Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)

Mamat Noorhidayah; Noor Azrizal-Wahid; Van Lun Low; Norma-Rashid Yusoff

doi:10.1371/journal.pone.0301392

. 2024 Apr 5;19(4):e0301392. doi: 10.1371/journal.pone.0301392

Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)

Mamat Noorhidayah ¹, Noor Azrizal-Wahid ^2,^*, Van Lun Low ³, Norma-Rashid Yusoff ¹

Editor: Tzen-Yuh Chiang⁴

PMCID: PMC10997100 PMID: 38578719

Abstract

Despite is known to have widespread distribution and the most active species of the family Chlorocyphidae, the molecular data of Rhinocypha fenestrella (Rambur, 1842) are relatively scarce. The present study is the first that examined the genetic diversity and phylogeographic pattern of the peacock jewel-damselfly R. fenestrella by sequencing the cytochrome C oxidase I (cox1) and 16S rRNA gene regions from 147 individuals representing eight populations in Malaysia. A total of 26 and 10 unique haplotypes were revealed by the cox1 and 16S rRNA genes, respectively, and 32 haplotypes were recovered by the concatenated sequences of cox1+16S. Analyses indicated that haplotype AB2 was the most frequent and the most widespread haplotype in Malaysia while haplotype AB1 was suggested as the common ancestor haplotype of the R. fenestrella that may arose from the Negeri Sembilan as discovered from cox1+16S haplotype network analysis. Overall haplotype and nucleotide diversities of the concatenated sequences were H_d = 0.8937 and P_i = 0.0028, respectively, with great genetic differentiation (F_ST = 0.6387) and low gene flow (N_m = 0.14). Population from Pahang presented the highest genetic diversity (H_d = 0.8889, P_i = 0.0022, N_h = 9), whereas Kedah population demonstrated the lowest diversity (H_d = 0.2842, P_i = 0.0003, N_h = 4). The concatenated sequences of cox1+16S showed genetic divergence ranging from 0.09% to 0.97%, whereas the genetic divergence for cox1 and 16S rRNA genes were 0.16% to 1.63% and 0.01% to 0.75% respectively. This study provides for the first-time insights on the intraspecific genetic diversity, phylogeographic pattern and ancestral haplotype of Rhinocypha fenestrella. The understanding of molecular data especially phylogeographic pattern can enhance the knowledge about insect origin, their diversity, and capability to disperse in particular environments.

Introduction

Rhinocypha (Cowley, 1937), from the family of Chlorocyphidae, is the most characteristic genus of damselflies in tropical Asia. Rhinocyphae is of a very particular nature in their habitat [1] and certain species can adapt to and tolerate disturbed habitats [2]. This characteristic makes them good “thermometers” of environment quality, thus good study subjects for phylogeography [3]. Rhinocypha fenestrella (Rambur, 1842), syn, Aristocypha fenestrella, also known as a peacock jewel, is the most widespread species in the genus, and one of the most active species of the family, which occurs in Peninsular Malaysia, Thailand, Burma, Laos, Vietnam, and southern China [4]. It is one of several common mountain stream damselflies and is usually found in the primary forests [1].

Malaysia is known to be one of the three mega-biodiversity countries in Southeast Asia, however, the phylogeographic pattern of damselflies has been relatively scarce, especially for this particular species. Several phylogeographic studies have been published concerning odonates [5–7], however, until now, no comparable research related to odonate species in Malaysia has been conducted. Instead, the species with a widespread distribution that includes dragonflies normally will have complexes of multiple lineages or variations in the genetic diversity with the geographic region [8–12]. From a point of zoogeography, the Rhinocyphae are of substantial importance, for instance, in the Malay Archipelago, each large island, mostly has its own group of endemic species [1].

A phylogeographic analysis is a powerful method of obtaining insights into the historical processes that have shaped the species’ temporal distribution and genetic variation [13]. Recent phylogeographic research on widely distributed odonatan species has indicated different responses to historical climatic changes in space and time [14,15]. With the advances in molecular techniques, mitochondrial DNA has been identified as an excellent genetic marker of gene flow in matrilineal inheritance [16] and it is the most widely used marker to study the molecular ecology in animal taxa [17,18].

Particularly, cytochrome C oxidase subunit I (cox1) and 16S ribosomal RNA (16S) are known to be reliable genetic markers and the commonly applied markers in Odonata [7,19–21]. In addition to nuclear markers, mitochondrial markers were also provided well-resolved and supported trees from species to family level [22,23]. Given the high resolution of mitochondria-encoded cox1 and 16S genes reported in odonates [24], this study for the first time attempts to characterize the genetic diversity and population structure of R. fenestrella, across its range in Malaysia.

Materials and methods

Sample collection

A total of 147 Rhinocypha fenestrella individuals were collected from eight populations representing eight states in Peninsular Malaysia from the period of 2014 to 2015 with the permission of the Forestry Department Peninsular Malaysia (Permit Number: JH/100 Jld.7 (12)) (Fig 1 and Table 1). The identification of R. fenestrella was performed according to morphological descriptions and taxonomic keys as described by Orr and Hamalainen (2003) [25], and through personal experience. Methods of sampling and preservation of Odonata were based on previously described standard methods [26]. Generally, samples were caught by using a sweep net and were dried preserved for morphological identification, while the legs were removed from each individual and stored in a vial containing 80% ethanol for molecular works.

Table 1. Sampling localities and geographic position of sampling sites of Rhinocypha fenestrella.

State	District	Specific Locality	Geographic position
N. Sembilan	Jelebu	Jeram Toi Waterfall	N 02 51’ 52.7" E 102 00’ 52.0"
Johor	Bekok	Sungai Bantang Waterfall	N 02 20’ 46.7" E 103 09’ 23.9"
Kedah	Baling	Lata Bayu Waterfall	N 05 43’ 02.9" E 100 48’ 50.9"
Perak	Tapah	Lata Iskandar Waterfall	N 04 19’ 27.18" E 101 19’ 31.36"
Kelantan	Machang	Jeram Linang Waterfall	N 05 44’ 33.4" E 102 22’ 26.2"
Terengganu	Kuala Berang	Sekayu Waterfall	N 04 57’ 45.8" E 102 57’ 11.9"
Pahang	Bentong	Chamang Waterfall	N 03 30’ 34.2" E 101 51’ 36.3"
Selangor	Hulu Selangor	Sungai Sendat Waterfall	N 03 24’ 14.4" E 101 41’ 00.5"

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DNA extraction and amplification

Genomic DNA was extracted from four to six legs of each ethanol-preserved specimen using the i-genomic CTB DNA Extraction Mini Kit (iNtRON Biotechnology Inc., Seongnam, South Korea). The DNA amplifications of both cox1 and 16S genes were conducted using an Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems Inc., Foster City, CA, USA) with the amplification protocol consisting of 30 sec at 94°C followed by 35 cycles of 50 sec at 94°C, 50 sec at 50°C and 50 sec at 72°C, and a final 7 min at 72°C. Primers for amplification of the cox1 gene were 5’- GGT CAA CAA ATC ATA AAG ATA TTG G – 3’ for forward primer [27] and 5’- GGA TGG CCA AAA AAT CAA AAT AAA TG –3’ for reverse primer [28]. For the 16S gene, ODO 12852 and ODO 13393 primer sets (forward primer, 5’- AGA AAC CGA CCT GGC TTA AA -3’; reverse primer, 5’- CGC CTG TTT ATC AAA AAC AT -3’) were utilized [7]. Each PCR amplification was performed in a reaction mixture containing 50–100 ng of genomic DNA, 25 μl of NEXpro e-PCR 2X Master Mix (Genes Labs Inc., Gyeonggi-do, South Korea), and 10 pmol of each forward and reverse primer.

DNA purification, sequencing, and alignment

The amplified samples were then electrophoresed on 2% agarose gel pre-stained with SYBR Safe^™ (Invitrogen Corp., Carlsbad, CA, U.S.A.), and the PCR products were sent outsource to a commercial company (Apical Scientific Sdn. Bhd. Selangor, Malaysia) for DNA sequencing in both forward and reverse directions. The samples were sequenced using the BigDyeH Terminator 3.1 Sequencing Kit.

All sequences were assembled and edited using Molecular Evolutionary Genetics Analysis (MEGA) software Version 11.0 [29] and BioEdit 7.2 [30] and preliminarily aligned using CLUSTALX [31]. The aligned sequences were then subjected to a BLAST search (www.ncbi.nlm.nih.gov/blast/) for species validation.

Genetic diversity and haplotype analyses

The aligned cox1 and 16S sequences at first were analysed separately, then were concatenated to yield a total length for further analysis. Molecular characteristics such as the number of haplotypes (N_h), haplotype diversity (H_d), nucleotide diversity (P_i), the number of segregating sites (S), and average number of sequence differences (K) were determined with the program DNASP^® (DNA Sequence Polymorphism) v6.12.03 [32]. DNASP^® was also used to perform neutrality tests including Tajima’s D [33] and Fu’s F_s [34].

The genetic differentiation (F_ST) and gene flow (N_m) pairwise among the R. fenestrella populations were determined using DNASP^®. Whereas the significant level of F_ST was determined using ARLEQUIN v3.5 [35]. The levels of genetic differentiation are defined as F_ST > 0.25 (great differentiation), 0.15 < F_ST < 0.25 (moderate differentiation), and F_ST < 0.15 (negligible differentiation) according to the classification criteria by Wright (1978) [36]. The levels of gene flow are categorized as N_m > 1 (high gene flow), 0.25 < N_m < 0.99 (intermediate gene flow), and N_m < 0.25 (low gene flow) [37]. Molecular variance analysis (AMOVA) between populations was performed using ARLEQUIN v3.5 with 1000 permutations. Uncorrected pairwise distances (p-distance) were assessed using PAUP 4.0B10 to measure the genetic divergence of R. fenestrella. Moreover, the observed and expected distributions of the number of pairwise genetic differences (mismatch distributions) were performed using DNASP^®.

Furthermore, to visualize the phylogeographic pattern between the populations, and to calculate the minimum number of mutational steps between the sequences, haplotype networks were constructed using TCS 1.13^® [38] with a 95% parsimony criterion for both cox1 and 16S respectively, and concatenated cox1+16S genes sequences.

Results

Genetic diversity of Rhinocypha fenestrella

The final lengths of aligned sequence fragment were 614 bp, 534 bp, and 1148 bp for cox1, 16S, and concatenated cox1+16S, respectively. The generated sequences that exhibit unique haplotypes in this study were deposited in the National Center for Biotechnology Information (NCBI) GenBank database under accession numbers KY678719–KY678744 for cox1, and KY678745–KY678754 for 16S genes.

The haplotype diversity (H_d) in a population for the cox1 gene ranged from 0.1947 in the Kedah population to 0.8889 in the Pahang population. Nonetheless, for the 16S gene, it ranged from 0.0000 found in three populations (Johor, Perak, and Selangor) to 0.2842 in the Kelantan population. In concatenated cox1+16S sequence analyses, the overall value of haplotype diversity was 0.8937, and nucleotide diversity (P_i) was 0.0028. Kedah population showed the lowest diversity for both nucleotide and haplotype diversities (H_d = 0.2842, P_i = 0.0003), while the highest haplotype diversity was shown by Pahang (H_d = 0.8889), and the highest nucleotide diversity was presented by the Kelantan population (P_i = 0.0023) (Table 2). In total average data estimates, the cox1 gene revealed higher for both haplotype and nucleotide diversities (H_d = 0.8846, P_i = 0.0051) than did the 16S gene (H_d = 0.1292, P_i = 0.0013).

Table 2. Genetic diversity indices and neutrality test based on cox1, 16S, and concatenated cox1+16S sequences of Rhinocypha fenestrella from eight different populations in Peninsular Malaysia.

Markers	n	Nh	Hd	Pi	S	K	D	Fs
16S rRNA
Johor	20	1	0.0000	0.0000	0	0	np	np
Kedah	20	2	0.1000	0.0002	1	0.1000	-1.1644	-0.879
Kelantan	20	4	0.2842	0.0010	4	0.4947	-1.9857^*	-1.589
N. Sembilan	19	2	0.1053	0.0002	1	0.1053	-1.1648	-0.838
Pahang	20	3	0.1947	0.0054	15	2.7526	-1.2996	5.371
Perak	20	1	0.0000	0.0000	0	0.0000	np	np
Selangor	11	1	0.0000	0.0000	0	0.0000	np	np
Terengganu	20	4	0.2842	0.0031	15	1.5947	-2.4439^*	1.627
OVERALL	150	10	0.1292	0.0013	22	0.6850	-2.3808	-5.488
*cox1*
Johor	20	4	0.4316	0.001	3	0.4684	-1.1914	-1.713
Kedah	20	3	0.1947	0.000	2	0.2000	-1.5128	-1.863
Kelantan	20	4	0.7263	0.003	5	2.0158	1.3167	1.812
N. Sembilan	19	3	0.4328	0.0007	2	0.4561	-0.4849	-0.421
Pahang	18	9	0.8889	0.0040	9	2.4641	-0.2066	-2.838
Perak	20	5	0.5579	0.0013	5	0.7947	-1.3344	-1.711
Selangor	11	3	0.5636	0.0013	3	0.8000	-0.7494	0.158
Terengganu	19	6	0.7778	0.0028	6	1.7193	0.0049	-0.780
OVERALL	147	26	0.8846	0.0051	28	3.0981	-1.1123	-9.824
cox1+16S
Johor	20	4	0.4316	0.0004	3	0.4684	-1.1914	-1.713
Kedah	20	4	0.2842	0.0003	3	0.3000	-1.7233	-2.749
Kelantan	20	8	0.8211	0.0023	10	2.6105	-0.5605	-1.297
N. Sembilan	19	4	0.5088	0.0005	3	0.5614	-0.9407	-1.355
Pahang	18	9	0.8889	0.0021	9	2.4641	-0.2066	-2.838
Perak	20	5	0.5579	0.0007	5	0.7947	-1.3344	-1.711
Selangor	11	3	0.5636	0.0007	3	0.8000	-0.7494	0.158
Terengganu	19	6	0.7778	0.0017	8	1.9298	-0.5366	-0.460
OVERALL	147	32	0.8937	0.0028	37	3.2341	-1.5732	-16.523

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Notes: n = number of sequences; Nh = number of haplotypes; Hd = haplotype diversity; Pi = nucleotide diversity; S = number of segregating sites; K = average number of sequence differences; D = Tajima’s; F_s = Fu’s.

*significant at p<0.05

The results of AMOVA showed that the genetic variation among the R. fenestrella populations accounted for 64.33% of the total variation, which was higher than the 35.67% of genetic variation observed within the populations, indicating that the genetic variation in R. fenestrella mainly occurred among populations (Table 3). The genetic divergence (0.6387), which was measured by the fixation index (FST) showed a great degree of genetic differentiation among R. fenestrella samples in Malaysia.

Table 3. Analysis of molecular variance (AMOVA) for Rhinocypha fenestrella collected from eight populations in Peninsular Malaysia.

Source of Variation	df	Sum of Squares	Variance Components	Percentage of Variance (%)
Among populations	7	149.073	1.12884 Va	64.33
Within populations	139	87.015	0.62601 Vb	35.67
Total	146	236.088	1.75485
Fixation Index	FST	0.63872

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Haplotype variation and distribution

A set of aligned sequences of 147 taxa of the cox1 revealed 26 haplotypes (A1 –A26). For cox1, the most prevalent haplotype was A2 (n = 40) and was found in five populations. The second most-frequent haplotypes were A1, A4, and A14 (n = 15) while the least prevalent haplotypes presented as singleton were A3, A5, A7 –A11, A18 –A20, A23, and A25. Notably, based on the star-like pattern of the haplotype network (Fig 2), the haplotypes A2 –A26 from the cox1 gene were considered to be originated from haplotype A1.

Fig 2 — Each circle represents a haplotype variation, and the size of a circle is proportional to the number of sequences assigned to that haplotype. Circles of the same colour represent haplotypes from the same population. A small black square represents median vectors.

For the 16S gene, the sequences revealed 10 haplotypes (B1 –B10). The most prevalent haplotype was B1 (n = 140) while all other haplotypes except B8 (n = 2), appeared as singleton haplotypes. Haplotype B1 was found in all populations. The haplotype network of 16S gene showed a star-like pattern suggesting haplotype B1 as a common ancestor of all the others (Fig 3). Nevertheless, a median-joining network showed no obvious geographical pattern in haplotype distribution.

Fig 3 — Each circle represents a haplotype variation, and the size of a circle is proportional to the number of sequences assigned to that haplotype. Circles of the same colour represent haplotypes from the same population. A small black square represents median vectors.

The concatenated cox1+16S sequences revealed 32 haplotypes (AB1 –AB32). Haplotype AB2 was the most frequent and the most widespread haplotype based on its prevalence in Malaysia, while there were 18 singleton haplotypes (AB3, AB4, AB6, AB8 –AB12, AB15, AB18, AB20, AB21, AB 23 –AB26, AB29, and AB31 (Table 4). Furthermore, the Kelantan population presented as the most diverse population that had the highest number of haplotypes (N_h = 30) while the least number of haplotypes was presented in the Selangor population (N_h = 11). Haplotype AB1 appeared to be as a common ancestor of the other haplotypes as indicated by the star-like pattern of concatenated cox1+16S haplotype network (Fig 4). Consequently, based of sampling sites, this study revealed that a recent common ancestor of the R. fenestrella species in Malaysia existed from the Negeri Sembilan population that constituted the most numbers of haplotype AB1.

Table 4. Haplotype distribution of Rhinocypha fenestrella (n = 147) from Peninsular Malaysia based on the concatenated cox1+16S sequences corresponding to the eight populations.

Population	n	Haplotype cox1+16S
Population	n	AB1	AB2	AB3	AB4	AB5	AB6	AB7	AB8	AB9	AB10	AB11	AB12	AB13	AB14	AB15	AB16	AB17	AB18	AB19	AB20	AB21	AB22	AB23	AB24	AB25	AB26	AB27	AB28	AB29	AB30	AB31	AB32
N. Sembilan	19	13	4	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Johor	20	-	-	-	-	15	1	3	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Kedah	20	-	17	-	-	-	-	-	-	1	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Perak	20	-	13	-	-	-	-	-	-	-	-	-	1	1	4	1	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Kelantan	20	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	13	7	1	5	1	1	1	1	-	-	-	-	-	-	-	-	-
Terengganu	19	-	4	-	-	-	-	-	-	-	-	-	-	-	-	-	8	-	-	3	-	-	2	-	1	1	-	-	-	-	-	-	-
Pahang	18	1	1	-	-	-	-	-	-	-	-	-	-	-	-	-	2	-	-	-	-	-	-	-	-	-	1	3	3	1	5	1	-
Selangor	11	-	-	-	-	-	-	-	-	-	-	-	-	1	-	-	-	-	-	-	-	-	-	-	-	-	-	7	-	-	-	-	3
TOTAL	147	14	39	1	1	15	1	3	1	1	1	1	1	2	4	1	23	7	1	8	1	1	3	1	1	1	1	10	3	1	5	1	3

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n = number of sequences

Fig 4 — Each circle represents a haplotype variation, and the size of a circle is proportional to the number of sequences assigned to that haplotype. Circles of the same colour represent haplotypes from the same population. A small black square represents median vectors.

Haplotype genetic divergence

The p-distance between R. fenestrella haplotypes for cox1 ranged from 0.16%– 1.63% with the highest value showed pairwise between haplotypes A5 with A15, A17, A19, and between haplotypes A7 with A15, A17, A19 (S1 Table). On the other hand, the genetic divergence of 16S haplotypes ranged from 0.01%– 0.75% with the highest value presented pairwise between haplotypes B5 and B7 (S2 Table). For the concatenated cox1+16S, the genetic divergence ranged from 0.09% to 0.97% (S3 Table).

In concatenated cox1+16S sequences, the neutrality test of Fu’s F_s showed negative values in all populations with an exception for Selangor. Additionally, Tajima’s D tests for all populations were indicated by negative values and were not statistically significant. The overall F_ST is 0.6387 and N_m is 0.14, suggesting that the genetic differentiation was great while there was a low gene flow among the populations of R. fenestrella in Malaysia. Based on Table 5, the highest F_ST value (0.92494) appeared between Negeri Sembilan and Johor populations while the lowest value (0.03561) was between the populations of Kelantan and Terengganu. Moreover, in mismatch distribution analysis, the actual distribution curve showed a bimodal characteristic indicating no recent population expansion. Nevertheless, based on the graph pattern, historically, the population distribution undergone sharply increased, sudden decreased and then increased (Fig 5).

Table 5. Pairwise genetic differentiation (F_ST) among Rhinocypha fenestrella in eight distinct populations of Peninsular Malaysia.

Population	S1	S2	S3	S4	S5	S6	S7	S8
[S1] N. Sembilan		+	+	+	+	+	+	+
[S2] Johor	0.92494		+	+	+	+	+	+
[S3] Kedah	0.58987	0.93701		+	+	+	+	+
[S4] Perak	0.49448	0.90132	0.08772		+	+	+	+
[S5] Kelantan	0.50648	0.81452	0.41789	0.39192		–	+	+
[S6] Terengganu	0.50694	0.84408	0.37966	0.34860	0.03561		+	+
[S7] Pahang	0.32321	0.80936	0.16887	0.09112	0.28709	0.23649		+
[S8] Selangor	0.69758	0.91882	0.65154	0.49765	0.48615	0.49847	0.23087

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F_ST value on the below diagonal, and upper diagonal showing the significance (+, p<0.05)

Fig 5 — Note: The solid red line represents the actual observed distribution; the blue dashed line represents the expected distribution.

Discussion

Genetic diversity of Malaysian Rhinocypha fenestrella

Throughout the last five decades, the understanding of the ecology and evolution of odonates has increased dramatically. Recent advances in molecular techniques have inspired several phylogeographical studies of Odonata using genetic data [24,39,40]. As is known, most of the odonates have varying levels of dispersal abilities [41] that could influence the genetic diversity and phylogeographic structures of the populations. A less-mobile species may be expected to show some evidence of haplotype clustering according to geographic region [42]. Therefore, in this study, we defined the intraspecific genetic diversity, phylogeographical patterns, and mitochondrial variations of the Rhinocypha fenestrella using cox1 and 16S genes.

The variability of obtained sequences among all studied individuals for both markers ranged from 1.5–4.6% reflected in 28 variable nucleotide sites in 614 sequenced base pairs from cox1 gene, and eight variable nucleotides in 534 base pairs from 16S gene. The base composition of both cox1 and 16S gene sequences showed a significant A+T nucleotide bias which is consistent with insect mitochondrial genes [39,43,44].

In the present study, overall genetic diversity (H_d = 0.8937; P_i = 0.0028) of Malaysian R. fenestrella based on the concatenated cox1+16S sequences was slightly lower if compared to the study that did on the damselfly Matrona basilaris populations from mainland China [45]. The study reported higher values of haplotype diversity (H_d = 0.9750) and nucleotide diversity (P_i = 0.0049) by using cox1 sequences. Nevertheless, the genetic diversity of R. fenestrella in Malaysia can be considered as high when compared to other damselflies species from the same sub-order Psolodesmus mandarinus (MacLahlan, 1870) from Taiwan that reported haplotype diversity as 0.43 and nucleotide diversity as 0.0017 based on cox1+16S sequences [46].

Among the sampling locations, Pahang population showed the highest haplotype numbers. Pahang, being a largest state in Malaysia, may have diverse geographic features such as rivers, mountains, and forests that probably contributed to the isolation of populations, hence promoting genetic divergence, and increasing the likelihood of different haplotypes. Geographical barriers known to restrict the movement of individuals between populations, reducing the gene flow. Consequently, limited gene flow allows for the accumulation of genetic differences between populations, leading to increased genetic diversity over time [47].

Haplotype variation and distribution of Rhinocypha fenestrella

In this study, 26 haplotypes were revealed by cox1, 10 haplotypes by 16S and 32 haplotypes by concatenated sequences. The number of haplotypes detected in this study was higher compared to other similar studies by Lin et al. (2012) [46] that reported only 14 haplotypes variation in the damselfly population of Psolodesmus mandarinus from Taiwan. The high numbers of haplotypes are due to a high degree of cox1 and 16S gene polymorphisms in R. fenestrella samples in Peninsular Malaysia. In fact, the polymorphism that occurred in Odonata were widely reported in other studies [48,49]. Whereas this study showed extremely low haplotype numbers when compared to study done by Jiang et al. (2023) [50] on the population of damselfly Ischnura senegalensis (Rambur, 1842) that revealed of 51 haplotype variations.

The haplotype AB2 appeared as the most dominant haplotype, while the limited geographical distribution of some of the haplotypes including those with singleton sites suggests the existence of genetic differentiation within the populations. In this study, haplotype AB1 was suggested as the common ancestor which appear as central haplotype producing a star-like radiation in the haplotype network, indicating the divergence of other haplotypes from its polymorphic sequence. This haplotype probably had eventually evolved over time into the numerous haplotypes (AB2 –AB15) to adapt to the habitat and demographic changes and consequently distributed across states in Peninsular Malaysia. Likewise, the most recent common ancestor for R. fenestrella in Malaysia may be derived from Negeri Sembilan as this state is constituted by high frequencies of the AB1 haplotypes.

Nevertheless, the network revealed no apparent geographical pattern, indicating a lack of genetic structuring across different populations in Peninsular Malaysia. The median-joining network revealed a close relationship among haplotypes, suggesting that R. fenestrella populations in Malaysia shared a recent history without long-term genetic isolation. Nevertheless, the ancestral haplotype AB1 of concatenated cox1+16S dataset was having a limited distribution with restricted geographic division. This indicates that geographic barriers and climatic factors could also have little influence on the dispersion of this damselflies into different habitats in Malaysia besides their less ability to disperse due to weak flyers [51].

In mismatch distribution analysis, the peak curves indicated that the R. fenestrella populations had underwent an expansion process though the graph represented a bimodal characteristic which indicate no recent population expansion. The hypothesized that the bimodal pattern of mismatch distribution shown by the R. fenestrella populations could probably be due to the low migration rate of the samples within the studied locations. This aligned with the behaviour of the species that known as weak flyer and lack of dispersion capability [51].

Genetic distance and differentiation

In our dataset, the highest genetic distance value based on cox1 gene was 0.16%, while 16S gene was 0.75%. The data shows a higher genetic distance among Malaysian R. fenestrella as compared to the populations of Rhinocypha taiwana (Wang & Chang, 2013), Rhinocypha uenoi (Asahina, 1964), and Rhinocypha drusilla (Needham, 1930) from China where their highest genetic distances obtained based on cox1 gene were 0.00%, 0.00% and 0.50%, respectively [52]. However, the genetic distance of Malaysian R. fenestralla was relatively lower if compared to other dragonfly populations of Trithemis stictica (Burmeister, 1839) in Namibia [9] and Nannophya pygmaea (Rambur, 1842) in Malaysia [53] that presented the genetic distance of cox1 gene of up to 9.00% and 12.00%, respectively.

Based on AMOVA analysis, the major variation observed in this study was among the populations, indicating that samples have differentiated into separate genetic pools that could lead to genetic fragmentation, hence, genetic variation has led to a high level of differentiation among populations. Additionally, N_m<1 indicates of insufficient gene flow between populations leading to genetic differentiation and genetic isolation. Rhinocypha fenestrella is known as a weak flyer [51,54] and is unlikely to migrate over large-scale regions, which likely contributes to its overall high genetic differentiation to happen among the populations. Therefore, it is not surprising that genetic differentiation happened in the species R. fenestrella in which their populations were separated up to more than 500 km. In fact, the migratory behaviour of odonate could homogenize genetic differentiation among populations by the exchange of individuals and genes among populations with high genetic differentiation as observed in the population of migratory dragonflies Libellula quadrimaculata (Linnaeus, 1758) [39] and Pantala flavescens (Fabricius, 1798) [55] respectively, which are known as strong fliers than the damselflies.

Dispersal ability and long-distance migration are the most important factors contributing to a high level of gene flow and consequent slowing or limiting of geographic differentiation [56,57]. A less-mobile species may be expected to show some evidence of haplotype clustering according to geographic region due to less ability to disperse and therefore resulted in genetic isolation by distance. In this study, calculated F_st value shows high genetic differentiation in the populations of R. fenestrella in Malaysia. This high value is consequently supported by the low rate of gene flow found amongst the R. fenestrella populations in Malaysia as well as between the different sites.

When all eight populations were regarded as a whole, Tajima’s D statistic was statistically not significant. The results showed that these populations were in a stable state with no demographic expansion and no recent bottleneck. Whereas, in the Selangor population, Fu’s F_S statistic values were negative with p-values being significant (p < 0.02) which shows this population had experienced a recent population expansion. Our results suggested that R. fenestrella population in Selangor could successfully colonize and adapt to new habitats and were able to disperse randomly and exchange genes with local populations. Maintaining genetic diversity is noteworthy as it related to population viability [58–60] and also to the transformative potential of a species to react to the environmental changes [61,62].

Overall, this study found strong evidence for intraspecific patterns of haplotype variation among populations of R. fenestrella in Malaysia though the weak dispersal abilities of the species. The study also revealed high genetic differentiation within the populations and a low rate of gene flow among the geographically difference populations of Malaysian R. fenestrella. Moreover, a high haplotype number was observed in R. fenestrella population, indicates the existence of genetic isolation within the populations of the sampling sites.

Although many phylogeographical mechanisms have been proposed using damselflies as model organisms, more detailed sampling and a larger variety of ecological investigations are required to promote better understanding. Therefore, this study contributed new insight for an advanced understanding of the evolution and phylogeography of damselfly. Nevertheless, additional sequence data from other regions outside of Malaysia (e.g.: Thailand, Burma, Laos, Vietnam, and southern China), which is considered a gap of knowledge due to the lack of genetic data on this particular species, may prove useful in detecting phylogenetic relationships and phylogeography patterns, and revealing common ancestors of these populations between the other regions and continents.

A comprehensive understanding of genetic diversity is crucial for effective biodiversity conservation. The findings from this study may contribute to broader initiatives aimed at preserving biodiversity in the region especially in Peninsular Malaysia. Different populations may have unique ecological adaptations, and management efforts can consider these variations for more effective conservation. Moreover, monitoring genetic diversity over time can help assess the impact of environmental changes on the damselfly population. The information obtained from this study could have implications for understanding the species’ ability to adapt to changing environments.

Supporting information

S1 Table. Percentage (%) of uncorrected “p” distance matrix among the 26 representative cox1 haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s001.pdf^{(127.7KB, pdf)}

S2 Table. Percentage (%) of uncorrected “p” distance matrix among the 10 representative 16S rRNA haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s002.pdf^{(73.2KB, pdf)}

S3 Table. Percentage (%) of uncorrected “p” distance matrix among the 32 representatives for the concatenated cox1+16S haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s003.pdf^{(166.1KB, pdf)}

Acknowledgments

The authors would like to thank Mr. Mohaiyidin Mohamed and Mr. Mohd Fauzi Abd Hamid for their assistance in the fieldwork.

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

Author who received the funding award= Norma-Rashid Yusoff; Grant numbers awarded to the author= 1; The full name of funder= The Universiti Malaya Research Grant (UMRG) PG065 – 2015 and ST061-2022; URL of each funder website= https://umhvl.um.edu.my/grants-and-projects-awarded; Role of funder: Study design, conceptualization, data collection and analysis, preparation of the manuscript draft.

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PLoS One. doi: 10.1371/journal.pone.0301392.r001

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25 Sep 2023

PONE-D-23-28970Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Odonata: Chlorocyphidae)PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: No

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: No

Reviewer #2: No

**********

5. Review Comments to the Author

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

Reviewer #1: The number of populations and sequences seem appropriate, and the analyses are acceptable. However, MANOVA and Mantel tests should also be performed. There are no conclusions about the divergence pattern. The discussion is somewhat chaotic and not sound, a lot more should be said on one hand, and several repeated statements should be eliminated, also some contradictory opinions. Please see my comments in the text. I am not a native speaker, but English should be corrected - at some parts it is simply impossible to understand the text. I have made some suggestions, but these are only suggestions, and much more should be corrected. Also some strange terminology can be found, like "uncorrected genetic diversity".

Reviewer #2: In the present study the genetic diversity and phylogeography of the Rhinocypha fenestrella were studied. The authors used the cytochrome C oxidase I and 16S rRNA gene regions from 147 individuals representing eight populations in Malaysia. The research is interesting, but to make it suitable for printing, additions and corrections are needed, however, I believe that not all of them are possible. The work has flaws that are difficult to correct. Eight places are not enough to draw phylogeographic conclusions ( I am of course aware of all the problems that may arise during field work), however, the number of individuals per population is sufficient to conduct basic population analyses. The second problem can be removed - the authors used only fragments of mtDNA, so they are actually analyzing the phylogeny of the maternal line. Perhaps it would be worth adding markers for nuclear genes, histone, 18S, 28S, they should be sequenced relatively quickly, and they would undoubtedly significantly increase the value of the work. There is also a problem with the low observed diversity. In fact, the work describes small variability within undoubtedly one species based only on mtDNA and it is not possible to draw general conclusions on this basis. Taking the above into account, it may be worth considering publishing the results in less reputable journals than PlosOne

Other comments:

- authors should also use BOLD Systems for sequences identification

- MUSCLE is better than CLUSTAL X

- the authors should perform a full phylogenetic analysis using maximum likelihood and Bayesian approaches (maybe some mOTUs will be showed) with comparison to closely related species and detailed analysis. As I have already mentioned, small genetic diversity and a small number of sites may make such analyzes much more difficult

- the sequence length is given unnecessarily in two different places

- number of sequences and the GB numbers are results not methods

- comparing genetic distances and Fst with geographic distances between populations should enrich the results

- sites should be marked on map, this same for haplotypes, or maybe mOTUs?

- based on their data, the authors drew too far-reaching conclusions about the evolutionary history of the species under study

I am not native spiker, but in my opinion some linguistic corrections should be made

**********

6. 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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Reviewer #1: Yes: Andrzej Falniowski

Reviewer #2: No

**********

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Attachment

Submitted filename: PONE-D-23-28970_reviewer_af.pdf

pone.0301392.s004.pdf^{(2.3MB, pdf)}

PLoS One. 2024 Apr 5;19(4):e0301392. doi: 10.1371/journal.pone.0301392.r002

Author response to Decision Letter 0

8 Oct 2023

The detail responses to the editors and reviewers have been provided as attached file (see .doc file with title "Response to Reviewers". Thank you.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0301392.s005.docx^{(34.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0301392.r003

Decision Letter 1

Tzen-Yuh Chiang

12 Dec 2023

PONE-D-23-28970R1Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)PLOS ONE

Dear Dr. Abdul Wahid,

Please submit your revised manuscript by Jan 26 2024 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'.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

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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 #3: (No Response)

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

Reviewer #6: All comments have been addressed

Reviewer #7: All comments have been addressed

**********

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

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Partly

Reviewer #6: Yes

Reviewer #7: Partly

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

6. Review Comments to the Author

Reviewer #3: The manuscript contains very useful information regarding the genetic population structure of the damselfly Rhinocypha fenestrella in Malaysia. However, there are several issues that should be addressed by the authors before the manuscript is accepted for publication

1. Include the country where the study was conducted in the title

2. The abstract does not contain a statement of the problem

3. Include in the abstract the implications of these results

4. Page 2, line 16, you are talking about dragonflies - the title is about damselflies - note that damselflies are completely different from dragonflies

5. The introduction is very short – the biology, particularly life history of the studied species is not explained in the introduction

6. Page 4, line 9, what was the concentration of ethanol used to preserve the samples

7. Page 6, line 9 – 12, focus on the p-value. Check whether there Fst values are significant – forget about the classification

8. Correct grammatical errors in page 6, lines 23 – 24

9. Table 2, the measured Hd for Johor, Perak and Selangor based on 16S is zero – this could be attributed to low sample size. Example, looking at Table 3, it appears, you only sampled 11 individuals from Selangor. Do you this this number is enough to give you meaningful comparisons of the indices of genetic diversity between populations?

10. Subtitles in page 6 line 22 and page 8 line 2 have the same meaning

11. State whether the Fst values in Table 4 are significant. If they are not significantly different from zero – then your explanations in page 12, lines 2 – 7 do not make any sense

12. Page 12 line 8, was the Fus’ values significant?

13. Page 12, line 11 to 12, correct grammatical errors

14. Page 12, line 12 to 15, look at the values you are calling high and low – (0.0100 and 0.0010). Are they significantly different from zero? If they are not significantly different from zero, then it suggests that these populations are genetically homogenous. Please – revisit the interpretation of your results

15. Correct grammatical errors in page 12 line 18

16. Page 13, line 21, comparing diversity indices from different markers does not make sense

17. Page 14, line 5, high compared to what?

18. Page 14, line 11, you are comparing different and unrelated species

19. Page 14, line 14 – 15, check the interpretation – it is not correct. Having singletons in a population does not necessarily mean the population is isolated from the others

20. Page 15, lines 7 – 15 requires serious revisions – interpretations given here are not correct

21. Page 16, lines 7 - 8, revisit your interpretations

22. Page 16, lines 12 and 13, interpretations of Tajima values should be rechecked

23. Interpretations of results in page 16, lines 23 – 25 are conflicting each other

24. Phylogenetic analysis of Rhinocypha fenestrella is missing in the entire document. Revise the title or do phylogenetic analysis of the studied species. Note - This was advised by the previous reviewer. The author just ignored this comment but his defense of this omission lacks coherence and fails to address the critical need for this analysis. Further attention is warranted to rectify this deficiency and enhance the study's comprehensiveness.

25. What are the implications of this study on the management of aquatic ecosystems?

Reviewer #4: Dear Editor in chief,

I have reviewed the manuscript; several flaws must be must properly be taken care of.

1. Why did the author use a concatenated sequence for their analysis? All the discussion and interpretation of the results were based on concatenated sequences.

2. In Table 2, The number of sequences (N) should also be used in each sampling site for each marker.

3. In Table 3, edit (n) the number of sequences. Also, they should mention the number of haplotypes for each site on the right side of the table.

4. Please discuss the possible reason for the highest number of haplotypes in Pahang.

5. Please discuss why the diversity in Selangor, with the lowest number of haplotypes, was higher than in Kedah.

6. Why didn't the authors consider sequences from other countries? They should include other sequences from neighboring countries to show the gene flow and genetic diversity between Malaysia and other countries.

7. They should also use a phylogenetic tree to illustrate their result better.

Reviewer #5: This manuscript aimed to explore the genetic diversity and observed phylogeogrphic pattern of Rhinocypha fenestrella using two different markers along Malay peninsula. Frankly, the revised manuscript appears to be much better. However, I still find some points can be improved and some analysis need to be added into this MS, and I believe that it will make this MS more attractive and informative. I have added some comments into the file, please see the attached. Also some major concerned are listed below:

1. I think instead of showing only haplotype network (Figure 2-4), It must be nice if authors can add the map showing haplotype distribution in to each figure. It will be easy to see clearly about the haplotype diversity and promotion in each population.

2. As I found author responded to one of reviewer that this paper aim to emphasize on genetic diversity and population structure analysis. I think AMOVA analysis is really needed to be done. Also mismatch distribution or skyline plot should be calculated to see demographic history of this species in Malaysia too.

3. I don't think DNAsp is suitable to use for Fst calculations as it did not provide significant level. Please rerun Fat in Alrequin or GenALEx.

4. Author mentioned low gene flow, but table 4 show very low genetic differentiation between the population. I think it is incongruous. Please reanalyze.

Good luck

Reviewer #6: REF. Manuscript Number: PONE-D-23-28970R1

The manuscript entitled “Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)” by Mamat-Noorhidayah et al. demonstrated the study on population structure and haplotype diversity of the damselfly, R. fenestrella. The revised manuscript has been made accordingly to the editor and reviewer’s comments. The current form of the paper is well written with acceptable English present. The rationale, scientific logic, methods, and analysis of data are appropriate.

Reviewer #7: The findings about the intraspecific genetic diversity, phylogeographic trend, and the ancestral haplotype of the peacock jewel-damselfly are remarkable. However, the authors need to address the following concerns:

1. Deviation from a standard neutral model is restrictive per gene and more informative when each gene is analyzed separately. Concatenated data may give a false impression of Tajima’s D, Fu’s and Li, Fu’s or Fu and Li’s D*, Fu and Li’s F*, and Fu’s FS statistic.

Abstract

2. Page 1, Line 24-25 needs to be rephrased for clarity.

Methodology

3. Confirm the 300 sec on pcr protocol.

4. …..The aligned cox1 and 16S sequences at first were analysed respectively…… were analysed separately….

5. The FST and Nm pairwise values were calculated to access genetic differentiation and gene flow among the R. fenestrella population and were calculated using DNASP®… Rephrase the sentence for clarity.

Discussion

1. …..The obtained sequences for cox1 and 16S genes from eight populations ranged from 1.5……….. Rephrase the sentence for clarity.

2. In this study, the number of haplotypes detected was considered high with 26 haplotypes were revealed by cox1 and 10 haplotypes by 16S. Rephrase the sentence for clarity.

3. Page 14 Line 6-7 The high number is due to and not “may indicate” ..... It is an indicative of high diversity in species.

4. Page 14 Line 14 …..with singleton sites…….. also include Reference attributing presence of singleton sites to current genetic differentiation.

5. …..to star-like radiation in……This is not an indicator of common ancestry…….Is a common ancestry due divergence of haplotype from its polymorphic sequence.

6. ….Likewise, according to location20 based, the most recent common ancestor for R. fenestrella in Malaysia may be derived from Negeri Sembilan as this state is constituted by high frequencies of the common ancestor AB1 haplotypes. Rephrase the sentence to avoid repetition.

7. ….The data shows a higher genetic distance among Malaysian R. fenestrella as compared to the populations of Rhinocypha taiwana (Wang & Chang, 2013), Rhinocypha uenoi (Asahina, 1964), and Rhinocypha drusilla (Needham, 1930) from China where the highest genetic distances based on cox1 gene were 0.00%, 0.00% and 0.50%, respectively……….0.00% is not high

8. ……cox1 gene of up to 9.00% and 12.00%, respectively……………..

9. …..Zygopterans are known as weaker fliers since many species do not diffusing outside several kilometres……rewrite for clarity.

**********

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Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: No

Reviewer #7: Yes: Kevin O. Ochwedo

**********

Attachment

Submitted filename: comments.docx

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Attachment

Submitted filename: PONE-D-23-28970_R1-Review.pdf

pone.0301392.s007.pdf^{(1.6MB, pdf)}

PLoS One. 2024 Apr 5;19(4):e0301392. doi: 10.1371/journal.pone.0301392.r004

Author response to Decision Letter 1

29 Feb 2024

The authors would like to thank to all reviewers for acknowledge our great efforts on preparing this manuscript. We highly appreciated all the given comments to improvise the writing and content of the manuscript in which we believe that our revised manuscript is now have met the standard requirements to be published in PLOS ONE journal where the knowledge and information rehashed and discussed in this manuscript will be of great value to readers and researchers alike. We have addressed all the comments and the responses to the comments point to point are available in attached document.

Attachment

Submitted filename: Response to Reviewers-R2.docx

pone.0301392.s008.docx^{(42KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0301392.r005

Decision Letter 2

Tzen-Yuh Chiang

14 Mar 2024

Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)

PONE-D-23-28970R2

Dear Dr. Abdul Wahid,

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

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at http://www.editorialmanager.com/pone/ and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #4: All comments have been addressed

Reviewer #6: All comments have been addressed

Reviewer #7: All comments have been addressed

**********

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

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

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

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: Yes

**********

6. Review Comments to the Author

Reviewer #4: As they did not include the sequences of other countries, it would be better to mention the country name in the title.

Reviewer #6: The authors have response to all comments and suggestions. The paper has been improved and it current form is accepted for publication.

Reviewer #7: The authors have adequately addressed all the comments raised. The current form of the manuscript, "Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)," is well written, and all the previously noted typos have been corrected.

**********

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 #4: Yes: Elham Kazemirad

Reviewer #6: No

Reviewer #7: Yes: Kevin Omondi Ochwedo

**********

PLoS One. doi: 10.1371/journal.pone.0301392.r006

Acceptance letter

Tzen-Yuh Chiang

20 Mar 2024

PONE-D-23-28970R2

PLOS ONE

Dear Dr. Azrizal-Wahid,

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

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

* All relevant supporting information is included in the manuscript submission,

* There are no issues that prevent the paper from being properly typeset

If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps.

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If we can help with anything else, please email us at customercare@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Tzen-Yuh Chiang

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Percentage (%) of uncorrected “p” distance matrix among the 26 representative cox1 haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s001.pdf^{(127.7KB, pdf)}

S2 Table. Percentage (%) of uncorrected “p” distance matrix among the 10 representative 16S rRNA haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s002.pdf^{(73.2KB, pdf)}

S3 Table. Percentage (%) of uncorrected “p” distance matrix among the 32 representatives for the concatenated cox1+16S haplotypes of Rhinocypha fenestrella in Malaysia.

(PDF)

pone.0301392.s003.pdf^{(166.1KB, pdf)}

Attachment

Submitted filename: PONE-D-23-28970_reviewer_af.pdf

pone.0301392.s004.pdf^{(2.3MB, pdf)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0301392.s005.docx^{(34.2KB, docx)}

Attachment

Submitted filename: comments.docx

pone.0301392.s006.docx^{(14.1KB, docx)}

Attachment

Submitted filename: PONE-D-23-28970_R1-Review.pdf

pone.0301392.s007.pdf^{(1.6MB, pdf)}

Attachment

Submitted filename: Response to Reviewers-R2.docx

pone.0301392.s008.docx^{(42KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Genetic diversity and phylogeographic patterns of the peacock jewel-damselfly, Rhinocypha fenestrella (Rambur, 1842)

Mamat Noorhidayah

Noor Azrizal-Wahid

Van Lun Low

Norma-Rashid Yusoff

Roles

Abstract

Introduction

Materials and methods

Sample collection

Fig 1. Map of sampling sites and haplotype distribution (AB1–AB15) of concatenated cox1+16S sequences for Rhinocypha fenestrella from eight sampling locations representing eight states of Peninsular Malaysia.

Table 1. Sampling localities and geographic position of sampling sites of Rhinocypha fenestrella.

DNA extraction and amplification

DNA purification, sequencing, and alignment

Genetic diversity and haplotype analyses

Results

Genetic diversity of Rhinocypha fenestrella

Table 2. Genetic diversity indices and neutrality test based on cox1, 16S, and concatenated cox1+16S sequences of Rhinocypha fenestrella from eight different populations in Peninsular Malaysia.

Table 3. Analysis of molecular variance (AMOVA) for Rhinocypha fenestrella collected from eight populations in Peninsular Malaysia.

Haplotype variation and distribution

Fig 2. Median joining haplotype network of Rhinocypha fenestrella of cox1 sequences isolated from eight different states in Peninsular Malaysia.

Fig 3. Median joining haplotype network of Rhinocypha fenestrella of 16S sequences isolated from eight different states in Peninsular Malaysia.

Table 4. Haplotype distribution of Rhinocypha fenestrella (n = 147) from Peninsular Malaysia based on the concatenated cox1+16S sequences corresponding to the eight populations.

Fig 4. Median joining haplotype network of Rhinocypha fenestrella of concatenated cox1+16S sequences isolated from eight different states in Peninsular Malaysia.

Haplotype genetic divergence

Table 5. Pairwise genetic differentiation (FST) among Rhinocypha fenestrella in eight distinct populations of Peninsular Malaysia.

Fig 5. Distribution curves of mismatch distribution for cox1+16S analysis of Rhinocypha fenestrella populations based on pairwise differences among haplotypes.

Discussion

Genetic diversity of Malaysian Rhinocypha fenestrella

Haplotype variation and distribution of Rhinocypha fenestrella

Genetic distance and differentiation

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Tzen-Yuh Chiang

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Author response to Decision Letter 0

Decision Letter 1

Tzen-Yuh Chiang

Roles

Author response to Decision Letter 1

Decision Letter 2

Tzen-Yuh Chiang

Roles

Acceptance letter

Tzen-Yuh Chiang

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Table 5. Pairwise genetic differentiation (F_ST) among Rhinocypha fenestrella in eight distinct populations of Peninsular Malaysia.