Taxonomic diversity in Lake Bangweulu and Lake Mweru

We caught and identified over 600 cichlids in Lake Bangweulu at eight sites, of which we preserved 404. We also caught and identified over 1200 cichlids in Lake Mweru at 12 sites and at six sites on the Kalungwishi and Luongo/Luapula Rivers that flow into Lake Mweru, of which we preserved 1066. Our samples from Lake Bangweulu were readily identified morphologically to ten described species28. These included six haplochromine cichlids, five of which are widespread in the Zambezi. The sixth species is restricted to Lakes Bangweulu and Mweru and the Luapula River but is closely related to Zambezi taxa (Fig. 1, Supplementary Note 2). In contrast to the modest diversity in Lake Bangweulu, we encountered a spectacular diversity of haplochromine cichlids with mostly previously unknown species in Lake Mweru. In Lake Mweru and two inflowing rivers, the Kalungwishi and the Luongo, a tributary of the Luapula (Fig. 1), we recorded six non-haplochromine cichlids (all previously described) (Supplementary Figs. 2, 3). We also found each of the haplochromine species that are also represented in Lake Bangweulu (Supplementary Fig. 3). In addition, we discovered about 40 putative species of haplochromine cichlids previously unknown to science and endemic to Lake Mweru or its inflowing rivers (Supplementary Figs. 2, 4, Supplementary Note 3). Genomic and morphological data revealed that they evolved in four larger radiations, all confined to Lake Mweru (hereafter called lacustrine radiations, red circles in Fig. 1), and four smaller radiations with endemic species in the lake and inflowing rivers (Fig. 1). The radiations form part of two highly divergent groups, the serranochromines and a second group including the genera Orthochromis and Pseudocrenilabrus as well as some undescribed relatives, that we informally collectively refer to subsequently as “orthochromines” (Fig. 1). “Orthochromines” encompass four smaller radiations with species of the genus Orthochromis in the lake and adjacent rivers and a lacustrine Pseudocrenilabrus radiation, which is the largest of all radiations in the Mweru system with 15 putative species, and the only lacustrine radiation of Pseudocrenilabrus known in all of Africa (Supplementary Figs. 2, 4, Supplementary Note 3). We (R.B.S. and O.S.) performed mate choice experiments in aquaria with three of these putative species and showed that they mate strongly species-assortatively37. We also bred these putative species in aquaria and found them to breed true. Serranochromines contain the three other lacustrine radiations comprising six Sargochromis putative species and two radiations in the genus Serranochromis. One Serranochromis radiation includes four mostly inshore predators with large teeth, which we named ‘large-tooth serranochromines’. The second radiation, which we call ‘small-tooth serranochromines’, includes six littoral or bentho-pelagic putative species with noticeably smaller teeth (Supplementary Figs. 2, 4, Supplementary Note 3).

We identified individuals to putative species in the field and subsequently analysed the radiations in the Lake Mweru catchment morphologically and genetically (Supplementary Fig. 4). ‘Species’ as a factor explained highly significant amounts of variance on one or more morphological and also on one or more genomic PC axes within each lacustrine radiation except for the genomic PCA of the large-tooth serranochromines, but sample sizes of the latter were small (Supplementary Fig. 4, Supplementary Note 3).

Mweru radiations are complementary in ecology and morphospace

The radiations of Lake Mweru show remarkable complementarity with each other in morphology and ecology (Figs. 1, 2). Each radiation occupies different and unique sectors in morphospace that mirror trophic and habitat niche space (Fig. 1). We inferred the most likely trophic niche from dentition and morphology38, and the habitat occupancy from presence/absence and abundance at our sampling sites. Orthochromis species in the lake (O. sp. “red-cheek” and O. polyacanthus) are confined to rocky shores, where they scrape algae and other Aufwuchs (periphyton and benthic invertebrates). Orthochromis in the rivers (O. sp. “Kalungwishi” and species of the Orthochromis kalungwishiensis complex) are confined to lotic (running water) habitats taking algae and insects from rocky substrate. The two species of the “New Kalungwishi” complex feed on insect larvae in lentic (lake-like) extensions of the Kalungwishi and the Luongo River. Species of the Pseudocrenilabrus radiation feed on insect larvae, and small benthic and planktonic crustaceans in a wide range of habitats. Members of the Sargochromis radiation are littoral molluscivores or feed on large insect larvae. Large-tooth and small-tooth serranochromines feed in several different habitats on large insect larvae and smaller fish, likely including the “orthochromine” species, their eggs and fry, and small cyprinid fish. Whereas for some of the genera, these trophic niches are typical across much of their African range, the largest Mweru radiation occurred in the genus Pseudocrenilabrus which otherwise contains exclusively trophically unspecialized microinvertivorous wetland species39,40. In Lake Mweru, however, they have diversified into all available habitats (littoral soft substrates, rocky shores and demersal offshore) and have adopted a variety of feeding styles and prey.

Fig. 2 Morphological variation and morphospace partitioning between Lake Mweru radiations. a Morphological complementarity of the four intralacustrine cichlid radiations, the lacustrine members of the Orthochromis assemblage, the non-endemic Serranochromis altus and Se. angusticeps (Se. alt/ang), the nonendemic Se. robustus and Se. thumbergi (Se. rob/thumb) and Orthochromis stormsi (O. stormsi) in Lake Mweru. The first two principal components of all haplochromine cichlid species we found in Lake Mweru show nearly perfect complementarity in morphospace occupation among the radiations. Of the Orthochromis species, only the taxa occurring in Lake Mweru, O. polyacanthus and O. sp. “red-cheek” are shown, but not the seven riverine species of the Kalungwishi and Luongo Rivers. Sister taxa of each radiation are indicated with filled symbols. Their centroids or that of the radiations themselves (in the absence of sister taxa) are connected by thin lines to each phenotype of the corresponding radiation to visualize approximate trajectories of phenotypic divergence and diversification. The underlying data and the R script for all panels are provided at Zenodo with doi: 10.5281/zenodo.3435419. b Mweru taxa predicted onto morphospace of serranochromines of the radiation of ancient Lake Makgadikgadi. Makgadikgadi Sargochromis include Chetia and Pharyngochromis species which are nested in the genus Sargochromis42. In the presence of a diverse Pseudocrenilabrus radiation in Lake Mweru, the serranochromine radiations altogether are confined to a subset of the morphospace this lineage occupies elsewhere (such as in the Okavango region, the modern centre of Makgadikgadi-derived diversity). This is mostly due to much reduced morphological diversity in Sargochromis of Lake Mweru. Compared to Pseudocrenilabrus of Lake Bangweulu, the Pseudocrenilabrus radiation in Lake Mweru expanded into serranochromine morphospace. c In the presence of the Pseudocrenilabrus radiation in the lake, Orthochromis are confined to the epilithic algae and Aufwuchs scraper niche in the rocky wave washed littoral of the lake, an extreme feeding niche that the Pseudocrenilabrus radiation has not invaded. In the rivers, where Pseudocrenilabrus have not radiated and only the ancestral type P. philander is present, two species with partial Orthochromis and partial Pseudocrenilabrus ancestry evolved in lentic, i.e. lake-like, parts of the river. These “New Kalungwishi” species overlap in morphospace with littoral Pseudocrenilabrus from Lake Mweru. Full size image

“Orthochromines” are generally smaller than serranochromines (Supplementary Fig. 5). We measured ecologically relevant morphological traits41 to assess morphospace occupation as a proxy for niche partitioning between species and among radiations. In morphological PCA space of all Lake Mweru haplochromines combined, we observed nearly no overlap in morphospace among the lacustrine radiations (Fig. 2a). Investigating the trajectories of phenotypic diversification of each radiation compared to ancestor proxies, we identified patterns that are suggestive of limits to divergence and diversification imposed by the presence of the other sympatric radiations (Fig. 2a). In comparison to the radiation of serranochromines that arose in paleolake Makgadikgadi (many of which persist today in the Okavango region)42, the serranochromines in Lake Mweru lack representatives in a large part of the Makgadikgadi/Okavango morphospace which in Mweru is occupied by “orthochromines” (Fig. 2b). In the Makgadikgadi/Okavango region, in contrast, the “orthochromines” are represented by a single wetland generalist, Pseudocrenilabrus philander. Moreover, patterns of diversity in Orthochromis and Pseudocrenilabrus in Lake Mweru are consistent with evolutionary effects of competition in sympatry: in Lake Mweru, these genera do not overlap in morphospace (Fig. 2a). While Pseudocrenilabrus have diversified into a wide range of insectivores and planktivores in many different habitats, Orthochromis in the lake are confined to shallow water rocky shore algae/Aufwuchs scraper niches. However, in the Kalungwishi River, where the only Pseudocrenilabrus is a wetland generalist, the “New Kalungwishi” lineage which mitochondrially belongs to Orthochromis, and is genomically admixed between Orthochromis and Pseudocrenilabrus, formed two endemic species that occupy lake-like habitats with quiet water and soft bottoms associated with morphologies that converge in morphospace on the littoral members of the Pseudocrenilabrus radiation (Fig. 2c).

The complementarity in morphospace among the radiations is consistent with competition among lineages having constrained the adaptive radiations and having shaped niche partitioning among them in sympatry. At the same time, ecological interactions among the most distantly related and ecologically most distinct haplochromine lineages may have facilitated radiation in Lake Mweru. The Pseudocrenilabrus species invaded a variety of littoral, rocky-shore and offshore habitats and may have facilitated radiation of the two lineages of large piscivorous Serranochromis into these diverse habitats by serving as prey, a hypothesis that should be tested with ecological data in the future.

All Lake Mweru radiations evolved rapidly and recently

Next, we wanted to know if the Lake Mweru radiations evolved before or after the Chambeshi/Luapula River capture event and if the different radiations were of similar age. We found that within all four lacustrine radiations of Lake Mweru, mitochondrial branch lengths are short and mitochondrial lineage sorting between putative species is highly incomplete. We estimated the age of the population expansions associated with the radiations using BEAST v. 2.5.043 on subsets of the six most divergent mitochondrial haplotypes for each radiation, excluding individuals with cyto-nuclear mismatch among radiations. We added the closest relative from outside each radiation and some outgroups. Note that the deepest split in a haplotype clade may predate the onset of the corresponding species radiation, as some of the haplotype diversity may already have been present in the ancestral population. As the correct calibration of cichlid trees is controversial44, we used four different calibration sets with priors on the age of the Lake Malawi cichlids and on the modern haplochromines (see Supplementary Table 1).

The 95% confidence intervals around the age of the radiations overlap broadly with each other and with the age estimate of the Lake Victoria Region Superflock of haplochromine cichlids, which is thought to be 100,000–200,000 years old45,46,47 (Supplementary Fig. 6, Supplementary Note 4). This suggests that the radiations may have occurred largely contemporaneously and recently. Depending on the calibration set used, the deepest splits in the mitochondrial haplotype clades of Pseudocrenilabrus, the large-tooth serranochromines, the Sargochromis radiation, and the small-tooth serranochromines are dated to 0.27–0.35, 0.43–0.56, 0.27–0.94, 0.72–0.94 Mya, respectively. These age estimates may be overestimates due to the time-dependency of the molecular clock in the most recent 1 Mya15,48,49,50. The absence of mitochondrial haplotype sorting amongst putative species within any of the lake radiations (Supplementary Fig. 6, Supplementary Data 1) suggests that speciation must be considerably more recent than the split of the most divergent mitochondrial haplotypes in each radiation. Yet, the age estimates of the haplotype radiations are all younger than the Luapula-Chambeshi River capture event which is estimated to about 1 Mya35,36. The radiations both in Congolese and Zambezian lineages of Lake Mweru thus appear to have begun only after the Zambezian lineages could have arrived in Lake Mweru. Similarly, we estimated the crown age of the group including O. sp. “red-cheek” and O. sp. “Kalungwishi” as 0.36–0.47 Mya depending on the calibration set used. In contrast, the entirely riverine O. kalungwishiensis complex seems to be older (1.20–1.57 Mya) and likely predates the origin of the Mweru–Bangweulu connection. The species in this group are exclusively allopatrically distributed (different rivers) and qualify as a non-adaptive radiation rather than as an adaptive radiation51,52.

Despite considerable overlap in the confidence intervals of all lake radiations, the mitochondrial clade of the small-tooth serranochromines might be slightly older than those of the three other lake radiations (the mean age estimate of the deepest split is roughly twice that in the other radiations). This deeper mitochondrial divergence in the small-tooth serrranochromines may reflect larger ancestral haplotype diversity. This scenario is likely because the mitochondrial lineage of the small-tooth serranochromines does not have any close relatives outside the lake and may thus have been present in the Lake Mweru drainage long before the connection to the Bangweulu drainage became established, and possibly long before the beginning of adaptive radiation.

A single individual of the Pseudocrenilabrus radiation of Lake Mweru has a mitochondrial haplotype that groups it with P. philander of Lake Bangweulu and Lake Chilwa. This individual split from those P. philander 0.40–0.52 Mya (Supplementary Fig. 6), after the Luapula-Chambeshi capture event. This time also coincides with the estimated age of the beginning of the expansion of the majority mitochondrial clade in the Pseudocrenilabrus radiation, consistent with introgression of the Bangweulu/Chilwa mitochondrial haplotype before or around the time of the onset of the Pseudocrenilabrus radiation in Lake Mweru (see below).

Hybrid origins of every Lake Mweru radiation

Next, we wanted to understand how the radiations could all have emerged so rapidly within less than a million years in Lake Mweru, and why none evolved in Lake Bangweulu. For this, we tested the hypothesis that rapid radiations may have been preceded by introgressive hybridization between distinct colonizing lineages and that lineages that did not radiate had not received introgression.

Like the classical cichlid radiations in other African lakes, the individual radiations in Lake Mweru are largely monophyletic in the mitochondrial tree and fully monophyletic in the genome-wide nuclear tree (Supplementary Fig. 3). However, large cytonuclear discordance (Supplementary Fig. 3), rare cases of mitochondrial haplotype sharing across radiations (Supplementary Fig. 3) and genomic tests of hybridization (D statistics, f4 tests53 and MixMapper54, Supplementary Data 2–9) revealed evidence for rampant ancestral hybridization at or near the origins of every Lake Mweru radiation (Fig. 3, Supplementary Note 5). The hybridization tests were robust to the choice of outgroups (Supplementary Data 2–4, 7, 8). Genomic clustering analyses with ADMIXTURE55 revealed no evidence for recent gene flow between radiations (Supplementary Fig. 7). In addition, signatures of excess allele sharing were consistent and homogeneous across species within each radiation (Supplementary Data 3, 7) suggesting that hybridization took place before the radiation processes started, consistent with the hypothesis that it could have fuelled diversification. Haplotype sharing patterns identified by fineRADstructure broadly support the absence of recent gene flow and the ancient hybridization events inferred with D statistics (Supplementary Figs. 8, 9). The admixture graph method MixMapper56 confirmed the evidence for hybrid origins of the radiations and provided estimates for ancestry proportions (Supplementary Data 6, 9). All four lacustrine radiations in the melting pot Lake Mweru seem to be of hybrid origin between Zambezian and Congolese lineages. In addition, the two riverine species in the “New Kalungwishi” clade show strong signatures of hybrid ancestry between Orthochromis and Pseudocrenilabrus (Fig. 3, Supplementary Data 8). This suggests that introgression from Pseudocrenilabrus may have facilitated ecological niche expansion in a lineage that is Orthochromis in its mitochondrial lineage. Other riverine Orthochromis species also show evidence for past hybridization (Supplementary Data 8) but this did not seem to have spurred further diversification, probably due to lack of ecological opportunity in the lotic river habitat17.

Fig. 3 Cytonuclear discordance and tests of hybridization reveal reticulate ancestry of all Mweru radiations. Nuclear cartoon topology of the “orthochromines” (a) and serranochromines (c) with green vertical lines showing mitochondrial sister relationships that deviate from the nuclear tree (solid lines) and sharing of mitochondrial haplotypes (dashed lines). Lake Mweru taxa are highlighted with red edges and radiations are shown as triangles, whereas Lake Bangweulu taxa are shown with blue edges. Riverine taxa in the drainage system of Lake Mweru are shown with orange edges. Black vertical lines indicate evidence for hybridization from D statistics (numbered as shown in b and d) and other tests of hybridization. Where data allow, arrow heads indicate the putative direction of gene flow. b and d D statistic results supporting hybridization edges shown in a and c for “orthochromines” and serranochromines, respectively. Error bars indicate three standard deviations from the mean and are depicted in grey if overlapping with 0 (non-significant, |z| < 3). D statistics and z-scores are averaged across multiple tests with different outgroups. Tests for edges 4–6 in b show averages of tests using Pseudocrenilabrus from Lake Mweru, Bangweulu or from the Zambezi/Cunene as P2 as they were all very similar. Likewise, tests where two groups are given for P2 or P3 revealed very similar results for both groups and have thus been averaged. All D statistics used to compute the averages are provided as Supplementary Data 2–4 and 7–8. R scripts and the data underlying the figures are provided at Zenodo with https://doi.org/10.5281/zenodo.3435419. Full size image

Lack of radiations and of hybridization in Lake Bangweulu

Whereas in Lake Mweru cichlids cytonuclear discordance at the base of each radiation is strong and genome-wide tests of hybridization support admixed ancestry of every radiation (Fig. 3, Supplementary Fig. 3), much less evidence for hybridization was detected in Lake Bangweulu (Fig. 4, Supplementary Data 10). No mismatches between phenotypic taxonomy and mitochondrial haplotype clade were observed among Lake Bangweulu taxa (Supplementary Fig. 3). The only hybridization signals involving Lake Bangweulu taxa are signals of excess allele sharing and cytonuclear discordance that affect Sargochromis of both Lake Mweru and Lake Bangweulu (Fig. 4): Compared to Serranochromis robustus/thumbergi of the Kalungwishi River, Se. robustus of Lake Bangweulu shows excess allele sharing with Sargochromis of both Lakes Mweru and Bangweulu (Fig. 4, Supplementary Data 10). However, the facts that Sargochromis from both lakes show equally strong allele sharing with Se. robustus and that Se. thumbergi from Lake Bangweulu also shows slightly elevated allele sharing with both Sargochromis clades, suggest that introgression was likely from Se. robustus into the common ancestor of both Sargochromis clades. This likely reflects a hybridization event in the relatively distant past rather than hybridization restricted to Lake Bangweulu. None of the Lake Bangweulu taxa show any additional evidence for hybridization (Fig. 4, Supplementary Data 10).

Fig. 4 Lake Bangweulu taxa show no signatures of hybridization. Comparisons of Bangweulu taxa (as P1) with their closest relatives (sister taxon in Lake Mweru as P2) reveals no evidence for excess allele sharing between any Bangweulu taxon with any other taxon (P3) in our dataset, which would lead to positive D statistics. The only exception is Se. robustus, which shows excess allele sharing with Sargochromis both of Lake Mweru and of Lake Bangweulu. Given that the two Sargochromis lake clades do not differ in allele sharing with Se. robustus of Bangweulu (see first test of ‘SaB vs SaM’ in this figure) and that slight excess allele sharing is also observed in the closely related Se. thumbergi of Lake Bangweulu, gene flow likely occurred from Se. robustus Bangweulu into the common ancestor of both Sargochromis lake clades. Therefore, the onxly signature of hybridization involving a Bangweulu taxon is shared by both Bangweulu and Mweru taxa and may reflect hybridization in the distant past. Hence, there is no additional event of hybridization in Lake Bangweulu, whereas there is rampant evidence for hybridization among multiple different Lake Mweru taxa (see Fig. 3). Abbreviations and colour scheme follow those in Fig. 3. Exact values are given in Supplementary Data 10. The R script and the input file used to make this figure are provided at Zenodo with https://doi.org/10.5281/zenodo.3435419. Full size image

The absence of hybridization signals in Pseudocrenilabrus from Lake Bangweulu is easily explained by the absence of close relatives in the catchment. However, with the one exception discussed above, we also found no evidence of hybridization among serranochromines, even though all species are sufficiently closely related to hybridize. This may be due to their long history of sympatric coexistence and the evolution of behavioural reproductive isolation. In contrast, in Lake Mweru, lineages came into contact that had previously evolved in allopatry, separated in different drainage systems, and thus may hybridize more readily. In line with this argument of larger potential for hybridization between allopatrically evolved lineages, Pseudocrenilabrus from Lake Mweru, when tested in mate choice experiments, showed no premating isolation against phenotypically similar Pseudocrenilabrus from Lake Bangweulu. However, they did show premating isolation against other sympatric Pseudocrenilabrus species from Lake Mweru despite much smaller genetic distance compared to the allopatric lineages tested37.