Transcriptome sequencing reveals the evolutionary histories and gene expression evolution in two related Pagurus species

Zakea Sultana; Isaac Adeyemi Babarinde; Masafumi Nozawa; Kazuho Ikeo; Akira Asakura; Tomoyuki Nakano

doi:10.1371/journal.pone.0330170

. 2025 Aug 20;20(8):e0330170. doi: 10.1371/journal.pone.0330170

Transcriptome sequencing reveals the evolutionary histories and gene expression evolution in two related Pagurus species

Zakea Sultana ¹, Isaac Adeyemi Babarinde ^2,^*, Masafumi Nozawa ^3,⁴, Kazuho Ikeo ^5,⁶, Akira Asakura ⁷, Tomoyuki Nakano ^7,^*

Editor: Feng ZHANG⁸

PMCID: PMC12367144 PMID: 40834015

Abstract

Pagurus lanuginosus (De Haan, 1833–1850) and Pagurus maculosus (Komai and Imafuku, 1996) are two closely related species of the genus Pagurus, generally referred to as “right-handed hermit crabs”. Previously thought to be color morphs of the same species, recent studies have itemized their unique features. To investigate the molecular and gene expression evolution that have followed the divergence of these two species, we performed transcriptome sequencing and de novo transcriptome assembly using cephalothorax (head) and pereopod (leg) of the two species. Synonymous and nonsynonymous divergence between the two species was estimated to be 0.0246 and 0.0066, respectively. About 5% of the protein-coding transcripts had signatures of positive selection. The phylogenetic analyses showed that the two species are indeed closely related and diverged about 6.24 million years ago. Comparison of tissue expression showed that head expressions were more conserved, and that more genes were upregulated in the legs of P. maculosus leg; while more pigmentation genes were found to be upregulated in the legs of P. lanuginosus. Genes associated with the extracellular matrix and space, chitin binding and chitin-based larval cuticles were found to have higher expression in the legs of P. maculosus, suggesting the roles in morphological differences. This study sheds light on the evolutionary history of the two species at the molecular level.

Introduction

The genus Pagurus [1], generally referred to as ‘right-handed hermit crabs’, is widely distributed in almost all marine habitats ranging from shallow to deep waters, worldwide. The genus is found in diverse biotopes along hard rocky shores to intertidal muddy flats. Thus, the species of this genus exhibit extremely high morphological diversity [2]. The morphological diversity and the ecological heterogeneity associated with the species of this genus make it difficult to identify and classify Pagurus species. Consequently, many sibling species remain cryptic species that are difficult to recognize using classic systematic methods [3]. Many new species are being described and added to this genus continuously, even as many more are yet to be recognized [4]. Therefore, the taxonomic classification of this ever-proliferative genus Pagurus is far from being resolved [5].

This concern is exemplified in two Japanese water intertidal hermit crab species, Pagurus lanuginosus [6] and P. maculosus [7]. Because of their superficial similarities, the two species were previously considered to be two color morphs of one species, P. lanuginosus [8]. The major difference was the color of their chromatophores scattered on the pereopods (legs) and cephalothorax (head). The chromatophores are white in P. maculosus and black in P. lanuginosus [7,8]. They both inhabit intertidal rocky shores and sometimes exhibit cohabitation [9]. However, the distribution of P. lanuginosus is broader than that of P. maculosus. Additional differences in morphological characters such as the number of calcareous teeth on the dactyl and the presence or absence of a large tubercle on the merus of the right cheliped in adults were described by Komai and Imafuku [7]. Thus, Komai and Imafuku concluded that these hermit crabs are two different species [7]. Further, careful studies of the larval and post-larval development of these two species revealed differences in the number of setae or spines on the antennule, maxillule and maxilla, and the presence of chromatophores on the carapace [10,11].

Morphological characters have been classically used for the identification and descriptions of decapod crustaceans. However, in some cases, morphological characters were very similar between congeners, raising uncertainty about their independent species status. With the advancement in nucleic acid extraction [12] and sequencing technologies [13], recent attention has been focused on the evolutionary studies of diverse decapod groups, utilizing the molecular approaches to disentangle various unresolved questions on the origins of diversity, habitat shifts, and diversification [14–17]. Molecular evolution has now been used as the basis for investigating morphological evolution [18–20].

Previous molecular evolution attempts using mitochondrial loci showed that P. maculosus and P. lanuginosus were indeed phylogenetically very close [9,21]. In this study, we aimed to investigate the phylogenetic relationships of the two species with other related species with additional focus on the gene expression evolution using transcriptome data. To achieve this, we generated head and leg transcriptomes of the two species. We conducted de novo transcript assembly and functionally annotated the newly assembled transcripts. We used the assembled transcripts to reveal some insights into the molecular evolution, phylogenetic relationship and gene expression evolution in the two hermit crab species.

Materials and methods

Sampling location and permit

Specimens of Pagurus lanuginosus and P. maculosus were collected from the same location at a rocky shore at Shirahama (33°41’ N, 135°23’ E), Wakayama Prefecture, along the North Pacific, Japan, in November 2023, and were transported to the laboratory alive. Sample collection permit for experimental research with number 5−2 was granted by Wakayama Prefecture, Japan. Researches on Pagurus species do not require any special approval. All experiments complied with institutional regulations and Japanese policy on animal use [22].

RNA extraction and sequencing

Three individuals of each species (P. maculosus and P. lanuginosus) were sampled for RNA extraction. For each individual, RNA was extracted from two distinct tissues in which pigmentation is most prevalent: the cephalothorax (head) and pereopod (leg), as illustrated in Fig 1A and Fig 1B. This resulted in three biological replicates per tissue type for each species. Total RNA was extracted by homogenizing approximately 25 mg of head or leg in 500 µl of Binding Buffer (4 M Guanidine Thiocyanate; 25 mM Na Citrate; 0.5% SDSC; 0.1 M Mercaptoethanol) with a tissue homogenizer (BEADS CRUSHER µT-12, TAITEC). The homogenate was processed following the manufacturer’s protocol of PureLink RNA Mini Kits (Qiagen). RNA purity and integrity were assessed visually in agarose gels, and quantified in spectrophotometry (Nanodrop ND-1000, NanoDrop Technologies, USA). High-quality total RNA samples were sent to an outside facility (Macrogen, Japan) for subsequent steps including library preparation and next generations sequencing. Library preparation was done using TruSeq Stranded Total RNA Library Preparation Kit (Illumina) and the sequencing was done on the NovaSeq 6000 system (Illumina) platform with 150 base pair paired-end (PE) reads. Head and leg of each species were sequenced in triplicates, with 10Gb of data per sample. All raw reads generated in this study are available in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB35803. The data sets supporting the results presented here are available with the accession numbers DRR708008-DRR708019.

Fig 1 — A. The picture of *P. maculosus* showing the parts from which RNA was extracted. B. The picture of *P. lanuginosus* showing the parts from which RNA was extracted. C. Alignment rates of reads that could be mapped back to the assembled transcripts. D. The distribution of coding and noncoding transcripts and expression. The transcript pie charts show the distribution of the transcript counts while the expression pie charts show contributions to the expression levels. E. Homology of a species in the other species. Unique transcripts did not have significant similarity with any transcripts in the other species. Transcripts with homology were divided into “one-to-one” or “other homologs” based on one-to-one correspondence in the two species. F. Coverage of one-to-one transcripts in the other species.

Retrieval of publicly available annotation data and crustacean short-read data

Publicly available data were retrieved for annotation and evolutionary analyses. Nonredundant (nr) amino acid sequences were retrieved from the NCBI database. The crustacean species with the highest homology were selected. The short-read raw RNA-seq data of the top homologous crustacean species, with at least 20,000 homologous transcripts, were then retrieved from the NCBI short-read archives. In addition, we retrieved the short-read RNA-seq data from the project PRJNA562428 [23]. Both newly generated RNA-seq data and publicly available data were then independently assembled using the same de novo transcript assembly pipeline.

Transcriptome assembly and its evaluation

The quality of the sequenced reads was examined using version v0.11.9 of fastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc). For transcript assembly, raw reads of all tissues and replicates of the same species were concatenated. De novo transcript assembly was performed using the default settings of the version v2.13.2 of Trinity [24,25]. Initial transcript expression quantification was done using bowtie2 in RSEM [26], with the settings “--bowtie2 --paired-end --bowtie2-sensitivity-level very_sensitive”. Transcripts with less than 2 RSEM’s expected counts were discarded. To evaluate the quality of the assembly, bowtie2 [27] was used to map the raw reads back to the assembled transcripts to quantify percentage of reads that were used for the actual assembly. The filtered transcript sets were then used for the downstream analyses.

Transcript expression quantification

Transcript expression quantification was performed using RSEM with bowtie2 alignment option. First, RSEM reference was built for the transcript assembly of each species. The raw reads were cleaned with fastp [28] with the settings “-q 20 -u 10”. The fastp-filtered reads were then used for the expression quantification. The RSEM settings used were “--bowtie2 --paired-end --bowtie2-sensitivity-level very_sensitive”.

Transcript annotation

Transcript annotation was focused on the predicted protein-coding transcripts. First, the TransDecoder [25] was used to extract all the six open reading frames (ORFs) with at least 50 amino acids. The most likely protein-coding ORF was then predicted by TransDecoder in Trinity Package. TransDecoder-predicted amino acid sequences were then used as queries for homology search to identify the homologous annotated proteins in NCBI Reference Sequence (RefSeq) non-redundant (nr) [29,30], Uniprot reference (UniRef90) [31] and UniProtKB/TrEMBL [32] databases. Homology search was done using DIAMOND [33] with “--sensitive” mode.

Functional annotation of the predicted amino acids was done using InterProScan 5 [34]. The predicted gene ontology (GO) terms and pathways were also included in the output. For the annotation of pigmentation-related genes, we employed a published strategy [35]. Briefly, the term “pigmentation” was searched in the gene ontology database [36] to obtain the list of genes with pigmentation functions in any species. The sequences of the genes were then obtained from UniProtKB/TrEMBL database to obtain the pigmentation gene reference sequences. Predicted amino acids were then searched in the pigmentation reference using BLASTP [37] with “--evalue 0.001” to obtain the list of pigment-related crab genes.

Gene ontology enrichment analyses

Gene ontology (GO) enrichment analyses were performed using the results of InterProScan. Genes with no annotated GO term was not included in the enrichment analyses. Similarly, GO terms with no annotated gene were excluded. GO analyses were computed for positively selected gene set and differentially expressed genes. For each GO term, the enrichment and hypergeometric probability were computed. Multiple testing was corrected with Bonferroni procedure [38]. Adjusted p value < 0.05 was set as the threshold for significantly enriched GO term.

Evolutionary analyses

Evolutionary analyses were conducted at both the nucleotide and amino acid levels. Nucleotide homology search was done with BLASTN [37] while amino acid searches were done with DIAMOND or BLASTP, depending on the size of the database. Reciprocal best hits, assessed by the bit scores, between the two species were termed “one-to-one”. The identification of the coding sequences (CDS) was done with TransDecoder. Stop codons were striped from the CDS. Each cluster of one-to-one genes were aligned with CLUSTALW [39]. Custom python scripts were used to concatenate the sequences, and partition them into different codon positions. The best amino acid and nucleotide models were estimated using maximum likelihood models in MEGA [40]. The rates of synonymous (dS) and nonsynonymous (dN) substitutions using both Nie-Gojobori [41] and Yang-Nielsen [42] methods were computed with yn00 in version 4.10.6 of PAML [43]. The evolutionary pressures acting on the transcripts were estimated using the estimated and error values of dS and dN computed from Yang-Nielsen method [42]. For positively selected transcripts, dN-dN_err > dS + dS_err, where dN_err and dS_err are error estimates for dN and dS, respectively. For purifying selection, dS-dS_err> dN + dN_err. Other transcripts were classified as neutrally evolving.

Phylogenetics and divergence time estimates

Because of the computational requirements of the maximum likelihood, only one fourth of the amino acid positions of the concatenated amino acids were used for the phylogenetic tree. To extract the alignment subset, the amino acids of the first position in 4-step moving window were extracted using a custom Python script. The phylogenetic analyses were conducted using maximum likelihood method in MEGA. Aligned sites with gaps or unknown amino acids were completely deleted. Gamma distribution with invariant sites (G + I) and gamma = 5, was used for the rate among sites. The initial tree was made with NJ/BioNJ method. Weak branch swap filter was set. LG model with frequencies were used. Branch support was evaluated with 1,000 bootstrap replicates.

Divergence time estimates were computed using MCMCtree program available in version 4.10.6 of PAML following previously published methods [44–46]. First, the overall substitution rates were estimated using baseml in PAML with the root age set to 450 million years ago (MYA). Next, the gradient (g) and Hessian (H) were estimated using the phylogenetic tree and alignments of the multiple species. Finally, the divergence times were estimated using the computed gradient, Hessian, alignments and the fossil calibrations. Nine calibration points were retrieved from published studies [17,47–49]. To assess the convergence, the divergence time estimation was repeated three times with different random seed numbers.

Differential gene expression

The consistencies of the replicates were first examined by principal component analyses and clustering analyses using the expression data normalized by DESeq2 [50]. The two most consistent samples for each species-tissue combination were used for differential gene expression analyses. Differential gene expressions were computed for each tissue between the two species using DESeq2. Differential gene expression was done independently for head and leg. Statistical significance was set at adjusted p value of 0.05.

Statistical tests and graphical representations

Statistical tests were computed using Scipy package in Python or R statistical package. For gene enrichment analyses, hypergeometric tests were conducted to test the significance of the enrichment of specific gene ontology and term. Multiple tests were corrected using Bonferroni method [38]. Graphical representations were done mostly by Matplotlib in Python and occasionally by R.

Results

Head and leg transcriptome assemblies of P. maculosus and P. lanuginosus

RNA sequencing was performed with three biological replicates from each tissue type and species. For each replicate, ~ 21,000,000–27,000,000 paired-end reads were sequenced (S1 Table). A total of 139,881,387 P. lanuginosus and 148,561,226 P. maculosus paired-end reads were generated (Table 1). Transcriptome assembly produced 373,660 and 468,052 initial transcripts for P. maculosus and P. lanuginosus, respectively. Deeper sequencing has been reported to be positively correlated with the number of assembled transcripts, including potentially noisy transcripts [51]. Removal of potentially noisy transcripts produced 203,793 transcripts, including 140,954 genes for P. maculosus, and 259,375 transcripts, including 179,685 genes for P. lanuginosus (Table 1). More than 95% of raw reads could be mapped back to the filtered assembled transcripts (Fig 1C), showing that the transcriptomes were sufficiently represented in the transcript assemblies.

Table 1. De novo transcript assembly statistics.

Metrics	Pagurus lanuginosus	Pagurus maculosus
Total raw read count	139881387	148561226
Assembled gene count	179685	140954
Assembled transcript count	259375	203793
Transcript N50	1029	1084
Transcript median length (bp)	470	498
Transcript average length (bp)	765	804
Predicted coding transcripts*	87037	69511

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*Transcripts with open reading frame (orf) with at least 50-amino acid were predicted to be protein-coding.

Transcriptome assembly has the potential to retrieve both coding and noncoding transcripts. We therefore checked the proportion of both coding and noncoding transcripts in our transcriptome assembly (see Materials and Methods). In both species, about 34% of the transcripts were coding (Fig 1D). However, expression quantifications showed that coding transcripts constituted about 79% and 75% of the P. maculosus and P. lanuginosus transcriptomes, respectively, reflecting higher expression levels of coding transcripts, as previously reported in human [52,53].

Next, we checked the homology between the assembled P. maculosus and P. lanuginosus transcripts. At the nucleotide levels, > 95% of P. lanuginosus transcripts had homologs in P. maculosus (Fig 1E). On the other hand, > 97% P. maculosus transcripts had homologs in P. lanuginosus. At the amino acid levels, 83% of the P. lanuginosus transcripts and 86% of P. maculosus transcripts had detectable homologs in the other species, at the specified search thresholds. We defined one-to-one homology as the reciprocal best hit in the two species in reciprocal BLAST searches. Interestingly, although the majority of the transcripts had homologs in the two species, one-to-one relationship could not be established for many transcripts. The inability to establish one-to-one relationship could suggest the presence of transcriptional noise, false positives or false negatives reflecting the limitation of transcript assembly and annotation procedures from short-read RNA-seq [51,53]. This observation could also highlight the roles of independent evolution of gene duplications, losses and/or splicing events after the divergence of the two species. For transcripts with one-to-one correspondence, a substantial proportion of the transcripts had 90–100% alignment coverage in the two species (Fig 1F), highlighting the confidence of our assembly and annotation, at least for transcripts with one-to-one relationship. These results highlight the properties of the assembled transcripts and the evolution of gene expressions after the divergence of the two species.

Annotation of the assembled Pagurus transcripts

To functionally annotate the assembled transcripts, we searched for the predicted amino acids in NCBI RefSeq nr, UniRef90 and UniProtKB TrEMBL amino acid databases. We defined homology by significant similarity with the annotated proteins. About 48,000 P. lanuginosus and about 39,000 P. maculosus transcripts had homology in the databases (S2 Table, upper panel of Fig 2A). Interestingly, 48,712 P. lanuginosus transcripts had homolog in at least one of the investigated databases, including 47,653 (97.8%) with homology in the three investigated databases (S2 Table). Similarly, 39,639 P. maculosus transcripts had homolog in at least one of the three databases, 38,805 (97.9%) of which were found in all three databases. The consistency across databases was also found at gene levels. By attributing the assembled transcript to the annotated protein with the highest score, P. lanuginosus transcripts had homology with 26,006 unique proteins in RefSeq nr, 24,027 for UniRef90 and 24,178 for UniProtKB TrEMBL database (lower panel of Fig 2A). For P. maculosus transcripts, homology was found for 21,228, 19,574 and 19,721 unique proteins in NCBI nr, UniRef90 and UniProtKB TrEMBL, respectively. Majority of the amino acid sequences with homology had very high coverage with the annotated proteins in the database (Fig 2B). These data showed that a substantial number of predicted amino acid sequences had homology with known proteins.

Fig 2 — A. The number of assembled transcripts (upper panel) with significant homologs in the listed database and the corresponding number of unique homologous proteins in the database (lower panel). B. Coverage of the annotated proteins in the assembled transcripts. C. The number of transcripts found in the top 30 represented species. D. The list of the top annotated species with at least 20,000 homologs in the assembled transcripts.

In order to more efficiently annotate the assembled transcripts, we decided to identify the phylogenetically closely related well-annotated species that could potentially be used as the reference. To identify the closest species with known amino acid sequences, we identified the top 30 species with homologous proteins in both P. lanuginosus and P. maculosus. Eleven species each had homology to more than 20,000 predicted P. lanuginosus and P. maculosus transcripts (Fig 2C). Interestingly, the same species were the top closest known species to both P. lanuginosus and P. maculosus. Based on the number of homologous amino acid sequences, the closest two species were American lobster, Homarus americanus and Porcelain crab, Petrolisthes cinctipes. The two species probably diverged from Pagurus sp. more than 200 million years ago (MYA) [54]. Therefore, the species with the highest numbers of homologous proteins do not share recent evolutionary histories with Pagurus species. These results highlight the need for concentrated efforts on the annotation of the transcripts of Pagurus sp.

Phylogenetic analyses and divergence time estimates

The divergence times between two Pagurus species and the closest crustacean species suggests that the closest species have not been sequenced or annotated to be included in the databases. We therefore, retrieved the raw reads of multiple crustacean species published as part of the crustacean annotated transcriptome (CAT) database [23]. Using the same pipeline, we assembled the transcripts and predicted amino acids sequences. To have a uniform processing, we also retrieved the short-read sequences of the top 11 species from the database (Figs 2B and 2C) and perform de novo transcriptome assembly. In total, 20 species had proteins with homology to more than 20,000 Pagurus transcripts. We then extracted transcripts with one-to-one homology between P. maculosus and each of the 21 species, including P. lanuginosus and the 20 other species. The transcripts were aligned, concatenated and used for phylogenetic analyses. To ensure that the same sites were used in all the species, positions with alignment gaps in any species were completely deleted. A total of 2,035 one-to-one transcripts covering 147,186 gapless amino acids were extracted. These amino acid sequences were used for phylogenetic analyses.

The phylogenetic analyses revealed that P. maculosus and P. lanuginosus were sister species (Fig 3), consistent with the previous results with the mitochondrial genomes [9,21]. We also found that the next closest species to P. maculosus and P. lanuginosus was a red king crab, Paralithodes camtschaticus which was found to be more closely related than another Pagurus species, Pagurus longicarpus. This result further confirmed the previous studies reporting “hermit-to-king” hypothesis that Paralithodes species evolved from Pagurus lineage [55–58]. Importantly, we confirmed that the closest species to the P. maculosus and P. lanuginosus are not well annotated in the public amino acid databases, highlighting the gap being filled by this study.

Next, we computed the divergence times involving 22 species using MCMC tree. Nine calibration points (S3 Table) were used. Our analysis showed that P. maculosus and P. lanuginosus diverged around 6.24 (4.43–8.16 95% confidence interval) MYA (Fig 3). The two species diverged from the red king crab, Par. camtschaticus around 47.63 (36.14–59.77 95% confidence interval) MYA. The closest identified species pair was two porcelain crabs, Pet. cinctipes and Pet. manimaculis which were found to have diverged less than 1 MYA. These two Petrolisthes species were estimated to have diverged from Pagurus species 249.86 (227.16–272.02 95% confidence interval) MYA. Even more diverged was the American lobster, H. americanus which was found to have diverged from Pagurus species more than 350 MYA. These results showed that P. maculosus and P. lanuginosus are sister species, and highlight the phylogenetic relationships between Pagurus species and selected crustacean species with transcriptome data.

Selection pressures after P. maculosus and P. lanuginosus divergence

After confirming that both Pagurus species were sister species, we proceeded to investigate the evolutionary pressures that have shaped the evolution of the two species after their divergence. We first computed the synonymous (dS) and nonsynomous (dN) substitutions between the two species using 22,516 transcripts with a minimum gapless length of 50 amino acids. The estimations of dS and dN from both Yang-Nielsen [42] and Nei-Gojobori [41] methods were very consistent with very high correlation coefficients (Fig 4A). The median dS and dN values from Nei-Gojobori method were 0.0284 and 0.0062, respectively while the vales from Yang- Nielsen method were 0.0246 and 0.0066, respectively (Fig 4B). These results showed that both methods produced similar results.

Fig 4 — A. Scatter plots showing the relationships between Yang-Nielsen and Nei-Gojobori methods for synonymous (left) and nonsynonymous (right) substitutions. r = Pearson’s correlation coefficient. B. The distributions of synonymous and nonsynonymous substitutions computed with Yang-Nielsen and Nei-Gojobori methods. C. The distributions of dN/dS computed from Yang-Nielsen method. The transcripts were grouped into neutral, negative and positive based on the values and error estimates of the dN and dS. D. Pie charts showing the distributions of the transcripts based on the selection pressures. E. The percentages of positively selected transcripts with homology to pigmentation genes. F. Venn diagram showing the overlap between one-to-one and pigmentation genes. G. Venn diagram showing the positively selected pigmentation genes. H. Gene ontology enrichments for positively selected transcripts.

Having established the consistencies of the two methods, we decided to use Yamg-Nielsen method for the subsequent analyses because the method provides error estimate for each value. We computed the dN/dS ratio to identify the evolutionary pressures acting on each predicted protein-coding transcript. Evolutionary selections of the transcripts were classified into neutral, negative and positive based on the value and error estimates of dS and dN (see Materials and Methods section for details). For neutral transcripts, the estimates of dN and dS and the corresponding errors overlapped. Positively selected transcripts had higher dN values (including the error estimates) while transcripts under purifying selection had higher dS values (including the error estimates). Although higher dN than dS values could also result from relaxed purifying selection [59,60], we simply referred to transcripts with significantly higher dN than dS values as positively selected. The distribution of the dN/dS ratio revealed that dN/dS ratio was less than one for most of the transcripts. (Fig 4C). Because of the inclusion of error estimates in classifying the transcripts into different categories, transcripts from different categories may have similar dN/dS values. Interestingly, dN/dS values for some negatively and positively selected transcripts overlapped the values for some neutrally evolving transcripts while other evolutionarily selected transcripts had substantially higher values, possibly reflecting two distinct levels of selection as presented in Ohta’s nearly neutral theory of molecular evolution [61]. The distribution of selection pressures showed that about 55% of the transcripts were under purifying selection while only 5% were under positive selection (Fig 4D), suggesting that purifying selection dominates the evolutionary pressures shaping the gene evolution.

The two species were initially thought to be color morphs of the same species. We therefore checked if the pigmentation genes were positively selected. We first identified assembled Pagurus genes that were homologous to known pigmentation genes (S4 Table). Indeed, we found 52 pigmentation genes to be positively selected (Fig 4E). However, the 52 genes only represented about 1% of total one-to-one pigment-related genes (Figs 4E and 4F). Specifically, while 4,260 one-to-one pigmentation genes were found (Fig 4F), only 52 one-to-one pigmentation genes were found to be positively selected (Figs 4E and 4G). The GO enrichment analyses showed that the most significantly enriched term in the positively selected genes was extracellular region (GO:0.0005576) (Fig 4H). These results revealed the dynamics of evolutionary selection after the divergence of P. maculosus and P. lanuginosus.

Evolution of gene expressions in P. maculosus and P. lanuginosus

To investigate the evolution of expression levels in the two species, we took advantage of the high sequence similarity of the two species. It is important to note that the median synonymous divergence was lower than 3%, and the non-synonymous divergence was even lower (0.6%) (Fig 4B). We wanted to see if the assembled transcripts from a species could be used as reference for the quantification of the tissues from the other species. We first asked if the proportion of the homologous genes in the transcriptome of both species were substantial enough. Interestingly, close to 100% of the transcriptomes in both species came from homologous genes (Fig 5A). However, the percentages of transcriptomes with one-to-one homology were lower, with the value dropping to 70% and 79% in a P. maculosus leg and head, respectively. This suggests that gene duplications and/or alternative splicing events might have shaped gene evolutions in these two species. Furthermore, the transcriptome percentages when transcripts were considered were even lower, reiterating the potential contribution of splicing to the evolution of gene expression in the two species.

Fig 5 — A. The percentages of *P. maculosus* and *P. lanuginosus* reads that can be mapped to the genes of other species. Close to 100% percent of the reads could be mapped to the transcripts of the other species. However, when genes/transcripts with one-to-one relationships were used, the percentages dropped. B. 2-D hexagonal binning plots showing the relationships between gene expression patterns of the *P. maculosus* (PM) and *P. lanuginosus* (PL) in the head (upper panel) and the leg (lower panel). r = Pearson’s correlation coefficient while the red lines represent the linear regression lines. Log₂-transfromed TMM-normalized expressions were used for the plots C. Volcano plots showing differential gene expression analyses between *P. maculosus* (PM) and *P. lanuginosus* (PL) in the head (upper) and leg (lower). D. Venn diagram showing genes that were consistently upregulated in *P. maculosus* (PM, left panel) and *P. lanuginosus* (PL, right panel) head and leg. E. Percentages of pigmentation genes in head (upper) and leg (lower) differentially expressed genes. P values were computed using Fisher exact tests. F. Gene ontology enrichment analyses for genes upregulated in *P. maculosus* leg. The bar color corresponded to the Bonferroni-corrected hypergeometry p value.

Having established the high sequence similarity and percentages of transcriptomes in the two species, we performed expression quantification again using P. lanuginosus reference. We clustered the samples using principal component analyses and heatmaps to identify the two most consistent samples. Using the average expression of the most consistent duplicates, we found that the expression correlation in the head (Pearson’s correlation coefficient = 0.76) of the two species was higher than the leg correlation (Pearson’s correlation coefficient = 0.52), suggesting higher expression evolution in the genes expressed in the leg (Fig 5B). When only the predicted protein-coding genes were used for the correlations (S1A Fig), the correlations became stronger (Pearson’s correlation coefficients were 0.84 and 0.60, respectively for head and leg), suggesting divergence expression in the legs across the two species. Interestingly, the trend of higher head correlation persisted when only positively selected genes were used (S1B Fig). Differential gene expression analyses further showed more differentially expressed genes in the leg, compared to the head (Fig 5C), further confirming more divergent expression in the leg and conserved expression in the head across these two species. While 4,985 differentially expressed genes (DEGs) were found in the head, 7,585 DEGs were found in the leg. Also, the patterns of head DEGs were similar across the two species (Fisher exact p value = 0.155) while more genes were upregulated in P. maculosus leg (Fisher exact p value = 3.66 × 10^-22) (Figs 5C and 5D), suggesting tissue-specific evolution of gene expression patterns.

We next asked if the expressions of the same set of genes evolved in both head and leg. Log-fold changes for differentially expressed genes in leg and head were correlated (Spearman’s rank coefficient = 0.54) (S1C Fig). For genes that were significantly differentially expressed in both leg and head, the patterns of differential expression were mostly consistent. For genes with higher expression in P. maculosus, 896 were found to be common to both head and leg differential expression (Fig 5D). However, a larger proportion of the genes were found to be specific to head or leg comparison. Similar patterns were found for genes with higher expression in P. lanuginosus, suggesting that Pagurus gene expression evolution involved both shared and tissue-specific components.

To identify the potential functions of the differentially expressed genes, we checked the enrichments of pigmentation genes. Head DEGs included 149 P. maculosus-enriched pigmentation genes and 167 P. lanuginosus-enriched pigmentation genes (Fig 5E). However, there was no significant bias in pigmentation genes differentially expressed in the head across the two species (p value = 0.64). More pigmentation genes were found in the leg differentially expressed genes. Importantly, more pigmentation genes were found to have higher expression in P. lanuginosus leg (p value = 1.76 × 10⁻⁰⁸). We checked the enrichment of positively selected genes in deferentially expressed gene sets but found no bias in either of the species (S1D Fig), suggesting that positive selection on gene expression was not specifically biased to either species.

Finally, we performed gene ontology enrichments for the differentially expressed genes. As the assembled genes have not been annotated, we first performed GO annotation of the genes using InterProScan 5 to predict the likely gene functions and the potential GO terms for the assembled protein-coding transcripts. Of the 55,714 P. lanuginosus with InterProScan annotation, 29,186 could be mapped to at least one GO term (S5 Table). For P. maculosus, 23,703 of the 45,038 annotated transcripts could be mapped to at least one GO term. In total, 5,916 (P. lanuginosus) and 5,326 (P. maculosus) GO terms were found.

GO enrichment analyses showed that the largest enrichments were found in the genes that were upregulated in P. maculosus leg (Fig 5F). Gene involved in extracellular activities were significantly enriched. Additionally, genes involved in the chitin-based larval cuticle and chitin binding were upregulated in P. maculosus leg. On the contrary, genes upregulated in P. lanuginosus leg were enriched in the nucleus, and in peptidase inhibitor and catalytic activities (S1E Fig). Next, we checked the GO enrichment of P. maculosus-enriched head DEGs and found that the genes were enriched in ion exchange (S1F Fig). Finally, we checked the GO enrichment of P. lanuginosus-upregulated head genes and found that genes involved in sulfation, sulfotransferase and transmembrane transporter activities were enriched (S1E Fig). These results showed that genes upregulated in different species and different tissues performed different functions. Taken together, the results highlight how gene expressions have evolved after the divergence of P. maculosus and P. lanuginosus.

Discussion

Pagurus maculosus and P. lanuginosus are two intertidal hermit crab species found in the Japanese water. They were initially thought to be color morphs of the same species. However, subsequent studies demonstrated that they are two separate species with unique features [7,9–11,21]. In this study, we report the analyses of the head (cephalothorax) and leg (pereopod) transcriptomes of the two species. We performed independent de novo transcript assembly for the two species, and confirmed that our transcript assembly did not lead to substantial loss of transcriptome data. Although almost all reads of one species could be mapped to the transcripts of the other species, a sizable proportion of the transcripts did not have one-to-one correspondence between the two species. Also, consideration at gene levels had higher proportion of one-to-one correspondence between the two species. These suggest that gene duplication and alternative splicing have played important roles in shaping the evolution of gene expressions in the two species.

We quantified the genetic distance between the two species. Using Yang-Nielsen method [42], the synonymous and nonsynonymous substitution rates for the two species were estimated to be 2.46% and 0.66%, respectively. These values were lower than 3.4–4.1% reported from nucleotide sequences of selected mitochondrial loci [21]. This discrepancy might be due to the differences in the evolutionary rates of mitochondrial and nuclear genes [62]. In addition, while the mitochondrial value was computed using Kimura 2-parameter on mitochondrial regions without segregation into synonymous and nonsynonymous rates, the evolutionary distances computed in this study were computed from predicted protein-coding transcripts. Based on synonymous and nonsynonymous substitutions, majority of the genes were found to undergo purifying selection, and only 5% were found to be undergoing positive selection. Interestingly, we found that substantial number of genes under selection are undergoing slightly neutral evolution [61,63]. This observation is interesting as the two species are sometimes found in the same ecosystem, suggesting that the two species might not have been subjected to drastically different pressures. The role of geographical isolation in shaping speciation and molecular evolution after speciation would be an interesting future study.

The search of public databases revealed that the closest annotated species to P. lanuginosus was the porcelain crab, Pet. cinctipes, which was estimated to have diverged around 250 MYA. However, our analyses revealed that the pair of P. lanuginosus and P. maculosus diverged from Par. camtschaticus about 36.1–59.8 MYA. These suggest that species closer to Pagurus have not been well annotated or their annotations have not been integrated into public protein databases. Supporting the later possibility, indeed transcriptome data of a number of species have recently been published [23,64–66]. Using the transcriptome of multiple related species, we estimated the divergence time of P. lanuginosus and P. maculosus to be 6.24 MYA (4.43–8.16; 95% confidence interval). This divergence time gives the rate of synonymous substitutions of 3.9 × 10⁻⁹, a rate similar to what was found in mammals [46]. Consistent with the previous reports that genus Pagurus is not monophyletic [4,9,67,68], we showed that the red king crab, Par. camtschaticus is more closely related to P. maculosus than P. longicarpus.

Gene expression evolution was found to be different across tissues with the head having higher correlation, suggesting slower gene expression evolution in the head when compared to the leg. This observation was similar to mammalian studies that investigated conserved noncoding sequences to infer that gene expression evolution varied across tissues [69–71]. Interestingly, we found that gene expression evolution was not symmetric. Genes involved in pigmentation were found to be significantly upregulated in P. lanuginosus. We found that genes upregulated in P. maculosus leg were enriched in extracellular space, larval cuticle, chitin binding and extracellular matrix, suggesting relationships between the gene expression changes and previously reported morphological changes [7,10,11]. Taken together, this study uncovers the molecular changes that have ensued the divergence of P. maculosus and P. lanuginosus and the potential relationships with the reported morphological changes.

Supporting information

S1 Fig. Gene expression evolution and the nature of genes enriched across the species.

A. 2-D hexagonal binning plots showing the relationships between protein-coding gene expression patterns of the P. maculosus (PM) and P. lanuginosus (PL) in the head (upper panel) and the leg (lower panel). B. 2-D hexagonal binning plots showing the relationships between the positively selected protein-coding gene expression patterns of the P. maculosus (PM) and P. lanuginosus (PL) in the head (upper panel) and the leg (lower panel). For A and B, r = Pearson’s correlation coefficient while the red lines represent the linear regression lines. Log₂-transfromed TMM-normalized expressions were used for the plots C. Scattered plot showing the relationship between the log-fold changes of genes differentially expressed in head and/or leg. Each dot represents a gene. Negative values represent P. lanuginosus enrichment while positive values represent P. maculosus enrichment. Rho = Spearman’r correlation coefficient. D. Species bias of positively selected differentially expressed head (upper) and leg (lower) genes. P values were computed using Fisher exact tests. Gene ontology enrichments of genes are upregulated in P. lanuginosus leg (E), P. maculosus head (F) and P. lanuginosus head (G). The bar color corresponded to the Bonferroni-corrected hypergeometry p value.

(TIF)

pone.0330170.s001.tif^{(2.4MB, tif)}

S1 Table. Details of Transcriptome sequencing.

(XLSX)

pone.0330170.s002.xlsx^{(10.8KB, xlsx)}

S2 Table. Number of homologous transcripts and genes in different databases.

(XLSX)

pone.0330170.s003.xlsx^{(10.3KB, xlsx)}

S3 Table. Calibrations used for divergence time estimates.

(XLSX)

pone.0330170.s004.xlsx^{(10.6KB, xlsx)}

S4 Table. The list of known pigmentation genes.

(XLSX)

pone.0330170.s005.xlsx^{(41.3KB, xlsx)}

S5 Table. InterproScan annotation statistics for the assembled transcripts.

(XLSX)

pone.0330170.s006.xlsx^{(10.1KB, xlsx)}

Acknowledgments

The authors are thankful to Mr. Nakano, R., Ms. Nakano, M., and the members of SMBL for their help in collecting the study animals.

Data Availability

All data generated in the study have been made publicly available and deposited to the DNA Data Bank of Japan under the project accession number PRJDB35803, and can be accessed on the NCBI database (https://www.ncbi.nlm.nih.gov/bioproject/PRJDB35803/). All short-read data have been described in S1 Table.

Funding Statement

This study was supported by the funding of Research Organization of Information and Systems (ROIS) for special collaboration research student program of National Institute of Genetics (NIG) and Kyoto University, Japan awarded to ZS, and by the Operating Expenses (運営費) from Kyoto University, Japan awarded to AA and TN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0330170.r001

Decision Letter 0

Feng ZHANG

27 Jun 2025

Dear Dr. Babarinde,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: No

Reviewer #2: Yes

**********

Reviewer #1: I believe that this paper is valuable for publication. However, as mentioned below, it needs some revisions.

L.38, 39: leg of xxx leg?

L.40: chin-based?

L.52: what is “those species”?

L.52: “Sibling species and cryptic species are sometimes not properly identified”. Because taxa not known are cryptic species.

L.93: “RNA samples were extracted separately from the head and leg” Both parts contain muscle, membrane, and exoskeleton or something else, but RNA was extracted from muscle. First, the material must be properly identified.

BLAST not blast

L.211-212: This must be placed in Materials and Methods.

L.215: remove “for each sample” How many individuals per species were used?

L215-216: This must be placed in Materials and Methods.

L.219-221: This must be placed in Materials and Methods.

L.242-244: This must be placed in Materials and Methods.

L.321-324: References 9 and 18 addressed only the genus Pagurus. Therefore, if the authors are referring to the non-monophyletic status of Pagurus in relation to the family Lithodidae, they should cite other references (e.g., Cunningham et al., 1992; Zaklin, 2001; Morrison et al., 2002; Blacken-Grissom et al., 2013).

L.505: respectively

The generic abbreviations for Pagurus, Paralithodes, and Petrolisthes should be clear. For example, they could be Pag., Par., and Pet.

L.505-: I guess that almost all nucleotide substitutions in protein-coding mitochondrial DNA between the two Pagurus species are synonymous.

L.519: “found” should be “estimated”

L.525: specify the two species.

L.529: A recent study on ITS1 sequence divergence and length (Chow et al. 2023, Crust. Res. 52) showed well separate status between Pagurus and Lithodid. No discussion for this?

Reviewer #2: The MS investigates the molecular and gene expression evolution of two closely related Pagurus species using transcriptome sequencing. The results sheds light on the evolutionary history of the two species at the molecular level. This MS demonstrates substantive content, sound data analysis, and reliable results, presented through well-structured writing that meets the criteria for publication readiness.

**********

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Reviewer #2: No

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PLoS One. 2025 Aug 20;20(8):e0330170. doi: 10.1371/journal.pone.0330170.r002

Author response to Decision Letter 1

25 Jul 2025

We appreciate the editor and the reviewers for the time and efforts spent in reviewing our manuscript. We are particularly grateful for the suggestions towards the improvement of the manuscript. We have meticulously revised the manuscript based on the suggestions and comments from the editor and the reviewers. We have also corrected other typographical or grammatical errors that we encountered. All changes have been marked in one of the uuploaded documents. Specific response and changes made in response to each comment are itemized below (in blue font).

Response to the editor

We are grateful to the editor for the comments. We have responded to each raised comment with blue font below.

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

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We appreciate the editor for pointing this out. The first page has been revised to be consistent with PLOS ONE’s style. We have also revised the listing of supplementary figures and tables to be consistent with the PLOS ONE’s style.

We have updated the Materials and Methods section by adding the following sentences. “Sample collection permit for experimental research with number 5-2 was granted by Wakayama Prefecture, Japan. Researches on Pagurus species do not require any special approval. All experiments complied with institutional regulations and Japanese policy on animal use [22].” We also corrected the sampling date to reflect the actual date when the specific sequenced samples were collected.

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As suggested by the editor, we have deleted the funding-related text from the manuscript. And we would like to leave the funding statement as it is, that is, “This study was supported by the funding of Research Organization of Information and Systems (ROIS) for special collaboration research student program of National Institute of Genetics (NIG) and Kyoto University, Japan”

We have made the raw read data publicly available (as from July 22nd, 2025) using the new accessions provided in the manuscript. The data can now be assessed from https://www.ncbi.nlm.nih.gov/bioproject/PRJDB35803. We would like to point out that we discovered that the biological replicates were not correctly entered in the original submission (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJDB7354). That is why we had to revise the submission and now have new accessions for the project and the samples. The manuscript has been appropriately revised to state “All raw reads generated in this study are available in the DDBJ Sequence Read Archive (DRA) under the BioProject accession number PRJDB35803. The data sets supporting the results presented here are available with the accession numbers DRR708008-DRR708019.”

ORCID ID has been linked and validated in Editorial Manager.

8. We are unable to open your Supporting Information file [HermitCrab_SupplementaryTables.xlsx and S1Fig.eps]. Please kindly revise as necessary and re-upload.

We have confirmed that the files are still in good shape. We suspect that the problem might have arisen during file uploading. We will re-upload the files during the submission of the revised manuscript. To ensure that the files can be opened, supplementary figure will be saved in two formats (.eps and .tiff). An additional compressed file of the supplementary figure and table files will also be attached. Multiple uploading of the same files is to ensure the ability of the files to be opened.

The revised manuscript now included references 18-20 supporting the growing use of molecular barcodes. We have listed reference 22 about institutional regulations and Japanese policy on animal use. In addition, we have cited new papers (references 55-58) suggested by a reviewer.

Response to reviewers

Reviewer #1: I believe that this paper is valuable for publication. However, as mentioned below, it needs some revisions.

We appreciate the reviewer for the time and efforts towards the improvement of our manuscripts. We are sincerely grateful for the multiple corrections that the reviewer has suggested. We have incorporated the reviewer’s suggestions and corrections in the revised version of the manuscript. We have also listed point-by-point response to the reviewer’s suggestion and comments.

L.38, 39: leg of xxx leg?

This has been revised by deleting “leg”.

L.40: chin-based?

“chin-based” has been changed to “chitin-based”

L.52: what is “those species”?

“those species” has been changed to “Pagurus species”

L.52: “Sibling species and cryptic species are sometimes not properly identified”. Because taxa not known are cryptic species.

This sentence has been changed to “Consequently, many sibling species remain cryptic species that are difficult to recognize using traditional classic methods”

We apologize for this confusion. RNA extraction was actually done using the whole leg or head as pointed out in Figures 1A and B, and not just for the muscle. We have replaced “muscle” with “head or leg” in the Methods section.

BLAST not blast

All instances of “blast” to “BLAST”, including “BLASTN” and “BLASTP”.

L.211-212: This must be placed in Materials and Methods.

This has been moved.

L.215: remove “for each sample” How many individuals per species were used?

This has been revised. Three individuals were used per species.

L215-216: This must be placed in Materials and Methods.

This has been removed.

L.219-221: This must be placed in Materials and Methods.

This has been deleted. We have revised the following sentence to read “Removal of potentially noisy transcripts produced 203,793 transcripts, including 140,954 genes for P. maculosus, and 259,375 transcripts, including 179,685 genes for P. lanuginosus (Table 1)”

L.242-244: This must be placed in Materials and Methods.

This has been deleted. We have added the following to give the background to the result: “Transcriptome assembly has the potential to retrieve both coding and noncoding transcripts. We therefore checked the proportion of both coding and noncoding transcripts in our transcriptome assembly (see Materials and Methods)”

We appreciate the reviewer. We have rephrased the sentence to focus on the evolution of Paralithodes species from Pagurus lineage. As suggested by the reviewer, we have added Cunningham et al. (1992), Morrison et al. (2002) and Bracken-Grissom et al. (2013), as suggested by the reviewer. We also added Chow et al. (2023) which supports the same hypothesis. The sentence now reads “This result further confirmed the previous studies reporting “hermit-to-king” hypothesis that Paralithodes species evolved from Pagurus lineage [55–58].”.

L.505: respectively

“respective” has been changed to “respectively”

The generic abbreviations for Pagurus, Paralithodes, and Petrolisthes should be clear. For example, they could be Pag., Par., and Pet.

We appreciate the reviewer for this suggestion. Since our focus is on Pagurus, we have retained P. for Pagurus genus while using Par. and Pet. for Paralithodes, and Petrolisthes, respectively, as suggested by the reviewer.

L.505-: I guess that almost all nucleotide substitutions in protein-coding mitochondrial DNA between the two Pagurus species are synonymous.

In Sultana et al., two rRNAs, two ATPs, Cox1, Cox2, Cox3, Nn2, and Nd3 mitochondrial regions were analyzed to compute overall nucleotide distance. Although the mitochondrial genes included those that code and those that do not code for proteins, all regions were analyzed together without considering nucleotide substitution type. Indeed, one of the motivations for this study was to more systematically investigate nucleotide evolution. In this study, we have separately computed synonymous and nonsynonymous substitution rates in larger gene set.

L.519: “found” should be “estimated”

This has been corrected.

L.525: specify the two species.

“the two species” has been changed to “P. lanuginosus and P. maculosus”

L.529: A recent study on ITS1 sequence divergence and length (Chow et al. 2023, Crust. Res. 52) showed well separate status between Pagurus and Lithodid. No discussion for this?

We appreciate the reviewer for calling our attention to this paper. Interestingly, the paper showed that lithodoid species had unique internal transcribed spacer 1 (ITS1), which evolved from those found among paguroid species. Their results support the evolution of monophyletic Lithodid from Pagurus lineage. In other words, their findings support the “hermit-to-king” crab hypothesis, as our findings do. Please see Fig. 8 of Chow et al. (2023) which presents the hypothetical evolutionary relationship among hermit crab families. We have included the reference in our manuscript.

Again, we thank the reviewer for their contribution. We hope all the concerns of the reviewer has been addressed.

We appreciate the reviewer for the positive feedback about our manuscript. Thank you.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0330170.s008.docx^{(39.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0330170.r003

Decision Letter 1

Feng ZHANG

29 Jul 2025

Transcriptome sequencing reveals the evolutionary histories and gene expression evolution in two related Pagurus species

PONE-D-25-30439R1

Dear Dr. Isaac Adeyemi BABARINDE,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0330170.r004

Acceptance letter

Feng ZHANG

PONE-D-25-30439R1

PLOS ONE

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Associated Data

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

Supplementary Materials

S1 Fig. Gene expression evolution and the nature of genes enriched across the species.

(TIF)

pone.0330170.s001.tif^{(2.4MB, tif)}

S1 Table. Details of Transcriptome sequencing.

(XLSX)

pone.0330170.s002.xlsx^{(10.8KB, xlsx)}

S2 Table. Number of homologous transcripts and genes in different databases.

(XLSX)

pone.0330170.s003.xlsx^{(10.3KB, xlsx)}

S3 Table. Calibrations used for divergence time estimates.

(XLSX)

pone.0330170.s004.xlsx^{(10.6KB, xlsx)}

S4 Table. The list of known pigmentation genes.

(XLSX)

pone.0330170.s005.xlsx^{(41.3KB, xlsx)}

S5 Table. InterproScan annotation statistics for the assembled transcripts.

(XLSX)

pone.0330170.s006.xlsx^{(10.1KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0330170.s008.docx^{(39.2KB, docx)}

Data Availability Statement

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PERMALINK

Transcriptome sequencing reveals the evolutionary histories and gene expression evolution in two related Pagurus species

Zakea Sultana

Isaac Adeyemi Babarinde

Masafumi Nozawa

Kazuho Ikeo

Akira Asakura

Tomoyuki Nakano

Roles

Abstract

Introduction

Materials and methods

Sampling location and permit

RNA extraction and sequencing

Fig 1. Transcriptome sequencing of Pagurus maculosus and P. lanuginosus.

Retrieval of publicly available annotation data and crustacean short-read data

Transcriptome assembly and its evaluation

Transcript expression quantification

Transcript annotation

Gene ontology enrichment analyses

Evolutionary analyses

Phylogenetics and divergence time estimates

Differential gene expression

Statistical tests and graphical representations

Results

Head and leg transcriptome assemblies of P. maculosus and P. lanuginosus

Table 1. De novo transcript assembly statistics.

Annotation of the assembled Pagurus transcripts

Fig 2. Annotation of the assembled transcripts with predicted coding abilities.

Phylogenetic analyses and divergence time estimates

Fig 3. Phylogenetic analyses and divergence times estimates of the investigated species.

Selection pressures after P. maculosus and P. lanuginosus divergence

Fig 4. Evolutionary analyses of the assembled transcripts with protein-coding potentials.

Evolution of gene expressions in P. maculosus and P. lanuginosus

Fig 5. Pagurus gene expression evolution.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Feng ZHANG

Roles

Author response to Decision Letter 1

Decision Letter 1

Feng ZHANG

Roles

Acceptance letter

Feng ZHANG

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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