Bull. Natl. Mus. Nat. Sci., Ser. B, 47(2), pp. 103–108, May 21, 2021

Phylogenetic Position of Lagotis kunawurensis(Plantaginaceae) in Himalaya,a Species of Boreal L. glauca Aggregate
Koh Nakamura1,*, Rinchen Yangzom2, Atsushi Sugano3 and Yoshinori Murai4
1

Botanic Garden, Field Science Center for Northern Biosphere, Hokkaido University,
Sapporo, Hokkaido 060–0003, Japan
2
National Biodiversity Centre, Ministry of Agriculture and Forests, Post Box 875,
Serbithang, Thimphu, Bhutan
3
Division of Environmental Resources, Graduate School of Agriculture, Hokkaido University,Sapporo, Hokkaido 060–8589, Japan
4
Department of Botany, National Museum of Nature and Science, 4–1–1 Amakubo, Tsukuba,Ibaraki 305–0005, Japan*E-mail: kohnakamur@gmail.com(Received 10 February 2021; accepted 24 March 2021)

Abstract Phylogenetic position of a Himalayan species Lagotis kunawurensis is elucidated. Thisspecies is recognized as a member of L. glauca aggregate. The idea of close relationship between
L. kunawurensis in Himalaya and L. glauca in the north paciﬁc rim, however, contradicts with thescenario of the migration of the genus from Himalaya to the boreal region indirectly via Central
Asian highlands. In our phylogenetic analysis of the genus based on chloroplast DNA sequencedata, L. kunawurensis and L. glauca were placed together in a clade of ser. Lagotis. Within theclade, L. kunawurensis formed a subclade with other six Himalayan species, among which
L. clarkei was its sister species. Phylogenetic aﬃnity of L. kunawurensis to L. glauca is notsupported, disagreeing with the view that L. kunawurensis is a member of L. glauca aggregate
and being compatible with the migratory scenario.
Keywords : alpine plant, Bhutan, Himalaya, Lagotis, molecular phylogeny, series assignment.

Introduction
Lagotis J.Gaertn. (Plantaginaceae) comprises
ca. 30 species of perennial herbs (Hong et al.,1998) distributed in Himalaya, Central Asia,
boreal Asia, and northwest America (Lu, 1992).
In the genus, two sections (Lagotis and Acaules
Maxim.) and three series (Lagotis, Pharicae
X.F.Lu, and Ramalanae X.F.Lu) in sect. Lagotis
are recognized (Lu, 1992). Molecular phylogenyof Lagotis was published including ca. 80% ofthe described species (Li et al., 2014), and sect.
Acaules and the three series were reported to be© 2021 National Museum of Nature and Science

respectively monophyletic. Besides, the molecular analysis well covered the geographic range ofthe genus and inferred the migratory history (Liet al., 2014); Lagotis have likely originated in theTibetan Plateau in the Miocene and diversiﬁed
there from the Miocene to Pleistocene, andspread to the boreal region via Central Asian
highlands.
Among the unanalyzed species in the molecular study (Li et al., 2014) is L. kunawurensis(Royle ex Benth.) Rupr. (Fig. 1A). This species
is broadly distributed in Himalaya (Bhutan,India, Nepal, and Pakistan) at around 2,700–
5,600 m asl. and grows on wet semi-stable screeand marshland (Polunin et al., 1987; Grierson

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Fig. 1. Habit of Lagotis kunawurensis (A; Mt. Jomolhari range, western Bhutan) and L. glauca (B; Rebun Island,northern Japan). Scale bars indicate 5 cm.

and Long, 2001; GBIF.org, 2021). Lagotis
kunawurensis is recognized as a member of L.
glauca aggregate, a group of species with highlyvariable and overlapping morphology (Griersonand Long, 2001) and was previously named as L.
glauca Gaertn. var. sikkimensis Hook.f., whichname is now treated as its synonym (Griersonand Long, 2001). Lagotis glauca is a boreal species and widely distributed in Japan (Rebun
Island in northernmost Japan and alpine rangesof central Honshu), the Kuriles, Kamchatka, theAleutian, and Alaska (Yamazaki, 1993; Takahashi, 2015) (Fig. 1B). The idea of close relationship between L. kunawurensis in Himalaya andL. glauca in the north paciﬁc rim, however, contradicts with the above-mentioned “Central Asiatic Highland Corridor” scenario for migrationfrom Himalaya to the boreal region (Li et al.,2014). The species relationship should be clariﬁed with molecular analysis. This study aims toelucidate the phylogenetic position of L. kunawurensis and to discuss its series assignment andthe relationship with L. glauca.
Materials and Methods
In our collaborative ﬁeld research in Mt.
Jomolhari range, Paro, Bhutan, a sample of L.
kunawurensis was collected. The voucher specimen (voucher number: THIM16153) was deposited in the National Herbarium (THIM) ofNational Biodiversity Centre, Bhutan. DNAsequence data obtained from the sample wereanalyzed together with GenBank data used in Liet al. (2014), that included 22 species fromChina, northeastern Russia, Kazakhstan, Armenia, Alaska, and India (Table 1). For monophyletic species in Li et al. (2014), one sample each
was randomly selected and used (in L. integrifolia, L. integra, L. alutacea, L. brevituba, L.
angustibracteata, L. decumbens, L. globosa, andL. brachystachya). Wulfenia carinthiaca Jacq.
was selected as an outgroup following Li et al.
(2014).
Genomic DNA was extracted from the silica
gel-dried leaf sample of L. kunawurensis using
the cetyltrimethyl ammonium bromide (CTAB)
method (Doyle and Doyle, 1990). Four chloroplast DNA regions (matK, rps16, trnG–trnS, andtrnL–F) used in Li et al. (2014) were PCR ampliﬁed using common primers (Taberlet et al., 1991;
Johnson and Soltis, 1994; Steele and Vilgays,1994; Oxelman et al., 1997; Hamilton, 1999).
PCR ampliﬁcation was performed in 20 μl total
volume: 10 μl of Taq DNA polymerase 2× master mix (Amplicon, Rødovre, Denmark), 0.8 μl ofeach forward and reverse primers (10 pmol/μl),
0.4 μl of dimethyl sulfoxide (DMSO), 0.8 μl of
total DNA (ca. 20 ng/μl) and 7.2 μl of distilledwater. The PCR cycle conditions were as follows: initial template denaturation for 4 min at95°C; 25 cycles of 50 s at 94°C, 40 s at 50°C, and40 s at 72°C; a ﬁnal extension for 10 min at 72°C.
The puriﬁed PCR products were ampliﬁed using

L. alutacea W.W.Sm.
L. angustibracteata Tsoong & H.P.Yang
L. brachystachya Maxim.
L. brevituba Maxim.
L. cashmeriana Rupr.
L. clarkei Hook.f.
L. crassifolia Prain
L. decumbens Rupr.
L. glauca Gaertn.
L. globosa Hook.f.
L. humilis Tsoong & H.P.Yang
L. integra W.W.Sm.
L. integrifolia (Willd.) Schischk.
L. kongboensis T. Yamaz.
L. korolkowii Maxim.
L. kunawurensis (Royle ex Benth.) Rupr.
L. macrosiphon Tsoong & H.P.Yang
L. minor (Willd.) Standl.
L. pharica Prain
L. praecox W.W.Sm.
L. ramalana Batalin
L. stolonifera Maxim.
L. yunnanensis W.W.Sm.
Wulfenia carinthiaca Jacq.

Taxa
Lagotis/Lagotis
Lagotis/Lagotis
Acaules/—
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Pharicae
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Acaules/—
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Lagotis
Lagotis/Pharicae
Lagotis/Rhamalanae
Lagotis/Rhamalanae
Acaules/—
Lagotis/Lagotis—

Section/series
Yunnan, China
Qinghai, China
Qinghai, China
Qinghai, China
Rohtang, India
Tibet, China
Tibet, China
Xinjiang, China
Commander Island, Russia
Xinjiang, China
Tibet, China
Qinghai, China
KazakhstanTibet, China
Armenia
Paro, Bhutan
Tibet, China
Alaska, USASichuan, China
Sichuan, China
Qinghai, China
Kazakhstan
Yunnan, China—

Sample locality
KC413450
KC413452
KC413454
KC413459
KC413461
KC413462
KC413463
KC413474
KC413465
KC413467
KC413469
KC413470
KC413478
KC413479
KC413480
LC314879
KC413481
KC413466
KC413482
KC413484
KC413485—
KC413486
AY492169

matK
KC413529
KC413531
KC413533
KC413538—
KC413540
KC413541
KC413552
KC413543
KC413545
KC413547
KC413548
KC413556
KC413557
KC413558
LC314814
KC413559
KC413544
KC413560
KC413562
KC413563
KC413564
KC413565
AY218804

rps16

KC413608
KC413610
KC413612
KC413617—
KC413619
KC413620
KC413632
KC413622
KC413624
KC413626
KC413628
KC413634
KC413635
KC413636
LC314909
KC413637
KC413623
KC413638
KC413640
KC413641
KC413642
KC413643—trnG–trnS

KC413568
KC413570
KC413572
KC413577
KC413579
KC413580
KC413581
KC413592
KC413583
KC413585
KC413587
KC413588
KC413596
KC413597
KC413598
LC314849
KC413599
KC413584
KC413600
KC413602
KC413603
KC413604
KC413605
AF486409

trnL–F

Table 1. Section/series assignment, sample locality, and GenBank accession numbers of Lagotis species in the phylogenetic analysis. A dash in GenBank accession
indicates missing data

Phylogenetic position of Lagotis kunawurensis
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ABI PRISM Big Dye Terminator v.3.1 (AppliedBiosystems, Foster, CA, USA) with the sameprimers as those used for the PCR. DNA
sequencing was performed on an ABI Prism
3730 DNA analyzer (Applied Biosystems). Automatic base-calling was checked manually using
Chromas 2.6.2 (http://technelysium.com.au/wp/chromas/). Sequence alignment was conductedusing ClustalX v.2.1 (Larkin et al., 2007) andthen edited manually using SeaView v. 4.6.1(Gouy et al., 2010).
Phylogenetic analyses were conducted using
Bayesian (BI) and maximum parsimony (MP)inferences. To perform BI analyses, the bestmodel of nucleotide substitution was estimatedas SYM＋G with MrModeltest (Nylander, 2004)
using Akaike information criterion (AIC; Akaike,1974). BI analysis was conducted using MrBayes
3.2.6 (Ronquist et al., 2012). Analyses were runfor 10 million generations, sampling every 1,000generations. Convergence and eﬀective sample

size (＞ 200 after burn-in) of all parameters werechecked with Tracer v.1.6 (Drummond and Rambaut, 2007) and then the ﬁrst 2,500 trees werediscarded as burn-in. The 50% majority rule consensus tree of all the post-burn-in trees andposterior probability (PP) was calculated. TheMP analysis was performed with PAUP* v.4.0a(Swoﬀord, 2002) using heuristic searches of
1,000 replicates with random taxon addition andtree bisection reconnection (TBR) branch swapping, with the MulTrees and steepest descentoptions on, and the MaxTrees option set to10,000. Indels were treated as missing data
because they were diﬃcult to align among thewide range of taxa. Bootstrap support (BS) wasestimated from 1,000 replicates with randomtaxon addition and TBR branch swapping, withthe MulTrees option oﬀ and the MaxTrees option
set to 200. The BI and MP trees were displayedusing Figtree v.1.3.1 (Drummond and Rambaut,2007).

Fig. 2. Bayesian phylogenetic tree of 23 Lagotis species based on cpDNA. The outgroup Wulfenia carinthiaca isomitted from the ﬁgure for simplicity. Numerals on branches indicate Bayesian posterior probability (left) andbootstrap support (right) in the MP analysis. –means ＜ 50%. Scale bar indicates 0.002 substitutions per site.

Phylogenetic position of Lagotis kunawurensis

Results and Discussion
The aligned length of cpDNA was 2,623 bp. In
this study, clades with PP≥0.95 and/or BS≥70%
were considered statistically supported. There
was no topological incongruence between BI andMP trees (data not shown). Therefore, only BI
tree is presented with BS. In the BI phylogenetictree (Fig. 2), the two sections Lagotis (PP/BS＝1.00/—) and Acaules (1.00/99.1%) werereciprocally monophyletic. For each of the threeseries Lagotis, Ramalanae, and Pharicae, monophyly was not recovered. In the previous study(Li et al., 2014), the three series were respectively monophyletic based on concatenated dataof cpDNA and nuclear ribosomal DNA (nrITS).
We tested the phylogenetic congruence between
cpDNA and nrITS using the incongruence length
diﬀerence test (Farris et al., 1994) with PAUP
and the test revealed signiﬁcant heterogeneity
between the cpDNA and nrITS data sets
(P＜0.05). Thereby, the concatenation approach
may be inappropriate and the monophyly of thethree series needs further veriﬁcation.
Series Lagotis was divided into two clades:
one clade including L. kunawurensis and L.
glauca plus other 11 species (1.00/58.3%) andthe other clade comprising the other three species(0.99/—). Although the monophyly of ser. Lagotis was not indicated but L. glauca is the typespecies of the series and thereby the result supports the idea that L. kunawurensis is placed inser. Lagotis (Lu, 1992). Within the former clade,
one subclade including L. kunawurensis andother six species (1.00/90.3%) was recognized,
where L. glauca was not included. These six arespecies of eastern Himalaya, i.e., Bhutan, Sikkim, Nepal, and Tibet (L. clarkei, L. crassifolia,
L. humilis, L. kongboensis, and L. macrosiphon)and of western Himalaya and Pakistan (L. cashmeriana) (Hong et al., 1998; Grierson and Long,2001; Mill, 2015), and in this Himalayan clade,
L. kunawurensis clustered with L. clarkei(1.00/91.7%). Phylogenetic aﬃnity of L.
kunawurensis to L. glauca is not supported, disagreeing with the view that L. kunawurensis is a107

member of L. glauca aggregate (Grierson andLong, 2001). The present result provides further
support for Himalayan diversiﬁcation of thegenus and is compatible with the scenario ofmigration from Himalaya to the boreal region
indirectly via Central Asian highlands (Li et al.,2014).
Acknowledgments
This study was supported in part by Grants-inAid for Scientiﬁc Research, KAKENHI to K.N.
(20K060870003) and Discretionary expenses ofDirector General of the National Museum ofNature and Science.
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