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. 2023 Jun 14;15(6):evad110. doi: 10.1093/gbe/evad110

Evolution of MHC IIB Diversity Across Cichlid Fish Radiations

Carlos Lozano-Martín 1,✉, Seraina E Bracamonte 2, Marta Barluenga 3,✉
Editor: Bonnie Fraser
PMCID: PMC10306275  PMID: 37314153

Abstract

The genes of the major histocompatibility complex (MHC) are among the most polymorphic genes in vertebrates and crucial for their adaptive immune response. These genes frequently show inconsistencies between allelic genealogies and species phylogenies. This phenomenon is thought to be the result of parasite-mediated balancing selection maintaining ancient alleles through speciation events (trans-species polymorphism [TSP]). However, allele similarities may also arise from postspeciation mechanisms, such as convergence or introgression. Here, we investigated the evolution of MHC class IIB diversity in the cichlid fish radiations across Africa and the Neotropics by a comprehensive review of available MHC IIB DNA sequence information. We explored what mechanism explains the MHC allele similarities found among cichlid radiations. Our results showed extensive allele similarity among cichlid fish across continents, likely due to TSP. Functionality at MHC was also shared among species of the different continents. The maintenance of MHC alleles for long evolutionary times and their shared functionality may imply that certain MHC variants are essential in immune adaptation, even in species that diverged millions of years ago and occupy different environments.

Keywords: adaptive radiation, major histocompatibility complex, balancing selection, convergent evolution, functional supertypes

Significance.

Cichlid fish are one of the most diverse groups of vertebrates, with elevated genetic diversity in a key component of the immune system, the genes of the major histocompatibility complex (MHC). We report extensive MHC allele similarities across cichlids on different continents that have prevailed over millions of years, predating divergence. This process has also resulted in different alleles having similar functions across radiations.

Introduction

Host–parasite interactions promote genetic diversity within and among host populations (Haldane 1949; Summers et al. 2003; Ebert and Fields 2020). Parasites impose selective pressures on their hosts that might differ across environments, affecting specifically the immune system (Acevedo-Whitehouse and Cunningham 2006; Lazzaro and Little 2009). The major histocompatibility complex (MHC) is a gene family directly involved in the activation of the adaptive immune response in jawed vertebrates (Klein 1986). Classical MHC genes are among the most polymorphic genes in vertebrates (Radwan et al. 2020). Evidence suggests that balancing selection mediated by the interaction with parasites drives the evolution of allelic diversity and sequence divergence (Piertney and Oliver 2006; Spurgin and Richardson 2010), although sexual selection has also been invoked to play a role (Milinski 2006; Ejsmond et al. 2014).

Sequence polymorphism at the MHC is mostly concentrated in the exons containing the antigen-binding sites (ABS), which are the sites that interact directly with the parasitic antigens and experience the strongest parasite-mediated selection (Wegner 2008). The antigen-binding specificity of MHC alleles is determined by the physicochemical properties of the amino acids at these sites. Alleles with similar biochemical properties are assumed to bind to similar sets of parasitic antigens, and they can be clustered into functional groups, known as supertypes (Doytchinova and Flower 2005; Schwensow et al. 2007). The presence of specific supertypes has been associated with higher resistance to parasites in fish (Smallbone et al. 2021), birds (Sepil et al. 2013), and reptiles (Hacking et al. 2018). Lighten et al. (2017) proposed that balancing selection might act to maintain supertypes rather than alleles, preserving lineages of functionally similar alleles. As a result, alleles would face rapid turnover governed by demographic processes. However, this hypothesis has been questioned (Ejsmond et al. 2018), and later studies using different species were not able to support the role of selection maintaining supertype diversity (Herdegen-Radwan et al. 2021; Talarico et al. 2021).

A remarkable evolutionary feature of MHC genes is that sometimes alleles are more similar between related species than within species, resulting in inconsistencies between MHC allelic genealogies and species phylogenies (Klein et al. 1998, 2007). When allele similarities are the product of common ancestry, this is defined as trans-species polymorphism (TSP; Klein 1987). TSP is caused by parasite-mediated balancing selection, retaining alleles longer than expected under neutral evolution (Klein 1987; Klein et al. 2007), causing MHC alleles to persist through speciation events (Klein et al. 1998). However, MHC allele similarity might also be driven by two alternative postspeciation mechanisms, convergence and introgression. Convergent evolution at the molecular level is a mechanism in which similar MHC alleles arise independently due to adaptation to equivalent selective pressures in different settings, specifically the exposure to parasites (Gustafsson and Andersson 1994; Kriener et al. 2000). Similarity patterns might also occur due to introgression of MHC alleles between hybridizing species (Wegner and Eizaguirre 2012). Convergent evolution was found as the underlying mechanism of MHC allele similarities in certain mammal species (Kriener et al. 2000; Srithayakumar et al. 2012), whereas introgression was argued as the mechanism explaining MHC allele similarities in sympatric species of newts (Nadachowska-Brzyska et al. 2012; Dudek et al. 2019). TSP is considered the mechanism that better explains MHC allele similarities and polymorphism in several vertebrate taxa (Kiemnec-Tyburczy et al. 2010; Eimes et al. 2015; Gillingham et al. 2016; Kaesler et al. 2017; Sagonas et al. 2019; reviewed in Těšický and Vinkler 2015), including teleost fish such as African cichlids (Klein et al. 1993), cyprinids (Ottová et al. 2005), sticklebacks (Lenz et al. 2013), and percids (Million and Lively 2022). Allele similarity at the MHC across species is mainly found in the exons encoding the ABS. Therefore, most studies on allele similarity, lineage sharing, and TSP across species have focused on those exons, specifically on the exon 2 of MHC IIB (reviewed in Těšický and Vinkler 2015). Distinguishing between the mechanisms driving MHC IIB similarities is challenging, and cichlids are an excellent system for the study of these mechanisms, given their enormous diversity and tendency to radiate (Salzburger 2018).

Cichlid fish (family Cichlidae) represent one of the largest groups of vertebrates, distributed in the tropical and subtropical freshwaters of Africa, America, the Middle East, India, and Madagascar (Salzburger and Meyer 2004; Salzburger 2018; fig. 1A). Cichlids have greatly diversified over the last 80 Myr, with African and American lineages separating around 60 Ma, with a long-debated dispersal across marine waters after the separation of those continents (Matschiner et al. 2020). The largest and most diverse adaptive radiations are described in the East African Great Lakes Victoria, Tanganyika, and Malawi (e.g., Salzburger and Meyer 2004; Ronco et al. 2021). Overall, cichlid adaptive radiations have resulted in an astonishing diversity of body forms, trophic adaptations, coloration patterns, and behaviors (Salzburger 2018) and have been accompanied by recurrent convergent evolution in some of these traits (Muschick et al. 2012; Burress 2015). Striking examples of the evolution of convergent phenotypes are found not only in African cichlids (Kocher et al. 1993; Muschick et al. 2012) but also in cichlid species across continents (e.g., Colombo et al. 2013). This convergence may also occur at the molecular level, including the MHC.

Fig. 1.

Fig. 1.

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(A) Map showing the approximate distribution of cichlid species worldwide. Shaded areas refer to the distribution of the different geographical lineages: India, Madagascar, Africa, and America. (B) Neighbor-Net network based on MHC class IIB exon 2 sequences from different cichlid taxa. The shapes at the tip of the branches relate to the geographic regions in the map: triangle for India, pentagon for Madagascar, circle for Africa, and square for the Neotropics.

The genes of the MHC have been characterized in different cichlid species. Several studies described MHC diversity patterns among species and populations (Klein et al. 1993; Ono et al. 1993; Málaga-Trillo et al. 1998; Figueroa et al. 2000; Hofmann et al. 2017; Bracamonte et al. 2022). This has revealed that cichlids possess a high MHC diversity, and a large number of MHC loci were estimated for the Neotropical Midas cichlid and the African tilapia, with up to 13 and 17 loci, respectively (Málaga-Trillo et al. 1998; Hofmann et al. 2017). Other studies correlated MHC variation to parasite communities assessing its potential role in promoting local adaptation, divergence, and ultimately speciation (Blais et al. 2007; Hablützel et al. 2016; Meyer et al. 2019). The MHC IIB genes of African cichlids are monophyletic (Hablützel et al. 2013), and within the African lineage, exon 2 shows a pattern consistent with TSP (Klein et al. 1993; Hablützel et al. 2013).

Here we aimed to disentangle the different mechanisms underlying the evolution of MHC IIB genes in cichlid fish radiations across continents by using exons with different properties and subjected to different selective forces. We collected available MHC class IIB exon 2 and exon 3 sequences of African and Neotropical cichlids, as well as some from Indian and Madagascar cichlids, and we analyzed MHC IIB sequence polymorphism and diversity. For exon 2, we used phylogenetic reconstructions to assess sequence similarity and lineage sharing among cichlids from different geographic regions. Focusing on cichlid species from Africa and the Neotropics, we explored whether sequence similarity was due to TSP, maintaining alleles through radiations and speciation events, or alternatively, if it was due to convergence in response to comparable selective pressures, as observed for other cichlid traits. Although introgression may explain MHC sequence similarity between closely related cichlid species, available data did not allow to test for this mechanism. We clustered exon 2 sequences into supertypes to determine the extent of putatively shared functionality between African and Neotropical species. For exon 3 sequences, a more conserved region of the gene, we performed phylogenetic reconstructions including several noncichlid teleost orders, to provide a broader framework of cichlid MHC IIB evolution. Finally, we discussed the adaptive significance of shared and exclusive MHC evolutionary lineages and functional supertypes in cichlid species from different continents.

Results

Sequence Data Set

The final MHC IIB exon 2 data set consisted of 155 unique sequences belonging to 28 cichlid taxa (tables 1 and S1, Supplementary Material online). The nucleotide alignment was trimmed to 137 bp that was covered by all sequences. This represents a partial fragment of the cichlid exon 2, which has a total length of 273 bp in both African and Neotropical cichlids (Klein et al. 1993; Hofmann et al. 2017). The alignment included 113 polymorphic sites, with five sequences from African species presenting a 3 bp deletion at position 118 of the alignment. We observed identical sequences (excluded from the final data set) among species of the same continent, but never between species from different continents. African species belonging to the same radiation (Melanochromis auratus and Maylandia zebra, Lake Malawi) and to different radiations (Cyphotilapia frontosa and Maylandia emmiltos, Lake Tanganyika and Lake Malawi, respectively) shared identical MHC IIB exon 2 sequences. For exon 3, the data set with only cichlid sequences included 40 sequences from 17 species (supplementary table S2, Supplementary Material online), and the data set including cichlids and teleost taxa had a total of 148 sequences from 13 orders and 68 taxa (supplementary table S3, Supplementary Material online). The nucleotide alignment of the exon 3 sequences was 214 bp long and presented no deletions. Taxa information, number of sequences, and GenBank accession numbers for exon 2 and exon 3 are provided in supplementary tables S1 and S3, Supplementary Material online.

Table 1.

Taxa by Region and Number of Sequences Used in MHC IIB Exon 2 Analyses

Taxon Distribution Source Number of Sequences
India
 Etroplus canarensis Karnataka Ronco et al. (2021) a 1
Madagascar
 Paratilapia polleni North Madagascar Ronco et al. (2021) a 1
Africa
 Haplochromis nyererei Lake Victoria Figueroa et al. (2000)
Brawand et al. (2014) a 		7
 Haplochromis cinctus Lake Victoria Figueroa et al. (2000) 2
 Haplochromis xenognathus Lake Victoria Figueroa et al. (2000) 1
 Haplochromis velifer Lake Nabugabo Figueroa et al. (2000) 2
 Alcolapia alcalica Lake Natron Figueroa et al. (2000) 2
 Tropheus sp. Lake Tanganyika Hablützel et al. (2013) 26
 Astatotilapia burtoni Lake Tanganyika Baldo et al. (2011) a 6
 Neolamprologus brichardi Lake Tanganyika Brawand et al. (2014) a 5
 Simochromis diagramma Lake Tanganyika BioProject PRJNA704661a 4
 Cyphotilapia frontosa Lake Tanganyika Ono et al. (1993) 4
 Tropheus moorii Lake Tanganyika Hablützel et al. (2014) 4
 Haplochromis katavi Lake Rukwa Figueroa et al. (2000) 1
 Maylandia zebra Lake Malawi Klein et al. (1993)
Figueroa et al. (2000)
Brawand et al. (2014) a 		13
 Melanochromis auratus Lake Malawi Klein et al. (1993)
Figueroa et al. (2000) 		5
 Astatotilapia calliptera Lake Malawi BioProject PRJNA(4888)11a 4
 Maylandia fainzilberi Lake Malawi Blais et al. (2007) 4
 Aulonocara hansbaenschi Lake Malawi Ono et al. (1993) 3
 Maylandia emmiltos Lake Malawi Blais et al. (2007) 3
 Oreochromis aureus Nile River Tao et al. (2021) a
BioProject PRJNA(5898)54a 		8
 Oreochromis niloticus Nile River Conte et al. (2017) a 7
Neotropics
 Amphilophus spp. Nicaraguan Lakes Hofmann et al. (2017)
Bracamonte et al. (2022) 		31
 Archocentrus centrarchus Nicaraguan Lakes BioProject PRJNA559550a 5
 Bujurquina vittata Paraná River Ronco et al. (2021) a 2
 Australoheros scitulus Rosario River Ronco et al. (2021) a 2
 Andinoacara biseriatus Colombian rivers Ronco et al. (2021) a 2
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Note.—Source refers to the paper where sequences were published, or the genome project information from which the sequences were extracted.

a

MHC information was obtained from whole genome sequences.

Phylogenetic Analyses

The Neighbor-Net network of the MHC IIB exon 2 clustered the cichlid sequences into at least seven major lineages (fig. 1B), with only the sequences of the two species of India and Madagascar not clustering with any of them. Two lineages were continent-specific, including sequences from either African or Neotropical species. The other five lineages included sequences from species of both continents. The phylogenetic inferences were generally congruent with the Neighbor-Net network, recovering similar relationships between the MHC IIB alleles and a clearer discrimination of the lineages (Bayesian inference: fig. 2; maximum likelihood [ML]: supplementary fig. S1, Supplementary Material online).

Fig. 2.

Fig. 2.

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Consensus topology from Bayesian inference constructed with MHC class IIB exon 2 sequences. PP are indicated for the major branches. The shapes at the tips of the branches relate to geographic regions as in figure 1.

The phylogenetic reconstruction of only cichlid MHC IIB exon 3 separated fish from the different continents with Neotropical alleles forming a strongly supported subcluster of African alleles (Bayesian inference; supplementary fig. S3, Supplementary Material online). The analysis of exon 3 including the teleostean species clustered sequences according to taxonomic orders and confirmed the monophyly of cichlid MHC IIB (Neighbor-Net network; fig. 3).

Fig. 3.

Fig. 3.

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Neighbor-Net network based on MHC class IIB exon 3 sequences comprising cichlids and different teleost orders.

Selection Analyses

The nucleotide alignment of MHC IIB exon 2 translated into 45 amino acid sites, of which 39 were polymorphic (supplementary fig. S2, Supplementary Material online). The nucleotide sequences translated into 139 unique amino acid sequences. We found no evidence for exon-wide positive selection (Z = 0.72, P = 0.36). However, we found evidence of positive selection acting at the codon level. The different methods identified between six positively selected sites (PSS; CODEML) and 11 PSS (Fixed Effects Likelihood [FEL]; supplementary table S4, Supplementary Material online). In total, we considered ten sites to be under positive selection that were identified by at least two different methods (supplementary fig. S2 and table S4, Supplementary Material online). These PSS were largely consistent with the human ABS. Six sites were identical to human ABS, and the remaining four sites were in positions in close proximity to human ABS (supplementary fig. S2, Supplementary Material online). Reducing exon 2 sequences to only the PSS resulted in 136 unique amino acid sequences. The exon 3 nucleotide alignment translated into 70 amino acid sites, of which 63 were polymorphic. We also found no exon-wide positive selection (Z = 2.38, P = 0.02). The analyses of positive selection at the codon level identified only one site under positive selection (Fast Unconstrained Bayesian AppRoximation [FUBAR] and FEL).

Allele Classification Into Supertypes

We obtained 13 functional supertypes based on clustering the exon 2 sequences by putative ABS (fig. 2). The number of exon 2 sequences per supertype ranged from 2 to 25 (table 2). Most of the supertypes (11 out of 13) occurred in both African and Neotropical species, except for supertype 8 (nine sequences) that was exclusive to African species and supertype 14 (two sequences) that was exclusive to Neotropical species (table 2). The number of supertypes found in cichlids from both continents was the same, with a total of 12 supertypes in cichlids of each continent. The sequences of the Indian and the Malagasy species were not assigned to exclusive supertypes, but to supertype 11 and supertype 3, respectively. The 13 supertypes were not forming monophyletic groups in the phylogeny, except for supertypes 6 and 7 (fig. 2).

Table 2.

Distribution of MHC IIB Supertypes in the Different Geographic Regions

Supertype Africa America Total
Supertype 1 21 4 25
Supertype 2 15 5 20
Supertype 3 13 4 18a
Supertype 4 5 9 14
Supertype 5 12 1 13
Supertype 6 9 4 13
Supertype 7 7 3 10
Supertype 8 9 — 9
Supertype 9 8 1 9
Supertype 10 6 3 9
Supertype 11 4 2 7b
Supertype 12 2 4 6
Supertype 13 — 2 2
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a

Includes a sequence from a species of Madagascar.

b

Includes a sequence from a species of India.

TSP Versus Convergent Evolution

The results of codon usage analysis were congruent with the TSP hypothesis. Of the 13,385 amino acids at the putative ABS of African and Neotropical cichlid species, 11,762 were encoded by the same codon. This number was closer to those simulated under a coancestry scenario (mean = 11,611 ± 38; P < 0.001) than those simulated under a convergent evolution scenario (mean = 7,292 ± 56; P < 0.001). Hence, identical amino acids at ABS showed higher codon similarity than expected under convergent evolution and support TSP.

The phylogenetic reconstructions of the partition comprising only ABS (supplementary fig. S4, Supplementary Material online) and the partition comprising only putatively neutral sites (excluding ABS and sites under purifying selection; supplementary fig. S5, Supplementary Material online) showed a pattern in which sequences of African and Neotropical species clustered together, similar to the phylogeny of the exon 2, lending support to a TSP scenario. However, the short length of the final alignments of both partitions (30 and 68 bp, respectively) in relation to the number of sequences led to low resolution of the resulting phylogenies, as indicated by low node support. Comparing the phylogenetic reconstruction of exon 2 with the more limited phylogeny of exon 3 showed that for both, sequences of African cichlids are paraphyletic, clustering together with sequences of Neotropical cichlids. The limited number of available exon 3 sequences, particularly for Neotropical cichlids, prevents a more detailed comparison.

Discussion

Cichlid fish have remarkably high levels of polymorphism at the immune genes of the MHC IIB. Most of this diversity is concentrated in exon 2 (Klein et al. 1993; Hofmann et al. 2017; Bracamonte et al. 2022). Here, we studied MHC IIB evolution across African and Neotropical cichlid radiations by a comprehensive review of existing MHC IIB sequence information focusing on exon 2 and including information about exon 3. We aimed to understand the distribution of MHC IIB diversity across cichlid radiations and the patterns of shared MHC IIB exon 2 allelic lineages and allele similarity between species across continents. We explored whether allele similarities at the nucleotide level were due to TSP or convergent evolution and found that polymorphism at exon 2 in cichlids is most likely the result of TSP. We also found that cichlids present at least 13 groups of functionally similar MHC IIB alleles, which are classified as supertypes. These supertypes were extensively shared among cichlid radiations of the different continents. Additionally, we aimed to provide a framework of cichlid MHC IIB evolution by analyzing the more conserved exon 3 from cichlid fish and other teleost orders. This confirmed the monophyly of cichlid MHC IIB.

Previous studies have shown that MHC IIB exon 2 lineages are extensively shared among radiations of African cichlids (Klein et al. 1993; Hablützel et al. 2013). Here, we demonstrated that this phenomenon extends to cichlid species from different continents. Our analyses recovered MHC lineages that were shared by both African and Neotropical cichlids. This pattern could be indicative not only of TSP but also of alternative scenarios such as convergence. Distinguishing between TSP and convergent evolution based solely on phylogenetic reconstructions of short nucleotide sequences can be challenging (Kriener et al. 2000), and additional analyses are required for inferring the most likely evolutionary scenario (Lenz et al. 2013; Gillingham et al. 2016; Kaesler et al. 2017). By modeling codon usage patterns, we showed that TSP is the most likely explanation for MHC allele similarity and lineage sharing among cichlid fish radiations from different continents. Comparisons of the phylogenetic reconstructions of exon 2 and its partitions of ABS and neutral sites are also in line with TSP. Since these sites are in close linkage and do not evolve entirely independent, we further compared the phylogeny of exon 2 with the phylogeny of the more conserved exon 3. Although only a limited number of exon 3 sequences were available, particularly for Neotropical cichlids, the emerging pattern indicates that Neotropical sequences are nested within African sequences, thus suggesting certain degree of shared ancestry. This indicates that balancing selection has maintained MHC lineages through speciation events.

TSP in MHC genes is a generalized feature, identified in representatives of all major vertebrate groups (i.e., fish, Ottová et al. 2005; Lenz et al. 2013; Million and Lively 2022; amphibians, Kiemnec-Tyburczy et al. 2010; reptiles, Sagonas et al. 2019; birds, Gillingham et al. 2016; and mammals, Kaesler et al. 2017). However, the persistence of MHC polymorphisms due to TSP appears to differ considerably in timescales in different taxa. TSP was found to be maintained for longer times in fish, amphibians, and reptiles than in mammals (reviewed in Těšický and Vinkler 2015). Indeed, paddlefish and Chinese sturgeon, two fish species that diverged more than 180 Ma, share lineages of MHC class I alleles, which is the oldest case of MHC lineage sharing described so far (Wang et al. 2010). Here, we found evidence that TSP across cichlid lineages from Africa and the Neotropics was maintained for at least 60 Myr (Matschiner et al. 2020).

We also analyzed MHC IIB exon 3 sequences from cichlid and noncichlid teleosts. This exon is more conserved than exon 2, and accordingly, we did not find clear signs of positive selection, as found in other vertebrates as well (Gillingham et al. 2016). Therefore, the exon 3 phylogeny, in contrast to exon 2, recovered the phylogenetic history of the species. Cichlid sequences all clustered into a single lineage, supporting previous evidence of monophyly of cichlid MHC IIB reported for African species (Hablützel et al. 2013) and monophyly of the other teleost orders included in the analysis. In cichlids, the apparent segregation of sequences from the different continents suggests that postspeciation events drove divergence of MHC exon 3 among radiations.

A hallmark of cichlid adaptive radiations is trait convergence, found in phenotypic as well as in molecular traits (Salzburger 2018). Parallel parasite-mediated selection pressures across hosts could drive convergent evolution at the immune genes of the MHC (Lenz et al. 2013), although evidence for this is scarce. Some studies on sympatric mammal species have found patterns of molecular convergent evolution at the MHC (Kriener et al. 2000; Srithayakumar et al. 2012). However, other studies integrating parasite and MHC IIB data on sympatric lemur species (Kaesler et al. 2017) and on two sympatric stickleback species (Lenz et al. 2013) did not find evidence of convergent evolution. In this study, we found no evidence of MHC IIB convergent evolution in cichlids.

Using different computational methods, we identified several sites under positive selection in cichlid MHC IIB exon 2. Although a crystallographic approach is necessary to determine the exact sites interacting with parasitic antigens (Brown et al. 1993), the inference of ABS with positive selection analyses provides a good approximation and has been used by numerous studies (Kaesler et al. 2017; Minias et al. 2018; Talarico et al. 2021; Li et al. 2023). Accordingly, several of the PSS that we inferred for exon 2 of the cichlid MHC IIB overlap with ABS described for humans using crystallography (Brown et al. 1993). Inferring ABS allowed us to establish putative functional groups (supertypes) according to amino acid similarity at these sites. Previous studies have associated the presence of MHC functional supertypes with higher resistance to parasites (Sepil et al. 2013; Hacking et al. 2018), reduced parasite loads (Biedrzycka et al. 2018; Smallbone et al. 2021), or better body condition (Talarico et al. 2021), suggesting that supertypes could relate to susceptibility to parasites at individual and population level. In our study, supertypes were largely shared among cichlid species from different continents although in diverse proportions. The large majority of supertypes (11) were present in species of both continents, whereas one was exclusive of African cichlids and another exclusive of Neotropical cichlids. Exclusive supertypes could reflect a response to continent-specific pressures. The number of MHC supertypes found in this study is the same as that found in the radiation of the Neotropical Midas cichlid (Bracamonte et al. 2022). It is interesting to note that when combining MHC sequences from phylogenetically distant taxa, the total number of MHC supertypes is maintained and this could suggest that a certain number of supertypes are essential for cichlid fish to resist the full diversity of infecting parasites they encounter throughout their geographical distribution. However, in order to settle the number of functional supertypes in cichlids, additional detailed studies would be required.

It was hypothesized, based on simulations, that balancing selection could act to maintain functional supertypes rather than alleles (Lighten et al. 2017). Under this scenario, MHC allelic variation would be governed by demographic processes, experiencing constant turnover, whereas supertype variation would be maintained by strong balancing selection (Lighten et al. 2017). However, empirical work on MHC with guppy fish and brown trout did not find evidence of stronger selection on supertypes than alleles (Herdegen-Radwan et al. 2021; Talarico et al. 2021). Theoretical models suggest that balancing selection on supertypes will only maintain TSP if these are monophyletic, whereas a paraphyletic pattern would be indicative of functional convergence of alleles within supertypes (Ejsmond et al. 2018). In cichlids, we found that the majority of the supertypes were not monophyletic suggesting convergent evolution of allele functionality.

Parasites are considered the major drivers of MHC evolution and diversity. Allele similarity and functional sharing at MHC in cichlid fish are expected to be correlated with comparable parasite pressures across continents. Parasite communities and their impact on cichlid fish are still not fully understood. In recent years, there have been important efforts at describing cichlid parasites worldwide, although knowledge is still incomplete (Vanhove et al. 2016; Cruz-Laufer et al. 2022; Santacruz et al. 2022). Monogenean gill parasites have been extensively studied in Africa, and they appear to be the most diverse parasite group for cichlids in this region (Vanhove et al. 2016). In Central American cichlids, different species of gill monogeneans from the same family have also been recognized (Santacruz et al. 2022). Whether MHC supertypes interact with individual parasites or combinations of them, or if combinations of MHC alleles respond in concert, is still unknown. Analyses of convergence in parasite–MHC interactions between Neotropical and African systems would be facilitated by similar approaches of parasite research in both systems.

Besides TSP and convergence, introgression of MHC alleles between hybridizing species has been proposed as an alternative mechanism producing MHC allele sharing (Wegner and Eizaguirre 2012). Introgression of MHC alleles has been found in sympatric species of newts (Nadachowska-Brzyska et al. 2012; Dudek et al. 2019) and lizards (Sagonas et al. 2019). Hybridization has played a very important role in cichlid evolution and is thought to have fueled the adaptive radiations of the East African Great Lakes (Meier et al. 2017; Irisarri et al. 2018; Malinsky et al. 2018; Svardal et al. 2020). Our analyses cannot rule out the role of introgression on MHC allele similarities and lineage sharing between some closely related species. Indeed, we found identical MHC IIB exon 2 alleles shared among African species, even among species inhabiting different lakes. We also found identical alleles shared among Neotropical species within the Nicaraguan lakes. However, the presence of identical exon 2 sequences in different species appears to be restricted to cichlids inhabiting the same geographic region or belonging to the same radiation. We found no shared alleles between continents and within lineage alleles segregated by continent. It seems unlikely that introgression can explain lineage sharing among species that diverged more than 60 Ma and inhabit different continents. Still, a detailed analysis of additional MHC sequences of closely related species and the inclusion of neutral markers would be necessary to completely rule out this mechanism.

Despite the extensive sharing of MHC allelic lineages across species from Africa and America, we identified two lineages that were exclusive to cichlids of either continent. Given that MHC polymorphism commonly derives from standing genetic variation, the continent-specific lineages may have arisen by sorting of these ancestral alleles or by uneven retention of alleles due to differential selection regimes after speciation events. However, other mechanisms that are frequently invoked in MHC evolution such as gene duplication, mutation, recombination, or gene conversion (Reusch and Langefors 2005; Spurgin et al. 2011; Bentkowski and Radwan 2019) can also have generated new allelic lineages following the split between the continents in response to divergent continent-specific selective pressures. However, the retention of specific allelic lineages over long evolutionary periods may imply that a core set of MHC IIB alleles is necessary for protection against parasites (Těšický and Vinkler 2015).

Conclusions

In this study, we presented a comprehensive representation of cichlid MHC IIB diversity. We documented TSP between MHC IIB exon 2 alleles of African and Neotropical cichlid species that diverged around 60 Ma and subsequently underwent several adaptive radiation and speciation events. This adds to a growing body of evidence that TSP is a common pattern at the MHC of related species and that ancestral polymorphism is maintained over long evolutionary periods. Meanwhile, we found that MHC functionality, defined by supertypes, is widely shared among different cichlid lineages in Africa and the Neotropics. Therefore, MHC IIB exon 2 alleles in cichlids experienced TSP at the sequence level, and this polymorphism is also maintained at the functional level. We also provided further evidence to the monophyly of MHC IIB in cichlids based on exon 3. We acknowledge that we did not recover the full MHC IIB exon 2 diversity for cichlid fish that some lineages may be missing, and that there is an overrepresentation of African cichlid species in both MHC IIB sequences and whole genome sequence data. However, adding more sequences is unlikely to change the general pattern, although resolving the relationship of MHC from cichlid species of India and Madagascar may provide further insight into the evolution of MHC in cichlids.

Materials and Methods

Sequence Selection

We compiled sequences of MHC IIB exon 2 and exon 3 from cichlid fish species from GenBank. For exon 2, we retrieved sequences from two cichlid genera for which MHC IIB was extensively characterized: the African Tropheus from Lake Tanganyika (Hablützel et al. 2013) and the Neotropical Amphilophus from the Nicaraguan lakes (Hofmann et al. 2017; Bracamonte et al. 2022). We blasted the nucleotide sequences of Tropheus and Amphilophus against available cichlid genomes in GenBank, aiming at exon 2 sequences of species representing the major radiations of the East African Great Lakes Victoria, Tanganyika, and Malawi, as well as the Neotropical Nicaraguan lakes. We also retrieved exon 2 nucleotide sequences from genomes of species from South America, India, and Madagascar. To further increase exon 2 sequence representation and species sampling, we used a keyword search using the terms “MHC” and “class IIB” and “cichlids” in GenBank (accessed on November 1, 2021). We retained a maximum of four exon 2 sequences per species when generated by the same study, to avoid overrepresentation of largely studied species that may bias the data set. We aimed at the most divergent sequences, selecting these sequences based on percent identity of the BLAST search to maximize diversity. We used a final data set consisting of 155 unique sequences from 28 cichlid taxa (table 1). Of these, 111 belonged to African and 42 to Neotropical cichlids, with two additional sequences of species from India and Madagascar. Information about species included and sources of sequence data is provided in table 1. African cichlids were represented by taxa from the East African radiations of the Great Lakes Victoria, Tanganyika, and Malawi, adjacent lakes, and by two riverine species of the genus Oreochromis. Neotropical cichlids were represented by two genera inhabiting the Nicaraguan lakes in Central America (Amphilophus and Archocentrus) and three riverine species from South America. GenBank accession numbers of exon 2 sequences are provided in supplementary table S1, Supplementary Material online.

For MHC IIB exon 3, we retrieved sequences belonging to both cichlid and noncichlid teleost species to investigate the presumed monophyly of MHC IIB in cichlids (Hablützel et al. 2013). We blasted available nucleotide sequences of exon 3 from Amphilophus (Hofmann et al. 2017) in GenBank. We collected 40 sequences from 17 cichlid species (supplementary table S2, Supplementary Material online). When possible, we selected exon 3 sequences from the same cichlid individuals used for the exon 2 data set. Additionally, we retrieved available exon 3 sequences from other teleostean orders. The final data set including cichlids and other teleosts comprised 13 orders and 68 taxa, with a total of 148 sequences (supplementary table S2, Supplementary Material online). The MHC IIB exon 3 nucleotide alignment was 214 bp long and presented no deletions. Taxa information, number of sequences, and GenBank accession numbers are provided in supplementary table S3, Supplementary Material online. For both exon 2 and exon 3 data sets, we kept only unique sequences.

Phylogenetic Analyses

We aligned the exon 2 and exon 3 data sets separately with the ClustalW algorithm (Thompson et al. 1994) implemented in MEGA X (Kumar et al. 2018). We further checked the alignments using TranslatorX (Abascal et al. 2010) that aligns protein-coding nucleotide sequences following translation into amino acids.

Prior to the phylogenetic analyses, we tested whether nucleotide substitutions might have experienced saturation, which would decrease phylogenetic information. We used this approach because African and Neotropical cichlid lineages diverged about 60 Ma (Matschiner et al. 2020), which may have caused nucleotide substitution saturation. We used DAMBE7 (Xia 2018) to compare the observed saturation (Iss) with the critical saturation (Iss.c; Xia et al. 2003). As this test provided no evidence of substitution saturation in both MHC IIB exon 2 and exon 3 data sets, with Iss being significantly lower than Iss.c, we proceeded to infer phylogenetic relationships.

We analyzed the phylogenetic relationships of MHC IIB exon 2 and exon 3 sequences separately using SplitsTree v4.15.1 (Huson and Bryant 2006), applying uncorrected p-distances. Neighbor-Net networks are appropriate for genes with intricate evolutionary histories, including the MHC (Gillingham et al. 2016). To further investigate phylogenetic relationships for cichlid exon 2 and exon 3 sequences (i.e., excluding other teleostean orders) and to obtain nodal support for the allelic lineages, we performed both Bayesian inference and ML analyses. For the Bayesian inference analysis, we used MrBayes v3.2.7a (Ronquist et al. 2012) as implemented in the CIPRES Science Gateway (Miller et al. 2010). To estimate posterior probabilities (PP), we performed two independent runs with four chains each and 107 generations. Trees were sampled every 1,000 generations, discarding the first 25% as burn-in. We evaluated convergence among chains with Tracer v1.7.1 (Rambaut et al. 2018). For the ML analysis, we used IQ-TREE v.1.6.12 (Nguyen et al. 2015). The TIM2e + G4 nucleotide substitution model was selected with ModelFinder (Kalyaanamoorthy et al. 2017) under the Bayesian information criterion. We assessed branch support through 1,000 bootstrap replicates.

Selection Analyses

We tested for historical positive selection acting on cichlid MHC IIB exon 2 and exon 3. We performed a Z-test for gene-wide positive selection in MEGA X, applying the Nei–Gojobori method using Jukes–Cantor correction and 10,000 replicates. An excess of nonsynonymous substitutions (dN) over synonymous substitutions (dS) at coding sites indicates positive selection (dN/dS > 1). We also used different methods to identify codons under positive selection. First, signatures of positive pervasive selection at codons were evaluated with two tests: FEL (Kosakovsky Pond and Frost 2005) and FUBAR (Murrell et al. 2013), both implemented in the Datamonkey web server (Weaver et al. 2018). For FUBAR, we considered codons with PP above 0.9 to be positively selected, whereas for FEL, we considered a P value below 0.1 for inferring positive selection. We also performed a Wu–Kabat analysis on the amino acid alignment. This test identifies sites of high variability dividing the number of amino acids at a position by the frequency of the most common amino acid (Wu and Kabat 1970). We considered codons to be positively selected when the Wu–Kabat index was twice the mean of the Wu–Kabat index for all sites (Bos and Waldman 2006). For exon 2, we additionally used CODEML implemented in PAML v4.9j (Yang 2007) to compare site models assuming no positive selection to those allowing for positive selection using different codon substitution models: M1a (nearly neutral) versus M2a (positive selection) and M7 (beta distribution) versus M8 (beta distribution + positive selection). PSS were inferred with the Bayes empirical Bayes method. The ML tree (see above) was used as input tree. For exon 2, we considered sites to be positively selected when identified by at least two different methods. We will subsequently refer to these sites as PSS and assume that the inferred PSS correspond to the ABS (Hughes and Yeager 1998). MHC class II molecules are well conserved across vertebrates, and we corroborated our assumption by comparing the position of these cichlid PSS to the position of human ABS (Brown et al. 1993). Additionally, FUBAR and FEL methods, using the same conditions as indicated above, were used to identify sites under purifying (negative) selection on exon 2 and sites under positive selection in exon 3.

Allele Classification Into Supertypes

We classified MHC IIB exon 2 sequences into functional supertypes, according to the specificity of the residues at the putative ABS (Doytchinova and Flower 2005). We aligned the amino acids corresponding to the PSS and assigned five values to each amino acid that describe the physicochemical properties: z1 (hydrophobicity), z2 (steric bulk), z3 (polarity), z4 and z5 (electronic effects; Sandberg et al. 1998). Subsequently, we grouped MHC sequences with similar physiochemical properties at the PSS into supertypes with putatively similar binding properties, following the procedure described by Sepil et al. (2012). We applied a K-means clustering and a discriminant analysis of principal components (DAPC) to identify clusters of alleles. We used the function “find.clusters” and “xvalDAPC,” respectively, implemented in the R package “adegenet” (Jombart 2008; Jombart et al. 2010). The number of clusters was selected based on Bayesian information criterion values. We assigned a preliminary cluster to each allele based on the consensus of ten rounds of clustering. We repeated the procedure of clustering and preliminary assignment five times and assigned the final supertypes based on a consensus of the five preliminary assignments.

TSP Versus Convergent Evolution

We used two different methods to discern TSP from convergent evolution between cichlid species from Africa and the Neotropics. First, we analyzed codon usage patterns between African and Neotropical species. This analysis compares the number of identical codons at the ABS between alleles of two species to theoretical distributions of identical codons expected under TSP and under convergent evolution (Lenz et al. 2013). Under TSP, identical amino acids at the ABS should be encoded by the same codon as a trace of coancestry, whereas under convergent evolution, this pattern is not expected. High levels of codon similarity at the ABS between species are congruent with a TSP scenario (Lenz et al. 2013). We used modified custom scripts provided by Lenz et al. (2013) upon request. We performed the analysis at continent level. For this, we assigned alleles either to Neotropical or African origin. We calculated the number of identical codons per identical amino acid at PSS for all pairwise comparisons of Neotropical to African alleles. To obtain the distribution of identical codons expected under TSP, we recorded whether identical amino acids were encoded by identical or different codons for all sites of all pairwise comparisons of Neotropical alleles and of African alleles. We then drew 1,000 random samples of identical amino acid sites maintaining the observed frequency of each amino acid between Neotropical and African PSS and counted the number of times they were encoded by identical codons. For obtaining the distribution of identical codons expected under convergent evolution, we used a similar approach but recording whether identical amino acids were encoded by identical codons between all sites of Neotropical versus African alleles. We then determined whether the observed number of identical codons fell within the 95% confidence interval of either distribution.

Second, we split the MHC IIB exon 2 sequences into two partitions. One partition included only the codons of the putative ABS, whereas the other included the putative neutral codons outside the ABS and the sites under purifying selection. We performed ML phylogenetic reconstructions of both partitions with IQ-TREE v.1.6.12 under the same conditions used for the reconstruction of the complete exon 2 sequence phylogeny (see above). Comparison of the resulting phylogenies and the clustering patterns of the sequences allows discerning between TSP and convergence (Kriener et al. 2000). Under TSP, the phylogenetic reconstruction of the partition excluding the ABS and the purifying selection sites would retain the coancestry signatures and display a pattern more similar to the exon 2 reconstruction (Klein et al. 1998; Lenz et al. 2013). Alternatively, under convergent evolution, the phylogenetic reconstruction with the putative ABS, but not the neutral sites, is expected to be more similar to the exon reconstruction (Yeager and Hughes 1999). Since ABS and non-ABS sequences of exon 2 may not evolve independently, we also compared clustering patterns of the phylogenetic reconstructions of exon 2 and exon 3 of African and Neotropical cichlids.

Supplementary Material

evad110_Supplementary_Data
Click here for additional data file. (997KB, docx)

Acknowledgments

Funding was provided by Spanish Ministry of Science and Innovation (MCIN)/Spanish Research Agency (AEI) and European Regional Development Fund (ERDF) “A way to make Europe” through projects CGL2017-82986-C2-1-P and PID2020-115481GB-I00 to M.B. C.L.M. was supported by a predoctoral fellowship from MCIN/AEI and European Social Funds (FPI-PRE2018-085797). S.E.B. was supported by a Swiss National Science Foundation Early PostDoc.Mobility fellowship (P2SKP3_191312) and a Juan de la Cierva training contract from MCIN/AEI and NextGeneration EU/PRTR (FJC2020-042779-I). We thank Ana Santacruz for useful discussions. We are grateful to Bonnie Fraser and three anonymous reviewers for their constructive comments, which helped to improve the manuscript.

Contributor Information

Carlos Lozano-Martín, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.

Seraina E Bracamonte, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.

Marta Barluenga, Museo Nacional de Ciencias Naturales, CSIC, Madrid, Spain.

Supplementary Material

Supplementary data are available at Genome Biology and Evolution online (http://www.gbe.oxfordjournals.org/).

Author Contributions

C.L.M., S.E.B., and M.B. conceived the study, C.L.M. collected the sequence data, C.L.M. and S.E.B. analyzed the data, C.L.M. wrote the initial draft of the manuscript, and all authors contributed to the final version.

Data Availability

All accession numbers for the data used in this study can be found in the Supplementary Tables.

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Associated Data

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Supplementary Materials

evad110_Supplementary_Data
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Data Availability Statement

All accession numbers for the data used in this study can be found in the Supplementary Tables.

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