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Assembly and annotation of the chloroplast genomesAssembly resulted in a whole cp genome sequence of C. hirtinoda with a length of 139, 561 bp (Fig. 1), consisting of 83, 166 bp large single-copy region, 20, 811 bp small single-copy regions, and two 21,792 bp IR regions, comprising the typical quadripartite structure of terrestrial plants. The cp genome of C. hirtinoda was annotated with 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes (Table 1). Most of the 15 genes in the C. hirtinoda cp genome contain introns. Of these, 13 genes contain one intron (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rps16, trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, trnV-UAC) and only the gene cyf3 includes two introns, and the gene clpP intron was deleted (Supplementary Table S1). The rps12 gene contained two copies, and the three exons were spliced into a trans-splicing gene18.Figure 1Chloroplast genome map of C. hirtinoda. Different colors represent different functional genes groups. Genes outside the circle indicate counterclockwise transcription, and genes inside the clockwise transcription. The thick black line on the outer circle represents the two IR regions. The GC content is the dark gray area within the ring.Full size imageTable 1 Summary of the chloroplast genome of C. hirtinoda.Full size tableThe accD, ycf1, and ycf2 genes were missing in the cp genome of C. hirtinoda, and the introns in the genes clpP and rpoC1 were lost. This phenomenon is consistent with previous systematic evolutionary studies on the genome structure of plants in the Poaceae family19. The phenomenon of missing genes is reported in other plants20,21,22,23.The total GC content in the C. hirtinoda cp genome was 38.90%, and the content for each of the four bases, A, T, G, and C, was 30.63%, 30.46%, 19.57%, and 19.33%, respectively (Table 2). The LSC region (36.98%) and SSC region (33.21%) exhibited much lower values than the IR region (44.23%), indicating a non-uniform distribution of the base contents in the cp genome, probably because of four rRNAs in the IR region, which in turn makes the GC content higher in the IR region. These values were similar to cp genome results previously reported for some Poaceae plants24,25.Table 2 Base composition in the C. hirtinoda choloroplast genome.Full size tableRepeat sequences and codon analysisSSR consists of 10-bp-long base repeats and is widely used for exploring phylogenetic evolution and genetic diversity analysis26,27,28,29.In total, 48 SSRs were detected in C. hirtinoda, including 27 mononucleotide versions, accounting for 56.25% of the total SSRs, primarily consisting of A or T. Additionally, four dinucleotide repeats consisting of AT/TA and TC/CT repeats, and 3 tri, 13 tetra, and 1penta-repeats (Fig. 2A). From the SSRs distribution perspective, the majority (79%) of SSRs (38) were observed in the LSC area, whereas 6 SSRs in the IR region (13%) and 4 SSRs in the SSC region (8%) were discovered (Fig. 2B). Previous research suggests that the distribution of SSRs numbers in each region and the differences among locations in GC content are related to the expansion or contraction of the IR boundary30.Figure 2Analysis of simple sequence repeats in C. hirtinoda cp genome. (A) The percentage distribution of 45 SSRs in LSC, SSC, and IR regions. (B).Full size imageThe REPuter program revealed that the cp genome of C. hirtinoda was identified with 61 repeats, consisting of 15 palindromic, 19 forward and no reverse and complement repeats (Fig. 3). We noticed that repeat analyses of three Chimonobambusa genus species exhibited 61–65 repeats, with only one reverse in C. hejiangensis. Most of the repeat lengths were between 30 and 100 bp, and the repeat sequences were located in either IR or LSC region31 (Supplementary Table S2).Figure 3Information of chloroplast genome repeats of Chimonobambusa genus species.Full size imageWe identified 20,180 codons in the coding region of C. hirtinoda (Fig. 4, Supplementary Table S3). The codon AUU of Ile was the most used, and the TER of UAG was the least used codon (817 and 19), excluding the termination codons. Leu was the most encoded amino acid (2,170), and TER was the lowest (85). The Relative Synonymous Codon Usage (RSCU) value greater than 1.0 means a codon is used more frequently32. The RSCU values for 31 codons exceeded 1 in the C. hirtinoda cp genome, and of these, the third most frequent codon was A/U with 29 (93.55%), and the frequency of start codons AUG and UGG used demonstrated no bias (RSCU = 1).Figure 4Amino acid frequencies in C. hirtinoda cp genome protein coding sequences. The column diagrams indicate the number of amino acid codes, and the broken line indicates the proportion of amino acid codes.Full size imageComparative analysis of genome structureThe nucleotide variability (Pi) values of the three cp genomes discovered in the Chimonobambusa genus species ranged from 0 to 0.021 with an average value of 0.000544, as demonstrated from DnaSP 5.10 software analysis. Five peaks were observed in the two single-copy regions, and the highest peak was present in the trnT-trnE-trnY region of the LSC region (Fig. 5). The Pi value for LSC and SSC is significantly higher than that of the IR region. In the IR region, highly different sequences were not observed, a highly conserved region. The sequences of these highly variable regions are reported in other plants during examinations for species identification, phylogenetic analysis, and population genetics research33,34,35.Figure 5Sliding window analysis of Chimonobambusa genus complete chloroplast genome sequences. X-axis: position of the midpoint of a window, Y-axis: nucleotide diversity of each window.Full size imageThe structural information for the complete cp genomes among three Chimonobambusa genus species revealed that the sequences in most regions were conserved (Fig. 6). The LSC and SSC regions exhibit a remarkable degree of variation, higher than the IR region, and the non-coding region demonstrates higher variability than the coding region. In the non-coding areas, 7–9 k, 28–30 k, 36 k and other gene loci differed significantly. Genes rpoC2, rps19, ndhJ and other regions differ in the protein-coding region. However, the agreement between the tRNA and rRNA regions is 100%. A similar phenomenon has also been reported by others36.Figure 6Visualization of genome alignment of three species chloroplast genome sequences using Chimonobambusa hejiangensis as reference. The vertical scale shows the percent of identity, ranging from 50 to 100%. The horizontal axis shows the coordinates within the cp genome. Those are some colors represents protein coding, intron, mRNA and conserved non-coding sequence, respectively.Full size imageIR contraction and expansion in the chloroplast genomeDue to the unique circular structure of the cp genome, there are four junctions between the LSC/IRB/SSC/IRA regions. During species evolution, the stability of the two IR regions sequences was ensured by the IR region of the chloroplast genome expanding and contracting to some degree, and this adjustment is the primary reason for chloroplast genome length variation37,38.The variations at IR/SC boundary regions in the three Chimonobambusa genus chloroplast genomes were highly similar in the organization, gene content, and gene order. The size of IR ranges from 21,797 bp (C. tumidissinoda) to 21,835 bp (C. hejiangensis). The ndhH gene spans the SSC/IRa boundary, and this gene extended 181–224 bp into the IRa region for all three Chimonobambusa genus. The gene rps19 was extended from the IRb to the LSC region with a 31–35 bp gap. The rpl12 gene was located in the LSC region of all genomes, varied from 35–36 bp apart from the LSC/IRb (Fig. 7).Figure 7Comparison of LSC, SSC and IR boundaries of chloroplast genomes among the three Chimonobambusa species. The LSC, SSC and IRs regions are represented with different colors. JLB, JSB, JSA and JLA represent the connecting sites between the corresponding regions of the genome, respectively. Genes are showed by boxes.Full size imageThree chloroplast genomes of the Chimonobambusa genus were compared using the Mauve alignment. The results showed that all sequences show perfect synteny conservation with no inversion or rearrangements (Fig. 8).Figure 8The chloroplast genomes of three Chimonobambusa species rearranged by the software MAUVE. Locally collinear blocks (LCBs) are represented by the same color blocks connected by lines. The vertical line indicates the degree of conservatism among position. The small red bar represents rRNA.Full size imagePhylogenetic analysisWe performed a phylogenetic analysis using the complete chloroplast genomes and matK gene reflecting the phylogenetic position of C. hirtinoda. The maximum likelihood (ML) analysis based on the complete chloroplast genomes indicated seven nodes with entirely branch support (100% bootstrap value). However, the three Chimonobambusa genera exhibited a moderate relationship due to fewer samples used, supporting that C. hirtinoda is closely related to C. tumidissinoda with a 62% bootstrap value more than C. hejiangensis. A phylogenetic tree based on the matK gene revealed that Chimonobambusa species clustered in one branch was consistent with the phylogenetic tree constructed by the complete cp genome tree (Fig. 9). The results show that the whole chloroplast genome identified related species better than the former, consistent with the previous study39.Figure 9Maximum likelihood phylogenetic tree based on the complete chloroplast genomes (A) and matK gene (B).Full size image More

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Fisheries overexploitation is a problem in all oceans and seas globally. Authorities and administrations in charge of assigning quotas have very little fine-grained information on the fish captures, and instead use large-scale, coarse data to assess the health level of fisheries. Thus, being able to cross-match fish species and sizes, to the sea regions they were captured from, can be helpful in this regard, providing finer-grained information.Previous attempts at assembling datasets for fish detection and classification exist, ranging from fish detection or counting in underwater images and video streams1,2,3, to counting on belts on trawler ships4, to classification in laboratory conditions5,6, or in underwater preprocessed images of single fish7,8,9, or single fish in free-form pictures10, as well as simultaneous detection and classification of several fish11,12. However, none of the works found in the literature addresses the topic of simultaneous instance segmentation and species classification, along with fish size estimation, in a fish market environment, as is the aim of this paper. Instance segmentation refers to the extraction of pixel-level masks for each individual object (in this case fish specimens), rather than bounding boxes (object detection), or class label masks (e.g. a single mask for all fish specimens of the same species, also referred to as semantic segmentation). Moreover, works in the literature use pictures taken in laboratory conditions (with a single fish per image, shown from the side), or in underwater conditions. Only French et al.4 uses pictures of fish catches on a belt, for counting purposes. Table 1 shows a summary of the datasets identified in the literature, along with their characteristics, including how the proposed dataset compares.Table 1 Summary of previous datasets found in the literature, and comparison to proposed dataset.Full size tableThe DeepFish project (website: http://deepfish.dtic.ua.es/) is aimed at providing fish species classification and size estimation for fish specimens arriving at fish markets, both for the automation of fish sales, and the retrieval of fine-grained information about the health of fisheries. For a period of six months (April to September 2021), images have been captured at the fish market in El Campello (Alicante, Spain). Images of market trays show a variety of fish species, including targeted as well as accidental captures from the ‘Cabo de la Huerta’, an important site for protection and preservation of marine habitats and biodiversity as defined by the European Comission Habitats Directive (92/43/EEC). From the pictures, a total of 59 different species are identified with 12 species having more than 100 specimens and 25 with more than 10 specimens, as shown in Table 2. There is a high imbalance of species captured due to the natural variation in fish species populations according to seasonality and other ecological factors (rarity of the species, i.e. total population count, etc). Due to some species showing sexual dimorphism (i.e. Symphodus tinca), this species is split into two separate class labels, leading to a different number of species, and class labels (59 species, but 60 class labels). The dataset presents a high temporal imbalance too. As shown in Fig. 1, the capture of new fish tray images was not evenly distributed during the six month study period. Several factors contributed to this: wholesale fish market operating days (e.g. no weekend data, holidays and stop periods, etc.), fish species variability (one of the aims was to be able to capture at least 100 specimens from several species, and seasonality meant some could not be available for capture in later months), as well as the time availability of research group members to attend the fish arrival, tray preparation and auctioning in the evenings.Table 2 Distribution of fish species in the dataset.Full size tableFig. 1Temporal distribution of fish tray images captured. It can be observed that April (04) and May (05) were much more active than the rest of months. This is due to several contributing factors.Full size imageThe resulting DeepFish dataset introduced here contains annotated images from 1,291 fish market trays, with a total of 7,339 specimens (individual fish instances) which were labelled (species and mask) using a specially-adapted version of the Django labeller instance segmentation labelling tool13. Subsequently, another JSON file is generated, following the Microsoft Common Objects in Context (MS COCO) dataset format14, which can be directly fed to a neural network. This is done via a script that is also provided15. Figure 2 shows the distribution of individuals for the selected species within the dataset. Furthermore, Fig. 3 shows examples of the trays, with instance segmentation (ground truth silhouette, i.e. as an interpolation from human-provided points) along with species labelling (different colour shading).Fig. 2Graphical view of the distribution of fish species in the DeepFish dataset for species above 10 specimens. Note, Symphodus tinca is considered separately due to sexual dimorphism (211 male; 335 female samples).Full size imageFig. 3Examples of ground truth fish instance masks with class labelling, showing the 12 species (13 labels) with more than 100 specimens (in bold in Table 2).Full size imageFrom the point of view of research, this data is important for the classification of fish species, instance segmentation, as well as specimen size estimation (e.g. as a regression problem, or otherwise). From an end-results perspective, data automatically labelled with fish instance segmentation accompanied by species name and estimated size is useful to different stakeholders, namely: fishing authorities (to understand how much of each species is being caught per zone), maritime conservation (to calculate depletion of fisheries), but also managers of the markets themselves, as well as clients (digitized sales, e-commerce), etc.The usage of the provided data can be manifold, as it can be used for several problems, namely: object detection and classification, which involves finding objects (in this case fish specimens) providing a bounding box, and a class for each of these boxes; additionally, the data can also be used for semantic segmentation, which can provide a pixel-wise segmentation of the image providing labels (in this case species labels) to different pixel regions of the image; furthermore, also instance segmentation is possible, in which not just a single label for all instances of the same species is provided, but each specimen is provided with a mask (specimen segmentation), as well as a label (species). Furthermore, several measurements of each fish are provided, which can also be used to estimate their size, since they have been shown to be correlated with each other16. These are estimated from the calculated homography (given the tray size is known), given the burden of measuring each fish due to the large amount of specimens in the dataset. More