Morphological and molecular studies of Sphaerospermopsis torques-reginae(Cyanobacteria, Nostocales) from South American water blooms

VERA REGINA WERNER1*, HAYWOOD DAIL LAUGHINGHOUSE IV2,3, MARLI FATIMA FIORE

4, CELIA LEITE SANT’ANNA5,

CAROLINE HOFF4, KLEBER RENAN DE SOUZA SANTOS

5, EMANUEL BRUNO NEUHAUS1, RENATO JOSE REIS MOLICA

6,

RICARDO YUKIO HONDA4

AND RICARDO OMAR ECHENIQUE7

1Natural Sciences Museum, Zoobotanical Foundation of Rio Grande do Sul, Porto Alegre (RS), Brazil2Department of Botany, National Museum of Natural History, Smithsonian Institution, Washington, DC 20560, USA3MEES Program, College of Computer, Mathematics and Natural Sciences, University of Maryland, College Park,

MD 20742, USA4Center for Nuclear Energy in Agriculture, University of Sao Paulo, Piracicaba (SP), Brazil

5Institute of Botany, Section of Phycology, Sao Paulo (SP), Brazil6Federal Rural Universtity of Pernambuco, Garanhuns (PE), Brazil

7Phycology Scientific Department, College of Natural Sciences and Museum (UNLP) and Scientific Research Commission of

Buenos Aires Province, La Plata, Argentina

WERNER V.R., LAUGHINGHOUSE IV H.D., FIORE M.F., SANT’ANNA C.L., HOFF C., SANTOS K.R.D.S., NEUHAUS E.B.,MOLICA R.J.R., HONDA R.Y. AND ECHENIQUE R.O. 2012. Morphological and molecular studies of Sphaerospermopsis

torques-reginae (Cyanobacteria, Nostocales) from South American water blooms. Phycologia 51: 228–238. DOI:10.2216/11-32.1

Sphaerospermopsis torques-reginae (Komarek) Werner, Laughinghouse IV, Fiore & Sant’Anna comb. nov. was originallydescribed as Anabaena torques-reginae Komarek from planktonic populations of Cuban eutrophic environments,characterized by twisted trichomes with spherical akinetes adjacent to the heterocytes. Recently, using molecularanalyses, all planktonic Anabaena Bory ex Bornet & Flahault morphospecies were transferred into the genusDolichospermum (Ralfs ex Bornet & Flahault) Wacklin et al., including Dolichospermum torques-reginae (Komarek)Wacklin et al. However, by a polyphasic characterization of strains of Anabaena reniformis Lemmermann andAphanizomenon aphanizomenoides (Forti) Horecka & Komarek (5Anabaena aphanizomenoides Forti), these planktonicspecies were reclassified into Sphaerospermopsis Zapomelova et al. Our study’s main objective was to characterizemorphologically and molecularly cyanobacterial populations identified as Dolichospermum torques-reginae, observed indifferent aquatic ecosystems in South America. The 16S rRNA gene of two Dolichospermum torques-reginae strains(ITEP-024 and ITEP-026) was sequenced and phylogenetically analyzed for the first time. The morphological andphylogenetic analyses demonstrated the affiliation of the studied populations with the genus Sphaerospermopsis and,consequently, were denominated as Sphaerospermopsis torques-reginae. Furthermore, geographic distribution, ecology,and toxicity of the species are discussed. It was observed in different aquatic environments, natural and artificial,tropical and subtropical in Brazil, temperate in Argentina, and tropical in Colombia, suggesting a wide distribution inSouth America. It normally occurred in dense freshwater blooms, although it was also found in water with low salinity.Sphaerospermopsis torques-reginae toxic blooms have been reported in tropical water bodies in northeastern Brazil.

KEY WORDS: 16S rRNA gene, Anabaena torques-reginae, Cyanobacteria, Dolichospermum, Ecology, Geographicdistribution, Morphology, Phylogeny, Toxicity

INTRODUCTION

Cyanobacteria present many taxonomical and nomenclatural

problems, mostly due to several different morphotypes occurr-

ing in various species. Currently, polyphasic taxonomy has

been used to characterize cyanobacterial taxa more precisely.

Molecular and phenotypic approaches have contributed in

forming coherent phylogenetic clusters and have solved many

of the problems in these organisms’ classification. With the

advance of molecular studies, based mainly on the 16S

rRNA gene sequence, the taxonomy of the group, especially

of nostocaceans, has been widely discussed. The studies

vary from generic levels (Anabaena – Aphanizomenon –

Cylindrospermopsis – Raphidiopsis – Trichormus – Nostoc) to

the differentiation of morphotypes (Rajaniemi et al. 2005a,

b; Willame et al. 2006; Melcher 2007; Ballot et al. 2008;

Ferreira 2008; Komarek 2008, 2010a, b; Honda 2009;

Wacklin et al. 2009; Zapomelova et al. 2009, 2010a, b).

During a study of natural bloom-forming and other

planktonic Anabaena Bory ex Bornet & Flahault morpho-

species with twisted trichomes from subtropical southern-

most Brazilian water bodies, a peculiar species, identified as

Anabaena oumiana Watanabe (Werner & Laughinghouse IV

2009), that had not been previously reported in South

America, was encountered. However, according to Ko-

marek & Zapomelova (2007), this morphospecies is

synonymous with Anabaena torques-reginae Komarek.

Anabaena torques-reginae was originally described by

Komarek (1984) from planktonic Cuban populations in

eutrophic environments, characterized by presenting twisted* Corresponding author (vera-werner@fzb.rs.gov.br).

Phycologia (2012) Volume 51 (2), 228–238 Published 12 March 2012

228

trichomes with spherical akinetes adjacent to the heterocytes.

Recently, based on molecular analyses, planktonic species of

Anabaena were transferred to the genus Dolichospermum

(Ralfs ex Bornet & Flahault) Wacklin et al., including

Dolichospermum torques-reginae (Komarek) Wacklin et al.

Subsequently, Zapomelova et al. (2009) established the

genus Sphaerospermum based on the polyphasic analyses of

two planktonic species [Anabaena reniformis Lemmermann

and Aphanizomenon aphanizomenoides (Forti) Horecka &

Komarek (5Anabaena aphanizomenoides Forti)], and basi-

cally characterized the genus by the form and position of its

akinetes (spherical, on one or both sides of the heterocytes),

including species with coiled or straight trichomes. Howev-

er, this name was illegitimate so the genus Sphaerospermum

was renamed Sphaerospermopsis Zapomelova et al. Thus,

the main aim of our study was the morphological and

molecular analyses of populations identified as Sphaeros-

permopsis torques-reginae (Komarek) Werner, Laughing-

house IV, Fiore & Sant’Anna comb. nov. from different

aquatic systems in South America. In addition to taxonomic

studies, the study aimed at contributing to the geographic

distribution, ecology, and toxicity of Sphaerospermopsis

torques-reginae.

MATERIAL AND METHODS

Sampling

The studied populations were obtained from water bodies

in Colombia (Cesar Department, Loma de Calenturas, La

Pachita swamp – 9u38923.40S, 73u37917.70W); Brazil in the

states of Pernambuco (Recife, Tapacura Resorvoir –

8u029140S, 35u099460W), Mato Grosso do Sul (Corumba,

Pantanal da Nhecolandia, lake – 19u189320S, 57u039180W), Sao

Paulo (Lake Peixes – 21u129420S, 47u489240W), and Rio

Grande do Sul [Lake Violao – 29u209070S, 49u439370W, Patos

Lagoon – 32u029060S, 52u059550W, in artificial ponds at the

University Foundation of Rio Grande (FURG) – 32u049430S,

52u109030W, and at the Zoo of the Zoobotanical Foundation

of Rio Grande do Sul (FZBRS) – 29u479410S, 51u109140W];

and Argentina (Buenos Aires Province, Ensenada, lake of the

Aeroclub La Plata – 34u529460S, 57u579350W and Los Patos

lake – 34u509440S, 57u579230W) (Fig. 1). Samples were either

collected with bottles directly from the water’s surface or using

plankton nets (20 or 30 mm). They were preserved with

formaldehyde (4%), Transeau (1:1), or lugol’s solution. Some

of these samples are deposited in the Prof. Dr Alarich R.H.

Schultz Herbarium (HAS) at the Natural Sciences Museum of

FZBRS, Porto Alegre, Brazil, and in the Maria Eneyda P.

Kauffmann Fidalgo Herbarium (SP) of the Institute of

Botany, Sao Paulo, Brazil.

Morphological studies

The samples were studied using light microscopy and at least

30 individuals of each population were analyzed. The

specimens were drawn with the aid of a camera lucida and

the photos were taken using a digital camera. The mucilag-

inous envelope structure was observed using India ink. The

Hoffmann et al. (2005) classification system was adopted.

Cyanobacterial isolates strains

The two isolates of the species Sphaerospermopsis torques-

reginae (strains ITEP-024 and ITEP-026) are maintained in

ASM-1 medium (Gorham et al. 1964) under light:dark cycle

with white fluorescent illumination (40 mmol photon

mm22 s21). These strains are kept in the culture collections

at the Institute of Technology of Pernambuco (PE) (12:12h

at 26 6 1uC), at CENA/USP in Piracicaba (SP) (14:10h at

25 6 1uC), and at the Natural Sciences Museum of FZBRS

(RS), Brazil (8:16h at 25 6 1uC).

Phylogenetic analysis

Total genomic DNA was isolated from cells of cultured

Sphaerospermopsis torques-reginae strains ITEP-024 and

ITEP-026 using a modified CTAB (cetyl-trimethyl-

ammonium bromide) based extraction method adapted for

cyanobacteria (Fiore et al. 2000). The almost complete 16S

rRNA gene from the genomic DNA of both Sphaerosper-

mopsis torques-reginae strains was amplified by PCR using

the oligonucleotide primers 27F1 and 1494Rc (Neilan et al.

1997), corresponding to the E. coli 16S rRNA gene positions

27 to 1494. The PCR amplification and sequencing were

carried out as described previously (Fiore et al. 2007).

The 16S rRNA gene sequences obtained in this study and

reference sequences retrieved from GenBank were aligned,

refined, and used to generate phylogenetic trees. Trees were

reconstructed with neighbor-joining (NJ) and maximum-

likelihood (ML) algorithms implemented by the MEGA

version 5.0 program package (Tamura et al. 2011) using the

Kimura 2 and Tamura-Nei parameters, respectively, as

nucleotide substitution models. The robustness of the trees

was estimated by bootstrap percentages using 1000 replica-

tions. For Bayesian analysis, substitution models for nucleo-

tide evolution were determined using jModeltest (Guindon &

Gascuel 2003; Posada 2008). Under the Akaike Information

Criterion (AIC), the GTR + I + C model was utilized. The

software MrBayes v3.2 (Ronquist & Huelsenbeck 2003) was

used for determining Bayesian inference. For priors, we

assumed no prior knowledge on the data, thus a Dirichlet

(1,1,1,1,1,1) prior for substitution rate parameters was

estimated, in addition to, a uniform (0,1) prior for the pinvar

parameter. A uniform (0, 200) prior was set on the gamma

shape parameters and for branch lengths, an unconstraine-

d:exponential (10) prior. Two runs of four chains (three heated

and one cold) were run for 1.5 3 106 generations, sampling

every 100 trees. In each run, the first 25% of samples were

discarded as the burn-in phase. The figures presented in the

tree were taken from different sources (Fremy 1930; Komarek

1996; Watanabe 1996; Werner 2002; Werner & Laughinghouse

IV 2009). The DNA sequences from Sphaerospermopsis

torques-reginae strains ITEP-024 and ITEP-026 were

deposited in the NCBI GenBank database under accession

numbers HQ730086 and HQ730087, respectively.

RESULTS AND DISCUSSION

Morphological characterization

Nostochophycidae

Nostocales

Werner et al.: Sphaerospermopsis torques-reginae (Cyanobacteria) in South America 229

Nostocaceae

Sphaerospermopsis Zapomelova, Jezberova, Hrouzek, Hisem,

Rehakova & Komarkova Journal of Phycology 46: 415.

2010.

SYNONYMOUS: Sphaerospermum Zapomelova, Jezberova,

Hrouzek, Hisem, Rehakova & Komarkova, Journal of

Phycology 45: 1371. 2009 (nomen non legitiums).

Sphaerospermopsis torques-reginae (Komarek) Werner, Laugh-

inghouse IV, Fiore & Sant’Anna comb. nov.

BASIONYM: Anabaena torques-reginae Komarek, Acta Bo-

tanica Cubana 19:14–16. 1984.

SYNONYMOUS: Dolichospermum torques-reginae (Komarek)

Wacklin, Hoffmann & Komarek, Fottea 9(1): 62. 2009.

FIGS 2–12: Trichomes solitary, coiled, usually with a

mucilaginous envelope (1.0–10.2 mm); coils irregular or

regular, (17.5)20–54.5 mm wide, 9–22.5 mm distance;

vegetative cells rounded; (4)4.3–7(8) mm in diameter, 4–

6.4(8) mm long (R w:l 5 0.6–1.2:1); cell content blue-green,

with aerotopes; terminal cells rounded; heterocytes round-

ed, (4–5)5.4–9.5 mm in diameter, (5)5.5–9 mm long; akinetes

rounded with a smooth epispore, single or in pairs, attached

to one or both sides of the heterocytes, (7)7.9–13 mm in

diameter.

Fig. 1. Geographical distribution of Sphaerospermopsis torques-reginae (N), identified as Anabaena torques-reginae (*), Anabaena spiroides(m), and Anabaena (¤).

230 Phycologia, Vol. 51 (2), 2012

Sphaerospermopsis torques-reginae was originally de-

scribed as Anabaena torques-reginae by Komarek (1984),

from planktonic Cuban populations in eutrophic environ-

ments, though recently transferred to the genus Dolichos-

permum (Ralfs ex Bornet & Flahault) Wacklin et al. 2009

[5Dolichospermum torques-reginae]. Using molecular anal-

yses, mainly the 16S rRNA gene sequence, all planktonic

morphospecies of Anabaena were transferred to the genus

Dolichospermum. However, Zapomelova et al. (2009)

undertaking a polyphasic characterization of strains of

Anabaena reniformis Lemmermann and Aphanizomenon

aphanizomenoides (Forti) Horecka & Komarek (5Anabae-

na aphanizomenoides Forti), reclassified these two plank-

tonic species into the genus Sphaerospermum, though this

genus was subsequently renamed Sphaerospermopsis since

the prior was an illegitimate name because it is a homonym

of Sphaerospermum Cleve (Nova Acta Regiae Societatis

Scientiarum Upsaliensis. Serie 3, 6(11): 12, 35, 1868),

presently considered a heterotypic synonym of the genus

Mougeotia C. Agardh (Systema Algarum xxvi, 83, 1824),

nomen conservandum (Zapomelova et al. 2010b). This genus

is characterized mainly by the shape and posi-

tion of its akinetes (spherical, on one or both sides

adjacent to the heterocytes), including species with

solitary coiled or straight trichomes and an obligatory

presence of aerotopes. In the present study we confirm

that the populations observed in the studied South

American water bodies, initially identified as Dolichosper-

mum torques-reginae, actually belong to the genus Sphaer-

ospermopsis, mainly by the placement of our strains in the

phylogenetic tree based on the 16S rRNA gene sequences

(Fig. 13), as well as, the formation of rounded akinetes at

one or both sides of the heterocytes. Thus, we reclassify and

denominate the species Sphaerospermopsis torques-reginae

(Komarek) Werner, Laughinghouse IV, Fiore & Sant’Anna

comb. nov.

Taxonomic changes on the generic level in nostocacean

cyanobacteria were proposed at the end of 2008, based on

molecular studies (especially 16S rRNA gene sequencing)

together with cytological and morphological analyses of

Figs 2–7. Sphaerospermopsis torques-reginae. General aspect of the filaments, showing the twisting variation of the trichomes, thickness ofthe mucilaginous envelope, and number and position of the akinetes. Bars: 10 mm. (After Werner & Laughinghouse IV 2009, asAnabaena oumiana.)

Werner et al.: Sphaerospermopsis torques-reginae (Cyanobacteria) in South America 231

Figs 8–12. General aspect of a mixed cyanobacterial bloom with Sphaerospermopsis torques-reginae. Figs 9–12. General aspect ofSphaerospermopsis torques-reginae filaments, showing the twisting variation of the trichomes, thickness of the mucilaginous envelope, andnumber and position of the akinetes. Bars: Figs 8, 11 5 30 mm; Figs 9, 12 5 10 mm; Fig. 10 5 20 mm.

232 Phycologia, Vol. 51 (2), 2012

these cyanobacteria (Komarek 2010a). A comparison among

characteristic features of Dolichospermum, Sphaerospermop-

sis, Anabaena, Aphanizomenon, and Cuspidothrix is present-

ed in Table 1.

In general, the analyzed populations of Sphaerospermopsis

torques-reginae agree with those from the Cuban population

described by Komarek (1984), except for coiled parallel

filaments (double helix) described for Anabaena torques-

reginae. This type of filament was not seen in the studied

South American populations or in the populations reported

in the revised literature, agreeing with the type material

of Anabaena oumiana described by Watanabe (1996).

However, according to Komarek (personal communication)

parallel filaments are actually exceptions, only forming

when the species occurs in mass development.

Although Komarek & Zapomelova (2007) treated

Anabaena oumiana (5Sphaerospermopsis oumianum) as a

synonym of Anabaena torques-reginae (5Sphaerospermop-

sis torques-reginae) based on morphological analyses, the

16S rRNA gene sequences indicated that they are different

but closely related species, since the phylogenetic analyses

positioned them in different internal clusters together in a

major cluster (Fig. 13) and the similarity between sequences

of these two species was 97.9%. This finding is in agreement

Fig. 13. Maximum likelihood phylogenetic tree based on the 16S rRNA gene sequences (1336 bp) showing the relationships of the studiedSphaerospermopsis torques-reginae strains ITEP-024 and ITEP-026 (in bold). Bootstrap tests involving 1000 resamplings were performedand bootstrap values greater than 50% are given in front of the relevant nodes for ML and NJ analyses followed by Bayesian posteriorprobabilities . 0.5 using Bayesian analysis.

Werner et al.: Sphaerospermopsis torques-reginae (Cyanobacteria) in South America 233

Table

1.

Main

chara

cter

isti

cso

fth

en

ost

oca

ccea

ngen

era

Doli

chosp

erm

um

,S

phaer

osp

erm

opsi

s,A

nabaena,

Aphaniz

om

enon

an

dC

usp

idoth

rix

reco

gn

izab

leacc

ord

ing

toth

em

ole

cula

rap

pro

ach

an

dm

orp

ho

logic

al

featu

res.

Doli

chosp

erm

um

(Ralf

sex

Bo

rnet

&F

lah

au

lt)

Wack

lin

et

al.

2009

Sphaero

sper

mopsi

sZ

ap

om

elo

va

et

al.

2010b

Anabaen

aB

ory

ex

Bo

rnet

&F

lah

au

lt1886–1888

Aphaniz

om

enon

Mo

rren

ex

Bo

rnet

&F

lah

au

lt1886–1888

Cusp

idoth

rix

(Usa

cev)

Raja

nie

mi

et

al.

2005b

Basi

on

ym

(;)

;A

nab

aen

aB

ory

ex

Bo

rnet

&F

lah

au

lt-

Sec

tio

IID

oli

chosp

erm

um

Ralf

sex

Bo

rnet

&F

lah

au

lt

=S

phaer

osp

erm

um

___

___

;A

nabaen

ais

sats

chenk

oi

Usa

cev

5A

ph.

issa

tschenk

oi

(Usa

cev)

Pro

sk.-

Lavr.

Syn

on

ym

(5)

Zap

om

elo

va

et

al.

Tri

cho

me

-so

lita

ry(o

rsm

all

clu

ster

s)-s

oli

tary

-in

mats

-so

lita

ryo

rin

fasc

icle

s-s

oli

tary

-co

iled

or

stra

igh

t-c

oil

edo

rst

raig

ht

-fle

xu

ou

so

r6

coil

ed-s

traig

ht

-str

aig

ht

or

coil

ed-m

etam

eric

-met

am

eric

-met

am

eric

-su

bsy

met

ric

-su

bsy

met

ric

Cel

l-a

pic

al

cell

sim

ilar

toth

eveg

etati

ve

cell

s-a

pic

al

cell

slig

htl

ym

od

ifie

d-a

pic

al

cell

sim

ilar

toth

eveg

etati

ve

cell

so

rsl

igh

tly

mo

dif

ied

-ter

min

al

cell

scy

lin

dri

cal,

elo

ngate

d,

hyali

ne,

no

to

ro

nly

ver

ysl

igh

tly

narr

ow

ed

-cle

arl

yn

arr

ow

edan

dp

oin

ted

at

the

end

-ap

ical

cell

elo

ngate

d,

acu

min

ate

,h

yali

ne

Aer

oto

pes

-ob

ligato

ry-o

bli

gato

ry-n

ever

occ

ur

-ob

ligato

ry-f

acu

ltati

ve

Het

ero

cyte

-in

terc

ala

rly

-in

terc

ala

rly

-in

terc

ala

rly

-in

terc

ala

rly

-in

terc

ala

rly

-usu

all

yso

lita

ry-u

suall

yso

lita

ry-u

suall

yso

lita

ry-u

suall

yso

lita

ry-s

oli

tary

Ak

inet

e-d

ista

nt

fro

mth

eh

eter

ocy

te(p

ara

het

ero

cyti

cal)

-6el

on

gate

d

-att

ach

edto

on

eo

rb

oth

sid

eso

fth

eh

eter

ocy

te-r

ou

nd

ed

-dis

tan

to

ratt

ach

edto

the

het

ero

cyte

(para

het

ero

cyti

cal)

-dis

tan

to

ratt

ach

edto

the

het

ero

cyte

(para

het

ero

cyti

cal)

-dis

tan

to

ratt

ach

edto

the

het

ero

cyte

(para

het

ero

cyti

cal)

Hab

itat

-pla

nk

ton

-pla

nk

ton

-ben

tho

s,p

erip

hyto

n,

met

ap

hyto

n,

edap

hic

-pla

nk

ton

-pla

nk

ton

Ref

eren

ceW

ack

lin

et

al.

(2009)

Zap

om

elo

va

et

al.

(2009,

2010b

)G

ugger

et

al.

(2002)

Ko

mare

k(2

010a)

Raja

nie

mi

et

al.

(2005b

)R

aja

nie

mi

et

al.

(2005a)

Ko

mare

k(2

010a)

Ko

mare

k(2

010a)

Ko

mare

k(2

010a)

234 Phycologia, Vol. 51 (2), 2012

Table

2.

Mo

rph

om

etri

cch

ara

cter

isti

cs(m

m)

of

natu

ral

po

pu

lati

on

s(N

P)

an

dst

rain

s(I

TE

P-0

24

an

dIT

EP

-026)

of

Sphaero

sper

mopsi

sto

rques-

regin

ae

fro

md

iffe

ren

tS

ou

thA

mer

ican

site

s.(D

ept.

5D

epart

men

t;*

5n

ot

ob

tain

ed).

CO

LO

MB

IAB

RA

ZIL

AR

GE

NT

INA

Dep

t.D

elC

esar

Per

nam

bu

coM

ato

Gro

sso

do

Su

lS

ao

Pau

loR

ioG

ran

de

do

Su

lB

uen

os

Air

esP

rovin

ceN

PIT

EP

-024

ITE

P-0

26

Co

il wid

thir

regu

lar

—(4

2.9

67.1

)—

(42.8

66.3

)—

(14.3

63.7

)20.5

–54.5

(33.7

66.7

)17.5

–31.6

(25.1

63.6

)20–46

(24.5

62.9

)38.6

–50

(46.2

64.5

)d

ista

nce

—(3

2.6

611.8

)—

(30.2

611)

—(3

26

3.8

)10.2

–19.7

(13.7

62.3

)10–18.1

(14

62.8

)9–20

(13.3

63.4

)12.5

–22.5

(17.5

67.1

)

Cel

l dia

met

er4.5

–6.5

(5.9

60.6

)5–6

(5.5

60.5

)5–7

(66

0.6

)4–6

(5.6

60.5

)4.4

–6.2

(5.5

60.3

)4.3

–6.6

(5.3

60.5

)4.5

–8

(6.3

60.7

)4.5

–5,8

(5.3

60.4

)le

ngth

5–6.1

(5.6

60.4

)4–6

(4.5

60.7

)4–6

(5.3

60.5

)4–6

(56

0.4

)4–6.4

(5.4

60.5

)4–6

(56

0.6

)4.5

–8

(5.9

61)

4.5

–5.8

(56

0.4

)R

l:w

0.8

–1.1

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Werner et al.: Sphaerospermopsis torques-reginae (Cyanobacteria) in South America 235

with another study that reports the existence of these two

species (Watanabe 1996).

Sphaerospermopsis reniforme (Lemmermann) Zapome-

lova et al. (5Anabaena reniformis Lemmermann emend

Aptekar) is the species that most closely resembles Sphaer-

ospermopsis torques-reginae and they are easily confused due

to the shape of their vegetative cells. Although the cells of

both species are round (spherical or slightly elongated), they

differ essentially by their cell length:width ratio. It was verified

that the cell length:width relation of the analyzed specimens are

0.6–1.2:1, while the cells of Sphaerospermopsis reniforme are

longer (R l:w 0.67–2.50 / 3–6 3 2.5–10.0 mm - Watanabe et

al. 2004 as Anabaena reniformis). In addition, the 16S rRNA

gene sequences of the studied populations clearly show their

different position in the phylogenetic tree (Fig. 13), assuring

our present identification.

Table 2 shows the metric limits obtained for Sphaeros-

permopsis torques-reginae populations registered in the

studied South American aquatic environments.

The number of akinetes attached to the heterocytes varied

considerably, forming one or two akinetes at one or both

sides of the heterocytes. The nature of the coil twists also

varied greatly among the analyzed populations, where some

trichomes had regular coils, while others irregular, and some

with both regular and irregular coils in the same trichome.

Mucilage was not originally characterized for the Cuban

populations identified as Anabaena torques-reginae (5

Sphaerospermopsis torques-reginae), yet our studied popu-

lations demonstrated variations in this characteristic. The

thickness of mucilage around trichomes was generally

consistent between populations; however, in one population

specimens with thick mucilage were observed (Figs 3, 8, 9,

11) and others practically without mucilage (Figs 5, 6, 12).

Considering the mucilaginous structure variable, subject to

environmental conditions and the age of the microorganism,

the taxonomic value is constituted only on the ability of the

same developing mucilaginous envelopes.

Sphaerospermopsis torques-reginae can be easily confused

with Dolichospermum spiroides (Klebahn) Wacklin et al.

(5Anabaena spiroides) due to their morphological similarity

and because their cell, heterocyte, and coil size overlap;

however, the latter differs from the former by usually

producing ellipsoidal akinetes, sometimes slightly bent, and

remote from the heterocytes. Therefore, due to this

morphometric similarity, reliable identification of these

species is only possible when akinetes and heterocytes are

present, avoiding, in this manner, incorrect identifications

and consequently, imprecise interpretations on the geo-

graphic distribution of this species.

Molecular characterization

Near-full-length (1411 bp) fragments of 16S rRNA gene

were amplified and sequenced from the Sphaeropermopsis

torques-reginae strains ITEP-024 and ITEP-026. The

BLAST analyses among the 16S rRNA sequences obtained

in this study with sequences from GenBank showed the

highest similarities to Anabaena reniformis 07-01 (97.9%

identity, 98% coverage) isolated from a Vysehrad fishpond,

Czech Republic and Aphanizomenon aphanizomenoides 04-

43 (98.4% identity, 96% coverage) originating from Svet

fishpond, Czech Republic. In the phylogenetic tree, the two

Table 3. Environmental conditions of aquatic ecosystems where the studied populations of Sphaerospermopsis torques-reginae wereregistered in South America (* 5 not obtained).

Sites

Environmental conditions

Water temperature (uC) pH Conductivity (mS cm21) Salinity (%)

Colombia

Del Cesar DepartamentLa Loma de CalenturasLa Pachita swamp 33 * * 0

Brazil

Pernambuco StateRecifeTapacura Reservoir * 7.3–8.9 430–460 0

Mato Grosso do Sul State

CorumbaParque Pantanal da Nhecolandia Road

lake of Bridge 45 23.7 8.9 254 0

Rio Grande do Sul State

TorresLake Violao 24.8–32.0 7.5–8.5 241–320 0

Rio GrandePatos Lagoon 26–30 * * 0–4

Sapucaia do SulMato Lake 26 7.1 * 0

Argentina

Buenos Aires ProvinceEnsenada, La Plata 12–26 7.2–9.8 631–800 0

lake of the Aeroclub La PlataPatos pond 24–31 8.5–10.5 537–742 0

236 Phycologia, Vol. 51 (2), 2012

Sphaeropermopsis torques-reginae 16S rRNA gene sequenc-

es fall within a highly supported (bootstrap values of 74%,

89% and 0.99 for ML, NJ, and Bayesian algorithms,

respectively) cluster (Fig. 13) containing a group of

planktonic members originally classified in the genus

Anabaena but recently included into the newly described

genus Sphaeropermopsis according to genetic and morpho-

logical traits (Zapomelova et al. 2009, 2010a). This study

represents the first report of sequencing of nearly complete

16S rRNA gene from the cyanobacterial morphospecies

Anabaena torques-reginae and confirms its affiliation to the

newly erected genus Sphaeropermopsis.

Distribution and ecology

Sphaerospermopsis torques-reginae was originally described

as Anabaena torques-reginae by Komarek (1984) from

blooms recorded in Cuban eutrophic water bodies.

According to Komarek & Zapomelova (2007), the species

was also observed in El Salvador, Brazil, and Argentina;

however, the specific sites were not mentioned.

In South America, the species’ presence had already been

reported in a tropical northeastern Brazilian drinking water

supply reservoir, by Molica et al. (2005), but was misiden-

tified as Anabaena spiroides. Furthermore, populations of

this cyanobacterium were also found in other Brazilian

tropical and subtropical regions, and in temperate areas of

Argentina, both in natural and artificial aquatic ecosystems.

Its occurrence was also confirmed in a tropical Colombian

swamp. Werner & Laughinghouse IV (2009) reported the

microorganism as a bloom-former in many subtropical

bodies of water in southernmost Brazil; however, these

populations were identified as Anabaena oumiana due to the

analyzed populations not presenting parallel filaments, thus

differing from the iconotypus of Anabaena torques-reginae.

The species was usually reported in fresh water but in South

America, in a coastal lagoon of the extreme south of Brazil, it

was also found in a sample with a 4% salt concentration. It

was generally observed in eutrophic systems, usually in dense

blooms, predominating or accompanying blooms of other

cyanobacterial species, such as Microcystis aeruginosa

Kutzing, Microcystis protocystis Crow, Microcystis panni-

formis Komarek et al., Sphaerocavum brasiliense Azevedo &

Sant’Anna, Radiocystis fernandoi Komarek & Komarkova-

Legnerova, Cylindrospermopsis raciborskii (Wolosynska)

Seenayya & Subba Raju, Anabaenopsis circularis (G.S.

West) Wolosynska & Miller, Pseudanabaena sp., or by

chlorophyceans, especially Scenedesmus spp. Figure 1 pre-

sents the worldwide geographic distribution of Sphaeros-

permopsis torques-reginae.

Sphaerospermopsis torques-reginae predominated in

South American alkaline waters, with a pH between 7.1 to

10.5 (X 5 8.4 6 1.2). The conductivity was relatively low,

oscillating between 241 and 800 mS cm21 (X 5 490 6 204).

The highest conductivities (537–800 mS cm21) and the

highest pH values (9.8–10.5) were registered in the temperate

Argentinean lakes. The species stood out as a component in

mixed cyanobacterial blooms when water temperature was

higher than 23.7uC (X 5 26.3 6 5.8); the Colombian

population was found in a water temperature of 33uC.

Nonetheless, it has also been observed in colder water

(12uC), in a lake of the Aeroclub La Plata (Argentina). The

environmental conditions of the aquatic ecosystems where

the studied populations were found are expressed in Table 3.

The observation of Sphaerospermopsis torques-reginae in

different aquatic environments (natural and artificial lakes,

a lagoon, reservoirs, and a swamp) in both fresh and

brackish water, of tropical, subtropical, and temperate

zones suggests a wide distribution of the species in South

America and also that it probably presents broad ecological

plasticity. This fact implies that, until now, populations

of Sphaerospermopsis torques-reginae have probably been

improperly identified as other species of Dolichospermum

with twisted filaments, such as Dolichospermum spiroides

which is usually cited in literature for different locations in

South America. Thus, Sphaerospermopsis torques-reginae

should have a wider distribution than presented in literature.

Toxicity

Toxic blooms of the species with production of anatoxin-a(s)

have been reported in tropical water bodies in northeastern

Brazil (Molica et al. 2005; Dorr et al. 2010). According to

Molica et al. 2005, three isolated cultures (identified as

Anabaena spiroides) from Tapacura Reservoir (Recife,

Pernambuco State) were toxic in a mouse bioassay, the

time of death varied from 3 to 12 minutes and anatoxin a(s)-

like anticholinesterase symptoms, including salivation and

limb fasciculation, were observed. Moreover, Mullor (1945)

reports the intoxication and death of more than 1000 Indian

Runner ducks, associated to the occurrence of Anabaena

blooms in Bedetti Lagoon, Santa Fe Republic (Argentina).

However, the description and illustration presented by

this author suggests that this population corresponds to

Sphaerospermopsis torques-reginae. Additionally, toxicolog-

ical analyses were run on samples of a mixed bloom, formed

by Sphaerospermopsis torques-reginae (identified as Anabae-

na oumiana), Microcystis protocystis, Microcystis pannifor-

mis, Sphaerocavum brasiliense, and Anabaenopsis circularis

from Lake Violao, a freshwater subtropical coastal lake

from southernmost Brazil, and the mass spectrum con-

firmed the following substances: anabaenopeptins F and B,

as well as microcystins-LR and -RR (Carvalho et al. 2008).

ACKNOWLEDGEMENTS

We especially thank Dr Jiri Komarek and Dr Masayuki

Watanabe (in memoriam) for their valuable comments; to

CNPq (PIBIC - MCN/FZBRS) for financial support to

H.D. Laughinghouse IV and E.B. Neuhaus, graduate

scholarship to C. Hoff (142749/2009-5), and research

fellowship to M.F. Fiore (308299/2009-4); to FAPESP for

financial support to M.F. Fiore (2007/07075-5); to the

Ministry of Education Agency (CAPES) for the graduate

scholarship to R.Y. Honda; to Dr Clarisse Odebrecht and

Dr Marli Bergesch (FURG - Rio Grande, Brazil), and to Dr

Juan Pablo Alvarez Silva (National University of Colom-

bia) for providing some of the studied samples; to Rejane

Rosa and to Arlete Ieda Pasqualetto and Everton Luis Luz

de Quadros from MCN/FZBRS, for passing Indian ink on

the line drawings for final print and for designing the maps,

respectively; to Felipe Secco Richter for his technical help.

Werner et al.: Sphaerospermopsis torques-reginae (Cyanobacteria) in South America 237

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Received 21 March 2011; accepted 8 August 2011

Associate editor: Dale Casamatta

238 Phycologia, Vol. 51 (2), 2012