Fresh records on Ichthyofaunal diversity of River Panjkora Dir, Khyber Pakhtunkhwa, Pakistan.

This study was designed to assess the ichthyofaunal diversity of River Panjkora, Khyber Pakhtunkhwa, Pakistan. For this purpose, a total of 1189 fish from six different sites were collected along the river and identified using standard keys. The fish collected and identified were representing 38 species, belonging to 7 families. The investigation spanned a year, from July 2021 to May 2022. The most dominant family was Cyprinidae 76% (n=906/1189), followed by Nemacheilidae 5.8% (n=69/1189), Channidae 5.2% (n=62/1189), Sisoridae 5.1% (n=61/1189), Mastacembelidae 4.9% (n=58/1189), Salmonidae 2.6% (n=31/1189) and least was Bagridae 0.17 (n=2/1189). The most abundant speices was Schizothorax plagiostomus with relative density of 16.8. Family Cyprinidae was represented by 21 species, Sisoridae by 7 species, Nemacheilidae by 5 species, Channidae by 2 species, while Bagridae, Salmonidae and Mastacembelidae, were each represented by a single species. PAST 3, XLSTAT and EXCEL 2019 were used for principal component analysis to study correlation of fish diversity and richness. Eigenvalue obtained from Kumrat to Busaq were 3.32, 1.01, 0.80, 0.44, 0.31 and 0.10 respectively. The higher value at Kumrat shows higher diversity. The water quality assessment showed average value of water temperature 10.4 ͦC, pH 7.0, electrical conductivity 184 mg/L, dissolved oxygen 7.9 mg/L, turbidity 43.73 mg/L, total dissolved solids 101 mg/L, total suspended solids 34.72 mg/L, total solids 135.53 mg/L, total alkalinity 75.77 mg/L, total hardness 58.37 mg/L, ammonia 0.46 mg/L, sulphate 26.03 mg/L, chloride 14.67 mg/L, calcium 69.11 mg/L, chromium 0.18 mg/L, copper 0.03, cobalt mg/L 0.04, nickel 0.039 mg/L, lead 0.02 mg/L and Zinc 0.35 mg/L. The findings of this study indicated that most of the physicochemical parameters remained within the acceptable limits throughout the study period. Analysis of fish gut contents included; nymphs, insect larvae, the presence of algae, protozoans and macroinvertebrates in the river ecosystem.

Population Genomics of Commercial Fish Sebastes schlegelii of the Bohai and Yellow Seas (China) Using a Large SNP Panel from GBS.

Sebastes schlegelii is one of the most commercially important marine fish in the northwestern Pacific. However, little information about the genome-wide genetic characteristics is available for S. schlegelii individuals from the Bohai and Yellow Seas. In this study, a total of 157,778, 174,480, and 188,756 single-nucleotide polymorphisms from Dalian (DL), Yantai (YT), and Qingdao (QD) coastal waters of China were, respectively, identified. Sixty samples (twenty samples per population) were clustered together, indicating shallow structures and close relationships with each other. The observed heterozygosity, expected heterozygosity, polymorphism information content, and nucleotide diversity ranged from 0.14316 to 0.17684, from 0.14035 to 0.17145, from 0.20672 to 0.24678, and from 7.63 × 10-6 to 8.77 × 10-6, respectively, indicating the slight difference in genetic diversity among S. schlegelii populations, and their general genetic diversity was lower compared to other marine fishes. The population divergence showed relatively low levels (from 0.01356 to 0.01678) between S. schlegelii populations. Dispersing along drifting seaweeds, as well as the ocean current that flows along the western and northern coasts of the Yellow Sea and southward along the eastern coast of China might be the major reasons for the weak genetic differentiation. These results form the basis of the population genetic characteristics of S. schlegelii based on GBS (Genotyping by Sequencing). In addition to basic population genetic information, our results provid a theoretical basis for further studies aimed at protecting and utilizing S. schlegelii resources.

Complete Mitochondrial Genome of Four Peristediidae Fish Species: Genome Characterization and Phylogenetic Analysis.

The systematic revision of the family Peristediidae remains an unresolved issue due to their diverse and unique morphology. Despite the popularity of using mitochondrial genome research to comprehensively understand phylogenetic relationships in fish, genetic data for peristediid fish need to be included. Therefore, this study aims to investigate the mitochondrial genomic characteristics and intra-family phylogenetic relationships of Peristediidae by utilizing mitochondrial genome analysis. Therefore, this study aims to investigate the phylogenetic relationship of Peristediidae by utilizing mitochondrial genome analysis. The mitochondrial genome of four species of Peristediidae (Peristedion liorhynchus, Satyrichthys welchi, Satyrichthys rieffeli, and Scalicus amiscus) collected in the East China Sea was studied. The mitochondrial gene sequence lengths of four fish species were 16,533 bp, 16,526 bp, 16,527 bp, and 16,526 bp, respectively. They had the same mitochondrial structure and were all composed of 37 genes and one control region. Most PCGs used ATG as the start codon, and a few used GTG as the start codon. An incomplete stop codon (TA/T) occurred. The AT-skew and GC-skew values of 13 PCGs from four species were negative, and the GC-skew amplitude was greater than that of AT-skew. All cases of D-arm were found in tRNA-Ser (GCT). The Ka/Ks ratio analysis indicated that 13 PCGs were suffering purifying selection. Based on 12 PCGs (excluding ND6) sequences, a phylogenetic tree was constructed using Bayesian inference (BI) and maximum likelihood (ML) methods, providing a further supplement to the scientific classification of Peristediidae fish. According to the results of divergence time, the four species of fish had apparent divergence in the Early Cenozoic, which indicates that the geological events at that time caused the climax of species divergence and evolution.

An integrated spatio-temporal view of riverine biodiversity using environmental DNA metabarcoding.

Anthropogenically forced changes in global freshwater biodiversity demand more efficient monitoring approaches. Consequently, environmental DNA (eDNA) analysis is enabling ecosystem-scale biodiversity assessment, yet the appropriate spatio-temporal resolution of robust biodiversity assessment remains ambiguous. Here, using intensive, spatio-temporal eDNA sampling across space (five rivers in Europe and North America, with an upper range of 20-35 km between samples), time (19 timepoints between 2017 and 2018) and environmental conditions (river flow, pH, conductivity, temperature and rainfall), we characterise the resolution at which information on diversity across the animal kingdom can be gathered from rivers using eDNA. In space, beta diversity was mainly dictated by turnover, on a scale of tens of kilometres, highlighting that diversity measures are not confounded by eDNA from upstream. Fish communities showed nested assemblages along some rivers, coinciding with habitat use. Across time, seasonal life history events, including salmon and eel migration, were detected. Finally, effects of environmental conditions were taxon-specific, reflecting habitat filtering of communities rather than effects on DNA molecules. We conclude that riverine eDNA metabarcoding can measure biodiversity at spatio-temporal scales relevant to species and community ecology, demonstrating its utility in delivering insights into river community ecology during a time of environmental change.

CoSFISH: a comprehensive reference database of COI and 18S rRNA barcodes for fish.

Fish, being a crucial component of aquatic ecosystems, holds significant importance from both economic and ecological perspectives. However, the identification of fish at the species level remains challenging, and there is a lack of a taxonomically complete and comprehensive reference sequence database for fish. Therefore, we developed CoSFISH, an online fish database. Currently, the database contains 21 535 cytochrome oxidase I sequences and 1074 18S rRNA sequences of 21 589 species, belonging to 8 classes and 90 orders. We additionally incorporate online analysis tools to aid users in comparing, aligning and analyzing sequences, as well as designing primers. Users can upload their own data for analysis, in addition to using the data stored in the database directly. CoSFISH offers an extensive fish database and incorporates online analysis tools, making it a valuable resource for the study of fish diversity, phylogenetics and biological evolution. Database URL:  http://210.22.121.250:8888/CoSFISH/home/indexPage.

The Tree of Life eDNA metabarcoding reveals a similar taxonomic richness but dissimilar evolutionary lineages between seaports and marine reserves.

Coastal areas host a major part of marine biodiversity but are seriously threatened by ever-increasing human pressures. Transforming natural coastlines into urban seascapes through habitat artificialization may result in loss of biodiversity and key ecosystem functions. Yet, the extent to which seaports differ from nearby natural habitats and marine reserves across the whole Tree of Life is still unknown. This study aimed to assess the level of α and ß-diversity between seaports and reserves, and whether these biodiversity patterns are conserved across taxa and evolutionary lineages. For that, we used environmental DNA (eDNA) metabarcoding to survey six seaports on the French Mediterranean coast and four strictly no-take marine reserves nearby. By targeting four different groups-prokaryotes, eukaryotes, metazoans and fish-with appropriate markers, we provide a holistic view of biodiversity on contrasted habitats. In the absence of comprehensive reference databases, we used bioinformatic pipelines to gather similar sequences into molecular operational taxonomic units (MOTUs). In contrast to our expectations, we obtained no difference in MOTU richness (α-diversity) between habitats except for prokaryotes and threatened fishes with higher diversity in reserves than in seaports. However, we observed a marked dissimilarity (ß-diversity) between seaports and reserves for all taxa. Surprisingly, this biodiversity signature of seaports was preserved across the Tree of Life, up to the order. This result reveals that seaports and nearby marine reserves share few taxa and evolutionary lineages along urbanized coasts and suggests major differences in terms of ecosystem functioning between both habitats.

Stochastic process is main factor to affect plateau river fish community assembly.

Plateau river ecosystems are often highly vulnerable and responsive to environmental change. The driving mechanism of fish diversity and community assembly in plateau rivers under changing environments presents a significant complexity to the interdisciplinary study of ecology and environment. This study integrated molecular biological techniques and mathematical models to identify the mechanisms influencing spatial heterogeneity of freshwater fish diversity and driving fish community assembly in plateau rivers. By utilizing environmental-DNA metabarcoding and the null model, this study revealed the impact of the stochastic process on fish diversity variations and community assembly in the Huangshui Plateau River of the Yellow River Basin (YRB) in China. This research identified 30 operational taxonomic units (OTUs), which correspond to 20 different fish species. The findings of this study revealed that the fish α-diversity in the upstream region of Xining is significantly higher than in the middle-lower reach (Shannon index: P = 0.017 and Simpson: P = 0.035). This pattern was not found to be related to any other environmental factors besides altitude (P = 0.023) that we measured. Further, the study indicated that the assembly of fish communities in the Huangshui River primarily depends on stochastic ecological processes. These findings suggested that elevation was not the primary factor impacting the biodiversity patterns of fish in plateau rivers. In plateau rivers, spatial heterogeneity of fish community on elevation is mainly determined by stochastic processes under habitat fragmentation, rather than any other physicochemical environmental factors. The limitations of connectivity in the downstream channel of the river could be taken the mainly responsibility for stochastic processes of fish community in Huangshui River. Incorporating ecological processes in the eDNA approach holds great potential for future monitoring and evaluation of fish biodiversity and community assembly in plateau rivers.

Suitable habitat shifts and ecological niche overlay assessments among benthic Oplegnathus species in response to climate change.

Climate change has had a significant impact on many marine organisms. To investigate the effects of environmental changes on deep-water benthic fishes, we selected the genus Oplegnathus and applied species distribution modeling and ecological niche modeling. From the last glacial maximum to the present, the three Oplegnathus species (O. conwayi, O. robinsoni, and O. peaolopesi) distributed in the Cape of Good Hope region of southern Africa experienced fitness zone fluctuations of 39.9%, 13%, and 5.7%, respectively. In contrast, O. fasciatus and O. punctatus, which were primarily distributed in the western Pacific Ocean, had fitness zone fluctuations of -6.5% and 11.7%, respectively. Neither the O. insignis nor the O. woodward varied by more than 5% over the period. Under future environmental conditions, the range of variation in fitness zones for the three southern African Oplegnathus species was expected to be between -30.8% and -26.5%, while the range of variation in fitness zones for the two western Pacific stonefish species was expected to remain below 13%. In addition, the range of variation in the fitness zones of the O. insignis was projected to be between -2.3% and 7.1%, and the range of variation in the fitness zones of the O. woodward is projected to be between -5.7% and -2%. The results indicated that O. fasciatus and O. punctatus had a wide distribution and high expansion potential, while Oplegnathus species might have originated in western Pacific waters. Our results showed that benthic fishes were highly adaptable to extreme environments, such as the last glacial maximum. The high ecological niche overlap between Oplegnathus species in the same region suggested that they competed with each other. Future research could explore the impacts of environmental change on marine organisms and make conservation and management recommendations.

Using low volume eDNA methods to sample pelagic marine animal assemblages.

Environmental DNA (eDNA) is an increasingly useful method for detecting pelagic animals in the ocean but typically requires large water volumes to sample diverse assemblages. Ship-based pelagic sampling programs that could implement eDNA methods generally have restrictive water budgets. Studies that quantify how eDNA methods perform on low water volumes in the ocean are limited, especially in deep-sea habitats with low animal biomass and poorly described species assemblages. Using 12S rRNA and COI gene primers, we quantified assemblages comprised of micronekton, coastal forage fishes, and zooplankton from low volume eDNA seawater samples (n = 436, 380-1800 mL) collected at depths of 0-2200 m in the southern California Current. We compared diversity in eDNA samples to concurrently collected pelagic trawl samples (n = 27), detecting a higher diversity of vertebrate and invertebrate groups in the eDNA samples. Differences in assemblage composition could be explained by variability in size-selectivity among methods and DNA primer suitability across taxonomic groups. The number of reads and amplicon sequences variants (ASVs) did not vary substantially among shallow (<200 m) and deep samples (>600 m), but the proportion of invertebrate ASVs that could be assigned a species-level identification decreased with sampling depth. Using hierarchical clustering, we resolved horizontal and vertical variability in marine animal assemblages from samples characterized by a relatively low diversity of ecologically important species. Low volume eDNA samples will quantify greater taxonomic diversity as reference libraries, especially for deep-dwelling invertebrate species, continue to expand.

Harnessing eDNA metabarcoding to investigate fish community composition and its seasonal changes in the Oslo fjord.

In the face of global ecosystem changes driven by anthropogenic activities, effective biomonitoring strategies are crucial for mitigating impacts on vulnerable aquatic habitats. Time series analysis underscores a great significance in understanding the dynamic nature of marine ecosystems, especially amidst climate change disrupting established seasonal patterns. Focusing on Norway's Oslo fjord, our research utilises eDNA-based monitoring for temporal analysis of aquatic biodiversity during a one year period, with bi-monthly sampling along a transect. To increase the robustness of the study, a taxonomic assignment comparing BLAST+ and SINTAX approaches was done. Utilising MiFish and Elas02 primer sets, our study detected 63 unique fish species, including several commercially important species. Our findings reveal a substantial increase in read abundance during specific migratory cycles, highlighting the efficacy of eDNA metabarcoding for fish composition characterization. Seasonal dynamics for certain species exhibit clear patterns, emphasising the method's utility in unravelling ecological complexities. eDNA metabarcoding emerges as a cost-effective tool with considerable potential for fish community monitoring for conservation purposes in dynamic marine environments like the Oslo fjord, contributing valuable insights for informed management strategies.

Recent Advances in Closed-Tube Barcoding for FastFish-ID.

FastFish-ID for rapid and accurate identification of fish species was conceived at Brandeis University based on pioneering work on Closed-Tube Barcoding (Rice et al., Mitochondrial DNA Part A 27(2):1358-1363, 2016; Sirianni et al., Genome 59:1049-1061, 2016). FastFish-ID was subsequently validated and commercialized at Thermagenix, Inc. using a portable device and high-precision PCR (Naaum et al., Food Res Int 141:110035, 2021). The motivation for these efforts was the pressing need for a technology that could be widely used throughout the seafood supply chain to combat IUU Fishing (Helyar et al., PLOS ONE 9, 2014) and overfishing (FAO, State of the World Fisheries and Aquaculture 2018. http://www.fao.org/documents/card/en/c/I9540EN/ , 2018), along with seafood fraud and mislabeling (Watson et al., Fish Fish 17:585-595, 2015). These destructive practices are wasting fish stocks, frustrating attempts to achieve seafood sustainability, endangering oceanic ecosystems, and causing consumers billions of dollars each year (Porterfield et al., Oceana: February, 2022). During the past three Covid19 pandemic years, EcologeniX, LLC has taken over further development and optimization of FastFish-ID. The present chapter provides an overview of the improvements introduced throughout the FastFish-ID process.

Global distribution patterns and geographic range of Cymothoa Fabricius, 1793 (Isopoda: Cymothoidae) associated with host fish.

Over recent years, fish parasites of the genus Cymothoa Fabricius, 1793, have received increased attention due to both their ecological and their economic importance to aquaculture and fishery. As the studies about Cymothoa have increased this improve our understanding on the host specificity and distribution of these parasites. The aim of this paper was to review the current global geographic distribution, distribution patterns and parasite-host interactions patterns of Cymothoa spp. associated with fish from marine and brackish water bodies around the world. A total of 144 samples were analyzed, from which 23 species of Cymothoa were found parasitizing 84 teleost fish species of 35 families and 20 orders. Most of these parasites were found in the mouth of the host fish, including in wild fish. The highest occurrence of parasites was found in host species belonging to the families Carangidae and Lutjanidae. Host specificity was an important factor in the geographic distribution of Cymothoa species as also environmental temperature. Cymothoa indica, Cymothoa exigua and Cymothoa excisa were the species with lowest specificity for host family and widest geographic distribution.

Gars truly are 'living fossils,' massive DNA data set shows.

The fish's genomes change so slowly that species separated since the dinosaurs can produce fertile hybrids today.

Resurrection and redescriptions of nominal species previously regarded as synonyms of Thrissina mystax (Bloch and Schneider, 1801) (Teleostei: Clupeiformes: Engraulidae).

Examination of the original descriptions and available type specimens of nominal species previously regarded as synonyms of Thrissina mystax (Bloch and Schneider, 1801), and many non-type specimens representing an extensive geographic range, confirmed the validities of T. mystax, Thrissina porava (Bleeker, 1849), and Thrissina valenciennesi (Bleeker, 1866). Additionally, Engraulis poorawah Bleeker, 1872, a nominal species previously regarded as a junior synonym of T. mystax, is recognized as a junior synonym of Thrissina malabarica (Bloch, 1795). Diagnoses and detailed color descriptions are given for all of the valid species, in addition to clarification of their taxonomic histories, and neotype designation for T. porava. The phylogenetic relationships among 15 species of Thrissina (including T. porava and T. valenciennesi but not T. mystax) were reconstructed from the mitochondrial cytochrome oxidase I (COI) gene. T. porava and T. valenciennesi were not recovered as a monophyletic group, instead being divergent from each other and the other species of Thrissina by 12.4% and >11.7% mean uncorrected distances, respectively, confirming their reciprocal validity.

A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

Biogeography of Greater Antillean freshwater fishes, with a review of competing hypotheses.

In biogeography, vicariance and long-distance dispersal are often characterised as competing scenarios. However, they are related concepts, both relying on collective geological, ecological, and phylogenetic evidence. This is illustrated by freshwater fishes, which may immigrate to islands either when freshwater connections are temporarily present and later severed (vicariance), or by unusual means when ocean gaps are crossed (long-distance dispersal). Marine barriers have a strong filtering effect on freshwater fishes, limiting immigrants to those most capable of oceanic dispersal. The roles of vicariance and dispersal are debated for freshwater fishes of the Greater Antilles. We review three active hypotheses [Cretaceous vicariance, Greater Antilles-Aves Ridge (GAARlandia), long-distance dispersal] and propose long-distance dispersal to be an appropriate model due to limited support for freshwater fish use of landspans. Greater Antillean freshwater fishes have six potential source bioregions (defined from faunal similarity): Northern Gulf of México, Western Gulf of México, Maya Terrane, Chortís Block, Eastern Panamá, and Northern South America. Faunas of the Greater Antilles are composed of taxa immigrating from many of these bioregions, but there is strong compositional disharmony between island and mainland fish faunas (>90% of Antillean species are cyprinodontiforms, compared to <10% in Northern Gulf of México and Northern South America, and ≤50% elsewhere), consistent with a hypothesis of long-distance dispersal. Ancestral-area reconstruction analysis indicates there were 16 or 17 immigration events over the last 51 million years, 14 or 15 of these by cyprinodontiforms. Published divergence estimates and evidence available for each immigration event suggests they occurred at different times and by different pathways, possibly with rafts of vegetation discharged from rivers or washed to sea during storms. If so, ocean currents likely provide critical pathways for immigration when flowing from one landmass to another. On the other hand, currents create dispersal barriers when flowing perpendicularly between landmasses. In addition to high salinity tolerance, cyprinodontiforms collectively display a variety of adaptations that could enhance their ability to live with rafts (small body size, viviparity, low metabolism, amphibiousness, diapause, self-fertilisation). These adaptations likely also helped immigrants establish island populations after arrival and to persist long term thereafter. Cichlids may have used a pseudo bridge (Nicaragua Rise) to reach the Greater Antilles. Gars (Lepisosteidae) may have crossed the Straits of Florida to Cuba, a relatively short crossing that is not a barrier to gene flow for several cyprinodontiform immigrants. Indeed, widespread distributions of Quaternary migrants (Cyprinodon, Gambusia, Kryptolebias), within the Greater Antilles and among neighbouring bioregions, imply that long-distance dispersal is not necessarily inhibitory for well-adapted species, even though it appears to be virtually impossible for all other freshwater fishes.

Influence of abiotic factors on the composition of fish assemblages in the Aracá-Demeni sub-basin of the middle Negro River.

We evaluated the influence of flooding and receding comparison and spatial variation on the composition and structure of fish assemblages in blackwater and clearwater rivers of the Aracá-Demeni sub-basin in the middle Negro River, Amazonas state, Brazil. The collections were carried out during the falling-water period (November 2018) and the rising-water period (April 2019) using gillnets with meshes ranging from 30 to 120mm stretched mesh size. Ecological estimates of richness, equitability, the Shannon-Wiener diversity index, and the Berger-Parker index showed no significant differences between the periods. The composition of the ichthyofauna showed variations between the falling-water and rising-water periods. Beta diversity in the Aracá-Demeni sub-basin is almost completely caused by species substitution, with spatial turnover accounting for most of the estimate. The identification of these patterns and of the responsible factors are fundamental for the definition of conservation strategies, especially in an ecosystem whose dynamics can be influenced by climate change through changes in the intensity of the flood pulse and connectivity.

Marine heatwaves are not a dominant driver of change in demersal fishes.

Marine heatwaves have been linked to negative ecological effects in recent decades1,2. If marine heatwaves regularly induce community reorganization and biomass collapses in fishes, the consequences could be catastrophic for ecosystems, fisheries and human communities3,4. However, the extent to which marine heatwaves have negative impacts on fish biomass or community composition, or even whether their effects can be distinguished from natural and sampling variability, remains unclear. We investigated the effects of 248 sea-bottom heatwaves from 1993 to 2019 on marine fishes by analysing 82,322 hauls (samples) from long-term scientific surveys of continental shelf ecosystems in North America and Europe spanning the subtropics to the Arctic. Here we show that the effects of marine heatwaves on fish biomass were often minimal and could not be distinguished from natural and sampling variability. Furthermore, marine heatwaves were not consistently associated with tropicalization (gain of warm-affiliated species) or deborealization (loss of cold-affiliated species) in these ecosystems. Although steep declines in biomass occasionally occurred after marine heatwaves, these were the exception, not the rule. Against the highly variable backdrop of ocean ecosystems, marine heatwaves have not driven biomass change or community turnover in fish communities that support many of the world's largest and most productive fisheries.

Are vertebrates constrained to two sets of paired appendages? The morphology, development, and evolution of pre-pelvic claspers in the Holocephali.

Holocephalans exhibit auxiliary appendages called pre-pelvic claspers (PPCs) that are located anterior to the pelvic fins, while pelvic claspers are pelvic fin modifications located posteriorly as modified metapterygia. Articulation points of the PPCs have not previously been imaged or evaluated in a comparative context, therefore, they may represent modified pelvic fin structures if they articulate with the propterygium. Alternatively, they could represent the only example of an independent third set of paired appendages in an extant taxon, if they articulate independently from any pelvic fin basal cartilages, challenging the current paradigm that extant jawed vertebrates are constrained to two sets of paired appendages. Two extinct groups, including Placoderms and Acanthodians, exhibit variation in the number of paired appendages, suggesting this may be a plesiomorphic trait. We evaluated PPC developmental growth rates, morphology, and articulation points in spotted ratfish (Hydrolagus Colliei, Holocephali). We also compared variation in PPC morphology among representatives of the three extant holocephalan families. Both, the pre-pelvic and pelvic claspers exhibit a dramatic surge in growth at sexual maturity, and then level off, suggesting synchronous development via shared hormonal regulation. While mature females are larger than males, pelvic fin growth and development is faster in males, suggesting a selective advantage to larger fins with faster development. Finally, microcomputed tomography scans revealed that PPCs are not modified propterygia, nor do they articulate with the propterygium. They articulate with the anterior pre-pelvic process on the anterior puboischiadic bar (or pelvic girdle), suggesting that while they are associated with the pelvic girdle, they may indeed represent a third, independent set of paired appendages in extant holocephalans.