Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini across Indonesia and the Western Indian Ocean

Sutanto Hadi; Noviar Andayani; Efin Muttaqin; Benaya M Simeon; Muhammad Ichsan; Beginer Subhan; Hawis Madduppa

doi:10.1371/journal.pone.0230763

. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini across Indonesia and the Western Indian Ocean

Sutanto Hadi ¹, Noviar Andayani ^2,³, Efin Muttaqin ³, Benaya M Simeon ³, Muhammad Ichsan ³, Beginer Subhan ¹, Hawis Madduppa ^1,^*

Editor: Susana Caballero⁴

PMCID: PMC7529310 PMID: 33002022

Abstract

Scalloped Hammerhead shark (Sphyrna lewini) is an endangered species which its populations have been declining globally including in Indonesia, the world’s top shark fishing country. However, there is a lack of information on the recent population structure of this species to promote proper management and its conservation status. This study aimed to investigate the genetic diversity, population structure, and connectivity of the S. lewini population, in three major shark landing sites: Aceh (n = 41), Balikpapan (n = 30), and Lombok (n = 29). Meanwhile, additional sequences were retrieved from West Papua (n = 14) and the Western Indian Ocean (n = 65) populations. From the analyses of the mitochondrial CO1 gene, a total of 179 sequences of S. lewini, with an average size of 594 bp, and 40 polymorphic loci in four and eight haplotypes for the Indonesian population and the Western Indian Ocean population were identified. The overall values of genetic diversity were high (h = 0.717; π = 0.013), with the highest values recorded in Aceh (h = 0.668; π = 0.002) and the lowest in Papua (h = 0.143; π = 0.000). On the contrary, the overall value was fairly low in the Western Indian Ocean (h = 0.232; π = 0.001). Furthermore, AMOVA and F_ST showed three significant subdivisions in Indonesia (F_ST = 0.442; P < 0.001), with separated populations for Aceh and West Papua, and mixed between Balikpapan and Lombok (F_ST = 0.044; P = 0.091). In contrast, genetic homogeneity was observed within the population of the Western Indian Ocean (F_ST = –0.013; P = 0.612). The establishment of a haplotype network provided evidence of a significantly different population and a limited genetic distribution between the Indonesian and the Western Indian Ocean populations (F_ST = 0.740; P < 0.001). This study showed the presence of a complex population of S. lewini with limited connectivity only in Indonesia separated from the Western Indian Ocean and requiring specific management measures based on the population structure at the regional level.

Introduction

The scalloped hammerhead shark, Sphyrna lewini is considered a coastal species because of its need for nursery areas. Globally, it is distributed in tropical waters as well as on the mainland, islands, and near the coastal region [1, 2]. This species has the unique modification of a lateral head which improves the ability to navigate and follow geomagnetic orientations across the ocean [3–5]. S. lewini can move in high rates of dispersal, and its female show allegiance to single nursery areas and exhibit no evidence of continued inter-oceanic migration. On the contrary, male is spread over a large area across ocean waters, with clear evidence of cross-reproduction and gamete transmission [6].

Scalloped hammerhead is one of the most exploited and threatened sharks. Around one to three million sharks are killed each year because of fishing and the shark fin trade around the world [7, 8]. This species was considered to be underexploited in 1999. However, in 2009 the International Union for Conservation of Nature (IUCN) listed the species on the Red List with Endangered (EN) status [9]. Five years later, the Convention on International Trade in Endangered Species (CITES) listed hammerhead sharks in Appendix II, and in 2019 the status was upgraded to Critically Endangered (CR) [10].

High exploitation of the S. lewini has an impact on its population structure, reducing the fecundity of the species and the genetic diversity [11]. Hammerhead sharks are viviparous with a yolk-sac placenta with an annual number of 12–30 young per litter. This species has a slow growth rate, late sexual maturity, long gestation period, and a long lifespan in nature [12–15]. The combination of high pressure and their biological properties makes this species vulnerable to overexploitation.

The study of population genetics has become an important tool for understanding population connectivity, supporting fisheries management, and improving conservation strategies. Furthermore, genetic information can be used to define the conservation effort and course of action by studying the structure of shark populations [16–19].

Shark fin product, including from scalloped hammerhead are very popular in Hong Kong [20], where trade regulations for endangered species and effective regulations are promoted [16, 20, 21]. The population structure of S. lewini, which is important for fisheries stock management, has been widely investigated in different coastal areas and ocean basins on a global and regional scale [17–19, 22, 23]. Duncan et al. [22] reported a global phylogeographic study of S. lewini that indicated that the Indo-West Pacific region is the center of diversity for tropical sharks, such as S. lewini, with a high and unique genetic diversity; however, no samples from Indonesia were included in that study, or in the study reported by Ovenden et al. [23], which included limited samples from Indonesia.

This study aimed to investigate the genetic diversity, population structure, and connectivity of S. lewini, where the populations of this species are affected by fishing activities at a regional scale in the Western Indian Ocean. Finally, the implications of these results for species management and conservation were examined.

Material and methods

Ethics statement

All samples were already dead when collected, and therefore, no approval from any institutional animal ethics committee was required. The sample collection and transportation followed the regulation of the Ministry of Marine Affairs and Fisheries of the Republic of Indonesia (Number 5/PERMEN-KP/2018) on the prohibition of cowboy and hammerhead shark export from Indonesia. Furthermore, this study was approved by the Ministry of Marine Affairs and Fisheries of the Republic of Indonesia, under permission numbers 276/BPSPL.03/PRL/X/2018 and 319/PNK/BPSPL.03/PK.230/REKOM/X/2018.

Tissue sample collection

From October 2017 to November 2018, a total of 100 tissue samples were obtained from S. lewini, including 41 from the fishing ports of Meulaboh and Aceh Jaya, 30 from a local shark landing in Manggar, and 29 from the fishing port of Tanjung Luar (Table 1). The samples (~0.5 cm³) were dissected and preserved in sample bottles containing 96% ethanol.

Table 1. Sampling collection sites at major shark landing sites in Indonesia.

Site	Geographic coordinate	Number of samples
Aceh (ACH)
Meulaboh	N 04° 08’ 29” E 96° 07’ 55”	33
Aceh Jaya	N 04° 38’ 34” E 95° 34’ 58”	8
Balikpapan (BPN)
Manggar	S 01° 12’ 53” E 116° 58’ 24”	30
Lombok (LOM)
Tanjung Luar	S 08° 46’ 39” E 116° 31’ 01”	29
Total		100

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DNA extraction, amplification, and sequencing

DNA extraction was performed at the Biodiversity and Biosystematics Laboratory, IPB University, according to the protocol of the gSYNC DNA extraction kit product. A fragment of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene was amplified using the forward primer fish-BCL (5'–TCA ACY AAT CAY AAA GAT ATY GGC AC–3′) and the reverse fish-BCH (5'–ACT TCY GGG TGR CCR AAR AAT CA–3′) [24, 25] in a 24 μL reaction mixture consisting of 3 μL of DNA template, 12.5 μL of MyTaq HS Red Mix, 9 μL of ddH₂O, 1.25 μL each of forward and reverse primers. Meanwhile, the reaction mixture was processed in a polymerase chain reaction (PCR) on a thermocycler using modified cycling conditions [26, 27]: pre-denaturation at 94°C for 15 s. This process was followed by 38 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 45 s; as well as a final extension at 72°C for 10 min. In addition, the amplicons were visualized by 1.5% agarose gel electrophoresis added with ethidium bromide at 100 V for 20 min. The gel was observed under UV light to identify bands showing the presence of DNA fragments. Sequencing was also performed using a machine with an optimized protocol of Sanger method [28].

All laboratory protocols on sampling and DNA identification methods were deposited in protocols.io platform with a digital object identifier (DOI) available at dx.doi.org/10.17504/protocols.io.bfwmjpc6.

Data analysis

Genetic diversity

Over 179 mitochondrial CO1 DNA sequences with an average length of 594 bp were edited and aligned using the ClustalW algorithm [29] implemented in MEGA 6.06 [30]. Genetic diversity parameters, such as the number of haplotypes and diversity (h) as well as nucleotide diversity (π), were calculated using the DNASp v6 [31] and Arlequin v.3.5 program [32]. Furthermore, additional CO1 sequencing data of S. lewini from West Papua retrieved from GenBank (Table 2) (n = 14) were reanalyzed. These days were obtained by Sembiring et al. [33], sequences from previous studies performed in India (n = 6) [34], the United Arab Emirates (n = 30) [35], and Madagascar (n = 29) [36] to assess the genetic diversity in Indonesian and Western Indian Ocean populations.

Table 2. Localities, the total number (n), and accession number of CO1 gene sequences of S. lewini from Aceh, Balikpapan, Lombok, and Western Papua (Indonesia), India, the United Arab Emirates, as well as Madagascar (Western Indian Ocean).

Locality	n	Accession number	Author
Indonesia
Aceh	41	MT324149-156, MT324187-219	This study
Balikpapan	30	MT324157-186	This study
Lombok	29	MT324220-248	This study
West Papua	14	KF590254-55, KF590271-76, KF793729, KF793738-42	[33]
Western Indian Ocean
India	6	KF899746-51	[34]
United Arab Emirates	30	KP177238-41, KP177241, KP177254, KP177262, KP177272, KP177285-99, KP177300-07	[35]
Madagascar	29	HQ171735-47, HQ171761-76	[36]

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Population structure

An analysis of molecular variance (AMOVA) and fixation index (F_ST) [37] was performed for three major groups: 1) within and among the four populations from Indonesia, 2) within and among the three populations from the Western Indian Ocean, and 3) comparison between populations from Indonesia and Western Indian Ocean using the Arlequin v.3.5 program (set up, 1000 permutations; significance level threshold, α = 0.05). These two analyses allowed the estimation of the overall extent of the genetic variation and differentiation level in Indonesia and the Western Indian Ocean. Furthermore, population differentiation and its significance between sampling sites were also calculated with pairwise estimates [38–40].

Genetic connectivity

A haplotype network was constructed with a median-joining method in the Network v5.1.1.0 program [41] for all haplotypes detected in Indonesia and the Western Indian Ocean. This network aimed to obtain haplotype connectivity following a broader spatial connection in the regional area of the Indian Ocean. The distribution of haplotypes for each location was also provided in a proper map to show the clear distributions and genetic connectivity among the populations.

Results

Genetic diversity

All sequences of S. lewini obtained were deposited in the BOLD System with the Barcode Index Number (BIN) registry of BOLD: AAA2403 and database of GenBank with accession numbers MT324149-248 (Table 2). A total of 179 sequences of 594 bp mitochondrial CO1 gene was obtained from three sampling sites (Aceh, Balikpapan, and Lombok). Meanwhile, additional sequences of samples from West Papua and the Western Indian Ocean region were used to generate a total of 11 haplotype variations with 40 polymorphic loci (Table 3).

Table 3. Forty polymorphic loci of 11 haplotypes from 179 sequences of the mitochondrial CO1 gene of S. lewini samples from four localities in Indonesia and three localities in the Western Indian Ocean region.

Haplotypes	Locus Position
Haplotypes	1	15	24	27	33	45	48	63	69	105	120	138	141	156	165	174	186	198	229	279	289	303	306	325	339	342	346	366	396	420	453	456	459	483	495	498	522	531	534	543
H1^*	A	T	A	C	T	T	G	T	T	A	T	C	G	T	T	C	C	T	T	C	G	T	T	C	C	A	C	T	T	C	C	T	T	C	T	C	C	T	C	T
H2^*	.	.	.	A	.	.	.	.	.	G	.	A	T	.	C	T	T	C	C	T	.	C	C	T	T	C	.	C	C	T	.	.	.	A	.	T	.	C	.	C
H3	.	.	.	A	.	.	.	.	C	G	.	A	T	.	C	T	T	C	C	T	.	C	.	T	T	C	.	C	C	T	.	.	.	A	.	T	.	C	.	.
H4^*	.	.	.	A	.	.	.	.	.	G	.	A	T	.	C	T	T	C	C	T	.	C	C	T	T	C	.	C	C	T	.	.	.	A	.	T	.	C	.	.
H5	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.	G	.	.	.	.	.
H6	.	.	.	.	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H7	.	.	C	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H8	.	C	.	.	C	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H9	.	.	.	.	.	.	.	.	.	.	G	.	.	.	.	.	.	G	.		T	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	G	.
H10	G	.	.	.	.	.	.	G	.	.	.	.	.	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
H11	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	T	.	.	.	T	.	.	.	C	.	T	.	.	.

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Notes:

Nucleobase at each position is given for H1 while those different are written for all other haplotypes. Nucleobases identical to H1 are indicated with dots (.)

*Three original haplotypes of the S. lewini populations were obtained from the areas of study (Aceh, Balikpapan, and Lombok) in Indonesia. The remaining haplotypes were reanalyzed from previous studies.

The comparison of the genetic diversity of S. lewini following haplotype and nucleotide diversity showed the presence of variation (Table 4). The haplotype diversity (h) among the samples obtained from Indonesia ranged from 0.143 to 0.668, while the nucleotide diversity (π) ranged from 0.000 to 0.020. The highest genetic diversity was observed for the samples from Aceh (h = 0.668; π = 0.020), followed by the Balikpapan population, which exhibited a lower haplotype and nucleotide diversity (h = 0.646; π = 0.002). On the contrary, the lowest genetic diversity was detected in West Papua (h = 0.143, π = 0.000). Similarly, the S. lewini population from Lombok exhibited a fairly low genetic diversity (h = 0.362; π = 0.001). However, the overall diversity in Indonesia was relatively high (h = 0.717; π = 0.013) since the average in the Western Indian Ocean region was low and ranged from 0.000 to 0.467. Therefore, it is reasonable to conclude that the overall diversity in the Western Indian Ocean region was also low (h = 0.232; π = 0.001).

Table 4. Genetic diversity of S. lewini based on sample size (n), haplotype number (Hn), haplotype diversity (h), and nucleotide diversity (π) in samples from each site in Indonesia and the Western Indian Ocean region.

Population	n	Genetic Diversity
Population	n	Hn	h	Π
Indonesia
Aceh (ACH)	41	4	0.668	0.020
Balikpapan (BPN)	30	3	0.646	0.002
Lombok (LOM)	29	3	0.362	0.001
West Papua (WEP)	14	2	0.143	0.000
Overall Indonesia	114	4	0.717	0.013
Western Indian Ocean
India (IND)	6	1	0.000	0.000
United Arab Emirates (UAE)	30	8	0.467	0.002
Madagascar (MDG)	29	1	0.000	0.000
Overall Western Indian Ocean	65	8	0.232	0.001

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Population structure

The analysis of the fixation index (F_ST) and the corresponding P-values between and within the four S. lewini populations (ACH, BPN, LOM, and WEP) from Indonesia and three populations (IND, UAE, and MDG) from the Western Indian Ocean region are shown in Table 5. The overall F_ST value in Indonesia was significantly higher than that observed in other regions (F_ST = 0.442; P < 0.001) due to the presence of multiple subdivisions.

Table 5. Analysis of molecular variance (AMOVA) for the percentage of variation (%), F_ST value, and significance level (P-value) in S. lewini samples from Indonesian, the Western Indian Ocean, and between Indonesian and Western Indian Ocean populations.

Source of variation	df	Percentage of variation (%)	F_ST value	P-value
Indonesia
Among Populations	3	44.15	0.442	0.000
Within Populations	110	55.85	0.442	0.000
Total	113
Western Indian Ocean
Among Populations	2	−1.31	−0.013	0.612
Within Populations	62	101.31	−0.013	0.612
Total	64
Indonesia vs. Western Indian Ocean
Among Populations	1	74.04	0.740	0.000
Within Populations	177	25.96	0.740	0.000
Total	178

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A genetic homogeneity was observed in the population from the Western Indian Ocean region (F_ST = –0.013; P = 0.612). However, a comparison of the population structure between Indonesia and the Western Indian Ocean region yielded significant differentiation (F_ST = 0.740; P < 0.001). The pairwise F_ST values between the populations from the four locations in Indonesia and the Western Indian Ocean population are shown in Table 6. Furthermore, the overall pairwise analysis through the distance method showed the presence of significant differentiation among the four populations. In contrast, BPN and LOM (F_ST = 0.044; P = 0.091) showed fairly low F_ST values and no significant P-values. Furthermore, among the populations from Indonesia, the ACH showed a trend of being closer to the Western Indian Ocean since it exhibited a lower F_ST and significant P-value (F_ST = 0.509; P < 0.001).

Table 6. Pairwise F_ST values (below the diagonal) and P-values (above the diagonal) between the S. lewini populations from Aceh (ACH), Balikpapan (BPN), Lombok (LOM), West Papua (WEP), and Western Indian Ocean (WIO).

Sample Sites	ACH	BPN	LOM	WEP	WIO
ACH	-	0.000	0.000	0.000	0.000
BPN	0.427	-	0.091	0.000	0.000
LOM	0.438	0.044	-	0.000	0.000
WEP	0.398	0.495	0.736	-	0.000
WIO	0.509	0.965	0.973	0.975	-

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Genetic connectivity

Network analysis of the haplotype identified two main groups of haplotypes (Fig 1) referred to as clade A and B. Clade A consisted of haplotype H1, which was observed in several regions, i.e., Aceh, India, United Arab Emirates, and Madagascar, while H5, H6, H7, H8, H9, H10, and H11 were present only in samples from the United Arab Emirates. Clade B consisted of three haplotypes (H2, H3, and H4), which were spread evenly in Indonesia. Haplotypes H1, H3, and H4 were predominant in Aceh, West Papua, and Balikpapan-Lombok, respectively (Fig 2).

Discussion

Genetic diversity

The overall genetic diversity of S. lewini at the haplotype and nucleotide levels was relatively high for populations in Indonesia. These findings are consistent with the results reported by Ovenden et al. [23] regarding the mitochondria control region from three localities in Indonesia [22]. The scalloped hammerhead sharks are a highly migratory species with a wide distribution in tropical and warm-temperate waters. This specie can move across oceanic waters to a distance of up to 1671 km [12]. Due to its migratory ability and broad ecological niches, this species tends to have higher genetic diversity than others [42]. Generally, high levels of genetic diversity are associated with large population size [43] and are promoted by several factors, such as local population sizes, fast generation times [44], high nucleotide substitution rates [45], and high gene flow between geographically distant populations.

The finding of a relatively high genetic diversity for S. lewini appears to be inconsistent on the assumption that overexploitation of this species as both a target of fishing and bycatch led to the decline of its populations on a global scale [46]. However, the results obtained from Lombok may be relevant since the lower genetic diversity detected was probably driven by continuous fishing pressure. S. lewini species are the top three targeted sharks at the Tanjung Luar fishing port in Lombok and have faced high fishing pressure over more than 40 years with recent exploitation rates (E) reaching 0.59 [47].

Furthermore, according to the global fisheries information system on a global scale and Indonesia by FAO [48], the hammerhead sharks (Sphyrnidae), including S. lewini, are very important. These species were highly exploited in the last two decades, with an estimated rapid increase in global capture, from 220 tons per year in 1985 up to approximately 10,362 in 2016. During the same period, the capture level also increased significantly, reaching approximately 1,492 tons in 2016. Meanwhile, Indonesia recorded one of the highest numbers of sharks and rays caught on the global catches reported in 2000–2011 [49].

Clarke et al. [50] reported similar findings regarding the mitochondrial DNA of the silky shark, Carcharhinus falciformis, which exhibited a high genetic diversity under circumstances of overexploitation. Elasmobranchs exhibit adaptability to environmental and anthropogenic stresses, which causes genetic bottlenecks because of their particular life histories [8]. However, the population decline caused by recent fishery activities might be insufficient to reduce genetic diversity, particularly for species with a long life span (13–20 years), such as S. lewini [51, 52].

The persistent decline, as predicted for the Lombok population of S. lewini, correlated positively with the loss of genetic diversity and created a bottleneck [52, 53], as reported by Pinsky and Palumbi in a meta-analysis of several marine fish [54].

Population structure

The obtained results showed the presence of homogeneity among the S. lewini populations from Balikpapan and Lombok. The pairwise F_ST analysis detected no significant genetic differentiation in these two populations with the lowest value. These findings complement previous studies conducted in Indo-Australian waters. Similarly, Ovenden et al. [23] reported evidence for the mitochondrial control region regarding the structure, with no differentiation between two populations of S. lewini (Lombok and northern Australia). This pattern of single-stock population suggests that these localities are a migration zone of S. lewini and a reproductive movement may occur in their coastal areas. However, there are strong Indonesian through flow currents between Kalimantan and Sulawesi Island. Adult S. lewini specimens are highly migratory, with a large body supporting the high dispersal ability of this species. Consequently, they show the possibility of overcoming that geographical barrier. In contrast, different results from the comparison among these two populations from central Indonesia and one from Aceh (western Indonesia) as well as from West Papua (eastern Indonesia), which exhibited strong genetic differentiation were obtained. Furthermore, these regions were spatially separated by a long distance, with the possibility of the existence of more complex barriers. These barriers can be inter-island or anthropogenic factors on a high commercial and artisanal fishing pressure along the southern and northern coasts of Java.

Genetic connectivity

Regarding the significant value of F_ST among the population (F_ST = 0.740; P < 0.001) as well as haplotype network and distribution shown in Figs 1 and 2, two restricted haplogroups which separated the S. lewini population in Indonesian with the Western Indian Ocean population were observed. The two haplogroups were separated with 19 different nucleotide bases due to monomorphic and polymorphic mutations. Furthermore, limited gene flow that occurs among populations in Indonesia forms a different pool with the Western Indian Ocean population. However, this was expected because of the complex geographic barrier in Indonesia’s marine ecosystem, and the global distribution pattern of S. lewini, with significant separation population across ocean basin as well as discontinuous coastline habitat [6].

Generally, S. lewini populations from Balikpapan, Lombok, and West Papua appear to be isolated from the Western Indian Ocean and shared a haplotype network exclusively only in eastern Indonesian waters. However, an interesting result regarding genetic sharing between the populations from Aceh and the Indian Ocean population was obtained. H1 is a unique haplotype that was only obtained in Aceh. However, the F_ST value observed significant differences between the population in Aceh and the Western Indian Ocean, and there was an indication of genetic sharing between those localities (F_ST = 0.509; P < 0.001). The similarity between the predominant haplotype (H1) of S. lewini from Aceh and that of the populations from India, the United Arab Emirates as well as Madagascar reflected a genetic sharing process in the Indian Ocean region. This showed the presence of past historical gene flow between the populations in spatially separated regions driven by ancestral interaction [17]. However, recent studies reported that the scalloped hammerhead demonstrated a strong differentiation in population structure across ocean basins e.g. Indian Ocean and discontinuous continental coastlines, as shown by the separation between Aceh and Indian coastline [6, 22].

Conservation implications

The high diversity of the S. lewini populations in Indonesia shows that this species has not experienced a genetic loss because of exploitation pressure. However, the lower genetic diversity of S. lewini from Lombok and West Papua showed a higher risk of loss, which probably was the result of high fisheries pressure. Furthermore, the genetic assessment of S. lewini samples from four localities showed that a single stock exists between Lombok and Balikpapan. On the contrary, a separate stock was observed for Aceh and West Papua, showing that the management of this species should occur on a stock-based approach at least on three mitochondrial-stock conservation units. The complex population of S. lewini with limited connectivity observed in Indonesia and the Western Indian Ocean region demonstrated the importance of promoting specific collaborative management strategies among Indonesian, and in conjunction with Western Indian Ocean agencies at the regional scale.

Conclusion

This study provided important findings on the population structure of S. lewini in Indonesia, with a high genetic diversity and three significant subdivisions. The results showed the capability of the population to adapt to rapid environmental changes and pressure, including fishing activities. In addition, the lower genetic diversity in Lombok and West Papua was also considered. The restricted genetic sharing detected among the species obtained from Indonesia showed unique features among these populations. Therefore, a specific collaborative action across regions is needed to promote sustainable management and conservation purposes, both in Indonesia and at the regional scale in the Western Indian Ocean area.

Acknowledgments

The authors are grateful to the institutions and individuals that have made the study possible: colleagues from Marine Biodiversity and Biosystematics Lab (BIODIVSI), Faculty of Marine Science and Technology, IPB University for their help during this study. The authors are further grateful to BPSPL Satker Balikpapan, Listian Nova, and Mr. Hery, Head of TPI Manggar, Balikpapan for their help during the sample’s collection, and to reviewers and proofreaders that have provided highly constructive suggestions for better writing of the study.

Data Availability

All relevant data are within the paper. All sequences of S. lewini obtained were deposited in the BOLD System with the Barcode Index Number (BIN) registry of BOLD: AAA2403 and database of GenBank with accession numbers MT324149-248.

Funding Statement

This research was supported by Wildlife Conservation Society (WCS)-Indonesia, in collaboration with Faculty of Fisheries and Marine Sciences IPB University. SH received a scholarship from Indonesia Endowment Fund for Education (LPDP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0230763.r001

Decision Letter 0

Susana Caballero

18 Mar 2020

PONE-D-20-06557

Genetic connectivity and diversity of endangered species the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population in Indonesia and Western Indian Ocean

PLOS ONE

Dear Dr. Madduppa,

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.

This is a very interesting manuscript and it will be definitely useful in the future for comparisons for example in market surveys. However, overall the quality of the english needs to be improved. I think once this has been taken care of other details regarding presentation of results, etc will be much clearer to review. Also, figures need to be improved, since resolution is low and they look grainy. Attached you will find a file with some suggestions. I work on this particular subject (conservation genetics of aquatic vertebrates) and I have a lot of experience in sharks, so I can really see the potential in this manuscript, but english needs to be improved a lot.

We would appreciate receiving your revised manuscript by 20th of April. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Susana Caballero, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

This is a very interesting manuscript and it present very important information that will help understanding the genetic diversity of scalloped hammerhead in this region and this information will be likely used for future assignment comparisons for example for market surveys etc. However, the english NEEDS to be improved. I really hope the authors can find the help of a native english speaker that can help them with this. I wrote some things in the PDF I am attaching but it definitely needs an overall improvement of the english.

The resolution of the figures needs to be improved because they look grainy.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript
A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)
A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

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

"This research was supported by Wildlife Conservation Society (WCS)-Indonesia, in collaboration with Faculty of Fisheries and Marine Sciences IPB University. Sutanto Hadi received a scholarship from Indonesia Endowment Fund for Education (LPDP)."

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

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

"NO - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

5. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

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Reviewers' comments:

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: PONE-D-20-06557REVed.pdf

Click here for additional data file.^{(1.5MB, pdf)}

PLoS One. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763.r002

Author response to Decision Letter 0

7 Apr 2020

Dear Susana Caballero, PhD

Academic Editor

PLOS ONE

Thank you for reviewing and suggesting our manuscript:

PONE-D-20-06557

Genetic connectivity and diversity of endangered species the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population in Indonesia and Western Indian Ocean

PLOS ONE

I would like to response your comments:

Additional Editor Comments (if provided):

1. This is a very interesting manuscript and it present very important information that will help understanding the genetic diversity of scalloped hammerhead in this region and this information will be likely used for future assignment comparisons for example for market surveys etc. However, the english NEEDS to be improved. I really hope the authors can find the help of a native english speaker that can help them with this. I wrote some things in the PDF I am attaching but it definitely needs an overall improvement of the english.

Thank you for your interest and suggestion. We have improved overall of the English for better manuscript after proof read by native English speaker.

2. The resolution of the figures needs to be improved because they look grainy.

Thank you for your suggestion. We have improved the resolution of figures and generated in PACE to meet PLOS ONE requirement.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

We have read and followed the format as The PLOS ONE style templates

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

We have provided additional location information including geographic coordinates in a table in manuscript

We have asked our colleague to check and edit our manuscript,

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

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

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:

"NO - The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Thank you for your suggestion. We have removed any funding-related statement from manuscript.

https://www.youtube.com/watch?v=_xcclfuvtxQ

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

Reviewers' comments:

Thank you for your suggestion. We have uploaded and adjusted the pictures to PACE to meet PLOS ONE figures requirement. We attached the picture in submission.

Attachment

Submitted filename: 1. Response to Reviewers (1).docx

Click here for additional data file.^{(19.3KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0230763.r003

Decision Letter 1

Susana Caballero

10 Apr 2020

PONE-D-20-06557R1

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and Western Indian Ocean

PLOS ONE

Dear Dr. Maddupa,

There are still a number of grammatical english mistakes along the manuscript that need to be corrected. I would suggest looking into a service that revises manuscripts. sometimes local Universities have this service in place in language schools. I can see some mistakes for example in the results section, when you present results from the AMOVA. This is very important because these results need to be very clear for the readers.

Also, I think it is good to submit your sequences not only to BOLD but also to Genbank, since for genetic studies, it is high likely that researchers will look for information for comparisons on Genbank.

We would appreciate receiving your revised manuscript by 10 of May, particularly considering the current situation regarding Covid-19. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Susana Caballero, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

There are still a number of grammatical errors. I think it is important to have a service look over this manuscript.

Please submit the sequences you generated to Genbank as well as BOLD, since probably more people will have an opportunity to look for these sequences in this database

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

PLoS One. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763.r004

Author response to Decision Letter 1

25 Apr 2020

Dear Susana Caballero, PhD

Academic Editor

PLOS ONE

Thank you for reviewing and suggesting our manuscript:

“Genetic connectivity and diversity of endangered species the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and the Western Indian Ocean”

I would like to response your comments:

Additional Editor Comments (if provided):

This is a very interesting manuscript and it present very important information that will help understanding the genetic diversity of scalloped hammerhead in this region and this information will be likely used for future assignment comparisons for example for market surveys etc. However, the English NEEDS to be improved. I really hope the authors can find the help of a native English speaker that can help them with this. I wrote some things in the PDF I am attaching but it definitely needs an overall improvement of the English

Thank you for your interest and suggestion. We have improved overall of the English for better manuscript after proof read by professional native English speaker.

The resolution of the figures needs to be improved because they look grainy.

Thank you for your suggestion. We have improved the resolution of figures and generated in PACE to meet PLOS ONE requirement.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: ttp://journals.plos.org/plosone/s/submission-guidelines#loc laboratory-protocols

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). This letter should be uploaded as separate file and labelled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labelled 'Revised Manuscript with Track Changes'.

•An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labelled 'Manuscript'.

Journal Requirements:

When submitting your revision, we need you to address these additional requirements:

http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliat

ions.pdf

Thank you for your suggestion. We have read and followed the format as The PLOS ONE style templates.

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

Thank you for your suggestion. We have provided additional location information including geographic coordinates in a table in manuscript

Thank you for your suggestion. We have sent and proof read our manuscript to a professional scientific editor to check and enhance our manuscript grammar, spelling and language use.

Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

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

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:

"NO - The funders had no role in study design, data collection and analysis, decision to

publish, or preparation of the manuscript."

Thank you for your suggestion. We have removed any funding-related statement from the

manuscript.

Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account:

https://www.youtube.com/watch?v=_xcclfuvtxQ

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

Reviewers' comments:

Thank you for your suggestion. We have uploaded and adjusted the pictures to PACE to meet PLOS ONE figures requirement. We attached the picture in submission.

Please send them (sequence data) to GenBank as well.

Thank you for your suggestion. We have deposited our all sequences data to Genbank as well with accession number MT324149 to MT324248.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0230763.r005

Decision Letter 2

Susana Caballero

30 Apr 2020

PONE-D-20-06557R2

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and the Western Indian Ocean

PLOS ONE

Dear Dr.Madduppa,

The English still needs further improvement and I think the authors need to further analyze their genetic connectivity section. In the discussion, it does not reflect some of the findings you get, particularly from your haplotype network. Please read my suggestions on the attached PDF.

We would appreciate receiving your revised manuscript by 15th May 2020. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Susana Caballero, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

This is a very interesting paper, but I am still finding mistakes in the english. Also, the authors need to do further interpretation of their results, particularly regarding the population connectivity. I am including the text with my suggestions.

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

Attachment

Submitted filename: PONE-D-20-06557_R2SCcomm.pdf

Click here for additional data file.^{(2.8MB, pdf)}

PLoS One. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763.r006

Author response to Decision Letter 2

7 May 2020

To:

Susana Caballero, PhD

Academic Editor

PLOS ONE

Thank you for reviewing and suggesting our manuscript:

“Genetic connectivity and diversity of endangered species the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and the Western Indian Ocean”

I would like to response your comments in the following systematic table format:

1st SUBMISSION RESPONSE

No. REVIEWER COMMENT AUTHOR RESPONSES

Additional Editor Comments

(if provided)

1. This is a very interesting manuscript and it present very important information that will help understanding the genetic diversity of scalloped hammerhead in this region and this information will be likely used for future assignment comparisons for example for market surveys etc. However, the English NEEDS to be improved. I really hope the authors can find the help of a native English speaker that can help them with this. I wrote some things in the PDF I am attaching but it definitely needs an overall improvement of the English

Thank you for your interest and suggestion. We have improved overall of the English for better manuscript after proof read by professional native English speaker.

2. The resolution of the figures needs to be improved because they look grainy Thank you for your suggestion. We have improved the resolution of figures and generated in PACE to meet PLOS ONE requirement.

3. We would appreciate receiving your revised manuscript by 20th of April. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

4. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: ttp://journals.plos.org/plosone/s/submission-guidelines#loc laboratory-protocols

5. 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). This letter should be uploaded as separate file and labelled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labelled 'Revised Manuscript with Track Changes'.

•An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labelled 'Manuscript'.

No. Journal Requirements Author Responses

1. When submitting your revision, we need you to address these additional requirements:

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliat

ions.pdf

Thank you for your suggestion. We have read and followed the format as The PLOS ONE style templates.

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

Thank you for your suggestion. We have provided additional location information including geographic coordinates in a table in manuscript.

Thank you for your suggestion. We have sent and proof read our manuscript to a professional scientific editor to check and enhance our manuscript grammar, spelling and language use.

4. Upon resubmission, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

• A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

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

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:

"NO - The funders had no role in study design, data collection and analysis, decision to

publish, or preparation of the manuscript."

Thank you for your suggestion. We have removed any funding-related statement from the

manuscript.

6. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.

Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account:

https://www.youtube.com/watch?v=_xcclfuvtxQ

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

2nd SUBMISSION RESPONSE

No. Reviewers' comments Author Responses

1. 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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Thank you for your suggestion. We have uploaded and adjusted the pictures to PACE to meet PLOS ONE figures requirement. We attached the picture in submission.

2. which populations have been declining worldwide Thank you for your correction, we have revised the sentence

3. use h instead of Hd Thank you for your correction, we have replaced all of term “Hd” with “h”

4. is considered coastal due to their need for nursery areas Thank you for your suggestion, we used term “semi-oceanic” as stated in some journals which have the similar meaning that you suggested, so we agreed and have revised the sentence

5. shorter phrases (Line 47-51) Thank you for your correction. We have shortened the phrases.

6. were already dead when collected Thank you for your correction. We have revised the sentence.

7. sample (Line 83) Thank you for your correction

8. 100 samples of which length?

Thank you for your question, we used 594 bp of nucleotide and 179 sequences in total, not 100. We have revised it in the sentence

9. you previously mention a 100 (sequences)

Thank you for your correction. We used 179 sequences in total, we have revised in previous statement

10. please send them (sequence data) to genbank as well Thank you for your suggestion. We have deposited our all sequences data to Genbank as well with accession number MT324149 to MT324248.

11. use identifiers particular for your area of study so that they dont get confused with other previously published Thank you for your suggestion. We used * (asterisk) as identifiers of original haplotype from area of study at notes section below the table.

12. repeat “find and finding” (Line 290)

Thank you for your correction. We have revised the sentence.

remove any funding-related text from the manuscript Thank you for your correction. We have removed any funding-related text.

No. Reviewers' comments Author Responses

2nd Submission

1. like before: usually 13-16 words,

title is too long Thank you for your correction. We have tried to shorter the title

2. which will make co-management actions necessary across

regions (Line 38-39) Thank you for your correction in Line 38-39.

3. I think the introduction too short.

According to your title you could write some more about the

management used now and what are the points of

improvement or change in your eyes for S. lewini Thank you for your correction. We have tried to enrich the introduction with what this study can give suggestion for better shark management and conservation

What?

..additional.. (Line 177) Thank you for your correction in Line 177.

What..? were displayed? That’s not a

sentence here (Line 181) in Table Thank you for your suggestion. We used * (asterisk) as identifiers of original haplotype from area of study at notes section below table.

One or more? Than add an s Thank you for your correction. We have revised the sentence.

One or more? Add s Thank you for your correction. We have revised the sentence.

What?? Not a sentence here.. (Line 280) Thank you for your correction. We have revised the sentence.

Here smaller font size 11 before 12!

check Thank you for your correction. We have fixed the font size.

Check fond size here again its

smaller!! (Line 290,291,302) Thank you for your correction. We have fixed the font size.

One or more? Add s Thank you for your correction.

Strange phrase here..I dont know

what that should mean..

Thank you for your correction. We have revised the sentence.

3rd SUBMISSION RESPONSE

No. Additional Editor Comments

(if provided) Author Responses

1. 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.

2. The English still needs further improvement and I think the authors need to further analyze their genetic connectivity section. In the discussion, it does not reflect some of the findings you get, particularly from your haplotype network. Please read my suggestions on the attached PDF. Thank you for your correction. We have added some sentences in discussion to reflect our genetic data particularly in connectivity section.

3. We would appreciate receiving your revised manuscript by 15th May 2020. When you are 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.

4. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

5. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Laboratory protocols

To enhance the reproducibility of your results, we recommend and encourage you to deposit laboratory protocols in protocols.io, where protocols can be assigned their own persistent digital object identifiers (DOIs).

To include a link to a protocol in your article:

1. Describe your step-by-step protocol on protocols.io

2. Select Get DOI to issue your protocol a persistent digital object identifier (DOI)

3. Include the DOI link in the Methods section of your manuscript using the following format provided by protocols.io: http://dx.doi.org/10.17504/protocols.io.[PROTOCOL DOI]

At this stage, your protocol is only visible to those with the link. This allows editors and reviewers to consult your protocol when evaluating the manuscript. You can make your protocols public at any time by selecting Publish on the protocols.io site. Any referenced protocol(s) will automatically be made public when your article is published. Thank you for your suggestion. We have deposited a short laboratory protocol in protocols.io platform and added the DOI number at the end of Method section, Line 125-127.

6. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

Thank you for your suggestion. We have tried to reorganize all comments and our responses in this table to make better history of publication process.

No. Reviewers' comments Author Responses

1. of a haplotype (typo A) Thank you for your correction. We have fixed it.

2. explain how genetic information can be used to trave back the origin of shark find from this species found in markets, particularly in Asia. Cite Fields et al 2020, in Animal Conservation, a manuscript that came out this week.

Thank you for your suggestion. We have added introduction in Line 53-67 and cited from Fields et al and Jabado et al in Line 68-75.

DNA extraction

Thank you for your correction. We have fixed it.

3. I dont think you need to inlude this, as this is know by population geneticists and also because it will also depend of the probability of significance.

Thank you for your suggestion. We agreed with your suggestion, we have omitted the sentence.

4. No significant differentiation was observed

(Line 203) Thank you for your correction. We have revised the sentence.

5. significant differentiation Thank you for your correction. We have revised the phrase.

6. differentiation Thank you for your correction. We have revised it.

7. include number of Fst and p value

Thank you for your suggestion. We have added value of Fst and p in the sentence Line 210.

8. is a highly Thank you for your correction. We have revised the phrase.

9. what type of fisheries, be more specific

Thank you for your suggestion. We have added more specific information in Line 275.

“according to the global fisheries information system on hammerhead shark capture production 1950-2017 on global scale and Indonesia by FAO”

10. you have many nucleotide changes between your haplotypes from Indonesia and those from the Western Indian Ocean...what could be happening there? is there really gene flow with the West Indian Ocean? I think gene flow will be restricted, otherwise you would not find such differentiated haplotypes. Re -think this section and go deeper into your results

Thank you for your correction. We have stated some sentences in Line 318-330 regarding haplotype differentiation between two haplogroup between Indonesia and western Indian Ocean, and we also highlight in next sentence 331-347, an indication on genetic sharing between population in Aceh and Western Indian Ocean which have similarity in H1 (haplotype one).

11. and gene flow

Thank you for your correction. We have added the phrase in Line 339.

Attachment

Submitted filename: 1. Response to Reviewers (1).docx

Click here for additional data file.^{(31.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0230763.r007

Decision Letter 3

Susana Caballero

13 May 2020

PONE-D-20-06557R3

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and the Western Indian Ocean

PLOS ONE

Dear Dr.Madduppa,

There are still issues with the way some of the results, particularly in the abstract, are written. You use, for example, insignificant, that are not the proper way to refer to your results from a population genetics perspective. Also, be careful when you make assumptions such as the fact that haplotypes being shared in a network are proof of current gene flow. This result could also be the result of ancestral sharing of haplotypes. I am including a PDF with these observations and I ask you to consider them carefully and improve these points.

We would appreciate receiving your revised manuscript by 25th of May 2020. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Susana Caballero, PhD

Academic Editor

PLOS ONE

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

Attachment

Submitted filename: PONE-D-20-06557_R3 (1)SCComm.pdf

Click here for additional data file.^{(3MB, pdf)}

PLoS One. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763.r008

Author response to Decision Letter 3

25 Jun 2020

4th SUBMISSION RESPONSE

No. Reviewers' comments Author Responses

1. be careful when you explain your findings. You never talk about insignificant differentiation. You say non-significant and siginificantly different. This mistake was already in the previous version. You can look as example sifferentpapers on genetics to see examples on how to write this.

Thank you for your correction.

We have tried to look other paper as a reference to fix it.

2. in a network you can say haplotype sharing but be careful when you say gene flow...haplotype sharing can be the result of gene flow OR from ancestra sharing of alleles.

Thank you for your suggestions.

We have detected haplotype sharing between population in Aceh and the Western Indian Ocean, but as you said maybe it is not as a result of gene flow. We have added some sentences to reflected our result in abstract and discussion in Genetic Connectivity section.

3. The combination (Line 65) Thank you for your correction. We have fix it.

4. protocols (plural) (Line 125)

Thank you for your correction. We have fixed it.

5. what about the probability? was it significant or non-significant?

(Line 214) Thank you for your question.

It was significant, we have written the value of Fst and P.

Attachment

Submitted filename: 1. Response to Reviewers.docx

Click here for additional data file.^{(34.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0230763.r009

Decision Letter 4

Susana Caballero

2 Jul 2020

PONE-D-20-06557R4

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini (Griffith & Smith 1834) population across Indonesia and the Western Indian Ocean

PLOS ONE

Dear Dr. Madduppa,

Your submission still requires substantial editing for English grammar and usage. We ask that you please have the manuscript copyedited by either a native-English speaking colleague or a professional copy-editing service. While you may approach any qualified individual or any professional scientific editing service of your choice, PLOS has partnered with American Journal Experts (AJE) to provide discounted services to PLOS authors. AJE has extensive experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. If the PLOS editorial team finds any language issues in text that AJE has edited, AJE will re-edit the text for free. To take advantage of this special partnership, use the following link: https://www.aje.com/go/plos/.

Please submit your revised manuscript by Aug 16 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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

We look forward to receiving your revised manuscript.

Kind regards,

Susana Caballero, PhD

Academic Editor

PLOS ONE

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

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.

PLoS One. 2020 Oct 1;15(10):e0230763. doi: 10.1371/journal.pone.0230763.r010

Author response to Decision Letter 4

10 Sep 2020

Dear Editor and Reviewers,

Thank you very much for your opportunity to improve our manuscript. We have followed the instructions and address all reviewers comments and suggestions. We have also asked professional native speaker to check our manuscript, as this the main comment from last submitted revision. Thanks again and we hope now the manuscript is suitable for publication.

Please kindly see the document entitled "Response to Reviewers" for detail on how addressed questions and suggestions.

Attachment

Submitted filename: 1. Response to Reviewers.docx

Click here for additional data file.^{(35KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0230763.r011

Decision Letter 5

Susana Caballero

15 Sep 2020

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini across Indonesia and the Western Indian Ocean

PONE-D-20-06557R5

Dear Dr. Madduppa

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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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,

Susana Caballero, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you very much for submitting this revised version and thanks for following the recommendation to have your manuscript professionally proof-read by a service. It reads WAY better!!!! Now it not only presents super interesting results but also they are easier to follow and understand!

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0230763.r012

Acceptance letter

Susana Caballero

21 Sep 2020

PONE-D-20-06557R5

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini across Indonesia and the Western Indian Ocean

Dear Dr. Madduppa:

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.

If we can help with anything else, please email us at plosone@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. Susana Caballero

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

Attachment

Submitted filename: PONE-D-20-06557REVed.pdf

Click here for additional data file.^{(1.5MB, pdf)}

Attachment

Submitted filename: 1. Response to Reviewers (1).docx

Click here for additional data file.^{(19.3KB, docx)}

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file.^{(19.7KB, docx)}

Attachment

Submitted filename: PONE-D-20-06557_R2SCcomm.pdf

Click here for additional data file.^{(2.8MB, pdf)}

Attachment

Submitted filename: 1. Response to Reviewers (1).docx

Click here for additional data file.^{(31.8KB, docx)}

Attachment

Submitted filename: PONE-D-20-06557_R3 (1)SCComm.pdf

Click here for additional data file.^{(3MB, pdf)}

Attachment

Submitted filename: 1. Response to Reviewers.docx

Click here for additional data file.^{(34.4KB, docx)}

Attachment

Submitted filename: 1. Response to Reviewers.docx

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Data Availability Statement

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PERMALINK

Genetic connectivity of the scalloped hammerhead shark Sphyrna lewini across Indonesia and the Western Indian Ocean

Sutanto Hadi

Noviar Andayani

Efin Muttaqin

Benaya M Simeon

Muhammad Ichsan

Beginer Subhan

Hawis Madduppa

Roles

Abstract

Introduction

Material and methods

Ethics statement

Tissue sample collection

Table 1. Sampling collection sites at major shark landing sites in Indonesia.

DNA extraction, amplification, and sequencing

Data analysis

Genetic diversity

Table 2. Localities, the total number (n), and accession number of CO1 gene sequences of S. lewini from Aceh, Balikpapan, Lombok, and Western Papua (Indonesia), India, the United Arab Emirates, as well as Madagascar (Western Indian Ocean).

Population structure

Genetic connectivity

Results

Genetic diversity

Table 3. Forty polymorphic loci of 11 haplotypes from 179 sequences of the mitochondrial CO1 gene of S. lewini samples from four localities in Indonesia and three localities in the Western Indian Ocean region.

Table 4. Genetic diversity of S. lewini based on sample size (n), haplotype number (Hn), haplotype diversity (h), and nucleotide diversity (π) in samples from each site in Indonesia and the Western Indian Ocean region.

Population structure

Table 5. Analysis of molecular variance (AMOVA) for the percentage of variation (%), FST value, and significance level (P-value) in S. lewini samples from Indonesian, the Western Indian Ocean, and between Indonesian and Western Indian Ocean populations.

Table 6. Pairwise FST values (below the diagonal) and P-values (above the diagonal) between the S. lewini populations from Aceh (ACH), Balikpapan (BPN), Lombok (LOM), West Papua (WEP), and Western Indian Ocean (WIO).

Genetic connectivity

Fig 1. Haplotype network of the S. lewini (n = 179) population from Indonesia and the Western Indian Ocean region, which was constructed using the median joining method.

Fig 2. Distribution of the 11 haplotypes of the S. lewini population from Indonesia and Western Indian Ocean at the regional scale.

Discussion

Genetic diversity

Population structure

Genetic connectivity

Conservation implications

Conclusion

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Susana Caballero

Roles

Author response to Decision Letter 0

Decision Letter 1

Susana Caballero

Roles

Author response to Decision Letter 1

Decision Letter 2

Susana Caballero

Roles

Author response to Decision Letter 2

Decision Letter 3

Susana Caballero

Roles

Author response to Decision Letter 3

Decision Letter 4

Susana Caballero

Roles

Author response to Decision Letter 4

Decision Letter 5

Susana Caballero

Roles

Acceptance letter

Susana Caballero

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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Table 5. Analysis of molecular variance (AMOVA) for the percentage of variation (%), F_ST value, and significance level (P-value) in S. lewini samples from Indonesian, the Western Indian Ocean, and between Indonesian and Western Indian Ocean populations.

Table 6. Pairwise F_ST values (below the diagonal) and P-values (above the diagonal) between the S. lewini populations from Aceh (ACH), Balikpapan (BPN), Lombok (LOM), West Papua (WEP), and Western Indian Ocean (WIO).