A new preferentially outcrossing monoicous species of Volvox sect. Volvox (Chlorophyta) from Thailand.

Volvox sect. Volvox is an interesting group of green algae; it comprises mostly monoicous species, but evidence suggests an evolution towards dioicy. Based on cultured strains originating from Thailand, we describe Volvox longispiniferus, a novel species in Volvox sect. Volvox. This species is distinguished from others in the section by the large number of sperm packets in its monoicous sexual spheroids and by the long spines on its zygote wall. Phylogenetic analyses indicate that V. longispiniferus is distinct from the other species of two monophyletic groups within Volvox sect. Volvox. In addition, the novel species produces more zygotes when different cultures are combined compared with a single culture, suggesting a preference for outcrossing.

Introduction

Sex is recognized in various eukaryotic lineages and contributes to the mixing of genomes between two individuals, normally designated as male or female based on the production of sperm or eggs, respectively. In some invertebrates, flowering plants, fungi, and algae, both sperm and eggs are produced by the same individual; this is known as “hermaphroditism”, “monoecism”, “monoicy”, or “homothallism” [1-3]. Monoecism in flowering plants has been of interest to evolutionary biologists since the time of Charles Darwin, as self-fertilization rapidly leads to inbreeding depression [4].

Within the green algal genus Volvox, Volvox sect. Volvox exhibits interesting sexual features, with seven monoicous and three dioicous species [5-7], and ancestral state reconstruction suggests its evolution from monoicy toward dioicy [3]. Recently, Hanschen et al. [8] studied a dioicous species of Volvox sect. Volvox (V. perglobator) and further explored the evolution of dioicy in this section.

During field surveys in Thailand, WM collected water samples from which strains of Volvox sect. Volvox were cultured. Under experimental culture conditions, we induced the production of monoicous sexual spheroids, which in turn produce zygotes. Intriguingly, this species exhibited an apparent preference for outcrossing. Based on morphological characteristics and molecular phylogeny, the strains were identified as a novel species, Volvox longispiniferus Nozaki & Mahakham sp. nov. Here we describe the morphology, phylogeny, taxonomy, and outcrossing preference of this species.

Materials and methods

Ethics statement

WM collected colonial volvocine green algae from the water column in a marsh in Thailand. Collection was undertaken in accordance with the Plant Variety Protection Act, B.E. 2542 (1999), Section 53, Department of Agriculture, Thailand, which addresses the collection of plants for research, study, or experimentation for non-commercial purposes. Analysis of Thai materials was conducted in accordance with a Memorandum of Understanding between the University of Tokyo and Khon Kaen University for international cooperative research on the systematics, phylogenetics, and evolution of freshwater green algae in Thailand (2017–2021). A Material Transfer Agreement was arranged between Khon Kaen University and the University of Tokyo, with WM as the provider scientist and HN as the recipient scientist.

Establishment of cultures and morphological observations

Water samples (pH 6.85; temperature 30.5°C) were collected from a marsh in Nong Ya Ma, Yang Talat District, Kalasin Province, Thailand (16° 28ʹ 14.55ʹʹ N, 103° 16ʹ 25.55ʹʹ E), on 1 November 2019 (Table 1). Clonal cultures of V. longispiniferus (strains 1101-NZ-3, 1101-NZ-4, 1101-NZ-5, 1101-NZ-14, and 1101-RM-5) were established from the water sample using the pipette washing method [9]. Cultures were grown in 18 × 150 mm screw-cap tubes containing 10–11 mL artificial freshwater-6 (AF-6) or Volvox thiamin acetate (VTAC) medium [10] at 25°C on a 14:10 hour light: dark (L:D) schedule, under cool-white fluorescent lamps (with color temperature of 5000 K) at an intensity of 80–130 μmol m−2 s−1. They were at first maintained in AF-6 medium. For observing morphological details, possible bacterial contamination was removed from the cultures by picking up a young spheroid still within the parental spheroid and washing the young spheroid several times with fresh medium using a micropipette; the young spheroid was then grown in 10–11 mL VTAC medium. Asexual spheroids were observed in actively growing cultures in VTAC medium, as described previously [11]. To induce production of sexual spheroids, 0.3–0.8 mL actively growing culture in VTAC medium (the volume depending upon the number and density of the spheroids in the culture) was inoculated with 10–11 mL urea soil Volvox thiamine (USVT) medium [12]. This culture was grown at 20°C on a 14:10 hour L:D schedule under cool-white fluorescent lamps at an intensity of 50–70 μmol m−2 s−1. Sexual spheroids typically developed within 5 days. To enhance formation and maturation of zygotes, immature sexual spheroids cultured under the aforementioned conditions were transferred to fresh USVT medium and cultured at 25°C on a 14:10 hour L:D schedule. For measurements of rates of zygote formation (fertilization rates) in eggs per spheroid, three types of cultures of sexual spheroids (“sexual cultures 1–3”; see below) were prepared and grown at 25°C on a 14:10 hour L:D schedule.

Table 1

List of species/strains used in the present phylogenetic analyses (Figs 4–6).

Species	Sample/strain designation	Origin of sample/strain	DDBJ/ENA/GenBank Accession number
			ITS-1, 5.8S rDNA and ITS-2	rbcL	psbC
Volvox longispiniferus sp. nov. from Thailand	1101-NZ-3a (= NIES-4432)	Water sample collected from a marsh in Nong Ya Ma, Yang Talat District, Kalasin Province, Thailand (water temperature 30.5°C; pH 6.85; 16° 28ʹ 14.55ʹʹ N, 103° 16ʹ 25.55ʹʹ E) in 1 November 2019.	LC546055b	LC546060a	LC546065b
	1101-NZ-4a (= NIES-4433)		LC546056b	LC546061b	LC546066b
	1101-NZ-5a (= NIES-4434)		LC546057b	LC546062b	LC546067b
	1101-NZ-14a (= NIES-4435)		LC546058b	LC546063b	LC546068b
	1101-RM-5a (= NIES-4436)		LC546059b	LC546064b	LC546069b
Volvox barberi	UTEX 804	USA	AB663341	D86835	AB044477
Volvox capensis	NIES-3874	USA	LC034074	LC033870	LC033872
Volvox kirkiorum	NIES-2740	Japan	AB663324	AB663322	AB663323
Volvox ferrisii	NIES-2736	Japan	AB663336	AB663334	AB663335
Volvox globator	SAG 199.80 (= UTEX 955)	USA	AB663340	D86836	AB044478
Volvox perglobator	TucsonVspTf	USA	MG429137	KY489662	KY489659
Volvox rousseletii from South Africa	UTEX 1862 (= NIES-734)	South Africa	AB663342	D63448	AB044479
Volvox rousseletii from Japan	NIES-4336	Japan	LC493797	LC493808	LC493810
Volvox sp. Sagami	NIES-4021	Japan	LC191308	LC191316	LC191326
Colemanosphaera angeleri	NIES-3382	Japan		AB905592	AB905598
Colemanosphaera charkowiensis	NIES-3383	Japan		AB905591	AB905598
Platydorina caudata	NIES-728 (= UTEX 1658)	USA		D86828	AB044494

a Established in this study.

b Sequenced in this study.

Sexual culture 1: 1.0 mL cultures including immature sexual spheroids from two different cultures were mixed and inoculated into 10–11 mL fresh USVT medium in Petri dishes (57 × 16 mm) (60mm/Non-treated Dish, IWAKI AGC TECHNO GLASS, Shizuoka, Japan).

Sexual culture 2: 2.0 mL culture including immature sexual spheroids from a single culture was inoculated into 10–11 mL fresh USVT medium in Petri dishes (57 × 16 mm).

Sexual culture 3: a single immature sexual spheroid was isolated by a micropipette and inoculated into 0.5 mL fresh USVT medium in a tissue culture plate (MULTIWELL™ 48 well, Becton Dickinson, NJ, USA).

After 6–8 days, formation of zygotes was examined under the light microscope. For statistical analyses, the Kolmogorov-Smirnov test of normality was performed for the three data sets “sexual cultures 1–3” by Social Science Statistics https://www.socscistatistics.com/tests/kolmogorov/default.aspx (S1 Table). Student’s t-test of Social Science Statistics was subjected to pairs of the data sets because they did not differ significantly from that which is normally distributed (S1 Table).

Light microscopy was conducted using the BX60 microscope (Olympus, Tokyo, Japan) equipped with Nomarski interference optics. Spheroid cells were counted as described previously [5,13].

Molecular experiments

To infer the phylogenetic position of V. longispiniferus within Volvox sect. Volvox, we evaluated the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA ((rDNA); ITS-1, 5.8S rDNA, and ITS-2) and two chloroplast genes (the large subunit of Rubisco (rbcL) and photosystem II CP43 apoprotein (psbC) genes) as described previously [7]. Sequences were determined based on direct sequencing of polymerase chain reaction (PCR) products from a disrupted cell solution, using KOD One PCR Master Mix (Toyobo, Osaka, Japan). The PCR parameters for rbcL and psbC amplification were 2 minutes at 94°C, followed by 45 cycles of 10 seconds at 98°C, 30 seconds at 50°C, and 30 seconds at 68°C; those for rDNA ITS amplification were 2 minutes at 94°C, followed by 40 cycles of 10 seconds at 98°C, 30 seconds at 66°C, and 30 seconds at 68°C.

To assess the phylogeny of rDNA ITS regions, chloroplast genes (rbcL plus psbC), and a combined dataset from rDNA ITS regions and the two chloroplast genes, we analyzed the operational taxonomic units of the species, samples, and strains listed in Table 1. Sequences were aligned as described previously [6,12,14], and the alignments are available in TreeBASE (www.treebase.org/treebase-web/home.html; Study ID S26186). We followed previously described methods [6] for outgroup designation in the rbcL-psbC phylogeny and used the tree topology from this phylogeny to designate the root of Volvox sect. Volvox in the analyses of rDNA ITS regions and the combined dataset. Maximum-likelihood (ML) analyses based on sequence alignments from all three datasets were performed using MEGA X [15] with best-fitted models (K2+G for rDNA ITS, GTR+G for rbcL-psbC, and T92+G for the combined data set) selected by MEGA X and 1000 bootstraps [16]. In addition, Bayesian inference (BI) of the alignments was determined using MrBayes 3.2.6 [17], as described previously [14]. A partitioned analysis was implemented in BI of rbcL-psbC and the combined data set. Models for BI were SYM+G for rDNA ITS, and F81, JC, and GTR+I+G for first, second, and third codon positions of rbcL-psbC, respectively, selected by hierarchical likelihood ratio test using MrModeltest 2.3 [18].

The secondary structures of ITS-2 were predicted as described previously [6,12,14,19].

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN constitutes a published work according to the International Code of Nomenclature for algae, fungi, and plants (Article 29.1) [20]; hence, the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

Results

Asexual spheroids

Mature asexual spheroids of V. longispiniferus in the present cultures were subspherical or ovoid in shape, with 5600–8200 somatic cells embedded in individual sheaths at the periphery of the gelatinous matrix (Fig 1A–1C). Spheroids were up to 830 μm long. Somatic cells had two flagella and a cup-shaped chloroplast with a single basal pyrenoid and a single eyespot up to 10 μm long and were connected by cytoplasmic bridges (Fig 1C and 1D). Cytoplasmic bridges were thicker than the flagella (Fig 1C). The anterior somatic cells of the spheroids were laterally subspherical or trapezoidal, and the cell length was shorter than or nearly equal to the cell width (Fig 1D). In general, 7–11 gonidia, or developing embryos, were present in the posterior two-thirds of asexual spheroids (Fig 1A and 1B). Gonidia of the next generation were not evident in the developing embryo either during or immediately after inversion (Fig 1E).

Fig 1

Asexual spheroids of Volvox longispiniferus sp. nov. strain 1101-NZ-5 from Thailand.

(A, B, E) Bright-field microscopy. (C, D) Nomarski differential interference contrast microscopy. (A) Spheroid with developing embryos (d). (B) Fully matured spheroid with inverting daughter spheroids (d). (C–E) Part of spheroids. (C) Front view of somatic cells with thick cytoplasmic bridges (b) and individual sheaths (asterisks). (D) Side view of anterior somatic cells showing eyespot (e) and pyrenoid (p) in the chloroplast. (E) Surface view of compact embryo just after inversion. Note that differentiation of gonidia of the next generation is not evident.

Sexual reproduction

Induced sexual spheroids were monoicous, forming both sperm packets and eggs (Fig 2A–2C). Mature sexual spheroids were subspherical or ovoid in shape and up to 800 μm long, with 4900–7800 somatic cells arranged at the periphery. Spheroids had 25–58 eggs and 5–16 sperm packets distributed randomly within the posterior four-fifths of the spheroid. Differences in proportion of eggs and sperm packets in a sexual spheroid were not seen between isolates of V. longispiniferus. Sperm packets often swam outside the spheroid and attached to the surface of the sexual spheroid; packets then gradually dissociated to become individual sperm that penetrated the spheroid, potentially for fertilization. Irrespective of the presence or absence of fertilization, eggs matured nearly simultaneously within a single sexual spheroid (Fig 2D). Mature zygotes were reddish–brown in color with a spiny cell wall (Fig 2E), but some mature eggs did not secret a cell wall possibly due to the lack of fertilization. Fully developed spines were 12–14 μm long and were straight or slightly curved, with an acute apex (Fig 2E). Zygotes were 40–48 μm in diameter, excluding spines. Hatching of the zygotes was not observed.

Fig 2

Sexual reproduction of Volvox longispiniferus sp. nov. from Thailand.

(A, B, D, E) Bright-field microscopy. (C) Nomarski differential interference contrast microscopy. (A) Young monoicous spheroid showing eggs (eg) and sperm packets (sp). Strain 1101-NZ-4. (B) Sperm packet in monoicous spheroid. Strain 1101-NZ-4. (C) Egg in monoicous spheroid. Strain 1101-NZ-4. (D) Sexual spheroid with matured zygotes. Strains 1101-NZ-4 x 1101-NZ-5. (E) Matured zygotes with long, acute spines on zygote walls. Strains 1101-NZ-4 x 1101-NZ-5.

Under the sexual induction conditions used here, abundant zygotes were formed when the two different cultures including immature sexual spheroids were mixed and grown in USVT medium at 25°C. More than 50% of eggs in sexual spheroids developed into matured zygotes in such mixed culture when the strains were newly established (three months after the establishment; Figs 2D and 3A–3C). However, ratios of zygote formation in sexual spheroids decreased during the maintenance of cultures (four months after the establishment).

Fig 3

Comparison of development of potential zygotes (walled cells, arrows) in sexual spheroids of Volvox longispiniferus sp. nov. between mixture of two different cultures (A–C) and a single clonal culture (D–F).

(A–C) Strains 1101-NZ-4 x 1101-NZ-5. Note more than half of eggs developing into zygotes. (A) Four days after mixture of sexually induced cultures. (B) Six days after the mixture. (C) Eight days after the mixture. (D) Eight-day-old sexual spheroid in a single culture (strain 1101-NZ-5). (E, F) Six-day-old, singly isolated sexual spheroids of strain 1101-NZ-4 (E) and strain 1101-NZ-5 (F). Note less formation of zygotes in sexual spheroids that were isolated singly.

Comparative measurements of ratios of zygote formation in eggs per sexual spheroid were carried out after four months from the strain establishment. At that time, 6–25% (average 17%) of eggs in a spheroid developed into potential zygotes (walled cells) when two different clonal cultures were mixed and grown in USVT medium for zygote formation and maturity (sexual culture 1) (S1 Table). By contrast, 0–19% (average 12%) of eggs in a spheroid developed into potential zygotes within a single sexually induced culture (sexual culture 2) (Fig 3D). When a single immature sexual spheroid was isolated and inoculated into fresh USVT medium (sexual culture 3), 3–19% (average 11%) of eggs developed into potential zygotes (Fig 3E and 3F). Based on the zygote formation rates in eggs per sexual spheroid from these three types of sexual cultures, statistical t-tests were carried out. Significant difference [corrected probability values (based on Bonferroni correction) < 0.05] in the rate was detected between sexual cultures 1 and 3 (S1 Table).

Molecular phylogeny

All five strains of V. longispiniferus exhibited identical psbC and rbcL sequences; however, the nuclear rDNA ITS region of the five strains comprised two distinct types that differed with respect to three nucleotides in the ITS-1 region. The phylogenetic positions of V. longispiniferus based on the ITS region and rbcL-psbC gene sequences are shown in Figs 4 and 5, respectively. Phylogenetic relationships within Volvox sect. Volvox were essentially identical between the two trees, and two robust sister clades were resolved, one clade composed of V. globator and V. capensis and the other (clade A) containing V. rousseletii, V. perglobator, V. sp. Sagami, V. ferrisii, V. kirkiorum, V. barberi, and V. longispiniferus. Within clade A, a large monophyletic group composed of V. rousseletii, V. perglobator, V. sp. Sagami, V. ferrisii, and V. kirkiorum was resolved, with 68–90% bootstrap values in ML and 0.99–1.00 posterior probability in BI. However, the phylogenetic positions of V. longispiniferus and V. barberi were not robustly resolved within clade A. The combined dataset demonstrated moderate support for the sister relationship between V. longispiniferus and others within clade A, with a 73% bootstrap value in ML and 0.92 posterior probability in BI (Fig 6).

Fig 4

Phylogenetic analysis of Volvox longispiniferus sp. nov. from Thailand, based on Maximum Likelihood (ML) method of the Internal Transcribed Spacer (ITS) regions of nuclear ribosomal DNA (rDNA) (ITS-1, 5.8S rDNA, and ITS-2).

Branch lengths are proportional to the evolutionary distances that are indicated by the scale bar. Numbers in left and right sides at branches represent bootstrap values (50% or more) based on 1000 replications of ML and posterior probabilities (0.90 or more) by Bayesian inference, respectively.

Fig 5

Phylogenetic analysis of Volvox longispiniferus sp. nov. from Thailand, based on Maximum Likelihood (ML) method of two chloroplast genes (rbcL and psbC).

Branch lengths are proportional to the evolutionary distances that are indicated by the scale bar. Numbers in left and right sides at branches represent bootstrap values (50% or more) based on 1000 replications of ML and posterior probabilities (0.90 or more) by Bayesian inference, respectively.

Fig 6

Phylogenetic analysis of Volvox longispiniferus sp. nov. (strains 1101-NZ-4, 1101-NZ-5 and 1101-RM-5) from Thailand, based on Maximum Likelihood (ML) method of combined data set from the Internal Transcribed Spacer (ITS) regions of nuclear ribosomal DNA (rDNA) (ITS-1, 5.8S rDNA, and ITS-2) and two chloroplast genes (rbcL and psbC).

Branch lengths are proportional to the evolutionary distances that are indicated by the scale bar. Numbers in left and right sides at branches represent bootstrap values (50% or more) based on 1000 replications of ML and posterior probabilities (0.90 or more) by Bayesian inference, respectively.

When the secondary structures of ITS-2 were compared, two compensatory base changes were recognized between V. longispiniferus and V. barberi and three between V. longispiniferus and V. perglobator (S1 and S2 Figs).

Discussion

Seven monoicous species and one monoicous morphological type had previously been recognized in Volvox sect. Volvox (S2 Table) [6]. Among these, V. longispiniferus is morphologically similar to V. merrillii by virtue of its long zygote spines (> 10 μm) and the production of asexual spheroids with more than 10 gonidia or daughter spheroids (S2 Table). However, V. longispiniferus differs from V. merrillii with respect to the number of eggs and sperm packets in monoicous sexual spheroids. Volvox merrillii typically produces sexual spheroids with 60 or more eggs and up to eight sperm packets (S2 Table). By comparison, the sexual spheroids of V. longispiniferus have 25–58 eggs and 5–16 sperm packets. In addition, sizes of zygotes in V. longispiniferus are larger than that of V. merrillii (S2 Table). Zygotes of V. longispiniferus measure 40–48 μm in diameter (without spines) whereas those of V. merrillii are 37–40 μm in diameter (S2 Table). When numbers of eggs in sexual spheroids are focused (S2 Table), V. longispiniferus (with 25–58 eggs) is similar to V. kirkiorum (with 20–80 eggs). However, V. kirkiorum has short zygote spines (< 10 μm) and pear-shaped to ovoid anterior somatic cells (S2 Table). Anterior somatic cells of V. longispiniferus are subspherical or trapezoidal in shape (Fig 1D). Therefore, V. longispiniferus is clearly distinguished from other monoicous species in Volvox sect. Volvox by the long (12–14 μm) acute spines on the zygote wall and the production of fewer than 60 eggs in monoicous spheroids (S2 Table). Although V. longispiniferus was examined in only clonal cultured materials, no clonal cultures have been established and molecular data are lacking for V. merrillii (S2 Table). V. amboensis also lacks clonal cultural studies and molecular data (S2 Table). But this species is morphologically distinct from other monoicous species in Volvox sect. Volvox in having a large sexual spheroid measuring up to 2000 μm long with usually more than 200 eggs (S2 Table). Thus, eight morphological species and one morphological type with monoicous sexual spheroids are now recognized in Volvox sect. Volvox (S2 Table). However, further cultural studies and molecular data of V. merrillii and V. amboensis are needed to resolve their detailed taxonomic and phylogenetic positions within Volvox sect. Volvox.

Our phylogenetic analyses demonstrate that V. longispiniferus is weakly to moderately separated from other species within Volvox sect. Volvox (Figs 4–6). Strictly speaking, the phylogenetic relationship between V. longispiniferus and V. barberi is not well resolved, but both species belong to the robust clade A in this section (Figs 4–6). Furthermore, V. barberi and V. perglobator represent two major lineages within the clade A excluding V. longispiniferus (Fig 6). Comparison of the secondary structures of ITS-2 rDNA demonstrated two or three compensatory base changes between V. longispiniferus and V. barberi or between V. longispiniferus and V. perglobator, respectively (S2 Fig). These values suggest enough genetic differentiation to recognize V. longispiniferus as different species within clade A. Thus, V. longispiniferus comprises a new, morphologically and phylogenetically distinct species within Volvox sect. Volvox.

Monoicous sexual spheroids in V. capensis from the United States have 2–6 sperm packets and 70–100 eggs (S2 Table). Although sperm packets in this species do not escape from the sexual spheroids, nearly all eggs in the sexual spheroids develop into spiny-walled zygotes [12]. A similar phenomenon has been observed in V. ferrisii, another monoicous species that has 3–5 sperm packets and 100–150 eggs in the monoicous spheroids (S2 Table) [14]. By comparison, the ratio of sperm packets to eggs in V. longispiniferus is relatively high (5–16 sperm packets and 25–56 eggs; S2 Table); in addition, V. longispiniferus exhibits produces more zygotes when different cultures are combined compared with a single isolated spheroid (Fig 3; S1 Table). Therefore, V. longispiniferus may preferentially outcross via fertilization by sperm from other sexual spheroids. Preferential outcrossing also occurs in flowering plants [21], in which effective fertilization in outcrossing populations of monoicous plants relies on substantial production and transfer of pollen or sperm to other individuals. Conversely, self-fertilizing individuals do not require the same abundant production of pollen or sperm because of the higher probability of fertilization within a single individual [21]. Thus, the preferential outcrossing exhibited by V. longispiniferus may be indicative of the initial stages of transition to an outcrossing monoicous species.

Taxonomic treatment

Volvox longispiniferus Nozaki & Mahakham sp. nov.

Asexual spheroids subspherical or ovoid in shape, containing 5600–8200 somatic cells embedded in individual sheaths of the gelatinous matrix, with seven to 11 gonidia or developing embryo in the posterior two-thirds of the spheroid, measuring up to 830 μm long. Cells connected by thick cytoplasmic bridges. Anterior somatic cells of the spheroid were laterally subspherical or trapezoidal. Gonidia of the next generation not evident in the developing embryo either during or immediately after inversion. Sexual spheroids monoicous, 4900–7800-celled, with 25–58 eggs and 5–16 sperm packets distributed randomly within the posterior four-fifths of the spheroid, measuring up to 830 μm long. Eggs maturing nearly simultaneously within a single sexual spheroid. Mature zygotes with a spiny cell wall, measuring 40–48 μm in diameter (excluding spines). Fully developed spines 12–14 μm long, straight or slightly curved with an acute apex.

Holotype: Fig 2A, depicting a monoicous spheroid with eggs and sperm packets of strain 1101-NZ-4. This strain is available as NIES-4433 from the Microbial Culture Collection at the National Institute for Environmental Studies, Japan [10]. The holotype of a name of this new species is an effectively published illustration since it is impossible to preserve a specimen that would show the features, especially the monoicous sexual spheroid, attributed to V. longispiniferus (Article 40.5 of International Code of Nomenclature for algae, fungi, and plants [20]).

Strains examined: 1101-NZ-3 (= NIES-4432), 1101-NZ-4 (= NIES-4433), 1101-NZ-5 (= NIES-4434), 1101-NZ-14 (= NIES-4435) and 1101-RM-5 (= NIES-4436) (Table 1).

Etymology: The species epithet “longispiniferus” meaning “with long spines” for zygote wall morphology.

Type locality: A marsh in Nong Ya Ma, Yang Talat District, Kalasin Province, Thailand (16° 28ʹ 14.55ʹʹ N, 103° 16ʹ 25.55ʹʹ E). Water samples were collected by WM on 1 November 2019.

The secondary structure of nuclear ribosomal DNA (rDNA) Internal Transcribed Spacer 2 (ITS-2) transcript of five strains of Volvox longispiniferus sp. nov., including the 3’ end of the 5.8S ribosomal RNA (rRNA) and the 5’ end of the large Subunit of rRNA (LSU rRNA).

(DOCX)

Click here for additional data file.

Comparison of helices of the secondary structure of nuclear ribosomal DNA internal transcribed spacer 2 transcripts between Volvox longispiniferus sp. nov. and its related strain/species (Figs 4–6).

(DOCX)

Click here for additional data file.

Results of zygote formation in eggs per sexual spheroid from three types of sexually induced cultures of Volvox longispiniferus sp. nov.

(DOCX)

Click here for additional data file.

Comparison of Volvox longispiniferus and previously described monoicous morphological type and species of Volvox sect. Volvox.

(DOCX)

Click here for additional data file.

4 Jun 2020

PONE-D-20-14342

A new preferentially outcrossing monoecious species of Volvox sect. Volvox (Chlorophyta) from Thailand

PLOS ONE

Dear Dr. Nozaki,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I and two experts have read and reviewed your manuscript.  All of us are in agreement that it is suitable for publication pending minor revision and adequate responses to the Reviewers' comments. If you intend to include data on out-crossing please make sure to describe the experiments adequately in the Methods and pay attention to replication and statistics needed to support your conclusions.

I have two additional minor comments.

1. Please indicate in your Methods whether the cultures were maintained axenically. If not, could the presence of microbial contaminants have affected the outcomes of the crossing experiments?

2. Terminology in the field is changing.  Although traditionally monoecy/monecious dioecy/dioecious have been used for volvocine algae, these could be replaced by monoicy/monoicous and dioicy/dioicous that are used for plants that have haploid-phase sex determination (e.g. bryophytes); while the former terms are reserved for plant species with diploid-phase sex determination. 10.1016/j.tplants.2018.06.005. It is fine if you leave your terminology as is, but wanted to bring this alternative nomenclature system to your attention.

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'W.M. gratefully acknowledge financial support from Department of Biology & Applied Taxonomic Research Center, Faculty of Science, Khon Kaen University, Thailand.'

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

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'This study was supported by a Grants-in-Aid for Scientific Research (grant numbers 16H02518 for HN and 19K22446 for HN) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI (https://www.jsps.go.jp/english/e-grants/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript'

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Reviewers' comments:

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1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review of Nozaki et al; A new preferentially outcrossing monoecious species of Volvox sect. Volvox (Chlorophyta) from Thailand.

This manuscript describes a new monoecious, or hermaphroditic, species of green algae in the Volvox section Volvox of the volvocine green algae, Volvox longispiniferus. The authors provide clear and detailed methods and results regarding both asexual and sexual lifecycles of V. longispiniferus, and provide very interesting results on the preferential outcrossing of this hermaphrodite. This is good research and forms a workhorse species description that is worth publication upon a few improvements. My only major comment is that the preferential outcrossing suggestion is not sufficiently discussed and should be expanded upon.

This experiment is very interesting, as well as the suggestion that V. longispiniferus may be on a transition towards heterothallic obligate outcrossing. For the suggestion that V. longispiniferus may be transitioning to heterthallic obligate outcrossing, please clarify whether or not differences in proportion of eggs and sperm are seen between isolates of V. longispiniferus.

However, these results, despite making up the title and much of the discussion are in the supplemental file, not the main document. The authors should move Figure S1 into the main text. Furthermore, they may have enough quantitative data regarding egg number and fertilization rates to perform a statistical test, likely t test or Mann-Whitney U test depending on whether the data is normal or not, on the differences between fertilization rates between single colony and mixed colony experiments. Lastly, for these experiments, please add to the methods section clarifying whether a similar number of colonies and density of colonies could be expected based on adding 0.3-0.8 mL of culture to the experiment.

Minor comments:

1. Hanschen et al 2018 (see below) also described a Volvox section Volvox species and further explored the evolution of dioecy in Volvox section Volvox.

2. Did the authors observe hatching of the fertilized or unfertilized zygotes?

3. Please provide (brief) justification that this species is not V amboensis or V. kirkiorum in the main text.

4. Is it possible to add known geographical distribution to Table S1?

5. Page 5, line 94, please clarify which model of evolution was used in phylogenetic analyses and if a partitioning scheme was implemented in the psbC/rbcL phylogeny.

6. Figure 1, panel D, please ensure consistency of labelling eyespot (figure) or stigma (figure legend).

Reviewer #2: This is a nice, brief paper describing a new species of Volvox with some interesting features. It is well-written, understandable, and rigorous. I have just a few concerns, all but one of which are trivial.

My one substantive concern is regarding the justification for considering the new strains a separate species from V. merrillii. The phylogenetic position of V. merrillii is unclear and genetic data are unavailable (understandably, since — I think— this species is not available for examination), so we have to make do without those lines of evidence. The only justification that is given is the number of eggs and sperm packets in sexual colonies. Table S1 indicates a few other differences, for example number of cells in asexual spheroids, shape of anterior cells, and number of cells in sexual spheroids, but these are not discussed in the main text. The reason these differences don’t convince me that V. longispiniferus and V. merrillii are distinct species is that these traits are known to vary in response to culture conditions and by the length of time the strain has been cultured. The descriptions of V. merrillii are based on two very old (but authoritative) papers, and it’s not clear whether the culture conditions and the age of the strains are similar to the present study. Dr. Nozaki is, by far, the world’s leading expert on the phylogeny and taxonomy of Volvox and its relatives, and if he says they are distinct species they probably are, but the current version of the manuscript does not sufficiently explain the justification. I realize that the unfortunate lack of V. merrillii cultures limits the information that can be obtained. I would nevertheless like to see a much expanded discussion of the differences between V. longispiniferus and V. merrillii that addresses intraspecific variation, variation in response to strain age and culture conditions, and any other factors that might inform the distinctness of the putative new species.

Minor comments:

Regarding V. merrillii, a short explanation of the reason genetic and more recent morphological data are not available would be great.

The preference for outcrossing figures prominently in the abstract, but it is barely mentioned in the main text, with no methods provided and the relevant figure relegated to a supplement. Since this is the aspect of the paper that is most likely to interest the poor benighted souls who study organisms less interesting than Volvox, I urge the authors to consider discussing the experiment in more detail and moving the figure to the main text.

Line 19: I would like to know what Charles Darwin said about inbreeding depression, but Correspondence Volume 9, at 645 pages, is a bit much to wade through in search of the passage the authors have in mind. I know page numbers are generally only provided for direct quotes, but I hope the editors will allow an exception.

23: water samples IN which strains were cultured, or water samples FROM which strains were cultured?

53: please provide the color temperature in K of the lamps.

Table 1: I love that the authors have included the NIES strain designations. This is a service to future Volvox researchers.

110: it’s not clear to me whether these numbers are based on collected or cultured spheroids. The world’s leading authority on the phylogeny and taxonomy of Volvox and its relatives once told me that spheroids in culture rarely obtain the sizes of their wild-caught sisters.

139-140: What happened to the unfertilized eggs?

223: If the branching order in Fig. 3 is correct, V. longispiniferus is equally related to all of the other species in Clade A. There is no reason to focus this comparison on V. barberi in particular, since V. ferrisii (for example) is just as closely related to V. longispiniferus.

224: Some discussion of the value of compensatory base changes in ITS-2 in distinguishing species is needed.

**********

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Reviewer #1: No

Reviewer #2: No

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11 Jun 2020

PONE-D-20-14342

A new preferentially outcrossing monoecious species of Volvox sect. Volvox (Chlorophyta) from Thailand

PLOS ONE

Dear Dr. Nozaki,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

I and two experts have read and reviewed your manuscript. All of us are in agreement that it is suitable for publication pending minor revision and adequate responses to the Reviewers' comments. If you intend to include data on out-crossing please make sure to describe the experiments adequately in the Methods and pay attention to replication and statistics needed to support your conclusions.

Response: The data have been statistically examined and detailed methods for outcrossing have been described in the revised manuscript.

I have two additional minor comments.

1. Please indicate in your Methods whether the cultures were maintained axenically. If not, could the presence of microbial contaminants have affected the outcomes of the crossing experiments?

Response: We cannot say that the cultures were maintained axenically because of lack of examination of cultures grown in bacterial media. However, the cultures used for crossing experiments (S1 Table of the revised manuscript) did not show apparent bacteria when grown in acetate-containing medium VTAC. Thus, there is little possibility that microbial contaminants have affected the outcomes of the crossing experiments. For clarifying the removal of bacteria from the original cultures, details of the culture methods have been described in the Method section of the revised manuscript.

2. Terminology in the field is changing. Although traditionally monoecy/monecious dioecy/dioecious have been used for volvocine algae, these could be replaced by monoicy/monoicous and dioicy/dioicous that are used for plants that have haploid-phase sex determination (e.g. bryophytes); while the former terms are reserved for plant species with diploid-phase sex determination. 10.1016/j.tplants.2018.06.005. It is fine if you leave your terminology as is, but wanted to bring this alternative nomenclature system to your attention.

Response: The terminology has been revised as suggested.

Please submit your revised manuscript by Jul 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Response: Done as suggested.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

'W.M. gratefully acknowledge financial support from Department of Biology & Applied Taxonomic Research Center, Faculty of Science, Khon Kaen University, Thailand.'

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

'This study was supported by a Grants-in-Aid for Scientific Research (grant numbers 16H02518 for HN and 19K22446 for HN) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI (https://www.jsps.go.jp/english/e-grants/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript'

Response: We have removed any funding-related text from the manuscript. We would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

'This study was supported by a Grants-in-Aid for Scientific Research (grant numbers 16H02518 for HN and 19K22446 for HN) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT)/Japan Society for the Promotion of Science (JSPS) KAKENHI (https://www.jsps.go.jp/english/e-grants/), and the Applied Taxonomic Research Center (grant number: ATRC KKUR6309; http://atrc.sc.kku.ac.th/), Department of Biology, Faculty of Science, Khon Kaen University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript'

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response: Done as suggested.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Response: For outcross experiments, statistical analyses by Student’s t-test based on the Kolmogorov-Smirnov test of normality have been carried out in the revised manuscript.

________________________________________

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Review of Nozaki et al; A new preferentially outcrossing monoecious species of Volvox sect. Volvox (Chlorophyta) from Thailand.

This manuscript describes a new monoecious, or hermaphroditic, species of green algae in the Volvox section Volvox of the volvocine green algae, Volvox longispiniferus. The authors provide clear and detailed methods and results regarding both asexual and sexual lifecycles of V. longispiniferus, and provide very interesting results on the preferential outcrossing of this hermaphrodite. This is good research and forms a workhorse species description that is worth publication upon a few improvements. My only major comment is that the preferential outcrossing suggestion is not sufficiently discussed and should be expanded upon.

This experiment is very interesting, as well as the suggestion that V. longispiniferus may be on a transition towards heterothallic obligate outcrossing. For the suggestion that V. longispiniferus may be transitioning to heterthallic obligate outcrossing, please clarify whether or not differences in proportion of eggs and sperm are seen between isolates of V. longispiniferus.

Response: Differences in proportion of eggs and sperm packets in a sexual spheroid were not seen between isolates of V. longispiniferus. This situation has been described in the revised manuscript.

However, these results, despite making up the title and much of the discussion are in the supplemental file, not the main document. The authors should move Figure S1 into the main text. Furthermore, they may have enough quantitative data regarding egg number and fertilization rates to perform a statistical test, likely t test or Mann-Whitney U test depending on whether the data is normal or not, on the differences between fertilization rates between single colony and mixed colony experiments.

Response: In the revised version, Figure S1 has been moved into the main text (Figure 3), and statistical tests have been performed to detect differences in fertilization rates between single spheroid and mixed culture experiments, as well as between the unmixed and mixed culture experiments. Based on the Kolmogorov-Smirnov test of normality, all of the three data sets of fertilization rates per spheroid (mixed culture, unmixed culture and single spheroid experiments) do not differ significantly from those which is normally distributed. Thus, Student’s t-tests have been carried out to detect significant difference (p < 0.05) in fertilization rates per spheroid between single colony and mixed culture experiments, as well as between the unmixed and mixed culture experiments. These experimental procedures and results have been described in the main text and S1 Table newly prepared in the revised manuscript.

Lastly, for these experiments, please add to the methods section clarifying whether a similar number of colonies and density of colonies could be expected based on adding 0.3-0.8 mL of culture to the experiment.

Response: Cultures of Volvox are very unstable in growing conditions principally due to the pre-culture. Thus, a similar number of spheroids and density of spheroids could be expected based on adding 0.3-0.8 mL of culture to the experiment by using a loupe or naked eye. The Method section has been revised to clarify this point.

Minor comments:

1. Hanschen et al 2018 (see below) also described a Volvox section Volvox species and further explored the evolution of dioecy in Volvox section Volvox.

Response: The following paper has been cited in the Introduction section in the revised manuscript.

Hanschen ER, Davison DR, Ferris　PJ, Michod RE. On the rediscovery of Volvox perglobator (Volvocales, Chlorophyceae) and the evolution of outcrossing from self-fertilization. Evol Ecol Res. 2018; 19: 299–318.

2. Did the authors observe hatching of the fertilized or unfertilized zygotes?

Response: No. This situation has been described in the revised manuscript.

3. Please provide (brief) justification that this species is not V amboensis or V. kirkiorum in the main text.

Response: Discussions regarding justification that V. longispiniferus is not V amboensis or V. kirkiorum have been described in the Discussion section of the revised manuscript.

4. Is it possible to add known geographical distribution to Table S1?

Response: Geographical distribution has been added to S2 Table in the revised manuscript.

5. Page 5, line 94, please clarify which model of evolution was used in phylogenetic analyses and if a partitioning scheme was implemented in the psbC/rbcL phylogeny.

Response: Details of ML analyses and BI, especially models used, have been described in the Method section of the revised manuscript.

6. Figure 1, panel D, please ensure consistency of labelling eyespot (figure) or stigma (figure legend).

Response: Stigma (s) has been changed eyespot (e) throughout the text.

Reviewer #2: This is a nice, brief paper describing a new species of Volvox with some interesting features. It is well-written, understandable, and rigorous. I have just a few concerns, all but one of which are trivial.

My one substantive concern is regarding the justification for considering the new strains a separate species from V. merrillii. The phylogenetic position of V. merrillii is unclear and genetic data are unavailable (understandably, since — I think— this species is not available for examination), so we have to make do without those lines of evidence. The only justification that is given is the number of eggs and sperm packets in sexual colonies. Table S1 indicates a few other differences, for example number of cells in asexual spheroids, shape of anterior cells, and number of cells in sexual spheroids, but these are not discussed in the main text. The reason these differences don’t convince me that V. longispiniferus and V. merrillii are distinct species is that these traits are known to vary in response to culture conditions and by the length of time the strain has been cultured. The descriptions of V. merrillii are based on two very old (but authoritative) papers, and it’s not clear whether the culture conditions and the age of the strains are similar to the present study. Dr. Nozaki is, by far, the world’s leading expert on the phylogeny and taxonomy of Volvox and its relatives, and if he says they are distinct species they probably are, but the current version of the manuscript does not sufficiently explain the justification. I realize that the unfortunate lack of V. merrillii cultures limits the information that can be obtained. I would nevertheless like to see a much expanded discussion of the differences between V. longispiniferus and V. merrillii that addresses intraspecific variation, variation in response to strain age and culture conditions, and any other factors that might inform the distinctness of the putative new species.

Response: We are very happy to know that the reviewer realizes that the unfortunate lack of V. merrillii cultures limits the information that can be obtained. Based on the comment, much expanded discussion of the differences between V. longispiniferus and V. merrillii has been described in the Discussion section of the revised manuscript. However, there have been no clonal culture studies of Volvox merrillii, we cannot address intraspecific variation, variation in response to strain age and culture conditions.

Minor comments:

Regarding V. merrillii, a short explanation of the reason genetic and more recent morphological data are not available would be great.

Response: Explanation of the reason genetic and more recent morphological data are not available has been described in the Discussion section of the revised manuscript.

The preference for outcrossing figures prominently in the abstract, but it is barely mentioned in the main text, with no methods provided and the relevant figure relegated to a supplement. Since this is the aspect of the paper that is most likely to interest the poor benighted souls who study organisms less interesting than Volvox, I urge the authors to consider discussing the experiment in more detail and moving the figure to the main text.

Response: In the revised version, Figure S1 has been moved into the main text (Figure 3), and statistical tests have been performed to detect differences in fertilization rates between single spheroid and mixed culture experiments, as well as between the unmixed and mixed culture experiments. Our Student’s t-tests have detected significant difference (p < 0.05) in fertilization rates per spheroid between single colony and mixed culture experiments, as well as between the unmixed and mixed culture experiments. These procedures and results have been described in the main text and S1 Table newly prepared in the revised manuscript.

Line 19: I would like to know what Charles Darwin said about inbreeding depression, but Correspondence Volume 9, at 645 pages, is a bit much to wade through in search of the passage the authors have in mind. I know page numbers are generally only provided for direct quotes, but I hope the editors will allow an exception.

Response: The citation is based on the PLoS ONE Journal style. Thus, revision is not needed.

23: water samples IN which strains were cultured, or water samples FROM which strains were cultured?

Response: Revised as suggested.

53: please provide the color temperature in K of the lamps.

Response: the color temperature in K (5000 K) has been described in the revised manuscript.

Table 1: I love that the authors have included the NIES strain designations. This is a service to future Volvox researchers.

Response: Thank you!

110: it’s not clear to me whether these numbers are based on collected or cultured spheroids. The world’s leading authority on the phylogeny and taxonomy of Volvox and its relatives once told me that spheroids in culture rarely obtain the sizes of their wild-caught sisters.

Response: In the revised manuscript, we have clarified that the numbers are based on cultured spheroids.

139-140: What happened to the unfertilized eggs?

Response: Possible unfertilized eggs have been described in the revised manuscript.

223: If the branching order in Fig. 3 is correct, V. longispiniferus is equally related to all of the other species in Clade A. There is no reason to focus this comparison on V. barberi in particular, since V. ferrisii (for example) is just as closely related to V. longispiniferus.

Response: V. barberi and V. perglobator were selected as two phylogenetically separated representatives of clade A (excluding V. longispiniferus). Since we found two or three CBC in ITS-2 secondary structure between V. longispiniferus and V. barberi or between V. longispiniferus and V. perglobator, respectively, we consider that there is no need to compare V. longispiniferus with other species in clade A to discuss the genetic independency of V. longispiniferus within clade A. In the revised manuscript, such discussion has been added in the Discussion section.

224: Some discussion of the value of compensatory base changes in ITS-2 in distinguishing species is needed.

Response: Some discussion of the values of compensatory base changes in ITS-2 has been added to discuss the genetic independency of V. longispiniferus within Volvox sect. Volvox in the revised manuscript.

________________________________________

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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19 Jun 2020

A new preferentially outcrossing monoicous species of Volvox sect. Volvox (Chlorophyta) from Thailand

PONE-D-20-14342R1

Dear Dr. Nozaki,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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James G. Umen, Ph. D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

24 Jun 2020

PONE-D-20-14342R1

A new preferentially outcrossing monoicous species of Volvox sect. Volvox (Chlorophyta) from Thailand

Dear Dr. Nozaki:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

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