Genomic Differences Between the Sexes in a Fish Species Seen Through Satellite DNAs

Neotropical fishes have highly diversified karyotypic and genomic characteristics and present many diverse sex chromosome systems, with various degrees of sex chromosome differentiation. Knowledge on their sex-specific composition and evolution, however, is still limited. Satellite DNAs (satDNAs) are tandemly repeated sequences with pervasive genomic distribution and distinctive evolutionary pathways, and investigating satDNA content might shed light into how genome architecture is organized in fishes and in their sex chromosomes. The present study investigated the satellitome of Megaleporinus elongatus, a freshwater fish with a proposed Z1Z1Z2Z2/Z1W1Z2W2 multiple sex chromosome system that encompasses a highly heterochromatic and differentiated W1 chromosome. The species satellitome comprises of 140 different satDNA families, including previously isolated sequences and new families found in this study. This diversity is remarkable considering the relatively low proportion that satDNAs generally account for the M. elongatus genome (around only 5%). Differences between the sexes in regards of satDNA content were also evidenced, as these sequences are 14% more abundant in the female genome. The occurrence of sex-biased signatures of satDNA evolution in the species is tightly linked to satellite enrichment associated with W1 in females. Although both sexes share practically all satDNAs, the overall massive amplification of only a few of them accompanied the W1 differentiation. We also investigated the expansion and diversification of the two most abundant satDNAs of M. elongatus, MelSat01-36 and MelSat02-26, both highly amplified sequences in W1 and, in MelSat02-26’s case, also harbored by Z2 and W2 chromosomes. We compared their occurrences in M. elongatus and the sister species M. macrocephalus (with a standard ZW sex chromosome system) and concluded that both satDNAs have led to the formation of highly amplified arrays in both species; however, they formed species-specific organization on female-restricted sex chromosomes. Our results show how satDNA composition is highly diversified in M. elongatus, in which their accumulation is significantly contributing to W1 differentiation and not satDNA diversity per se. Also, the evolutionary behavior of these repeats may be associated with genome plasticity and satDNA variability between the sexes and between closely related species, influencing how seemingly homeologous heteromorphic sex chromosomes undergo independent satDNA evolution.

The critical role of natural history museums in advancing eDNA for biodiversity studies: a case study with Amazonian fishes

Ichthyological surveys have traditionally been conducted using whole-specimen, capture-based sampling with varied but conventional fishing gear. Recently, environmental DNA (eDNA) metabarcoding has emerged as a complementary, and possible alternative, approach to whole-specimen methodologies. In the tropics, where much of the diversity remains undescribed, vast reaches continue unexplored, and anthropogenic activities are constant threats; there have been few eDNA attempts for ichthyological inventories. We tested the discriminatory power of eDNA using MiFish primers with existing public reference libraries and compared this with capture-based methods in two distinct ecosystems in the megadiverse Amazon basin. In our study, eDNA provided an accurate snapshot of the fishes at higher taxonomic levels and corroborated its effectiveness to detect specialized fish assemblages. Some flaws in fish metabarcoding studies are routine issues addressed in natural history museums. Thus, by expanding their archives and adopting a series of initiatives linking collection-based research, training and outreach, natural history museums can enable the effective use of eDNA to survey Earth’s hotspots of biodiversity before taxa go extinct. Our project surveying poorly explored rivers and using DNA vouchered archives to build metabarcoding libraries for Neotropical fishes can serve as a model of this protocol.

Hyperspectral data as a biodiversity screening tool can differentiate among diverse Neotropical fishes

Hyperspectral data encode information from electromagnetic radiation (i.e., color) of any object in the form of a spectral signature; these data can then be used to distinguish among materials or even map whole landscapes. Although hyperspectral data have been mostly used to study landscape ecology, floral diversity and many other applications in the natural sciences, we propose that spectral signatures can be used for rapid assessment of faunal biodiversity, akin to DNA barcoding and metabarcoding. We demonstrate that spectral signatures of individual, live fish specimens can accurately capture species and clade-level differences in fish coloration, specifically among piranhas and pacus (Family Serrasalmidae), fishes with a long history of taxonomic confusion. We analyzed 47 serrasalmid species and could distinguish spectra among different species and clades, with the method sensitive enough to document changes in fish coloration over ontogeny. Herbivorous pacu spectra were more like one another than they were to piranhas; however, our method also documented interspecific variation in pacus that corresponds to cryptic lineages. While spectra do not serve as an alternative to the collection of curated specimens, hyperspectral data of fishes in the field should help clarify which specimens might be unique or undescribed, complementing existing molecular and morphological techniques.

The critical role of natural history museums in advancing eDNA for biodiversity studies: a case study with Amazonian fishes

Ichthyological surveys have traditionally been conducted using whole specimen capture-based sampling with varied, but conventional fishing gear. Recently, environmental DNA (eDNA) metabarcoding has emerged as a complementary, and possible alternative, approach to whole specimen approaches. In the tropics, where much of the diversity remains undescribed, vast reaches remain unexplored, and anthropogenic activities are constant threats; there have been few eDNA attempts for ichthyological inventories. We tested the discriminatory power of eDNA using the 12S rRNA MiFish primers with existing public reference libraries and compared this with capture-based methods in two distinct ecosystems in the megadiverse Amazon basin. eDNA provided an accurate snapshot of the fishes at higher taxonomic levels and corroborated the effectiveness of eDNA to detect specialized fish assemblages. Some flaws in fish metabarcoding studies are routine issues addressed in natural history museums. Thus, by expanding their archives to include eDNA and adopting a series of initiatives linking collection-based research, training and outreach, natural history museums can enable the effective use of eDNA to survey Earth′s hotspots of biodiversity before taxa go extinct. Our project surveying poorly explored rivers and using DNA vouchered archives to build metabarcoding libraries for Neotropical fishes can serve as a model of this protocol.

Challenges in assessing the Neotropical fishes using DNA metabarcoding

Species richness is a metric of biodiversity usually used in fish community assessment for monitoring programs. This metric is often obtained using traditional fisheries methods that rely on capture of target organisms, resulting in underestimation of fish species. DNA metabarcoding has been recognized as a powerful noninvasive alternative tool for fish biomonitoring and management. Despite the increasing popularity of this method for the assessment of aquatic megadiverse ecosystems, its implementation for studying the highly diverse Neotropical ichthyofauna still presents some challenges. One of them is to devise what primer set could reliably amplify the DNA of all fish species from a megadiverse river basin and have enough resolution to identify them. In order to identify and overcome these drawbacks, we have investigated the efficiency of the metabarcoding approach on Neotropical fishes using a mock sample containing genomic DNA of 18 fish species from the Jequitinhonha River basin, Eastern Brazil. We compared three primer sets targeting the 12S rRNA gene: two universal and widely used markers for fish metabarcoding [MiFish (~170bp) and Teleo_1 (~60bp)], and NeoFish (~190bp), recently developed by our research group specifically for the identification of Neotropical fishes (Milan et al., 2020). Two samples amplified using three primers were sequenced in a single multiplexed Illumina MiniSeq run, using normalized and non-normalized pools. Bioinformatic analyses were performed using a DADA2/Phyloseq based pipeline to perform filtering steps and to assign Amplicon Sequence Variants (ASVs). We used a custom 12S reference sequence database that included 190 specimens representing 101 species and 70 genera from the Jequitinhonha and São Francisco river basins. A total of 187 ASVs were recovered: 79, 66 and 42 for NeoFish, MiFish and Teleo_1, respectively. ASVs of unexpected species were identified for both pools (Fig. 1), though each of these ASVs had an abundance of less than 50 copies. In addition, species of the Hoplias and Prochilodus genera could not be identified at the species level, due to identical sequences within each genus, possibly because of the insufficient variation within the 12S region recovered by these primers’ amplicons. Unexpectedly, although a single individual of each species was placed in the pools, more than one ASV was identified for some species, likely caused by PCR biases. Overall, all primer sets displayed similar taxonomic resolution for the DNA pools and recovered all species, except for NeoFish, which could not detect Steindachneridion amblyurum due to an incompatibility in the 3’ of the NeoFish forward primer and Teleo_1, which could not identify Steindachnerina elegans. These results highlight the need of reliable databases in order to enable the full assignment of ASVs and OTUs to species level, and the importance of calibrating the DNA metabarcoding approach with mock samples to identify weaknesses and pivotal steps prior to the application on large scale DNA based biodiversity evaluation, that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Radiotagging a long-distance migratory characid fish: reproduction after surgery, tag losses, and effects in weight

Although biotelemetry studies bring useful information, tagging is a highly invasive procedure. For this reason, we assess if intracoelomic tagging alters survivorship, weight gain and/or reproductive capacity of a neotropical migratory fish. In August 2016, 60 fish were equally and randomly distributed among 3 treatments: Control (anesthesia), sham surgery (anesthesia and surgery), and true surgery (anesthesia, surgery and tagging). Surveys for weight and tag expulsion were performed periodically from surgery through spawning (November/2016), with a final survey occurring in May/2017. Mortality was higher in true surgery (25% died) and for males. Twenty percent expelled tags, and initial weight loss followed by gain were verified for all treatments. Females of all treatments spawned, and fecundity and fertility were not different. Then, future studies should consider the losses due to death and tag expulsion when defining the sample number. We also encourage further investigations about differences in mortality between sex and weight variation, as well as any other factor that leads to increase in mortality. This is the first study that investigated tagging effects in reproduction of neotropical fishes, and since the methods and tags used are not species-specific, we infer that similar effects could occur in other species.

Cross species amplification and characterization of microsatellite markers in Leporinus friderici (Characiformes, Anostomidae)

ABSTRACT: Leporinus friderici is a migratory neotropical fish with elevated ecological and economic importance in Brazil. Microsatellite markers are highly important in population genetic studies, management, and conservation programs; however, no markers are available for this species. In this study, seven microsatellite loci, previously developed for Megaleporinus obtusidens, were successfully cross-amplified in L. friderici. Among these loci, five presented moderate to high genetic variability levels, with four to seven alleles per loci and expected heterozygosities varying from ≥ 0.574 to 1.000. These markers represent a valuable tool for the future management and ecological studies involving this species and group of neotropical fishes.

First fossil of the Giant Armored Catfish Acanthicus (Siluriformes, Loricariidae) from the Late Miocene of Entre Ríos Province, Argentina

Loricariidae is a very diverse lineage of Neotropical fishes, being the most speciose family of the order Siluriformes. However, the loricariid fossil record is still very sparse. The aim of the present contribution is to describe in detail several bones belonging to the loricariid genus Acanthicus coming from late Miocene beds located at the Paraná River cliffs, Entre Ríos province, Argentina. Fishes of the Acanthicus clade are currently restricted to the northern half of South America, being mainly distributed in the Orinoco and Amazonas basins, with a single genus reaching the Paraná-Plata basin. Acanthicus adds to the list of several taxa that are shared by the Miocene of Paraná, Amazonas and Orinoco basins, but that nowadays are absent in the former (e.g., Colossoma, Phractocephalus, and Brachyplatystoma), and are typical of large river channels. This report of Acanthicus supports a close affinity between freshwater faunas of the Paraná, Orinoco, and Amazonas basins. Miocene fossils of freshwater fishes recorded in Paraná beds shed light on the connections between ancient basins of South America and also indicate that several fish clades suffered regional extinctions during the late Neogene or the beginning of the Quaternary.

Reproductive Biology of Neotropical Fishes: A Guide to Identification to the Gonadal Morphology During the Reproductive Cycle of Catfish Rhamdia quelen (Siluriformes: Heptapteridae)

In most Teleostei fish, the gametogenesis is a cyclical and seasonal event. The renewal of gametes, through their differentiation, development, maturation and release causes several changes in the morphological characteristics of the ovaries and testes throughout the annual reproductive cycles. These alterations are used to recognize different phases in the reproductive cycles. However, as the number of studies of fish reproduction increased, the number of types of gonadal classification and nomenclatures also diversified. This may make it difficult to communication between researchers and the aquaculture activity, since the recognition of these phases is one of the most important parameters applied in the management of fishery resources. In addition, the terminologies proposed in most of the current studies refer to reproductive stages applicable to marine fish, with marked and defined seasonality characteristics. In this way, this report presents a recent proposal for the recognition of reproductive phases originally developed for the Perciformes and here adapted for the Neotropical Siluriformes, using as a biological model a freshwater catfish Rhamdia quelen. In addition, we describe a brief characterization of the gametogenesis in this species. We hope that the material presented can be used as an easy and practical guide of identification for reproductive phases applicable to other neotropical freshwater fish, more especially Otophysi, such as Siluriformes.

New Insights into the Distribution, Physiology and Life Histories of South American Galaxiid Fishes, and Potential Threats to This Unique Fauna

South American galaxiids occupy both Patagonia and the ichthyogeographic Chilean Province, encompassing glacial Andean deep lakes, shallow plateau lakes, reservoirs, short Pacific rivers and long Atlantic rivers. The total fish fauna includes 29 species, comprising Neotropical fishes (siluriforms and characids), galaxiids, percichthyids, atherinopsids and mugilids, two lamprey species, and several exotic fishes (salmonids, Gambusia spp. and common carp). The family Galaxiidae shares a common ancestry with the Gondwanan temperate fish fauna, played a major role in the post-glacial colonization of Andean lakes and streams, and contributes key species to the food webs. Galaxiid species occupy an enormous latitudinal gradient, show a wide variety of life history patterns and are the southernmost native freshwater fishes of the world. Knowledge of South American galaxiids has improved notably, but new challenges arise due to climate change, biological invasions, damming, aquaculture and contamination. In this changing environment, the future of South American galaxiids should be carefully considered as a legacy of the old Gondwana and a unique attribute of the freshwaters of southern South America.