Thorea Indica Sp. Nov. (Thoreales, Rhodophyta) from Uttar Pradesh, India

Research Article

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

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, Brazil 2School of Biosciences 2, University of Melbourne, Parkville, VIC 3010, Australia 3Instituto Oceanográfico, Universidad de Oriente, Cumaná 6101, Venezuela 411 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 ofThorea . 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 ofT. 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- two genera, Nemalionopsis and Thorea (Starmach 1977, guished from the closely related Batrachospermales by Sheath et al. 1993, Müller et al. 2002). These two genera its multiaxial rather than uniaxial gametophytes (Müller of the Thoreaceae are distinguished by the positioning of et al. 2002). The thallus consists of a central medulla of the reproductive structures (sporangia) and arrangement colorless filaments surrounded by determinate assimi- of the assimilatory filaments in the outer region (Sheath latory filaments. The order is monotypic and includes et al. 1993, Necchi and Zucchi 1997, Entwisle and Foard

This is an Open Access article distributed under the Received September 18, 2015, Accepted October 20, 2015 terms of the Creative Commons Attribution Non-Com- Corresponding Author mercial License (http://creativecommons.org/licenses/by-nc/3.0/) which * permits unrestricted non-commercial use, distribution, and reproduction E-mail: [email protected] in any medium, provided the original work is properly cited. Tel: +55-17-3221-2406, Fax: +55-17-3221-2374 aPresent address: 3-A Srinivas Terrace, 52, II Main Road, Gandhi Nagar, Adyar, Chennai, 600020, Tamil Nadu, India

1999, Carmona and Necchi 2001): assimilatory filaments T. violacea in distinct geographic origins. Necchi et al. are densely arranged and sporangia are located on outer (2010a) found that Thorea had four major clades based on assimilatory filament inNemalionopsis , whereas in Tho- sequences of rbcL and SSU rDNA, each one representing rea the filaments are loosely arranged and sporangia are a distinct species: 1) T. gaudichaudii from Asia (Japan and located in the inner assimilatory filaments. Sexual repro- Philippines); 2) T. violacea from Asia (Japan) and North duction and carposporophytes have been described in America (USA and Dominican Republic); 3) T. hispida Thorea (Yoshizaki 1986, Necchi 1987, Sheath et al. 1993, from Europe (England) and Asia (Japan); 4) T. bachman- Necchi and Zucchi 1997, Entwisle and Foard 1999, Car- nii from South America (Brazil). In addition, they pointed mona and Necchi 2001) and carpogonia and putative out that Thorea species recognized by molecular data re- spermatangia were only recently reported in Nemalion- quire additional characters (e.g., reproductive details and opsis from Indonesia (Johnston et al. 2014). chromosome numbers) to allow consistent and reliable Thorea species have been reported in several regions taxonomic circumscriptions. of the world but tend to be more common in tropical, Singh (1960) reported briefly on the occurrence ofT. subtropical and warm temperate areas with hard waters hispida (= T. ramosissima) for the first time from North (Sheath and Hambrook 1990, Sheath et al. 1993, Necchi India. Khan (1978) added some details on thallus anato- et al. 1999, Carmona and Necchi 2001). Taxonomic char- my and noted the presence of a single monosporangium, acters used for species delineation are mostly vegetative which Desikachary et al. (1990) doubted. Bhatnagar and features (Sheath et al. 1993, Carmona and Necchi 2001, Chaturvedi (1987) made some important observations on Necchi et al. 2010a): plant and medulla diameter, branch- the sexual apparatus of T. hispida (as T. ramosissima). Im- ing frequency (abundant or sparse secondary branches), portantly they stated clearly that ‘spermatangia and car- size and shape of assimilatory filaments (clavate or non- pogonia are found to form on the same plant, i.e., their clavate), size and shape of sporangia. However there is specimens were bisexual or monoecious. Dhanalakshmi considerable overlap in these characters, causing prob- et al. (2009) studied T. hispida (as T. ramosissima) collect- lems in species identification (Carmona and Necchi 2001, ed from a waterfall in the Andaman and Nicobar islands, Necchi et al. 2010a). Characteristics of sexual reproduc- but did not specify whether their plants were uni- or bi- tion (carpogonia and spermatangia) and carposporo- sexual. A noteworthy collection of T. hispida (as T. ramo- phytes may provide a more reliable set of diagnostic char- sissima) is from a land locked lake at a high altitude (4,266 acters but as yet these features are poorly documented for m) in Ladakh, Indian Himalayas (Bhat et al. 2011). Suseela most species. and Toppo (2015) reported T. siamensis from the Sai River, Guiry and Guiry (2015) list 12 accepted species of Tho- Uttar Pradesh, India among other three freshwater red al- rea. Twenty-two years ago Sheath et al. (1993) recognized gal species. Feng et al. (2015) analyzed T. hispida from two only four species of Thorea worldwide: T. clavata Seto et cool temperate sites in Shanxi, China based on sequences Ratnasabapathy, T. hispida (Thore) Desvaux, T. violacea of two genes (rbcL and SSU rDNA). Bory, and T. zollingeri Schmitz. Since then T. siamensis To identify the newly collected material of Thorea re- Kumano & Traichaiyaporn (Traichaiyaporn et al. 2008) quired morphological and molecular comparisons with from Thailand, T. conturba Entwisle & Foard (1999) from not only Indian collections (where possible) but with Australia, and six species (including some taxa consid- those from elsewhere in the world. 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 MATERIALS AND METHODS Ratnasabapathy et Seto, and T. riekei Bischoff. Studies using molecular data are still inadequate for Thorea specimens were collected by K. Toppo (collec- the genus. Müller et al. (2002) found the following se- tion No. SAI 201102) on March 13, April 3, and April 24, quence divergence among species of Thorea for two mo- 2014 from the Sai River, Uttar Pradesh, India (26°39′00.7″ lecular markers: 5.4-13.3% for rbcL (plastid gene encod- N, 80°47′38.3″ E) at an elevation of 106 m. For morpholog- ing the large RuBisCO subunit) and 2.5-13.3% for small ical studies, the materials were preserved in 4% formalin subunit (SSU) rDNA (nuclear gene encoding the large ri- with voucher specimens mounted on herbarium paper bosomal subunit). They recognized four species based on and lodged at Herbaria MEL, MICH, and SJRP (herbarium these two markers, which could not be properly named acronyms follow Thiers 2015). Specimens for DNA anal- because it was impossible to distinguish T. hispida from ysis were blotted dry with tissue and preserved in silica

http://dx.doi.org/10.4490/algae.2015.30.4.265 266 Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)

desiccant. Morphological analyses were carried out for all determined as the best-fit model of sequence evolution characters previously used as diagnostic in relevant stud- by the Akaike Information Criterion using jModelTest ies of the genus (Yoshizaki 1986, Necchi 1987, Sheath et al. 2.1.4 (Darriba et al. 2012). Maximum-likelihood (ML) 1993, Necchi and Zucchi 1997, Kumano 2002). As a rule, topologies and bootstrap values from 10,000 replicates twenty measurements or counts were taken for each char- were inferred using RAxMLGUI (Silvestro and Michalak acter in each sample (Necchi and Zucchi 1997). For mi- 2012) and bayesian analysis (BA) was performed using croscopic observations mounting media including stains MrBayes 3.2 (Huelsenbeck and Ronquist 2001) as plugin were prepared as follows: 1) 0.02% aniline blue WS in 50% for Geneious. BA consisted of three runs of five chains corn syrup and 0.5% phenol (Ganesan et al. 2015), speci- of Metropolis coupled Markov Chain Monte Carlo for 10 mens added for 30 min, removing water carefully with × 106 generations. The first 500 trees were discarded as blotting paper, adding a drop of mounting medium and burn-in. arranging the specimens with a fine needle and forceps The cox1 marker sequence was compared to other red tip; 2) a double staining of 1% aqueous methylene blue algal barcodes available in databases: the barcode of life (Grimstone and Skaer 1972) and 1% alcian blue (Sheath data systems (BOLD) database (Ratnasingham and He- and Cole 1990) by placing small pieces for 10 min in each bert 2007) and GenBank (Benson et al. 2013). solution. Surface views, cross and longitudinal sections were prepared on microscope slides after staining. Photo- graphs were taken with Canon G3 camera (Canon, Tokyo, RESULTS Japan) adapted to a Zeiss binocular research compound microscope (Carl Zeiss, Jena, Germany) and a Leica DFC Molecular analyses 320 digital camera with a LAS capture and image analysis software coupled to a Leica DM 5000 microscope (Leica Comparison of the cox1 barcode sequence from our In- Microsystems, Wetzlar, Germany). dian specimen with those in GenBank database returned For DNA extraction, samples were ground with a Pre- the following close matches (Appendix 1): 90.8% with cellys 24 tissue homogeneizer (Bertin Technologies, Mon- Thorea sp. (KC130141) (Scott et al. 2013) from Australia; tigny-le-Bretonneux, France), followed by DNA extrac- 90.4% with T. hispida from Hawaii (KC596320) (Carlile tion using NucleoSpin plant II mini kit (Macherey-Nagel, and Sherwood 2013) and from China (KC511076) (un- Duren,̈ Germany). A 1,282 bp fragment of the plastid-en- published). BOLD database returned no close matches coded ribulose-1,5-bisphosphate carboxylase–oxygenase (>85%). These results indicate that the sequence from the large-subunit gene (rbcL) was polymerase chain reaction Indian specimen belongs to a species quite distinct from (PCR) amplified using the set of primers specified by Vis any previously sequenced samples. et al. (1998). The 664 bp barcode region near the 5′ end of The rbcL gene sequence from India was compared with the cox1 gene was PCR amplified using the M13 F and R 18 previously published sequences of Thorea obtained primers (Saunders and Moore 2013). PCR reactions were from GenBank (Appendix 1). BA and ML analyses of the tested for the two markers as follows: 25 μL Top Taq Mas- rbcL alignment yielded similar trees, showing strong ter Mix (Qiagen GmbH, Hilden, Germany), 2.0 μL each support for Nemalionopsis and Thorea as monophyletic primer, 4.0 μL dH2O, and 2.0 μL extracted DNA. The PCR groups (Fig. 1). Within the genus Thorea nine clades were protocols for the rbcL and cox1 barcode region gene fol- revealed, that we interpreted as different species. The lowed Vis et al. (1998) and Saunders and Moore (2013), sequence of Indian specimen was positioned in a major respectively. PCR products were purified using the Nu- clade with high support containing two other species: T. cleoSpin Extract II (MN, Macherey-Nagel) PCR clean up okadae from Japan and T. hispida from continental USA, or Gel Extraction. Sequencing was performed using the Hawaii, the UK, and China. The divergences among these amplification primers for both markers, and in addition sequences were as follows: India vs. T. okadae (2.8%, 35 the internal rbcL primers R897 and F650 (Vis et al. 1998). bp); India vs. T. hispida (2.9-3.4%, 37-44 bp). The two Sequencing reactions were run using the ABI PRISM Big samples of T. okadae are identical and the reference of Dye v3.1 Terminator Cycle Sequencing Ready Reaction sequence AF506269 as T. violacea (Appendix 1) was prob- kit and the ABI PRISM 3130xl Genetic Analyzer (Applied ably a misidentification. These divergence levels are high Biosystems, Foster City, CA, USA). Sequence alignments enough to consider the Indian specimen as a distinct spe- were assembled in Geneious 7 (Kearse et al. 2012). For cies that we describe below. phylogenetic analyses of the rbcL data a GTR + I + G was

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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.

Thorea indica Necchi, E. K. Ganesan et J. A. West ments in an outer whorl at right angle to the main shoot. sp. nov. (Fig. 2A-I) Short assimilatory filaments 25-45 µm long, composed of 4-8, short barrel-shaped to cylindrical cells, 5-8 µm diam- Diagnosis. Plants dioecious, moderately mucilaginous, eter; long assimilatory filaments, sparsely branched, with 4-12 cm in length, dark brown to brown-greenish, mod- 10-22 cylindrical cells of uniform diameter (4.5-7.5 µm) erately to abundantly branched, multiaxial, attached to along each filament length, 380-500 µm in length. Each substrata by discoid holdfasts. Male plants slender and cell appears to have a single multi-lobed peripheral chlo- abundantly branched, 500-900 µm in diameter; female roplast. Spermatangia arising from the short assimilatory plants larger and moderately branched, 700-1,250 µm in filaments, usually developing in clusters or less often in diameter. Thallus structure consisting of a central medul- two, obovoid or elliptical, 8.5-12.0 µm in length, 5.0-7.5 la, with interwoven colorless branched and twisted fila- µm in diameter. Carpogonia bottle-shaped or ovoid, 5.0- ments with short often irregularly shaped cells. Medulla 9.5 µm in diameter, inserted directly on the basal cell of 150-400 µm in diameter. Outer medulla a ring of long par- assimilatory filaments or on one discoid or barrel-shaped allel filaments, with irregularly sized cells 2-4 µm in diam- cell; trichogynes elongate and filiform, 2.5-4.5 µm in di- eter. Cortex composed of short and long assimilatory fila- ameter and 150-225 µm in length. Monosporangia, car-

http://dx.doi.org/10.4490/algae.2015.30.4.265 268 Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)

A B C

D E F

G H I

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.

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posporangia and ‘Chantransia’ stage not observed. ments. Although monosporangia are produced terminal- Holotype. India, Uttar Pradesh, Sai River, coll. K. Top- ly at the bases of assimilatory filaments, they have been po, Mar 13, 2014 (MEL2389295); isotype SJRP 31508. reported at the apex of the long assimilatory filaments Habitat. Moderately flowing, clear stream; alga grow- (Entwisle and Foard 1999, Xie and Shi 2003). The former ing at depth of 1-2.5 ft, attached to submerged stones. authors termed them as archeospores, as defined by Nel- son et al. (1999). Necchi et al. (2010a) found that four species of Thorea DISCUSSION could be recognized based on sequences of rbcL and SSU rDNA: 1) T. gaudichaudii from Asia (Japan and Philip- The two genera in the Thoreales can be separated on pines); 2) T. violacea from Asia (Japan) and North America reliable morphological characters, but species of Thorea (USA and Dominican Republic); 3) T. hispida from Europe are hardly distinguishable on the basis of morphologi- (England) and Asia (Japan); 4) T. bachmannii from South cal features, as pointed out in previous studies (Carmona America (Brazil). They pointed out that T. okadae from and Necchi 2001, Necchi et al. 2010a). Wide overlap of Japan (Hanyuda et al. unpublished data) did not form morphological and morphometric characters are usually a separate branch, but grouped together with samples observed among species, leading to difficulties for spe- from Japan and England (Müller et al. 2002). Thus, they cies circumscriptions. Plant and medulla diameter are proposed that T. okadae should be treated as a synonym largely used but are not reliable criteria to differentiate of T. hispida and not of T. violacea. However, in this study species as Thorea is apparently seasonal in tropical and we found that T. okadae forms a quite distinct clade, sister temperate regions with dimensions changing according of the new species T. indica (Table 1, Fig. 1). Both formed to the season (see Simić et al. 2014). Branching pattern, a sister clade of T. hispida from the UK, the continental i.e., abundant vs. sparse secondary branches, has also USA and Hawaii. In summary, nine species can be recog- been demonstrated to be unreliable for species delinea- nized from molecular data: T. bachmannii from Brazil, T. tion (Carmona and Necchi 2001). One of the few vegeta- gaudichaudii (from Japan and the Philippines), T. hispida tive characters that seems to be useful for species identi- (from the UK, the continental USA, Hawaii, and China), fication is the shape of assimilatory filaments, i.e., clavate T. okadae (from Japan), T. riekei (from USA), T. violacea vs. non-clavate, the former being exclusive of T. clavata (from USA), T. indica and an undetermined species–Tho- and T. zollingeri. rea sp. from Hawaii (Carlile and Sherwood 2013) and the Monosporangia, spermatangia, carpogonia, and car- continental USA reported as T. violacea (AF506268) (Mül- posporophytes can be potentially valuable as additional ler et al. 2002). A clear geographic distribution is evident diagnostic characters for species identification in the for most Thorea species based on molecular data. This genus. However, detailed descriptions of these charac- could be used as an additional criterion to distinguish ters have been poorly documented in most studies. In them, considering the little value of the currently used addition, monosporangia are probably misinterpreted morphological characters discussed above. by some authors as spermatangia or carposporangia. The comparison with previous reports from India Monosporangia and carposporangia frequently have (Table 1) is not conclusive, mostly due to the inadequate similar shape (obovoid to elliptical) and overlapping size descriptions provided and lack of molecular evidence. ranges, thus care should be taken to distinguish between The specimens previously reported as T. hispida fit within these two. Monosporangia can be easily differentiated the circumscription of T. indica and we recommend their from spermatangia by their granular content and larger tentatively placement in this species. The report of T. sia- size (Carmona and Necchi 2001). The existence of mono- mensis by Suseela and Toppo (2015) from the same local- sporangia in Thorea as the sole means of reproduction ity as T. indica has no morphological basis, either in com- was questioned by Necchi (1987) and Necchi and Zucchi parison with the original description (Traichaiyaporn (1997), as they could so easily be mistaken for spermatan- et al. 2008) or data from this study (Table 1). Relevant gia or carposporangia. More recently the coexistence of vegetative characters were not presented, particularly monosporangia with sexual reproductive structures (car- plant diameter and short assimilatory filaments. They pogonia and spermatangia), as well as carposporangia, described assimilatory filaments as being clavate (cell di- was confirmed by Carmona and Necchi (2001). They were ameter enlarging from proximal to distal part), which is borne singly or in pairs on undifferentiated branches, exclusive of T. clavata and T. zollingeri, and quite differ- arising from the proximal cells of the assimilatory fila- ent of T. siamensis. In addition, the reported value of as-

http://dx.doi.org/10.4490/algae.2015.30.4.265 270 Necchi et al. Thorea indica sp. nov. (Thoreales, Rhodophyta)

similatory filament length (170 μm long) is much shorter matangia (5.5-7.5 µm × 4.0-5.0 µm in diameter), larger than reported for T. siamensis (200-250 μm) (Traichai- carpogonia (5.0-9.5 µm × 3.0-4.0 µm of basal diameter) yaporn et al. 2008) and other species reported from In- and longer (150-225 µm × 70-150 µm long) and wider dia or phylogenetically related (Table 1). We presume the (2.5-4.5 µm × 2.0 µm in diameter) trichogynes (Table 1). measurement was taken in mistake or from young parts. In summary, the description of specimens from the They made no reference to spermatangia, carpogonia or Sai River by Suseela and Toppo (2015) is very incomplete, carposporophytes. whereas the data reported fit within our description of Thorea indica is comparable to T. siamensis in being T. indica. The specimens of T. hispida and T. okadae are dioecious and in plant size (length and diameter), me- sister to our new species and are distinguished by minor dulla diameter, shape and number cells of assimilatory morphometric characters and sexuality (dioecious vs. filaments (Table 1). However, T. indica has longer assimi- monoecious). 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 Dioecious Plant size Length (cm) Up to 30 8-15 4-12 Up to 300 4-12 Diameter (µm) 1,900-5,000 800 - 2,100-4,000 500-1,250 Medula diameter (µm) 150-445 250 200-400 - 150-400 Short assimilatory filaments No. of cells - - - 3-6 4-8 Length (µm) - - - <150 25-45 Cell diameter (µm) - - - 5.0-18.0 5.0-8.0 Long assimilatory filaments Shape - Not clavate Clavate Not clavate Not clavate No. of cells 13-30 10-18 11-14 10-21 10-22 Length (µm) - 200-250 170 Up to 400 380-500 Cell diameter (µm) - 2.2-5.7 - 8.0-12.0 4.5-7.5 Spermatangia Shape - Elliptical - Ovoid Obovoid or elliptical Size Length (µm) - 8.0-10.0 - 10.0-13.0 8.5-12.0 Diameter (µm) - 4.0-5.0 - 5.0-6.0 5.0-7.5 Carpogonia basal diameter (µm) - 3.0-4.0 - 6.0-7.0 5.0-9.5 Trichogyne Diameter (µm) - 2.0 - 3-4 2.5-4.5 Length (µm) - 70-150 - 160-350 150-225 Carposporangia Shape - Club-shaped, obovoid - Obovoid Not observed Size Length (µm) - 16-19 - 10-26 - Diameter (µm) - 8-9 - 7-18 - Monosporangia Shape Ovoid Not observed - Obovoid Not observed Size Length (µm) (4-9) 15-26 - - 8-16 - Diameter (µm) (12-13) 16-23 - - 6-12 - References Bhatnagar and Traichayaporn Suseela and Yoshizaki (1986), This study Chaturvedy et al. (2008) Toppo (2015) Kumano (2002) (1987), Dhanalakshmi et al. (2009)

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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, Scott et al. (2013) cox1 Australia 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 Necchi et al. (2010b) rbcL National Park, MG, Brazil AB159658 Nemalionopsis shawii locality not specified, Japan Hanyuda et al. unpublished rbcL AF506266 Nemalionopsis shawii Lower Barton Creed, Müller et al. (2002) rbcL Wake Co., NC, USA KC596164 Nemalionopsis shawii Maui, Hawaii Carlile and Sherwood (2013) rbcL KF557550 Nemalionopsis shawii Indonesia Johnston et al. (2014) rbcL EF116878 Nemalionopsis sp. Maui, Hawaii Chiasson et al. (2005) rbcL ‘Chantransia pygmaea’ EF116879 Nemalionopsis sp. Maui, Hawaii Chiasson et al. (2005) rbcL ‘Chantransia pygmaea’ AB159659 Nemalionopsis tortuosa Locality not specified, Japan Hanyuda et al. unpublished rbcL AF506267 Nemalionopsis tortuosa Hanabusa at Kikuchi City, Müller et al. (2002) rbcL Kumamota Prefecture, Japan GU953247 Thorea bachmannii Tributary of Sorocaba River, Necchi et al. (2010a) rbcL Jumirim, SP, Brazil AB159649 Thorea gaudichaudii Kucha-ga, Yonashiro Cho, Hanyuda et al. unpublished rbcL Okinawa Prefecture, Japan AB159650 Thorea gaudichaudii Nugusuky-ga, Okinawa, Japan Hanyuda et al. unpublished rbcL AB159651 Thorea gaudichaudii Kawasan River, Cebu Island, Hanyuda et al. unpublished rbcL Philippines AB159653 Thorea hispida Locality not specified, Japan Hanyuda et al. unpublished rbcL AF506270 Thorea hispida River Thames, Cookham, Müller et al. (2002) rbcL England GU169076 Thorea hispida West-central Ohio, USA Fuelling et al. (2012) rbcL ‘Chantransia’ stage 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, Feng et al. (2015) rbcL China AB159655 Thorea okadae Yahagi-furu River, Nisio, Hanyuda et al. unpublished rbcL 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, Vis et al. (1998) rbcL TX, USA AF506268 Thorea violacea Hudson River, Saratoga Co., NY, Müller et al. (2002) rbcL USA AF506269 Thorea violacea Kikuchi River, Yamaga, Müller et al. (2002) rbcL Kumamota Prefecture, Japan AF506271 Thorea violacea Near San Cristobal, Müller et al. (2002) rbcL Dominican Republic

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