens var. intermedia and its allies with morphological analysis and molecular phylogenetic analyses of nuclear ribosomal DNA (internal and external transcribed spacer regions) and plastid DNA (ycf1-rps15 spacer, rbcL, and trnL-F) sequences. Both morphological analysis and molecular phylogenetic analyses showed that S. lutescens var. intermedia from the Kinki region and var. lutescens were closely related to each other. However, var. intermedia from the Kanto region exhibited an association with S. lutescens var. crenata and var. stolonifera, which also grew in eastern Japan, rather than var. intermedia in the Kinki region. These results indicated that S. lutescens var. intermedia is not a taxon with a disjunctive distribution, but a combination of two or more allopatric taxa. Present study also suggested that S. akiensis was most closely related to S. omerocalyx.

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Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 87
Taxonomic study on Japanese Salvia (Lamiaceae):
Phylogenetic position of S. akiensis, and polyphyletic
nature of S. lutescens v ar. intermedia
Atsuko Takano1
1 Division of Natural History, Museum of Nature and Human Activities, Hyogo. Yayoigaoka 6, Sanda, Hyogo
669-1546, Japan
Corresponding author: Atsuko Takano (takano@hitohaku.jp)
Academic editor: E. Fischer |  Received 7 February 2017 |  Accepted 8 May 2017  |  Published 5 June2017
Citation: Takano A (2017) Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis, and
polyphyletic nature of S. lutescens var. intermedia. PhytoKeys 80: 87–104. https://doi.org/10.3897/phytokeys.80.11611
Abstract
Both Salvia akiensis and S. lutescens (Lamiaceae) are endemic to Japan. Salvia akiensis was recently de-
scribed in 2014 in the Chugoku (= SW Honshu) region, and each four varieties of S.lutescens distributed
allopatrically. Among varieties in S.lutescens, var. intermedia show a disjunctive distribution in the
Kanto
(=E Honshu) and Kinki (= W Honshu) regions. Recent eld studies of S.lutescens var. intermedia
revealed several
morphological dierences between the Kanto and Kinki populations. Here, I evalu-
ated these dierences
among Salvia lutescens var. intermedia and its allies with morphological analysis
and molecular phylogenetic analyses of nuclear ribosomal
DNA (internal and external transcribed spacer
regions) and plastid DNA (ycf1-rps15 spacer, rbcL, and trnL-F)
sequences. Both morphological analysis
and molecular phylogenetic analyses showed that S.
lutescens var. intermedia from the Kinki region and
var. lutescens were closely related to each other.
However, var. intermedia from the Kanto region exhibited
an association with S. lutescens var. crenata and
var. stolonifera, which also grew in eastern Japan, rather
than var. intermedia in the Kinki region. ese results indicated that S. lutescens var. intermedia is not
a
taxon with a disjunctive distribution, but a combination of two or more allopatric taxa. Present study also
suggested that S. akiensis was most closely related to S.
omerocalyx.
Keywords
cpDNA, Lamiaceae, nrDNA, Phylogenetics, Salvia akiensis, Salvia lutescens
PhytoKeys 80: 87–104 (2017)
doi: 10.3897/phytokeys.80.11611
http://phytokeys.pensoft.net
Copyright Atsuko Takano. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Introduction
e genus Salvia L. (tribe Mentheae) is the largest genus in Lamiaceae; it comprises
nearly 1,000 species.
Salvia has radiated extensively into three regions of the world:
Central and South America (500 spp.),
West Asia (200 spp.), and East Asia (100 spp.)
(Alziar, 1988–1993). In Japan, twelve species, eight
varieties, and one putative hybrid
have been described since unberg’s (1784) rst account. e genus
was classied
into three subgenera (subg.), including Allagospadonopsis Briq., Salvia, and Sclarea
(Moench) Benth, (Hihara et al. 2001, Inoue 1997, Murata and Yamazaki 1993, Ta-
kano et al. 2014). Most
of the taxa are endemic to Japan, with the exception of S.
japonica unb., S. nipponica Miq. and S. plebeia R. Br. (Murata and Yamazaki 1993).
There are four varieties known in S. lutescens (Koidz.) Koidz.: var. crenata,
var.
intermedia, var. lutescens, and var. stolonifera (Murata 1952, Yonekura and Kajita 2003
onwards). Fukuoka and
Kurosaki (1982) noticed distribution of each taxon does not
overlap and claried that the distribution of
var. crenata on the Japan Sea side of Cen-
tral to Northern Honshu, var. stolonifera on the Pacic side of
Central Honshu, var.
lutescens around the Suzuka Mountain range (Mie Pref., W Honshu), and the disjunc-
tive distribution of var.
intermedia in the Kanto (E Honshu) and Kinki regions (W
Honshu) based on herbarium works.
Takano and Okada (2011) conducted molecular phylogenetic analyses of Japa-
nese Salvia and found that
the species were distributed among three subclades: (1)
S. plebeia (subg. Sclarea), (2) subg. Salvia, and (3) subg. Allagospadonopsis. ey also
found four varieties of S. lutescens that did not form a
monophyletic group; instead,
they were dispersed among several clades in phylogenetic trees, based on
both plastid
DNA (cpDNA) and nuclear ribosomal DNA (nrDNA) data, and their topologies
were not concordant with each other. In addition, they became
paraphyletic in the
phylogenetic trees based on combined cpCNA and nrDNA data (Takano and Okada
2011). Furthermore, during a recent eld survey, I noticed that S. lutescens var. inter-
media in the Kanto
and Kinki regions had dierent morphological characteristics. e
basal part of the anther connective was
generally glabrous in the Kanto population,
but it was pilose in the Kinki population. Also, in the Kanto
population, the stalk of
the inorescence declinated toward the ground after owering, and it typically
became
proliferous; in contrast, in the Kinki population, the inorescence grew erect, and it
was never
proliferous.
Recently, a new species of Japanese Salvia, S. akiensis A.Takano, T.Sera et Kurosa-
ki has been described from Shimane and Hiroshima Prefectures (Takano et al. 2014).
At the moment, this species shows disjunctive distribution, ca. 40 km away from each,
and the habitat is also very dierent between Hiroshima and Shimane: it grows among
bamboo by roadsides and on slopes below evergreen mixed forests and plantations in
Shimane (Sakoda et al. 2014), but it is found in moist, shallow soil on rock walls by
streams in deciduous forests in Hiroshima (Takano et al. 2014). erefore, it may
wonder if the species be monophyletic. Takano et al. (2014) discussed relationships
among S. akiensis, S. isensis Nakai ex H.Hara, S. lutescens and S. omerocalyx Hayata

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 89
based on morphological characters, but molecular phylogenetic position of S. akiensis
remains unclear.
As a step toward taxonomic revision of variety of S. lutescens and to conrm mono-
phyly and phylogenetic position of S. akiensis, morphological and molecular phyloge-
netic analyses were conducted. Takano and Okada (2011) followed the Murata and
Yamazaki (1993) system in which treated var.
intermedia as a forma f. lobatocrenata
and var. lutescens as f. lutescens, however, here I follow the Murata
(1952) system (=Y-
list, Yonekura and Kajita 2003 onwards), and each infraspecic taxon of S. lutescens is
treated as a variety.
Materials and methods
Morphological analyses on S.lutescens in herbaria
Murata (1952) studied morphological variations in the plants of subgen.
Allagospadonopsis in Japan and found hairiness, number or shape of leaets, presence
/absence of glandular hairs were so variable and could not be used as diagnostic
characters. Diagnostic characters separated each variety of S. lutescens are indumen-
tums of the basal part of the anther connective and oral color (Nakai 1950, Mu-
rata 1952). Among varieties, var. lutescens shows pale yellow owers and pilose at the
base of anther connective, var. intermedia shows deep violet corolla and pilose at the
base of anther connective, var. crenata does purple corolla and glabrous base of anther
connective. Floral color and indumentums of var. stolonifera is same as var. crenata,
however, var. stolonifera extends its stolon after anthesis (Nakai 1950). Since it is dif-
cult to know exact oral color by examining dry specimens, the indumentums at
the base of the anther connective were observed for glabrousity in selected
specimens,
which bore at least several owers. A total of 89 specimens of S. lutescens var. interme-
dia, including its syntypes, of the 34 specimens are from Kanto region and 55 from
Kinki, were examined in the following herbaria: the Museum of Nature and Human
Activities, Hyogo (HYO); the Kanagawa Prefectural Museum (KPM); Kyoto Univer-
sity (KYO); Tokyo
Metropolitan University (MAK), the Osaka Museum of Natural
History (OSA), and e University of
Tokyo (TI) (Appendix 1). Additionally, all the
specimens of S. lutescens var. lutescens including its holotype at KYO were examined on
the same characters, since no information on that character is available.
DNA extraction, PCR, and DNA sequencing
e protocols for DNA extraction, polymerase chain reaction (PCR), purication, and
DNA sequencing
were described previously by Takano and Okada (2011). e PCR
conditions and the PCR and sequencing
primers for rbcL, the trnL-F intergenic spacer
region of cpDNA (trnL-F), and the internal transcribed
spacer (ITS) region of nuclear

Atsuko Takano / PhytoKeys 80: 87–104 (2017)
90
ribosomal DNA (nrDNA) were also described previously by Takano and
Okada
(2011). To amplify in the ycf1-rps15 spacer region found in cpDNA (ycf1-rps15),
5711f and
rps15r (both from Drew and Sytsma 2011) were used as PCR primers in
PCR assays, and ETS-bdf1 (Drew and Sytsma 2011) and 18S-E (Baldwin and Markos
1998) were used to amplify the external transcribed
spacer (ETS) sequence from
18S-26S ribosomal DNA. e four PCR primers were also used for
sequencing. e
PCR conditions for amplifying the two loci were: denaturation at 95 °C for 3 min,
followed by 40 cycles at 95 °C for 30 s, 54 °C for 30 s, and 72 °C for 30 s; and a nal
extension at 72°C
for 5 min.
Sequence alignment and phylogenetic analysis
Raw sequence data were assembled and edited manually, with BioEdit software (ver.
7.2.5 Hall 1999)
DNA sequences were aligned with the CLUSTALW 1.83 software package, with
default settings and
multiple alignments (ompson et al. 1994). Alignments of the
rbcL, trnL-F, and ycf1-rps15 sequences
of cpDNA, and the ITS and ETS sequences of
nrDNA were combined. Gaps were deleted.
Compared to Takano and Okada (2011), the ETS (Baldwin and Markos 1998)
and ycf1-rps15 of cpDNA
(Dong et al. 2015) were newly sequenced for all samples.
Further, two individuals of S. akiensis and three
of S. lutescens var. intermedia, three of S.
lutescens var. crenata, and one each of S. isensis, S.
japonica var. japonica, S. lutescens var.
lutescens, and S. plebeia were newly added for the analysis. e
sampling sites of S. lutes-
cens group were shown in Fig. 1. A total of 36 individuals of Salvia were used, including
all the Salvia
taxa from Japan and one Taiwanese Salvia (S. arisanensis Hayata). Salvia
polystachya M. Martens et
Galeotti and S. plebeia were selected as outgroup; the former
species belonged to clade II sensu Maria and Classen-Bockho (2014), which became a
sister to group IV and contained the East Asian Salvia; the latter
species became a sister
to a species of the subgenus Allagospadonopsis and Salvia (Hu 2015).
Materials, acces-
sion numbers for the sequences, vouchers, and references to the literature are presented
in
Table 1. e sampling sites for the varieties of S. lutescens are shown in Fig. 1.
e incongruence length dierence (ILD) test (Farris et al. 1994) was used to
evaluate congruence between the chloroplast and the nuclear data sets. 100 replications
were performed using PAUP*4.010b (Swoord 2002). As the ILD test (P < 0.01)
suggested incongruence between the two datasets, and the topologies also exhibited
discordance, I performed separate analyses for the cpDNA and the nrDNA data.
Maximum Likelihood (ML) and Bayesian inference (BI) were used. Nucleotide substi-
tution model parameters were determined for each partition by gene was evaluated with
KAKUSAN 4.0 (Tanabe 2007), and the corrected Akaike information criterion (AICc)
(Sugiura 1978) was used for model selection. For the cpDNA partitions KAKUSAN
suggested the HKY85 (rbcL) and GTR+G (trnL-F, ycf1-rps15spacer) models, and the
HKY85 model for ETS and GTR+G model for ITS for the nrDNA partitions. e ML

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 91
Akita
Shiga
Nara
Aichi Mt.Mikuni Mt.Kintoki
Yushin
Tanzawa
Fukui
var. intermedia
var. crenata
var. lutescens
var. stolonifera
distribution of var. intermedia
(modified Fukuoka & Kurosaki 1982)
KANTO Reg.
CHUGOKU Reg.
KINKI Reg.
KANTO Reg.
Figure 1. Map of Japan shows the sites where Salvia lutescens taxa were sampled. Open circle = var.
crenata,
lled circle = var. intermedia, open square = var. lutescens, lled square = var. stolonifera. e areas
encircled
with dotted lines show the Kinki and Kanto regions, as indicated.
analyses were completed using TREEFINDER version March 2011 (Jobb et al. 2004).
A replicated (500 iterations) partitioned analysis was performed with bootstrap (1000
rounds) using AICc separated model for nrDNA dataset and AICc proportional model
for cpDNA dataset. Bayesian evolutionary analysis using partitioned datasets were
run in BEAST v.1.8.3 (Drummond et al. 2012, Heled and Drummond 2010) with
20 million Markov Chain Monte Carlo (MCMC) iterations, under an uncorrelated
relaxed clock (Drummond et al. 2006), Yule process of speciation with a random
starting tree for each partition. Convergence of the chains was checked using the
program Tracer1.6 (Rambaut et al. 2014). High eective sample sizes were observed
for all parameters (posterior ESS values > 200 for the combined analyses). Maximum
clade credibility trees with divergence times means and 95% highest probability
densities (HPDs) were produced using Tree Annotator (Drummond et al.2012).
Results
Morphological characteristics
Among the 89 specimens of S. lutescens var. intermedia examined, 52 specimens from
the Kinki region were pilose at the base of the
anther connective (Fig. 2), and no speci-

Atsuko Takano / PhytoKeys 80: 87–104 (2017)
92
Table 1. Taxa, Genbank accession number, and voucher specimens/references used in this study. Newly sequecned data are shown bold.
Name Pop. Code rbcL trnL-F ycf1-rps15 ETS ITS Voucher / References
S. akiensis A.Takano, T.Sera et Kurosaki
HIR
(HirohsimaPref.) LC124176 LC124188 LC060529 LC060825 LC060729 A.Takano and N.Kurosaki with T.Sera 130606-
1(HYO)
S1(Shimane Pref.) LC124177 LC124189 LC060530 LC060826 LC060728 M.Sakoda et al. 1 (HYO,KYO)
S. arisanensis Hayata AB295063 AB295074 LC060531 LC060827 AB295085 Sudarmono and Okada (2007)
S. glabrescens (Franch. et Sav.) Makino
var. glabrescens FS (Wakasa, Fukui) AB541134 AB541148 LC060532 LC060828 AB541120 Takano and Okada (2011)
var. repens (Koidz.) Kurosaki KY (Kyoto) AB295064 AB295075 LC060533 LC060829 AB295086 Sudarmono and Okada (2007)
S. isensis Nakai ex Hara
MIE AB266221 AB266231 LC060534 LC060830 AB266241 Sudarmono and Okada (2007)
AICHI LC124178 LC124190 LC060535 LC060831 LC060730 A-200933 (living material at Hiroshima Bot.Gard.
Originally from Owariasahi city, Aichi Pref.)
S. japonica unb.
f. albiora Hiyama AB266220 AB266230 LC060536 LC060832 AB260240 Sudarmono and Okada (2007)
f. japonica Osaka AB266219 AB266229 LC060537 LC060833 AB266239 Sudarmono and Okada (2007)
f. japonica Gotenba LC124179 LC124191 LC060538 LC060834 LC060731 A.Takano 140806-5 (HYO)
f. longipes (Nakai) Sugimoto AB266218 AB266228 LC060539 LC060835 AB266238 Sudarmono and Okada (2007)
S. koyamae Makino AB541128 AB541142 LC060540 LC060836 AB541114 Takano and Okada (2011)
S. lutescens Koidz.
var. crenata (Makino) Murata
AICHI AB266223 AB266233 LC060541 LC060837 AB266243 Sudarmono and Okada (2007)
Yushin AB353202 AB353198 LC060542 LC060838 AB353206 Takano and Okada (2011)
Akita LC124180 LC124193 LC124205 LC124201 LC124203 Y. Horhii, S. Nishida et al. 2015026 (HYO)
Fukui LC124181 LC124194 LC124204 LC124200 LC124202 A.Takano 150702-1a (HYO)
var. intermedia (Makino) Murata
Nara LC124182 LC124195 LC060544 LC060840 AB295097 Sudarmono and Okada (2007)
Shiga LC124183 LC124196 LC060546 LC060842 LC060735 A.Takano 140821-1 (HYO)
Mt.Mikuni LC124184 LC124197 LC060547 LC060843 LC060733 A.Takano 140806-4 (HYO)
Tanzawa LC124185 LC124198 LC060548 LC060844 LC060734 A.Takano 140622-2 (HYO)
var. lutescens Koidz.
MIE AB266222 AB266232 LC060549 LC060845 AB266242 Sudarmono and Okada (2007)
Aoyama LC124186 LC128192 LC060550 LC060846 LC060737 a201241 (living material at Hiroshima Bot.Gard.
Originally from Aoyama Kogen, Mie Pref.)

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 93
Name Pop. Code rbcL trnL-F ycf1-rps15 ETS ITS Voucher / References
var. stolonifera G.Nakai AB541139 AB541153 LC060551 LC060847 AB541125 Takano and Okada (2011)
S. nipponica Miq.
var. nipponica
TOKU (Tokushima) AB541132 AB541146 LC060552 LC060848 AB541118 Takano and Okada (2011)
KUMA
(Kumamoto) AB541127 AB541141 LC060553 LC060849 AB541113 Takano and Okada (2011)
var. kisoensis K.Imai NAK AB541136 AB541150 LC060554 LC060850 AB541122 Takano and Okada (2011)
S. omerocalyx Hayata
var. omerocalyx HI (Hidaka, Hyogo) AB353204 AB353196 LC060555 LC060851 AB353200 Takano and Okada (2011)
HYO (Yabu, Hyogo) AB353205 AB353197 LC060556 LC060852 AB353201 Takano and Okada (2011)
var. prostrata Satake AB541138 AB541152 LC060557 LC060853 AB541124 Takano and Okada (2011)
S. pygmaea Matsum.
var. pygmaea AB295072 AB295083 LC060558 LC060854 AB295094 Sudarmono and Okada (2007)
var. simplicior Hatus. ex T.Yamaz. AB541140 AB541154 LC060559 LC060855 AB541126 Takano and Okada (2011)
S. ranzaniana Makino AB287373 AB287374 LC060560 LC060856 AB287375 Sudarmono and Okada (2007)
S. ×sakuensis Naruh. et Hihara AB541129 AB541143 LC060561 LC060857 AB541116 Takano and Okada (2011)
Outgroup
S. plebeia R.Br. KIZU AB295073 AB295084 LC060563 LC060858 AB295095 Sudarmono and Okada (2007)
KAMI LC124187 LC124199 LC060562 LC060859 LC060738 A.Takano and N.Kurosaki 090607-2 (HYO)
S. Polystachya M.Martens et Galeotti AY570435 JF301399 JF289067 JF301334 JF301356 Drew and Systma (2011)

Atsuko Takano / PhytoKeys 80: 87–104 (2017)
94
Figure 2. Photographs of S. lutescens var. intermedia owers. a Flower of A. Takano 140806-4-2 (HYO),
from
Mt. Mikuni, Susono-shi, Shizuoka Pref. (Kanto region). Arrows indicate the base of the anther connec-
tive. No hairs are visible b Flower of A. Takano 140813-1 (HYO), from Mt. Yamatokatsuragi, Gose-shi, Nara
Pref. (Kinki region). e
red open circle indicates the base of the anther connective. White hairs are visible.
mens from the Kanto region shared this characteristic (Appendix
1). Ten specimens
collected from the Kanto region had at least one, but less than 10 hairs. Twenty-four
specimens from the Kanto region (Fig. 2) and three specimens from the Kinki region
(Y.Kato s.n. [KYO], T.Kobayashi 23369 [KYO], and A.Takano 140821-1 [HYO]) were
glabrous at the base of the anther
connective. However, a duplicate of T.Kobayashi
23369 (KYO) examined at HYO was pilose at the base
of the anther connective (Ap-
pendix 1).
Totally, 18 specimens of S. lutescens var. lutescens were deposited at KYO and
examined, 13 of these had pilose at the base of the anther connective (Appendix 1).
Four of these had no owers, and only one specimen, M.Hara s.n., collected from Mt.
Takami, Maze-Mura, Iinan-gun, Mie Pref. showed glabrousity.
Phylogenetic positions of Japanese taxa in the genus Salvia
A
likelihood analysis using the concatenate cpDNA datasets (rbcL+trnL-F+ycf1-rps15
spacer) for 36 individuals in 23 taxa resulted in a ML tree with –lnL = 5295.264. e
ML and Bayesian trees had similar topology; the Bayesian maximum clade credibility
tree is shown with ML bootstrap (ML-BS) and Bayesian posterior probability (BI-PP)
in Figure 4. e Japanese and Taiwanese species of subg. Allagospadonopsis formed
a well supported clade (ML-BS/BI-PP, 100/0.97). Two subclades were found in the
subg. Allagospadonopsis clade: (1) S. japonica + S. pygmaea + one S. akiensis + S. arisan-
ensis + ve individuals of S. lutescens in E Japan subclade, and (2) one S. akiensis
(S1),
two S. isensis, S. lutescens in Kinki + S. ranzaniana + two S. lutescens in the Kanto region

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 95
0
5
10
15
20
25
no. of specimens
Locality
mixed
>10 hairs
0-10 hairs
glabrous
Kanto region Kinki region
Figure 3. A graph shows the number of specimens examined indumentums at the base of anther
connective.
+ S.
omerocalyx. e latter group of taxa, minus the S. omerocalyx (HYO), consisted of a
strongly supported
subclade, with high ML-BS/BI-PP values (98/0.99). S. lutescens in
E Japan
were scattered between both subclades, but the S. lutescens in the Kinki region
consisted a cluster with the weak support (--/0.70).
A concatenate nrDNA datasets (ETS+ITS) yielded a ML tree with –lnL =
3789.114. e ML and
Bayesian trees had similar topology; the Bayesian maximum
clade credibility tree is shown with ML-BS and BI-PP
in Figure 5. e Japanese and
Taiwanese species of subg. Allagospadonopsis formed a well supported clade (ML-BS/
BI-PP, 100/1.00). ere were four subclades in the
Allagospadonopsis clade: (1) S. lute-
scens group in E Japan + S. isensis (ML-BS/BI-PP, --/0.69), (2) S. lutescens in Kinki +
S. ranzaniana (ML-BS/BI-PP, 61/0.57), (3) S. arisanensis + S. omerocalyx + S. akien-
sis (ML-BS/BA-PP, 76/0.99), and (4) one S. lutescens var. crenata + S. japonica + S.
pygmaea (ML-BS/BA-PP, 58/0.97). us,
Salvia lutescens and its allies apparently be-
came polyphyletic. Salvia ranzaniana became a sister group to S. lutescens in the Kinki
region but the ML-BS /BA-PP support was weak (61/0.57). Salvia isensis became a
sister group to S. lutescens in the Kanto region with strong ML-BS/BA-PP support
(86/1.00). Salvia akiensis formed a strongly supported subclade with S. omerocalyx
group (89/1.00).

Atsuko Takano / PhytoKeys 80: 87–104 (2017)
96
Salvia polystachya
S. akiensis (HIR)
S. akiensis (S1)
S. omerocalyx (HYO)
S. omerocalyx v. prostrata
S. isensis (Mie)
S. ranzaniana
S. lut. v. intermedia (Nara)
S. lut. v. intermedia (Shiga)
S. arisanensis
S. lut. v. crenata (Aichi)
S. pygmaea
S. japonica f. albiflora
S. japonica f. japonica (Osaka)
S. japonica f. longipes
S. nipponica v. nipponica (TOKU)
S. sakuensis
S. glabrescens v. glabrescens (FS)
S. glabrescens v. repens (KY)
S. plebeia (KIZU)
S. lut. v. lutescens (Aoyama)
S. lut. v. stolonifera
S. lut. v. intermedia (Tanzawa)
S. lut. v. crenata (Yushin)
S. lut.v. intermedia (Mt.Mikuni)
S. pygmaea v. simplisior
S. nipponica v. nipponica (KUMA)
S. nipponica v. kisoensis (NAK)
S. omerocalyx (HI)
S. lut. v. lutescens (Mie)
S. koyamae
S. lut. v. crenata (Akita)
S. lut. v. crenata (Fukui)
S. isensis (Aichi)
S. japonica f. japonica (Gotenba)
S. plebeia (KAMI)
E JAPAN
anther connective
glabrous
Subg. Allagospadonopsis
anther connective
pilose
KINKI Reg.
100/1.00
72/1.00
99/1.00
100/1.00
64/0.99
69/1.00
92/0.99
100/0.97
--/0.93
98/0.99
--/0.54
69/0.79
72/0.88
--/0.7
73/0.61
87/0.95
Figure 4. e Bayesian maximum clade credibility tree derived from plastid DNA
(concatenate dataset
of rbcL, trnL-F, ycf1-rps15). ML-bootstrap/Bayesian PP numbers are shown near the
corresponding
branch. ick lines denote a clade that was strongly supported, with ML-
bootstrap and/or Bayesian-PP
greater than 95 %. ML: maximum likelihood; PP:
posterior probability.
Discussion
is study suggests that S. lutescens var. intermedia is polyphyletic. Four individuals of
var. intermedia, two from the Kanto and two from the Kinki region fell into dierent
subclades in both molecular phylogenetic trees using cpDNA and nrDNA datasets,
although the two from the Kinki region were always in the same subclade (Figs 4, 5).
The plants of var. intermedia from the Kanto region (Tanzawa and Mt.Mikuni) fell
into the same subclade in the nrDNA tree together with var. crenata, var. stolonifera,

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 97
Figure 5. e Bayesian maximum clade credibility tree derived from nuclear ribosomal
DNA (con-
catenate dataset of ETS and ITS). ML-bootstrap/Bayesian-PP numbers are shown above or below the cor-
responding branch. ick lines denote a clade
that was strongly supported with ML-bootstrap and/or
Bayesian-PP values greater than
95 %. ML: maximum likelihood; PP: posterior probability.
S. lut. v. lutescens (Aoyama)
S. lut. v. lutescens (Mie)
Subg. Allagospadonopsis
anther connective
pilose
KINKI Reg.
S. akiensis (HIR)
S. akiensis (S1)
S. omerocalyx (HYO)
S. omerocalyx v. prostrata
S. arisanensis
S. lut. v. crenata (Aichi)
S. pygmaea
S. japonica f. albiflora
S. japonica f. japonica (Osaka)
S. japonica f. longipes
S. nipponica v. nipponica (TOKU)
S. sakuensis
S. glabrescens v. glabrescens (FS)
S. glabrescens v. repens (KY)
S. plebeia (KIZU)
S. pygmaea v. simplisior
S. nipponica v. nipponica (KUMA)
S. nipponica v. kisoensis (NAK)
S. omerocalyx (HI)
S. koyamae
S. japonica f. japonica (Gotenba)
S. plebeia (KAMI)
Salvia polystachya
S. isensis (Mie)
S. ranzaniana
S. lut. v. intermedia (Nara)
S. lut. v. intermedia (Shiga)
S. lut. v. stolonifera
S. lut. v. intermedia (Tanzawa)
S. lut. v. crenata (Yushin)
S. lut.v. intermedia (Mt.Mikuni)
S. lut. v. crenata (Akita)
S. lut. v. crenata (Fukui)
S. isensis (Aichi)
E JAPAN
anther connective
glabrous
98/1.00
96/1.00
61/0.57
--/0.69
86/1.00
70/0.67
76/0.99
89/1.00
97/1.00
95/1.00
--/0.93
58/0.97
88/1.00
98/1.00
98/1.00
59/0.91
82/1.00
100/1.00
97/1.00
71/0.87
100/0.93
100/1.00
86/0.59
91/0.99
and S. isensis whereas they fell into dierent subclades in the cpDNA tree. Such a
contradiction might indicate that var. intermedia from the Kanto region have multiple
origins, or might have originated via hybridization or introgressive gene ow between
nighbouring taxa (e.g., Sudarmono and Okada 2007). e discordance between nr
DNA and cpDNA data is common in the mint family (Trusty et al. 2004, Moon et
al. 2010, Drew and Sytsma 2013, Deng et al. 2015), and chloroplast-based phylogeny
often does not reect their morphological relationships, which can be explained by

Atsuko Takano / PhytoKeys 80: 87–104 (2017)
98
chloroplast capture (Rieseberg and Soltis 1991). Morphological analysis also supports
the contention that var. intermedia is polyphyletic, as the specimens of var. intermedia
studied showed in the indumentums at the base of the anther connective: pilose in
the plants from the Kinki region, and glabrous in the plants from the Kanto region
(Fig.3).erefore it is clear that var. intermedia from the Kinki region and the taxon
from the Kanto region are dierent entities, suggesting that var. intermedia is not a
taxon that shows disjunctive distribution, but is instead admixture of two or more
biological entities. Additionally, as mentioned in introduction, after owering the
stalk of the inorescence becomes declinate to ground and usually proliferous in case
of the plants from the Kanto region, but never become declinate in the plants from
the Kinki region. e indumentums at the base of anther connective is eective to
select pollinators to avoid intrusion of insects which could not be eective pollinators
(R.Classen-Bockho pers. Comm..) However, pollinators of var. intermedia in the
Kinki and the Kanto region are not dierent (=Bombus (Diversobombus) diversus di-
versus, some Halictidae, and Syrphidae. Takano 2017). Habitat is also similar: half-
shaded, on mesic soils along streamlet on the forest oor of deciduous forests. ey
might have begun to be diverged from each other after long geographical isolation.
On the contrary, present morphological and molecular phylogenetic analyses
indicated that S. lutescens var. lutescens and var. intermedia from the Kinki region are
closely related to each other. In molecular phylogenetic analysis, they formed a cluster
in both cpDNA- and nrDNA trees, though ML-BP/BI-PP support was not strong in
cpDNA tree. e morphological study revealed var. lutescens is pilose at the base of the
anther connective: therefore, S. lutescens var. intermedia in the Kinki region share the
same morphological status with var. lutescens. The distribution of var. lutescens is very
near to that of var. intermedia in the Kinki region (Mie, Shiga, Nara Prefs.), although
var. lutescens and populations of the Kinki regions of var. intermedia have never been
found to grow together.
Salvia lutescens var. intermedia in the Kanto region may be more closely related to var.
crenata and var. stolonifera. Murata (1952) mentioned that the base of anther connective is
glabrous in var. stolonifera and var. crenata. e present study revealed that var. intermedia
in the Kanto region shares this character with those two taxa. Salvia lutescens var. intermedia
in the Kanto region formed a strongly supported sucblade with var. crenata, var. stolonifera
and S. isensis in nrDNA phylogenetic tree. In the cpDNA phylogenetic tree, S.lutescens
var.intermedia from the Kanto region (Mt.Mikuni) was included in the subclade containg
S. akiensis, S. japonica, S. lutescens var. crenata, and S. pygmaea whereas S. lutescens var.
intermedia (Tanzawa) formed a subclade with var.
stolonifera and was included in the
subclade containing S. akiensis, S. omerocalyx, S. ranzaniana, and S.lutescens var. intermedia
from the Kinki + S. isensis. ese ndings suggest a close relationship among var. crenata,
var. stolonifera, and var. intermedia from the Kanto region. Var. intermedia from the Kanto
region may belong to var. stolonifera and var. crenata. e identity of var. intermedia and
other varieties of S. lutescens are needed to be re-evaluated, and further study is necessary
towards revision of varieties of S. lutescens.

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 99
e phylogenetic analyses also suggest that S. akiensis comprises a monophyletic
group, as indicated by nrDNA tree, and that most of the species allied to S. akiensis was
the S. omerocalyx group. Salvia akiensis and S. omerocalyx group comprised a subclade in
nrDNA (ML-BS/BI-PP: 89/1.00). ese two taxa did not form a subclade in cpDNA,
but it may be of introgression/chloroplast capture /hybridization as mentioned above.
In contrast, S. akiensis and S. omerocalyx share following characters: bearing the largest
owers among species in the subg.
Allagospadonopsis, ower from May to June, and
exhibit gynodioecy (Takano 2013; Takano
et al. 2014). These characters are assumed
to be symapomorph.
Acknowledgements
I am grateful to Teruo Katsuyama (KPM), Nobuhira Kurosaki (Hyogo Museum/ Uni-
versity of Hyogo), Masahiro Sakoda (Chugai Technos Co.), Tetsuya Sera (Hiroshima
City Agricultural
Committee Secretariat), and Sachiko Nishida (Nagoya Univ.) for
providing plant materials, references,
and information. Dr. Hiroshi Okada (University
of Hyogo), and anonymous reviewers provided constructive comments on
an early
draft of the manuscript. I also thank the curators of KPM, KYO, MAK, OSA, and
TI, for
allowing me to use their collections and facilities. is study was supported,
in part, by the New
Technology Development Foundation and by a Grant-in-Aid for
Scientic Research (C) (no. 26440227)
from the Japan Society for the Promotion of
Science (JSPS).
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Appendix 1
Specimens examined Salvia lutescens var. intermedia. And var. lutescens.
Salvia lutescens var. intermedia
Specimens glabrous at base of anther connective (27 sheets)
KANTO Region. Yamagata Pref.: Mt. Kushigata, S.Kigawa s.n., July 10, 2001 (KPM
NA0151444, NA015445). Ibaragi Pref.: Tsukuba, C.Owatari s.n., July 8, 1895 (syn-
type, TI); Ibidem, C.Owatari s.n., July 25, 1895 (syntype, TI). Gunma Pref.: Mt.
Akagi, H.Hara s.n., July 12, 1928 (TI); Akagi, Shikisimadori, Chubu, H.Hara s.n.,
11 July 1928 (TI), Kouzuke, Tone, Yunogoya-daira, H.Hara s.n., 13 July, 1928(TI);
Joshu, Tone-gun, Tokura, S.Saito 145 (TI). Kanagawa Pref.: Hakone, S.Ohkubo s.n.,
July 26, 1881 (syntype TI); Ibidem, unknown collector, July 26, 1881 (syntype, TI);
ibidem, S.Tamaki s.n., July 14, 1914 (TI); Minami pass, Hakone, T.Makino 62582
(KYO); Yoduku, Yamakita-cho, T.Katsuyama et al., July 23, 2005 (KPM NA0124794);
Mikuniyama-rindo, Hakone, Inoue et al., June 18, 1998 (KPM NA0112995); Tan-
zawa-Ohyama, T.Nishio 1489 (KPM); Summit of Mt. Kintoki, Hakone, S.Kigawa
s.n., July 3, 1980 (KPM NA1020531); Ishigoya-Ochiai, Kiyokawa-mura, H.Takahashi
20563 (KPM); Ishigoya-Ochiai, Kiyokawa-mura, H.Takahashi 20565 (KPM); Mine-
saka Pass to Myojin Pass, Yamakita-cho, S.Mori 20536 (KPM); Kaminokawa, Tsukui-
machi, S.Kigawa 20559 (KPM); Kurokura, Yamakita-machi, M.Furuse 45371 (KPM);
Ogawadani, Yamakita-cho, A.Takano 140622-2 (HYO). Shizuoka Pref.: Mt.Mikuni,
Fukayoshi, Susono-shi, A.Takano 140806-4-1 (HYO); ibidem, A.Takano 140806-4-2
(HYO).
KINKI Region. Nara Pref.; Yoshino, Yamato, Y.Kato s.n., Aug. 8. 1936 (KYO).
Hyogo Pref.:Taki-gun, Nishiki-cho, T.Kobayashi 23369 (KYO). Shiga Pref.: Ikadachi-
tochu, A.Takano 140821-1 (HYO).
Specimens showed one to several hairs at base of anther connective (10 sheets)
KANTO Region. Pref.Sizuoka: Mt. Higane, Prov. Izu, S. Shimazu s.n., July 18,
1920(KPM);
Pref.Kanagawa: Sirogane rindo, Yugawara-cho, Y.Hasegawa 14263 (KPM); Oga-
wadani-rindo, Yamakita-cho, T.Katsuyama s.n., Aug. 22, 1995 (KPM NA0100397);
Marudake, Hakone, M.Tashiro s.n.,July 18, 1956 (KPM NA0157166); Tounomine,
Hakone, T.Deguchi 80495 (KPM); Tougadake, Yamakita-cho, S.Kigawa s.n., July 3,
1980 (KPM NA1020531); Ishigoya-Ochiai, Kiyokawa-mura, H.Takahashi 20564
(KPM), Mt.Ohmuro, Tsukui-machi, S.Kigawa s.n., June 10, 1979 (KPM NA1020566);
Hayatogawa Rindo, Tsukui-machi, S.Kigawa 20541 (KPM), Youkizawa, Yamakita-
machi, S.Kigawa 20567 (KPM).

Taxonomic study on Japanese Salvia (Lamiaceae): Phylogenetic position of S. akiensis... 103
Specimens which showed long pilose at base of anther connective (52 sheets)
KINKI Region. Kyoto Pref.: Rakuhoku, Ohara, Otonashi W.F., S.Hajacava s.n., Aug.
1896 (TI), Kyoto, Ohara, T.Tsuyama s.n., Sep.7, 1934 (TI); Kiyotaki-Takao, Ukyo-ku,
Kyoto, S.Tsugaru & T.Takahashi 26448 (KYO); Mt.Hyotankuzure-yama, near Ohara,
G.Nakai 5401 (KYO); ibidem, G.Nakai s.n., July 25, 1951 (KYO); Kadono-gun, Naka-
gawa-mura, M.Tagawa 887 (KYO, two sheets); Maesaka Takanomine to Shimosugisaka,
S.Okamoto s.n., July 14, 1932 (KYO), Bodai W.F., Nakagawa, G.Nakai 6305 (KYO);
Mt. Kibune, unknown collector, June 28, 1921 (KYO); Kyoto-shi, Nakagawa to Bodai
no Taki, M.Hutoh 10515 (OSA); ibidem, M.Hutoh 9264 (OSA); ibidem, M.Hutoh
10528 (OSA); ibidem, M.Hutoh 3465 (OSA); Mt.Hiei-san, S.Tanaka s.n., June 30,1932
(OSA). Hyogo Pref.: Taki-gun, Nishiki-cho, T.Kobayashi 23369 (HYO); Youtakuji,
Sanda, T.Makino 62583 (KYO); Moshi, Sanda, A.Takano 140813-1 (HYO). Nara Pref.:
Yamato, Sanjo-ga-dake to Gyojagaeri, Y.Momiyama s.n., July 16, 1955(TI) (three sheets),
Ibidem, H.Hara s.n., July 16, 1955 (TI) ; ibidem, T.Kobayashi 30611 (OSA); near the
temple Kongo, Kashiwagi, Yamato, K.Kondo s.n., June 8, 1928 (TI); Kosei River, Ten-
kawa Village, K.Seto 44248 (OSA); Mt. Omine to Mt.Sanjogadake, H.Hara 4683 (TI);
en route from Wasamata hut to Mt.Nihon-dake, Kamikitayama-mura, M.Okamoto
1966 (OSA); Mt.Daifugendake, T.Kodama 10833 (OSA); Shonoiwaya-Mt.Wasamata
Kamikitayama-mura, K.Kodama 14356 (OSA); Irinami, Yamato, S.Sakaguchi s.n., June,
1930 (KYO); Mt.Ohmine, S.Sakaguchi s.n., Aug. 4, 1931 (KYO); enroute from Mt.Sanjo
to Mt.Daihugen, T.Kodama s.n., Aug.6,1959 (KYO); Mt.Sanjo, G.Koizumi s.n., July
13, 1922 (KYO); Mt.Yamatokatsuragi, Gose, A.Takano 140819-1 (HYO). Osaka Pref.:
Mt.Izumi-katsuragi, S.Nakanishi s.n., July 30, 1968 (OSA); Ibidem, T.Nakajima s.n.,
Aug.21, 1952 (OSA); Ibidem, C.Satonaka s.n., July 12, 1981 (OSA). Wakayama Pref.:
Ryujin-Mura, Koya, T. Nakajima s.n., July 31, 1931 (two sheets, TI); Doro Hacho,
G.Nakai 5213 (KYO); Ibidem, T.Kodama s.n., May 30, 1951 (OSA); ibidem, M.Hori
s.n., May 30, 1951 (OSA); Hidaga-gun, Ooze, S.Sakaguchi s.n., July 27,1932 (KYO);
Mt. Sukuyama, Katsuragi-cho, Ito-gun, K.Seto 29839 (KYO, OSA); Mt. Kurosawa,
Sayiki-mura, Y.Ogawa s.n., Aug.30, 1950 (KYO). Shiga Pref.: Otsu, N.Takemura s.n.,
June 1901 (Lectotype MAK); Omi, Tochu, M.Togashi 1205 (TI); Tochu to Ikadachi,
M.Umebayashi 737 (KYO); Mt. Hiei, G.Murata 11415 (KYO); Ukawa, Shiga-cho,
M.Tanimoto s.n., June 9, 1973 (KYO); Benzaiten to Sakamoto, Mt.Hiei, S.Tanaka s.n.,
June 30, 1932 (KYO). Mie Pref.: Wada, Kiwa-cho, Minami-murogun, H.Takahashi
21040 (KYO); Taki-gun, Miyagawa-mura, Shimomate (cult.), K.Seto 17303 (OSA).
Salvia lutescens var. lutescens
Specimens which showed long pilose at base of anther connective (13 sheets)
Mie Pref.: Itaya, Kata, Kameyama, S.Tsugaru & T.Sawada 34155(KYO); Notoyama,
Suzuka-gun, T.Hattori s.n., Aug. 5, 1928 (KYO); Kozu-mura, Naga-gun, G.Nakai

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4772 (KYO);Shinzan kokuyu-rin, Iinan-gun, Z. Tashiro s.n., 4.Aug., 1934 (KYO),
Mt. Gozaisyo, G.Koizumi s.n., 11 Jun. 1922 (KYO); Kozu-mura, Myouga-gun, (cult.
at KYO) G.Nakai 5402 (KYO); Ibidem, G.Nakai 4773 (KYO); Nagaishi-dani, Mt.
Kamagadake, Komono-cho, Mie-gun, N. Fukuoka 4948 (KYO); Onsen-do, Mt. Go-
zaisho, G.K. & S.F. s.n., June 1922 (KYO, holotype).
Shiga Pref.: Nasugahara, Ohara-Mura, Kouga gun, G.Koizumi s.n., 2 July, 1939
(KYO), Kurotaki, Tsuchiyama-cho, Koga-gun, T. Murase 47897 (KYO); Koga-gun,
Suzuka-Pass, H. Koyama & N. Fukuoka 55 (KYO); Nara Pref.: Kamide, Momomata,
Mitsue-mura, Uda-gun, K.Kawabata 9994 (KYO)