Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region.

The genus Halophila shows the highest species diversity within the seagrass genera. Southeast Asian countries where several boundary lines exist were considered as the origin of seagrasses. We hypothesize that the boundary lines, such as Wallace's and Lydekker's Lines, may act as marine geographic barriers to the population structure of Halophila major. Seagrass samples were collected at three islands in Vietnamese waters and analyzed by the molecular maker ITS. These sequences were compared with published ITS sequences from seagrasses collected in the whole region of interest. In this study, we reveal the haplotype and nucleotide diversity, linking population genetics, phylogeography, phylogenetics and estimation of relative divergence times of H. major and other members of the Halophila genus. The morphological characters show variation. The results of the ITS marker analysis reveal smaller groups of H. major from Myanmar, Shoalwater Bay (Australia) and Okinawa (Japan) with high supporting values. The remaining groups including Sri Lanka, Viet Nam, the Philippines, Thailand, Malaysia, Indonesia, Two Peoples Bay (Australia) and Tokushima (Japan) showed low supporting values. The Wallacea region shows the highest haplotype and also nucleotide diversity. Non-significant differences were found among regions, but significant differences were presented among populations. The relative divergence times between some members of section Halophila were estimated 2.15-6.64 Mya.

Introduction

Seagrasses are a polyphyletic group of monocotyledonous angiosperms that play an important ecological role and provide important ecosystem services in various coastal regions [1]. Approximately 72 seagrass species have been identified around the world [2]. Among six global regions, the Indo-Pacific region shows the largest number of seagrass species worldwide with 24 taxa that form vast meadows of mixed species stands [3]. Within the genus Halophila, eight sections based on their geographic distribution consisting of approximately 24 species have been described [4]. Halophila ovalis (Brown.) Hooker 1858 shows a global distribution whereas other members occur only in specific areas [5]. The Halophila section including H. major (Zollinger) Miquel 1856, H. ovalis, H. bullosa (Setch.) Kuo, n. comb., H. minor (Zollinger) Hartog 1957, H. gaudichardii Kuo 2006, H. ramamurthiana (Ravikumar et Ganesan) Kuo, n. com, H. mikii Kuo 2006, H. linearis den Hartog 1957, H. nipponica Kuo 2006, H. okinawensis Kuo 2006, H. johnsonii Eiseman 1980, and H. madagascariensis Steud. ex Doty et Stone 1967 is known to present one the most complex challenges in plant taxonomy [5].

Halophila major differs from closely related species by two main characteristics, the number of cross veins and the ratio of the distance between the intramarginal vein, with the lamina margin at the half-way point along the leaf length [6]. The species commonly occurs in Sri Lanka [7] Japan [8], Australia [8, 9], Southeast Asian countries such as Indonesia [10], Philippines [11], Malaysia, Myanmar [12] and Thailand [8]. In Viet Nam, H. major was misidentified as H. ovalis in the off-shore islands from Nha Trang Bay [13]. Defining taxonomic boundaries within the Halophila section has continued to present a real challenge due to leaf morphological traits that overlap among species and due to a high plasticity within species and even within populations [3]. Therefore, molecular markers could provide promising approaches for an unambiguous classification. Among the markers applied, the nuclear ribosomal internal transcribed spacer (ITS1-5.8S-ITS2) region was used to identify H. ovalis and closely related species, and species resolution was higher than by the analysis of the concatenated sequences of genes encoding the large subunit of ribulose-1,5-bisphosphate-carboxylase-oxygenase (rbcL) and chloroplast maturase K (matK) [14]. Kurniawan et al. [10] found that H. major populations in Indonesia seem to split into two groups based on the ITS marker. In addition, the study of Tuntiprapas et al. [15] revealed four different haplotypes of H. major within the Andaman Sea based on the ITS marker. Hence, the diversity of haplotypes may be higher than what we currently know.

For the time-calibrated phylogeny of seagrass, both nuclear (ITS) and chloroplast loci (rbcL, matK) were used to estimate the divergence of seagrass taxa. Coyer et al. [16] combined both nuclear and chloroplast loci to show the divergence within the seagrass family Zosteraceae. The result based on multi-locus marker analysis revealed that the most recent common ancestor of the Hydrocharitaceae family existed in Asia during the Late Cretaceous and Palaeocene (54.7–72.6 Mya) [17]. The authors also showed that the divergence time for the Halophila genus was 19.41 Mya ago. Recently, Kim et al. [18] indicated that the species H. nipponica diverged 2.95 ± 1.08 Mya from H. ovalis, and the divergence times for H. ovalis and H. major were similar, around 3.5 Mya. Our previous study on H. ovalis populations along the Egyptian coastline showed that the Red Sea H. ovalis populations did not group with the H. ovalis assemblage worldwide. H. major, H. ovalis and H. ovalis collected from the Red Sea were sister clades [19].

Among the six defined seagrass bioregions of the world, the Indo-West Pacific bioregion (Bioregion 5) is the largest and most diverse [11]. Within this bioregion, the seagrass beds in Southeast Asia have been separated into 22 marine provinces and ecoregions [20]. The Wallacea is located between the Sunda and the Sahul Shelf. It is a distinct region because it comprises many endemic, drought-tolerant floristic elements. The flora of the two shelves is more homogeneous than the Wallacean flora [21]. Several biogeographic barriers and boundary lines are found in this bioregion. The Sunda Shelf is a barrier that restricts the exchange of fish populations between the tropical Indian Ocean and the Pacific Ocean [22] while Wallace’s Lines and the modification of Wallace’s Line (Huxley’s Line) were considered as boundary line for several marine organisms such as the seagrass species Syringodium isoetifolium (Ascherson) Dandy 1939 [23], terrestrial vertebrates [24], and seagrass-associated fungal communities [25]. However, another seagrass species Thalassia hemprichii (Ehrenberg) Ascherson 1871 shows a genetically distinguishable cluster located within the Wallacea [23]. There are no any reports about the genetic structure of seagrass found between Wallacea and the Sahul Shelf. The Lydekker’s Line seems to be a marine barrier for the populations of blue swimming crab (Portunus pelagicus Linnaeus 1758) between the Sunda Shelf/Wallacea and Sahul Shelf [26]. Oceanic currents can act to both promote and limit gene-exchange. For example, the Kuroshio Current influences genetically homogeneous populations of Enhalus acoroides (Linnaeus) Royle 1839 between Yaeyama (Japan) and north‐east Philippines [27].

These findings lead to the hypothesis that the seagrass species H. major may form a monophyletic group in the interesting area. In this study, we analysed genetic diversity and link population genetics of H. major populations in the different bioregions. In addition, divergence times of members of the genus Halophila were also estimated.

Materials and methods

Sampling and species identification

The seagrass materials were collected at different locations in Viet Nam including Ly Son (15.376°N; 109.135°E), Con Dao (08.684°N; 106.626°E), and Phu Quoc (10.227°N; 104.684°E) Island (Fig 1). Ly Son Islands locate in Central Viet Nam, about 30 km from the shore. The Islands consist of two off-shore volcanic islands in the South China Sea, and a few islets. The previous report of Quang et al. [28] indicated that the distribution of seagrass from this area was 188.9 ha. Con Dao Islands is a national park located in the South of Viet Nam. It consists of 120 km2 of sea area and 14 islands. The seagrass beds from this off-shore islands were estimated at 200 ha, that mainly contribute to the main island [29]. Phu Quoc Islands which are located in the Bay of Thailand in the South of Viet Nam are the biggest island of Viet Nam. Seagrass beds at Phu Quoc are known for the largest area (more than 10,000 ha) and their species diversity compared to the other off-shore islands. In this location, nine species including putative Halophila ovalis were recorded [29]. The field surveys from the three above described locations were permitted by the People’s Committee of Ly Son, Con Dao and Phu Quoc in response to letters from the Institute of Oceanography, Viet Nam.

Fig 1

The map shows the sampling sites in Viet Nam and sequences of other regions were obtained from GenBank.

Region I (Sunda Shelf, sites = solid rounds) includes sampling sites in Viet Nam, Thailand, Malaysia and Myanmar. Region II (Wallacea, sites = solid squares) includes sampling sites in the Philippines and Indonesia. Region III (Sahul Shelf, sites = solid stars) includes sampling sites in Australia. Region IV (site = solid triangle) and V (sites = solid diamond) are sampling sites in the Bay of Bengal and the Coast of Japan, respectively. IDN = Indonesia, MAS = Malaysia, MYA = Myanmar, THA = Thailand, VIE = Viet Nam. LS = Ly Son Island, CD = Con Dao Island, PQ = Phu Quoc Island. Dotted line is the boundary line. Source of digital map: The National Oceanic and Atmospheric Administration (NOAA), USA, public domain data. Wallace’s and Lydekker’s Lines were adapted from Van Welzen et al. [30].

In the present study, SCUBA diving and snorkelling were used to collect seagrass samples in deep (6–10 m) and shallow water (1–3 m), respectively. At each site, five different plants were collected. At each site, these five different plants were randomly collected across the beds with a distance of 20–25 m interval between two plants. For each plant, one to two young leaves were fixed in DESS solution (20% dimethyl sulfoxide, 0.25 M disodium EDTA, and saturated NaCl) for DNA extraction and morphological observation. The remaining part was pressed as herbarium voucher specimens. Sample information is presented in S1 Table. Voucher specimens were deposited in the Institute of Oceanography (ION), Nha Trang City, Viet Nam. Specimens were identified using the keys of den Hartog [31], Kuo [4], Kuo et al. [6] and Kurniawan et al. [10]. The morphological characters used for measurements were cross veins (CV), branching cross veins (BCV), space between cross veins (SC), the angle between cross veins and midveins (AG), leaf width (LW), and leaf length (LL).

DNA extraction, PCR amplification and sequencing

The fixed materials (five plants/site) in DESS solution were separately homogenized in liquid nitrogen by mortar and pestle. Of the finely powdered plant material 100 mg was used for DNA extraction using the Quick-DNATM Miniprep Plus Kit (Zymo Research, CA, USA) following the manufacturer’s instructions. The region selected for PCR amplification was the ITS1-5.8S-ITS2 region. The primers ITS5a (5’-CCTTATCATTTAGAGGAAGGAG-3’ [32] and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) [33] were used to amplify sequences of 700 bp. For the ITS1-5.8S-ITS2 region, the total volume of 25 μl included 2x OneTag® Master Mix (New England Biolabs, Ipswich, MA, USA), 10–30 ng template DNA, and 1 pmol of each primer. PCRs consisted of an initial denaturation step at 95° C for 4 min, 35 cycles consisting of denaturation at 95° C for 40 s, annealing at 520 C for 30 s, and elongation at 72° C for 35 s. The 35 cycles were followed by a final extension at 72° C for 5 min, terminated by a final hold at 10° C. PCR was performed in an Applied Biosystems 2720 thermocycler (Applied Biosystems, Foster, CA, USA) with a heated lid. PCR products were cleaned using a GenEluteTM PCR Clean-Up kit (SigmaAldrich, St. Louis, MO, USA) following the manufacturer’s instruction. Direct Sanger sequencing of PCR products in both directions was done by 1ST BASE (Selangor, Malaysia) from both directions. The consensus sequence was achieved by Clone Manager 9 (Sci-Ed, Cary, NC, USA).

Phylogenetic analyses

There were no nucleotide differences in sequence of the five plant samples collected at each site. Therefore, only one of the sequences per site was used in the phylogenetic analysis. For the phylogenetic analysis, the dataset of ITS sequences, including three sequences obtained in this study and 73 sequences of known Halophila species retrieved from GenBank (https://www.ncbi.nlm.nih.gov/), were used for analysis (S1 Table). Among them, 69 ITS sequences from H. major were produced from samples collected in different regions (Fig 1). The sequences were aligned by the MAFFT algorithm with the selection of the q-ins-i option, considering the secondary structure for the alignment [34]. jModelTest version 2.1.6 [35] and the corrected AIC (Akaike Information Criterion) were used to find the best model for the analysis. Halophila beccarii Ascherson 1871 was used as the out-group. Two algorithms including Maximum Likelihood (ML) and Bayesian Inference (BI) were used for the phylogenetic analysis. Phylogenetic analyses were performed using RAxML version 8.1 [36] for Maximum Likelihood (ML) with model parameters fixed according to the values determined. The bootstrap values of the ML tree were estimated via the bootstrap algorithm with 1,000 replications. BI analyses were performed in MrBayes v.3.2.2 [37] using the same model as in the ML algorithm. In the BI, the two parallel runs with four chains each (three heated and one cold) were performed for 2 million generations, sampling a tree every 100 generations. The posterior probability values in each node were calculated by FigTree software (version 1.4.3). The consensus tree based on two different trees (achieved from the two methods) was constructed by Dendro Scope software, version 3.2.10 [38]. The average number of nucleotide differences between sampling locations for the full ITS fragment and per nucleotide was estimated in Mega X [39] using the Kimura 2-parameter model [40].

Population analysis and estimation of relative divergence times

For the population analysis, all sequences of H. major were included into the analysis. The number of haplotypes (N), haplotype diversity (h), and nucleotide diversity (π) were measured within each region using DnaSP version 6 [41]. Haplotype data were also used to construct a TCS network [42] performed by PopART [43] in order to generate haplotype networks for ITS1-5.8S-ITS2 sequences using their respective alignments. Significant genetic differences among 8 populations (Φ), among five regions (Φ) (Fig 1), and among individuals (Φ) were calculated by non-parametric analysis of molecular variance (AMOVA) to examine the hierarchical population genetic structure by grouping the samples of H. major with Arlequin version 3.5 [44].

The relative divergence times of the clades in the Halophila spp including H. ovalis, H. major, H. minor, H. nipponica, H. stipulacea, H. decipiens and members of the section Microhalophila (H. beccarii) were estimated based on the ITS sequence divergence to understand the evolutionary trend of H. major using Beast v2.5 [45]. The values between 1.72 × 10−9 and 1.71 × 10−8 mutations per site and year were used as the range for ITS mutation rates in plants [18, 46]. For Beast analyses, we used a Relaxed Clock Log Normal model. A General Time Reversible (GTR) substitution model with Gamma Categories set to 6 was adopted. The starting tree was randomly generated with a Calibrated Yule process prior. More than 90,000,000 generations of Markov Chain Monte Carlo (MCMC) were implemented of which every 1,000 generations were sampled. The Beast output was analyzed by Tracer v1.7 (Rambaut et al., 2018) [47] and uncertainty in parameter estimates was expressed as values of the 95% highest probability density (HPD). The effective sample sizes of all estimated parameters were also checked in Tracer v1.7 to ensure values were greater than 200. The consensus tree was generated with TreeAnnotator v1.7.3 (Drummond et al., 2012) [48], based on 64,801 trees.

Results

Halophila major in Vietnamese waters

The leaf shape of H. major collected at three different sites, Ly Son Island (Fig 2A), Phu Quoc Island (Fig 2B) and Con Dao Island (Fig 2C) showed a variability, either elliptic or oblong. Among the three populations, leaves collected at Ly Son Island (leaf length = 30.66 ±1 mm; leaf width = 10.12±0.4 mm) were larger than leaves of the two remaining populations (leaf length < 20.0 mm; leaf width < 9.0 mm). However, the number of cross veins of the samples collected at Con Dao (19–22) was higher than those from Ly Son (16–17) and Phu Quoc (14–17). The result also revealed that there were no differences in the branching cross veins between the three populations whereas the space between cross veins of samples collected in Ly Son (1.54 ±0.32 mm) was much wider than those from Con Dao (0.80±0.20 mm) and Phu Quoc (0.90±0.1 mm). Finally, there were no differences in the angle between cross veins and midveins between populations of Ly Son and Phu Quoc (45−600), but this parameter was higher in Con Dao (75−800) (Table 1).

Fig 2

Variation in morphology of Halophila major leaves collected from Vietnamese waters.

A: Ly Son Island; B: Phu Quoc Island; C = Con Dao Island. Scale in each figure = 1 mm.

Table 1

Leaf dimensions of Halophila major collected in the Vietnamese waters.

Sites	LW (mm)	LL (mm)	CV (vein)	BCV (vein)	CS (mm)	AG (0)
LS	10.12±0.36	30.66±1.09	16–17	3–5	1.54±0.32	45–60
PQ	8.32±0.30	18.10±0.26	14–17	2–4	0.91±0.10	45–60
CD	6.84±0.17	11.46±0.11	19–22	3–5	0.80±0.21	75–80

CV: cross veins, BCV: branching cross veins, SC: space between cross veins, AG: the angle between cross veins and mid-veins, LW: leaf width, and LL: leaf length. See Fig 1 for abbreviations of sampling sites.

A final alignment of 600 bp including gaps was generated for the ITS marker, of which 446 (74.3%) were conserved sites, 142 (23.7%) were variable sites, 59 (9.8%) were parsimony informative characters, and 81 (13.5%) were singletons. Results of the two algorithms applied (ML, BI) showed that all ITS sequences of H. major around the world showed eight smaller groups including Sri Lanka (1), Viet Nam/Philippines (2), Japan/Australia (3), Indonesia/Malaysia/Thailand (4) with low support values. Myanmar (5), Australia (6), and the samples of Thailand (7) and Japan (8) also formed the remaining groups (Fig 3). In general, H. major groupings are unsupported. Evolutionary divergence as measured by estimated total fragment and per nucleotide differences are 1–16 nucleotides and 0.03–0.28, respectively. In the comparison between samples collected in Viet Nam and other groups, there are 0–2 nucleotide differences among samples collected in Viet Nam and Philippines while the highest number of different nucleotides (14) was found between samples from Viet Nam and Shoalwater, Australia (Table 2).

Fig 3

Phylogenetic tree of members of section Halophila inferred from Maximum likelihood and Bayesian inference.

Data set based on 600 bp of ITS1-5.8S-ITS2. Bootstrap values and posterior probability of each method are shown at each node: (left) maximum likelihood; (right) Bayesian inference. *, full support (bootstrap value  =  100, posterior probability  =  1). 1–8 are smaller groups of Halophila major. See S1 Table for more information. The consensus tree was constructed by Dendro Scope software, version 3.2.10.

Table 2

Evolutionary divergence (un-shading cells) as measured by estimated total fragment and per nucleotide differences (shading cells) of Halophila major.

	1	2	3	4	5	6	7	8
1		0.003	0.03–0.012	0.05–0.015	0.05	0.021	0.002	0.005–0.007
2	2		0.007–0.015	0.009–0.019	0.009	0.024	0.005	0.009–0.010
3	2–7	4–9		0.005–0.021	0.009–0.014	0.019–0.022	0.003–0.010	0.003–0.012
4	3–9	4–11	3–12		0.010–0.021	0.019–0.028	0.007–0.017	0.010–0.022
5	3	5	4–8	6–12		0.019	0.003	0.003–0.005
6	12	12–14	11–12	11–16	11		0.019	0.015–0.017
7	1	3	2–6	4–10	2	11		0.003–0.005
8	3–4	5–6	2–7	6–13	2–3	9–10	2–3

See Fig 3 for abbreviation of groups.

Genetic diversity and phylogeography of Halophila major

A total of 69 ITS sequences (including three new sequences from the present study) of H. major collected in five geographic regions: Sunda Shelf (I), Wallacea (II), Sahul Shelf (III), Bay of Bengal (IV) and coast of Japan (V) generated 22 putative haplotypes (hap01-22). The nucleotide diversity (π) and haplotype diversity (Hd) of ITS within all regions were 0.00458 and 0.710, respectively. Among regions, region II revealed the highest haplotype diversity (1.0) and nucleotide diversity (0.01015) whereas regions I and V showed lower haplotype diversity (0.713 and 0.933, respectively) and nucleotide diversity (0.00478 and 0.00401, respectively). Region IV showed the lowest haplotype diversity (0.202) and nucleotide diversity (0.00035) (Table 3).

Table 3

Summarized Halophila major sample size, number of haplotypes observed, and estimates of genetic diversity.

Regions	N	h	Hd	π	S
I	17	7	0.713	0.00478	10
II	7	7	1.0	0.00583	20
III	2	2	na	na	na
IV	46	3	0.202	0.00035	2
V	6	5	0.933	0.00401	5
Over all	78	22	0.710	0.00458	37

N: Number of sequenced isolates, h: number of haplotypes, Hd: haplotype diversity, π: nucleotide diversity, S: number of segregating sites. See Fig 1 for abbreviations of regions, na: not available.

* p < 0.05.

A total number of 22 haplotypes (one haplotype from this study and 21 haplotypes deduced from previous studies) showed that seven haplotypes were found in geographic region II (hap01, 06, 08–12) whereas the region I contained five haplotypes (hap01-05). Notably, hap01 was shared by Viet Nam and the Philippines. Hap13-14 were found in the region III only. In the same way, hap15-19 were only distributed in the region V. Among haplotypes in the Bay of Bengal, frequencies of hap20 were highest, as it occupied 89% of the total number. Three haplotypes, hap20-22, were also found in the Bay of Bengal (region IV) (Fig 4). The haplotype network based on the ITS sequences failed to yield some clear phylogeographical separation among the regions (Fig 5). The most parsimonious network revealed two groups comprising two haplotypes, hap05 and hap20. Hap05 (the presumed ancestral haplotype) is at the centre Sunda Shelf and Wallacea (1), and six variants of H. major (hap06-12) were raised from hap05. There were 1–5 mutations between hap05 and hap06-12. Hap20 may be the central of the remaining groups. In this group, the data trend to form seven smaller groups including Bay of Bengal (1), Viet Nam–the Philippines (2), Japan-Australia (3), Thailand (5), Myanmar (6), Australia (7) and Japan (8). However, there was no clear phylogeographical separation between the coasts of Japan and Australia. The results of AMOVA based on the five regions explained 12.97% of the variation (or fixation index φ = 0.8, p-value < 0.01) (Table 4).

Fig 4

Distribution of haplotype frequency of Halophila major at different regions.

Twenty two haplotypes are defined by different colours/background. The data were processed by PopART software.

Fig 5

Haplotype network of 22 haplotypes and their distribution found for Halophila major worldwide.

Haplotypes are written beside or in the circles. Each short segment in the distance between two genotypes is a single mutation. Each dotted line rectangle presents each small group, numbers (1–8) following The data were processed by PopART software.

Table 4

AMOVA (analysis of molecular variance) results for ITS variation of Halophila major collected at five regions.

Source of variation	d.f.	SS	Ϭ2	% of variation	Fixation indices
Among populations	7	217.381	17.697	67.003	ΦSC = 0.769*
Among regions	4	261.376	3.425	12.966	ΦCT = 0.129
Within populations	11	58.200	5.291	20.031	ΦST = 0.799**
Total	22	536.957	26.413

d.f.: degree of freedom, SS: Sum of squares. See Fig 1 for the regions.

** p < 0.001

* p < 0.05.

Evolutionary trends and estimation of relative divergence times

The relative divergence times based on ITS1-5.8S-ITS2 for the Halophila genus revealed a sequential progression of diversification, from a fairly resolved split between section Microhalophila (H. beccarii) and the Halophila spp (H. ovalis and closely related species) (32.18 Mya, posterior probability value, p.p. = 1.0, 95% highest probability density, HPD = 13) to the most recent and highly resolved (p.p. = 1.0) divergence of H. minor and H. nipponica at 2.15 Mya (95% HPD: 0–4) (Fig 6). Within the section Halophila, the relative divergence times for H. decipiens and H. stipulacea were 11.72 and 10.47 Mya, respectively (p.p. = 1.0, 95% HPD: 7–8). In contrast, the most recent and highly resolved divergences of H. major, H. ovalis, H. minor and H. nipponica were 5.44–5.49 Mya (p.p. = 1.0, 95% HPD: 5). Notably, the relative divergence times of H. ovalis collected from the Red Sea was 5.44 Mya (p.p = 0.7, 95% HPD: 4). Among the H. major groups, the relative divergence times were 1.14–1.71 Mya (p.p. = 1.0, 95% HPD: 1–1.5) (Fig 6).

Fig 6

The relative divergence times based on ITS1-5.8S-ITS2 for Halophila spp.

The 95% Highest Density Probability (HPD) intervals are provided at each node; upper value = node divergence time (Mya), lower value = posterior probability values (p.p.). Time-calibrated phylogeny was processed by Beast v2.5.

Discussion

Our results reveal the high variation of leaf morphology among populations in Vietnamese waters. Kurniawan et al. [10] also indicated that the leaf dimensions of H. major collected from different sites in Indonesia also showed variation. The main different morphological character between those from Vietnamese waters and Indonesia is the number of branching cross veins: the number of branching cross veins of samples collected in Viet Nam is significantly lower than in the Indonesian samples (3–4 vs 6–8). The relatively closely related species, H. ovalis, showed morphological variability particularly in the leaves (leaf length and leaf width) in response to the different environmental factors in the various habitats [49]. In this present study, the phylogenetic tree based on ITS sequences reveals that all samples collected from the off-shore islands in Viet Nam were H. major. This result is in agreement with our previous study in Malaysia using samples collected from Mabul and Gusungan Islands, off the south eastern coast of Sabah [12]. The phylogenetic tree also showed the monophyletic group of the seagrass species H. major. Comparing different populations in the Southeast Asian countries and an adjacent region (Japan), the results of Kurniawan et al. [10] indicated two groups of H. major whereas samples collected in Malaysia, Indonesia and Thailand tend to form a single group.

The haplotype distribution showed that only hap01 was shared between Viet Nam (region I–Sunda Shelf) and the Philippines (region II) whereas there were no haplotypes shared with other regions. Therefore, boundary lines, such as the Wallace’s and Lydekker’s Lines, may play an important role as barrier between the Sunda Shelf, Wallacea and the Sahul Shelf, highlighting the strong effects of these geographical barriers also for the evolution of diverse seagrass taxa as was not shown before. Based on this fact of this area as living laboratory of evolution more samples of seagrasses and associated species need to be collected in future studies. A previous study on the haplotype distribution of the sister species H. ovalis also indicated that there are no haplotypes shared among Southeast Asian countries, the Bay of Bengal and the coast of Australia. However, a limited number of haplotypes were shared between Southeast Asian countries and the coast of Japan [19]. For other seagrass species, based on microsatellite analysis, Wainwright et al. [23] found that the manatee grass Syringodium isoetifolium formed a cluster that was exclusively located on the shallow Sunda Shelf and appears to follow the demarcation defined by the Wallace’s Line. The Wallace’s Line is known as the continental margin of the Sunda Shelf, and several other studies on marine snails [50], seahorses [51, 52], and crab [26] have shown similar results observed in our H. major study. In contrast, the marine brown alga Sargassum polycystum Agardh 1824 showed a homogeneous population throughout Southeast Asia [53]. Haplotype network and distribution of H. major showed significant differences between regions separated by the Malay Peninsula which is considered as a geographic barrier for several marine animals and plants. Similar results were also found with H. ovalis [12] and with the mangrove species Lumnitzera racemosa Willd. 1803 [54]. Within the samples collected from Australia, both phylogenetic analysis and haplotype network revealed two distinct groups and twelve mutations. This may be explained by the long distance between the two sampling sites and different oceanic systems. The average sea temperatures from sampling sites at Japan (between 30–35°N) and Australia (between 30–35°S) are similar, around 18–20°C (), and may explain the similarities of H. major in two regions.

There were several missing haplotypes, mainly in the Sunda Shelf (Fig 5). Unfortunately, data from the Java Sea, Natuna Archipelago and Singapore Strait were not available. Therefore, it is likely the missing haplotypes may occur in the above mentioned regions. Samples used in this study were stored by dried materials (for voucher specimens) and DESS solution. Fixing samples in DESS solution and store in -20°C may be the best way for DNA extraction later and morphological observation in the future. Hence, the sub-samples of herbarium voucher specimens should be fixed in DESS solution. More samples in the wider regions should be collected, and international collaboration studies are necessary in order to really get more complete data for better finding out its genetic diversity. The present study also revealed the highest nucleotide diversity of H. major in regions I (Sunda Shelf) and II (Wallacea). Previous studies on marine plants also indicated that the highest genetic diversity was also found in Southeast Asian countries, for example, H. ovalis [19] and for three species of the mangrove genus Rhizophora [55].

The relative divergence times of members of the genus Halophila were estimated for the first time based on the ITS marker was estimated. The relative divergence times of H. major and H. ovalis were similar (6.64 Mya). It is older than what Kim et al. [18] found (around 3.5 Mya). This difference may be based on technical aspects and the length of the sequences in the dataset used. By using multi-loci of the plastid genome, the divergence time estimates between H. ovalis and closely related species were 8.4 Mya [17]. Using the single ITS marker also revealed that the divergence time estimates among members of Halophila were 2.15 Mya (between H. minor and H. nipponica) and 11.72 Mya (between H. decipiens and remaining species). Moreover, an unexpected result revealed that H. ovalis collected form the Red Sea was split from H. major instead of H. ovalis, and its divergence time estimate was 5.44 Mya. This finding may lead to another hypothesis that H. ovalis collected from the Red Sea may be treated as a distinct species or a sub-species of H. major. The sequences of plastid genes, for example, rbcL, matK and psbA-trnH from other members of the Halophila genus are not available in GenBank. Therefore, our next study will apply multi-locus for the analysis, then the evolution of this genus may be understood in more details.

In conclusion, our findings in this study revealed the haplotype and genetic diversity of H. major in Southeast Asian countries and neighbouring regions. Halophila major shows variation in morphology in Viet Nam. Phylogenetic tree showed a monophyletic clade of H. major, with unique haplotypes occurring among regions but no or low support for regional groupings. Wallace’s and Lydekker’s Lines may indicate marine geographic barriers defining to the population structure of H. major observed today similar to the patterns seen in many marine organisms, while the Malay Peninsula acts as a geographic land barrier of this seagrass species in the two oceanic systems of the Pacific and Indian Oceans.

GenBank accession numbers of the sequences used in the analyses.

a: First identified as Halophila euphlebia. b: used in phylogenetic analysis, c: used in population genetic, d: used in time-calibrated phylogeny.

(DOCX)

Click here for additional data file.

20 Jan 2021

PONE-D-20-31468

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

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Dear Dr Nguyen,

We received the position of the three reviewers and you can see that the opinions are a little dissonant. Anyway, all the reviewers present very relevant considerations and so I chose to indicate a "Major Review". Please address any doubts and issues raised by the reviewers and I look forward to a new version of the manuscript.

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**********

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Reviewer #1: Ms. Number. : PONE-D-20-31468

Title : Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

General comments:

The article is very good and very interesting. This article discusses the species diversity of genus Halophila and tries to reveals a high genetic diversity of Halophila major in the Wallacea region using rDNA analysis. This is a good new insight because H. major still has big challenge, especially in Wallacea region. In order to provide a broad impact and strong research and data linkages, it would be better if it could involve cross-country researchers from regional areas, and could be used as a reference for the distribution of Halophila seagrass, especially H. major in ASEAN; I think in Indonesia, this species has just been rediscovered, and I believe it still has a wider range of life, and has a high genetic diversity, not only in the Wallacea region, as you mention in manuscript.

Also, there are several scientific names of species that are not italicized and according to the standard of writing, and should only be the scientific name of the species in italics. And then, there is a lot of writing that needs to be improved, especially related to continuity between sentences and between paragraphs, as well as table and unit. The units should use symbols that have been defined internationally, and are consistent.

Specific comments:

Abstract:

- Clear.

- “The morphological characters show variation, but based on the results of the ITS marker they fom a homogenous population.” Typo ‘fom’?

Introduction:

- Page 3: …“The Halophila section including H. ovalis, H. decipiens Ostenfeld, H. gaudichudii J. Kuo, H. major (Zoll.) Miquel, H. minor (Zollinger) den Hartog, H. nipponica J. Kuo, H. okinawensis J. Kuo and H. stipulacea (Forssk.) Ascherson is known to present as one the most complex challenges plant taxonomy [4,5].” … When biological species that is the first time mentioned in the text, it should attach the authority name and year.

- Page 4: Syringodium isoetifolium, Enhalus acoroides similar comment above.

Material and Methods:

- Page 6: Phylogenetic analysis section….”, including three sequences obtained in this study and 73 sequences of known Halophila sections retrieved from GenBank..”. Total 76 but in Result All sequences were 78.

Results

- Page 7: “Morphological observation and phylogenetic analysis of Halophila major in Vietnamese waters”. Suggestion to reduce, “Halophila major in Vietnamese waters.”

- Page 8: “A total of 78 ITS sequences (including three new sequences from the present study) of H. major collected in five geographic areas: Sunda Shelf (I), Wallacea (II), Sahul Shelf (III), Bay of Bengal (IV) and coast of Japan (V)”.Total sequences are 78 or 76?; this sentence prefer in phylogenetic analysis in method.

- Page 9: in Table 3, please consistent, areas or region is used based on the context.

- Page 10: First paragraph “Area” similar comment above.

Discussion

- Page 11: The number of branching veins for H. ovalis 3-4 from Indonesia samples. Generally, branching cross veins of H. ovalis is 3-4 pairs. How about the branching cross veins of H. ovalis in Vietnam?

- Page 11: “….whereas samples collected in Malaysia, Indonesia and Thailand tend to form a single clade”. Insert Fig. 3 and Why it placed in single clade, How it related with this finding?

- Page 12: Typo. “…..in regions II (Sunda Shelf) and III (Wallacea)”. it means Sunda Shelf (region I) and Wallacea (II)

Figure

- Improve the quality of all Figure (should be min. 350 DPI) follow the journal requirement.

- Figure 1. Suggestion: prefer polygon with different color or pattern to differentiate the region.

- Figure 2. Please insert the scale like 1 cm or 1 mm.

Recommendation: A major revision is required to improve the manuscript.

Reviewer #2: The manuscript entitled "Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea

region" by Nguyen et al. Using only "15" newly collected H. major plants from Viet Nam with other published ITS from GenBank to reveal the phylogentic relationship with other species in the genus Halophila as well the phylogeography of H. major. There are several flaws which are fatal to lead this rejection. First of all, the tile is not falling into their main conclusions(findings), because through out the manuscript, they only discussed about the high genetic diversity in Wallacea with only few sentences in page 12. Second, they seem to be misunderstood the the definition of the term "monophyletic", based on their phylogenetic tree, they mentioned there are 8 subclades in H. major and form nonmonophyletic groups. However, since the sub clades the authors defined in this studied are not well supported at the tips. They is "no" subclade in H. major, and sequences of H. major around the world formed" a "monophyletic clade with well support (supporting values :98/1). This is also supported by the TCS haplotype network which showed no sign of population partition. Third, they use same symbol to code geographic regions as well as 8 clades (I, II, III.....), which is really confusing while reading. Lastly, the resolution of the figures are generally low especially Figure 6.

There are some minor comments as following

Page 5, spell out "DESS"

Figure 1 caption: "where samples were collected change" to "where sequences were obtained from GenBank"

Page 7, "World type" "Red Sea type", you need to explain what are they or simply state previously been defined as "World type"and "Red Sea type" by someone....

There are many sentences developed from the erroneous statement "8 subclades", so the authors may need to reframe the manuscript based on the correct interpretations.

Reviewer #3: The authors analysed rDNA of three H. major collections from Vietnamese islands and similar molecular data from other SE Asia stored in geneBank to demonstrate the origin of this species was located in the Wallacea region. They further estimated the relative divergence times of certain taxa within the section Halophila and also that in section Halophila and section Microhalophila.

Comments:

a. The species Halophila major (H. euphlebia) was ‘re-established’ in 2006, therefore, using the prior to 2006 data from the geneBank to treat as “H. major” could be questionable. For example, the Halophila specimens from Shoalwater Bay, Australia have distinct linear leaf blades with L/W ratio 7-10; cross veins 8-12 (-14), unbranched…Based on the above morphological description, the specimens may not belong to H. major.

b. Abstract stated: “H major was misidentified as H. ovalis in Viet Nam”. However, this statement did not appear in other sections of this manuscript nor any reference was given.

c. Introduction: References [4, 5] did not use section Halophila. Furthermore, H. australis, H. capricorni, and H. sulawesii also include in this section. However, the latest Halophila taxonomy review shows that section Halophila does not include these three species and also not H. stipulacea and H. decipiens (see Kuo 2020).

d. Fig. 3 H. major has eight subclasses. Those from Japan belong to Class III and VIII; Australia belongs to III and VI; while Thailand had IV and VII. Why two different subclasses in the same country (Japan, Australia, Thailand)? Any morphological or environmental difference of the specimens in two different subclasses from the same country?

e. References: should list: Liu et al. (2020) Genetic identification and hybridizationin the seagrass genus Halophila (Hydrocharitaceae) in Sri Lanka waters. PeerJ 2020, 8, e10027

f. Authors estimated relative divergence of H. major and H. ovalis were 6.64 Mya, that was much older than Kim et al.’s estimation of 3.4 Mya. The authors of this manuscript explained these differences were due to ‘technical aspect’ and the length of sequences in the dataset used. They should explain what is ‘technical aspect’.

g. Authors estimated the divergence between section Halophila and section Microhalophila was 32.18 Mya, this estimation implies that the divergence of the genus Halophila would be much earlier than that estimated divergence of genus Halophila in the family Hydrocharitaceae was only 19.41 Mya by Chen et al. The authors of this manuscript should discuss these vast differences.

**********

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Reviewer #2: No

Reviewer #3: No

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4 Feb 2021

Recommendation Responses

Editor

Q1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming

R1: Thank you very much, the new version was formatted following guidelines of journal including the title, authors, affiliations, figure, table and support information.

Q2. In your Methods section, please provide additional location information of the sampling sites, including geographic coordinates for the data set if available.

R2: We mentioned the additional location information of the sampling sites, including geographic coordinates for the data set in the method section.

Q3. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the sampling sites access and, if no permits were required, a brief statement explaining why.

R3: We added the additional information regarding the permits our team obtained for the work at three locations

Q4. 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

Please include the updated Competing Interests Statement and Funding Statement in your cover letter. We will change the online submission form on your behalf

R4. Yes, we removed funding-related text from the manuscript. The cover letter is updated.

Q5. We note that Figures 1 and 4 in your submission contain map images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

R5. The background map used in the manuscript is from NOAA/NGDC. For our previous papers, we contacted the NOAA/NGDC for permission. They answered to us that it is public domain and available for use. Therefore, we wrote “Source of digital map: The National Oceanic and Atmospheric Administration (NOAA), USA, public domain data”

For Fig. 4. The map was integrated in the PopArt software that was used to produce Fig. 4. To be safe, we modified Fig. 4 with background map used in Figure 1.

Q6. Please include a caption for figures 5 and 6.

R6. Captions for figures 5 and 6 were added

Q7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information

R7. Caption for Supporting Information file was included at the end of your manuscript.

We updated citations to match accordingly.

Reviewer 1

Q1. There are several scientific names of species that are not italicized and according to the standard of writing, and should only be the scientific name of the species in italics. And then, there is a lot of writing that needs to be improved, especially related to continuity between sentences and between paragraphs, as well as table and unit. The units should use symbols that have been defined internationally, and are consistent.

R1. Thank you very much, the manuscript was improved. Please see the changes that were highlighted

Q2. Typos “fom”

R2. Thank you very much, we corrected

Q3. When biological species that is the first time mentioned in the text, it should attach the authority name and year R3. We added the authority name and year after scientific name

Q4. Page 6: Phylogenetic analysis section….”, including three sequences obtained in this study and 73 sequences of known Halophila sections retrieved from GenBank..”. Total 76 but in Result All sequences were 78

R4. We changed to …”73 sequences of known Halophila species” 69 ITS sequences of H. major. Please see the S1 Table,

Q5. Page 7: “Morphological observation and phylogenetic analysis of Halophila major in Vietnamese waters”. Suggestion to reduce, “Halophila major in Vietnamese waters.”

R5. Thank you very much, we agree to reduce

Q6. Page 8: “A total of 78 ITS sequences (including three new sequences from the present study) of H. major collected in five geographic areas: Sunda Shelf (I), Wallacea (II), Sahul Shelf (III), Bay of Bengal (IV) and coast of Japan (V)”.Total sequences are 78 or 76?; this sentence prefer in phylogenetic analysis in method.

R6. Yes, we checked and corrected the numbers, please see my answer at R4.

Q7. Page 9: in Table 3, please consistent, areas or region is used based on the context

R7. Thank you for your finding, we changed all “area” to “region” except where area was used for specific locations in Viet Nam

Q8. Page 10: First paragraph “Area” similar comment above.

R8. Please, see our answer in R7

Q9. Page 11: The number of branching veins for H. ovalis 3-4 from Indonesia samples. Generally, branching cross veins of H. ovalis is 3-4 pairs. How about the branching cross veins of H. ovalis in Vietnam?

R9. Thank you very much for your sharing, it is the same, 3-4 from the Vietnamese samples

Q10. Page 11: “….whereas samples collected in Malaysia, Indonesia and Thailand tend to form a single clade”. Insert Fig. 3 and Why it placed in single clade, How it related with this finding?

R10. We inserted Fig 3. The group IV contains materials from Malaysia, Indonesia and Thailand were performed by data. There are several articles mentioning long‐distance dispersal, positively buoyant shoots with attached rhizomes or seedlings having high potential for long‐distance dispersal. Therefore, DNA fingerprinting should be applied to investigate the population structure and gene flows among populations of Halophila ovalis or Halophila major from SEA in further research studies.

Q11. Page 12: Typo. “…..in regions II (Sunda Shelf) and III (Wallacea)”. it means Sunda Shelf (region I) and Wallacea (II) R11. Thank you very much, we corrected

Q12. Improve the quality of all Figure (should be min. 350 DPI) follow the journal requirement.

R12. We agree, all figures were improved

Q13. Figure 1. Suggestion: prefer polygon with different color or pattern to differentiate the region.

R13. Thank you for your suggestion. We tried polygon, but the land is polygon too. However, Figure 1 was modified following reviewer 2 by using different symbols coding different regions

Q14. Figure 2. Please insert the scale like 1 cm or 1 mm

R14. We agree, we edited the figure 2, the scale is 1 mm that was mentioned in figure caption.

Reviewer 2

Q1: the title is not falling into their main conclusions (findings), because throughout the manuscript, they only discussed about the high genetic diversity in Wallacea with only few sentences in page 12.

R1. We add more sentences, it may support to the tittle, please see it in the highlight.

Q2. Second, they seem to be misunderstood the the definition of the term "monophyletic", based on their phylogenetic tree, they mentioned there are 8 subclades in H. major and form nonmonophyletic groups. However, since the sub clades the authors defined in this studied are not well supported at the tips. They is "no" subclade in H. major, and sequences of H. major around the world formed" a "monophyletic clade with well support (supporting values: 98/1). This is also supported by the TCS haplotype network which showed no sign of population partition.

R2. Thank you very much for your suggestion, we changed to smaller groups, and statement “and sequences of H. major around the world formed" a "monophyletic clade”

Q3. Third, they use same symbol to code geographic regions as well as 8 clades (I, II, III.....), which is really confusing while reading

R3. We changed different symbols to different regions in Fig. 1. Eight small groups were also modified

Q4. the resolution of the figures are generally low especially Figure 6.

R4. Thank you, we improved the resolution all figure

Q5. Page 5, spell out "DESS"

R5. We explained DESS solution. It is 20% dimethyl sulfoxide, 0.25 M disodium EDTA, and saturated NaCl.

Q6 Figure 1 caption: "where samples were collected change" to "where sequences were obtained from GenBank"

R6. Thank you very much. We changed, and modified the Figure 1 caption. Please see our answer R13 to reviewer 1.

Q7. "World type" "Red Sea type", you need to explain what are they or simply state previously been defined as "World type"and "Red Sea type" by someone....

There are many sentences developed from the erroneous statement "8 subclades", so the authors may need to reframe the manuscript based on the correct interpretations.

R7. We corrected and removed Red Sea type, simply we changed to “samples collected from Red Sea”, and we changed “subclade” to “group”

Reviewer 3

Q1. The species Halophila major (H. euphlebia) was ‘re-established’ in 2006, therefore, using the prior to 2006 data from the geneBank to treat as “H. major” could be questionable. For example, the Halophila specimens from Shoalwater Bay, Australia have distinct linear leaf blades with L/W ratio 7-10; cross veins 8-12 (-14), unbranched…Based on the above morphological description, the specimens may not belong to H. major.

R1. Thank you very much, we agree with you. It is not easy to identify the Halophila major and closely related species based on morphological observation only. In this present study, first we used the sequence of H. major (Uchimura et al. 2008) for blasting in NCBI. All sequences with similarity more than 99% were used for the analysis. This group contained two sequences from Australia. Intra-species variation within H. nipponica were reported in Japan or H. ovalis from Malaysia.

Q2. Abstract stated: “H major was misidentified as H. ovalis in Viet Nam”. However, this statement did not appear in other sections of this manuscript nor any reference was given.

R2. The Halophila ovalis/major complex was reported from Viet Nam in 2013 (Ref 13), and H. major was only found at one off-shore island. Therefore, we are now checking the Halophila ovalis/major complex from three different locations.

Q3. Introduction: References [4, 5] did not use section Halophila. Furthermore, H. australis, H. capricorni, and H. sulawesii also include in this section. However, the latest Halophila taxonomy review shows that section Halophila does not include these three species and also not H. stipulacea and H. decipiens (see Kuo 2020).

R3. We modified these sentences, 13 species in section Halophila was updated, and the Ref. Kuo, 2020 was updated. Please see our answer to reviewer 1.

Q4. Fig. 3 H. major has eight subclasses. Those from Japan belong to Class III and VIII; Australia belongs to III and VI; while Thailand had IV and VII. Why two different subclasses in the same country (Japan, Australia, Thailand)? Any morphological or environmental difference of the specimens in two different subclasses from the same country?

R4. It is a really nice question. Your question is something that is related to Q11 from reviewer 1. In Australia, I think that the geographic distance between two sites from Australia, it may be about 3,000 km from the west side to the east site. In the case of Japan, one site is Okinawa where the water temperature is warmer than the mainland (the other site), but we cannot explain in the case of Thailand. Two haplotypes were found in the Andaman Sea. Hopefully, we will collect more materials from above locations, then we use SSRs approach to estimate the migration among populations in SEA and the other regions

Q5. References: should list: Liu et al. (2020) Genetic identification and hybridizationin the seagrass genus Halophila (Hydrocharitaceae) in Sri Lanka waters. PeerJ 2020, 8, e10027

R5. Thank you, we cited and added this Ref.

Q6. Authors estimated relative divergence of H. major and H. ovalis were 6.64 Mya, that was much older than Kim et al.’s estimation of 3.4 Mya. The authors of this manuscript explained these differences were due to ‘technical aspect’ and the length of sequences in the dataset used. They should explain what is ‘technical aspect’.

R6. Thank you for your interesting comment. The different estimated relative divergence in Kim et al. 2017 can be explained as follows: Figure 4A (estimated relative divergence = 3.5 Mya for H. ovalis), and Figure 4B (estimated relative divergence is about 8 Mya for H. ovalis clade). The software applied in their work was NETWORK 4.6 program. Therefore. the 95% Highest Density Probability (HPD) intervals and posterior probability values were not included. In this present study we used the popular software for estimating relative divergences Beast v2.5

Q7 Authors estimated the divergence between section Halophila and section Microhalophila was 32.18 Mya, this estimation implies that the divergence of the genus Halophila would be much earlier than that estimated divergence of genus Halophila in the family Hydrocharitaceae was only 19.41 Mya by Chen et al. The authors of this manuscript should discuss these vast differences.

R7. We agree with you. Chen et al used multilocus in plastid DNA while this present study used rDNA. Unfortunately, there are only limited data of plastid DNA from members of Halophila.

Submitted filename: responses to Reviewers.docx

Click here for additional data file.

19 Apr 2021

PONE-D-20-31468R1

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen

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.

==============================

Thank you very much for the new version of the manuscript. Note that one of the reviewers still expects a few changes before his manuscript is accepted for publication. Please make these adjustments so that I can proceed with the revision of the manuscript.

==============================

Please submit your revised manuscript by 04/30/2021. 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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Sergio N. Stampar, Dr.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Dear authors,

Thank you very much for the new version of the manuscript. Note that one of the reviewers still expects a few changes before his manuscript is accepted for publication. Please make these adjustments so that I can proceed with the revision of the manuscript.

Kind regards

Sergio

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. 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: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. 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

**********

5. 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

**********

6. 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: In the discussion section, suggestions should be made regarding the use of fresh specimens and adding locations, and things that become input and challenges in the future, including collaborative research in order to really get more complete data to better find out its genetic diversity.

Also, there is something that needs to be ascertained from the phylogenetic tree, is H. major in Australia really any specimens based on morphological evidence? If there is, it may be stated that the source of the citation is that H. major is found in Australia in Materials and methods.

Reviewer #2: Although authors have replied most of my comments, there is one exception which related to the symbol (I, II, III etc.) they used in the Figure 1 and in the phylogenetic tree to represent geographic areas and "groups", respectively.

They are several things that I concern while reviewing this revision. Here I list them point by point as follows.

Line 75-76: Delete "In addition, the study....on the IST marker."

Line 88-89: Unclear, had to rephrase.

Line 106-108: This sentence is odd, need to rewrite to clarify what you were trying to say.

Line 109-110:How the genetic partition occur "within" and "among" barrier ? The authors may need to clarify this or simply make this sentence more straight forward.

Line 129: Use "sequences of other regions" instead of "other regions sequences"

Line 140: Use "the" instead of "this".

Line 243-244:I would like the authors to provide the supporting values of each groups they defined. Because technically, if those groups are not support by supporting values they should not be defined as different group. I know the authors already used group instead of clade. One thing the authors can do is be honest with what they found (groups are not well-supported) and add up the information that the grouping of those haplotypes might affiliate with geographic location based on the haplotype network instead of define them as groups without substantial evidence.

Line 328-329:The authors should explain what is the "similar way" they mentioned here? Because the original sentence is not very clear. Additionally, in the tree generated by Waycott et al. 2002, those groups found in H. ovalis samples were not well-supported, therefore, they also over-interpreted their data almost 20 years ago. And I truly believe we should not follow the conclusion which is not correct.

Line 382-383:You only have the morphological data from Viet Nam, therefore, I don't think you can make this conclusion while you mentioned a broad area in previous sentence.

I strongly recommend the authors send their next revision for English editing before submitting back to PloS One, because I did not correct them across the manuscript. I am not a native speaker too, and I always send my manuscript for English editing before submitting, just because it actually help the audiences to read and receive the information that we are trying to deliver.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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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: Yes: Fery Kurniawan

Reviewer #2: No

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25 Apr 2021

Dear Editor

Thank you very much for forwarding the detailed suggestions and comments from the reviewers. All suggestions have been very helpful for us to improve the manuscript. All changes are highlighted in the newest version. Our responses are below:

List of revisions

Editor

Q1. Note that one of the reviewers still expects a few changes before his manuscript is accepted for publication. Please make these adjustments so that I can proceed with the revision of the manuscript.

R1: Thank you very much, please see our responses to the reviewers below.

Reviewer 1

Q1. In the discussion section, suggestions should be made regarding the use of fresh specimens and adding locations, and things that become input and challenges in the future, including collaborative research in order to really get more complete data to better find out its genetic diversity.

R1. Thank you very much for your suggestions, we add more information in discussion part (yellow highlight).

Q2. There is something that needs to be ascertained from the phylogenetic tree, is H. major in Australia really any specimens based on morphological evidence? If there is, it may be stated that the source of the citation is that H. major is found in Australia in Materials and methods.

R2. We agree with you, “H. major” was previously treated as H. ovalis by Waycott et al. (2002). Uchimura et al. (2008) suggested that it should be H. major. Therefore, we add Uchimura et al. (2008) and Waycott et al. (2002) when we mention that H. major occurs in Australia (yellow highlight. Line 63, page 3)

Reviewer 2

Q1: Although authors have replied most of my comments, there is one exception which related to the symbol (I, II, III etc.) they used in the Figure 1 and in the phylogenetic tree to represent geographic areas and "groups", respectively. R1. Thank you very much for your finding the conflicts between Fig. 1 and Fig. 3. We kept I, II, III in Fig. 1. We changed 1,2,3… in Figure 3

Q2 They are several things that I concern while reviewing this revision. Here I list them point by point as follows.

Line 75-76: Delete "In addition, the study....on the IST marker."

Line 88-89: Unclear, had to rephrase.

Line 106-108: This sentence is odd, need to rewrite to clarify what you were trying to say.

Line 109-110:How the genetic partition occur "within" and "among" barrier ? The authors may need to clarify this or simply make this sentence more straight forward.

Line 129: Use "sequences of other regions" instead of "other regions sequences"

Line 140: Use "the" instead of "this".

R2. Thank you for your recommendation

Line 75-76: Deleted

Line 88-89: Yes, we modified the sentence.

Line 106-108: Thank you, we rewrote the sentence.

Line 109-110: We changed this sentence.

Line 129: Yes, we modified.

Line 140: done

Q3. Line 243-244: I would like the authors to provide the supporting values of each groups they defined. Because technically, if those groups are not support by supporting values they should not be defined as different group. I know the authors already used group instead of clade. One thing the authors can do is be honest with what they found (groups are not well-supported) and add up the information that the grouping of those haplotypes might affiliate with geographic location based on the haplotype network instead of define them as groups without substantial evidence.

R3. Thank you, we agree with you, we added the supporting values to the tree. Therefore, the Fig. 3 was modified. We agree with you, H. major is a monophyletic group, but the trend seems to separate it into groups with low supporting values (not for all groups). You can follow our changes by highlighting them. For the haplotype network, we also agree with you and modified Fig. 5. We removed group A,B. The text was also modified, please see the highlighted passages.

Q4. Line 328-329: The authors should explain what is the "similar way" they mentioned here? Because the original sentence is not very clear. Additionally, in the tree generated by Waycott et al. 2002, those groups found in H. ovalis samples were not well-supported, therefore, they also over-interpreted their data almost 20 years ago. And I truly believe we should not follow the conclusion which is not correct.

R4. We removed two last sentences of this paragraph.

Q5. Line 382-383: You only have the morphological data from Viet Nam, therefore, I don't think you can make this conclusion while you mentioned a broad area in previous sentence.

R5. We changed to monophyletic group that was not changed in the previous version.

Q6 I strongly recommend the authors send their next revision for English editing before submitting back to PloS One, because I did not correct them across the manuscript. I am not a native speaker too, and I always send my manuscript for English editing before submitting, just because it actually help the audiences to read and receive the information that we are trying to deliver.

R6. Thank you for your recommendation, we will consider your suggestion. Our co-authors, especially Prof. Lawrence M. Liao and Prof. J. Papenbrock carefully checked and edited the manuscript in the final version.

We hopefully fulfilled all points raised.

Sincerely yours,

Xuan-Vy Nguyen

Submitted filename: responses to Reviewers.docx

Click here for additional data file.

28 Jul 2021

PONE-D-20-31468R2

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen,

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 below.

The revised manuscript is well written and reviewer 1 has indicated that their prior comments have all been met. As the stand-in Subject Editor for the manuscript, I appreciate the authors’ and prior reviewers’ efforts to ensure the accuracy and clarity of the manuscript. However, I feel there are several issues that need to be addressed prior to publication.

Principally, results as presented do not clearly support the primary conclusion of significant genetic isolation of Halophila major within the central Indo-Pacific (lines 42-43 and elsewhere). I base this specifically on the lack of phylogenetic support for putative H. major clades and the non-significant PhiCT values presented in the AMOVA table (table 4).

The lack of support for phylogenetic divergence within H. major is acknowledged in the text (eg. lines 381-383) yet elsewhere support for the putative groupings is implied (eg. lines 38-39). At a minimum, this confuses the discussion and should be clarified.

As described in Excoffier et al. 2010, AMOVA allows the hierarchical partitioning of genetic variation among populations (phiSC), among regions (phiCT), and among individuals within populations (phiST). As presented, table 4 indicates a non-significant phiCT. This may simply be a typo, as suggested by the lack of haplotype sharing among sites (table 2). If, however, this is not a typo, I feel the primary conclusions of the manuscript are unsupported and significant revisions would be needed before publication.

I am therefore recommending major revisions with the caveat that my concerns about project findings may be addressed if AMOVA results in table 4 are not correct as presented.

Below I describe additional recommendations and concerns that should be addressed.

Lines 38-39: Misidentification of Viet Nam H. major was described in a prior study and should not be highlighted here as a new finding.

Line 40: It is unclear who “they” is referencing. Viet Nam samples only?

Lines 42-43: If the AMOVA table is correct as presented this conclusion will need to be revised

Line 52: I’m unfamiliar with using “sections” to describe phylogenetic clades. Is that common in plants? Please correct if not.

Lines 54-59: Author taxonomy citations are inconsistent. My understanding is that presenting the authors last name is standard format. Please confirm and correct as needed.

Line 104-105: Sentence starting with “Oceanic currents…” is unclear. Please revise.

Line 118: Delete “area”

Line 146: Change “Information of the samples…” to “Sample information…”

Methods: Significant methodological details are missing…

How were pairwise nucleotide differences estimated? What mutational model was used?

What is a p-distance?

How were AMOVA groupings determined and what were they?

Highest Probability Density (BEAST?) is not described.

Lines 231-232: The described H. major groupings are unsupported and should be collapsed into a single branch. Alternatively, the text should be clarified here and elsewhere to avoid any suggestion that there is phylogenetic support for the groups. This does not alone refute the project findings, but it does add some uncertainty.

Table 3: Type “summared”

Lines 281-282: The AMOVA statement will need to be revised if table 4 is correct.

Line 309: Typo “highest density probability”

Please submit your revised manuscript by Sep 11 2021 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.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey A. Eble, Ph.D.

Academic Editor

PLOS ONE

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. 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

**********

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

Reviewer #1: Yes

**********

4. 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

**********

5. 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

**********

6. 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: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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

[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.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Aug 2021

Dear Editor

Thank you very much for your detailed suggestions and comments. All your suggestions are very helpful for us to improve the manuscript. All changes are highlighted in the newest version. Our responses are below:

List of revisions

Recommendation Responses

Editor

Q1. Principally, results as presented do not clearly support the primary conclusion of significant genetic isolation of Halophila major within the central Indo-Pacific (lines 42-43 and elsewhere)

R1: We agree with you, we changed to non-significant genetic isolation.

Q2. The lack of support for phylogenetic divergence within H. major is acknowledged in the text (eg. lines 381-383) yet elsewhere support for the putative groupings is implied (eg. lines 38-39). At a minimum, this confuses the discussion and should be clarified.

R2: We modified lines 38-39, and line 381-383

Q3. As described in Excoffier et al. 2010, AMOVA allows the hierarchical partitioning of genetic variation among populations (phiSC), among regions (phiCT), and among individuals within populations (phiST). As presented, table 4 indicates a non-significant phiCT. This may simply be a typo, as suggested by the lack of haplotype sharing among sites (table 2). If, however, this is not a typo, I feel the primary conclusions of the manuscript are unsupported and significant revisions would be needed before publication.

R3. We agree with you, it is typos, Table 4 was corrected

Q4. I am therefore recommending major revisions with the caveat that my concerns about project findings may be addressed if AMOVA results in table 4 are not correct as presented.

R4. We changed the results of Table 4. Non-significant differences were found among regions, but significant differences were presented among populations

Q5. Lines 38-39: Misidentification of Viet Nam H. major was described in a prior study and should not be highlighted here as a new finding

R.5 It was removed

Q6. Line 40: It is unclear who “they” is referencing. Viet Nam samples only?

R6. We modified the sentence.

Q7. Lines 42-43: If the AMOVA table is correct as presented this conclusion will need to be revised

R7. Please see our responses in R5 and R6

Q8. Lines 54-59: Author taxonomy citations are inconsistent. My understanding is that presenting the authors last name is standard format. Please confirm and correct as needed.

R8. We used authors last name, and they are consistent now

Q9. Line 104-105: Sentence starting with “Oceanic currents…” is unclear. Please revise.

R9. We used “sea currents”

Q10. Line 118: Delete “area”

R10. Done

Q11. Line 146: Change “Information of the samples…” to “Sample information…”

R11. Done

Q12. Methods: Significant methodological details are missing…How were pairwise nucleotide differences estimated? What mutational model was used? What is a p-distance? How were AMOVA groupings determined and what were they? Highest Probability Density (BEAST?) is not described.

R12. We added significant methodological details.

Q13. Lines 231-232: The described H. major groupings are unsupported and should be collapsed into a single branch. Alternatively, the text should be clarified here and elsewhere to avoid any suggestion that there is phylogenetic support for the groups. This does not alone refute the project findings, but it does add some uncertainty.

R13. Thank you very much, our changes to mention that the H. major groupings are unsupported.

Q14. Table 3: Type “summared”

R14. Done

Q15. Lines 281-282: The AMOVA statement will need to be revised if table 4 is correct.

R15. Done

Q16. Line 309: Typo “highest density probability”

R16. Done

We hopefully fulfilled all points raised.

Sincerely Yours

Xuan-Vy Nguyen

Submitted filename: Response to Reviewer.docx

Click here for additional data file.

23 Aug 2021

PONE-D-20-31468R3

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PLOS ONE

Dear Dr. Nguyen,

Thank you for submitting your revised manuscript to PLOS ONE. After careful consideration, we feel that you have successfully addressed comments on the previous draft manuscript (revision 2) and no additional changes are requested aside from grammar and wording issues listed below. We invite you to submit a revised version of the manuscript after addressing the issues listed below.

Lines 37-38: "show variation" is unclear. For clarity I recommend indicating among what groups variation was observed. With the addition of this information I as well recommend splitting this statement into two sentences, the first highlighting where variation is observed and the second indicating low support for regional H. major groupings.

Lines 105-106. Sorry for the confusion. My prior concerns about this sentence were not related to the term 'oceanic currents', rather I feel your statement that currents can "act as a gene-exchange line for the mirgration requiring long-distance dispersal abilities" is unclear. I'm pretty sure I understand your point, perhaps something like "Oceanic currents can act to both promote or limit gene-exchange" would be more clear.

Figure 1: The Myanmar  site currently shows a solid circle but instead should be a solid diamond based on the figure 1 legend.

Line 146: "Sample" not "Samples"

Lines 191-193: Suggested revision "The average number of nucleotide differences between sampling locations for the full ITS fragment and per nucleotide was estimated in Mega X (40) using the Kimura 2-parameter model (39)."

Lines 249-250: Which ML and base values are you referring to? I would not consider the values in general to be high given the lack of support for the H. major group. Please revise to avoid giving the false impression that there is significant support for H. major groupings.

Line 251 and elsewhere: Table 2 presents two different estimates of 'evolutionary divergence', one for the full fragment and one standardized estimate of the per nucleotide rate. I recommend changing how these measures are referred to here and elsewhere for clarity. Perhaps something like "Evolutionary divergence as measured by estimated total fragment and per nucleotide differences...".

Table 2 caption: See my note above.

Table 3 caption: "Summarized statistics of haplotypes" is unclear. I recommend something like "Summarized Halophila major sample size, number of haplotypes observed, and estimates of genetic diversity."

Table 3: I only just noticed that you've included estimates of Tajima's D and Fu's Fs here but they are not mentioned in the results nor discussion. I recommend deleting these measures from table 3 and the methods unless you can find a meaningful way to include the results.

Line 401: Recommend changing "but it trends to form..." to "with unique haplotypes occurring among regions but no or low support for regional groupings."

Please submit your revised manuscript by Oct 07 2021 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.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Jeffrey A. Eble, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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

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24 Aug 2021

List of revisions

Recommendation Responses

Editor

Q1. Lines 37-38 "show variation" is unclear. For clarity I recommend indicating among what groups variation was observed. With the addition of this information I as well recommend splitting this statement into two sentences, the first highlighting where variation is observed and the second indicating low support for regional H. major groupings

R1: We agree with you, we changed by splitting into two sentences.

Q2. Lines 105-106.. perhaps something like "Oceanic currents can act to both promote or limit gene-exchange" would be more clear.

R2: Thank you for your suggestion. We changed

Q3. Figure 1: The Myanmar site currently shows a solid circle but instead should be a solid diamond based on the figure 1 legend.

R3. I think it should by a solid circle, please see our highlight on the figure 1 legend

Q4. Line 146: "Sample" not "Samples"

R4. Done

Q5. Lines 191-193: Suggested revision "The average number of nucleotide differences between sampling locations for the full ITS fragment and per nucleotide was estimated in Mega X (40) using the Kimura 2-parameter model (39).

R.5 Thank you for your correction, we changes. We also changed in the Reference. Please see our highlight

Q6. Lines 249-250: Which ML and base values are you referring to? I would not consider the values in general to be high given the lack of support for the H. major group. Please revise to avoid giving the false impression that there is significant support for H. major groupings.

R6. We removed the sentence.

Q7. Line 251 and elsewhere: Table 2 presents two different estimates of 'evolutionary divergence', one for the full fragment and one standardized estimate of the per nucleotide rate. I recommend changing how these measures are referred to here and elsewhere for clarity. Perhaps something like "Evolutionary divergence as measured by estimated total fragment and per nucleotide differences...".

R7. We changed

Q8. Table 2 caption: See my note above..

R8. We changed

Q9. Table 3 caption: "Summarized statistics of haplotypes" is unclear. I recommend something like "Summarized Halophila major sample size, number of haplotypes observed, and estimates of genetic diversity

R9. Done

Q10. Table 3: I only just noticed that you've included estimates of Tajima's D and Fu's Fs here but they are not mentioned in the results nor discussion. I recommend deleting these measures from table 3 and the methods unless you can find a meaningful way to include the results

R10. We removed in both Table and in Method

Q11. Line 401: Recommend changing "but it trends to form..." to "with unique haplotypes occurring among regions but no or low support for regional groupings."

R11. Thank you, we changed

Submitted filename: responses to Editor.docx

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11 Oct 2021

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

PONE-D-20-31468R4

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Academic Editor

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13 Oct 2021

PONE-D-20-31468R4

Analysis of rDNA reveals a high genetic diversity of Halophila major in the Wallacea region

Dear Dr. Nguyen:

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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