First Look at Lutefish Chromosomes Reshapes Ideas of Characin Genome Evolution

For decades, one of the most species-rich branches of the fish tree of life has kept a curious secret: nobody had ever looked at the chromosomes of an Afrotropical lutefish. That gap has now been closed. In a study published in the journal Comparative Cytogenetics, researchers report the first karyotypes — complete chromosome inventories — for two species of the genus Citharinus, members of the family Citharinidae, a small but evolutionarily pivotal group of African freshwater fishes. The findings, though based on just two species, are already prompting a rethink of how chromosome numbers evolved across the entire order Characiformes, the sprawling group that includes tetras, piranhas, and their many relatives.

The order Characiformes is divided into two suborders with very different biogeographic stories. Citharinoidei is exclusively Afrotropical and contains only two families: the Citharinidae, with eight species in three genera, and the Distichodontidae, with 108 species in 16 genera. Characoidei, by contrast, is far larger, encompassing more than 2,000 species in two Afrotropical families and twenty Neotropical ones. Both suborders are considered monophyletic — each descends from a single common ancestor — although some molecular phylogenies have questioned whether the order as a whole holds together. The two lineages are ancient: their divergence is estimated to have occurred between 115 and 145 million years ago, deep in the Cretaceous period.

Despite this evolutionary significance, cytogenetic information on Citharinoidei was almost nonexistent. Until now, only a single species out of the suborder’s 116 — the distichodontid Distichodus affinis, studied in 1998 — had ever had its chromosomes analyzed. The new study, led by researchers working with material from West and Northeast Africa, doubles that number and adds the very first data for the family Citharinidae itself. Given that Citharinoidei sits at the base of the Characiformes phylogeny, even a handful of karyotypes from this lineage carries outsized weight for reconstructing the ancestral state of the whole order.

The team karyotyped seven individuals of Citharinus citharus, a species purchased through Nigerian aquarium fish dealers, and three individuals of Citharinus latus, collected in southwestern Ethiopia by the Joint Ethiopian-Russian Biological Expedition from the Alvero River, a tributary draining into the White Nile system. Chromosome preparations were made from kidney tissue using established protocols: the tissue was treated with colchicine to arrest dividing cells in metaphase, exposed to a hypotonic potassium chloride solution to swell the cells, fixed in methanol-acetic acid, and finally dropped onto slides to spread the chromosomes for staining with Giemsa. For each fish, at least ten complete metaphase plates were examined, with 101 metaphases analyzed in total for C. citharus and 42 for C. latus.

The results were strikingly clean. Citharinus citharus carries a diploid chromosome number of 2n = 40, composed of 26 metacentric and 14 submetacentric chromosomes, giving a fundamental number of 80. Citharinus latus has 2n = 44, with 30 metacentrics and 14 submetacentrics and a fundamental number of 88. Both karyotypes consist exclusively of bi-armed chromosomes — elements with centromeres positioned so that each chromosome has two distinguishable arms. No distinguishable sex chromosomes were observed in either species, mirroring the earlier finding in Distichodus affinis. Notably, C. citharus possesses nine pairs of conspicuously large chromosomes, while C. latus has seven such pairs.

How did these two closely related species come to differ by four chromosomes? The authors outline several possible scenarios. If the karyotype of C. latus resembles the ancestral condition, two fusion events — joining four pairs of smaller chromosomes — could have produced the complement of C. citharus. Alternatively, if C. citharus is taken as ancestral, two fissions of large chromosome pairs could have generated the C. latus karyotype. A third possibility is that the two karyotypes arose independently. The closest studied relative, Distichodus affinis, has 2n = 48, while diploid numbers of 50 to 54 are typical for the phylogenetically basal groups of Characoidei. This suggests that both Citharinus karyotypes may have descended, through different numbers of chromosome fusions, from an ancestral complement with a higher diploid number than either species displays today.

The authors caution that all of these scenarios remain speculative, because cytogenetic data are still lacking for most genera and species of Citharinoidei. Nevertheless, combined with the basal position of Citharinidae within the suborder’s phylogeny and the distant placement of Distichodus affinis within it, the new data point to a substantial role for chromosome fusions and fissions in shaping karyotypes across Citharinoidei. All three cytogenetically studied species of the suborder — both Citharinus species and Distichodus affinis — share the distinctive feature of exclusively bi-armed chromosomes, though the fragmentary sampling means it is too early to declare this a suborder-wide signature.

Placing the new findings in a broader context reveals a fascinating contrast with the better-studied Characoidei. In that suborder, karyotype structure varies widely even among its most basal lineages. Exclusively bi-armed karyotypes do occur in the family Crenuchidae, which sits at the root of the Characoidei tree, and in several other families — Anostomidae, Chilodontidae, Curimatidae, and Prochilodontidae — though these are nested far from the phylogenetic base. In contrast, two clusters of families that branch close to the basal nodes show karyotypes enriched with, or even dominated by, uni-armed chromosomes: the Neotropical Ctenoluciidae and Lebiasinidae on one hand, and the Afrotropical Hepsetidae and Alestidae together with the Neotropical Erythrinidae on the other. The exceptions are the erythrinid genera Hoplerythrinus and Hoplias, whose karyotypes are almost completely bi-armed. Against this backdrop, the uniformly bi-armed complements of the basal Citharinoidei stand out as a genuinely new data point for the order.

Perhaps the most consequential implication concerns the long-standing hypothesis about the ancestral chromosome number of Characiformes itself. Based almost entirely on cytogenetic data from Characoidei, researchers had proposed that 2n = 54 was the ancestral diploid number for the entire order. But because Citharinidae is basal within Citharinoidei, and Citharinoidei is basal within all Characiformes, the new karyotypes carry special weight in ancestral-state reconstructions. The authors argue that their data raise the possibility of a lower ancestral chromosome number for the order than previously assumed, and they emphasize the need for further cytogenetic studies in the phylogenetically basal groups of both suborders to clarify how chromosome number and karyotype structure evolved across characin history.

There is also a taxonomic twist. Some recent molecular phylogenies of ray-finned fishes have proposed splitting Citharinoidei out of Characiformes entirely, elevating it to its own order, Cithariniformes, as a sister group to Characiformes plus Siluriformes — the catfishes. Under that scenario, the karyotypes of the two lineages could have evolved more independently than the traditional classification implies. Yet even in that case, the authors note, the new data together with any future cytogenetic studies of the group will help reconstruct the evolutionary history of karyotypes within a key part of the superorder Ostariophysi, the vast assemblage that also includes carps and catfishes and dominates the freshwater fish faunas of every continent. For a family of African fishes that had never yielded a single chromosome count, the lutefishes have suddenly become an important voice in one of fish cytogenetics’ oldest debates.

Subject of Research: Cytogenetics and karyotype evolution of Afrotropical lutefishes (Citharinidae) within the order Characiformes

Article Title: First cytogenetic data on Afrotropical lutefishes ( Citharinidae ) in the light of karyotype evolution in Characiformes

Article References: Simanovsky, S. A., Medvedev, D. A., Tefera, F., & Golubtsov, A. S. (2022). First cytogenetic data on Afrotropical lutefishes (Citharinidae) in the light of karyotype evolution in Characiformes. Comparative Cytogenetics, 16(2), 143-150. https://doi.org/10.3897/compcytogen.v16.i2.79133

Image Credits: AI Generated

DOI: 10.3897/compcytogen.v16.i2.79133

Keywords: Citharinidae, Citharinus, Characiformes, karyotype evolution, cytogenetics, chromosome fusions, Citharinoidei, Characoidei, freshwater fishes, Africa, diploid number, Comparative Cytogenetics