Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

Adnan Shahdadi; Katharina von Wyschetzki; Hung-Chang Liu; Ka Hou Chu; Christoph D Schubart

doi:10.1371/journal.pone.0262122

. 2022 Jan 13;17(1):e0262122. doi: 10.1371/journal.pone.0262122

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

Adnan Shahdadi ¹, Katharina von Wyschetzki ², Hung-Chang Liu ³, Ka Hou Chu ^4,⁵, Christoph D Schubart ^6,^*

Editor: Benny KK Chan⁷

PMCID: PMC8757990 PMID: 35025933

Abstract

Due to the lack of visible barriers to gene flow, it was a long-standing assumption that marine coastal species are widely distributed, until molecular studies revealed geographically structured intraspecific genetic differentiation in many taxa. Historical events of sea level changes during glacial periods are known to have triggered sequential disjunctions and genetic divergences among populations, especially of coastal organisms. The Parasesarma bidens species complex so far includes three named plus potentially cryptic species of estuarine brachyuran crabs, distributed along East to Southeast Asia. The aim of the present study is to address phylogeography and uncover real and hidden biological diversity within this complex, by revealing the underlying genetic structure of populations and species throughout their distribution ranges from Japan to West Papua, with a comparison of mitochondrial COX1 and 16S rRNA gene sequences. Our results reveal that the P. bidens species complex consists of at least five distinct clades, resulting from four main cladogenesis events during the mid to late Pleistocene. Among those clades, P. cricotum and P. sanguimanus are recovered as monophyletic taxa. Geographically restricted endemic clades are encountered in southeastern Indonesia, Japan and China respectively, whereas the Philippines and Taiwan share two clades. As individuals of the Japanese clade can also be found in Taiwan, we provide evidence of a third lineage and the occurrence of a potential cryptic species on this island. Ocean level retreats during Pleistocene ice ages and present oceanic currents appear to be the main triggers for the divergences of the five clades that are here addressed as the P. bidens complex. Secondary range expansions converted Taiwan into the point of maximal overlap, sharing populations with Japan and the Philippines, but not with mainland China.

Introduction

The biogeography of marine species is generally determined by different abiotic and biotic factors. Historical events of sea level changes during glacial periods in different geological epochs [1, 2] have triggered sequential disjunction/junctions of coastal populations and consequently resulted in successive genetic divergence [3–6]. Former and current oceanic currents are also important agents for the”transport” of alleles between distant populations [7–9] or can act as barriers, preventing reciprocal gene flow among nearby populations [10–12]. The mode of reproduction, early developmental motility, and dispersal ability of different ontogenetic stages of marine organisms are additional important players in structuring their biogeography [12–14]. Some species have even been able to perform trans-Pacific dispersal, resulting in certain degree of genetic differentiation [15].

The field of molecular phylogeography has contributed significantly to the development of our present understanding of marine biogeographic patterns [14]. For decades, the lack of visible geographic barriers was responsible for the general belief that most marine species are widely, or some of them even globally, distributed and dispersed via oceanic currents [16]. Molecular comparisons, however, have revealed genetic disjunctions and phylogeographic structures among and within many marine animals [17], including decapod crustaceans [18]. Such genetic disconnections and locally structured patterns are also common among partly pelagic [19] and meroplanktonic (e.g. coastal crabs with planktonic larvae) species [20].

Brachyuran crabs of the family Sesarmidae Dana, 1851 are among the most important faunal components of tropical estuarine habitats, including marshes and mangroves, with a high species diversity, especially throughout the Indo-West Pacific (IWP) [21–25]. This zoogeographic region is well known for its high biodiversity, especially among marine taxa [26–29]. Several studies have attempted to understand and describe phylogeographic patterns of different representatives in this marine realm [9, 30–32]. Ragionieri et al. [33] studied the phylogeography of sesarmid crabs referred to as Neosarmatium meinerti (De Man, 1887), with a presumably wide distribution throughout the IWP. This taxon showed a clear genetic structure composed of four distinct clades (i.e. East Africa, western Indian Ocean, Southeast Asia and northern Australia), with the north Australian clade being most clearly separated from others. As these clades turned out to be similarly diverged from one another as from the West Pacific species N. fourmanoiri Serène, 1973, all four of them were recognized and described as valid species [33, 34].

With currently 54 extant species, Parasesarma De Man, 1895 is one of the two largest sesarmid genera [35]. Its representatives are distributed throughout the IWP, mostly in East and Southeast Asia [36]. In a recent phylogenetic analysis, we recovered several stable clades among species of Parasesarma [36]. One of these clades showed similar patterns as those reported for the N. meinerti species group, with representatives in East Africa (P. guttatum (A. Milne-Edwards, 1869) and P. capensis (Fratini, Cannicci and Innocenti, 2019), India (P. bengalense (Davie, 2003)), Southeast Asia (P. cricotum (Rahayu and Davie, 2002)), Australia (P. brevicristatum (Campbell, 1967), P. darwinense (Campbell, 1967) and P. holthuisi (Davie, 2010)), and East Asia (P. bidens (De Haan, 1835) and P. sanguimanus Li, Shih & Ng, 2019) [36].

Within the IWP, Southeast Asia sticks out as a general biodiversity hotspot, harbouring the highest species richness among all other marine phylogeographic provinces [37, 38]. Numerous studies have thus been conducted to uncover the mechanisms and history of the diversification in this area and to address the local barriers and reasons for lineages’ divergences [16, 39, 40]. On the other hand, the East Asian coastline was affected by many changes during its geological history, and therefore has been given increased attention in several phylogeographic studies [20, 41–43].

According to the presence of several marginal seas in the area and their role in genetic divergences during ice ages [41, 44], a thorough phylogeographic study can potentially uncover undetected biodiversity in this group and date potential divergence events. Moreover, such study could also map the distribution ranges and boundaries of the corresponding phylogenetic groups. With this intention, the present study focuses on the coastal crab species occurring in this area, i.e. Parasesarma cricotum from Southeast Asia vs. P. sanguimanus and P. bidens from East Asia, in order to reveal underlying causes for species divergence and biogeographic distribution patterns in these two Asian subregions. Parasesarma bidens was originally described from Japan [45], with a supposed wide distribution throughout East to Southeast Asia [46]. Within mainland China, P. bidens has been characterized by its genetic homogeneity [47], whereas morphological and genetic distinction of West Papuan [48] and Taiwanese [49] populations led to the relatively recent descriptions of the two regionally confined species P. cricotum [48] and P. sanguimanus [49].

The present study aimed to gain deeper insights into the phylogeography and genetic structure of these species and other populations still belonging to Parasesarma bidens, which will here be referred to as the P. bidens species complex. For this purpose, and to document overall diversity within this complex, we molecularly screened 142 individuals originating from a wide distribution range (from Japan to West Papua) by comparing DNA sequences corresponding to the mitochondrial COX1 and 16S rRNA genes. Furthermore, a molecular clock approach was applied to estimate divergence times for the genetic groups.

Materials and methods

Materials examined

Specimens of the P. bidens group (including P. bidens, P. cricotum and P. sanguimanus) were gathered from Japan (Hiroshima, Nagasaki, Iriomote), China (Hong Kong, Hainan), Taiwan (Pingtung, Taichung), Philippines (Bohol, Cebu, Luzon) and Indonesia (West Papua, Sulawesi) (Fig 1). Materials from Gaomei (Taiwan, Taichung) were loaned from the Zoological Collections of the Department of Life Science, National Chung Hsing University (Taichung, Taiwan). Some of the specimens from the Philippines and Japan were loaned from the Florida Museum of Natural History (Florida, USA) and the Ryukyus University Museum (Fujukan, Okinawa, Japan), respectively (see S1 Table). All other materials, which were collected during different official field expeditions between 1995 to 2011 from non-protected mangrove forests and estuaries were legally integrated in biological collections in Germany, Japan, Hong Kong and Singapore (see S1 Table for localities, museum voucher numbers, and DNA extraction code of the CD Schubart lab).

Fig 1 — Localities for materials of the present study (solid black squares) and distribution map of the five clades (from Fig 2B) comprised within the P. *bidens* complex, with percentages of each clade in each locality of the study. Map reprinted from Natural Earth (public domain) [http://www.naturalearthdata.com/] under a CC BY license.

Laboratory methods and sequence preparations

Genomic DNA was isolated using a modified Puregene method (Gentra Systems, Minneapolis, MN) from muscular leg tissue. A segment of nearly 800 base pairs (bp) from the 3’ end of the mitochondrial protein-coding gene cytochrome oxidase subunit 1 (COX1) was selected as the main molecular marker for our genetic analyses. After preliminary analyses of these sequences revealed the underlying phylogenetic relationships, for a subset of specimens an approximately 650 bp segment of the 5’ end of the COX1 gene, corresponding to the barcode region, as well as a segment of a more conserved mitochondrial gene, encoding the RNA of the large ribosomal subunit (16S rRNA), was also sequenced. Polymerase chain reactions (PCRs) were performed with the following profile: initial denaturation step for 4 min at 94°C; 40 cycles with 45 s at 95°C for denaturing, 60 s at 48°C for annealing, 60 s at 72°C for extension; and 5 min at 72°C as final extension step. To amplify the 3’ end of COX1, the primers COL8 (forward) and COH1b (reverse) were used. In case this combination did not work, the alternative forward primers COL1b or COL11 were used to amplify a segment of about 680 or 420 bp, respectively. To amplify the barcode region of COX1, the primer set COL6-COH6, and for the 16S rRNA gene the primer combination 16L29-16H10, was used (see S2 Table for primer information). PCR products were outsourced for sequencing to Macrogen Europe (for accession numbers see S1 Table). Sequences were proofread using Chromas Lite (v. 2.1.1) (Technelysium Pty Ltd, Queensland, Australia). Primer regions were removed and the remaining sequences were aligned with ClustalW [50] implemented in BioEdit 7.0.5 [51].

Phylogenetic and phylogeographic analyses

To uncover phylogeographic structure and genetic groups among the obtained sequences, two phylogenetic algorithms were applied for the sequences of our large COX1 dataset, corresponding to its 3’ end. A maximum likelihood (ML) tree was constructed using the software raxmlGUI v. 1.5b2 [52] with 1000 bootstrap replicates. To estimate the clades’ divergence times, we conducted a Bayesian inference (BI) analysis with the software BEAST 2.6.2 [53], using a strict clock model (Yule Model) with a rate of evolution for the COX1 of 2.33% per million years (my), following [54]. Markov chains were run for 10 million generations, sampling every 1000th iterations and discarding the first 25% as burnin. The remaining 7500 trees were used to calculate the maximum clade credibility tree in TreeAnnotator v.1.6.1 (part of the BEAST package). Phylogenetic analyses were based on the General Time Reverse plus Gamma (GTR + G, [55]) evolutionary model, as suggested by the AIC algorithm in jModelTest 0.1.1 [56]. A sequence of P. bengalense (Davie, 2003) was also included to the analyses as a representative of the sister clade, consisting of P. bengalense, P. capensis and P. guttatum, to the P. bidens group [36, 49, 57], in order to re-examine their phylogenetic relationship. Based on previous phylogenetic analyses [36, 57], sequences of three other species of the genus, P. eumolpe (De Man, 1895), P. indiarum (Tweedie, 1940) and P. peninsulare Shahdadi, Ng & Schubart, 2018, were included in the analyses as outgroups.

To obtain a better resolution of the genetic relationships among and within groups, a maximum parsimony haplotype network [58] was built via the software PopART [59]. Mean genetic distances (Kimura 2 Parameter = K2P) between phylogenetic groups (clades) were calculated with the software MEGA version X [60]. To find the phylogenetic positions of some sequences that were recovered from GenBank (https://www.ncbi.nlm.nih.gov/), a haplotype network was also constructed for the sequences of the barcode region of the COX1 gene. To check the genetic relationships in a second and more conserved marker, a maximum parsimony haplotype network [58] was also built for the 16S rRNA gene, using the software PopART [59].

Analyses of population genetics and demography

To calculate the genetic diversity indices (i.e. the values of haplotype diversity (Hd), nucleotide diversity (π), number of segregating sites (S), and average number of nucleotide differences (k)) of the large COX1 dataset, DnaSP 5.10 [61] was used. Demographic changes at levels of mitochondrial DNA lineages were analyzed by the neutrality test using estimation of Fu’s Fs [62] in ARLEQUIN 3.5 [63] with 1,000 permutations. To trace population size changes, we analyzed the distribution of pairwise differences (mismatch distribution) [64] in DnaSP 5.10 [61] with the model of constant population size for expected values, and the graphs were created in Microsoft Excel 2013 [65]. In order to measure the smoothness of the mismatch distribution, we also analyzed the distribution of pairwise differences in ARLEQUIN 3.5 [63] under sudden expansion models, with calculation of Harpending’s raggedness index (r) [66] under the null hypothesis of neutral evolution using 1,000 bootstrap iterations. The neutrality estimations and mismatch distribution were calculated for each of the five evolutionary clades (recovered from the phylogenetic analyses). The diversity indices were calculated for each of these clades, as well as for the populations comprised in each clade separately. To identify genetic differentiation among populations of each clade, the fixation index, F_ST (in mitochondrial DNA Φ_ST), and the analyses of molecular variance (AMOVA) were also performed with ARLEQUIN [63] and 1,000 permutations.

Sequences of two localities from the main islands of Japan had to be lumped, because of a low number of available sequences: Hiroshima (4 sequences) and Nagasaki (5 sequences).

Results

In total, COX1 sequences were obtained from 142 specimens of P. bidens species complex (Fig 2A, S1 Table), with variable lengths from different localities as the main ingroup of the present analyses. Most sequences had a length of 742 bp, after removal of the primer sequences and adjacent regions. Two phylogenetic trees, one BI with BEAST (Fig 2B, S1 Fig) and one ML (S2 Fig) were constructed with all available sequences of the 3’ end. 139 sequences had a length of at least 618 bp and were batched in a second alignment for building a haplotype network, calculating K2P distances, diversity indices and other demographic tests. For the barcode region (5’ end of COX1) 13 sequences (eight sequences from GenBank and five from the present study) with a length of 610 bp, and for the 16S gene 15 sequences (present study) with a length of 527 bp were available (See S1 Table) to build the haplotype networks.

For the COX1 sequences, no sequence contained stop codons or resulted in alignment indels, which may have indicated the presence of pseudogenes.

Phylogeny and phylogeography

The topologies of BI and ML phylogenetic trees were largely congruent (Fig 2B, S1 and S2 Figs) and allowed to recover five well supported clades (hereafter referred to as clades I–V) for the P. bidens group in the Western Pacific (Fig 2B, S1 and S2 Figs). These five clades form a monophyletic group, sister to P. bengalense. The five clades of the P. bidens group form two main clusters, one composed of clades I, II and III, and the other including clades IV and V. The sequences from China (Hainan and Hong Kong) (P. bidens Clade I) form a stable monophyletic group together with the closely related Clade II (P. bidens), which is composed of all Japanese specimens (main islands and Iriomote) together with few Taiwanese individuals. The monophylum consisting of clades I and II holds a sister position with the Indonesian (West Papua and Sulawesi) Clade III (= P. cricotum). Most Taiwanese sequences, together with some from the Philippines, cluster in a solid clade, separated from all others by a relatively long branch (Clade IV). This clade shows a sister relationship to P. sanguimanus (Clade V). Most specimens of Clade V were from the Philippines, with a single one (out of 17 sequences) from Taiwan.

The result from the divergence time estimation in the Bayesian analyses (Fig 2B) reveal that the five clades of the P. bidens species complex diverged from their sister group (P. bengalense and allies) approximately 2.27 million years ago (1.64–2.96 mya). The results further show that these five clades shared a last common ancestor about 1.56 million years ago (1.15–2.02 mya). Following the first divergence event at about 1.56 mya, which resulted in the two main clusters, there were two divergence events at seemingly different times. At about 1.14 (0.78–1.50) mya one of these events separated P. cricotum (Clade III) from P. bidens (clades I and II), and the second at about 1.08 (0.71–1.46) mya separated Clade IV from Clade V (P. sanguimanus). The divergence of Clade I from China and Clade II from Japan dates back to about 0.69 (0.45–0.94) mya. The earliest divergence events within each of these five clades are recorded at about 0.42 mya (Fig 2B).

The haplotype networks based on the COX1 gene (both segments) (Fig 3A and S2 and S3 Figs) recovered exactly the same five groupings as described above. In the main network (the 3’ end) (Fig 3A), all clades except for Clade III (P. cricotum) include a common haplotype and to some extent show a star-shape structure. In Clade III, specimens of the two localities (West Papua and Sulawesi) revealed a very close association, but without sharing haplotypes. In the haplotype network the 5’ end (the barcode region), the sequences from South Korea grouped with the Japanese specimens and the one from Palau Island clustered with those of Sulawesi and West Papua (S3 Fig).

To confirm these patterns of genetic differentiation with a more conserved mitochondrial marker 15 sequences with 527 bp of the 16S rRNA gene (after removing primer sequences and adjacent regions) were compared. Genetic relationships based on the 16S rRNA haplotype network (Fig 3B) were generally similar to those of COX1 (Fig 3A) and equivalent genetic groups were recovered, but with fewer mutation steps between groups (Fig 3B).

Among the geographic areas covered in this study, Taiwan holds a special position by hosting members of three clades, II, IV and V (Fig 1). The Philippines harbour specimens of two clades (IV and V), while other regions only include members of single clades (Fig 1). Clade IV is the most common clade in Taiwan, while in the Philippines Clade V is the more common one (Fig 1). With regard to the K2P distances (S3 Table), clades I and II show the least distance, 1.4%, while other clades are separated by K2P values of more than 2.4%. The largest distance is 4.0%, between clades III and IV. The mean K2P distance between two superimposed clusters is 3.6%. The mean distance between Clade III and clades I and II is 2.6%.

Population genetic and historical demography

Clades II and III show the highest total genetic diversity among all clades (Hd = 0.755, π = 0.00356, k = 2.194 in Clade II; Hd = 0.857, π = 0.00354, k = 2.186 in Clade III) (see Table 1), whereas Clade IV has the least genetic diversity (Hd = 0.672, π = 0.00160, k = 0.987), especially within Taiwan with even lower diversity indices (Hd = 0.350, π = 00061, k = 0.375). Among the studied localities, the Philippines revealed a high genetic diversity in Clade IV (Hd = 0.910, π = 0.00631, k = 3.897).

Table 1. Diversity indices.

Statistical diversity indices (in five here recovered phylogenetic clades and main localities of each clade) and neutrality tests (Fu’s Fs) (in the five clades) based on 618 bp of Cox1 (the 3’ end). Indices were calculated with DnaSP 5.10, and the neutrality test carried out with ARLEQUIN 3.5. Pairwise Φ_ST values among the main populations of each clade and Harpending’s raggedness index were calculated in ARLEQUIN 3.5.2.2.

Clade	Locality	n	h	Hd±SD	π	S	k	Fu’s Fs	Φs_T	Harpending’s r
I	Hainan	20	5	0.505±0.126	0.00103	4	0.637
	Hong Kong	14	8	0.868±0.068	0.00245	7	1.516
	Total	34	11	0.690±0.077	0.00167	9	1.032	-7.91287^*	0.07094	0.0877* (p = 0.1)
II	Jp. main islands	9	5	0.861±0.087	0.00252	5	1.556
	Iriomote	21	7	0.614±0.116	0.00371	12	2.286
	Total	30	11	0.755±0.077	0.00356	15	2.194	-3.66265^*	0.08285	0.0484* (p = 0.8)
III	West Papua	16	5	0.650±0.108	0.00195	4	1.483
	Sulawesi	14	6	0.780±0.081	0.00659	5	1.110
	Total	30	11	0.857±0.040	0.00354	9	2.186	-3.79208^*	0.56503^*	0.0668 (p = 0.05)
IV	Taiwan	16	4	0.350±0.148	0.00061	3	0.375
	Philippines	13	8	0.910±0.056	0.00631	22	3.897
	Total	29	10	0.672±0.097	0.00160	9	0.987	-7.25174^*	0.22907^*	0.0645* (p = 0.2)
V	Total	16	6	0.683±0.120	0.00191	6	1.183	-2.16352^*		0.1425* (p = 0.9)

Open in a new tab

n = Number of sequences; h = Number of haplotypes; Hd = Haplotype diversity; SD = Standard deviation; π = Nucleotide diversity; S = Number of segregating sites; k = Average number of nucleotide differences

* = P < 0.05 in Fu’s Fs and Φ_ST, with support values for population expansion.

Comparisons of Φ_ST values among populations (of each clade) revealed the lowest values in the two Chinese populations, Hainan and Hong Kong (Φ_ST = 0.07094), compared to all other population pairs (Table 1). In contrast, highest values were found between the Indonesian populations from Sulawesi and West Papua (both Clade III, Φ_ST = 0. 0.56503) (Table 1). The result of the AMOVA analysis revealed low levels of molecular variance among populations within clades I and II. A higher value of variation was found between the two Indonesian populations (Clade III) compared to the other examined population pairs (Table 2).

Table 2. AMOVA.

Summary of hierarchical analysis of molecular variance (AMOVA) based on 618 bp of Cox1 (the 3’ end).

Clades and populations	source of variation	d.f.	Sum of squares	Components of competence	Percentage of variation
I (Hainan & Hong Kong)	Among populations	1	1.122	0.03796 (0.05767)	7.09
I (Hainan & Hong Kong)	Within populations	32	15.907	0.49710	92.91
II (Iriomote & main islands of Japan)	Among populations	1	2.221	0.09382 (0.06647)	8.28
II (Iriomote & main islands of Japan)	Within populations	28	29.079	1.03855	91.72
III (West Papua & Sulawesi)	Among populations	1	13.361	0.85083 (0.00000)	56.50
III (West Papua & Sulawesi)	Within populations	28	18.339	0.65497	43.50
IV (Philippines & Taiwan)	Among populations	1	2.176	0.12738 (0.00000)	22.91
IV (Philippines & Taiwan)	Within populations	26	11.146	0.42869	77.09

Open in a new tab

The numbers in parentheses under the column of ‘Components of competence’ are P values after 1000 permutations.

Generally, the significant negative values of the neutrality test (Fu’s Fs) (Table 1) confirmed demographic expansions in all the five clades, with clades I and IV showing stronger signs of expansion with lower values (Table 1). In the analyses of mismatch distribution, all the five clades showed a unimodal pattern with one main peak (Fig 4). Clades II and V, however, showed signs of a small second peak (Fig 4). The values of Harpending’s r were statistically non-significant in the case of all clades, except for Clade III, implying that population expansion is evident in all clades, except for Clade III.

Fig 4 — Distribution of pairwise COX1 (618 bp of the 3’ end) differences (mismatch distribution graphs) for the five clades comprised within the P. *bidens* complex, calculated with DnaSP.

Discussion

During the Pleistocene (since 2.58 mya), more than 11 major and many minor glacial events could be identified [67]. As a consequence, global sea levels experienced sequential falling and rising throughout these successions of glacials and interglacials [68]. During the cold periods, sea basins were shallow and, depending on their depth, the global ocean shape or local sea ranges successively changed [69]. Furthermore, many organisms migrated to warmer areas in the vicinity of the equator. In consequence of these refugial retreats followed by geographic expansions during warmer periods, genetic divergences among isolated populations occurred repeatedly [70]. Present phylogeographic structures of the majority of tropical and subtropical marine species, including those in East and Southeast Asia, are therefore known to have been shaped during these Pleistocene climate fluctuations [2, 4, 71, 72]. Several studies attempted to time-calibrate genetic divergences among related groups from different phyla, using available fossil data and other geological evidence [73–75]. In the present study, we use the substitution rate of 1.66–2.6% (mean = 2.33) per my for COX1, calibrated by Schubart et al. [54] for sesarmid crabs, based on the closure of the Isthmus of Panama.

East and Southeast Asian marine waters consist of several deep basins and trenches surrounded by shallow waters (e.g. Sea of Japan, South China Sea, Philippine Sea, Sulu Sea, Celebes Sea and Banda Sea). These deeper water basins served as refugia for many marine species during periods of low sea level [5, 76], and several studies addressed phylogeographic patterns and the history of this area [16, 20, 39, 41, 77]. However, since animals have a wide variety of life cycles and dispersal capabilities [78], the present-day structures of marine taxa are a mosaic and do not follow a universal pattern [76]. Horne et al. [79] found no phylogeographic structure in two surgeonfish species across their Indo-Central Pacific ranges. A lack of divergence and genetic structure was also found among populations of the rock snail Thais clavigera (Küster, 1860) throughout the northwestern Pacific [44]. On the other hand, Chang et al. [80] recovered four lineages among the soft shore barnacle Fistulobalanus albicostatus (Pilsbry, 1916) in East Asia and estimated their divergence to have occurred during the Pleistocene, as a result of past and present oceanographic regimes. Congruently, Shin et al. [20] also detected a shallow genetic gap between Taiwanese and Korean-Japanese populations of two littoral crab species of the genus Hemigrapsus (Brachyura: Varunidae).

Previous phylogenetic analyses on species of Parasesarma [36, 49, 57, 81] revealed that members of the P. bidens species complex from the Western Pacific (P. bidens, P. cricotum and P. sanguimanus) hold a sister relationship with a clade from within the Indian Ocean consisting of P. bengalense, P. capensis and P. guttatum. Under a larger clade, these six species form two reciprocally monophyletic groups, a western clade versus an eastern one [36, 49, 57, 81]. This pattern highlights the importance of the Indo-Pacific barrier [31] in structuring phylogeny of these marine invertebrates. Present results also recovered representatives of the Western Pacific (the five clades of P. bidens group) as a monophyletic group under a supported clade (Fig 2B, S1 and S2 Figs). Based on the divergence time estimation, it seems that the eastern cluster (the five clades of P. bidens group from the West Pacific) has been separated from their western sister clade at about 2.27 mya (Fig 2B).

Phylogeny, phylogeography and evolutionary history of the P. bidens species complex

In comparison to the previously mentioned studies, the present one covers a larger geographic range along East to Southeast Asia, reaching southward to New Guinea and Sulawesi (Indonesia), in the north to Japan (Hiroshima), and west to Hainan Island (China), furthermore including major islands in eastern Asia like Bohol, Cebu, Luzon from the Philippines and Taiwan (Fig 1, S1 Table, S1 and S2 Figs). Our results reveal that members of the P. bidens species complex, which were considered to be a single species until 20 years ago, and so far include three described species (i.e. P. bidens, P. cricotum and P. sanguimanus), consist of at least five distinct lineages, with different genetic divergence (Figs 2B, 3A, 3B, S1 Table, S1–S3 Figs). The corresponding clades resulted from four main cladogenesis events at different time periods (Fig 2B). According to the estimated divergence times, the most recent common ancestor of the five clades dates back to about 1.56 (2.02–1.15) mya. This means that all divergences and formations of the clades must have happened during the mid to late Pleistocene (Calabrian to Chibanian). The first divergence at about 1.56 mya resulted in two main clusters. The smaller cluster, with representatives in the Philippines and Taiwan experienced another divergence event at about 1.08 (0.71–1.46) mya, giving rise to two currently geographically overlapping clades IV and V, with the latter corresponding to P. sanguimanus. It is very likely that this later divergence happened during one of the major glacials, with sea level at about 100 m below its current level. This large water retreat could have separated the Sulu Sea from surrounding water bodies (i.e. South China Sea in the north, Philippine Sea in the east and Celebes Sea in the south) (see fig. 1 in Voris, 2000). The two lineages corresponding to clades IV and V may have evolved as a result of isolation, but rising sea levels probably led to geographical expansion and subsequent secondary contact.

The phylogenetic history of the larger of the two main clusters seems to be more complex, as it resulted in a wider, but detached distribution of the three comprised clades. This cluster has representatives in southeast Indonesia (P. cricotum, Clade III), Japan and Taiwan (P. bidens, Clade II), and Hong Kong and Hainan (P. bidens, Clade I), but it is absent in the Philippines. Therefore, the history of this cluster can only be explained with the aid of a different scenario. It is conceivable that the origin of this cluster was to be found in its northern current range, for example in the Sea of Japan, as a refugial population during one of the mid-Pleistocene ice ages. Alternatively, it could also have evolved in one of the southern marginal seas like Celebes or Banda seas. However, at about 1.14 mya, so about 420,000 years after its origin, this early lineage split into two groups. These two groups (northern = clades I & II; southern = Clade III) do not overlap in their distribution, and they are not even geographically closest neighbours in their current ranges. Seemingly, a long-distance founding event must be purported, explaining the establishment of a new lineage. This colonization could have taken place via the Pacific Ocean, using the Micronesian Islands (e.g. Palau, Guam, and Mariana) as stepping stones. This hypothesis would be supported by the fact that P. cricotum (here Clade III) was recently recorded from Palau [82] (see also S3 Fig). Another possible explanation for the present-day disjunct distribution of P. bidens (clades I and II) vs. P. cricotum (Clade III) could be that there used to be a continuous distribution, but competition with crabs occupying similar ecological niches, like representatives of Clade IV and P. sanguimanus (Clade V), may have forced the first group into regions with different climatic or other ecological conditions. The most recent divergence leading to the five described clades, occurred about 0.69 mya and separated the Japanese Clade II from the Chinese Clade I. This divergence was probably triggered by another historic biogeographic event.

Among the studied areas, Taiwan with representatives from three clades (P. bidens Clade II, P. sanguimanus & Parasesarma sp. Clade IV) hosts crab populations from more clades than any other sampled regions (Fig 1). The majority of the examined materials from this island belongs to Clade IV (in Clade IV, 16 sequences out of 28, are from Taiwan), whereas one of the present sequences from Taiwan is placed in Clade II and one in clade of P. sanguimanus (Clade V) (Fig 1, S1 and S2 Figs). According to the distribution and frequency of the clades, it seems that Taiwan is not the region of origin for clades II and V, but was later reached by representatives from more northern (Japanese) and southern (Philippines) clades. This phenomenon of a “melting pot” with migrants from adjacent regions converts the island into a biogeographically interesting hotspot and explains the high biodiversity of Taiwanese waters as previously described [83, 84]. Members of Clade IV are distributed equally in Taiwan and the Philippines with low Φ_ST values (Table 1) and low genetic differentiation among the two island populations (Table 2), suggesting high connectivity and regular gene flow among the areas.

The Philippine Archipelago is home for crabs of clades IV and V, but at the same time acts as a barrier between relict populations of marginal seas (South China Sea, Sulu Sea, Philippine Sea, and Celebes Sea) during ice ages. This could have played a significant role in the formation of other evolutionary lineages. The corresponding area is already known to be the centre of Southeast Asian marine biodiversity [85]. West Papua and Sulawesi (both Indonesia) comprise the area of distribution of P. cricotum (Clade III), and at the same time the southernmost distribution range of this complex. Apparently, members of this clade have not dispersed to northern areas (with the exception of Palau, see Shahdadi et al. [82]), while the Philippine individuals of clades IV and V have not distributed to southern Indonesian islands. Thus, it appears that there is no reciprocal gene flow between islands from the Philippines and those from eastern Indonesia, possibly because of the Northern Equatorial Current that originates in the Pacific Ocean, flows through the Celebes Sea and Makassar Strait [85, 86] and may restrict reciprocal larval exchange. Members of Clade II are distributed in Japan (Hiroshima to southern islands) (Fig 2B and S1 Fig), South Korea [87] (S3 Fig), and likely Chinese coasts of the Yellow Sea. A single haplotype belonging to Clade II was also found in Taiwan (Fig 2B and S1 Fig). According to the geographical distribution of this clade and low frequency in Taiwan, it seems that members of this clade originated in Japan and reached the Taiwanese coast through rare migration event(s). A similar pattern was revealed by Tsang et al. [84] for an acorn barnacle Hexechamaesipho pilsbryi (Hiro 1936). Shin et al. [20] also revealed that Japanese and Taiwanese populations of two species of Hemigrapsus share no common haplotype and are genetically distinct, confirming gene flow restriction between Taiwan and Japanese islands. Congruently, crabs from Clade IV of the present study, which are abundant in Taiwan, were not found in Japanese islands. This gene flow restriction between Taiwan and Ryukyu in marine invertebrates were previously attributed to a combination of biological factors (e.g. spawning season) and oceanographic regime (e.g. the strong summer upwelling along the northeastern coastline of Taiwan and the Ryukyu Islands) (see Tsang et al. [88]). Clade I, distributed in mainland China westward to Hainan (present study) and eastward at least to Fujian [47] (see also S3 Fig), was apparently isolated from more northern areas of China, South Korea and main islands of Japan (range of clade II). Similar patterns of isolation were also discovered among other mangrove animals (e.g. in bubbler crabs [89], barnacles [80] and a mudskipper [90]). As previously hypothesized [80, 91, 92], the Yangtze River freshwater discharge could represent a possible barrier by interfering reciprocal gene flow between northern and southern areas (clade I vs. II). A similar case has been described for the role of the Orinoco freshwater discharge by restricting larval transport in western Atlantic fiddler crabs [93]. Amazon freshwater and sediment outflow are also known as a strong barrier responsible for most of the endemism found in Brazilian coastal reefs [12, 94]. While Taiwan hosts members of three clades of the P. bidens group, none of them were found in mainland China (Fig 1 and S1 Fig). Reciprocally, members of Clade I occurring in mainland China, apparently, have not colonized Taiwanese coasts. This pattern of isolation was also recorded for other marine invertebrates (e.g. Tetraclita squamosa [95] and Chthamalus malayensis [72]). In contrast, populations of some species seem to be identical in the two areas (e.g. [96, 97]). Two parallel northward summer currents (South China Sea Current & Taiwan Current) and the southward winter coastal current (Minzhe Current) passing through the Taiwan Strait [42, 44, 98] could be possible candidates for biogeographic barriers separating the populations of Taiwan and mainland China in some species. This diversity of phylogeographic structure has been attributed to differences in their reproductive strategies (e.g. spawning season), dispersal abilities (e.g. duration of larval stages) as well as their nesting ecology (e.g. utilization of a wide range of habitats) [44]. Members of the P. bidens group, however, are apparently not able to cross this hydrographic barrier, despite high offspring production during a prolonged spawning time (April to July [99] and January to October in Taiwan, H-C Liu, personal observation), probably because of their short planktonic larval phase (about 16 days for four zoeal stages [100]).

Population genetic and historical demography

Regarding the genetic diversity, the southern (III = Indonesian) and the northern (II = Japanese) clades show higher values of the different indices (Table 1). However, the differences among the five clades are not considerable, as for example the haplotype diversity (Hd) ranging from 0.85 in Clade III to 0.67 in Clade IV (Philippines & Taiwan). The Philippine specimens of Clade IV are characterized by the highest genetic diversity among all examined populations (Table 1). This might confirm that the Philippine islands are probably the centre of origin for this clade, and Taiwan was colonized after the population expansion, likely mediated by the Kuroshio Current [84]. However, for more conclusive inference a larger sample size from both areas is needed in order to run the corresponding statistical tests.

Concerning populations connectivity, it seems that regular gene flow is maintained between the two Chinese populations (Hainan and Hong Kong) (Clade I), with a low fixation index value (Φ_ST = 0.07094) (Table 1). Although this value is not significant, the low value of percentage of variation among the two populations in AMOVA (7.09%) (Table 2) could confirm the relatedness of these populations. This is congruent with the previous analyses by Zhou et al. [47] showing a high connectivity among Chinese mainland population of this species. A similar status could be inferred for the Japanese populations of the present study, based on our results (Tables 1 and 2). According to the fixation index (Φ_ST = 0.22907) and differentiation among populations in AMOVA (percentage of variation among population = 22.91%), the gene flow between the Philippines and Taiwan (in Clade IV) seems to be less common as in the cases of the Chinese and the Japanese populations. Sulawesi and West Papua (Clade III) also appear to be less connected, with a significantly high Φ_ST value and high differentiation between the two localities and with a high percentage of variation (Table 2). Like in many other animal lineages, according to present demographic analyses, clades of the here studied P. bidens species complex apparently have also been affected by recent historic events (e.g. the latest glacial maximum).

Conclusion

Our molecular data reveal that the here defined P. bidens species complex consists of at least five well separated phylogenetic groups, of which three have so far been described as nominal species, whereas two may be considered as cryptic. The discovery of this additional case of relatively recent sequential differentiation and speciation in East and Southeast Asia provides additional evidence for the amazingly high marine biodiversity in this region. Phylogenetic analyses indicate that these five lineages have originated not more than 1.6 mya, during the mid to late Pleistocene. Being relatively young lineages, and without any evidence of different habitat requirements, and thus external adaptations, they are not morphologically well differentiated. As occurring in many freshwater organisms that are isolated in different river systems [101, 102], here we can show that the real biodiversity is frequently underestimated and overlooked, in consequence of the fact that there are not enough taxonomic units to name all extant evolutionary significant lineages. Considering that all of these phylogenetic groups are unrepeatable and irreplaceable witnesses of unique evolutionary heritage, each of them merits separate management efforts when it comes to biodiversity conservation [103, 104].

Supporting information

S1 Fig. COX1 (the 3’ end; 742 bp) consensus Bayesian tree topology constructed with BEAST.

Values on tree branches refer to posterior probabilities in BI for each corresponding node. P. eumolpe, P. indiarum and P. peninsulare were selected as outgroups. Abbreviations: Ph, Philippines; Tw, Taiwan; Ch, China; Jp, Japan; In, Indonesia.

(TIF)

Click here for additional data file.^{(268.7KB, tif)}

S2 Fig. COX1 (the 3’ end; 742 bp) Maximum Likelihood (ML) tree topology constructed with RaxmlGUI.

Numbers are bootstrap values after 1000 pseudoreplicates. P. eumolpe, P. indiarum and P. peninsulare were selected as outgroups. Abbreviations: Ph, Philippines; Tw, Taiwan; Ch, China; Jp, Japan; In, Indonesia.

(TIF)

Click here for additional data file.^{(279.2KB, tif)}

S3 Fig. Maximum parsimony haplotype network of COX1 (the 3’ end; 610 bp) for a subset of specimens and sequences recovered from GenBank, constructed with PopART.

Hatch marks represent mutation steps.

(TIF)

Click here for additional data file.^{(160.6KB, tif)}

S1 Table. Material of Parasesarma examined for this study with locality, sex (M = male, F = female), size (maximum carapace width in millimeters), museum voucher number and year of collection, DNA extraction number and GenBank (NCBI) Accession number for COX1 (segments of the 5’end and the 3’ end, respectively) and 16S rRNA.

(DOCX)

Click here for additional data file.^{(29.9KB, docx)}

S2 Table. Primers used in the present study with corresponding DNA sequences (5´-3´) and references.

(DOCX)

Click here for additional data file.^{(14KB, docx)}

S3 Table. Mean pairwise K2P distances (expressed in %) based on 618 bp of the COX1 gene (the 3’ end) between five phylogenetic clades recovered in the present study and calculated with MEGA version X.

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

Acknowledgments

We appreciate the contribution of Niko Ramisch, who obtained preliminary data during his Diplom thesis work at the University of Regensburg. We thank Richard Landstorfer and Ling Ming Tsang for participation in collecting activities in Hong Kong, Hainan, and the Philippines, which was carried out during an academic exchange project from 2009 to 2010 between the Chinese University of Hong Kong and the University of Regensburg (DAAD project D/09/00532). In 2000, CDS collected mangrove crabs in Sulawesi, with the help of Dr. Daisy Wowor and Tse-Ming Leong and funding support from the National University of Singapore during a postdoc employment with Prof. Peter K.L. Ng. We also thank Dwi Listyo Rahayu, Hsi-Te Shih, Daisuke Uyeno and Tohru Naruse for sending additional materials. The latter as well as Roland Melzer and Stefan Friedrich from the Staatssammlung in Munich, Jose C.E Mendoza and Muhammad Dzaki Bin Safaruan from the Lee Kong Chian Natural History Museum in Singapore kindly facilitated new museum accession numbers, while Gustav Paulay from Florida Museum of Natural History loaned important material. The manuscript benefitted greatly from repeated comments by our esteemed colleague Ling Ming Tsang from the Chinese University of Hong Kong as well as by additional suggestions by two anonymous reviewers and the editor Dr. Benny Chan.

Data Availability

The datasets supporting the conclusions of this article are included within the article and in the additional supporting files S1–S3 Figs and S1–S3 Tables. The sequences have been submitted to the GenBank (NCBI) and are available in S1 Table. The materials examined are deposited in the zoological collections and the vouchers are available in S1 Table.

Funding Statement

Two travel grants under the Germany/Hong Kong Joint Research Scheme of the German Academic Exchange Service (DAAD) and Research Grants Council (RGC), Hong Kong in 2009-2010 (ID 50022239/G_HK008/08) and in 2012-2013 (ID 54385238/G-H022/11). The travel grants [German Academic Exchange Service (DAAD) and Research Grants Council (RGC)] were solely used to support travel expenses between universities and collection sites. Laboratory expenses were supported by departmental funds of: - University of Regensburg, Dept. Zoology & Evolution, chair: Prof. Jürgen Heinze - Hong Kong Research Institute of Textiles and Apparel (HK), HK008/08, chair: Prof. Ka Hou Chu. From those funding organizations, only Prof. KH Chu had a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Porter SC. Some geological implications of average Quaternary glacial conditions. Quaternary Research. 1989;32(3):245–61. [Google Scholar]
2.Woodruff DS. Biogeography and conservation in Southeast Asia: how 2.7 million years of repeated environmental fluctuations affect today’s patterns and the future of the remaining refugial-phase biodiversity. Biodiversity and Conservation. 2010;19(4):919–41. [Google Scholar]
3.Springer VG, Williams JT. Widely distributed Pacific plate endemics and lowered sea-level. Bulletin of Marine Science. 1990;47(3):631–40. [Google Scholar]
4.Hewitt G. The genetic legacy of the Quaternary ice ages. Nature. 2000;405(6789):907–13. doi: 10.1038/35016000 [DOI] [PubMed] [Google Scholar]
5.Barber PH, Erdmann MV, Palumbi SR. Comparative phylogeography of three codistributed stomatopods: origins and timing of regional lineage diversification in the coral triangle. Evolution. 2006;60(9):1825–39. [PubMed] [Google Scholar]
6.Beck SV, Carvalho GR, Barlow A, Rüber L, Hui Tan H, Nugroho E, et al. Plio-Pleistocene phylogeography of the Southeast Asian blue panchax killifish, Aplocheilus panchax. PLoS One. 2017;12(7):e0179557. doi: 10.1371/journal.pone.0179557 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Hellberg ME. Gene flow and isolation among populations of marine animals. Annual Review of Ecology, Evolution, and Systematics. 2009;40:291–310. [Google Scholar]
8.Dawson MN, Barber PH, González‐Guzmán LI, Toonen RJ, Dugan JE, Grosberg RK. Phylogeography of Emerita analoga (Crustacea, Decapoda, Hippidae), an eastern Pacific Ocean sand crab with long‐lived pelagic larvae. Journal of Biogeography. 2011;38(8):1600–12. [Google Scholar]
9.Borsa P, Durand J-D, Chen W-J, Hubert N, Muths D, Mou-Tham G, et al. Comparative phylogeography of the western Indian Ocean reef fauna. Acta Oecologica. 2016;72:72–86. [Google Scholar]
10.Silva IC, Mesquita N, Paula J. Genetic and morphological differentiation of the mangrove crab Perisesarma guttatum (Brachyura: Sesarmidae) along an East African latitudinal gradient. Biological Journal of the Linnean Society. 2010;99(1):28–46. [Google Scholar]
11.Shahdadi A, Davie PJ, Schubart CD. Systematics and phylogeography of the Australasian mangrove crabs Parasesarma semperi and P. longicristatum (Decapoda: Brachyura: Sesarmidae) based on morphological and molecular data. Invertebrate Systematics. 2018;32(1):196–214. [Google Scholar]
12.Thurman C, Alber R, Hopkins M, Shih H-T. Morphological and genetic variation among populations of the fiddler crab Minuca burgersi (Holthuis, 1967) (Crustacea: Brachyura: Ocypodidae) from shores of the Caribbean Basin and western South Atlantic Ocean. Zoological Studies. 2021;60(19). [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Grosberg R, Cunningham C. Genetic structure in the sea: From populations to communities. En: Bertness MD, Gaines SD, Hay ME(Eds.) Marine Community Ecology: 61–84. Sinauer Associates, Sunderland, MA, USA; 2001. [Google Scholar]
14.Bowen BW, Rocha LA, Toonen RJ, Karl SA. The origins of tropical marine biodiversity. Trends in Ecology & Evolution. 2013;28(6):359–66. doi: 10.1016/j.tree.2013.01.018 [DOI] [PubMed] [Google Scholar]
15.Hongjamrassilp W, Murase A, Miki R, Hastings PA. Journey to the west: trans-Pacific historical biogeography of fringehead blennies in the genus Neoclinus (Teleostei: Blenniiformes). Zoological Studies. 2020;59(09). [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Benzie J, Ballment E, Forbes A, Demetriades N, Sugama K, Moria S. Mitochondrial DNA variation in Indo‐Pacific populations of the giant tiger prawn, Penaeus monodon. Molecular Ecology. 2002;11(12):2553–69. doi: 10.1046/j.1365-294x.2002.01638.x [DOI] [PubMed] [Google Scholar]
17.Bickford D, Lohman DJ, Sodhi NS, Ng PK, Meier R, Winker K, et al. Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution. 2007;22(3):148–55. doi: 10.1016/j.tree.2006.11.004 [DOI] [PubMed] [Google Scholar]
18.Yednock BK, Neigel JE. Rethinking the mechanisms that shape marine decapod population structure. Phylogeography and Population Genetics in Crustacea. 2011:57–73. [Google Scholar]
19.Lai JC, Ng PKL, Davie PJ. A revision of the Portunus pelagicus (Linnaeus, 1758) species complex (Crustacea: Brachyura: Portunidae), with the recognition of four species. Raffles Bulletin of Zoology. 2010;58(2). [Google Scholar]
20.Shin J, Jung J, Kim W, Jung J. Phylogeographic studies on two shore crab species from East Asia: similar but different stories. Genes & Genomics. 2019;41(10):1127–34. doi: 10.1007/s13258-019-00831-9 [DOI] [PubMed] [Google Scholar]
21.Davie PJ. Variations in diversity of mangrove crabs in tropical Australia. Memoirs of the Queensland Museum Brisbane. 1994;36(1):55–8. [Google Scholar]
22.Tan CG, Ng PKL. An annotated checklist of mangrove brachyuran crabs from Malaysia and Singapore. Hydrobiologia. 1994;285(1):75–84. [Google Scholar]
23.Lee S. Ecological role of grapsid crabs in mangrove ecosystems: a review. Marine and Freshwater Research. 1998;49(4):335–43. [Google Scholar]
24.Guerao G, Anger K, Nettelmann U, Schubart CD. Complete larval and early juvenile development of the mangrove crab Perisesarma fasciatum (Crustacea: Brachyura: Sesarmidae) from Singapore, with a larval comparison of Parasesarma and Perisesarma. Journal of Plankton Research. 2004;26(12):1389–408. [Google Scholar]
25.De Grave S, Pentcheff ND, Ahyong ST, Chan T-Y, Crandall KA, Dworschak PC, et al. A classification of living and fossil genera of decapod crustaceans. Raffles Bulletin of Zoology. 2009. [Google Scholar]
26.Briggs JC. Centrifugal speciation and centres of origin. Journal of Biogeography. 2000;27(5):1183–8. [Google Scholar]
27.Briggs JC. The marine East Indies: diversity and speciation. Journal of Biogeography. 2005;32(9):1517–22. [Google Scholar]
28.Costello MJ, Coll M, Danovaro R, Halpin P, Ojaveer H, Miloslavich P. A census of marine biodiversity knowledge, resources, and future challenges. PLoS One. 2010;5(8):e12110. doi: 10.1371/journal.pone.0012110 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Wafar M, Venkataraman K, Ingole B, Khan SA, LokaBharathi P. State of knowledge of coastal and marine biodiversity of Indian Ocean countries. PLoS One. 2011;6(1):e14613. doi: 10.1371/journal.pone.0014613 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Puckridge M, Last PR, White WT, Andreakis N. Phylogeography of the Indo‐West Pacific maskrays (Dasyatidae, Neotrygon): A complex example of chondrichthyan radiation in the Cenozoic. Ecology and Evolution. 2013;3(2):217–32. doi: 10.1002/ece3.448 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Bowen BW, Gaither MR, DiBattista JD, Iacchei M, Andrews KR, Grant WS, et al. Comparative phylogeography of the ocean planet. Proceedings of the National Academy of Sciences. 2016;113(29):7962–9. doi: 10.1073/pnas.1602404113 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Ma KY, Chow LH, Wong KJ, Chen H-N, Ip BH, Schubart CD, et al. Speciation pattern of the horned ghost crab Ocypode ceratophthalmus (Pallas, 1772): an evaluation of the drivers of Indo-Pacific marine biodiversity using a widely distributed species (vol 45, pg 2658, 2018). Journal of Biogeography. 2019;46(4):830. [Google Scholar]
33.Ragionieri L, Fratini S, Vannini M, Schubart CD. Phylogenetic and morphometric differentiation reveal geographic radiation and pseudo-cryptic speciation in a mangrove crab from the Indo-West Pacific. Molecular Phylogenetics and Evolution. 2009;52(3):825–34. doi: 10.1016/j.ympev.2009.04.008 [DOI] [PubMed] [Google Scholar]
34.Ragionieri L, Fratini S, Schubart CD. Revision of the Neosarmatium meinerti species complex (Decapoda: Brachyura: Sesarmidae), with descriptions of three pseudocryptic Indo-West Pacific species. Raffles Bulletin of Zoology. 2012;60(1). [Google Scholar]
35.Shahdadi A, Davie PJ, Rahayu DL, Schubart CD. The synonymy of Parasesarma foresti (Rahayu & Davie, 2002) with P. indiarum (Tweedie, 1940) (Decapoda, Brachyura, Sesarmidae). Crustaceana. 2020;93(7):793–801. [Google Scholar]
36.Shahdadi A, Fratini S, Schubart CD. Taxonomic reassessment of Parasesarma (Crustacea: Brachyura: Decapoda: Sesarmidae) based on genetic and morphological comparisons, with the description of a new genus. Zoological Journal of the Linnean Society. 2020;190(4):1123–58. [Google Scholar]
37.Benzie J. Genetic structure of marine organisms and SE Asian biogeography. Biogeography and Geological Evolution of SE Asia. 1998;30:197–209. [Google Scholar]
38.Roberts CM, McClean CJ, Veron JE, Hawkins JP, Allen GR, McAllister DE, et al. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science. 2002;295(5558):1280–4. doi: 10.1126/science.1067728 [DOI] [PubMed] [Google Scholar]
39.Barber PH, Palumbi SR, Erdmann MV, Moosa MK. A marine Wallace’s line? Nature. 2000;406(6797):692–3. doi: 10.1038/35021135 [DOI] [PubMed] [Google Scholar]
40.Barber PH, Bellwood DR. Biodiversity hotspots: evolutionary origins of biodiversity in wrasses (Halichoeres: Labridae) in the Indo-Pacific and new world tropics. Molecular Phylogenetics and Evolution. 2005;35(1):235–53. Epub 2005/03/02. doi: 10.1016/j.ympev.2004.10.004 . [DOI] [PubMed] [Google Scholar]
41.Xu J, Chan T-Y, Tsang LM, Chu KH. Phylogeography of the mitten crab Eriocheir sensu stricto in East Asia: Pleistocene isolation, population expansion and secondary contact. Molecular Phylogenetics and Evolution. 2009;52(1):45–56. doi: 10.1016/j.ympev.2009.02.007 [DOI] [PubMed] [Google Scholar]
42.Cheng J, Wang Z, Song N, Yanagimoto T, Gao T. Phylogeographic analysis of the genus Platycephalus along the coastline of the northwestern Pacific inferred by mitochondrial DNA. BMC Evolutionary Biology. 2019;19(1):1–16. doi: 10.1186/s12862-018-1333-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Kojima S, Hayashi I, Kim D, Iijima A, Furota T. Phylogeography of an intertidal direct-developing gastropod Batillaria cumingi around the Japanese Islands. Marine Ecology Progress Series. 2004;276:161–72. [Google Scholar]
44.Guo X, Zhao D, Jung D, Li Q, Kong L-F, Ni G, et al. Phylogeography of the rock shell Thais clavigera (Mollusca): evidence for long-distance dispersal in the northwestern Pacific. PloS One. 2015;10(7):e0129715. doi: 10.1371/journal.pone.0129715 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Haan HMD. Crustacea. In: von Siebold P. F., P.F., Fauna Japonica sive Descriptio Animalium, quae in Itinere per Japoniam, Jussu et Auspiciis Superiorum, qui Summum in India Batava Imperium Tenent, Suspecto, Annis 1823–1830 Collegit, Notis, Observationibus et Adumbrationibus Illustravit. Leiden, the Netherlands: Published by the author; 1833–1850. 243 p. [Google Scholar]
46.Dai AY, Yang SL, Song YZ, Chen GX. Crabs of the China Seas. Beijing: China Ocean Press (Chinese edition); Berlin, Heidelberg, New York, Tokyo: Springer (English edition).1991. 682 p. [Google Scholar]
47.Zhou H, Xu J, Yang M, Wu B, Yan B, Xiong Y. Population genetic diversity of sesarmid crab (Perisesarma bidens) in China based on mitochondrial DNA. Mitochondrial DNA Part A. 2016;27(5):3255–62. doi: 10.3109/19401736.2015.1015002 [DOI] [PubMed] [Google Scholar]
48.Rahayu DL, Davie PJ. Two new species and a new record of Perisesarma (Decapoda, Brachyura, Grapsidae, Sesarminae) from Indonesia. Crustaceana. 2002:597–607. [Google Scholar]
49.Li J-J, Shih H-T, Ng PKL. Three new species and two new records of Parasesarma De Man, 1895 (Crustacea: Brachyura: Sesarmidae) from Taiwan and the Philippines from morphological and molecular evidence. Zoological Studies. 2019;58. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 1994;22(22):4673–80. doi: 10.1093/nar/22.22.4673 [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Hall TA, editor BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series; 1999: [London: ]: Information Retrieval Ltd., c1979–c2000. [Google Scholar]
52.Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML. Organisms Diversity & Evolution. 2012;12(4):335–7. [Google Scholar]
53.Bouckaert R, Vaughan TG, Barido-Sottani J, Duchêne S, Fourment M, Gavryushkina A, et al. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Computational Biology. 2019;15(4):e1006650. doi: 10.1371/journal.pcbi.1006650 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Schubart CD, Diesel R, Hedges SB. Rapid evolution to terrestrial life in Jamaican crabs. Nature. 1998;393(6683):363–5. [Google Scholar]
55.Rodriguez F, Oliver JL, Marín A, Medina JR. The general stochastic model of nucleotide substitution. Journal of Theoretical Biology. 1990;142(4):485–501. doi: 10.1016/s0022-5193(05)80104-3 [DOI] [PubMed] [Google Scholar]
56.Posada D, Buckley TR. Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Systematic Biology. 2004;53(5):793–808. doi: 10.1080/10635150490522304 [DOI] [PubMed] [Google Scholar]
57.Shahdadi A, Schubart CD, Mendoza JCE. Conspicuous genetic similarity within a widely distributed and newly described species of Parasesarma De Man, 1895 from Western Pacific oceanic islands, with notes on the allied P. calypso group (Crustacea: Brachyura: Sesarmidae). Invertebrate Systematics. 2021;35(5):542–69. [Google Scholar]
58.Templeton AR, Crandall KA, Sing CF. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics. 1992;132(2):619–33. doi: 10.1093/genetics/132.2.619 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Leigh JW, Bryant D. popart: full‐feature software for haplotype network construction. Methods in Ecology and Evolution. 2015;6(9):1110–6. [Google Scholar]
60.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;35(6):1547. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451–2. doi: 10.1093/bioinformatics/btp187 [DOI] [PubMed] [Google Scholar]
62.Fu Y-X. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics. 1997;147(2):915–25. doi: 10.1093/genetics/147.2.915 [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources. 2010;10(3):564–7. doi: 10.1111/j.1755-0998.2010.02847.x [DOI] [PubMed] [Google Scholar]
64.Rogers AR, Harpending H. Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution. 1992;9(3):552–69. doi: 10.1093/oxfordjournals.molbev.a040727 [DOI] [PubMed] [Google Scholar]
65.Carlberg C. Statistical analysis: Microsoft excel 2013. Indianapolis: Que Publishing; 2014. [Google Scholar]
66.Harpending H. Signature of ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Human Biology. 1994:591–600. [PubMed] [Google Scholar]
67.Richmond GM, Fullerton DS. Summation of Quaternary glaciations in the United States of America. Quaternary Science Reviews. 1986;5:183–96. [Google Scholar]
68.Murray-Wallace CV, Woodroffe CD. Quaternary sea-level changes: a global perspective: Cambridge University Press; 2014. [Google Scholar]
69.Voris HK. Maps of Pleistocene sea levels in Southeast Asia: shorelines, river systems and time durations. Journal of Biogeography. 2000;27(5):1153–67. [Google Scholar]
70.Marko PB, Hoffman JM, Emme SA, McGovern TM, Keever CC, Nicole Cox L. The ‘Expansion–Contraction’model of Pleistocene biogeography: rocky shores suffer a sea change? Molecular Ecology. 2010;19(1):146–69. doi: 10.1111/j.1365-294x.2009.04417.x [DOI] [PubMed] [Google Scholar]
71.Avise JC. Phylogeography: the history and formation of species: Harvard university press; 2000. [Google Scholar]
72.Tsang LM, Chan BKK, Wu TH, Ng WC, Chatterjee T, Williams GA, et al. Population differentiation in the barnacle Chthamalus malayensis: postglacial colonization and recent connectivity across the Pacific and Indian Oceans. Marine Ecology Progress Series. 2008;364:107–18. [Google Scholar]
73.Knowlton N. Sibling species in the sea. Annual Review of Ecology and Systematics. 1993;24(1):189–216. [Google Scholar]
74.Knowlton N, Weigt LA. New dates and new rates for divergence across the Isthmus of Panama. Proceedings of the Royal Society of London Series B: Biological Sciences. 1998;265(1412):2257–63. [Google Scholar]
75.Papadopoulou A, Anastasiou I, Vogler AP. Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration. Molecular Biology and Evolution. 2010;27(7):1659–72. doi: 10.1093/molbev/msq051 [DOI] [PubMed] [Google Scholar]
76.Wang J, Tsang LM, Dong Y-W. Causations of phylogeographic barrier of some rocky shore species along the Chinese coastline. BMC Evolutionary Biology. 2015;15(1):1–15. doi: 10.1186/s12862-015-0387-0 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Wu TH, Tsang LM, Chan BKK, Chu KH. Cryptic diversity and phylogeography of the island‐associated barnacle Chthamalus moro in Asia. Marine Ecology. 2015;36(3):368–78. [Google Scholar]
78.Kinlan BP, Gaines SD, Lester SE. Propagule dispersal and the scales of marine community process. Diversity and Distributions. 2005;11(2):139–48. [Google Scholar]
79.Horne JB, van Herwerden L, Choat JH, Robertson DR. High population connectivity across the Indo-Pacific: congruent lack of phylogeographic structure in three reef fish congeners. Molecular Phylogenetics and Evolution. 2008;49(2):629–38. doi: 10.1016/j.ympev.2008.08.023 [DOI] [PubMed] [Google Scholar]
80.Chang YW, Chan JS, Hayashi R, Shuto T, Tsang LM, Chu KH, et al. Genetic differentiation of the soft shore barnacle Fistulobalanus albicostatus (Cirripedia: Thoracica: Balanomorpha) in the West Pacific. Marine Ecology. 2017;38(2):e12422. [Google Scholar]
81.Shahdadi A, Schubart CD. Taxonomic review of Perisesarma (Decapoda: Brachyura: Sesarmidae) and closely related genera based on morphology and molecular phylogenetics: new classification, two new genera and the questionable phylogenetic value of the epibranchial tooth. Zoological Journal of the Linnean Society. 2017;182(3):517–48. [Google Scholar]
82.Shahdadi A, Schubart CD, Mendoza JCE. On the occurrence of Parasesarma cricotum (Rahayu and Davie, 2002 (Decapoda, Brachyura, Sesarmidae) in Palau and Sulawesi. Zootaxa. 2020;4803(2):zootaxa. 4803.2. 12-zootaxa. .2. 12. [DOI] [PubMed] [Google Scholar]
83.Ng PKL, Shih HT, Ho PH, Wang CH. An updated annotated checklist of brachyuran crabs from Taiwan (Crustacea: Decapoda). Journal of National Taiwan Museum. 2017;70:1–208. [Google Scholar]
84.Tsang LM, Chan BKK, Williams GA, Chu KH. Who is moving where? Molecular evidence reveals patterns of range shift in the acorn barnacle Hexechamaesipho pilsbryi in Asia. Marine Ecology Progress Series. 2013;488:187–200. [Google Scholar]
85.Carpenter KE, Springer VG. The center of the center of marine shore fish biodiversity: the Philippine Islands. Environmental Biology of Fishes. 2005;72(4):467–80. [Google Scholar]
86.Carpenter KE, Barber PH, Crandall ED, Ablan-Lagman M, Carmen A, Mahardika GN, et al. Comparative phylogeography of the Coral Triangle and implications for marine management. Journal of Marine Biology. 2011;2011. [Google Scholar]
87.Kim SY, Yi CH, Kim JM, Choi WY, Kim HS, Kim M-S. DNA Barcoding of the marine protected species Parasesarma bidens (Decapoda: Sesarmidea) from the Korean waters. Animal Systematics, Evolution and Diversity. 2020;36(2):159–63. [Google Scholar]
88.Tsang LM, Chan BKK, Ma KY, Chu KH. Genetic differentiation, hybridization and adaptive divergence in two subspecies of the acorn barnacle Tetraclita japonica in the northwestern Pacific. Molecular Ecology. 2008;17(18):4151–63. doi: 10.1111/j.1365-294x.2008.03907.x [DOI] [PubMed] [Google Scholar]
89.Wong KJ, Chan BKK, Shih H-T. Taxonomy of the sand bubbler crabs Scopimera globosa De Haan, 1835, and S. tuberculata Stimpson, 1858 (Crustacea: Decapoda: Dotillidae) in East Asia, with description of a new species from the Ryukyus, Japan. Zootaxa. 2010;2345(1):43–59. [Google Scholar]
90.Chen W, Hong W, Chen S, Wang Q, Zhang Q. Population genetic structure and demographic history of the mudskipper Boleophthalmus pectinirostris on the northwestern Pacific coast. Environmental Biology of Fishes. 2015;98(3):845–56. [Google Scholar]
91.Cheang CC, Chu KH, Ang J, Put O. Morphological and genetic variation in the populations of Sargassum hemiphyllum (phaeophyceae) in the northwestern Pacific. Journal of Phycology. 2008;44(4):855–65. doi: 10.1111/j.1529-8817.2008.00532.x [DOI] [PubMed] [Google Scholar]
92.Dong Y-W, Wang H-S, Han G-D, Ke C-H, Zhan X, Nakano T, et al. The impact of Yangtze River discharge, ocean currents and historical events on the biogeographic pattern of Cellana toreuma along the China coast. PLoS One. 2012;7(4):e36178. doi: 10.1371/journal.pone.0036178 [DOI] [PMC free article] [PubMed] [Google Scholar]
93.Laurenzano C, Costa TM, Schubart CD. Contrasting patterns of clinal genetic diversity and potential colonization pathways in two species of western Atlantic fiddler crabs. PLoS One. 2016;11(11):e0166518. doi: 10.1371/journal.pone.0166518 [DOI] [PMC free article] [PubMed] [Google Scholar]
94.Rocha LA. Patterns of distribution and processes of speciation in Brazilian reef fishes. Journal of Biogeography. 2003;30(8):1161–71. [Google Scholar]
95.Chan BKK, Tsang L, Chu K. Morphological and genetic differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita. Zoologica Scripta. 2007;36(1):79–91. [Google Scholar]
96.Shih H-T, Kamrani E, Davie PJ, Liu M-Y. Genetic evidence for the recognition of two fiddler crabs, Uca iranica and U. albimana (Crustacea: Brachyura: Ocypodidae), from the northwestern Indian Ocean, with notes on the U. lactea species-complex. Hydrobiologia. 2009;635(1):373–82. [Google Scholar]
97.Shih H-T, Hsu J-W, Wong KJ, Ng NK. Review of the mudflat varunid crab genus Metaplax (Crustacea, Brachyura, Varunidae) from East Asia and northern Vietnam. ZooKeys. 2019;877:1. doi: 10.3897/zookeys.877.38300 [DOI] [PMC free article] [PubMed] [Google Scholar]
98.Xu S, Song N, Zhao L, Cai S, Han Z, Gao T. Genomic evidence for local adaptation in the ovoviviparous marine fish Sebastiscus marmoratus with a background of population homogeneity. Scientific Reports. 2017;7(1):1–12. doi: 10.1038/s41598-016-0028-x [DOI] [PMC free article] [PubMed] [Google Scholar]
99.Kwok P-W. The ecology of two sesarmine crabs, Perisesarma bidens (De Haan) and Parasesarma plicata (Latreille) at the Mai Po Marshes Nature Reserve, Hong Kong. Hong Kong: The University of Hong Kong; 1995. [Google Scholar]
100.Islam MS, Shokita S. Larval development of a mangrove crab (Perisesarma bidens De Haan) (Crustacea: Brachyura: Sesarminae). Bangladesh Journal of Fisheries Research. 2000;4(1):43–56. [Google Scholar]
101.Poettinger T, Schubart CD. Molecular diversity of freshwater crabs from Sulawesi and the sequential colonization of ancient lakes. Hydrobiologia. 2014;739(1):73–84. [Google Scholar]
102.Copilaş‐Ciocianu D, Petrusek A. The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex. Molecular Ecology. 2015;24(15):3980–92. doi: 10.1111/mec.13286 [DOI] [PubMed] [Google Scholar]
103.Ryder OA. Species conservation and systematics: the dilemma of subspecies. Trends in Ecology & Evolution 1986;1:9–10. [DOI] [PubMed] [Google Scholar]
104.Crandall KA, Bininda-Emonds OR, Mace GM, Wayne RK. Considering evolutionary processes in conservation biology. Trends in Ecology & Evolution. 2000;15(7):290–5. doi: 10.1016/s0169-5347(00)01876-0 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0262122.r001

Decision Letter 0

Benny KK Chan

20 Aug 2021

PONE-D-21-19346

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

PLOS ONE

Dear Dr. Shahdadi,

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.

Please submit your revised manuscript by Oct 04 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

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

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

We look forward to receiving your revised manuscript.

Kind regards,

Benny K.K. Chan, Ph.D

Academic Editor

PLOS ONE

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

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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

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

4. In your manuscript, please provide additional information regarding the specimens used in your study. Ensure that you have reported specimen numbers and complete repository information, including museum name and geographic location.

If permits were required, please ensure that you have provided details for all permits that were obtained, including the full name of the issuing authority, and add the following statement:

'All necessary permits were obtained for the described study, which complied with all relevant regulations.'

If no permits were required, please include the following statement:

'No permits were required for the described study, which complied with all relevant regulations.'

For more information on PLOS ONE's requirements for paleontology and archaeology research, see https://journals.plos.org/plosone/s/submission-guidelines#loc-paleontology-and-archaeology-research.

5. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide

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

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

a. You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

b. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

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

Additional Editor Comments (if provided):

Dear authors,

Your MS will need minor revision. When you submit back your revised MS, please have a letter to state point by point how the comments were addressed.

Best wishes,

Benny

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

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

Reviewer #1: This manuscript examines the phylogeography of the mangrove crabs Perisesarma in the West Pacific. The result is clear and straightforward and should be published after some minor revision.

The MS is well written and the only aspects for improvement is it lacks references that are concerning on the phylogeography of mangrove organisms in the West Pacific for comparative purposes in the discussion. The authors consider phylogeography of intertidal species as a whole example. In fact, mangroves and rocky shores species may have different phylogeography patterns, because the local environment and currents in mangroves are much slower that rocky shore species. There are several major examples of phylogeography pattern of mangrove organisms which should be take into account for comparisons in the discussion. This includes:

1)Introduction: Lines 48, authors stated to state some factors to affect phylogeography of intertidal species. The formation of Isthmus of Panama have affect the genetic differentiation of Pacific and Caribbean crab populations (land mass mechanisms). The authors should cite some references concerning this mechanisms on mangrove crabs, including fiddler crabs:

Thurman CL, Alber RE, Hopkins MJ, Shih HT. 2021. Morphological and genetic variation among populations of the fiddler crab Minuca burgersi (Holthuis, 1967) (Crustacea: Brachyura: Ocypodidae) from shores of the Caribbean Basin and western South Atlantic Ocean. Zool Stud 60:19. doi:10.6620/ZS.2021.60-19.

2)In addition to land mass mechanisms, some species can perform trans-Pacific dispersal mechanisms and resulted in some degree of genetic differentiation. This should also be stated in the introduction.

Trans-Pacific mechanisms

Hongjamrassilp W, Murase A, Miki R, Hastings PA. 2020. Journey to the west: trans-Pacific historical biogeography of fringehead blennies in the genus Neoclinus (Teleostei: Blenniiformes). Zool Stud 59:9. doi:10.6620/ZS.2020.59-09

3)Below are some phylogeography of mangrove organisms in the West Pacific and I would suggest the authors to cite and use their results to compare with the pattern in Perisesarma, as these organisms almost share similar habitats.

Mangrove mudskippers:

Chen, W., Hong, W. S., Chen, S. X., Wang, Q., & Zhang, Q. Y. (2015).

Population genetic structure and demographic history of the mudskipper

Boleophthalmus pectinirostris on the northwestern pacific coast.

Environmental Biology of Fishes, 98(3), 845–856.

Mangrove snails:

Kojima, S., Hayashi, I., Kim, D., Iijima, A., & Furota, T. (2004). Phylogeography

of an intertidal direct-developing gastropod Batillaria cumingi around

the Japanese Islands. Marine Ecology Progress Series, 276, 161–172.

Kojima, S., Kamimura, S., Iijima, A., Kimura, T., Mori, K., Hayashi, I., & Furota,

T. (2005). Phylogeography of the endangered tideland snail Batillaria

zonalis in the Japanese and Ryukyu Islands. Ecological Research, 20(6),

686–694.

Kojima, S., Kamimura, S., Kimura, T., Hayashi, I., Iijima, A., & Furota, T. (2003).

Phylogenetic relationships between the tideland snails Batillaria flectosiphonata

in the Ryukyu Islands and B. multiformis in the Japanese

Islands. Zoological Science, 20(11), 1423–1433.

Mangrove barnacles:

Chang, Y. W., J. S. M. Chan, R. Hayashi, T. Shuto, L. M. Tsang, K. H. Chu and B. K. K. Chan* (2017). Genetic differentiation of the soft shore barnacle Fistulobalanus albicostatus (Cirripedia: Thoracica: Balanomorpha) in the West Pacific. Marine Ecology-An Evolutionary Perspective, 38(2): e12422.

Mangrove crabs:

Wong, K. J. H., Chan, B. K. K., & Shih, H. T. (2010). Taxonomy of the sand bubbler crabs Scopimera globosa De Haan, 1835, and S. tuberculate Stimpson, 1858 (Crustacea: Decapoda: Dotillidae) in East Asia, with description of a new species from the Ryukyus, Japan. Zootaxa, 2345, 43–59.

4)In the discussion, lines 438, the authors consider Philippines may be the origin of the clades that disperse northward in the West Pacific (sharing of genotype between Taiwan and the Philippines). Similar observation and conclusion was also addressed in the intertidal barnacle Hexechamaesipho and, Bresidium and Sesarmop crabs.

Please cite and refer to this example:

Tsang, L. M., B. K. K. Chan, G. A Williams and K. H. Chu (2013). Who is moving where? Molecular evidence reveals patterns of range shift in the acorn barnacle Hexechamaesipho pilsbryi in Asia. Marine Ecology Progress Series, 488: 187-200.

Li JJ, Shih HT, Ng PKL. 2020. The Taiwanese and Philippine species of the terrestrial crabs Bresedium Serène and Soh, 1970 and Sesarmops Serène and Soh, 1970 (Crustacea: Decapoda: Brachyura), with descriptions of two new species. Zool Stud 59:16. doi:10.6620/

ZS.2020.59-16.

Ng PKL, Li JJ, Shih HT. 2020. What is Sesarmops impressus (H. Milne Edwards, 1837) (Crustacea: Brachyura: Sesarmidae)? Zool Stud 59:27. doi:10.6620/ZS.2020.59-27.

5)Part of the above reference already show some of the glacial refugia. For more details on the glacial refugia in the West Pacific, the authors should refer:

Wu, T. H., Tsang, L. M., Chan, B. K. K., & Chu, K. H. (2015). Cryptic diversity

and phylogeography of the island-associated barnacle Chthamalus

moro in Asia. Marine Ecology, 36(3), 368–378.

Tsang, L. M., Chan, B. K. K., Wu, T. H., Ng, W. C., Chatterjee, T., Williams,

G. A., & Chu, K. H. (2008). Population differentiation in the barnacle

Chthamalus malayensis: Postglacial colonization and recent connectivity

across the Pacific and Indian Oceans. Marine Ecology Progress Series,

364, 107–118.

Chan, B. K. K., Tsang, L. M., & Chu, K. H. (2007). Morphological and genetic

differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita. Zoologica Scripta, 36(1), 79–91.

Reviewer #2: This is a study focusing on the phylogeography of the Parasesarma bidens species complex from West Pacific. While the methods of analyses were adequate, some biogeographical explanations based on the limited sample size should be more conservative.

Major comments:

* The included clades of this complex might be problematic. According to the trees in Shahdadi & Schubart (2017), Li et al. (2019), and Shahdadi et al. (2020), 3 spp. (bengalense, capensis, guttatum) in the Indian Ocean probably belong to the major clade of the bidens complex and at least closely related with the complex. The authors should include sequences of the above 3 spp. and might need to rewrite the part of "Phylogeny, phylogeography and evolutionary history" in Discussion.

* L391, L399, L438-440: As the authors also mentioned that large sample size from Taiwan and Philippines are needed (L440-441), it is apparently too early to say one area could be considered as the origin. Otherwise, it will be more convincing by citing related references that Philippines is the origin of some marine species.

* L428-430: There might be barriers between Taiwan and China for some crab species, however populations in both areas of some species might show identical. For example, Austruca lactea (Shih, H.-T., Kamrani, E., Davie, P. J. F. & Liu, M.-Y., 2009. Genetic evidence for the recognition of two fiddler crabs, Uca iranica and U. albimana (Crustacea: Brachyura: Ocypodidae), from the northwestern Indian Ocean, with notes on the U. lactea species-complex. Hydrobiologia 635: 373-382) and some Metaplax spp. (Shih, H.-T., Hsu, J.-W., Wong, K. J. H. & Ng, N. K. 2019. Review of the mudflat varunid crab genus Metaplax (Crustacea, Brachyura, Varunidae) from East Asia and northern Vietnam. ZooKeys 877: 1-29). It might be necessary to discuss the larval behavior/ecology of related organisms and the ocean currents in this region (e.g. Taiwan Strait).

Other comments:

* L30, L103: COX1 and 16S ?

* L95: "The latter species": It's clear to use the species name here.

* L149: [51] is a study of freshwater crabs with only 16S and H3. I will suggest the authors check all the references and see if they are really relevant.

* L15-156: It is better to use species from the sister clades as outgroup. See "major comments #1".

* L160: Will suggest to use the newest version of MEGA.

* L173-174: Try to use formal citation and reference for "Microsoft Excel (2013)".

* L175: ARLEQUIN => ARLEQUIN 3.5; and delete "3.5" in L182.

* L184 and others: Japan is composed by several large and small islands, but could not be considered as "mainland"! Will suggest to use "main islands of Japan" instead.

* L194-196: It is confused that ".... (COX1) 16 sequences (including those from GenBank) with a length of 610 basepairs.....". Just say how many sequences you have from this study and how many sequences from GenBank. Rewrite this sentence.

* L229: It should be confused whether it is necessary to provide 2 haplotype networks of COX1? One single tree of COX1 should be enough to provide strong evidence of the 5 clades and their relationship.

* L325: If the authors did not run the test of rate constancy or similar tests, it is not adequate to say that the substitute rate is "a relatively precise time estimate", compared with the relaxed method (fossil data and other geological evidences" (L322-323).

* L346: "major East Asian island" => "major islands in eastern Asia", because the Philippines is not a part of "East Asia".

* L440: samples size => sample size

* L393: Provide citation for "melting pot".

* L425-428: For freshwater discharge in eastern Americas, I might suggest cite two relevant papers: (1) Thurman, C. L. , Hopkins, M. J., Brase, A. L. & Shih, H.-T. 2018. The unusual case of the widely distributed fiddler crab Minuca rapax (Smith, 1870) from the western Atlantic: an exemplary polytypic species. Invertebrate Systematics 32(6): 1465-1490. (2) Thurman, C. L, Alber, R. E., Hopkins, M. J. & Shih, H.-T. 2021. Morphological and genetic variation among populations of the fiddler crab Minuca burgersi (Holthuis, 1967) (Crustacea: Brachyura: Ocypodidae) from shores of the Caribbean Basin and western South Atlantic Ocean. Zoological Studies 60: 19.

* Fig. 1: It will be clear for readers to mark the names for the major localities on the map.

* Fig. 2: A separated BI tree with all the haplotype names is suggested to be included in supporting information (similar to S3 Figure for ML tree). "P. Sanguimanus" => "P. sanguimanus".

* S1 Table: Full names of museums for the abbreviation should be provided. In "size", all values in CW should be round to one decimal place. "14,47"=>"14.5". "Honlong" => "Houlong". BTW I found several haplotypes in S1 Table were not appeared in S3 figure! Need to check them carefully.

**********

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.

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

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

Reviewer #1: No

Reviewer #2: No

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

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. 2022 Jan 13;17(1):e0262122. doi: 10.1371/journal.pone.0262122.r002

Author response to Decision Letter 0

4 Oct 2021

Response to reviewers:

We thank the esteemed reviewers for their nice and helpful comments. In the revised version of our manuscript we have tried to go through their comments and correct the manuscript accordingly. The changes are addressed here.

Reviewers' comments:

Answer. We thank you for introducing this relevant study. In the new version, we cited this study through the manuscript (Introduction, lines 48, 50, Discussion line 446).

Trans-Pacific mechanisms

Answer. The point is added now to the introduction (lines 50-52) and the study is cited (reference number 15).

Mangrove mudskippers:

Chen, W., Hong, W. S., Chen, S. X., Wang, Q., & Zhang, Q. Y. (2015).

Population genetic structure and demographic history of the mudskipper

Boleophthalmus pectinirostris on the northwestern pacific coast.

Environmental Biology of Fishes, 98(3), 845–856.

Mangrove snails:

Kojima, S., Hayashi, I., Kim, D., Iijima, A., & Furota, T. (2004). Phylogeography

of an intertidal direct-developing gastropod Batillaria cumingi around

the Japanese Islands. Marine Ecology Progress Series, 276, 161–172.

Kojima, S., Kamimura, S., Iijima, A., Kimura, T., Mori, K., Hayashi, I., & Furota,

T. (2005). Phylogeography of the endangered tideland snail Batillaria

zonalis in the Japanese and Ryukyu Islands. Ecological Research, 20(6),

686–694.

Kojima, S., Kamimura, S., Kimura, T., Hayashi, I., Iijima, A., & Furota, T. (2003).

Phylogenetic relationships between the tideland snails Batillaria flectosiphonata

in the Ryukyu Islands and B. multiformis in the Japanese

Islands. Zoological Science, 20(11), 1423–1433.

Mangrove barnacles:

Mangrove crabs:

Answer. We thank for introducing these relevant studies. They were indeed helpful and we have used them to add some more points and examples in our discussion (lines 335, 440,441). We have also cited them in our introduction (line 87). Reference numbers: 43,80,89, 90.

Please cite and refer to this example:

ZS.2020.59-16.

Ng PKL, Li JJ, Shih HT. 2020. What is Sesarmops impressus (H. Milne Edwards, 1837) (Crustacea: Brachyura: Sesarmidae)? Zool Stud 59:27. doi:10.6620/ZS.2020.59-27.

Answer. We thank you for introducing the interesting works. In the new version we have used and cited (ref no. 84) the study done by Tsang et al. (2013) in the discussion (lines 408, 429, 469). The other two study are valuable taxonomic works, but they did not discuss phylogeography and population genetic.

5)Part of the above reference already show some of the glacial refugia. For more details on the glacial refugia in the West Pacific, the authors should refer:

Wu, T. H., Tsang, L. M., Chan, B. K. K., & Chu, K. H. (2015). Cryptic diversity

and phylogeography of the island-associated barnacle Chthamalus

moro in Asia. Marine Ecology, 36(3), 368–378.

Tsang, L. M., Chan, B. K. K., Wu, T. H., Ng, W. C., Chatterjee, T., Williams,

G. A., & Chu, K. H. (2008). Population differentiation in the barnacle

Chthamalus malayensis: Postglacial colonization and recent connectivity

across the Pacific and Indian Oceans. Marine Ecology Progress Series,

364, 107–118.

Chan, B. K. K., Tsang, L. M., & Chu, K. H. (2007). Morphological and genetic

differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita. Zoologica Scripta, 36(1), 79–91.

Answer. These studied are cited in the new version of the manuscript. The two studies, Wu et al. (2015) (ref no. 77) and Tsang et al. (2008) (ref no. 72), are cited for the glacial refugia (Discussion lines 321,330, 450). The study done by Tsang et al. (2008) and the work of Chan et al. (2007) (ref no. 95) are cited for another discussion, the separation between Taiwan and China mainland (Discussion lines 330, 450).

Major comments:

Answer. We thank you for the nice comment. In the revised version we have added a sequence of P. bengalense as representative of the western clade (P. bengalense, P. capensis and P. guttatum), and reanalyzed the data. We made then a slight change in the method section (Materials & Methods, lines 157-160). We had slight changes in the results (new trees and Results lines 218, 229), and we made minor changes in the discussion according to the new results (Discussion lines 341-351). According to the trees in the abovementioned studies (Shahdadi & Schubart, 2017, Li et al., 2019 and Shahdadi et al., 2020) as well as in Shahdadi et al., 2021 (https://doi.org/10.1071/IS20046), this species group consists of two separate clades, the western clades (within the Indian Ocean) and the eastern clade (West Pacific). So the western species (P. bengalense, P. capensis and P. gutttum) form a monophyletic group, separated from the eastern species (P. bidens, P. cricotum and P. sanguimanus). During the time we were revising our manuscript, it was not possible for us to get material of all those western species in order to get the corresponding sequences, but we were able to add sequences of P. bengalense to our analyses. According to their monophyly, we believe that P. bengalense can represent the western clade and show the phylogenetic relationship between these two groups (eastern and western) (briefly discussed in the new version, Discussion, lines 341-351). As the new results (new trees) show, all five eastern clades stay under a bigger clade as a monophyletic group, separated from P. bengalense. After adding P. bengalense to our analyses as representative of the western clade, we are now able to calculate the divergence time (an estimate) between western and eastern clades. We therefore added a few sentences in this regards to our new version as addressed above.

we hope this change cover the issue, and the referee accept the manuscript in the present form, otherwise we need more time to ask for the material of P. guttatum and P. capensis and try to get new genetic data.

Answer. In the new version of our manuscript we have tried to soften this conclusion and we have given a relevant reference with the same conclusion (ref no. 84). At the end, however, we have admitted that larger sample size could give stronger support for the idea (see Discussion lines 469,470).

Answer. We thank you for this comment and in the new version we mentioned this diversity in phylogeographic structure and the possible reasons using these and others references (Discussion lines 446-456) (ref numbers 42, 44, 72, 95,96,97,98, 100).

Other comments:

* L30, L103: COX1 and 16S ?

Answer. Corrected now (line 30, 105).

* L95: "The latter species": It's clear to use the species name here.

Answer. Corrected now (line 95).

* L149: [51] is a study of freshwater crabs with only 16S and H3. I will suggest the authors check all the references and see if they are really relevant.

Answer. The reference 51 is deleted now from the manuscript. We have made other changes in the references in order to keep the relevancy and use the latest relevant studies.

* L15-156: It is better to use species from the sister clades as outgroup. See "major comments #1".

Answer. In the revised version of the manuscript, as suggested in the major comment 1, we have added a representative of the sister clade (P. bengalense) as one of the ingroups, in order to 1. Check their phylogenetic relationship, 2. To show monophyly of the eastern clade (Western Pacific clade) and 3. To calculate divergence time between eastern and western (within the Indian Ocean) clades (addressed in response to the major comments #1.

* L160: Will suggest to use the newest version of MEGA.

Answer. In the revised version we have used MEGA X to calculate the K2P distances (line 166) and the reference is also updated (ref no. 60). The slight changes in the values are applied in the table S5 for the new version of the manuscript.

* L173-174: Try to use formal citation and reference for "Microsoft Excel (2013)".

Answer. Corrected now (ref no. 65).

* L175: ARLEQUIN => ARLEQUIN 3.5; and delete "3.5" in L182.

Answer. Corrected now.

* L184 and others: Japan is composed by several large and small islands, but could not be considered as "mainland"! Will suggest to use "main islands of Japan" instead.

Answer. Corrected now.

Answer. To clarify and avoid confusion in the new version we have slightly changed the sentence (lines 200-203).

Answer. It is true that the haplotype networks confirm presence of five clades as the trees do. We, however, present the main haplotype network (for the 3’ end) as extra evidence for the clades showing the number of mutation steps between clades. It also gives visual evidence for haplotype relationship within each clade, which cannot be extracted from the trees. The haplotype network for the barcode region (for the 5’ end) is presented as a supplementary material in order to show the positions of the material from areas that we were not able to cover, but the data are available in GenBank. In order to avoid confusion we have tried to be clear in the Method section, as well as clarify the reason and our aim for presenting each illustration. So for this stage we prefer to keep both networks and, hoping that you accept our MS in this format.

Answer. It is true, and we have slightly change our state on this matter (Lines 324-325)

* L346: "major East Asian island" => "major islands in eastern Asia", because the Philippines is not a part of "East Asia".

Answer. Corrected now.

* L440: samples size => sample size

Answer. Corrected now.

* L393: Provide citation for "melting pot".

Answer. We see it as a fact and common term, so we see no need to add refrence.

Answer. We thank you for introducing the studies. In the new version, we have included more discussion and references (including Thurman et al., 2021) for this part (Discussion line 439-446) (reference numbers 12, 80, 89, 90, 91, 94).

* Fig. 1: It will be clear for readers to mark the names for the major localities on the map.

Answer. In the revised version names for the major localities are added in this figure.

* Fig. 2: A separated BI tree with all the haplotype names is suggested to be included in supporting information (similar to S3 Figure for ML tree). "P. Sanguimanus" => "P. sanguimanus".

Answer. In the new version we have added the BI tree as a supplementary figure (S3Fig). P. sanguimanus is also corrected now.

Answer. In the new version: Full names of museums for the abbreviation are added at the bottom of the S1 Table; values for the sizes are rounded; the spellings are checked and corrected now; the haplotypes in S1 Table that were not appeared in S3 figure are deleted now.

Attachment

Submitted filename: Comments from reviewers and answers.docx

Click here for additional data file.^{(30.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0262122.r003

Decision Letter 1

Benny KK Chan

27 Oct 2021

PONE-D-21-19346R1Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West PacificPLOS ONE

Dear Dr. Shahdadi,

Dear authors,

Most of the comments were addressed. But I noticed that in the references list of your MS, most of the author name Chan, BKK was mis-spelled as Chan BK. Please correct all the Chan BK into Chan BKK in the reference list.

Best wishes,

Benny

Please submit your revised manuscript by Dec 11 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Benny K.K. Chan, Ph.D

Academic Editor

PLOS ONE

Journal Requirements:

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

Additional Editor Comments:

Dear authors,

Best wishes,

Benny

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

Reviewers' comments:

PLoS One. 2022 Jan 13;17(1):e0262122. doi: 10.1371/journal.pone.0262122.r004

Author response to Decision Letter 1

16 Dec 2021

Referee comments and answers.

Dear Dr. Shahdadi,

Dear authors,

Answer.

We thank you for your kindness and the comment. We have corrected the mis-spelled names (Chan BK) in our new version (Chan BKK).

With best regards,

Adnan Shahdadi

Attachment

Submitted filename: Referee comments and answers.docx

Click here for additional data file.^{(11.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0262122.r005

Decision Letter 2

Benny KK Chan

19 Dec 2021

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

PONE-D-21-19346R2

Dear Dr. Shahdadi,

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,

Benny K.K. Chan, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The reviewer comments were addressed and it can be accepted for publication.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0262122.r006

Acceptance letter

Benny KK Chan

5 Jan 2022

PONE-D-21-19346R2

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

Dear Dr. Shahdadi:

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. Benny K.K. Chan

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 Fig. COX1 (the 3’ end; 742 bp) consensus Bayesian tree topology constructed with BEAST.

(TIF)

Click here for additional data file.^{(268.7KB, tif)}

S2 Fig. COX1 (the 3’ end; 742 bp) Maximum Likelihood (ML) tree topology constructed with RaxmlGUI.

(TIF)

Click here for additional data file.^{(279.2KB, tif)}

S3 Fig. Maximum parsimony haplotype network of COX1 (the 3’ end; 610 bp) for a subset of specimens and sequences recovered from GenBank, constructed with PopART.

Hatch marks represent mutation steps.

(TIF)

Click here for additional data file.^{(160.6KB, tif)}

(DOCX)

Click here for additional data file.^{(29.9KB, docx)}

S2 Table. Primers used in the present study with corresponding DNA sequences (5´-3´) and references.

(DOCX)

Click here for additional data file.^{(14KB, docx)}

(DOCX)

Click here for additional data file.^{(13.9KB, docx)}

Attachment

Submitted filename: Comments from reviewers and answers.docx

Click here for additional data file.^{(30.1KB, docx)}

Attachment

Submitted filename: Referee comments and answers.docx

Click here for additional data file.^{(11.8KB, docx)}

Data Availability Statement

[pone.0262122.ref001] 1.Porter SC. Some geological implications of average Quaternary glacial conditions. Quaternary Research. 1989;32(3):245–61. [Google Scholar]

[pone.0262122.ref002] 2.Woodruff DS. Biogeography and conservation in Southeast Asia: how 2.7 million years of repeated environmental fluctuations affect today’s patterns and the future of the remaining refugial-phase biodiversity. Biodiversity and Conservation. 2010;19(4):919–41. [Google Scholar]

[pone.0262122.ref003] 3.Springer VG, Williams JT. Widely distributed Pacific plate endemics and lowered sea-level. Bulletin of Marine Science. 1990;47(3):631–40. [Google Scholar]

[pone.0262122.ref004] 4.Hewitt G. The genetic legacy of the Quaternary ice ages. Nature. 2000;405(6789):907–13. doi: 10.1038/35016000 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref005] 5.Barber PH, Erdmann MV, Palumbi SR. Comparative phylogeography of three codistributed stomatopods: origins and timing of regional lineage diversification in the coral triangle. Evolution. 2006;60(9):1825–39. [PubMed] [Google Scholar]

[pone.0262122.ref006] 6.Beck SV, Carvalho GR, Barlow A, Rüber L, Hui Tan H, Nugroho E, et al. Plio-Pleistocene phylogeography of the Southeast Asian blue panchax killifish, Aplocheilus panchax. PLoS One. 2017;12(7):e0179557. doi: 10.1371/journal.pone.0179557 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref007] 7.Hellberg ME. Gene flow and isolation among populations of marine animals. Annual Review of Ecology, Evolution, and Systematics. 2009;40:291–310. [Google Scholar]

[pone.0262122.ref008] 8.Dawson MN, Barber PH, González‐Guzmán LI, Toonen RJ, Dugan JE, Grosberg RK. Phylogeography of Emerita analoga (Crustacea, Decapoda, Hippidae), an eastern Pacific Ocean sand crab with long‐lived pelagic larvae. Journal of Biogeography. 2011;38(8):1600–12. [Google Scholar]

[pone.0262122.ref009] 9.Borsa P, Durand J-D, Chen W-J, Hubert N, Muths D, Mou-Tham G, et al. Comparative phylogeography of the western Indian Ocean reef fauna. Acta Oecologica. 2016;72:72–86. [Google Scholar]

[pone.0262122.ref010] 10.Silva IC, Mesquita N, Paula J. Genetic and morphological differentiation of the mangrove crab Perisesarma guttatum (Brachyura: Sesarmidae) along an East African latitudinal gradient. Biological Journal of the Linnean Society. 2010;99(1):28–46. [Google Scholar]

[pone.0262122.ref011] 11.Shahdadi A, Davie PJ, Schubart CD. Systematics and phylogeography of the Australasian mangrove crabs Parasesarma semperi and P. longicristatum (Decapoda: Brachyura: Sesarmidae) based on morphological and molecular data. Invertebrate Systematics. 2018;32(1):196–214. [Google Scholar]

[pone.0262122.ref012] 12.Thurman C, Alber R, Hopkins M, Shih H-T. Morphological and genetic variation among populations of the fiddler crab Minuca burgersi (Holthuis, 1967) (Crustacea: Brachyura: Ocypodidae) from shores of the Caribbean Basin and western South Atlantic Ocean. Zoological Studies. 2021;60(19). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref013] 13.Grosberg R, Cunningham C. Genetic structure in the sea: From populations to communities. En: Bertness MD, Gaines SD, Hay ME(Eds.) Marine Community Ecology: 61–84. Sinauer Associates, Sunderland, MA, USA; 2001. [Google Scholar]

[pone.0262122.ref014] 14.Bowen BW, Rocha LA, Toonen RJ, Karl SA. The origins of tropical marine biodiversity. Trends in Ecology & Evolution. 2013;28(6):359–66. doi: 10.1016/j.tree.2013.01.018 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref015] 15.Hongjamrassilp W, Murase A, Miki R, Hastings PA. Journey to the west: trans-Pacific historical biogeography of fringehead blennies in the genus Neoclinus (Teleostei: Blenniiformes). Zoological Studies. 2020;59(09). [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref016] 16.Benzie J, Ballment E, Forbes A, Demetriades N, Sugama K, Moria S. Mitochondrial DNA variation in Indo‐Pacific populations of the giant tiger prawn, Penaeus monodon. Molecular Ecology. 2002;11(12):2553–69. doi: 10.1046/j.1365-294x.2002.01638.x [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref017] 17.Bickford D, Lohman DJ, Sodhi NS, Ng PK, Meier R, Winker K, et al. Cryptic species as a window on diversity and conservation. Trends in Ecology & Evolution. 2007;22(3):148–55. doi: 10.1016/j.tree.2006.11.004 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref018] 18.Yednock BK, Neigel JE. Rethinking the mechanisms that shape marine decapod population structure. Phylogeography and Population Genetics in Crustacea. 2011:57–73. [Google Scholar]

[pone.0262122.ref019] 19.Lai JC, Ng PKL, Davie PJ. A revision of the Portunus pelagicus (Linnaeus, 1758) species complex (Crustacea: Brachyura: Portunidae), with the recognition of four species. Raffles Bulletin of Zoology. 2010;58(2). [Google Scholar]

[pone.0262122.ref020] 20.Shin J, Jung J, Kim W, Jung J. Phylogeographic studies on two shore crab species from East Asia: similar but different stories. Genes & Genomics. 2019;41(10):1127–34. doi: 10.1007/s13258-019-00831-9 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref021] 21.Davie PJ. Variations in diversity of mangrove crabs in tropical Australia. Memoirs of the Queensland Museum Brisbane. 1994;36(1):55–8. [Google Scholar]

[pone.0262122.ref022] 22.Tan CG, Ng PKL. An annotated checklist of mangrove brachyuran crabs from Malaysia and Singapore. Hydrobiologia. 1994;285(1):75–84. [Google Scholar]

[pone.0262122.ref023] 23.Lee S. Ecological role of grapsid crabs in mangrove ecosystems: a review. Marine and Freshwater Research. 1998;49(4):335–43. [Google Scholar]

[pone.0262122.ref024] 24.Guerao G, Anger K, Nettelmann U, Schubart CD. Complete larval and early juvenile development of the mangrove crab Perisesarma fasciatum (Crustacea: Brachyura: Sesarmidae) from Singapore, with a larval comparison of Parasesarma and Perisesarma. Journal of Plankton Research. 2004;26(12):1389–408. [Google Scholar]

[pone.0262122.ref025] 25.De Grave S, Pentcheff ND, Ahyong ST, Chan T-Y, Crandall KA, Dworschak PC, et al. A classification of living and fossil genera of decapod crustaceans. Raffles Bulletin of Zoology. 2009. [Google Scholar]

[pone.0262122.ref026] 26.Briggs JC. Centrifugal speciation and centres of origin. Journal of Biogeography. 2000;27(5):1183–8. [Google Scholar]

[pone.0262122.ref027] 27.Briggs JC. The marine East Indies: diversity and speciation. Journal of Biogeography. 2005;32(9):1517–22. [Google Scholar]

[pone.0262122.ref028] 28.Costello MJ, Coll M, Danovaro R, Halpin P, Ojaveer H, Miloslavich P. A census of marine biodiversity knowledge, resources, and future challenges. PLoS One. 2010;5(8):e12110. doi: 10.1371/journal.pone.0012110 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref029] 29.Wafar M, Venkataraman K, Ingole B, Khan SA, LokaBharathi P. State of knowledge of coastal and marine biodiversity of Indian Ocean countries. PLoS One. 2011;6(1):e14613. doi: 10.1371/journal.pone.0014613 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref030] 30.Puckridge M, Last PR, White WT, Andreakis N. Phylogeography of the Indo‐West Pacific maskrays (Dasyatidae, Neotrygon): A complex example of chondrichthyan radiation in the Cenozoic. Ecology and Evolution. 2013;3(2):217–32. doi: 10.1002/ece3.448 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref031] 31.Bowen BW, Gaither MR, DiBattista JD, Iacchei M, Andrews KR, Grant WS, et al. Comparative phylogeography of the ocean planet. Proceedings of the National Academy of Sciences. 2016;113(29):7962–9. doi: 10.1073/pnas.1602404113 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref032] 32.Ma KY, Chow LH, Wong KJ, Chen H-N, Ip BH, Schubart CD, et al. Speciation pattern of the horned ghost crab Ocypode ceratophthalmus (Pallas, 1772): an evaluation of the drivers of Indo-Pacific marine biodiversity using a widely distributed species (vol 45, pg 2658, 2018). Journal of Biogeography. 2019;46(4):830. [Google Scholar]

[pone.0262122.ref033] 33.Ragionieri L, Fratini S, Vannini M, Schubart CD. Phylogenetic and morphometric differentiation reveal geographic radiation and pseudo-cryptic speciation in a mangrove crab from the Indo-West Pacific. Molecular Phylogenetics and Evolution. 2009;52(3):825–34. doi: 10.1016/j.ympev.2009.04.008 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref034] 34.Ragionieri L, Fratini S, Schubart CD. Revision of the Neosarmatium meinerti species complex (Decapoda: Brachyura: Sesarmidae), with descriptions of three pseudocryptic Indo-West Pacific species. Raffles Bulletin of Zoology. 2012;60(1). [Google Scholar]

[pone.0262122.ref035] 35.Shahdadi A, Davie PJ, Rahayu DL, Schubart CD. The synonymy of Parasesarma foresti (Rahayu & Davie, 2002) with P. indiarum (Tweedie, 1940) (Decapoda, Brachyura, Sesarmidae). Crustaceana. 2020;93(7):793–801. [Google Scholar]

[pone.0262122.ref036] 36.Shahdadi A, Fratini S, Schubart CD. Taxonomic reassessment of Parasesarma (Crustacea: Brachyura: Decapoda: Sesarmidae) based on genetic and morphological comparisons, with the description of a new genus. Zoological Journal of the Linnean Society. 2020;190(4):1123–58. [Google Scholar]

[pone.0262122.ref037] 37.Benzie J. Genetic structure of marine organisms and SE Asian biogeography. Biogeography and Geological Evolution of SE Asia. 1998;30:197–209. [Google Scholar]

[pone.0262122.ref038] 38.Roberts CM, McClean CJ, Veron JE, Hawkins JP, Allen GR, McAllister DE, et al. Marine biodiversity hotspots and conservation priorities for tropical reefs. Science. 2002;295(5558):1280–4. doi: 10.1126/science.1067728 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref039] 39.Barber PH, Palumbi SR, Erdmann MV, Moosa MK. A marine Wallace’s line? Nature. 2000;406(6797):692–3. doi: 10.1038/35021135 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref040] 40.Barber PH, Bellwood DR. Biodiversity hotspots: evolutionary origins of biodiversity in wrasses (Halichoeres: Labridae) in the Indo-Pacific and new world tropics. Molecular Phylogenetics and Evolution. 2005;35(1):235–53. Epub 2005/03/02. doi: 10.1016/j.ympev.2004.10.004 . [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref041] 41.Xu J, Chan T-Y, Tsang LM, Chu KH. Phylogeography of the mitten crab Eriocheir sensu stricto in East Asia: Pleistocene isolation, population expansion and secondary contact. Molecular Phylogenetics and Evolution. 2009;52(1):45–56. doi: 10.1016/j.ympev.2009.02.007 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref042] 42.Cheng J, Wang Z, Song N, Yanagimoto T, Gao T. Phylogeographic analysis of the genus Platycephalus along the coastline of the northwestern Pacific inferred by mitochondrial DNA. BMC Evolutionary Biology. 2019;19(1):1–16. doi: 10.1186/s12862-018-1333-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref043] 43.Kojima S, Hayashi I, Kim D, Iijima A, Furota T. Phylogeography of an intertidal direct-developing gastropod Batillaria cumingi around the Japanese Islands. Marine Ecology Progress Series. 2004;276:161–72. [Google Scholar]

[pone.0262122.ref044] 44.Guo X, Zhao D, Jung D, Li Q, Kong L-F, Ni G, et al. Phylogeography of the rock shell Thais clavigera (Mollusca): evidence for long-distance dispersal in the northwestern Pacific. PloS One. 2015;10(7):e0129715. doi: 10.1371/journal.pone.0129715 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref045] 45.Haan HMD. Crustacea. In: von Siebold P. F., P.F., Fauna Japonica sive Descriptio Animalium, quae in Itinere per Japoniam, Jussu et Auspiciis Superiorum, qui Summum in India Batava Imperium Tenent, Suspecto, Annis 1823–1830 Collegit, Notis, Observationibus et Adumbrationibus Illustravit. Leiden, the Netherlands: Published by the author; 1833–1850. 243 p. [Google Scholar]

[pone.0262122.ref046] 46.Dai AY, Yang SL, Song YZ, Chen GX. Crabs of the China Seas. Beijing: China Ocean Press (Chinese edition); Berlin, Heidelberg, New York, Tokyo: Springer (English edition).1991. 682 p. [Google Scholar]

[pone.0262122.ref047] 47.Zhou H, Xu J, Yang M, Wu B, Yan B, Xiong Y. Population genetic diversity of sesarmid crab (Perisesarma bidens) in China based on mitochondrial DNA. Mitochondrial DNA Part A. 2016;27(5):3255–62. doi: 10.3109/19401736.2015.1015002 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref048] 48.Rahayu DL, Davie PJ. Two new species and a new record of Perisesarma (Decapoda, Brachyura, Grapsidae, Sesarminae) from Indonesia. Crustaceana. 2002:597–607. [Google Scholar]

[pone.0262122.ref049] 49.Li J-J, Shih H-T, Ng PKL. Three new species and two new records of Parasesarma De Man, 1895 (Crustacea: Brachyura: Sesarmidae) from Taiwan and the Philippines from morphological and molecular evidence. Zoological Studies. 2019;58. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref050] 50.Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research. 1994;22(22):4673–80. doi: 10.1093/nar/22.22.4673 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref051] 51.Hall TA, editor BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series; 1999: [London: ]: Information Retrieval Ltd., c1979–c2000. [Google Scholar]

[pone.0262122.ref052] 52.Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML. Organisms Diversity & Evolution. 2012;12(4):335–7. [Google Scholar]

[pone.0262122.ref053] 53.Bouckaert R, Vaughan TG, Barido-Sottani J, Duchêne S, Fourment M, Gavryushkina A, et al. BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS Computational Biology. 2019;15(4):e1006650. doi: 10.1371/journal.pcbi.1006650 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref054] 54.Schubart CD, Diesel R, Hedges SB. Rapid evolution to terrestrial life in Jamaican crabs. Nature. 1998;393(6683):363–5. [Google Scholar]

[pone.0262122.ref055] 55.Rodriguez F, Oliver JL, Marín A, Medina JR. The general stochastic model of nucleotide substitution. Journal of Theoretical Biology. 1990;142(4):485–501. doi: 10.1016/s0022-5193(05)80104-3 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref056] 56.Posada D, Buckley TR. Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Systematic Biology. 2004;53(5):793–808. doi: 10.1080/10635150490522304 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref057] 57.Shahdadi A, Schubart CD, Mendoza JCE. Conspicuous genetic similarity within a widely distributed and newly described species of Parasesarma De Man, 1895 from Western Pacific oceanic islands, with notes on the allied P. calypso group (Crustacea: Brachyura: Sesarmidae). Invertebrate Systematics. 2021;35(5):542–69. [Google Scholar]

[pone.0262122.ref058] 58.Templeton AR, Crandall KA, Sing CF. A cladistic analysis of phenotypic associations with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics. 1992;132(2):619–33. doi: 10.1093/genetics/132.2.619 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref059] 59.Leigh JW, Bryant D. popart: full‐feature software for haplotype network construction. Methods in Ecology and Evolution. 2015;6(9):1110–6. [Google Scholar]

[pone.0262122.ref060] 60.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution. 2018;35(6):1547. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref061] 61.Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009;25(11):1451–2. doi: 10.1093/bioinformatics/btp187 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref062] 62.Fu Y-X. Statistical tests of neutrality of mutations against population growth, hitchhiking and background selection. Genetics. 1997;147(2):915–25. doi: 10.1093/genetics/147.2.915 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref063] 63.Excoffier L, Lischer HE. Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources. 2010;10(3):564–7. doi: 10.1111/j.1755-0998.2010.02847.x [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref064] 64.Rogers AR, Harpending H. Population growth makes waves in the distribution of pairwise genetic differences. Molecular Biology and Evolution. 1992;9(3):552–69. doi: 10.1093/oxfordjournals.molbev.a040727 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref065] 65.Carlberg C. Statistical analysis: Microsoft excel 2013. Indianapolis: Que Publishing; 2014. [Google Scholar]

[pone.0262122.ref066] 66.Harpending H. Signature of ancient population growth in a low-resolution mitochondrial DNA mismatch distribution. Human Biology. 1994:591–600. [PubMed] [Google Scholar]

[pone.0262122.ref067] 67.Richmond GM, Fullerton DS. Summation of Quaternary glaciations in the United States of America. Quaternary Science Reviews. 1986;5:183–96. [Google Scholar]

[pone.0262122.ref068] 68.Murray-Wallace CV, Woodroffe CD. Quaternary sea-level changes: a global perspective: Cambridge University Press; 2014. [Google Scholar]

[pone.0262122.ref069] 69.Voris HK. Maps of Pleistocene sea levels in Southeast Asia: shorelines, river systems and time durations. Journal of Biogeography. 2000;27(5):1153–67. [Google Scholar]

[pone.0262122.ref070] 70.Marko PB, Hoffman JM, Emme SA, McGovern TM, Keever CC, Nicole Cox L. The ‘Expansion–Contraction’model of Pleistocene biogeography: rocky shores suffer a sea change? Molecular Ecology. 2010;19(1):146–69. doi: 10.1111/j.1365-294x.2009.04417.x [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref071] 71.Avise JC. Phylogeography: the history and formation of species: Harvard university press; 2000. [Google Scholar]

[pone.0262122.ref072] 72.Tsang LM, Chan BKK, Wu TH, Ng WC, Chatterjee T, Williams GA, et al. Population differentiation in the barnacle Chthamalus malayensis: postglacial colonization and recent connectivity across the Pacific and Indian Oceans. Marine Ecology Progress Series. 2008;364:107–18. [Google Scholar]

[pone.0262122.ref073] 73.Knowlton N. Sibling species in the sea. Annual Review of Ecology and Systematics. 1993;24(1):189–216. [Google Scholar]

[pone.0262122.ref074] 74.Knowlton N, Weigt LA. New dates and new rates for divergence across the Isthmus of Panama. Proceedings of the Royal Society of London Series B: Biological Sciences. 1998;265(1412):2257–63. [Google Scholar]

[pone.0262122.ref075] 75.Papadopoulou A, Anastasiou I, Vogler AP. Revisiting the insect mitochondrial molecular clock: the mid-Aegean trench calibration. Molecular Biology and Evolution. 2010;27(7):1659–72. doi: 10.1093/molbev/msq051 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref076] 76.Wang J, Tsang LM, Dong Y-W. Causations of phylogeographic barrier of some rocky shore species along the Chinese coastline. BMC Evolutionary Biology. 2015;15(1):1–15. doi: 10.1186/s12862-015-0387-0 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref077] 77.Wu TH, Tsang LM, Chan BKK, Chu KH. Cryptic diversity and phylogeography of the island‐associated barnacle Chthamalus moro in Asia. Marine Ecology. 2015;36(3):368–78. [Google Scholar]

[pone.0262122.ref078] 78.Kinlan BP, Gaines SD, Lester SE. Propagule dispersal and the scales of marine community process. Diversity and Distributions. 2005;11(2):139–48. [Google Scholar]

[pone.0262122.ref079] 79.Horne JB, van Herwerden L, Choat JH, Robertson DR. High population connectivity across the Indo-Pacific: congruent lack of phylogeographic structure in three reef fish congeners. Molecular Phylogenetics and Evolution. 2008;49(2):629–38. doi: 10.1016/j.ympev.2008.08.023 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref080] 80.Chang YW, Chan JS, Hayashi R, Shuto T, Tsang LM, Chu KH, et al. Genetic differentiation of the soft shore barnacle Fistulobalanus albicostatus (Cirripedia: Thoracica: Balanomorpha) in the West Pacific. Marine Ecology. 2017;38(2):e12422. [Google Scholar]

[pone.0262122.ref081] 81.Shahdadi A, Schubart CD. Taxonomic review of Perisesarma (Decapoda: Brachyura: Sesarmidae) and closely related genera based on morphology and molecular phylogenetics: new classification, two new genera and the questionable phylogenetic value of the epibranchial tooth. Zoological Journal of the Linnean Society. 2017;182(3):517–48. [Google Scholar]

[pone.0262122.ref082] 82.Shahdadi A, Schubart CD, Mendoza JCE. On the occurrence of Parasesarma cricotum (Rahayu and Davie, 2002 (Decapoda, Brachyura, Sesarmidae) in Palau and Sulawesi. Zootaxa. 2020;4803(2):zootaxa. 4803.2. 12-zootaxa. .2. 12. [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref083] 83.Ng PKL, Shih HT, Ho PH, Wang CH. An updated annotated checklist of brachyuran crabs from Taiwan (Crustacea: Decapoda). Journal of National Taiwan Museum. 2017;70:1–208. [Google Scholar]

[pone.0262122.ref084] 84.Tsang LM, Chan BKK, Williams GA, Chu KH. Who is moving where? Molecular evidence reveals patterns of range shift in the acorn barnacle Hexechamaesipho pilsbryi in Asia. Marine Ecology Progress Series. 2013;488:187–200. [Google Scholar]

[pone.0262122.ref085] 85.Carpenter KE, Springer VG. The center of the center of marine shore fish biodiversity: the Philippine Islands. Environmental Biology of Fishes. 2005;72(4):467–80. [Google Scholar]

[pone.0262122.ref086] 86.Carpenter KE, Barber PH, Crandall ED, Ablan-Lagman M, Carmen A, Mahardika GN, et al. Comparative phylogeography of the Coral Triangle and implications for marine management. Journal of Marine Biology. 2011;2011. [Google Scholar]

[pone.0262122.ref087] 87.Kim SY, Yi CH, Kim JM, Choi WY, Kim HS, Kim M-S. DNA Barcoding of the marine protected species Parasesarma bidens (Decapoda: Sesarmidea) from the Korean waters. Animal Systematics, Evolution and Diversity. 2020;36(2):159–63. [Google Scholar]

[pone.0262122.ref088] 88.Tsang LM, Chan BKK, Ma KY, Chu KH. Genetic differentiation, hybridization and adaptive divergence in two subspecies of the acorn barnacle Tetraclita japonica in the northwestern Pacific. Molecular Ecology. 2008;17(18):4151–63. doi: 10.1111/j.1365-294x.2008.03907.x [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref089] 89.Wong KJ, Chan BKK, Shih H-T. Taxonomy of the sand bubbler crabs Scopimera globosa De Haan, 1835, and S. tuberculata Stimpson, 1858 (Crustacea: Decapoda: Dotillidae) in East Asia, with description of a new species from the Ryukyus, Japan. Zootaxa. 2010;2345(1):43–59. [Google Scholar]

[pone.0262122.ref090] 90.Chen W, Hong W, Chen S, Wang Q, Zhang Q. Population genetic structure and demographic history of the mudskipper Boleophthalmus pectinirostris on the northwestern Pacific coast. Environmental Biology of Fishes. 2015;98(3):845–56. [Google Scholar]

[pone.0262122.ref091] 91.Cheang CC, Chu KH, Ang J, Put O. Morphological and genetic variation in the populations of Sargassum hemiphyllum (phaeophyceae) in the northwestern Pacific. Journal of Phycology. 2008;44(4):855–65. doi: 10.1111/j.1529-8817.2008.00532.x [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref092] 92.Dong Y-W, Wang H-S, Han G-D, Ke C-H, Zhan X, Nakano T, et al. The impact of Yangtze River discharge, ocean currents and historical events on the biogeographic pattern of Cellana toreuma along the China coast. PLoS One. 2012;7(4):e36178. doi: 10.1371/journal.pone.0036178 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref093] 93.Laurenzano C, Costa TM, Schubart CD. Contrasting patterns of clinal genetic diversity and potential colonization pathways in two species of western Atlantic fiddler crabs. PLoS One. 2016;11(11):e0166518. doi: 10.1371/journal.pone.0166518 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref094] 94.Rocha LA. Patterns of distribution and processes of speciation in Brazilian reef fishes. Journal of Biogeography. 2003;30(8):1161–71. [Google Scholar]

[pone.0262122.ref095] 95.Chan BKK, Tsang L, Chu K. Morphological and genetic differentiation of the acorn barnacle Tetraclita squamosa (Crustacea, Cirripedia) in East Asia and description of a new species of Tetraclita. Zoologica Scripta. 2007;36(1):79–91. [Google Scholar]

[pone.0262122.ref096] 96.Shih H-T, Kamrani E, Davie PJ, Liu M-Y. Genetic evidence for the recognition of two fiddler crabs, Uca iranica and U. albimana (Crustacea: Brachyura: Ocypodidae), from the northwestern Indian Ocean, with notes on the U. lactea species-complex. Hydrobiologia. 2009;635(1):373–82. [Google Scholar]

[pone.0262122.ref097] 97.Shih H-T, Hsu J-W, Wong KJ, Ng NK. Review of the mudflat varunid crab genus Metaplax (Crustacea, Brachyura, Varunidae) from East Asia and northern Vietnam. ZooKeys. 2019;877:1. doi: 10.3897/zookeys.877.38300 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref098] 98.Xu S, Song N, Zhao L, Cai S, Han Z, Gao T. Genomic evidence for local adaptation in the ovoviviparous marine fish Sebastiscus marmoratus with a background of population homogeneity. Scientific Reports. 2017;7(1):1–12. doi: 10.1038/s41598-016-0028-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0262122.ref099] 99.Kwok P-W. The ecology of two sesarmine crabs, Perisesarma bidens (De Haan) and Parasesarma plicata (Latreille) at the Mai Po Marshes Nature Reserve, Hong Kong. Hong Kong: The University of Hong Kong; 1995. [Google Scholar]

[pone.0262122.ref100] 100.Islam MS, Shokita S. Larval development of a mangrove crab (Perisesarma bidens De Haan) (Crustacea: Brachyura: Sesarminae). Bangladesh Journal of Fisheries Research. 2000;4(1):43–56. [Google Scholar]

[pone.0262122.ref101] 101.Poettinger T, Schubart CD. Molecular diversity of freshwater crabs from Sulawesi and the sequential colonization of ancient lakes. Hydrobiologia. 2014;739(1):73–84. [Google Scholar]

[pone.0262122.ref102] 102.Copilaş‐Ciocianu D, Petrusek A. The southwestern Carpathians as an ancient centre of diversity of freshwater gammarid amphipods: insights from the Gammarus fossarum species complex. Molecular Ecology. 2015;24(15):3980–92. doi: 10.1111/mec.13286 [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref103] 103.Ryder OA. Species conservation and systematics: the dilemma of subspecies. Trends in Ecology & Evolution 1986;1:9–10. [DOI] [PubMed] [Google Scholar]

[pone.0262122.ref104] 104.Crandall KA, Bininda-Emonds OR, Mace GM, Wayne RK. Considering evolutionary processes in conservation biology. Trends in Ecology & Evolution. 2000;15(7):290–5. doi: 10.1016/s0169-5347(00)01876-0 [DOI] [PubMed] [Google Scholar]

PERMALINK

Molecular phylogeography reveals multiple Pleistocene divergence events in estuarine crabs from the tropical West Pacific

Adnan Shahdadi

Katharina von Wyschetzki

Hung-Chang Liu

Ka Hou Chu

Christoph D Schubart

Roles

Abstract

Introduction

Materials and methods

Materials examined

Fig 1. Sampling localities.

Laboratory methods and sequence preparations

Phylogenetic and phylogeographic analyses

Analyses of population genetics and demography

Results

Fig 2. BI tree.

Phylogeny and phylogeography

Fig 3. Haplotype networks.

Population genetic and historical demography

Table 1. Diversity indices.

Table 2. AMOVA.

Fig 4. Mismatch distribution.

Discussion

Phylogeny, phylogeography and evolutionary history of the P. bidens species complex

Population genetic and historical demography

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Benny KK Chan

Roles

Author response to Decision Letter 0

Decision Letter 1

Benny KK Chan

Roles

Author response to Decision Letter 1

Decision Letter 2

Benny KK Chan

Roles

Acceptance letter

Benny KK Chan

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases