Allozyme Types of Water Fern Azolla japonica and its Relatives (Azollaceae) Growing in Japan

2005·1.4 MB·English

by  Suzuki, Takeshi

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ISSN 1346-7565 ActaPhytotax.Geobot.56 (1)7:1-83(2005)

Allozyme[IbJpes Water FernAzolla its
of J'aponicaand

Relatives(AzollaceaGreo)wing inJapan

TAKESHI SUZUKIi,IWAO WATANABE2 TAKUMI SHIRAIWA3
and
)Division qfBiologica Rlesourees I,nstitu toefIVdt uannde Environment ,Universit oyfH))o 6g loh,yoigaoka, Sanda
669-1546,Jcrpan2,6-58-18 Jindaiji-kita-macChhiq,fai82-OOII,Jdpan,34-7-21-507 tsurt{kabuto,IVdda-ku,Kbbe

657-OOJI ,.lapan
Samples of the aquatic fioati nfgem Azollajaponic aand its relatives (see tAz.olla) growing in 73 local-
itie osf Japan were analyzed by alLozym eelectrepheresis. Six allozyme typ ewesre recognized: AWI BWI
BZ, TA, TO and YA types, Compared to the Intemation aRlice Research Institu (teIRR Iac)cessions of
Azolia by allozymes, the BZ and BW types had the same allozyrne patterns as those of A, carotiniana
and an artificial hybri dAzolta ,respectively; It was supposed that both types had escaped from Azotla
introduce tdo Japan for agricultural Ltse on ric efield sT,he YA and AW types had very similar allozyme
pattern sto those ofA. jilicul oaindd eA,s micmphylla, respectively. The TA and TO types shared all
allozyme bands except one. The TA and TO types were suggested to be closely related to A. rubra by
allozyme comparison. Morphologicall yi, twas suggested that numbers of septa in glochidi (aspikelets
on male massulae) differentiate tshe TA type from A. r"bra, although only one sample of the [IA type
was observed. The TA (an dpossibt TyO) type might be an independen stpecies of genus Azolla ,or true
A. j'aponica.
Key words: allozyme ,Azotla J'aponica ,glochidium i,ntroduced species
Azollais fern(AzollacPetaeer;idophyta) (Lumpki&n
an aquatic which are assemblages of microspores
that is found floatin gon the water surface of small Plucknett 1980). Whole plant shapes and trichomes
ponds and flooded rice fields o,r growing on wet on stems are usefu1 vegetative characteristics for dis-
soil. According to Saunder s& Fowler (1993 )t,he tinguishing the two sections (Watane bete al. 1992).
genus Azolta is diyide dint otwo subgenera, subgen. Plan tshape is ovate to fan-shape dand nichomes are
Azolta and subgen. 1letrasporocarpi aT,he latter only on leave sin sect. AzoUa; the shape is deltoi dto
contains only one species, A. nilotica Decne, with a triangular and trichomes are on both stems and
2n=52 four leavesin Rhizosperma.
chromosome number of and clusters of sect,
sporocarps, All species belongin gto the former In Japan, two species, AzoUa jqponi cFaranch,
subgenus have choromosome number of 2n;44 and et Sav. and A. imbricata (Roxb .ex Griff .N)akai,
clusters of two sporocarps. Moreover, subgenus have been recorded (Iwatsu 1k9i95). Azotla J'opon-
Alolla is separated into two sections, sect. Azolla ica belong sto sect, Azotla ,and A, imbricat baelongs
and sect. Rezosperma, based on the morphology to sect. Rhizosperma.
of megaspores and glochidia of male massulae, Currentl yA,zolla japoni chaas become rare,
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perhap sdue to the loss of it shabita tansd increased Japan, as Zimmerman et al, (1989 )suggested.
use of agricultural herbicides ;it is liste das an For Japanese Azolta, [Ibnaka (1995 r)eported
endangered species in Japan (Environm eAngetnacyl twe distinc ttypes of glochidi farom A. juponica.
of Japan 2000) ,On the other hand, Azotla is useful Shiraiw a(2004 )compared plants of A, japonica
for agriculture, as it ssymbiotic cyanobacterium from Yamato-Koriyam aCity CNar aPre£ ) with those
Anabaena azollae Strasb ,can fi xair nitrogen. from Ibyo-oka City (Hyogo Pref.) a,nd pointe dout
Traditional lAyz,olla has been used as a green several morphological and ecological differences
manure fbr rice paddy field isn southern China and between them. These studies suggested the presence
Vietnam (Moor e1969). The Internation aRlice of several cryptic species among A. j'aponica.
Research Institu t(eIRR Ii)n the Philippin ehsas Character sof reproductive featur e(micro-mor-
collected many accessions of Azolla from various phology of male massulae and macrospores) are
part sof wor]d and promoted utilization of selected very informati vfeor species recognition ofAzolta,
Azotla strains as biofertili izne rrice paddy fields but sperulation is so rare that it is diffic utlo tobserve
(Watana ebt eal. 1987) ,In Japan, a rice-duck-Azolla these characters. Specie sidentifica tbiy ovengetative
system (combin eusde of ducks for weeding and featur eiss also dithcu ldtue to the plastic iantdy sim-
Azolla as nitrogen nutrient for ric eas well as food plicit yofAzoUa morphology. For recognition and
for ducks) on rice field hsas been developed since identificati oofn cryptic species in ferns m,olecul ar
1993 and is now being rapidly adopted by organic tools have been shown to be very helpfu 1(Par iest
farrner(sFurun19o97,2001,Kishida& Utsunomiya 1989,Haufier I995,Yatabe 2001).
al, et at. et at.
1998) .Many stocks of exotic and artificial hybrid Our previous paper (Watanab eet al. 1999) sug-
Azolla accessions from IRRI in the Philippines gested the presence of two cryptic species in A.
have been introduce dfor such use. It is possib ltehat japonic ausing random amplified polymorphic
some have escaped or wil] escape and propagate DNAs (RAPD) as markers.
vegetative]y in nature. It is also necessary to check In this study, allozyme markers are used to
the effects in terms of behavio rand geneti cconta- distinguis hgeneti ctypes among Azolla ]'aponica
minatio nof introduce dAzotla upon native plants in and it srelatives naturally growing in Japan. The pur-
Japan. pose ef this paper was to reveal cryptic species in A.
The taxonomical status of AzoUa j'(rponi cias japoni acnda it srelatives naturally growing in Japan
also controversial. Japanes ebotanis ttsraditionally and to clarify their taxonomical status using
considered A. jq)oni ecndaemic to Japan (Kura t&a allozymes as genetic markers. We also discus sthe
Nakaike 1987, Iwatsuk i1995) .Some taxonomists situation arising from the inducti oonf exotic Azolla
have treated A. ,ioponica as synonymous with A. for agriculture use.
filiculoiLdaem,s (Moore 1969,Lumpkin &

P]ucknett 1980). In an allozyme study for acces- Materials and Methods
sions of sect. Azoll acultivated at IRRI, Zjmmerrnan
et al. (198 9re)ported that A. filic"l oAi. druebrsa , R, Samples
Br. andA, micmphyUa Kaulf. can be distinguishedLiving plan tmaterials ofAzollin japoni acnda it sre1-
by their allozyme pattern sI,n their study, four atives were collected from 73 localiti eisn Japan
Japanese accessions of Azolta, which had been shown as Fig. 1 and Appendix 1. The fan shape of
treated as A, ruhra at IRRI, were identifi aesd A, fil-the plant wsas used fbr prelimina rtayxonomic iden-
iculoides H,owever, they used only fbur accessions tificati oofn samples belongin tgo sect. Azolla .Some
from Japan; other Azolla species might still exist in IRRI collections of Azolla were obtained from
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eOagespt4S

-2oo
tso? ot'-L't 400km
Fia 1,Localitie osf plant materials ofAzolla,iaponica and gts rclativcs growin gin Japan. Locality numbers correspond to Appendix
]･
Osaka Prefectur eUniv. and/or Okayama Univ. as A total of eight enzymes were used: aspartate
listeidnAppendix2: FIIOOI(A.filicu- (AAT,E,C.2,6.1.1),
namely, aminotransferase aconitase
loide isn IRRI lis tan)d RU6502 (A .,ubra), CA3e04 (ACO, EC.4.2,1,3) ,hexokinase (HK, E,C.2,7,1.1),
(A .caroliniana Willd.) ,MI4138 (A .mic,ztphytta) isocitrat edehydrogenase (IDH, E.(Cl.1.1.42),
and MI4087 (artifi hcyibarlid betweenA. micro- leucine aminopeptidase (LAR E.C.3.4.11.1) p,hos-
phylla and A. filiculoi Sdaemsp)le.s were cultivated phoglucoisomera s(ePGI ,E.C.5,3.1.9) ,phospho-
at the greenhous eof the Museum of Nature and g]ucomutase (PGM, E.C.2.7.5. 1an)d shikirnate
Human Activiti eHsy,ogo by IRRIAzolla medium dehydrogena s(eSKD, E.(C1 . 1 . 1 .25) .Electrophoresis
(Watanab eet at. 1992) andlor kept cool in plastic was run on a horizonta lstarch gel system (Solt eits
bags until use for experiments. al. 1983) or venical acrylamide slab gel system
All vouchers were deposited in the herbari- (Shirai 1s9h8i8). The starch gel #8 buffer system of
um of Nature and Human Actiyiti eHsy,ogo (HYO). Solti set al. (1983 )was employed fbr HK, LAP
and PGI; the #10 system of Solti est al. (198 3wa)s
Alloayme analysis used for ACO and SKD; Histidin ebutfer system for
About O.3g of total plant sofAzolla were ground in starch gel (Card eyt al, 1981) was used for IDH and
O,9ml of cold extraction buffer (Solt eit sal. 1983); PGM. AM was resolved in an acrylamide system
O.IM [Ilt:is-H(CpIH.7.5l)m,M EDTA(4Na), 10mM (Shirai1s9h88i).

KCI, 10mM MgC12, O,2%(vlv )2-mercaptoethanol, Stainin gprocedures for these enzymes fol-
and 5%(w/v) polyvinylpyrrolid (oSniegm a40T), lowed Wendel & Weeden (1989 )L.oci were num-
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bered with the most anodal fbrm as ''i" and so on sulaeSamples
when more than one presen ftor an enzyme. Alleles of male massulae fbr glochidiu mobser-
were labele dat each loci ,with the most anodal vation were mainly obtained from cultivated plants
fbrm designat e"ad" and progressiv eslloywer forms at the greenhous eS,amples of 1ocali Ntoy.43, 62 and
as "b","c" and so en. In cases whei'e it was diMcult 63 were ebtained from natural populations.
to distinguis hloci ,the most anodal band was des- Glochidi aof male massulae of Azolla were also
ignate d"A" and progressive lsylower bands as examined using herbariu mspecimens kept in the
"B","C" and so on. herbari aof Kobe Gakuin Univ., Kyoto Univ.
(KYO) ,Osaka Museum of Natural History (OSA)
Pnetiminary observation qfglochidiaqfmale mas- and Uniy. of Tbkyo (TI) G,lochidi awere observed
AAT LAP
l

- - - ---- ------ ----a-b ---b -'-----l 1 -a -b
m 1-c
ta
1
TA To yA Aw g ZBAHIY Fi Ml RU T A T O YA AW g Z B AHI IY 1Fl Ml RU
ACO--m- 111--B-c
PGI-2--
-A
-- ----t l1--a
ll--E-D
-- '-F -- --b lII
i111
--c
-d
T A T O Y A A W BCAZBWHY F[ Ml RU T A T O Y A A W BCZA BWHY 1 iFI MI RU
HK
PGM
-
-la ---
'.-
tny :Lt --m. -. - .lc - - - - n y - -m l-a lny--1c lb
- - l2a-h - N>tww li
xu ---b ke -N rw .--- t---" l2'.. =2 mba - wwww twaw- -----I - i -"w-.- trwny w2-wb2c
gX:V l --2d
TA TO YA AW Fl MI RU T A T o y A A w BCZABWHY I 1 FI MI RU
IDH
l1I---- SKD
I1-A1
--- "- f1 -a - ---- - - -kp--
-b - -c-B
- - 1 1i -c - - l --D
- t- 1- .E

l,
T A T O Y A A W BCZABWHY Fl MI RU T A T O Y A A W BCZABWHY Fl MI RU
FiG. 2. Schematic zymograms of six allozyme types of Azolla jtu)on iancda it srelativeg and IRRI Azolla accessions examined in this
study. Ihe anodal side is the upper side of this figure A.t HK and PGM, black bands are interpret eads HK-1 and PGM-1; gray
bands are interpret easd HK-2 and PGM-2. In PGM-2, fainte brands (th ilinne )are treated as ghost bands .CA, FI, HY, MI and RU
are abbreviations for IRRI CA 3oo4, FI1OO1, MI4e87 (artifi hcyibar]id) M,I4 138 and RU6502, rcspectively. See text for ether six
abbreviatiens to allozyme types.
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TABLE 1. Al]ele sor bands ef six allozyme types ofAzolla.iaponica aiid it srelutiyes in Japan together with IRRI Azo"a accessions
examined in this study. Allele sare Iisted in ltalic ,bands in Reman. Abbreviation fsbr allozyme types and IRRI accessions cor-
respond to those ofFig. 2.
AatHk-1Hk-2 ldh lap Pgi-2Pgm-fPgm-2ACOSKD
TATORU aaa bbc hbb aaa bbb ccd b d CD BD
b d CD BBCD
c d CD
vaFI bb bcc aa aa cc aa bcbc bcbc BEBE AEAE
AWMI abab acac aa cc aa aba bcbc hcbc BEBE CDCD
BZCA bh cc aa bb aa bb aa bcbc AFAF AEAE
BWHY abab abab aa acac acac aa bebc acac BEBE ACDEACDE
by light microscope (OI;YMPUS ,HB-2 model). Six allozyme types (TA, TO, YA, AW, BZ
The number of septa in each glochidiu wmas count- and BW) were recognized in Japanes Aezolla ,These
ed in more than five glochidi eaach of more than types were fir sftound at localit iNeos. 49 (Tajima
seven male massulae, Prov. )N,o. 13 (Tbky Poref, N)o. 45 (Yama tPorov.),
No.67(AwaProv,)N,o.60(BizePnrov.)N,o,32

Results (Lak eBiwa shore), and were named after these
locali tnaymes respectively. The geologica ldistrib-
Altozyme analysis ution of the six allozyme types is shown in Fig .3,
Zymograms and interpret ealdleles are shown in The [[A and TO types shared al 1allozyme alle-
Fig. 2 and Tablel. Stergiano u& Fowler (1990) le sor bands except fbr a D band at SKD. Included
reperted that most accessions of Alolla J'aponic aand in these were many unique alleles or bands :Hk-2b,
it srelatives (speci oefs sect. Azolta )were cytologi- Lapb ,Pgi-2C P,gm-2d, CD bands at ACO, and B or
cally diploi dand that the three accessions from BD bands at SKD. Among the compared IRRI
Japan as well as IRRI FI1001 ,CA3004 and RU6502 accessions, RU6502 shared the larges tnumber of
used in this study were also diploid .Therefore, alleles (Aaf Hfk,-2b, Lapb, Pgm-2d) and bands (CD
sample plant swere assumed to be diploi dI,sozyme at ACO) with them. The TIA type was found at 36
numbers or band numbers were consistent with localit iien sHonshu and Shikoku ;the TO type was
those of a typic adliploi pdlant E,ight putati vleoc iof found at 7 localitie saround Kanto Distric tin
six enzymes were scorable and polymorphic A:at, Honshu.
Hk-1, Hk-2, ldh, Lap, Pgi-2, Pgm-I and Pgm-2. The YA type showed the same zymegram pat-
For ACO and SKD, only band patter nwesre shown terns as those of IRRI FIIOOI at alMoci except Hk-
in Fig. 2 and Tbblel becaus ezymograms could not 1. This type was distribu ttehdrough i9 localit jofes
be interpret etod loc iand genotypes F.or PGM-2, Honshu (mainl tyhe western part )and Kyushu.
fainte brands were detecte dand treated as ghost The BW and BZ types shared all alleles or
bands (th ibnands at PGM-2 in Fig. 2). bands with the IRRI MI 4087 and CA2038, respec-
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･/･
@ s

@ @g$)
"q @
#t-,
@ '--.
t"'x..

,as$s
.$
5
f ve
t:mjk:
TO@:

YAA:

AW

BZ"

BW

6?
".o
o 200 400km
FIG. 3, Geographica ldistributi oofn six allozyme types ofAzolta japonic aand it rselatives in Japan,
tivel Tyh.e AW typ ehad the same allel eors bands as produced sporangia in early summer of 1995-2001
those of IRRI MI4138 except at the Pgi-2 loci .The in the greenhouse T.heir glochidi haad mainly O-1
BW and BZ types were fOund in the western par tof septa (Fig 4.A, rllable 2). Male massulae of the BW
Honshu. The AW type was found in only one local- type were obtained in late autumn of 2002 in the
ity of Shikoku (local Nio,t y58). fiel dat twe localitie asn,d their glochidi haad O-1,
but rarely 2 septa lik ethose of the YA type (Fi g4.B
Pneliminary Observatio onfgtochi odjiCama lmaes- & Thble 2) .Glochidi aof male massu]ae of IRRI
sulaePhotographs FIIOOI (Azol ljailicuto iwedrees e)quipped with
of glochidiu m(spike lsetrtuctures on mainly one, sometimes no or two septa (Fi g4,C &
inFig.4. Tbble2).
surface of male massulae) are shown
Mean numbers of septa for each glochidiu mof Mcrosporocarp sof the TA type were obtained
allozyme samples ofAzolla are shown in [fabl 2e, only from Higashi-Osaka, Osaka Pref .(locality
those of herbariu mspecimens in Ffable 3. No.43) on June of l995. More than three septa
Some plant sof the YA type from 10 localitieswere present at the upper part of glochidia (Fig.
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Apnl 200S SUZUKI et alAllozlr me types ot A?otla Japontca 77
ll lndsutag.ts

-y
"trfv'"
"
tr K･str'j

'w"mutr
igel

ifaj:l,tst:.
'
it
iget:rv /kll,).･/
/f ･ L" V
:
E
-
FIG 4 Glochidi aofAzolla An arrow mdiLates edLh geptum in gtochidiu mA YA type, Kyoto Prct Kyo tanabe City ,Thkig i(loca]
ity No 36),B, IRR[ FIIeOl (A filicutoid TeAs )ty,pCe ,Osdkd Pre fHigashi osaka City ,Shukubo cho,Nu2 (loLal iNoty 43),
D, Herbanum spcumen ofA rzabra New Zealand (se eTlibi 1e), E, Herbarium specimen ot A Japontca Tokyo Pref Sh]muid,
,
(seTehble3),EBW type,OkayamaPrLf KurdshikiCityFutako(localiNtoy63) SLalebdrs 20prn
=
4D & Tbb]e 2) Eight herbanum specimens ofAzoUa congtant intervals (Fig4F & [Ibbl e3)
Japonica kept at the herbana of Kyoto Umv (KYO)
and Osaka Mugeum of Natural History (OSA) had Discussion
glochJdia with several gepta m the upper parts ,1ike
thoge ot the [[A type (Fi g4E & [[bbl 3e) A spe"- This study revealed the presenc eof gix allezyme
men ofA rubra at the herbamum of Kobe Gakum typeg in Azotla Japonica dnd its reldtives (sect
Umversity showed glochidia with tsevera lsepta at Azolla )naturally growing in Japan Our previoug
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TABLli 2. Numbers of septa for each glochidium on Ar,otta examined in this study. Allozyme types correspond to those of Fig, 2 and
Appendix 1.
Allozyme ND. Locality No.ofsepta No, of examined
type mean ±SD glochidia(massulae)
va 1719M3I6Y3A8G3I9,4Y0A4m4a4m5o4t6u626343 O.08±O.22 84 (7)77
YA CHIBA. Shonan O.18±O.28 (7)103
va MIE,Ueno O.16±O.32 (7)158
% KYOTO.Kyo-Tanabe O.13O.36 CIO)132
±
% OSAKA, Katano O.Ot±O.30 (10)100
YA OSAKA,Neyagawa O.l7±O.33 (7)128(10)166
YA OSAKA,Settsu O.14±O.37
wt OSAKA,Higashi-osaka O.05±025 (10)104
yn NARA, Yamato-koriyama O.03±O.15 (7)137
% NARA, Nara City O,23±0.43 (7)103
BW OKAYAMA, Okayarna City O.35±O.42 C7)123
BW OKAYAMA, Kurushikj O.42±O.52 (7)43
mIRRI OSAKA,Higashi-osaka 4.14±1,30 (8)
accessien
FIIOOI East Germany O.96±O.47 131 (10)
TABLE 3. Numbers of septa for each glochidiam observed in herbarium specimens ofAzella.
Locality Co]leetor Date No. No.
efseptamean ofexamined
±SD glochidia(massulae)
A. 1'apo nica
IBARAGI Pref,

Ibarag iTbwn, Naka-ishjzaki Noguchis.ll.(OSA) Jun, 25,1979 6.00±1,S2 S3 (7)49
Kita-ibaragiCity,Minami-nakago yasu4s43go,(osA) Sep.12,1979 3.47±1,37 (8)
CHIBA Pref,

SakuraCity,Oo-sakura Namegata2364.(OSA) JuL 03,1952 3.95±O.98 58 (7)102
Chose iTbwn Rigawa 8683,(OSA) May 29,1962 4.62±1.02 (9)
TOKYO Pref,

ltabashi-ku,Shimura Makino s.n. (OSA) May 11,1919 3.37±1.16 83 {10)42
Oeta-ku,Ooimachi Makino s.n. (KYO) May 9,1919 3.S5± l.20 (7)
KYOTO Pref,
'fagawa3S44(KYO)
Kyoto City, Tlakaragaike JuL 19,1951 5.70±2.02 83 (10)
OKAYAMAPref.
OkayamaCity ,Takamatsu Tsuboi s.n. (KYO) Jun.10,1937 3.95±O.98 58 (7)
A.
rubraNEWZEALAND
Mario Channei near Blenheim Mason3109{KGUa) Jan.21,19S5 2.54±O,71 61 (7)
" herbarium of Kobe Gakuin University
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paper using RAPD analysis showed two distinct Glochidia with no or one septa were observed
groups in A. ]'aponi c(aWatana beet ai. 1999), All in ten samples among all nineteen samples of the
samples designate ads group A (includ iAn. gfil-YA type, includin gthat from localit Nyo. 40 (Osaka
iculoides b)elonged to the YA type, and samples of Pref. ,Setts uCity) w,here Tanaka (1995 )observed
group B were divided into TA and TO types the same type of glochidi ain 1994 ,As for the mor-
(Append i1x), phology of glochidi at,hat of IRRI FIIOOI (Azolla
BZ type and BW type showed completely filiculo iwadse ssi)milar to that of the YA type,
identic aalllozyme pattern sto IRRI CA3004, which These findin wgere consistent with previous reports
was identifie das AzolXa caroliniana, and IRRI about A. .filiculoid ebsy Svenson (l944).
MI4087, which was an anificial hybrid between A. The TA and TO types shared almost the same
micmphyUa and A, respectively. The alleles or bands. which suggested that [l; Aand TO
.tiliculoides,
rice-duck-Azoll asystem has been applied in Japan types are very closely related, Plants of TO type
since 1993 (Furun 1o997) .Some IRRI Azotl aacces- were found only around Kanto District ,and the
sions, includin bgoth CA3004 and MI 4087, have differen caet SKD might be due to geographical
been provided to Japanese farmer sfrom Okayama variation. YA type and IRRI FIIOOI (Azottafiticu-
Univ ,(Kishi dpears .com.), In the prefectur wehsere toides )showed no similar allozyme pattern sto
BW and BZ types were fbund (Shig Waa,kayama, those ofthe TA and TO types. Among IRRI acces-
Hyogo, Okayama), many organic farmer shave used sions examined in thi sstudy, IRRI RU6502 (iden-
the rice-duck-Azolla system since 1997 (Kishidatified as A. rt{hra) had the allozyme pattern most
pers .com,). It was recorded that an organic farmer similar to those of TA and TO types W,e considered
of localit yNo.50 has been supplied IRRI Azolla that the [EA and TO types are more closely related to
accession CA3004 from Prof, Shiomi, Osaka A. rubra than to A. filicul oWhiedtheesr .or not the
Prefectu rUeniv. and has used it on his rice field TA and TO types should be attributed to A. ntbva, it
since 1993 (Shio mpeirs. com.). BZ type reported would not be accurate to attribute all Japanese
from logali tNyo.50 in this study were co]lected in native Azoll ato A. filiculoides.
a rice paddy fiel d8km apart from the organic Only one sample of male massulae of the TA
farmer's rice fiel din 2000 (Hamasa kcoanstmction type was obtained at the irrigat ipoonnd in Higashi-
oMce, Hyogo Pref .pers. com.). It is highl ylikely osaka City ,Osaka Pref. (local iNot,y 43), Several
that at leas stome of the BZ and BW types found in septa were present ,mainly in the upper part sof its
this study were plants that had escaped from rice gtochidia CFig .4C). Tanaka (1995 )observed
paddies and become naturalized in Japan, glochidia with several septa on samples from the
The YA type shewed nearly the same zymo- same pond. Glochidi awith several septa in upper
grtnm sas those of IRRI H1OO1, which was identified part were also observed in some herbariu mspeci-
as Azolla jiliculoi daneds ,the AW type showed mens collected between 1919 and 1979 (Fi g4.E,
very simi]ar zymograms to those of IRRI MI4138, [Ibbl 3e) .Seto & Nasu (]975 r)eported the same type
which was labele das A. micTztphylla. It would be of glochidia observed in the specimen kept in OSA
reasonable for the YA and AW types to be treated as (Tagaw a8683) ,which was also examined in this
A. fiticulo aindde As. microphytta, respectively, study (se eTable 3) .The existence of herbarium
based on the results of allozyme analysis. specimens with thi sglochidiu mmorphology suggest
Zimmerman et al, (198 9i)dentif ifebdur IRRI acces- that the TA type (an dpossibly the TO type) has
sions originating from Japan as A, fiticul oTihedyes.been present in Japan at leas tsince 19l9.
rnight have detecte odnly the YA type from Japan. Unfortunate lwye, could not fin dglochidium
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TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
80 APG Xlo1 .56
with no or one septa among herbarium specimens in Fernis Ctlub fbr their kind help in plant sampling and pro-
this study. Hence, there is no infbrmatio nabout viding valuable fiel idnformatio nW.e also thank Drs. Y/
YA type befor e1984, when Lumpkin has obtained Kishida ,N. Shiomi and S. Kitoh for sending samples of
IRRI FI 1603 (identi fasi Aezdollafiliculoide fsr)om the IRRI collection of AcoUa und giving us information
Kyo-tanabeCity,Kyoto Pref.(Watanabe personal] ayn,d the curators of Kobe Gakuin Univ., Kyoto
et al. Univ, (Kyo) ,Osaka Museum of Natura lHistor y(osA)
1992).
and Univ. of Tbkyo (TJ f)or permissio tno examine spec-
Glochidia of Azolla rubra were observed to imens .We would lik eto thank Dr. N. Murakami for
have seyeral septa at constant interva ](sFig.4F),helpfu lcomrnents on the manuscript. This study was
and they are clearly differe nftrom thoge of the [[A partl syupported by a Grant-i Anid for Scientif Riecsearch
tByrpaeg g(inFnis g(.149D 9C)3 h).ai]snon Qheaks (sh1ow9n98 )galndoc hLiadriga ewi th& Cf(urNlootm. ut0rh6ee,4 M5Ji4anp0ia7sn1t )rt y.oo fT SEd u(cNaot i0o6n7 ,8S0c4i5e0n)ce ,aSndpo rtto s aIndW
several septa at constant interval osn male massulae
of Australia nand New Zealand A. filicutoides, References
which should be treated as A. rubra by the taxo-
nomical treatment of Zimmerman et al. (1989).Cardy,B.J.,C. W. Stuber& M. N. Goodman. 198],

Allozyme data and glochidiu mmorphology sug- Techniques for Starch Gel Electrophoresis of
geste dthat the TIZ A(an dpossibl tyhe TO) type is nei- Enzymes from Maize (Ze amays L.) .Inst .Sratics
ther A. .tilicutoid neosr A. rttbra and shou]d be treat- Minmeograph Serie Nso.13 17, Nonh Carolina State,
Univ.
ed as an independent species, A type specimen ofA.
Chinnock,R.J.1998.Azollaceae.in:Mc.Carthy,R M.
japonica inYbkosuka(Kanagawa
was collected (ed. F)lora of Austra]ia ,vol.48. CSIRO Publishing,
Pref.,Kanto Dist.b)ySavatieirn1866-1876
when Victori a1 ,74- 176.
he stayed in Japan (Franch &e tSavatier 1876). Environmental Agency of Japan. 2000, Threatened
Herbarium specimens of Azolta suggest tha tAzolla WildLife of Japan :Red Data Book 2nd edition, vol,8,
with several septa had been distribut eidn Kanto Vascular Plants .Japan Wildlif eResearch Center,
Distric ton the time of Savatier s'tsay in Japan ,The Tbkyo. (i nJapanese)
Franchet,A. R. & P A. L.Savatie.1876,Enumeratio
TA (an dpossibl ythe TO) type might be true A. Plan tar um i n J ap oni a2 ,
japonicAad.ditional including
research, analysis Furuno. T. 1997. Duck-Rice system. Nousangyoson-
of cpDNAs and other morphological characters, bunka-kyokai [,Iloky o(,i nJapanese)
are necessary to make a conclusion on tbe taxo- . 2001, The Power of Duck. Tagari Publications,
nomical status of each allozyme type found in this Tasrnania.
Haufler,C.H,,D, E,Solti&s P S.Soltis1,995,Phylogeny
study, especially for the Tt- aLnd TO types,
The Japanes egovernment and regional gov- ocflo rtophleas tPoC MDpNoAdi" rmest rvitc{iltgiaonne sciotmep ldeaxt;a . SIynsst i.Bto etfs, r2e0m:
ernments in Japan have liste dAzoll a,joponica in 110-119.
their Red Data Books. However, "A, .japonica' 'in Iwatsuki ,K, 1995, Azollaceae .b= Iwatsuki ,K., T.
their lis tmight be a mixture of several allozyme Yarnazak iD,. E. Boutl'ord & H. Ohba (eds. M)o,ra of
types of Azolla. For the conservation of native Japan 1i 259. Kodansha, [[bkyo.
Kishida,YL&N. Utsunomiya,1998,Integratefdarming
Japanes eAzolla e,ach allozyme type should be dis-
tinguished separately in the Red Data Books. tsiyosnte imn poafd Adzy ofliteal- dA1. i: Egfafmeo dcut csofk amqeuata-triicce fe rpn rAozdoulcl-a
on growth of Aigamo duck and rice field,
The authors thank the collectors liste din Appendix 1 Sogonougaku 46: 19-23 .(i nJapanes ewith English
and Dr, K, Seto D,r. TZ N.akaik eM,r. A. Yamarnoto ,Mn S. summary)
Masuda, Mr, S, Hyodo, Ms. H, Kubo, Mr. M. Oota, Mr, Kurata, S, & T. Nakaike. 1987, Illustrati eofn sPterido-
M. Yamashita ,Dr. K. Hirata ,and members of Japan
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