Historical Field Records Reveal Habitat as an Ecological Correlate of Locomotor Phenotypic Diversity in the Radiation of Neotropical Geophagini Fishes.

AbstractPhenotypic macroevolutionary studies provide insight into how ecological processes shape biodiversity. However, the complexity of phenotype-ecology relationships underscores the importance of also validating phenotype-based ecological inference with direct evidence of resource use. Unfortunately, macroevolutionary-scale ecological studies are often hindered by the challenges of acquiring taxonomically and spatially representative ecological data for large and widely distributed clades. The South American cichlid fish tribe Geophagini represents a continentally distributed radiation whose early locomotor morphological divergence suggests habitat as one ecological correlate of diversification, but an association between locomotor traits and habitat preference has not been corroborated. Field notes accumulated over decades of collecting across South America provide firsthand environmental records that can be mined for habitat data in support of macroevolutionary ecological research. In this study, we applied a newly developed method to transform descriptive field note information into quantitative habitat data and used it to assess habitat preference and its relationship to locomotor morphology in Geophagini. Field note-derived data shed light on geophagine habitat use patterns and reinforced habitat as an ecological correlate of locomotor morphological diversity. Our work emphasizes the rich data potential of museum collections, including often-overlooked material such as field notes, for evolutionary and ecological research.

A Theoretical Comparison of Alternative Male Mating Strategies in Cephalopods and Fishes.

We used computer simulations of growth, mating and death of cephalopods and fishes to explore the effect of different life-history strategies on the relative prevalence of alternative male mating strategies. Specifically, we investigated the consequences of single or multiple matings per lifetime, mating strategy switching, cannibalism, resource stochasticity, and altruism towards relatives. We found that a combination of single (semelparous) matings, cannibalism and an absence of mating strategy changes in one lifetime led to a more strictly partitioned parameter space, with a reduced region where the two mating strategies co-exist in similar numbers. Explicitly including Hamilton's rule in simulations of the social system of a Cichlid led to an increase of dominant males, at the expense of both sneakers and dwarf males ("super-sneakers"). Our predictions provide general bounds on the viable ratios of alternative male mating strategies with different life-histories, and under possibly rapidly changing ecological situations.

Endoparasitic helminth fauna and diet of Geophagus sveni (Pisces) in Upper Paraná River basin.

In freshwater ecosystems, parasite infection patterns are influenced by factors including spatial-temporal variations, host diet, and habitat. Fish often change diets, affecting their parasite communities. This study focused on non-native host fish Geophagus sveni, aiming to characterize diet and endoparasitic helminth fauna patterns in the invaded area, investigating spatial and seasonal possible differences of endoparasite infections and correlating with host diet, in São José dos Dourados River and Tietê River areas. The host fish were collected in these areas during the dry and rainy season using gillnets. The endoparasites were collected and preserved in alcohol and identified using taxonomic methods, and stomach contents were examined for diet analysis. Parasitism descriptors were calculated and evaluated spatially and seasonally by ANOVA and the Kruskal-Wallis tests. PERMANOVA assessed G. sveni diet differences, and RDA correlated the endohelminth abundance with the host diet. Two endoparasites were recorded: metacercariae of Austrodiplostomum compactum (Trematoda) and larvae and adults of Raphidascaris (Sprentascaris) lanfrediae (Nematoda). Spatial differences were observed for the mean abundance and prevalence of R. (S.) lanfrediae and A. compactum prevalence. Seasonal variations of parasitic descriptors occurred for the nematode in the Tietê River area. The detritus and aquatic insects were the most consumed items by G. sveni. Detritus consumption positively correlates with nematode abundance. The findings indicate that factors such as artificial channels and rainfall, which can influence resource availability, may affect the fish's diet and potentially influence the structure of its endoparasite community. The study emphasizes the importance of understanding trophic chain-transmitted parasites and calls for further research in Neotropical environments.

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.

Distinct genetic origins of eumelanin intensity and barring patterns in cichlid fishes.

Pigment patterns are incredibly diverse across vertebrates and are shaped by multiple selective pressures from predator avoidance to mate choice. A common pattern across fishes, but for which we know little about the underlying mechanisms, is repeated melanic vertical bars. In order to understand genetic factors that modify the level or pattern of vertical barring, we generated a genetic cross of 322 F2 hybrids between two cichlid species with distinct barring patterns, Aulonocara koningsi and Metriaclima mbenjii. We identify 48 significant quantitative trait loci that underlie a series of seven phenotypes related to the relative pigmentation intensity, and four traits related to patterning of the vertical bars. We find that genomic regions that generate variation in the level of eumelanin produced are largely independent of those that control the spacing of vertical bars. Candidate genes within these intervals include novel genes and those newly-associated with vertical bars, which could affect melanophore survival, fate decisions, pigment biosynthesis, and pigment distribution. Together, this work provides insights into the regulation of pigment diversity, with direct implications for an animal's fitness and the speciation process.

Gene expression patterns associated with caudal fin shape in the cichlid Lamprologus tigripictilis.

Variation in fin shape is one of the most prominent features of morphological diversity among fish. Regulation of fin growth has mainly been studied in zebrafish, and it is not clear whether the molecular mechanisms underlying shape variation are equally diverse or rather conserved across species. In the present study, expression levels of 37 candidate genes were tested for association with fin shape in the cichlid fish Lamprologus tigripictilis. The tested genes included members of a fin shape-associated gene regulatory network identified in a previous study and novel candidates selected within this study. Using both intact and regenerating fin tissue, we tested for expression differences between the elongated and the short regions of the spade-shaped caudal fin and identified 20 genes and transcription factors (including angptl5, cd63, csrp1a, cx43, esco2, gbf1, and rbpj), whose expression patterns were consistent with a role in fin growth. Collated with available gene expression data of two other cichlid species, our study not only highlights several genes that were correlated with fin growth in all three species (e.g., angptl5, cd63, cx43, and mmp9), but also reveals species-specific gene expression and correlation patterns, which indicate considerable divergence in the regulatory mechanisms of fin growth across cichlids. Supplementary Information: The online version contains supplementary material available at 10.1007/s10750-022-05068-4.

The cichlid pharyngeal jaw novelty enhances evolutionary integration in the feeding apparatus.

The modified pharyngeal jaw system of cichlid fishes is widely viewed as a key innovation that substantially facilitated the evolutionary exuberance of this iconic evolutionary radiation. We conduct comparative phylogenetic analyses of integration, disparity, and rate of evolution among feeding-related, skeletal structures in Neotropical cichlids and North American centrarchids, which lack the specialized pharyngeal jaw. Contrasting evolutionary patterns in these two continental radiations, we test a classic decoupling hypothesis. Specifically, we ask whether the modified pharyngeal jaw in cichlids resulted in enhanced evolutionary independence of the oral and pharyngeal jaws, leading to increased diversity of trophic structures. Contrary to this prediction, we find significantly stronger evolutionary integration between the oral and pharyngeal jaws in cichlids compared to centrarchids, although the two groups do not differ in patterns of integration within each jaw system. Further, though we find no significant differences in disparity, centrarchids show faster rates of morphological evolution. Our results suggest that the modified pharyngeal jaw resulted in less evolutionary independence and slower rates of evolution within the feeding system. Thus, we raise the possibility that the cichlid novelty enhances feeding performance, but does not prompt increased morphological diversification within the feeding apparatus, as has long been thought.

Variation in parasite infection between replicates of speciation in Lake Victoria cichlid fish.

Because of potentially strong eco-evolutionary interactions with their hosts, parasites may initiate or enhance host diversification. The adaptive radiation of cichlid fish in Lake Victoria provides a good system to study the role of parasites at different stages of host speciation. We analyzed the macroparasite infection of four replicates of sympatric blue and red Pundamilia species pairs that vary in their age and extent of differentiation. Sympatric host species differed in parasite community composition and in the infection levels of some of these parasite taxa. Most infection differences were consistent between sampling years, indicating temporal consistency in parasite-mediated divergent selection between species. Infection differentiation increased linearly with genetic differentiation. However, significant infection differences between sympatric species were only found in the oldest, most strongly differentiated Pundamilia species pair. This is inconsistent with parasite-driven speciation. Next, we identified five distinct species of Cichlidogyrus, a genus of highly specific gill parasites that has radiated elsewhere in Africa. Infection profiles of species of Cichlidogyrus differed between sympatric cichlid species only in the oldest and most differentiated pair, again inconsistent with parasite-mediated speciation. To conclude, parasites may contribute to host differentiation after speciation, but do not initiate host speciation.

Genetic basis of ecologically relevant body shape variation among four genera of cichlid fishes.

Divergence in body shape is one of the most widespread and repeated patterns of morphological variation in fishes and is associated with habitat specification and swimming mechanics. Such ecological diversification is the first stage of the explosive adaptive radiation of cichlid fishes in the East African Rift Lakes. We use two hybrid crosses of cichlids (Metriaclima sp. × Aulonocara sp. and Labidochromis sp. × Labeotropheus sp., >975 animals total) to determine the genetic basis of body shape diversification that is similar to benthic-pelagic divergence across fishes. Using a series of both linear and geometric shape measurements, we identified 34 quantitative trait loci (QTL) that underlie various aspects of body shape variation. These QTL are spread throughout the genome, each explaining 3.2-8.6% of phenotypic variation, and are largely modular. Further, QTL are distinct both between these two crosses of Lake Malawi cichlids and compared to previously identified QTL for body shape in fishes such as sticklebacks. We find that body shape is controlled by many genes of small effect. In all, we find that convergent body shape phenotypes commonly observed across fish clades are most likely due to distinct genetic and molecular mechanisms.

Effects of temperature on metabolic rate and lower dissolved oxygen tolerance of juvenile speckled peacock bass Cichla temensis.

The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO2  kg-1 h-1 ), followed by 30°C (208.16 ± 12.45 mgO2  kg-1 h-1 ) and 25°C (144.09 ± 10.43 mgO2  kg-1 h-1 ). Correspondingly, the Q10 , or rate of increase in aerobic metabolic rate (MO2 ) relative to 10°C, for 30-35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO2 of 90 mmHg (4.13 mg l-1 ) at 35°C compared to pO2 values of 45 mmHg (2.22 mg l-1 ) and 30 mmHg (1.61 mg l-1 ) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25-30°C.

Comparison of seven complete mitochondrial genomes from Lamprologus and Neolamprologus (Chordata, Teleostei, Perciformes) and the phylogenetic implications for Cichlidae.

In this study, mitochondrial genomes (mitogenomes) of seven cichlid species (Lamprologuskungweensis, L.meleagris, L.ornatipinnis, Neolamprologusbrevis, N.caudopunctatus, N.leleupi, and N.similis) are characterized for the first time. The newly sequenced mitogenomes contained 37 typical genes [13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs) and 22 transfer RNA genes (tRNAs)]. The mitogenomes were 16,562 ~ 16,587 bp in length with an A + T composition of 52.1~58.8%. The cichlid mitogenomes had a comparable nucleotide composition, A + T content was higher than the G + C content. The AT-skews of most mitogenomes were inconspicuously positive and the GC-skews were negative, indicating higher occurrences of C than G. Most PCGs started with the conventional start codon, ATN. There was no essential difference in the codon usage patterns of these seven species. Using Ka/Ks, we found the fastest-evolving gene were atp8. But the results of p-distance indicated that the fastest-evolving gene was nad6. Phylogenetic analysis revealed that L.meleagris did not cluster with Lamprologus species, but with species from the genus Neolamprologus. The novel information obtained about these mitogenomes will contribute to elucidating the complex relationships among cichlid species.

Ecomorphology and Morphological Disparity of Caquetaia Kraussii (Perciformes: Cichlidae) in Colombia.

Understanding the interspecific morphological variability of Caquetaia kraussii (Perciformes: Cichlidae) between different localities in its distribution range is becoming essential, as this species constitutes a valuable resource for the economy and subsistence of the local human communities where it is endemic in Colombia and Venezuela. In order to develop efficient farming and handling plans for this species, a deep understanding of the factors and mechanisms generating morphological variability is crucial. This study analyzes the morphological variability of C. kraussii by using geometric morphometrics in four localities distributed between the Dique and North channels, which are part of the Bolívar department in Colombia. Likewise, the effect of environmental variables such as temperature (T°), dissolved oxygen (OD) and pH on morphological variability was analyzed using a partial least squares approach. The results show that environmental stress has an influence on ~10% of the body shape of C. kraussii, whereas ~90% of the body shape is not directly influenced by environmental parameters, suggesting an effect from stress related to sexual dimorphism. Similarly, the analyses show shape variation among localities, mainly between populations of lotic environments and those of lentic environments. This morphological disparity seems to be subject to environmental and sexual stresses in the different localities.

Fish gut-associated bacterial communities in a tropical lagoon (Aghien lagoon, Ivory Coast).

Aghien lagoon (Ivory Coast) is a eutrophic freshwater lagoon that harbors high biomasses of phytoplankton. Despite Increasing interest in fish gut microbiomes diversity and functions, little data is currently available regarding wild species from tropical west African lakes. Here, gut-associated bacterial communities are investigated in four fish species that are consumed by locale populations, namely the Cichlidae Hemichromis fasciatus, Tilapia guineensis and Sarotherodon melanotheron, and the Claroteidae Chrysichthys nigrodigitatus. Species-related differences are identified, that can be attributed to host phylogeny and diet. Important variations throughout the year are observed in T. guineensis and C. nigrodigitatus. This result emphasized the importance of time-series sampling and comparison with environmental variables even in tropical regions, that are not often conducted in wild populations. Effects of environmental factors (anthropogenic or not) on the microbiota and potential outcomes for fish health and populations sustainability need to be further explored. Interestingly, fish appear as major reservoirs of bacterial diversity, suggesting that they could contribute to the overall stability and resilience of bacterial communities present in the Aghien lagoon.

A new species of Geophagus (Teleostei: Cichlidae): Naming a cichlid species widely known in the aquarium hobby as 'Geophagus sp. Tapajos red head'.

A new species of Geophagus sensu stricto is described from the Tapajos River basin, Brazil, elevating the number of species of the genus to 21. The new species is of commercial importance and is known in the aquarist trade as Geophagus 'red head'. The new species is diagnosed using an integrative approach, based on mitochondrial DNA analysis along with morphological evidence. The new species is distinguished from all congeners by the absence of markings on the head, the bar pattern composed by nine vertical bars on the flanks and the presence of distinct longitudinal bands in the caudal fin. Additionally, it shows a genetic distance of at least 2.0% in cytochrome b gene sequences from its closest congeners. Molecular analysis including most genera of Cichlidae from South America corroborates that the new species belongs to the group of Geophagus sensu stricto.

A new species of deep-water Lethrinops (Cichlidae) from Lake Malawi.

A new species of cichlid fish, Lethrinops atrilabris is described from specimens collected by trawling at a depth of around 90 m off Monkey Bay, southern Lake Malawi. It is assigned to the genus Lethrinops on the basis of its vertical flank barring, lack of enlarged cephalic lateral line canal pores and the form of the lower jaw dental arcade. It can be distinguished from congeneric species by its male breeding dress of contrasting flank barring and dark ventral surface, most strikingly on the lips, throat and chest, its relatively small known maximum size [<75 mm standard length (SL)], large eyes (38%-41% head length), laterally compressed body (depth 2.5-2.7 times max head width) and lower gill raker count (13-14).

Comparative ontogenetic and transcriptomic analyses shed light on color pattern divergence in cichlid fishes.

Stripe patterns are a striking example for a repeatedly evolved color pattern. In the African adaptive radiations of cichlid fishes, stripes evolved several times independently. Previously, it has been suggested that regulatory evolution of a single gene, agouti-related-peptide 2 (agrp2), explains the evolutionary lability of this trait. Here, using a comparative transcriptomic approach, we performed comparisons between (adult) striped and nonstriped cichlid fishes of representatives of Lake Victoria and the two major clades of Lake Malawi (mbuna and non-mbuna lineage). We identify agrp2 to be differentially expressed across all pairwise comparisons, reaffirming its association with stripe pattern divergence. We therefore also provide evidence that agrp2 is associated with the loss of the nonstereotypic oblique stripe of Mylochromis mola. Complementary ontogenetic data give insights into the development of stripe patterns as well as vertical bar patterns that both develop postembryonically. Lastly, using the Lake Victoria species pair Haplochromis sauvagei and Pundamilia nyererei, we investigated the differences between melanic and non-melanic regions to identify additional genes that contribute to the formation of stripes. Expression differences-that most importantly also do not include agrp2-are surprisingly small. This suggests, at least in this species pair, that the stripe phenotype might be caused by a combination of more subtle transcriptomic differences or cellular changes without transcriptional correlates. In summary, our comprehensive analysis highlights the ontogenetic and adult transcriptomic differences between cichlids with different color patterns and serves as a basis for further investigation of the mechanistic underpinnings of their diversification.

Gill monogeneans of neotropical cichlid fish: diversity, phylogenetic relationships, and host-parasite cophylogenetic associations.

Host-parasite coevolution is one of the main topics of the evolutionary biology of host-parasite associations. The majority of monogeneans parasitizing fish exhibit a high degree of host specificity. As a result, their evolutionary history might be intertwined with that of their fish hosts. The Cichlidae represent a diverse group of secondary freshwater fish with disjunctive distribution. Host-specific dactylogyrid monogeneans commonly parasitize cichlid fish. Their high diversity is associated with the main areas of cichlid distribution, i.e., Neotropical America and Africa. Nevertheless, the parasite fauna of cichlids from Neotropical America is still underexplored. A total of 31 cichlid species were examined for the presence of monogeneans, with 20 of them being parasitized. On these cichlids, 30 monogeneans belonging to the genera Gussevia, Trinidactylus, and Scadicleithrum were identified, 17 of them potentially representing new species for science. Phylogenetic analyses revealed three monophyletic groups of Neotropic cichlid monogeneans. Genus Gussevia was monophyletic, while Sciadicleithrum resulted polyphyletic. Sciedicleithrum from South America and Sciadicleithrum from Mexico represented two divergent lineages. The plesiomorphic Neotropical cichlid host group for dactylogyrid monogeneans was Cichlini, from which the representatives of other Neotropical cichlid tribes were colonised. Cophylogenetic analyses revealed a statistically significant cophylogenetic signal in the investigated host-parasite system, with host switch and duplication representing the main coevolutionary events for monogeneans parasitizing Neotropical cichlids. This scenario is in accordance with previous studies focussed on dactylogyridean monogeneans parasitizing freshwater fish in Europe and Africa.

Explosive networking: The role of adaptive host radiations and ecological opportunity in a species-rich host-parasite assembly.

Many species-rich ecological communities emerge from adaptive radiation events. Yet the effects of adaptive radiation on community assembly remain poorly understood. Here, we explore the well-documented radiations of African cichlid fishes and their interactions with the flatworm gill parasites Cichlidogyrus spp., including 10,529 reported infections and 477 different host-parasite combinations collected through a survey of peer-reviewed literature. We assess how evolutionary, ecological, and morphological parameters determine host-parasite meta-communities affected by adaptive radiation events through network metrics, host repertoire measures, and network link prediction. The hosts' evolutionary history mostly determined host repertoires of the parasites. Ecological and evolutionary parameters predicted host-parasite interactions. Generally, ecological opportunity and fitting have shaped cichlid-Cichlidogyrus meta-communities suggesting an invasive potential for hosts used in aquaculture. Meta-communities affected by adaptive radiations are increasingly specialised with higher environmental stability. These trends should be verified across other systems to infer generalities in the evolution of species-rich host-parasite networks.

Morphological characters in light of new molecular phylogenies: the caudal-fin skeleton of Ovalentaria.

The Ovalentaria is a taxon of teleosts that has been proposed based on molecular analyses only. Previously widely separated families are assembled in this taxon. For the first time, the Ovalentaria are analysed using a comparative morphological approach. The caudal-fin skeleton of 355 species covering all 48 ovalentarian families are examined in cleared and stained specimens, µCT datasets and X-ray images as well as from the literature. A total of 38 morphological characters are evaluated and used for ancestral character state reconstructions and phylogenetic analyses. Results provide hypotheses for a scenario of the evolution of the caudal-fin skeleton and its ground plan in Ovalentaria. An evolutionary trend towards the reduction of skeletal elements in the caudal fin is observed. Connections between the evolution of the caudal-fin skeleton and modes of locomotion found in ovalentarian taxa are discussed. Phylogenetic analyses based on the caudal-fin morphology provide topologies for intra-ovalentarian relationships that largely agree with molecular hypotheses.

Benefits and limitations of a new genome-based PCR-RFLP genotyping assay (GB-RFLP): A SNP-based detection method for identification of species in extremely young adaptive radiations.

High-throughput DNA sequencing technologies make it possible now to sequence entire genomes relatively easily. Complete genomic information obtained by whole-genome resequencing (WGS) can aid in identifying and delineating species even if they are extremely young, cryptic, or morphologically difficult to discern and closely related. Yet, for taxonomic or conservation biology purposes, WGS can remain cost-prohibitive, too time-consuming, and often constitute a "data overkill." Rapid and reliable identification of species (and populations) that is also cost-effective is made possible by species-specific markers that can be discovered by WGS. Based on WGS data, we designed a PCR restriction fragment length polymorphism (PCR-RFLP) assay for 19 Neotropical Midas cichlid populations (Amphilophus cf. citrinellus), that includes all 13 described species of this species complex. Our work illustrates that identification of species and populations (i.e., fish from different lakes) can be greatly improved by designing genetic markers using available "high resolution" genomic information. Yet, our work also shows that even in the best-case scenario, when whole-genome resequencing information is available, unequivocal assignments remain challenging when species or populations diverged very recently, or gene flow persists. In summary, we provide a comprehensive workflow on how to design RFPL markers based on genome resequencing data, how to test and evaluate their reliability, and discuss the benefits and pitfalls of our approach.