Microb. Resour. Syst. 38
Dec.
（2）
：51
2022─ 62, 2022

Vol. 38, No. 2

Additional polyamine analysis of algal chlorarachniophytes,
euglenophytes, haptophytes, cryptophytes, dinoflagellates,
chromerids and heterokontophytes—Polyamine analysis of algae IV—

Koei Hamana1)＊, Takemitsu Furuchi2), Hidenori Hayashi1) and Masaru Niitsu2)
Faculty of Engineering, Maebashi Institute of Technology
460-1 Kamisadori-machi, Maebashi, Gunma 371-0816, Japan
2)
Faculty of Pharmacy and Pharmaceutical Sciences, Josai University
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan1)

Following the polyamine analysis of glaucophytes, green algae and red algae, to propose cellular polyamine distribution
catalogues of algae, polyamine components in other seven algal groups evolved by secondary and tertiary endosymbiosis
were analyzed. Acid extracts from an additional 42 organisms belonging to them were newly analyzed by HPLC and
HPGC-MS. The additional new polyamine data of the seven algal chlorarachniophytes (Rhizaria), euglenophytes(Excavata), haptophytes (Haptista), cryptophytes (Cryptista), dinoflagellates and chromerids (Alveolata), andheterokontophytes (Stramenopila), furthermore three protist-like cercomonads, imbricates and plasmodiophorids(Rhizaria) and the two fungal-like labyrinthulids and oomycetes (Stramenopila) were combined into our polyamine
catalogues of the previous 65 organisms. In the phylum Cercorozoa of Rhizaria, chlorarachniophytes contained
putrescine, norspermidine, spermidine, norspermine and spermine whereas the seven other species belonging to
cercomonads, imbricates or plasmodiophorids contained putrescine and spermidine. In the phylum Euglenida,
1,3-diaminopropane, putrescine, norspermidine, spermidine, norspermine were found in most of the Eutreptiella,
Cryptoglena, Euglena and Trachelomonas species, whereas norspermine was not detected in non-photosynthetic Astasia
longa. Norspermidine spermidine and norspermine were distributed in the phylum Haptophyta, and norspermidine andspermidine in the phylum Cryptophyta. In the phylum Dinophyta, photosynthetic Symbiodinium, Pyrocystis, Peridinium,
Glenodiniopsis and Amphidinium species and non-photosynthetic Noctiluca and Crypthecoidinium species contained
ubiquitously norspermidine and norspermine, furthermore spermine and thermospermine (and caldopentamine) were
found in the former two and the last species. A species of the phylum Chromerida, Chromera velia contained putrescine,norspermidine, spermidine, homospermidine, norspermine and thermospermine. In the phylum Heterokontophyta,
homospermidine, penta-amines and hexa-amines in addition to algal common diamines and triamines, were distributed inmost of the 12 species of diatoms including Nitzschia palea. Major polyamines of eustigmatophytes were putrescine andspermidine in the eight species and cadaverine, norspermidine, norspermine, spermine and thermospermine were
sporadically distributed. A freshwater brown alga Heribaudiella fluviatilis as well as six marine brown algae (seaweeds)
and a raphydophyte contained the penta-amines caldopentamine, homocaldopentamine and/or thermopentamine inaddition to norspermine, spermine and/or thermospermine. Putrescine, norspermidine, spermidine, norspermine,
spermine, thermospermine, caldopentamine, homocaldopentamine and thermopentamine were found in the phylum
Labyrinthulomycota and the phylum Oomycota, however species-specific differences were observed in their penta-aminelevels. 1,6-Diaminohexane, N-methylpolyamines and 2-hydroxypolyamines were detected sporadically in some algal
species, as a minor component.

Key words: alga, chlorarachniophyte, chromerid, dinoflagellate, polyamine

INTRODUCTION
Corresponding author＊

E-mail: koeihamana@gmail.com
Accepted: November 2, 2022

We have extensively analyzed cellular basic polyamines which are possible stabilize and control cellular nucleic acids DNA and RNA, along the phylogenetic tree of life. To propose the polyamine distribution catalogues of algae, furthermore to reveal the─ 51 ─

Polyamine analysis of algae

Hamana et al.

phylogenetic significance of polyamine components
in the varieties of algae by the symbiosis of a unicellular red alga and/or a green alga, we have analyzed cellular polyamines of various unicellular andmulticellular algae widespread in eukaryotic taxa
(Hamana, 2008; Hamana & Matsuzaki, 1982, 1985a;
Hamana & Niitsu, 2006; Hamana et al., 1988, 1990,
2004a, 2004b, 2013, 2016b, 2017, 2018a, 2018b).
Original photosynthetic algae belonging to thealgal phyla Glaucophyta, Rhodophyta, Chlorophyta
and Streptophyta have plastid(s) as a result of the
primary endosymbiosis of a phototrophic cyanobacterium, which evolved independently without secondary and tertiary endosymbiosis and are locatedtogether in algal Archaeplastida (unranked) of the
kingdom Plantae (Inouye, 2007, 2018). Chlorophyta
and Streptophyta include in algal Viridiplantae(unranked) of Plantae. Seven other algal groups
chrorarachniophytes (evolved after secondary endosymbiosis of a green alga) of cercozoans, euglenophytes (euglenoids) (secondary endosymbiosis of agreen alga), haptophytes (secondary endosymbiosis
of a red alga), cryptophytes (secondary endosymbiosis of a red alga), dinoflagellates (secondary endosymbiosis of a green alga, or tertiary endosymbiosis
of a haptophyte or heterokontophyte), chromerids(secondary endosymbiosis of a red alga) and heterokontophytes (secondary endosymbiosis of a red alga)
are located separately into the six different(unranked) Rhizaria, Excavata, Haptista, Cryptista,
Alveolata and Stramenopila locating outside Plantae,
as shown in Table 1. However, Cryptista (andHaptista) is most likely related to Archaeplastida(Yabuki et al., 2014; Yamazaki & Shimano, 2020).
Their classifications and the clade names for kingdom, phylum and class as their taxonomic ranks are
confusing and have been discussed, however wereferred to the taxonomy database of NCBI
(National Center for Biotechnology Information, US
National Library of Medicine, USA) (NCBI, 2021) andAlgaeBase, Ireland (2021) and the phylogenetic treeof life of MCC-NIES (Microbial Culture Collection,
National Institute for Environmental Studies, Japan)(MCC-NIES, 2021), furthermore International Code of
Nomenclature for algal, fungi, and plants (Shenzhen
Code) (JSPS ICN Translation Committee, 2019) and
Miyadoh et al., 2006.
As a contribution to advances in cellular polyamine
biosynthetic diversity in the phylogenetic evolutionof algae (Fuell et al., 2010; Lin & Lin, 2019; Minguet

et al., 2008; Shaw et al., 2010; Schweikert & Burritt,2015), we have analyzed cellular polyamines of various algae which evolved after primary, secondary
and tertiary symbiotic processes (Hamana, 2008;
Hamana & Matsuzaki, 1982, 1985a; Hamana & Niitsu,
2006; Hamana et al., 1990, 2004a, 2004b, 2013, 2016b,
2017, 2018a, 2018b) as a database of polyamine distribution profiles in eukaryotes, following to comprehensive polyamine analysis of cyanobacteria (Hosoya
et al., 2005; Hamana et al., 2008, 2016a) and higher
land plants (Hamana & Matsuzaki, 1985b; Hamana et
al. 2015, 2017, 2019).
Furthermore, since polyamines are considered toplay a role in the longevity of living animals, a comprehensive analysis of algal polyamines contributes
as nutritional information on polyamine ingestion bypolyamine-rich algal foods and supplements inhuman life (Handa et al., 2018).
In Archaeplastida, in addition to usual and common
diamines, triamines and tetra-amines, distributions ofaminobutylcadaverine, N 1-aminopentylspermidine,
N 8-aminopentylspermidine and penta-amines ingreen algae (Chlorophyta and Streptophyta) (Hamana
et al., 2018a), and distribution of 1,6-diaminohexane,
aminobutylcadaverine, canavalmine and aminopropylcadaverine in red algae (Rhodophyta) (Hamana etal., 2018b) have been shown. These novel polyamines
have never been found in Glaucophyta. In
Stramenopila, 1,6-diaminohexane, penta-amines andhexa-amines have been found in multicellular brown
algal seaweeds (Heterokontophyta) (Hamana et al.,2017), and N-methylated polyamines in addition topenta-amines and hexa-amines have been detectedin diatoms (Heterokontophyta), and penta-amines
have been found in raphidophytes (Heterokontophyta)
and fungal-like labyrinthulids (Labyrinthulomycota)(Hamana et al., 2016b).
In the present study, 44 organisms belonging tonine groups (phyla), cercozoans (including chlorarachniophytes) in Rhizaria, euglenophytes (euglenoids) in Excavata, haptophytes in Haptista, cryptophytes in Cryptista, dinoflagellates and chromerids
in Alveolata, heterokontophytes (diatoms, eustigumatophytes, raphidophytes and brown algae), labyrinthulids and oomycetes in Stramenopila, wereadditionally available and analyzed, therefore theirpolyamine profiles were combined into the polyamine profiles of 65 related species reported in ourprevious studies (Table 1). The organisms were
supplied from the micro-algal collections MCC-NIES,─ 52 ─

Microb. Resour. Syst. Dec. 2022

Table ₁Vol. 38, No. 2

Polyamine distribution profiles in cercozoans, euglenids, haptophytes, cryptophytes, dinoflagellates,
chromerids, heterokontophytes, labyrinthulids and oomycetes─ 53 ─

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Hamana et al.

Table ₁

Continued

Synonym rank and clade names are diverse and confusing. Dap (3) (abbreviations for the number of methylene chain unit between N), 1,3-diaminopropane; Put (4), putrescine; Cad (5), cadaverine; Dah (6), 1,6-diaminohexane;
NSpd (33), norspermidine; Spd (34), spermidine; HSpd (44), homospermidine; NSpm (333), norspermine; Spm (343), spermine; TSpm (334), thermospermine; APHSpd (344), aminopropylhomospermidine; HSpm (444), homospermine(aminobutylhomospermidine); APNSpd (3(3)3), N 4-aminopropylnorspermidine; APSpd (3(3)4), N 4-aminopropylspermidine; CPen (3333), caldopentamine; HCPen (3334), homocaldopentamine; TPen (3343), thermopentamine; CHex
(33333), caldohexamine; HCHex (33334), homocaldohexamine; THex (33343), thermohexamine; HTHex (33433), homothermohexamine; Agm, agmatine; 43OH, N-(4-aminobutyl)aminopropanol; 333OH, N,N-(3-aminopropyl)aminopropanol; H-Put (H-4), 2-hydroxputrescine; H-Spd (H-34), 2-hydroxyspermidine; ATCC, American Type Culture Collection, Manassas, Virginia, USA; DBT, Department of Biotechnology, Institute of Environmental Biology Co., Ltd.,
Yaizu, Shizuoka, Japan; IAM, IAM Culture Collection, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan (IAM algal collections have been transfered into NIES); KU, Kobe University
Macroargal Culture Collection (KU-MACC), KURCIS, Kobe Univ., Kobe, Japan; MBIC, Marine Biotechnology Institute Culture Collection, Kamaishi, Iwate, Japan (MBIC algal collections have been transfered into NBRC); NIES,
National Institute for Environmental Studies, Tsukuba, Japan; NBRC, Biological Resource Center, National Institute of Technology and Evaluation, Kisarazu, Japan; WDB, WDB Environmental and Biological Research Institute
Co. Ltd., Tokushima, Japan; -, not detected (<0.005 μmol/g wet weight). Our data pleviously published are cited from References and blanks are new data in the present study. Former algal names are shown in parenthsis.
N-Methylated polyamines found in the diatoms Skeletonema costatum, Skeletonema japonicum and Synedra acus in Hamana et al., 2016b and a diatom, Nitzschia palea NIES-2728 in the present study, are described in the text
but not shown in Table 1.

NBRC and DBT, and the macro-algal collection
KU-MACC, or collected from markets or fields inJapan. After 2015, we analyzed their polyamines indetail by our high-performance gas chromatography(HPGC) using a capillary column to detect methylpolyamines, hydroxypolyamines and alkanolpolyamines(polyaminoalkylalcohols) in addition to unusual longand branched polyamines. Some of the previous ourpolyamine data before 2014 (Hamana, 2008; Hamana& Matsuzaki, 1982, 1985; Hamana & Niitsu, 2006;
Hamana et al., 1990, 2004a, 2004b, 2013) by high-performance liquid chromatography (HPLC) and standard GC using a packed column, were cited in Table
1, as a reference.
Many other NIES and NBRC micro-algal strains
belonging to the seven former algal phyla were difficult to mass-culture, therefore not available for ourcellular polyamine analysis. In the two latter fungal-

like labyrinthulids and oomycetes, typical species
were analyzed. New polyamine datum was not
given in the class Raphidophyceae, a class of thephylum Heterokontophyta, in the present study,
however our previous data on the class were citedin Table 1 and re-described in the text as a reference. This is the fourth report of our researchseries "Polyamine analysis of algae" published in thisjournal and the first report on the polyamine analysis of chromerids, oomycetes and the photosynthetic
chromatophore (formerly cyanelle)-containing rizarian thecate amoeba Paulinella.

MATERIALS AND METHODS

Organisms deposited in culture collections were
supplied from Microbial Culture Collection, National
Institute for Environmental Studies (MCC-NIES),
Tsukuba, Japan and Biological Resource Center,─ 54 ─

Microb. Resour. Syst. Dec. 2022

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National Institute of Technology and Evaluation(NBRC), Kisarazu, Japan were cultivated in the light(10-14 h/24 h) at 20-25°C using 2-10 l of liquid medium designed by MCC-NIES (https://mcc.nies.go.jp/)
and NBRC (https://www.nite.go.jp/nbrc/). Medium
C and Medium AF-6 (for freshwater micro-algal culture), or Medium ESM, Medium f/2 and Medium
IMK (for marine micro-algal culture) were used.
Labyrinthula sp. was grown by a co-culture with
Vibrio on agar medium plates. “Shiomidoro”(Ectocarpus siliculosus) KU-1372 was supplied from
Kobe University Macroargal Culture Collection(KU-MACC), Kobe, Japan (https://ku-macc.nbrp.jp/).
Large-scale cultures of dinoflagellates were purchased from Department of Biotechnology, Instituteof Environmental Biology Co., Ltd. (DBT), Yaizu,
Shizuoka, Japan (http://www8.plala.or.jp/wamushiya/).
“Ishigaki shima no Euglena” (Euglena of Ishigaki
Island), a dried powder of Euglena glacilis containing four other chemicals, was purchased from
Kobayashi Pharm Co., Japan. “Midori no Shukan”(Custom of Green), a processed powder of Euglena
glacilis containing seven other plant materials waspurchased from Takeda Pharm Co., Japan. Dried
brown algal seaweed powder and fresh “Ishimozuku”(Sphaerotrichia divaricata) (their habitats are shownin Table 1) were purchased from a market in Japan.
Collection of “Izumi-ishinokawa” (Heribaudiella fluvi︲
atilis) in Takioka, Ohtawara, Tochigi, Japan was supported by Dr. Tominaga, T. and Education Board of
Ohtawara.
Unicellular organisms harvested at the stationary
phase in our laboratory (2-10 g wet weight), algae as
a powder (2-10 g wet weight) and fresh macro algae(100-200 g wet weight), were homogenized in 5%
perchloric acid (PCA). The PCA extract was subjected to a column containing a cation-exchange
resin, Dowex 50WX8, to concentrate the polyamines(Hamana, 2002). The polyamines were eluted with
6M HCl. The eluate was evaporated and then analyzed by high-performance liquid chromatography(HPLC) on a Hitachi L6000 using a column of cationexchange resin, using post-labeled fluorometry after
heating with o-phthalaldehyde (Hamana et al., 2016a,
2016b). After the heptafluorobutyrization (HFB) ofconcentrated polyamine samples, HPGC-mass spectrometry (HPGC-MS) on a JEOL JMS-700, equippedwith a capillary column of Inert Cap 1MS (GLSciences) were performed (Hamana et al., 2016a,
2016b; Niitsu et al., 2014). Two HPGC spectra ofoomycetes on a JEOL JMS-700 are shown in Fig. 1.
Mass spectra of the HFB derivatives of all the polyamines, 2-hydroxypolyamines and alkanolpolyamines
detected in HPGC-MS were obtained for their identification (Hamana et al., 2018a, 2018b, 2019; Niitsu etal., 2014). Quantification of week-acidic, acid-labile
acetylpolyamines was difficult because loss of themis possible in our extraction, concentration and analyses by HPLC and HPGC. Molar concentrations ofcellular polyamines per gram of wet weight of thestarting algae were roughly estimated from the
HPLC and HPGC were performed using authentic
polyamine standards and are shown in Table 1.

RESULTS AND DISCUSSION

Polyamines of cercozoans in Rhizaria
The phylum Cercozoa of Rhizaria tentatively consists of the four major classes Chlorarachniophyceae,
Cercomonadida (Sarcomonadea), Imbricatea andPhytomyxea, and others (MCC-NIES, 2021), and theclass Chlorarachniophyceae is known as a typicalalgal class. On the other hand, Phytomyxea is located in the Phylum Endomyxa and Imbricatea is independent as a phylum of Rhizaria in other research(NCBI, 2021). In our previous reports (Hamana, 2008;
Hamana et al., 2004b), Chlorarachnion species contained ubiquitously putrescine, norspermidine andspermidine, and norspermine and spermine weredetected in one-half of the two. In the newly analyzed Norrisiella, Lotharella, Bigelowiella andViridiuvalis species of the class, the detection of thetetra-amines norspermine, spermine and thermospermine, and a pentamine, caldopentamine, washeterogeneous (Table 1). However, it is suggested
that chlorarachniophytes evolved by secondary symbiosis of a green alga have the potential to produce
the tetra-amines and penta-amine from norspermidine and spermidine. On the other hand, norspermidine and tetra-amines were not detected in theseven organisms located in other three classes
Cercomonadida, Imbricatea and Phytomyxea, including the two chromatophore-containing Paulinella
species of the class Imbricatea. In the present
report, we did not include photosynthetic Paulinella
species into the typical algal groups, however they
are located in another distinct primary endosymbiotic
process of a cyanobacterium outside Archaeplastida(Inouye, 2007; Lee et al., 2017; Nakayama & Archibald,2012). It is possible that the occurrence of green
algal chroloplasts in chlorarachniophytes and the─ 55 ─

Polyamine analysis of algae

Fig. ₁

Hamana et al.

HPGC spectra of polyamines of (A) Saprolegnia diclina NBRC 32710 and (B) Pythium aphanider︲matum NBRC 100101 belonging to the phylum Oomycota. Numeric codes (abbreviations forpolyamines) are explained in Table 1. Arrows indicate the retention position of 2-hydroxypolyamines, alkanolpolyamines and tertiary-branched tetra-amines. Typical HPGC spectra of
N-methylated polyamines have been shown in Fig. 1 of Hamana et al., 2016b of this journal.

occurrence of chromatophores in Paulinella give a
differential effect for their cellular polyamine profiles.
The polyamine profiles found in the two Paulinella
species closely resemble that found in primary endosymbiotic glaucophytes (containing chromatophore
as its plastid) of Archaeplastida.
Since a pure culture of a Plasmodiophora brassicaeof the class Phytomyxea has never been reported,
we collected the cercozoans from the clubroot gallsof Brassica rapa “Komatsuna” (a Brassica crop plant)in our previous study (Hamana et al., 2015) and cited
its polyamine data in Table 1 of the present report.
Mataza and Trachyrhizium species belonging to theclass of Thecofilosea were not available.
Polyamines of euglenophytes (euglenoids) in
Excavata
The algal phylum Euglenida evolved by the second
endosymbiosis of a green alga within Excavata. Six
Euglena samples were newly analyzed in the present study and their polyamines are shown in comparison with our previous polyamine data in Table
1. Diaminopropane (1,3-diaminopropane), putrescine,norspermidine, spermidine and norspermine wereubiquitously distributed in Cryptoglena, Euglena andTrachelomonas strains of the order Euglenea. Variety
in diamine, triamine, and tetra-amine profiles wasfound within euglenophyte species; however, cadaver-ine, diaminohexane, homospermidine, spermine andthermospermine were sporadically distributed. It has
been reported that norspermine was not detected in aunique non-photosynthetic Astasia species belonging to
the class Aphagea, whereas spermine was not detected in a Eutreptiella species of the class Euglenophycea(Hamana, 2008; Hamana et al., 2004b). A tertiarybranched tetra-amine, N 4-aminopropylspermidine(3(3)4) was detected in a newly analyzed strain of E.
gracilis containing norspermine (333) and spermine(343) and lucking thermospermine (334), therefore itis suggested that synthetic ability of tetra-aminesfrom tri-amines are varied within euglenophyte species. Organisms belonging to another algal class
Heteronematina of the phylum Euglenida were notavailable.
Polyamines of haptophytes in Haptista
After the secondary endosymbiosis of a red alga,
haptophytes are located in Haptista and constitutethree classes of the only one phylum, Haptophyta(Table 1). In the previous analysis, the three speciesof haptophytes, Haptolina hirta (Chrysochromulina
hirta), Isochrysis galbana, and Pavlova pingus ubiquitously contained norspermidine (and norspermine),
in addition to spermidine found in the latter two
species (Hamana & Niitsu, 2006) (Table 1). Thoughmass culture of the haptophytes was difficult, fur-

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Vol. 38, No. 2

thermore polyamines were analyzed by HPLC alone,polyamines of Pavlova gyrans were newly analyzed
by HPLC and HPGC in the present study (Table 1).
Distribution of norspermidine, spermidine and norspermine within haptophytes is reliable. Algae
belonging to the algal class Rappephyceae were notavailable.
Polyamines of cryptophytes in Cryptista
By the loss of feeding ability and gain of photosynthetic ability, the algal phylum Cryptophyta is located in Cryptista (Yabuki et al., 2014). In previous polyamine analysis of non-photosynthetic Cryptomonas
paramecium (formerly Chilomonas paramecium),
spermidine and norspermidine were detected(Hamana & Niitsu, 2006). In the detail re-analysis in
the present study, spermine and thermospermine in
the addition to the two major polyamines weredetected in the organism, as shown in Table 1.
Polyamine profiles were not distinguished among
non-photosynthetic freshwater C. paramecium, four
photosynthetic freshwater Cryptomonas species anda photosynthetic freshwater Chroomonas species(Table 1). Norspermine was detected in marine
Hemiselmis andersenii as well as the marine
Rhodomonas species (Hamana & Niitsu, 2006), however high norspermidine level was observed in thetwo marine cryptophytes. Organisms belonging to
the non-photosynthetic phyla Goniononadea andKathablepharidea including a green flagellate, Hatena
arenicola, showing a secondary symbiosis in progress (Okamoto & Inouye, 2005), and Palpitomonas
bilix located a basal cryptist lineage (phylum),
Palpitia (Yabuki et al., 2014), were not available.
Polyamine of dinoflagellates in Alveolata
Dinoflagellates belonging to the algal phylum
Dinophyta were evolved by various secondary or
tertiary endosymbiotic processes within Alveolata(Keeling et al., 2004; Waller & Kořený, 2017). They
have an unusual condensed nucleus containing histone-like high basic proteins (Gornik et al., 2019).
Half of dinoflagellates are non-photosynthetic, heterotrophic species by parasitic or predacious ability,
furthermore, bioluminescent species have beenfound in dinoflagellates. Previously, many dinoflagellate strains were supplied from NIES and NBRC,
however mass-cultures were difficult to obtain,
therefore their polyamine analyses by HPLC andstandard GC were insufficient. However, some poly-amine data have been obtained (Hamana et al., 2004b)
and are cited in Table 1.
Four dinoflagellates were additionally available andthen analyzed by our new HPGC in the present
study. Polyamine profiles newly and previously analyzed in the four orders of the phylum are shown in
Table 1. Since Noctiluca miliaris “Yakouchuu” wasdifficult in cell collection and polyamine extraction
from the cells by its large size and watery cell, thepolyamine datum of the alga in Table 1 is insufficient. Ubiquitous distributions of norspermidine andnorspermine were found and their spermidine revels
were related to their putrescine levels as the precursor of spermidine in the dinoflagellates, therefore
differences in their whole polyamine profiles were
not clear. In non-photosynthetic Crypthecoidiniumsp., photosynthetic Symbiodinium voratum and photosynthetic, bioluminescent, large Pyrocystis fusifor︲
mis, spermine and thrmospermine (and caldopentamine) were detected as a minor polyamine inaddition to high spermidine level. Under use ofgood growing cells and HPGC analysis, various dinoflagellates without photosynthetic ability, bioluminescent ability and marine/freshwater growth condition, are possible to have similar polyamine profiles
to those of the three dinoflagellates. A novel
diamine, 1,6-diaminohexane, and an unusual triamine,homospermidine, were found in Pyrocystis fusiformis
possessing higher whole polyamine levels. We could
not suggest the phylogenetic significance of polyamine profiles within Dinophyta, because their endosymbiotic processes were varied in their evolutions.
Although long polyamines are able to bind to nucleic
acids DNA and RNA, relation of their cellular polyamine contents to their condensation of nuclear DNA
is not clear.
Polyamines of chromerids in Arveolata
Chromera velia and Vitrella brassicaformis of thealgal phylum Chromerida has typical feature of alveolates and contains a photosynthetic plastid (chloroplast) while apicomplexans of Arveolata have a nonphotosynthetic plastid called the apicoplast (Obornik& Lukes, 2013).
The present study is the first report on polyamine
analysis of chromerid. Putrescine, norspermidine,
spermidine, homospermidine, norspermine and thermospermine were detected in Chromera velia as
well as dinoflagellates (Dinophyta) within Alveolata.

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Polyamines of heterokontophytes in Stramenopila
Algal polyamine profiles in the four classes
Bacillariophyceae, Eustimatophyceae,
Raphidophyceae, and Phaeophyceae were show inTable 1. In diatoms, we found various mono-methylated and di-methylated diamines, triamines, tetraamines and penta-amines, in addition to long linear
polyamines such as penta-amines and hexa-amines,
in the acid-extracts of Skeletonema costatum,
Skeletonema japonicum and Synedra acus in Hamana
et al., 2016b and a diatom, Nitzschia palea NIES-2728
in the present study (cellular concentrations were
not shown in Table 1). Distribution of methylated
polyamines including methylated long polyamines
was specific for diatoms within the phylum
Heterokontophyta, furthermore within the nine
eukaryotic groups, analyzed in the present study.
Caldopentamine was found in Chaetoceros gracilis,
Cylindrotheca fusiformis and Phaeodactylum tricor︲
nutum, however hexa-amine and methylated polyamine were not detected in the three diatoms.
Aulacoseira and Asterionella species contained
putrescine, spermidine and homospermidine alone as
the major polyamines (Table 1). Distribution of norspermidine and norspermine are varied within the
diatoms analyzed in the previous (Hamana et al.,2016b) and present reports.
Although the muss-culture of non-photosynthetic(chloroplast-lost) Nitzschia sp. NIES-3576 (Kamikawa
et al., 2015) was difficult, putrescine and spermidine
alone were identified by HPLC, suggesting a simple
polyamine distribution of the diatom.
2-Hydroxyputrescine was detected in a few diatoms
and eustimatophytes of the phylum Heterokontophyta.
In eustigmatophytes, three strains (species) were
newly analyzed in the present study and additionally showed a simple polyamine profile (Table 1). The
old datum detected norspermidine in Vischeria species by HPLC alone without muss-cultivation, is notaccurate in the polyamine identification.
In raphidophytes, since cultivation of a largescale culture of Chattonella marina NIES-603 andFibrocapsa japonica NIES-462 failed in the present
study, the two previous data on Heterosigma akashi︲
wo alone were cited necessarily in Table 1. The
organisms contained norspermidine, spermidine, norspermine, spermine, thermospermine, caldopentamine and homocaldopentamine and lacked
homospermidine. Occurrence of caldopentamine andhomocaldopentamine in the red tide flagellates

Chattonella antiqua and Heterosigma akashiwo, wasreported by Nishibori et al. (2009).
In brown algae, polyamines of seven species belonging to the four orders of the class, including marine
Ectocarpus siliculosus KU-1372 “Shiomidoro” as amodel macro-brown alga cultivated axenically, were
newly analyzed in the present study. Caldopentamine,homocaldopentamine and (or) thermopentamine
were found ubiquitously in the seven brown algae(Table 1), as reported previously in 16 other brownalgal seaweeds (Hamana et al., 2017) (not cited in thetable). A unique freshwater micro brown-alga,
Heribaudiella fluviatilis “Izumi-ishinokawa” collectedfrom a liver was rich in putrescine, cadaverine, norspermidine, spermidine, homospermidine, norspermine and spermine, and contained a lower levelof penta-amines (Table 1). Although contaminationof microorganisms into the micro-algal sample collected from the field was not excluded, the occurrence of homospermidine and homospermine wasspecific within brown algae. The occurrence of thelong diamine, 1,6-diaminohexane and polycationicliner penta-amine and hexa-amine in the brown-algal
seaweeds might be related to their abundance ofpolyanionic acidic-polysaccharides such as alginicacid and fucoidan.
Polyamines of labyrinthulids in Stramenopila
Within the kingdom (subdomain) Stramenopila,
the phylogeny of the fungal-like phylum
Labrynthulomycota showed a lack of chloroplasts aswell as the fungal-like phylum Oomycota and theprotist-type phyla Bicosoecacea and Opalozoa, andgain of ectoplasmic gliding (Tsui et al., 2009). The
present study is the additional second report on thepolyamines of labyrinthulids. Penta-amine(s) has
been found as a minor polyamine in Labyrinthulaspecies of the order Labyrinthulida and as a major
polyamine in Oblongichytrium species of the order
Oblongichytrida and six species of the order
Thraustochytida including Aurantiochytrium(Hamana et al., 2016b). The present study indicatedthat Aplanochytrium species contain caldopentamine
as the minor polyamine, whereas Monorhizochytrium
and Thraustochytrium species contain caldopentamine as a major polyamine. Although two
types of penta-amine level were observed within thephylum, the occurrence of penta-amines seems to berelated to gain ectoplasmic gliding.

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Vol. 38, No. 2

Polyamines of oomycetes in Stramenopila
Fungal-like oomycetes in the phylum Oomycota
evolved from simple holocarpic marine parasites(Beakes et al., 2012). The present study is thefirst report on polyamine analysis of oomycetes.
Caldopentamine and homocaldopentamine were
found in Phytophthora nicotianae and Pythiumaphanidermatum but not detected in Aphanomyces
stellatus and Saprolegnia diclina (Table 1). The latter resembles to Labyrinthula and Aplanochytrium
species of another fungal-like phylum
Labyrinthulomycota, whereas the former resembles
nine other species of the phylum Labyrinthulomycota,in their polyamine profiles (Table 1). Organisms
belonging to the protist-type phyla Bicosoecacea andOpalozoa of Stramenopila were not available for thepresent polyamine analysis.
Distribution of diaminohexane, tertiary-branchedtetra-amines, hydroxypolyamines, alkanolpolyamines and methylpolyamines
1,3-Diaminopropane, putrescine (1,4-diaminobutane),cadaverine (1,5-diaminopentane) are known as acommon diamines within algal species. A novel long
diamine, 1,6-diaminohexane previously detected in
red algae (Rhodophyta) (Hamana et al., 2018b) andhigher land plants (Hamana et al., 2019). In the present study, 1,6-diaminohexane was widespread sporadically in seven species among the nine algal
phyla, as shown in Table 1. Although synthetic
pathways of 1,6-diaminohexane are unknown, thewide distribution of the diamine in algae as well as
higher land plants is interesting.
Tertiary branched tetra-amine, Na m i n o p r o p y l n o r s p e r m i d i n e (3(3)3) o r N aminopropylspermidine (3(3)4), was detected in aeuglenoid and a eustigmatophyte, as show in Table
1. The branched tetra-amines are widely distributed in prokaryotic bacterial and archaeal thermophiles. Phylogenetic significance of the sporadic distribution of the branched tetra-amines within algae
is not clear, however this is the first report on theoccurrence of branched polyamine in the nine
eukaryotic groups analyzed in the present study.
We have reported the occurrence of hydroxypolyamines (Hamana & Matsuzaki, 1993) and alkanolpolyamines (Hamana et al., 2019) in higher land plants,
respectively. In the present study, 2-hydroxyputrescine (H-Put, H-4) found in the five species of the phylum Heterokontophyta and 2-hydroxyspermidine(H-Spd, H-34) was detected in a species of an algal
class, Chlorarachniophyceae within the phylum
Cercozoa (Table 1). N-(4-aminobutyl)aminopropanol(43OH) and N,N-(3-aminopropyl)aminopropanol(333OH) were detected only in a powder of euglena
Co. strain, Takeda Pharm. of Euglena gracilis, containing seven other plant materials (Table 1). The
present study is the first report on the occurrenceof hydroxypolyamines and alkanolpolyamines inalgal samples, however phylogenetic significance of
these occurrences within the algae is not clear.
Various mono-N-methylated and di-N-methylated
diamines, tri-amines, tetra-mines and penta-amines
have been found in the diatoms Skeletonema costa︲tum, Skeletonema japonicum and Synedra acus inHamana et al. (2016b). In the present study, some of
the N-methylated polyamines were detected in a diatom, Nitzschia palea NIES-2728. Numerous andincalculable cellular-concentrations of the Nmethylated polyamines are not shown in Table 1.
The occurrence of various N-methylated polyamines
has been found in the roots as a major polyamine
derivative in higher land plants (Hamana et al., 2019;
Niitsu et al., 2014). The selective distribution of
N-methylated polyamines in diatoms within algae isphylogenetically valuable (Hamana et al., 2016b).
N-Methylated long polyamines have been found invarious diatoms and it has been known that long
(long chain) polyamines are essential for silica
biomineralization in diatoms (Kroger et al., 2000;
Sumper & Lehmann, 2006). In sponges, long polyamines related to biomineralization and peptide toxins were found but methylated polyamines were notdetected (Matsunaga et al., 2007, 2011).

ACKNOWLEDGEMENTS

We thank MCC-NIES, NBRC, KU-MACC and DBT
for supplying the organisms and advice for culture
conditions.

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クロララクニオン藻類，ユーグレナ藻類，ハプト藻類，クリプト藻類，渦鞭毛藻類，
クロメラ藻類，および不等毛藻類のポリアミンの追加分析
─藻類のポリアミン分析 IV ─
浜名康栄 1），古地壯光 2），林秀謙 1），新津勝 2）

前橋工科大学工学部，2）城西大学薬学部

1）

藻類のポリアミン成分構成をカタログ化するため，灰色藻植物，緑藻植物，紅藻植物の細胞内ポリアミン分析に引き続き，緑藻または紅藻の二次共生やその後の三次共生により進化した全 7 藻類グループと，これらと系統的に関連する 3 原虫様グループと 2 菌類様グループの細胞内ポリアミンを分析した．これらに属し，新規に分析対象とした 44 生物種から酸抽出したポリアミン画分を高性能液体クロマトグラフィーと高性能ガスクロマトグラフィー・質量分析により追加分析した．生物種
は，国内の公的保存機関の MCC-NIES，NBRC，KU-MACC および民間培養機関の DBT より提供された後に大量培養するか，国内の市場および自然界より収集した．新規に追加できたクロララクニオン藻類，ユーグレナ藻類，ハプト藻類，クリプト藻類，渦鞭毛藻類，クロメラ藻類，不等毛藻類，およびクロララクニオン藻類と近縁のリザリア類である原虫様ケルコ
モナス類，インブリカテア類，ネコブカピ類，および不等毛藻類と近縁な無色ストラメノパイル類である菌類様ラビリンチュ
ラ類と卵菌類のポリアミン分析データと，すでに分析済みの 65 関連生物種のポリアミン構成とも比較し，これらの生物群のポリアミン成分構成のデータベースの最終報告とした．ジアミノヘキサンを含むジアミン類の存在，ノルスペルミジン，スペルミジン，ホモスペルミジン，ノルスペルミン，スペルミン，サーモスペルミンなどの構成差，長鎖ペンタアミン類とヘキサアミン類の含有，についての各グループのポリアミン構成の特徴に系統分類関係や共生関係が反映されているのかを考察した．また，数種に，メチル化ポリアミンと水酸化ポリアミンが微量検出された．

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