Thorea indica sp. nov. (Thoreales, Rhodophyta) from Uttar ... · Necchi et al. Thorea indica sp....

Algae 2015, 30(4): 265-274http://dx.doi.org/10.4490/algae.2015.30.4.265

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Research Article

Copyright © 2015 The Korean Society of Phycology 265 http://e-algae.kr pISSN: 1226-2617 eISSN: 2093-0860

Thorea indica sp. nov. (Thoreales, Rhodophyta) from Uttar Pradesh, India

Orlando Necchi Jr.1,*, Monica O. Paiano1, John A. West2, E. K. Ganesan3,a and

Susan Loiseaux-de Goër4

1Department of Zoologia e Botânica, UNESP-Campus de São José do Rio Preto, Rua Cristóvão Colombo, 2265, 15054-000 S. Jose Rio Preto, São Paulo, Brazil2School of Biosciences 2, University of Melbourne, Parkville, VIC 3010, Australia3Instituto Oceanográfico, Universidad de Oriente, Cumaná 6101, Venezuela411 Rue des Moguerou, 29680 Roscoff, France

Thorea indica sp. nov. is described from the Sai River, Uttar Pradesh, India (26°39′00.7″ N, 80°47′38.3″ E). Its classifica-

tion is based on molecular sequences of the plastid-encoded RuBisCO large-subunit gene, rbcL and the barcode region

of the mitochondrial encoded cytochrome c oxidase subunit 1, cox1, and morphological data. The sequence analyses

confirm a new species of Thorea. The cox1 barcode sequence had 90.4-90.8% identity with Thorea sp. from Australia and

Thorea hispida from Hawaii and China. Based on rbcL sequences the Indian specimen was positioned in a major clade

with high support (>95 bootstrap and 0.95 posterior probability) containing two other species: T. okadae from Japan and

T. hispida from the continental USA, Hawaii, the UK, and China. The divergences among these sequences were T. indica

vs. T. okadae (2.8%) and T. indica vs. T. hispida (2.9-3.4%). The comparison of morphological characters of Thorea from

India was not conclusive due to the inadequate descriptions in previous reports: most specimens reported as T. hispida

fit within the circumscription of T. indica as described here. The previous report of T. siamensis from the Sai River is incor-

rect and the specimens fit within our description of T. indica. Thorea indica and T. okadae can be distinguished by minor

morphometric characters and sexuality (dioecious vs. monoecious).

Key Words: cox1; India; molecular systematics; morphology; rbcL; reproduction; Thorea indica sp. nov.; Thoreales

INTRODUCTION

The freshwater red algal order Thoreales is distin-

guished from the closely related Batrachospermales by

its multiaxial rather than uniaxial gametophytes (Müller

et al. 2002). The thallus consists of a central medulla of

colorless filaments surrounded by determinate assimi-

latory filaments. The order is monotypic and includes

two genera, Nemalionopsis and Thorea (Starmach 1977,

Sheath et al. 1993, Müller et al. 2002). These two genera

of the Thoreaceae are distinguished by the positioning of

the reproductive structures (sporangia) and arrangement

of the assimilatory filaments in the outer region (Sheath

et al. 1993, Necchi and Zucchi 1997, Entwisle and Foard

Received September 18, 2015, Accepted October 20, 2015

*Corresponding Author

E-mail: [email protected]: +55-17-3221-2406, Fax: +55-17-3221-2374aPresent address: 3-A Srinivas Terrace, 52, II Main Road, Gandhi Nagar, Adyar, Chennai, 600020, Tamil Nadu, India

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Com-

mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Algae 2015, 30(4): 265-274

http://dx.doi.org/10.4490/algae.2015.30.4.265 266

T. violacea in distinct geographic origins. Necchi et al.

(2010a) found that Thorea had four major clades based on

sequences of rbcL and SSU rDNA, each one representing

a distinct species: 1) T. gaudichaudii from Asia (Japan and

Philippines); 2) T. violacea from Asia (Japan) and North

America (USA and Dominican Republic); 3) T. hispida

from Europe (England) and Asia (Japan); 4) T. bachman-

nii from South America (Brazil). In addition, they pointed

out that Thorea species recognized by molecular data re-

quire additional characters (e.g., reproductive details and

chromosome numbers) to allow consistent and reliable

taxonomic circumscriptions.

Singh (1960) reported briefly on the occurrence of T.

hispida (= T. ramosissima) for the first time from North

India. Khan (1978) added some details on thallus anato-

my and noted the presence of a single monosporangium,

which Desikachary et al. (1990) doubted. Bhatnagar and

Chaturvedi (1987) made some important observations on

the sexual apparatus of T. hispida (as T. ramosissima). Im-

portantly they stated clearly that ‘spermatangia and car-

pogonia are found to form on the same plant, i.e., their

specimens were bisexual or monoecious. Dhanalakshmi

et al. (2009) studied T. hispida (as T. ramosissima) collect-

ed from a waterfall in the Andaman and Nicobar islands,

but did not specify whether their plants were uni- or bi-

sexual. A noteworthy collection of T. hispida (as T. ramo-

sissima) is from a land locked lake at a high altitude (4,266

m) in Ladakh, Indian Himalayas (Bhat et al. 2011). Suseela

and Toppo (2015) reported T. siamensis from the Sai River,

Uttar Pradesh, India among other three freshwater red al-

gal species. Feng et al. (2015) analyzed T. hispida from two

cool temperate sites in Shanxi, China based on sequences

of two genes (rbcL and SSU rDNA).

To identify the newly collected material of Thorea re-

quired morphological and molecular comparisons with

not only Indian collections (where possible) but with

those from elsewhere in the world.

MATERIALS AND METHODS

Thorea specimens were collected by K. Toppo (collec-

tion No. SAI 201102) on March 13, April 3, and April 24,

2014 from the Sai River, Uttar Pradesh, India (26°39′00.7″

N, 80°47′38.3″ E) at an elevation of 106 m. For morpholog-

ical studies, the materials were preserved in 4% formalin

with voucher specimens mounted on herbarium paper

and lodged at Herbaria MEL, MICH, and SJRP (herbarium

acronyms follow Thiers 2015). Specimens for DNA anal-

ysis were blotted dry with tissue and preserved in silica

1999, Carmona and Necchi 2001): assimilatory filaments

are densely arranged and sporangia are located on outer

assimilatory filament in Nemalionopsis, whereas in Tho-

rea the filaments are loosely arranged and sporangia are

located in the inner assimilatory filaments. Sexual repro-

duction and carposporophytes have been described in

Thorea (Yoshizaki 1986, Necchi 1987, Sheath et al. 1993,

Necchi and Zucchi 1997, Entwisle and Foard 1999, Car-

mona and Necchi 2001) and carpogonia and putative

spermatangia were only recently reported in Nemalion-

opsis from Indonesia (Johnston et al. 2014).

Thorea species have been reported in several regions

of the world but tend to be more common in tropical,

subtropical and warm temperate areas with hard waters

(Sheath and Hambrook 1990, Sheath et al. 1993, Necchi

et al. 1999, Carmona and Necchi 2001). Taxonomic char-

acters used for species delineation are mostly vegetative

features (Sheath et al. 1993, Carmona and Necchi 2001,

Necchi et al. 2010a): plant and medulla diameter, branch-

ing frequency (abundant or sparse secondary branches),

size and shape of assimilatory filaments (clavate or non-

clavate), size and shape of sporangia. However there is

considerable overlap in these characters, causing prob-

lems in species identification (Carmona and Necchi 2001,

Necchi et al. 2010a). Characteristics of sexual reproduc-

tion (carpogonia and spermatangia) and carposporo-

phytes may provide a more reliable set of diagnostic char-

acters but as yet these features are poorly documented for

most species.

Guiry and Guiry (2015) list 12 accepted species of Tho-

rea. Twenty-two years ago Sheath et al. (1993) recognized

only four species of Thorea worldwide: T. clavata Seto et

Ratnasabapathy, T. hispida (Thore) Desvaux, T. violacea

Bory, and T. zollingeri Schmitz. Since then T. siamensis

Kumano & Traichaiyaporn (Traichaiyaporn et al. 2008)

from Thailand, T. conturba Entwisle & Foard (1999) from

Australia, and six species (including some taxa consid-

ered as synonyms by Sheath et al. [1993]) summarized in

Kumano (2002): T. bachmannii Pujals, T. brodensis Klas,

T. gaudichaudii C. Agardh, T. okadae Yamada, T. prowsei

Ratnasabapathy et Seto, and T. riekei Bischoff.

Studies using molecular data are still inadequate for

the genus. Müller et al. (2002) found the following se-

quence divergence among species of Thorea for two mo-

lecular markers: 5.4-13.3% for rbcL (plastid gene encod-

ing the large RuBisCO subunit) and 2.5-13.3% for small

subunit (SSU) rDNA (nuclear gene encoding the large ri-

bosomal subunit). They recognized four species based on

these two markers, which could not be properly named

because it was impossible to distinguish T. hispida from

Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)

267 http://e-algae.kr

determined as the best-fit model of sequence evolution

by the Akaike Information Criterion using jModelTest

2.1.4 (Darriba et al. 2012). Maximum-likelihood (ML)

topologies and bootstrap values from 10,000 replicates

were inferred using RAxMLGUI (Silvestro and Michalak

2012) and bayesian analysis (BA) was performed using

MrBayes 3.2 (Huelsenbeck and Ronquist 2001) as plugin

for Geneious. BA consisted of three runs of five chains

of Metropolis coupled Markov Chain Monte Carlo for 10

× 106 generations. The first 500 trees were discarded as

burn-in.

The cox1 marker sequence was compared to other red

algal barcodes available in databases: the barcode of life

data systems (BOLD) database (Ratnasingham and He-

bert 2007) and GenBank (Benson et al. 2013).

RESULTS

Molecular analyses

Comparison of the cox1 barcode sequence from our In-

dian specimen with those in GenBank database returned

the following close matches (Appendix 1): 90.8% with

Thorea sp. (KC130141) (Scott et al. 2013) from Australia;

90.4% with T. hispida from Hawaii (KC596320) (Carlile

and Sherwood 2013) and from China (KC511076) (un-

published). BOLD database returned no close matches

(>85%). These results indicate that the sequence from the

Indian specimen belongs to a species quite distinct from

any previously sequenced samples.

The rbcL gene sequence from India was compared with

18 previously published sequences of Thorea obtained

from GenBank (Appendix 1). BA and ML analyses of the

rbcL alignment yielded similar trees, showing strong

support for Nemalionopsis and Thorea as monophyletic

groups (Fig. 1). Within the genus Thorea nine clades were

revealed, that we interpreted as different species. The

sequence of Indian specimen was positioned in a major

clade with high support containing two other species: T.

okadae from Japan and T. hispida from continental USA,

Hawaii, the UK, and China. The divergences among these

sequences were as follows: India vs. T. okadae (2.8%, 35

bp); India vs. T. hispida (2.9-3.4%, 37-44 bp). The two

samples of T. okadae are identical and the reference of

sequence AF506269 as T. violacea (Appendix 1) was prob-

ably a misidentification. These divergence levels are high

enough to consider the Indian specimen as a distinct spe-

cies that we describe below.

desiccant. Morphological analyses were carried out for all

characters previously used as diagnostic in relevant stud-

ies of the genus (Yoshizaki 1986, Necchi 1987, Sheath et al.

1993, Necchi and Zucchi 1997, Kumano 2002). As a rule,

twenty measurements or counts were taken for each char-

acter in each sample (Necchi and Zucchi 1997). For mi-

croscopic observations mounting media including stains

were prepared as follows: 1) 0.02% aniline blue WS in 50%

corn syrup and 0.5% phenol (Ganesan et al. 2015), speci-

mens added for 30 min, removing water carefully with

blotting paper, adding a drop of mounting medium and

arranging the specimens with a fine needle and forceps

tip; 2) a double staining of 1% aqueous methylene blue

(Grimstone and Skaer 1972) and 1% alcian blue (Sheath

and Cole 1990) by placing small pieces for 10 min in each

solution. Surface views, cross and longitudinal sections

were prepared on microscope slides after staining. Photo-

graphs were taken with Canon G3 camera (Canon, Tokyo,

Japan) adapted to a Zeiss binocular research compound

microscope (Carl Zeiss, Jena, Germany) and a Leica DFC

320 digital camera with a LAS capture and image analysis

software coupled to a Leica DM 5000 microscope (Leica

Microsystems, Wetzlar, Germany).

For DNA extraction, samples were ground with a Pre-

cellys 24 tissue homogeneizer (Bertin Technologies, Mon-

tigny-le-Bretonneux, France), followed by DNA extrac-

tion using NucleoSpin plant II mini kit (Macherey-Nagel,

Duren, Germany). A 1,282 bp fragment of the plastid-en-

coded ribulose-1,5-bisphosphate carboxylase–oxygenase

large-subunit gene (rbcL) was polymerase chain reaction

(PCR) amplified using the set of primers specified by Vis

et al. (1998). The 664 bp barcode region near the 5′ end of

the cox1 gene was PCR amplified using the M13 F and R

primers (Saunders and Moore 2013). PCR reactions were

tested for the two markers as follows: 25 μL Top Taq Mas-

ter Mix (Qiagen GmbH, Hilden, Germany), 2.0 μL each

primer, 4.0 μL dH2O, and 2.0 μL extracted DNA. The PCR

protocols for the rbcL and cox1 barcode region gene fol-

lowed Vis et al. (1998) and Saunders and Moore (2013),

respectively. PCR products were purified using the Nu-

cleoSpin Extract II (MN, Macherey-Nagel) PCR clean up

or Gel Extraction. Sequencing was performed using the

amplification primers for both markers, and in addition

the internal rbcL primers R897 and F650 (Vis et al. 1998).

Sequencing reactions were run using the ABI PRISM Big

Dye v3.1 Terminator Cycle Sequencing Ready Reaction

kit and the ABI PRISM 3130xl Genetic Analyzer (Applied

Biosystems, Foster City, CA, USA). Sequence alignments

were assembled in Geneious 7 (Kearse et al. 2012). For

phylogenetic analyses of the rbcL data a GTR + I + G was

Algae 2015, 30(4): 265-274


ments in an outer whorl at right angle to the main shoot.

Short assimilatory filaments 25-45 μm long, composed of

4-8, short barrel-shaped to cylindrical cells, 5-8 μm diam-

eter; long assimilatory filaments, sparsely branched, with

10-22 cylindrical cells of uniform diameter (4.5-7.5 μm)

along each filament length, 380-500 μm in length. Each

cell appears to have a single multi-lobed peripheral chlo-

roplast. Spermatangia arising from the short assimilatory

filaments, usually developing in clusters or less often in

two, obovoid or elliptical, 8.5-12.0 μm in length, 5.0-7.5

μm in diameter. Carpogonia bottle-shaped or ovoid, 5.0-

9.5 μm in diameter, inserted directly on the basal cell of

assimilatory filaments or on one discoid or barrel-shaped

cell; trichogynes elongate and filiform, 2.5-4.5 μm in di-

ameter and 150-225 μm in length. Monosporangia, car-

Thorea indica Necchi, E. K. Ganesan et J. A. West sp. nov. (Fig. 2A-I)

Diagnosis. Plants dioecious, moderately mucilaginous,

4-12 cm in length, dark brown to brown-greenish, mod-

erately to abundantly branched, multiaxial, attached to

substrata by discoid holdfasts. Male plants slender and

abundantly branched, 500-900 μm in diameter; female

plants larger and moderately branched, 700-1,250 μm in

diameter. Thallus structure consisting of a central medul-

la, with interwoven colorless branched and twisted fila-

ments with short often irregularly shaped cells. Medulla

150-400 μm in diameter. Outer medulla a ring of long par-

allel filaments, with irregularly sized cells 2-4 μm in diam-

eter. Cortex composed of short and long assimilatory fila-

Fig. 1. Maximum-likelihood topology of rbcL sequence data of the Thoreales. Support values at each node are bootstrap (BS) from RAxML (left) and Bayesian posterior probability (PP) (right). Nodes without values indicate BS ≤ 70 and PP ≤ 0.70.



Fig. 2. Thorea indica morphological characters. (A) General view of pressed holotype. (B) Mid portion of a mature plant showing assimilatory filaments (f ) and medulla (m). (C) Apex of a mature plant, showing assimilatory filaments (f ) and medulla (m). (D) Longitudinal section showing assimilatory filaments (f ) and branches (arrowheads). (E) Longitudinal section showing medulla (m), short (s) and long (l) assimilatory filaments. (F) Cross section showing medulla (m), short (s) and long (l) assimilatory filaments. (G) Spermatangia in clusters on short assimilatory filaments. (H) Detail of spermatangia (sp, arrows). (I) Carpogonium on 1-celled carpogonial branch. Staining: aniline blue (B-D, H & I); double methylene blue and alcian blue (E & F). Scale bars represent: A, 10 mm, B & C, 250 µm; D, 25 µm; E & F, 50 µm; G & I, 10 µm; H, 5 µm.

A C

D

B

E F

G H I

Algae 2015, 30(4): 265-274


ments. Although monosporangia are produced terminal-

ly at the bases of assimilatory filaments, they have been

reported at the apex of the long assimilatory filaments

(Entwisle and Foard 1999, Xie and Shi 2003). The former

authors termed them as archeospores, as defined by Nel-

son et al. (1999).

Necchi et al. (2010a) found that four species of Thorea

could be recognized based on sequences of rbcL and SSU

rDNA: 1) T. gaudichaudii from Asia (Japan and Philip-

pines); 2) T. violacea from Asia (Japan) and North America

(USA and Dominican Republic); 3) T. hispida from Europe

(England) and Asia (Japan); 4) T. bachmannii from South

America (Brazil). They pointed out that T. okadae from

Japan (Hanyuda et al. unpublished data) did not form

a separate branch, but grouped together with samples

from Japan and England (Müller et al. 2002). Thus, they

proposed that T. okadae should be treated as a synonym

of T. hispida and not of T. violacea. However, in this study

we found that T. okadae forms a quite distinct clade, sister

of the new species T. indica (Table 1, Fig. 1). Both formed

a sister clade of T. hispida from the UK, the continental

USA and Hawaii. In summary, nine species can be recog-

nized from molecular data: T. bachmannii from Brazil, T.

gaudichaudii (from Japan and the Philippines), T. hispida

(from the UK, the continental USA, Hawaii, and China),

T. okadae (from Japan), T. riekei (from USA), T. violacea

(from USA), T. indica and an undetermined species–Tho-

rea sp. from Hawaii (Carlile and Sherwood 2013) and the

continental USA reported as T. violacea (AF506268) (Mül-

ler et al. 2002). A clear geographic distribution is evident

for most Thorea species based on molecular data. This

could be used as an additional criterion to distinguish

them, considering the little value of the currently used

morphological characters discussed above.

The comparison with previous reports from India

(Table 1) is not conclusive, mostly due to the inadequate

descriptions provided and lack of molecular evidence.

The specimens previously reported as T. hispida fit within

the circumscription of T. indica and we recommend their

tentatively placement in this species. The report of T. sia-

mensis by Suseela and Toppo (2015) from the same local-

ity as T. indica has no morphological basis, either in com-

parison with the original description (Traichaiyaporn

et al. 2008) or data from this study (Table 1). Relevant

vegetative characters were not presented, particularly

plant diameter and short assimilatory filaments. They

described assimilatory filaments as being clavate (cell di-

ameter enlarging from proximal to distal part), which is

exclusive of T. clavata and T. zollingeri, and quite differ-

ent of T. siamensis. In addition, the reported value of as-

posporangia and ‘Chantransia’ stage not observed.

Holotype. India, Uttar Pradesh, Sai River, coll. K. Top-

po, Mar 13, 2014 (MEL2389295); isotype SJRP 31508.

Habitat. Moderately flowing, clear stream; alga grow-

ing at depth of 1-2.5 ft, attached to submerged stones.

DISCUSSION

The two genera in the Thoreales can be separated on

reliable morphological characters, but species of Thorea

are hardly distinguishable on the basis of morphologi-

cal features, as pointed out in previous studies (Carmona

and Necchi 2001, Necchi et al. 2010a). Wide overlap of

morphological and morphometric characters are usually

observed among species, leading to difficulties for spe-

cies circumscriptions. Plant and medulla diameter are

largely used but are not reliable criteria to differentiate

species as Thorea is apparently seasonal in tropical and

temperate regions with dimensions changing according

to the season (see Simić et al. 2014). Branching pattern,

i.e., abundant vs. sparse secondary branches, has also

been demonstrated to be unreliable for species delinea-

tion (Carmona and Necchi 2001). One of the few vegeta-

tive characters that seems to be useful for species identi-

fication is the shape of assimilatory filaments, i.e., clavate

vs. non-clavate, the former being exclusive of T. clavata

and T. zollingeri.

Monosporangia, spermatangia, carpogonia, and car-

posporophytes can be potentially valuable as additional

diagnostic characters for species identification in the

genus. However, detailed descriptions of these charac-

ters have been poorly documented in most studies. In

addition, monosporangia are probably misinterpreted

by some authors as spermatangia or carposporangia.

Monosporangia and carposporangia frequently have

similar shape (obovoid to elliptical) and overlapping size

ranges, thus care should be taken to distinguish between

these two. Monosporangia can be easily differentiated

from spermatangia by their granular content and larger

size (Carmona and Necchi 2001). The existence of mono-

sporangia in Thorea as the sole means of reproduction

was questioned by Necchi (1987) and Necchi and Zucchi

(1997), as they could so easily be mistaken for spermatan-

gia or carposporangia. More recently the coexistence of

monosporangia with sexual reproductive structures (car-

pogonia and spermatangia), as well as carposporangia,

was confirmed by Carmona and Necchi (2001). They were

borne singly or in pairs on undifferentiated branches,

arising from the proximal cells of the assimilatory fila-



matangia (5.5-7.5 μm × 4.0-5.0 μm in diameter), larger

carpogonia (5.0-9.5 μm × 3.0-4.0 μm of basal diameter)

and longer (150-225 μm × 70-150 μm long) and wider

(2.5-4.5 μm × 2.0 μm in diameter) trichogynes (Table 1).

In summary, the description of specimens from the

Sai River by Suseela and Toppo (2015) is very incomplete,

whereas the data reported fit within our description of

T. indica. The specimens of T. hispida and T. okadae are

sister to our new species and are distinguished by minor

morphometric characters and sexuality (dioecious vs.

monoecious).

similatory filament length (170 μm long) is much shorter

than reported for T. siamensis (200-250 μm) (Traichai-

yaporn et al. 2008) and other species reported from In-

dia or phylogenetically related (Table 1). We presume the

measurement was taken in mistake or from young parts.

They made no reference to spermatangia, carpogonia or

carposporophytes.

Thorea indica is comparable to T. siamensis in being

dioecious and in plant size (length and diameter), me-

dulla diameter, shape and number cells of assimilatory

filaments (Table 1). However, T. indica has longer assimi-

latory filaments (380-500 μm × 200-250 μm), larger sper-

Table 1. Comparative morphological features of Thorea species closely related phylogenetically or previously reported from India

Taxonomic characters T. hispida T. siamensis T. siamensis T. okadae T. indica

Sexuality Monoecious Dioecious - Monoecious DioeciousPlant size

Length (cm) Up to 30 8-15 4-12 Up to 300 4-12Diameter (μm) 1,900-5,000 800 - 2,100-4,000 500-1,250Medula diameter (μm) 150-445 250 200-400 - 150-400

Short assimilatory filaments 　　　　　

No. of cells - - - 3-6 4-8Length (μm) - - - <150 25-45Cell diameter (μm) - - - 5.0-18.0 5.0-8.0

Long assimilatory filaments 　　　　　

Shape - Not clavate Clavate Not clavate Not clavateNo. of cells 13-30 10-18 11-14 10-21 10-22Length (μm) - 200-250 170 Up to 400 380-500Cell diameter (μm) - 2.2-5.7 - 8.0-12.0 4.5-7.5

Spermatangia 　　　　　

Shape - Elliptical - Ovoid Obovoid or ellipticalSize

Length (μm) - 8.0-10.0 - 10.0-13.0 8.5-12.0Diameter (μm) - 4.0-5.0 - 5.0-6.0 5.0-7.5Carpogonia basal diameter (μm) - 3.0-4.0 - 6.0-7.0 5.0-9.5

Trichogyne 　　　　　

Diameter (μm) - 2.0 - 3-4 2.5-4.5Length (μm) - 70-150 - 160-350 150-225

Carposporangia 　　　　　

Shape - Club-shaped, obovoid - Obovoid Not observedSize

Length (μm) - 16-19 - 10-26 -Diameter (μm) - 8-9 - 7-18 -

Monosporangia 　　　　　

Shape Ovoid Not observed - Obovoid Not observedSize

Length (μm) (4-9) 15-26 - - 8-16 -Diameter (μm) (12-13) 16-23 - - 6-12 -

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ACKNOWLEDGEMENTS

Necchi and Paiano are grateful to Brazilian agencies

CNPq (487566/2012-2 and 306047/2013-6) and FAPESP

(2012/12016-6 and 2013/06436-5) for support by grants

and scholarship. West thanks the Geoff McFadden Labo-

ratory, School of Biosciences 2, University of Melbourne

for long term use of facilities and supplies to continue this

and other research projects. Authors are grateful to M. Su-

seela and K. Toppo for sending specimens.

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Appendix 1. Information on GenBank sequences used in this study

Accession No. Species Location Reference Molecular marker

KC130141 Thorea sp. Blue Lake, South Australia, Australia

Scott et al. (2013) cox1

KC596320 Thorea hispida Oahu, Hawaii Carlile and Sherwood (2013) cox1

KC511076 Thorea hispida locality not specified, China Ji et al. (2014) cox1

GQ368882 Kumanoa abilii Route MG-010, Serra do Cipó National Park, MG, Brazil

Necchi et al. (2010b) rbcL

AB159658 Nemalionopsis shawii locality not specified, Japan Hanyuda et al. unpublished rbcL

AF506266 Nemalionopsis shawii Lower Barton Creed, Wake Co., NC, USA

Müller et al. (2002) rbcL

KC596164 Nemalionopsis shawii Maui, Hawaii Carlile and Sherwood (2013) rbcL

KF557550 Nemalionopsis shawii Indonesia Johnston et al. (2014) rbcL

EF116878 Nemalionopsis sp. ‘Chantransia pygmaea’

Maui, Hawaii Chiasson et al. (2005) rbcL

EF116879 Nemalionopsis sp. ‘Chantransia pygmaea’

Maui, Hawaii Chiasson et al. (2005) rbcL

AB159659 Nemalionopsis tortuosa Locality not specified, Japan Hanyuda et al. unpublished rbcL

AF506267 Nemalionopsis tortuosa Hanabusa at Kikuchi City, Kumamota Prefecture, Japan


GU953247 Thorea bachmannii Tributary of Sorocaba River, Jumirim, SP, Brazil

Necchi et al. (2010a) rbcL

AB159649 Thorea gaudichaudii Kucha-ga, Yonashiro Cho, Okinawa Prefecture, Japan

Hanyuda et al. unpublished rbcL

AB159650 Thorea gaudichaudii Nugusuky-ga, Okinawa, Japan Hanyuda et al. unpublished rbcL

AB159651 Thorea gaudichaudii Kawasan River, Cebu Island, Philippines


AB159653 Thorea hispida Locality not specified, Japan Hanyuda et al. unpublished rbcL

AF506270 Thorea hispida River Thames, Cookham, England


GU169076 Thorea hispida ‘Chantransia’ stage

West-central Ohio, USA Fuelling et al. (2012) rbcL

KC511078 Thorea hispida Locality not specified, China Ji et al. (2014) rbcL

KC596169 Thorea hispida Oahu, Hawaii Carlile and Sherwood (2013) rbcL

KF746959 Thorea hispida Wulong Spring, Shanxi Province, China

Feng et al. (2015) rbcL

AB159655 Thorea okadae Yahagi-furu River, Nisio, Aichi Prefecture, Japan


AB159656 Thorea riekei Locality not specified, USA Hanyuda et al. unpublished rbcL

KC596168 Thorea sp. Kauai, Hawaii Carlile and Sherwood (2013) rbcL

AB159657 Thorea violacea Locality not specified, USA Hanyuda et al. unpublished rbcL

AF029160 Thorea violacea San Marcos River, San Marcos, TX, USA

Vis et al. (1998) rbcL

AF506268 Thorea violacea Hudson River, Saratoga Co., NY, USA


AF506269 Thorea violacea Kikuchi River, Yamaga, Kumamota Prefecture, Japan


AF506271 Thorea violacea Near San Cristobal, Dominican Republic


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