Algae 2015, 30(4): 265-274http://dx.doi.org/10.4490/algae.2015.30.4.265
Open Access
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
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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.
Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)
269 http://e-algae.kr
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
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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-
Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)
271 http://e-algae.kr
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
Müller et al. (2002) rbcL
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
Hanyuda et al. unpublished rbcL
AB159653 Thorea hispida Locality not specified, Japan Hanyuda et al. unpublished rbcL
AF506270 Thorea hispida River Thames, Cookham, England
Müller et al. (2002) rbcL
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
Hanyuda et al. unpublished rbcL
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
Müller et al. (2002) rbcL
AF506269 Thorea violacea Kikuchi River, Yamaga, Kumamota Prefecture, Japan
Müller et al. (2002) rbcL
AF506271 Thorea violacea Near San Cristobal, Dominican Republic
Müller et al. (2002) rbcL