Molecular Phylogenetic Analyses of Geranium robertianum Populations Recently Found in Japan

2012·0.71 MB·English

by  Nishida, Sachiko

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ISSN1346-7565 ActaPhytotax.Geebot.62(2/37)9:-87 (2012)

MolecularPhylogeneticAnalyses Geranium
of robertianum
PopulationsRecentlyFound inJapan

SACHIKO NISHIDAii HIRosHI AzuMA2, AKIyo NAIKI3 AND MAKoTo OGAwA4
'17ie 7Vtigoy aUhiversity thiseum. Nbg(o?a CJhiversi4 MFuro-cho, Chikusa-ku. Nagcp;a. 464-860J ,Jt{pan.
i' D qp a r t me n t "[email protected],ac,ip (2iuthorf coorrrespondencel;
qf'Botaiz BG"aduate School ofScienc tes,,' oCtlohiversi Staykyo-ku .ig,o t6o06-8502, Japan;
3Gradztate School qf'Education ,14Listerty Program, Oka.vama tbeiversi Kti.tya-ku ,Oka.vain 7a00-8530, Japan;
47bkushima PrqlZictztr Maugseum, Bunka-no-mori Park, Hbchiinan-cho, n)kushima 770-8070, mpan
Geranium robertiamtm L, is considered an endangered species jn Japan, while i ti sinvasiv ien several
countries outside Japan. Recently, plants ofG. robertianztm have been found at several localiti neonse, of
which are original statiens fbr G robertianum in Japan .Tb determin ewhether the recently found p]ants
are native Japanese plants or were naturalized from sources outside Japan, we conducted molecular phy-
logeneti canalyses using DNA sequences of the plasti drcgiens ot'the trn L intro nt,rnL3LtrnF and trnJI-
psbA intergen iscpacer regions, and the nuclear intcrna ltranscribed spacer (ITS )Am.ong 32 samples
collected froi n21 populations ,we recegnized th ree major groups ,consisting of 1) recently fbund plants,
2) indigenous plant sa,nd 3) plants from Britain ,Our results suggcst that recently discovere pdlant osf G.
robertianum in Japan are more likel ynaturalized from seurces outside Japan rather than haying cscaped
from indigenou Jsapanes epopulations .Thc origin ofthe new]y found plants is not known althuugh their
origin in Britain is unlikely.
Key words: endangered species, Geraniacea eG,eranium robertianztm, invasiv pelant,
molecu]arphylogeny
An endangered species is one at risk of ex- ning from Shiga Prefectur eto Mie Prefecture
tinctio nand usually fbund only in limite dlocali- through Gifu Prefecture ,and en Mt, Tsurugi lo-
ties or habitats ,In contrast, an invasiv eor natu- cated between Tokushima and Kochi prefectures,
ratized species is one that has extended it sdistri- These areas are both limeston eregions and G.
butio ninto nonnative localiti easnd usually grows robertianum is considered a calcicole in Japan.
in several habitats .We here report a case ofa Because of it snarrow distributio nand limited
plan tspecics that may be both endangered and habitat ,G. robertianum is liste ads endangered in
invasiv ein Japan ,a situation which we investi-Gifu, Mie, Tokushima, and Kochi prefectures
gated using molecular phylogenetic analyses. (Gif uPrefectur e2001, Tokushima Prefecture
Geranium robeFtianum L. (Geraniace ias ean) 2001. Mie Prefecture 2005, Kochi Prefecture
annual or biennia pllant that can be distinguished20IO). Outside ofJapan, however ,G robertianum
firo mother species of Geranium L, in Japan by its is widely distribute din Europe and northwestern
compound Ieaves d,istincti ovdeor and glandular Asia, and is naturalized in parts of North and
hairs covering most parts of the plant (ShimizuSouth America and New Zealand (Tof t2s004). In
1982, Akiyama 2001) .Geranium robertianum these areas, i tis also fbund in a wide range of
originaHy had a restricted distributio nin Japan: habitats ,e.g., woodlands, hedge banks, and open
on ML Ibu ki and the Suzuka mountain range run- habita tsincludin gon scree, rocks, and shingle
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([[bf 2t0s04). at NUM except for the specimen from Mt. Ibuki,
Recently ,specimens of Geranium robertia- Shiga, which was deposite dat Lake Biwa Muse-
num have been collected at localit iien sHokkaido um.
(#Goda1687in1994,KYO), Kyoto(#lamazaki

6550 in 1998, KYO), and Fukuoka (#Kbg 1a5820 DA44 extraction, PCR amplijication, andDAC4 se-
in 2006, KYO), none ofwhich are known to have quenelng
had populations of G. robertianum previously, Ibta lgenomic DNA was extracted from fresh
The newly fbund plants grow in various habitats leave sor leave sdried in silica gel using the mod-
and are indiffere tnot soil type (Nishi dpaersonal ifie dcetyltrimethylammonium bremide (CTAB)
observation), which might be evidence that they method of Doyle & Doyle <1987),
have naturalized fi;Q moutsidc Japan and diffbr The trnL intron plus trnL-trnF (tmL5L3LtrnF
from the original Japanes enative G robertia- hereafter t)r,nHLpsbA, and ITS regions were am-
num, The native Japanese plants ,however ,are plifie dusing polymeras echain reaction (PCR).
able to grow successfully in non-limestone soils The PCR mixture (20 yL) contained 1 ptL tem-
under cultivation (Nishi udnpaublished data) ,so plat eDNA, 2 "L dNTPs (2. m5M each), 1 ptL of
that habitat information alone cannot discrimi- each primer (1 0uM), 2 yL 10× 7lrq buffe r(con-
nate the origin of the newly fbund plants ;they taining 20 mM MgC12), O,5 U 7lae polymerase
co'utlldon bs.e derive dfrom original Japanes epopula- (ExTa qT;akara Bio Inc. ,Japan) .The PCR was
performed with a GeneAmp PCR System 960e or
We therefore conducted molecular phyloge- 2700 (Appli eBdiosystems Japan Ltd., Japan),
netic analyses on the piant sfrom outside the orig- starting at 940C (5 min), fo11owe dby 35 cycles of
inallkynown Japaneselocaliti(ehsereafter denaturatien940C(30 50eC(30
called at s), annealing at
Lrecently fbund plants' ),comparing them to s), and extension at 720C (3 0s or 1 min), and a fi-
plants in the original localit i(ehsercaf ctalelerd nal extension at 720C (7 min). After checking for
`indigenous plants' )and plants imported as gar- a single band using electrophoresis on 1% aga-
den ornamentals fi;o mBritai n(hereaf tcealrled rose TAE gel stained with ethidium bromide ,the
[British plants' )T.hrough these analyscs, we PCR product swere purifie wdith enzyme treat-
hoped to determine the erigin ofthe newly found ment. A 2-pL mixture containing O.3 U Exonu-
Geranium robertianum. clease I (faka rBaio Inc. )and O.3 U calf intestine
alkaline phosphatas e(Tbyob Coo. Ltd. ,Japan)
Materials and Methods was added to each PCR tube to degrade remain-
ing pr{mers and dephosphoryla taeny remaining
Plant materiat dNTPs, The tubes were heated at 370C fbr 30 min
DNA sequences of the plasti dregions of the then at 800C for 15 min.
trn L intron t,rnL3LtrnF and trnHipsh,4 intergen- Direct scquencing of both strands was con-
ic spacer regions, and the nuclear intern atlran- ducted on an ABI 3100 Geneti cAnalyzer (Ap-
scribed spacer (ITS )region of 32 samples of G. plie dBiosystems) using the BigDye Terminator
rebertianum, includin gsix samples from indige- vcrsion 3.1 Cycli cSequencing Ready Reaction
neus plants t,wo samples grown from imported Kit (Applie Bdiosystems Japan Ltd,) fo11owing
Britis hseeds, and another 24 samples from 16 re- the manufacturer's protocol .Primers fbr the am-
cently found plant populations (fabl e1) .A map plificati onandfor sequencing of trnL5L3LtrnF;
(Fi g1,) shows the sampling locatio nisn Japan. trnH-psbA, and ITS regions are liste d{n Table 2.
Since no close relatives of Ge"anium robertia- We manually aligned the DNA sequences ob-
num are in Japan, we used G. thunbeilgi Siieb .& tained en MEGA5 (Tamur eat al. 201 1).
Zucc. as the outgroup. Geranium thunbeFlgii is
the most common species of Geranium in Japan. Pltylogeneti canalysis
Voucher specimens fbr this study were deposited Maximum likelihoo danalysis (ML) was con-
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June2012 NIsHIDA & AL.-Phylogeny of Geraniuni robertiamtm 81
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FTa 1, Map of sampling sites o[' population sof C}eranittm robertianzm7 in Japan,
ducted in MEGA5 (Tamur eat at. 2011). The best analysis, gaps were partiall ydelete dusing the
substitution model was selected based on Bayes- 95% "site coverage cutofT' option. Nearcst-
ian information criteria (BIC )values of 24 possi- neighbor interchan g(eNNI w)as used as the ML
ble combinations of substitution models and rate heuristi cmethod and the initi atrlee "ras rnade au-
descriptio ann,d analyzed in MEGA5. Tamura's tomatically .Five hundred bootstra preplications
three-parameter model, with the lowest BIC val- were perfbrmed using the sarne settings. Acces-
ue, was chosen for all data sets, including the sion numbers of sequences obtained and used in
combined data set (trnL5'-3' +- ttrrnnHLFpsbA this study are liste idn Table 1.
and trnL5'-3'-trn F+ trnH-psbA t ITS). For ML
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thBLE 2. Primer sequences used in this study.
Region Forward Reverse Ref.
trnL5L3LtrnF CGAAATCGGTAGACGCTACG ATTTGAACTGGTGACACGAG (1)(2)(3)
trnH-psbA CGCATGGTGGATTCACAATC A(}ACCTAGCTGCTATCGAAG

ITS GGAAGTAAAAGTCGTAACAAGG TCCTCCGCTTATTGATATGC

{1 T)aberle ett al, (]991 (}2. )Azuma et al, (1999 )(.3 )W'hite et aL(l990).
Group C, consisting of two samples from Shiga,
Results although the bootstra spupport ofthese subclades
islow (639i6 68%,
and respectively).
DIVA
segzaence variation
The aligned data matrix of trnL5L3 '-trnF was Discussion
composed of 942 characters, of which 77 sites
(8.2% w)ere variable among all operational taxo- Our results show at leas tthree genetic lineag-
nomic units (OTUs )and seven (O.7% )varied es (Group sA-C) recognized among the G. era-
within ingroups. The aligned data matrix of nium robertiant{m samples examined (Fig .2).
trnH-psbA was composed of 328 characters, of Based on these results, we discus sthe relatien-
which 23 sites (7.0 %w)ere variable among all ships between recentiy fbund plant sand indige-
OTUs and one (O.39 v'a6ri)ed within ingroups, nous plant soi' G. rohertianum,
The aligned data matrix ofthe ITS was composed Among the samples examined in this study,
of 644 characters, of which 89 sites <13.8 9wetre6) all recently found plants belong to one clade
variable arnong all OTUs and six (O.99 /va6ri)ed (Grou Ap) that differ sfrom the clade containing
within ingroups, There was no variation within the indigenou splants (Grou pC). The clear sepa-
each population except fbr Hokkaido 2 and 3; Fu- ration of the two clades suggests that the recently
kushima1 and 2; Gunma 1,2 and 3; Kanagawa1 fbund plant swere introduced from outside of Ja-
and 2; Gifu 2 and 3; Shiga 4 and 5; and Fukueka pan rather than derived frem indigenous Japanese
1 and 2 (se eaccession nos, in Table 1). plants .Ofcourse, no definiti avneswer can yet be
given, since the possibi]it eyxists that the recently
Plp?logenet ainaclysis found plant sare derive dfrom an original popula-
Because no topological discordanc weas ob- tion with an ancestral genetic type fbr the two
served among the three phylogenetic trees clades in which we did not collect samples from
(trnL5 '-Ltrnl3l trnHLpsbA, and ITS), a combined the original populations .The populations from
data matrix of the three regions was used to con- which we did collect indigenous material are ac-
struct a molecular phylogenet itcree .The ML tree tually the only two regions (th Seuzuka Mountain
obtained is shown in Fig. 2. We recegnized three Range and the Tsurugi Mountain Range) that are
genetic groups among the Geranium robertia- thus far known for the original distributi (oInto
num samples, which wc named Groups A-C, 1932, Kitamura 1968 ,Shimizu 1982 ,Gifu Pre-
Group A consisted only of plants recently fbund fectur e2001; Fig, 1) .Given that the molecular
in Japan, although the bootstra pvalue was not analyses combined the indigenou ssamples from
high (7496 )Gr.oup B were plant sof Britis hlin- these two regions int oa distin ccltade, we v,ould
eage. Group C consisted of all remaining indige- not expect any other original populations with
nous plants .Phylogenet icrelationships among very differe ngteneti ctypes. It would be more
these groups are still uncertain, A subclade is reasonable to conclude that the recently found
recognized in Group A, consisting ofthree sam- plants are not derive dfrom indigenou slocalities,
ples from Hokkaido and one from Iwate, and in although furthe rstudics with more samples frem
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difTbre nltocaliti es(especi aflrlomy additional presumption h,owever, is prematur eand further
foreign countries) are needed for fina ldetermina- studies using additional molecular regions are
tion. needed. Also, whether the subclade in Group A
If the recently found plants are from outside had a differen torigin from other members ofthe
Japan, whether they entered by chance once or group is also unresolved. A bette rsupported phy-
several times must be determined .They might logeneti ctree tbr the recently found plant sis
havebecome from
naturalized several sources, peeded,
given the large rvariation in sequences within Thus far ,we have fbund no population osut-
Group A compared to the other two groups .This side Japan as the probabl eorigin of the recently
"the recently found plants"
"the indigenousplants"

bergii

FIG, 2. Maximum likelihoo dtree obtained from combincd data of trnL5L3Lirni71 trnHipsbA, and JTS regions of Geranium
robertianum. Nurnbcrs at branches indicat ebootstra ppcrcentab, e(s> 509{] )S,ee Tabl e1 fbr abbreviations of sample
names,
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fbund plants ,For population fsiro moutside Japan, various morphs, but all were combined into one
we include donly plants derive dfrom British subspecies by Baker (1956).
seeds in the analy$es. The seeds of Geraniblm Our results suggest that plants of Geranium
imported fromBritainl,ikely fbundinJapan
robeTtianum were robertianum recently are more
for medicinal use or ornament. They were once likel yto be naturalized from a source outside of
used fbr skin and kidney complaints and fbr red- Japan rather than being derived from indigenous
water fever in livesto cikn Irelan d(MabberleyJapanes epopulations ,because the clade consist-
2008), Our results showed that the Britis hplants ing of recently fbund plants is clearly distinct
fbrmed a distin ccltade, separated from both the from the clade of indigenous plants T.he origin of
recently found and the indigenou splants .That the recently fbund plant sis unknown, but they
the plants recently tbund in Japan originated from are unlikely to have been imported from Britain,
Britis himports is therefbre unlikely. Tracing Thus far ,no evidence indicat etshat recently
strains that have been intro duced over the years is fbund plants have invaded indigenou spopula-
difficu lTth,e Britis hsamples were the only seeds tions, although some of the ncw introductions
we were able to find in major trading markets, have been found in the same prefectu rase the in-
Geranizt mrobertianum was used in Chinese digenou spopulations ,Tb cQnserve the original
medicine, but less so than other species, such as biodiversi ttyhe, spread of recently fbund plants
G. thunbeJ:gii (e.g K.i,mura & Kimura 1981, Shi- should be monitored to prevent their hybridiza-
mizu 1989) ,We have not found seeds or plant sof tion with indigenou splants,
G robertianum in Japan that are sold as a Chi-
nese medicine. We thank the collectors ]iste din Table 1 for providing
Of the plants studied here ,none were culti- samples for our study. We are gratefu 1to Hidetoshi Na-
vated; all were wild or (le slsikely )recent garden ganadma sfuor fthber fmaolceieullairtat tiphnheg ysluorvgeeyn eotf i sacpneacliymsecnss. Waet KaYlOso
escapees, since no cultivated individua lwsere thank Natsuko Yoshino for managing the Iiving pLant ma-
fbund in the surrounding area according to the terials. This study was supported in part by a Grant-in-
collectors. Seeds of Geranium robertianum are Aid for Scientifi Rcesearch (2257008 f8r)om the Ministry
thought to be disperse bdy animals (Grim Ie988). of Education, Culture, Sports, Science and Tbchnology,
Japan.
The recently found plants may havc originated
frorn from
one or several populations outside of
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ReceivedJanuar.y IZ 20I2 ;aecepted Adbrch 31, 2012
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