Dwi Anggorowati Rahayu

Horseshoe crabs, ancient marine arthropods of the Limulidae family, are currently facing significant threats due to overexploitation driven by high demand for their eggs, which are widely consumed, and their hemolymph, which is critical for biomedical applications. This study aims to evaluate the molecular identification and phylogenetic placement of horseshoe crabs in East Java, a region previously unreported in this context. DNA barcoding was employed to analyze the mitochondrial COI gene region, complemented by detailed morphological identification, to provide a comprehensive understanding of the species in this area. A universal primer LCO/HCO was used for PCR, as described in a previous report, and Sanger sequencing was performed to confirm the approximately 655 bp amplification product. This research identifies horseshoe crabs from six distinct locations in East Java as Tachypleus gigas using molecular methods, with genetic analysis indicating>99% similarity to T. gigas sequences in the GenBank database. Morphological examinations of specimens from seven areas further corroborated the presence of T. gigas, distinguished by characteristic serrations along the telson and a single spine on the posterior region of the opisthosoma. The phylogenetic tree, constructed from our genetic data, shows that Tachypleus gigas populations in East Java are clustered in the same clade as those in Southeast Asia, as supported by GenBank records. This study underscores the importance of integrating genetic and morphological approaches for accurate species identification. Moreover, it provides crucial baseline data for developing conservation strategies to preserve Tachypleus gigas populations in East Java, thereby contributing to broader conservation efforts for this species.

Graphical Abstract Highlight Research 1. Combining DNA barcoding with morphological variation analysis can enhance the accuracy of Pufferfish species identification. 2. This is the first study to examine the genetic diversity and phylogenetic relationships pufferfish from Madura, East Java, using partial COI sequences. 3. DNA barcoding confirms the morphological identification of this group (96%-100%) 4. The reliability using targeted DNA barcoding and phenetic taxonomy to study genetic variation and identify species differences in pufferfish. Abstract Pufferfish are one of the most common fish species in the ocean particularly in Camplong Beach, Madura. Despite the abundant nature, there isn’t much information on this species in East Java, showing the need for identification. Therefore, this study sought to identify pufferfish species in Sampang Regency through morphological and molecular characterization utilizing DNA barcoding of the COI gene. Fins were kept in 96% absolute ethanol, and DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed using bioinformatics tools, including the BOLD system and the web-based Automatic Barcode Gap Discovery (ABGD) tool. To strengthen genetic study, phenetic taxonomy was again applied using Ntysc. Morphological identification revealed five distinct species of pufferfish, namely Chelonodontops patoca, Lagocephalus spadiceus, Arothron reticularis, Otracion rhinorhynchos, and Cyclichthys orbicularis. The high haplotype diversity (Hd) of 0.9 and nucleotide diversity (π) of 0.2 indicated substantial genetic variation. There were 11 polymorphic sites, and the overall ts/tv ratio was 2.2. The frequency of parsimonious informative sites was 21.6%. The phylogenetic tree displayed distinct branching patterns among species. Morphological and molecular identification data supported these results, demonstrating that five distinct pufferfish species occur at Camplong Beach in the Sampang District of Madura.

Pufferfish belong to the Tetraodontidae family, which consists of 28 genera and 184 species; however, there are limited studies on the species from East Java, especially Pasuruan. This indicates the need for the collection of more accurate morphological and genetic resources as an important step for its conservation. Therefore, this study was conducted to identify pufferfish species found in the Lekok Coastal Waters, Pasuruan District, through morphological characterization and molecular analysis using DNA barcoding of the COI gene. A total of 12 samples were collected from the Lekok Coastal Waters, Pasuruan Regency. Fin samples were stored in 96% absolute ethanol. DNA extraction, polymerase chain reaction (PCR), and sequence analysis were performed using bioinformatics tools, the BOLD system, and the Automatic Barcode Gap Discovery (ABGD) web platform. Phenetic taxonomy was further applied using Ntysc to enhance the robustness of genetic analysis. The results showed that there were identified five pufferfish species were identified: Lagocephalus spadiceus, Chelonodontops patoca, Cyclichthys orbicularis, Arothron stellatus, and Arothron reticularis. High haplotype diversity (Hd) of 1 and nucleotide diversity (π) of 0.1 indicate significant genetic variation. The frequency of parsimonious informative sites was 22.6%, with 13 polymorphic sites and an overall ts/tv ratio of 2.2. The phylogenetic tree showed unambiguous branching patterns among species. These findings are supported by morphological and molecular identification results, which indicate the presence of five species of pufferfish along the Lekok Coast, Pasuruan Regency.

Rahayu DA, Budijastuti W, Kuntjoro S, Winarsih, Nugroho ED, Ambarwati R, Faizah U, Sulung CAVT, Rusdianto R, Mamat NB. 2025. Morphological and genetic variation of local loach (Nemacheilus spp.) from Lumajang, East Java, Indonesia. Biodiversitas 26: 2896-2907. The genus Nemacheilus is a part of the family Nemacheilidae within the order Cypriniformes, comprising several morphologically similar fish species found in East Java, Indonesia. However, species identification in Lumajang remains unavailable. This study aimed to analyze morphological and genetic diversity of local loach species from Lumajang, East Java, contributing to the existing knowledge with new records. The experiment was carried out through diagnostic identification, meristic, and morphometric analyses, using Past.403 software for correlation assessments. Pectoral fin samples were preserved in high-purity ethanol for DNA extraction, followed by Polymerase Chain Reaction (PCR) targeting mitochondrial genomes, Recombinase-Activating Gene proteins 1 (RAG1), and mitochondrial Cytochrome C Oxidase I (COI) to explore phylogenetic relationships among Nemacheilidae species through maximum likelihood methods. Morphological identification showed two local loaches, Nemacheilus chrysolaimos (Valenciennes, 1846) and Nemacheilus fasciatus (Valenciennes, 1846) with two variants (dominant black color and yellow color). Several morphometric parameters showed high correlations, such as TL with SL, HL with Snout Length (SnL), and Dorsal Fin Length (DFL) with Anal Fin Length (AFL). The COI locus showed high genetic variation with 126 variable sites across 489 base pairs. Meanwhile, the RAG1 gene had a lower variation with 33 variable sites across 681 base pairs, with phylogenetic analysis showing two distinct clades. This comprehensive study enhanced the understanding of morphological and genetic variation in Nemacheilus species, related to conservation efforts and sustainable aquaculture.

Morphological plasticity in aquatic organisms is an adaptive phenomenon that enables species to respond to changing environmental conditions, particularly in native fish species such as the Uceng fish. This study aims to: (1) describe the morphological variation of the Uceng fish and water quality parameters in three river locations in East Java; (2) analyze the relationship between water quality parameters and the morphological characteristics of the Uceng fish; (3) identify the water quality parameters that most affect morphological variation of fish through PCA analysis; and (4) model the relationship between water quality parameters as independent variables and fish morphological characteristics as dependent variables. This research employed a correlational approach with a cross-sectional comparative design, comparing fish samples from three different geographic locations: Blitar, Pasuruan, and Lumajang. Data analysis was conducted using correlation analysis, PCA, and multiple regression modeling. Measurement of the Morphological Characteristics of the Uceng Fish, Morphometric Measurement (14 characters). The results showed a significant negative correlation (r = -0.155, p = 0.017) between environmental factors and fish morphology, with approximately 33% of the morphological variation explained by the combination of environmental factors (DO, temperature, COD, and TDS). PCA analysis identified DO as the parameter with the strongest positive influence, while temperature, COD, and TDS showed negative effects. These findings emphasize the species’ adaptive capacity and underline the importance of monitoring environmental quality to support biodiversity conservation in increasingly impacted river ecosystems of East Java.

Rahayu DA, Amarwati R, Faizah U, Nugroho ED, Rusdianto, Ajiningrum PS, Mamat NB. 2024. Genetic diversity and phylogenetic reconstruction of horseshoe crabs from East Java, Indonesia based on DNA barcode COI. Biodiversitas 25: 3789-3802. Horseshoe crabs or mimi mintuna (Indonesian) are an ancient arthropod with 4 species, but there are limited studies on the species from East Java. This indicates the need for the collection of more accurate genetic resources as an important step for its conservation. Therefore, this study aims to examine genetic diversity and phylogenetic reconstruction of horseshoe crabs from East Java using DNA barcode COI. A total of 20 samples were obtained from Beejay Bakau Resort, Probolinggo, Kenjeran Beach, Surabaya, Batah Timur Beach, Madura, and Lekok Beach, Pasuruan. The 621 bp CO1 gene sequence was used for genetic diversity analysis and phylogenetic tree reconstruction. Subsequently, muscle samples were preserved in pure-grade ethanol and subjected to DNA extraction, polymerase chain reaction (PCR), sequencing, and sequence analysis. The sequence data were analyzed using bioinformatics tools, Barcode of Life Data Systems (BOLD) Systems, the Automatic Barcode Gap Discovery (ABGD) web application, and haplotype network. Phenetic taxonomy was further applied using Ntysc to enhance the robustness of the genetic analysis. The results showed that the high haplotype diversity (Hd) of 0.957, 16 haplotypes, and nucleotide diversity (?) of 0.00097 indicated a wide range of genetic variations. The frequency of parsimony informative sites was 26.48%, with 19 polymorphic sites and an overall ts/tv ratio of 2.32. In addition, the phylogenetic trees showed a clear and unambiguous branching pattern for cluster Carcinoscorpius rotundicauda Latreille 1802 and Tachypleus gigas O.F.Müller 1785. This finding is a matter of concern for managing and conserving horseshoe crab species in East Java waters.

Sulung CAVT, Chair NE, Rahayu DA, Nugroho ED, Rusdianto. 2024. Morphology and COI barcodes reveal local loaches (Nemacheilus spp.) from Pasuruan River, East Java, Indonesia. Biodiversitas 25: 2806-2819. The family Nemacheilidae is one of the most diverse and widespread family of freshwater fishes. The nemacheilid loach in the Pasuruan river is relatively abundant; however, there is limited information available about this species. Therefore, further research is necessary to identify loaches from the Pasuruan area. This research aimed to identify the local loach species from Pasuruan District. The pectoral fins were stored in pure-grade ethanol and subjected to DNA extraction, polymerase chain reaction (PCR), and sequence analysis using bioinformatics tool, BOLD systems, and Automatic Barcode Gap Discovery (ABGD) web. The morphological identification revealed two local loaches, Nemacheilus chrysolaimos Valenciennes 1846 and Nemacheilus fasciatus Valenciennes 1846. The high haplotype diversity (Hd) of 0.9 and nucleotide diversity (?) of 0.1 indicate a wide range of genetic variations. The frequency of parsimony informative sites at 25.6% and 16 polymorphic sites and the overall ts/tv ratio of 2.3. The phylogenetic trees demonstrated a clear and unambiguous branching pattern for N. fasciatus and N. chrysolaimos forming their own monophyletic branches. Kimura's 2-parameter genetic distance of the COI gene sequences between these two species was 0.11. This finding was strengthened by morphological and molecular identification results, which indicated that there are two local loach species in Pasuruan District.