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\begin{document}

\title{A speciation continuum within repeated origin of hypertrophied lips in
cyprinid fish from East Africa}



\author[1]{Boris Levin}%
\author[1]{Aleksandra Komarova}%
\author[2]{Evgeniy Simonov}%
\author[3]{Alexei Tiunov}%
\author[1]{Marina Levina}%
\author[3]{Alexander Golubtsov}%
\author[4]{Fyodor Kondrashov}%
\author[5]{Axel Meyer}%
\affil[1]{I D Papanin Institute of Biology of Inland Waters RAS}%
\affil[2]{A N Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences}%
\affil[3]{A N Severtsov Institute of Ecology and Evolution RAS}%
\affil[4]{Okinawa Institute of Science and Technology Research Laboratories}%
\affil[5]{University of Konstanz}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Trophic specialization plays a major role in ecological speciation.
Multiple adaptive radiations among cyprinid fish of the genus
Labeobarbus were recently discovered in riverine environments of the
Ethiopian Highlands, East Africa. These radiations contain parallel
diversifications of trophic morphology, including thick-lipped or
`rubberlip' phenotype, whose trophic function remains largely
unexplored. To test the adaptive value of thickened lips, to identify
the ecological niche of the thick-lipped ecomorphs, and to test of these
ecomorphs are the products of speciation we studied six sympatric pairs
of ecomorphs with hypertrophied lips and the normal lip structure from
different riverine basins. Trophic morphology, diet, stable isotope (15N
and 13C) signatures as well as mtDNA markers and genome-wide SNP
variation were analyzed. Our results show that thick-lipped ecomorphs
partition trophic resources with generalized ecomorphs in only one half
of the examined sympatric pairs despite the pronounced divergence in lip
structure. In these thick-lipped ecomorphs that were trophically
diverged, the data on their diet along with the elevated 15N values
suggest an insectivorous specialization different from the basal
omnivorous-detritivouros feeding mode of generalized ecomorphs. Genetic
data confirmed an independent and parallel origin of all six lipped
ecomorphs. Yet, only one of those six thick-lipped ecomorphs had a
notable genetic divergence with sympatric non-lipped ecomorphs based on
nuclear SNPs data (FST = 0.21). Sympatric pairs can be sorted by
combinations of phenotypic, ecological, and genetic divergence within
the speciation continuum from an ecologically non-functional mouth
polymorphism via ecologically functional polymorphism to completed
speciation via divergent evolution.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Phenotypic variation provides the crucial basis for divergent selection
to act upon and is the source for further evolution of novel
morphological and ecological diversity. Variation in trophic morphology
is of particular interest to evolutionary biologists since the
partitioning of trophic resources is considered one of the main
prerequisites for ecological speciation and adaptive radiation (Meyer,
1987; Sibbing et al., 1998; R\selectlanguage{ngerman}über et al., 1999; Rundle et al., 2000).
Linking phenotypic traits to an adaptive ecological function requires
information at different levels of biological organization. Moreover, it
remains uncertain what is primary during adaptive radiation --
behavioral or ecological adaptation that precedes morphological
specialization or morphological novelty promoting filling of the new
ecological niches (reviewed in Schluter, 2000). Adaptive radiations
based on trophic resource partitioning have been studied in fishes such
as cichlids, coregonids, Arctic charr, three-spined sticklebacks, and
many other lineages of fish (Skulason, Smith, 1995; Schluter, 2000;
Sibbing, Nagelkerke, 2000; Barluenga et al., 2006; Martin and
Wainwright, 2011; Seehausen and Wagner, 2014; Burress, 2016). Cyprinid
fishes, Family Cyprinidae \emph{sensu lato} , is one of the most
diversified families of Actinopterygii (\textgreater{} 3000 species --
Fricke et al., 2022) that contains many adaptive radiations (e.g.,
Kornfield, Karpenter, 1984; Savvaitova et al., 1987; Nagelkerke et al.,
1994; Mina et al., 1996; Levin et al., 2020; 2021a; Komarova et al.,
2021), most of which, however, have not yet been studied using
genome-wide approaches.

Large African barbs of the genus \emph{Labeobarbus} Rüppell, 1835 are a
remarkably diverse lineage of polyploid cyprinid fishes (2n=150 --
Oellermann, Skelton, 1990; Golubtsov, Krysanov, 1993) with
\textgreater{} 130 species (Fricke, 2022) that display great diversity
as well as the distinct polymorphisms in mouth phenotypes (Banister,
1973). This diversity in trophic morphology is part of the explanation
for why in the genus \emph{Labeobarbus} numerous adaptive radiations
based on tropic resource partitioning evolved. Such radiations are found
in both lacustrine (e.g., the Lake Tana radiation is composed of 15
ecomorphs/species) as well as riverine environments (Nagelkerke et al.,
1994; Mina et al., 1996; Levin et al., 2019; 2020; Mironovsky et al.,
2019). Four major trophic phenotypes among \emph{Labeobarbus} have been
described: i) generalized; ii) algae scraping (with subtypes); iii) with
hypertrophied lips (thick-lipped); and iv) large-mouthed, or piscivorous
(with subtypes) (Banister, 1973; Nagelkerke et al., 1994; Mina et al.,
1998; Vreven et al., 2016; Levin et al., 2020). Remarkably, the very
similar mouth phenotypes repeatedly evolved throughout the range
of\emph{Labeobarbus} in Sub-Saharan Africa (Tsigenopoulos et al., 2010;
Levin et al., 2013; 2020; Vreven et al., 2016). One of the trophic types
-- the thick-lipped phenotype -- is of particular interest to us and is
characterized by hypertrophied lips with a well-developed fleshy lobe on
the lower jaw, which looks like a fleshy appendage on the chin (Fig. 1
and 2B). The feeding preferences of fishes with such hypertrophied lips
was most comprehensively studied in cichlids of both African and
American clades (e.g., Ribbink et al., 1983; Stiassny and Meyer 1999;
Colombo et al., 2012; Manousaki et al., 2013; Baumgarten et al., 2015;
Machado-Schiaffino et al.~2017; Torres-Dowdall \& Meyer, 2021; Sowersby
et al., 2021). The cichlid species or ecomorphs with hypertrophied lips
(also known as ``rubberlip'') display also other adaptive features (like
narrow and pointed head shape etc. -- see also Franchini et al. 2014;
Fruciano et al. 2016) evolved a new foraging strategy in rocky crevices
thereby occupying a new ecological niche. In particular, hypertrophied
lips might increase suction power by sealing cracks and grooves and also
protect the head from injury from prey on hidden benthic organisms
(Oliver \& Arnegard, 2010; Baumgarten et al., 2015). Thickened lips may
also allow these cichlid species to achieve higher numbers of taste
receptors (Schemmel, 1967; Oliver \& Arnegard, 2010; Manousaki et al.
2013).

The thick-lipped mouth phenotype is one of the most frequently occurring
within the \emph{Labeobarbus} lineage (Banister, 1973; Vreven et al.,
2016). Some individuals have such greatly hypertrophied lips that
pioneering investigator E. Rüppell assigned a new generic name to them
based on this conspicuous trophic feature (Rüppell, 1835). Genetic data
(mtDNA) support numerous parallel origins of the thick-lipped phenotype
among \emph{Labeobarbus} (Tsigenopoulos et al., 2010; Levin et al.,
2020; Decru et al., 2022). Nevertheless, the ecological role of the
thick-lipped ecomorphs in \emph{Labeobarbus} is not yet known. The
feeding preferences or trophic position of the thick-lipped ecomorph was
investigated so far for two cases only. One study (Sibbing \&
Nagelkerke, 2001) investigated the diet composition of the lacustrine
lipped phenotype of \emph{L.} cf. \emph{intermedius} (known also
as\emph{L. nedgia} Rüppell, 1835) in Lake Tana (East Africa, Ethiopia).
Another study (Levin et al., 2019) examined nitrogen and carbon stable
isotope signatures in riverine lipped phenotype of \emph{L.
gananensis}from the Genale River (Ethiopia). Their results provided weak
support for trophic resource partitioning between fish with
hypertrophied and normally developed lips. So weak trophic resource
partitioning between sympatrically co-occurring ecomorphs with
strikingly divergent trophic morphology turned out to be an intriguing
finding. In general, whether such mouth structure transition is just
non-adaptive phenotypic variation within highly polymorphic
\emph{Labeobarbus} lineage or thick lips are an adaptive trait involved
in trophic resource partitioning remains unclear so far. To address this
question, similar cases in a broader taxonomic and geographical context
have been studied by us. We could show that thick-lipped ecomorphs
evolved several times independently in several Ethiopian river basins.
Yet, paradoxically, the hypertrophied ecomorphs were indistinguishable
by mtDNA from sympatric ecomorphs with a generalized mouth phenotype
(Levin et al., 2020).

Our goals were two-fold. First, we aimed to test whether hypertrophied
lips are adaptive within \emph{Labeobarbus} as judged by information on
trophic resource partitioning, i.e., whether a thick-lipped ecomorph
occupies a separate trophic niche or whether these trophic variants
might be explained best by phenotypic plasticity alone. To do this, we
compared the trophic morphology, diet and stable isotopes signatures in
generalized and thick-lipped forms. Second, we tested the hypotheses
about genetic divergence between and reproductive isolation within
sympatric pairs of thick-lipped and generalized small-lipped ecomorphs
based on genome-wide genetic data obtained by double digest
restriction-site associated DNA (ddRAD).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1.} Phenotypes of generalized and thick-lipped ecomorphs
of the \emph{Labeobarbus} spp. from rivers of the Ethiopian Highlands.
Mature fish in ``alive'' coloration and their heads (bottom view) are
shown.

\textbf{Materials and methods}

\emph{Study Sites.} ~Sampling was done under the framework of the Joint
Ethiopian-Russian Biological Expedition (JERBE) in six rivers draining
the Ethiopian Highlands and belonging to its four major river basins
(Fig. 2): (1) the White Nile basin -- the Birbir R. -- 8.7364\selectlanguage{ngerman}° N
35.3518° E, and the Sore R. at two locations -- at the City of Metu --
8.3178° N, 35.5951° E -- and \textasciitilde{}35 km downstream along the
river course -- 8.3987° N, 35.4378° E; (2) the Blue Nile basin -- Blue
Nile at the City of Dejen -- 10.0775° N 38.1934° E -- and the Didessa
R., a tributary of the Blue Nile -- 8.6921° N 36.4144° E; (3) the
Omo-Turkana enclosed basin -- the Gojeb R., a tributary of the Omo R. --
7.2539° N 36.7943° E; (4) the Juba-Wabe-Shebelle basin in the Indian
Ocean catchment -- the Genale R. -- 5.7025° N 39.5446° E. Fish were
caught by gill and cast nets in March 2014 (Birbir), February--March
2011 (Didessa), March--April 2009 (Genale), February 2011 (Gojeb), April
2014 (Sore), and January 2022 (Blue Nile). Fish were killed with an
overdose of MS-222 anaesthetic and then photographed using a Canon EOS
50D camera (Canon Inc. Ota City, Tokyo, Japan). Standard length (SL, mm)
was measured with a ruler. Fish were preserved first in 10\% formalin
and then transferred to 70 \% ethanol. All specimens are deposited at
A.N. Severtsov Institute of Ecology and Evolution, the Russian Academy
of Sciences, Moscow under provisional labels of JERBE. In total, up to
296 samples were investigated by a set of various methods (Table 1).

\par\null

\textbf{Table 1.} Sample size of the generalized and thick-lipped
ecomorphs of \emph{Labeobarbus} for various analyses.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Ecomorphs} & \textbf{Morphology} & \textbf{Gut Length} &
\textbf{Diet} & \textbf{Stable Isotopes} & \textbf{mtDNA} &
\textbf{ddRAD}\tabularnewline
\textbf{Birbir R.} & \textbf{Birbir R.} & \textbf{Birbir R.} &
\textbf{Birbir R.} & \textbf{Birbir R.} & \textbf{Birbir R.} &
\textbf{Birbir R.}\tabularnewline
Generalized & 33 & 30 & -- & 17 & 25 & 4\tabularnewline
Thick-lipped & 2 & 1 & -- & 2 & 3 & 1\tabularnewline
\textbf{Blue Nile R.} & \textbf{Blue Nile R.} & \textbf{Blue Nile R.} &
\textbf{Blue Nile R.} & \textbf{Blue Nile R.} & \textbf{Blue Nile R.} &
\textbf{Blue Nile R.}\tabularnewline
Generalized & 31 & 15 & 15 & 23 & 21 & -\tabularnewline
Thick-lipped & 5 & 7 & 7 & 2 & 2 & -\tabularnewline
\textbf{Didessa R.} & \textbf{Didessa R.} & \textbf{Didessa R.} &
\textbf{Didessa R.} & \textbf{Didessa R.} & \textbf{Didessa R.} &
\textbf{Didessa R.}\tabularnewline
Generalized & 10 & 15 & 6 & 20 & 20 & 5\tabularnewline
Thick-lipped & 6 & 6 & 2 & 7 & 10 & 5\tabularnewline
\textbf{Genale R.} & \textbf{Genale R.} & \textbf{Genale R.} &
\textbf{Genale R.} & \textbf{Genale R.} & \textbf{Genale R.} &
\textbf{Genale R.}\tabularnewline
Generalized & 25 & 38 & 12 & 21 & 32 & 22\tabularnewline
Thick-lipped & 13 & 24 & 8 & 14 & 17 & 8\tabularnewline
\textbf{Gojeb R}. & \textbf{Gojeb R}. & \textbf{Gojeb R}. &
\textbf{Gojeb R}. & \textbf{Gojeb R}. & \textbf{Gojeb R}. &
\textbf{Gojeb R}.\tabularnewline
Generalized & 34 & 45 & 17 & 40 & 30 & 7\tabularnewline
Thick-lipped & 21 & 27 & 14 & 19 & 17 & 4\tabularnewline
\textbf{Sore R.} & \textbf{Sore R.} & \textbf{Sore R.} & \textbf{Sore
R.} & \textbf{Sore R.} & \textbf{Sore R.} & \textbf{Sore
R.}\tabularnewline
Generalized & 59 & 75 & -- & 59 & 25 & 1\tabularnewline
Thick-lipped & 13 & 13 & -- & 13 & 11 & 6\tabularnewline
\textbf{Total} & \textbf{252} & \textbf{296} & \textbf{81} &
\textbf{237} & \textbf{213} & \textbf{63}\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. A} : Map with sampling sites of
\emph{Labeobarbus}sympatric pairs (generalized and thick-lipped
ecomorphs) from rivers of the Ethiopian Highlands; the map was created
using QGIS v.3.16.4.\textbf{B} : Scheme of lips measurements: LLL --
lower lip length, MLL -- middle lobe length, ULW -- upper lip width.

\emph{Morphology.} The head length (\emph{C} ), middle lobe length
(\emph{MLL} ), lower lip length (\emph{LLL} ), and upper lip width
(\emph{ULW} ) were measured in CorelDRAW 2017 (v. 19.1.0.419) using
photographs. Scheme of measurements is given on Fig. 2B. The indexes
of\emph{MLL} , \emph{LLL} , and \emph{ULW} to head length (\emph{C} )
were used for subsequent analyses.

\emph{Gut Length.} Gut length (\emph{GL} ) was measured with a ruler (to
the nearest 1 mm). A ratio \emph{GL} to \emph{SL} expressed as \% was
used for analyses.

\emph{Diet} . Gut content was dried on filter paper and weighed using a
Pioneer PX84/E balance with 0.0001 g accuracy. The diet particles were
identified using Olympus CX41 microscope (100--1000\selectlanguage{ngerman}× magnification) and
Motic DMW-143-N2GG stereomicroscope (100--400× magnification). The diet
components were grouped into: (i) detritus, (ii) invertebrates, (iii)
macrophytes and (iv) fish. The group `Invertebrates' included the larvae
of amphibiotic insects, and their fragments. The group `Macrophytes'
included any fragments of plants---such as leaves, stems or seeds. A
composite measure of diet, an index of relative importance (\emph{IR} )
{[}Natarajan, Jhingran, 1961; Popova, Reshetnikov, 2011{]}, was used to
assess the contribution of different components to the diet.
The\emph{IR} index was calculated as follows:\(IR\ =\ \ \frac{Fi\ \times\ Pi}{\sum(Fi\ \times\ Pi)}\ \times\ 100\%\), where
\emph{Fi} = the frequency of occurrence of each food group, and\emph{Pi}
= its part by weight; the value of \emph{i} itself changes from 1 to
\emph{n} (\emph{n} = the part of food organisms in the food bolus).

\emph{Stable Isotope Composition.} For stable isotope (SI) analyses,
white muscle tissue from the dorsal side of the body under the dorsal
fin was sampled from freshly collected specimens. White muscle samples
were dried at 60 °C for subsequent SI analyses. The samples were weighed
using a Mettler Toledo MX5 microbalance (Mettler Toledo, Columbus, OH,
United States) with 2 \selectlanguage{greek}μ\selectlanguage{english}g accuracy, and were wrapped in tin capsules. The
weight of the fish tissue samples varied from 250 to 500 \selectlanguage{greek}μ\selectlanguage{english}g. SI analysis
was conducted at the Joint Usage Center of the A.N. Severtsov Institute
of Ecology and Evolution RAS, Moscow. Briefly, a Thermo Delta V Plus
continuous-flow IRMS was coupled to an elemental analyzer (Flash 1112)
equipped with a Thermo No-Blank device. The isotopic composition of N
and C was expressed in the %0. Along with the isotopic analysis, the
nitrogen and carbon content (as \%) and C/N ratios were determined. In
total, 237 white-muscle samples were analyzed.

\emph{Statistical Analyses of Morphological and Ecological Data.}Several
R packages and functions were used for the statistical analyses and plot
construction. Basal descriptive statistics was obtained using the
\emph{summarytools} library {[}Comtois, 2022{]}. The Mann-Whitney U test
was applied for pairwise comparison of the lipped and generalized
ecomorphs in lip characters \emph{MLL} , \emph{LLL} , \emph{ULW} , gut
length \emph{GL} , and SI composition within each locality using the
function \emph{wilcox.test} (in package \emph{stats} ) {[}R Core Team,
2021{]}. The Pearson correlation and the violin boxplots were obtained
using the \emph{ggplot2} library {[}Wickham, 2022{]}. Principal
component analysis (PCA) was done using the \emph{prcomp} function. For
visualization of PCA results, the packages \emph{factoextra} library
{[}Kassambara, Mundt, 2020{]}, \emph{ggfortify} library {[}Tang et al.,
2016{]}, and \emph{ggplot2} library {[}Wickham, 2022{]} were used. The
package SIBER v.2.1.6 {[}Jackson et al., 2011{]} was used to assess the
differences in the isotopic trophic niche features. The total convex
hull areas (TA), core trophic niche breadths, and sample size-corrected
standard ellipse area (SEAc) were estimated. The trophic overlap for
95\% TA was estimated using nicheROVER {[}Lysy et al., 2021{]}, a method
that is insensitive to the sample size and incorporates statistical
uncertainty using Bayesian approach {[}Swanson et al., 2015{]}.

\emph{DNA sampling, extraction, amplification, sequencing, and analysis
-- mtDNA data.} DNA samples (\emph{n} ~=~213) were collected from both
generalized and lipped ecomorphs of~\emph{Labeobarbus} ~from the same
six localities in Ethiopian Highlands (Fig. 2B; Supplementary
Table~S1~for details). Total genomic DNA was extracted from
ethanol-preserved fin tissues using the QIAamp DNA Micro~kit (Qiagen).
Sequences of the mitochondrial gene cytochrome~\emph{b} ~(\emph{cytb} ),
1038~bp in length, were amplified (polymerase chain reaction {[}PCR{]}
conditions were taken from: Palumbi,~1996; Perdices \& Doadrio,~2001).
PCR products were visualized on 1.5\% agarose gels, purified with
ExoSAP-IT and sequenced using an ABI 3500~sequencer. Some sequences were
obtained previously (Levin et al., 2020; GenBank accession nos. are
given in Table~S1) while new sequences obtained in this study were
deposited in GenBank under accession nos.~OQ604627-OQ604649~(see
Table~S1~for details). All sequences were aligned and edited using
the~muscle~algorithm (Edgar,~2004) as implemented in~mega~6.0 (Tamura et
al.,~2013). The data final set is comprised of 213~\emph{cytb}
~sequences.~A haplotype network was constructed using the median joining
algorithm (Bandelt et al.,~1999) in~popart~1.7 (Leigh \& Bryant,~2015)
with the default value of epsilon (0).

\emph{ddRAD-seq library preparation} . High-molecular-weight DNA was
isolated from fin tissue preserved in ethanol using a QIAamp DNA Mini
Kit (Qiagen) or obtained with a salt-based DNA extraction method
(Aljanabi \& Martinez,~1997) followed by purification using a CleanUp
Standard kit (Evrogen). The quantity of dsDNA was measured using a dsDNA
HS Assay Kit for fluorometer Qubit 3 (Life Technologies). A
ddRAD-library was constructed following the quaddRAD protocol (Franchini
et al.,~2017) using restriction enzymes~\emph{Pst} I and~\emph{Msp} I.
In total, 63 DNA samples of~\emph{Labeobarbus} ~ecomorphs from five
riverine basins (see Table~2) were sequenced by two runs of Illumina
HiSeq2500 and Illumina X Ten (2~\selectlanguage{ngerman}×~150~bp paired-end reads). The raw
sequencing data from 63 samples were demultiplexed by the sequencing
provider using outer Illumina TruSeq dual indexes.

\emph{Processing of RAD-seq data.} Read quality was assessed
with~fastqc~0.11.7 (Andrews \& Krueger,~2010) and~multiqc~1.13 (Ewels et
al.,~2016). Further demultiplexing of individually barcoded samples,
construction and cataloging of RAD-loci and single nucleotide
polymorphism (SNP) calling were done with~stacks~2.62 (Rochette et
al.,~2019). Identification and removal of PCR duplicates were done using
the~\emph{``clone\_filter''} ~module of stacks. The stacks module
\emph{``process\_radtags''} was used to demultiplex reads by the dual
index inner barcodes and obtain separate fastq files for each
individual. Demultiplexed reads were trimmed at their 5\selectlanguage{english}'- and 3\selectlanguage{english}'- ends
by 5 bp to a uniform length of 140~bp using fastp 0.20.1 (Chen et al.,
2018) to reduce the influence of sequencing error (due to decreased base
quality at both ends). Samples that failed to produce more than 100,000
reads were excluded from further processing. The `\emph{integrated'}
approach of~stacks~was used to assemble loci \emph{de novo} and perform
genotype calling after mapping assembled loci to a heterospecific
high-quality reference genome of \emph{Barbus barbus} (GenBank assembly
accession: GCA\_936440315.1) and filtering. We selected optimal
parameters for \emph{de novo} loci assembly using the approach suggested
by Paris et al. (2017). Following the aforementioned procedure, we found
that a minimum stack depth (-\emph{m} ) of 6, distance allowed between
stacks (-\emph{M} ) of 1 and maximum distance required to merge catalog
loci (-\emph{n} ) of 1 provided the best balance between data quality
and quantity for our data set. We also set --max\_locus\_stacks
parameter to 7 to improve binning and avoid paralogs (Stobie et al.
2017). We align the loci catalog generated in the \emph{de novo}
assembly to the reference genome using bwa mem v.0.7.17 (Li \& Durbin,
2009) with default settings. To avoid spurious alignments of \emph{de
novo} loci `stacks-integrate-alignments' was run with minimum alignment
coverage and percent identity are both set to 0.8.

\emph{Population genomic and phylogenomic analyses} . To prove that
sympatric ecomoprhs of \emph{Labeobarbus} from different basins are
independent evolutionary units we used maximum likelihood phylogenetic
analysis based on SNPs in iq-tree~1.6.12 (Minh et al.,~2020). Multiple
sequence alignment (MSA) of SNPs were created using the ``--phylip-var''
option of the ``\emph{populations} '' module of~stacks with retention of
loci genotyped in at least 70\% of all samples and SNPs with a minor
allele count above 3. Heterozygous sites within each individual were
encoded using IUPAC notation. Invariant sites (arising due to missing
data) were excluded from the MSA by IQ-TREE, resulting in 15,820
nucleotide sites. To take into account the absence of constant sites an
ascertainment bias correction (+ASC) model (Lewis, 2001) was applied to
all substitution models in a best-fit model selection process with
ModelFinder (REF). Branch support values were obtained using an
ultrafast bootstrap procedure (Hoang et al.,~2018) with 1,000
replicates. The phylogenetic tree was visualized using~figtree~1.4.4
(Rambaut,~2014).

STRUCTURE 2.3.4 (Pritchard et al., 2000) was used to examine population
structure of the whole dataset and within each pair of ecomorphs (i.e.
basin). First, all individual genotypes were filtered and tested for
deviations from Hardy--Weinberg Equilibrium (HWE) using the
``\emph{populations} '' module of~stacks~with the following settings:
(i) loci genotyped in at least 80\% of all samples were kept; (ii) SNPs
with a minor allele count (--min-mac) less than 3 were pruned. Next, we
whitelisted loci that are in HWE for all populations and created a
structure-formatted file with ``\emph{populations} '' while retaining a
single random SNP per locus to avoid inclusion of closely linked SNPs.
This dataset consisted of 2,411 SNPs. We performed 16 independent runs
(100,000 chains as burn-in plus 100,000 MCMC chains) of STRUCTURE for
each K between 1 and 8 using the admixture model with correlated allele
frequencies. Structure\_threader (Pina-Martins et al., 2017) was used to
parallelize the runs. Results of the Structure runs were summarized
using CLUMPAK webserver (Kopelman et al., 2015) with default settings.
The optimal K-value was determined by the approaches of Pritchard et al.
(2000) and Evanno et al. (2005). The same protocol was followed for
consecutive hierarchical STRUCTURE~runs for the identified clusters
until no subdivision was revealed (i.e., K = 1). For these runs we
created new SNP sets using the same procedure as described above but
limiting genotypes to the individuals from identified genetic clusters.
In addition, Principal Component Analysis (PCA) was performed using the
glPca function of the ADEGENET 2.1.1 R-package (Jombart and Ahmed,
2011). Pairwise Reich--Patterson~\emph{F} \textsubscript{ST}~values
(Reich et al.,~2009) with respective 95\% confidence intervals for
ecomorphs/genetic clusters were calculated using the R script from
Junker et al. (2020). The PCA and Reich--Patterson~\emph{F}
\textsubscript{ST}~calculations were done on the aforementioned 2,411
SNPs dataset.

\textbf{Results}

\textbf{Morphology}

\emph{Lip size.} Principal component analysis confirmed significant
divergence between sympatric pairs of lipped and generalized ecomorphs
in PC1 and PC2 space in all studied rivers (Fig. 3A). In three of the
six ecomorph comparisons non overlap was detected and the distributions
in PC-space were only weakly overlapping for other three comparisons of
sympatric ecomorphs. PC1 explained from 45.4 (Sore R.) to 71.7\%
(Didessa R.) of the variance, while PC2 explained less than 36\%.
Eigenvectors of the characters for all PCs are given in Supplementary
Table S2.~ Middle lobe length (MLL) and upper lip width (ULW) had the
highest contribution to divergence of sympatric ecomorphs.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. A} : PCAs of sympatric pairs of generalized vs.
thick-lipped ecomorphs of \emph{Labeobarbus} spp. that live
sympatrically in each of six rivers: Birbir, Blue Nile, Didessa, Genale,
Gojeb, and Sore. \textbf{B} : Food spectra (IR: the index of relative
importance) of the sympatric ecomorphs (G -- generalized, L --
thick-lipped) of the \emph{Labeobarbus} spp. from the Blue Nile,
Didessa, Genale, and Gojeb.

The thick-lipped ecomorph was characterized by greatly developed middle
lobes on the lower lip (MLL) that was significantly longer compared to
the generalized ecomorph in all studied rivers (Supplementary Figure
S3). Individual MLL values varied from 10 to 33\% in the thick-lipped
ecomorph (averaging -- 21\%) while in the generalized ecomorph they were
much lower -- from 4 to 16\% (averaging -- 9\%). Largest MLL values
within thick-lipped ecomorph were detected in the Birbir River (27\%)
while the smallest one -- in the Sore River (16\%) and Blue Nile River
(16\%).

\emph{Gut Length.} The guts of thick-lipped ecomorph were shorter than
in generalized ecomorph from all studied rivers, sometimes significantly
(Supplementary Figure S3). The gut length of thick-lipped ecomorph
varied from 140.0 to 507.8\% SL, averaging -- 305.1\%. The gut length of
generalized ecomorph varied from 157.2 to 646.1\%, averaging -- 332.9\%.
Significant differences between sympatric thick-lipped and generalized
ecomorphs were detected in the Blue Nile (p\textless{}0.01), Genale
(p\textless{}0.05) and Gojeb (p\textless{}0.01) (Wilcoxon test).

\textbf{Trophic divergence}

\emph{Diet.} Food spectra of sympatric ecomorphs of
\emph{Labeobarbus}spp. were rather diverse and composed of i) detritus,
ii) invertebrates (mainly insects: Ephemeroptera, Hemiptera,
Trichoptera, Coleoptera, Hymenoptera, and Diptera), iii) macrophytes
(remnants of helophytic and semi-aquatic plants, represented by seeds,
leaves, stems, and flower parts, sometimes coupled with filamentous
algae), and occasionally iv) fish (juvenile parts or scales). Feeding of
sympatric ecomorphs in all four rivers studied was divergent but at
various degree as estimated by index of relative importance (Fig. 3B).

Detritus was the main component among food items in both generalized
(63.8-92.1\%) and thick-lipped ecomorphs (27.1--79.0\%) (Fig. 3B).
Generally, the diet of the thick-lipped ecomorph was more insectivorous
compared to generalized ecomorph in all rivers studied (Figure 3B).
Invertebrates in the diet of both ecomorphs were represented by larvae
of Ephemeroptera and Trichoptera, larvae and imagoes of Diptera and
Coleoptera as well as imagoes of Hemiptera, and Hymenoptera.
Contributions of invertebrates for fish feeding might be underestimated
due to their quick digestion. This is why we also provide data on N and
C stable isotope composition (see below). Detailed description of the
diet of each ecomorph in certain rivers see in Supplementary material
S4.

\emph{Stable Isotope Composition.}Basic statistics for both
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N and \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values is given in
Supplementary Table S5. The thick-lipped ecomorph had higher
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N values that generalized one in all comparisons
with statistically significant difference in four of six paired
comparisons: Birbir, p \textless{} 0.05; Didessa, p \textless{} 0.01;
Gojeb, p \textless{} 0.01; and Sore, p \textless{} 0.05 (Fig. 4 and
Supplementary Fig. S6). When significant, the difference in mean
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N values between sympatric generalized and
thick-lipped ecomorphs varied from 1.9%0 (Didessa) to 0.9%0 (Sore).
Significant differences in \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C values between
sympatric pairs were found in the Didessa, Gojeb, and Sore Rivers. In
all these cases, the lipped ecomorph had higher \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{13}C
values (the difference varied from 1.6%0 to 1.2%0).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} SI Bayesian ellipses showing trophic niche widths and
overlaps in sympatric ecomorphs of the \emph{Labeobarbus} spp. from the
Birbir, Blue Nile, Didessa, Genale, Gojeb, and Sore Rivers. Ellipses
with 95\% confidence intervals are based on standard ellipses corrected
for small sample sizes (SEAc; isotopic niche metrics; SIBER package).
Each point corresponds to the isotopic value.

The total area (TA), standard ellipse area (SEA), and corrected standard
ellipse area (SEAc) were analyzed for all sympatric ecomorphs except for
thick-lipped ecomorphs from the Blue Nile and Birbir Rivers because of
small sample size. The thick-lipped ecomorph had largest SEAc values in
all rivers apart the Genale River (Supplementary Table S7). The niche
overlap between generalized and thick-lipped ecomorphs consisted 25.7\%
in the Didessa River, 57.2\% in the Gojeb River, and was considerably
larger in the Sore and Genale Rivers (81.6 and 82.3\%, respectively)
(Supplementary Table S8).

\textbf{Mitochondrial DNA divergence}

Among the 213 individuals sampled, 49 different haplotypes were
detected. The haplotype network is complex (Fig.~5) and composed of five
main haplogroups that correspond to i) Blue Nile basin (including the
Didessa R.), ii) Genale R. (Indian Ocean basin), iii) Gojeb R.
(Omo-Turkana basin), iv) Birbir R. (White Nile basin), and v) Sore R.
(White Nile basin) (Fig. 5). A few samples from the Didessa R. and
Birbir R. were close to central putative haplotypes interconnecting the
analyzed haplogroups. Genetic \emph{p} -distances between the geographic
basins varied from 0.009\selectlanguage{ngerman}±0.003 to 0.031±0.005 (Supplementary Table S9).
No haplotype sorting between sympatric generalized and thick-lipped
ecomorphs within each basin was revealed (Fig. 5).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig. 5. Median-joining haplotype network of the generalized and
thick-lipped ecomorphs of the \emph{Labeobarbus} spp. from four main
drainages of the Ethiopian Highlands constructed on the basis of
213~cyt\emph{b} ~sequences. Haplotypes of the generalized ecomorphs are
colored more intensively in each case of sympatry.

\textbf{Phylogenetic relationships and genetic population structure
inferred from nuclear genome}

Among 595,163 \emph{de novo}catalog loci generated with STACKS \emph{de
novo} pipeline and mapped to the \emph{B. barbus} genome 251,956 loci
had multiple alignments, 322,820 had one alignment and 20,387 were
unmapped. The `stacks-integrate-alignments' procedure filtered out
371,899 loci due to mapping quality, 97,561 due to alignment coverage,
and 12,020 due to percent identity, resulting in 93,296 loci retained
for downstream analyses (detailed statistics on each sample including
raw reads is given in Supplementary material S10).

The phylogeny of Ethiopian \emph{Labeobarbus} (Figure 6A) based on
15,820 SNPs is generally congruent with that based on mtDNA data (Levin
et al., 2020 and Figure 6A). Sympatric generalized and thick-lipped
ecomorphs clustered together and form monophyletic lineages with high
support for all studied basins/subbasins (90-100 bootstrap values)
except for the Gojeb (62). The \emph{Labeobarbus} lineages subdivided
into two clades representing i) Eastern (\emph{L. gananensis} from
Genale River, Indian Ocean basin) and ii) Western (\emph{L.}
cf.\emph{intermedius} sensu lato from the Didessa, Gojeb, Sore, and
Birbir Rivers) parts of the Ethiopian Highlands (Fig. 6A). The \selectlanguage{russian}сlade of
the Western Plateau is further subdivided for the Northern subclade
(Didessa River in the Blue Nile basin) and the Southern subclade that
combines the Gojeb (Omo-Turkana basin), Sore, and Birbir (White Nile
basin) populations. The Southern subclade is further subdivided into
lineages according to geographical basin belonging to the Gojeb
(Omo-Turkana) lineage and White Nile lineage comprising the Sore and
Birbir populations. Remarkably, the Sore and Birbir populations also
form the sister monophyletic sublineages within White Nile lineage (Fig.
6A).

An analysis of the population genetic structure revealed an optimum of
four (\emph{K} ) clusters that correspond to the (i) Genale, (ii)
Didessa, (iii) Gojeb, and iv) White Nile populations from the Sore and
Birbir Rivers (Figure 6A). Five samples from the Gojeb River have a
little admixture (\textasciitilde{}10\%) from the White Nile cluster.
When each basin with sympatric ecomorphs was analyzed independently,
only one river with subdivision by ecomorphs was revealed -- the Didessa
in the Blue Nile basin (Fig. 6A). Individuals from White Nile basin are
further subdivided by geographical populations from the Sore and Birbir
Rivers (Fig. 6A). PCA of the 2,411 SNPs confirmed six well-defined
clusters that correspond to the phylogenetic and population genomics
results including subdivision of generalized and thick-lipped ecomorphs
in the Didessa (Fig. 6B).

All Reich\emph{F} \textsubscript{ST} pairwise comparisons between
inferred genetic clusters were statistically significant with values
ranging from 0.21 (0.18-0.25 95\%CI) between Didessa generalized
ecomorph and Didessa thick-lipped ecomorph to 0.66 (0.63-0.68) between
Sore and Genale (Supplementary Data S11).\emph{F}
\textsubscript{ST}pairwise comparisons of the pairs of sympatric
ecomorphs within each basin (Supplementary Data S12) was significant
only for Didessa, where ecomorphs constitute well segregated genetic
pools.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6} . A: Maximum likelihood SNP-based phylogeny and
genetic structure of generalized and thick-lipped ecomorphs
of\emph{Labeobarbus} from the different basins of Ethiopia (Genale,
Didessa, Gojeb, Sore, and Birbir Rivers). The tree inferred with
IQ-TREE2 using 15,820 variant SNPs with the TVM+F+ASC+R3 substitution
model. Heterozygous sites within each individual were encoded using
IUPAC notation. The thick-lipped individuals in the tree are depicted in
yellow color. Individuals are labeled by their voucher numbers.
Ultrafast bootstrap values above 70\% are shown as numbers near the
corresponding nodes, while the black points in the nodes designate
95-100\% support. The genetic cluster proportions inferred by
hierarchical STRUCTURE analysis are shown to the right of sample
numbers: 1 - analysis of all samples with 2,411 SNPs revealed best K=4;
2 - analysis of the Genale genetic cluster with 7,905 SNPs revealed no
further subdivision; 3 - analysis of the Didessa genetic cluster with
6,932 SNPs revealed subdivision on two clusters (best K=2) corresponding
to mouth phenotypes; 4 - analysis of the Gojeb genetic cluster with
4,471 SNPs revealed no further subdivision; 5 - analysis of the
Sore/Birbir (White Nile basin) genetic cluster with 2,020 SNPs revealed
subdivision on two clusters (best K=2) corresponding to the river
basins. B: Principal Component Analysis (PCA) plot with points
representing individuals and 95\% confidence intervals colored by
genetic clusters inferred with STRUCTURE.

\textbf{Discussion}

The obtained results provide evidence for a continuum from phenotypic
polymorphism \emph{via} trophic resource partitioning to ecological
speciation as exemplified from six cases of independently evolved
thick-lipped \emph{Labeobarbus} ecomorphs from Ethiopian Highlands.

\textbf{Thick-lipped mouth and trophic divergence}

Although sympatric generalized and thick-lipped ecomorphs were clearly
divergent in lip size in all six pairs, not all have partitioned trophic
resources as might be expected from their phenotypic divergence.
Generally, the diet of lipped ecomorph was enriched with benthic
invertebrates compared to that of the generalized ecomorph with normally
developed lips. However, the difference in amount of consumed benthic
invertebrates between lipped and non-lipped ecomorphs varied
significantly in different rivers. As one might expect, the sympatric
pairs with larger differences in diet also showed significant divergence
in SI signatures (e.g., in the Didessa and Gojeb) that confirmed usage
of SI composition as diet proxy. Lipped ecomorphs had higher \selectlanguage{greek}δ\selectlanguage{english}15N values
compared to the generalized ecomorphs in all six comparisons (extra
0.2-1.9%0). Significant enrichment of the lipped ecomorph in \selectlanguage{greek}δ\selectlanguage{english}15N (4
pairs: extra 0.9-1.9%0) was usually accompanied by significant enrichment
in \selectlanguage{greek}δ\selectlanguage{english}13C (3 of 4 pairs: extra 1.3-1.7%0). Nevertheless, SI Bayesian
ellipses showed great trophic niche widths and overlaps in half of the
comparisons of pairs of sympatric ecomorphs (Fig. 4). When thick-lipped
ecomorph partitioned trophic resource with co-occurring generalized
ecomorph, it was more specialized to feed on aquatic invertebrates
similar to the feeding mode of thick-lipped cichlids that provides more
examples of convergent evolution among fishes. The variation in diet
divergence between sympatric pairs of thick-lipped and
generalized\emph{Labeobarbus} ecomorphs is similar to other cases of
sympatrically co-occurring thick-lipped and thin-lipped cichlid fish
such as ecomorphs/species of the genus \emph{Amphilophus} Agassiz, 1859
from various Nicaraguan lakes (Elmer et al., 2010; Kautt et al., 2012;
Manousaki et al., 2013).

Thus, hypertrophied lips in the \emph{Labeobarbus} as a phenotypic trait
is not sufficient to predict the diet of fish. The same was found not
only for riverine populations but also for lacustrine. For instance, the
thick-lipped ecomorph in Lake Tana had almost the same food spectrum as
sympatric generalized ecomorph (Sibbing \& Nagelkerke, 2001). This is
within Liem's paradox, i.e., that even species with specialized trophic
morphologies have dietary flexibility (Liem, 1980). Many examples
corroborate Liem's paradox suggesting it is a common phenomenon among
fishes (e.g., Sturmbauer et al., 1992; Robinson \& Wilson, 1998; Binning
et al., 2009; Wagner et al., 2009; Golcher-Benavides et al., 2019). This
phenomenon reduces the prediction of diet by phenotype and provides
evidence for the greater trophic plasticity of specialists (see also
recent examples from scraping feeders in Komarova et al., 2021; 2022).
Previous experimental studies on various diets exposed to Neotropical
and East African cichlid species showed significant phenotypic
plasticity provoked by diet (Meyer, 1987; Muschick et al. 2011,
Schneider et al., 2014). It suggests that not only certain phenotypes
considered as trophically specialized may be plastic in relation to diet
but that phenotypic plasticity may also rise from a different diet. This
coincides with a flexible stem hypothesis on the origin of adaptive
radiations from ancestral flexible stem (West-Eberhard, 2003; Wund et
al., 2008; Schneider et al., 2014; Schneider \& Meyer, 2017; Gibert,
2017).

What conditions can enable a thick-lipped mouth to function? Both
abiotic, (\emph{e.g} ., type of bottom substrate \emph{etc} .), or
biotic, (\emph{e.g} ., food resources availability), factors might
promote the adaptive value of the thick-lipped phenotype in some studied
localities. Unfortunately, information on the phenotype-environment
correlation is hard to collect for thick-lipped \emph{Labeobarbus} .
Moreover, existing data on association of thick-lipped phenotype with
bottom substrate are unclear. Some studies reported stony habitats as
preferred (Groenewald, 1958; Matthes, 1963) while another study
(Kisekelwa et al., 2021) reported that species \emph{L. longifillis}
with thick-lipped phenotype (Congo basin) appears to be linked to muddy
substrates without pebbles, cobbles and boulders but in warmer
localities with a relatively low electrical conductivity compared to
sympatrically co-occurring species with a ``generalized'' mouth. The
presence of the thick-lipped ecomorph on muddy substrate is reported by
F.N. Shkil (personal communication) for the Lake Tana basin. Due to an
unstable hydrological regime in mountain rivers of the Ethiopian
Highlands, the environment varies significantly from season to season.
Our assessment of diet and trophic position using SI analyses was done
based on the material collected during the dry season while the
conditions may change during the wet season. One may suggest that
thick-lipped trophic morphologies may serve as trade-off between
consumption of commonly available food (detritus) and benthic
invertebrates in unstable riverine environment, e.g., when seasonally
common prey is rare or even absent in some seasons. Additional
ecological (natural and experimental) studies need for the clarification
of the mechanisms of ecological functionalization of thick-lipped
phenotype.

\textbf{Evolution of adaptive divergence: from repeated polymorphisms to
ecological speciation}

This study provides first genome-wide results on phylogeny and
population genetic structure of \emph{Labeobarbus} from East Africa
(genome-wide data were previously obtained only for South
African\emph{Labeobarbus} by Stobie et al., 2018). Our phylogenetic
results are in agreement with previously obtained mtDNA phylogenies
(Beshera, Harris, 2014; Levin et al., 2020) and confirmed that
thick-lipped ecomorphs have evolved independently several times. The
repeated origins of thick-lipped ecomorphs in the river systems of the
Ethiopian Highlands suggests that this polymorphism in trophic
morphology might be an old developmental and genetic system that might
be facilitated by genetic assimilation (Schneider et al., 2014; Gunter
et al. 2017). Although results on genetic population structure confirmed
absence of genetic divergence between sympatric ecomorphs with and
without hypertrophied lips in the most of populations, one sympatric
pair was genetically divergent (in the Didessa River). To sort out the
cases of the sympatric pairs in terms of polymorphism or divergent
evolution, we analyzed data on trophic morphology, trophic niche
segregation along with genetic data (Table 2).

Table 2. Heat-map of ranking the sympatric pairs of generalized and
thick-lipped ecomorphs in different rivers based on divergence in
trophic morphology, SI composition, and genetics: 0 -- non-significant
difference; 1 -- significant difference (0.5 is given for SI composition
if only one of two analyzed elements is different). The populations were
placed from left to right according to their total ranking from lower
value to higher. Cells in rows of variables are colored according to
points: 0 -- no color, 0.5 -- blue, 1 -- intense blue; `Total points'
row are marked by various intense of yellow color corresponded to number
of cumulative points from 1 to 3. P -- polymorphism (n -- ecologically
non-functional; sf -- ecologically semi-functional; f - ecologically
functional), S -- speciation completed.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
Divergence / Rivers & Blue Nile & Genale & Birbir & Sore & Gojeb &
Didessa\tabularnewline
1. Morphological (lip size) & 1 & 1 & 1 & 1 & 1 & 1\tabularnewline
2. Trophic (SI composition) & 0 & 0 & 0.5 & 1 & 1 & 1\tabularnewline
3. Genetic & 0* & 0 & 0 & 0 & 0 & 1\tabularnewline
\textbf{Total points} & \textbf{1} & \textbf{1} & \textbf{1.5} &
\textbf{2} & \textbf{2} & \textbf{3}\tabularnewline
Interpretation & Pn & Pn & Psf & Pf & Pf & S\tabularnewline
\bottomrule
\end{longtable}

* assessed by mtDNA only.

The cases of divergence between sympatric ecomorphs in various rivers
are ranked from 1 to 3 points. The results can be treated as a row of
situations from ecologically non-functional polymorphism (1
point)\emph{via} ecologically functional polymorphism (2 points) to
divergent evolution or completed speciation (3 points in the presence of
genetic divergence). The case of the Sore and Gojeb (2 points) showing
the morphological and trophic divergence but no genome-wide divergence
can be considered an initial stage of speciation with only few loci
involved. Studies on genomic differentiation of thick-lipped phenotype
in \emph{Amphilophus} cichlids from Nicaraguan lakes showed that only a
small set of loci are responsible for this phenotype (Kautt et al.,
2012; Machcado-Schiaffino et al., 2017; Kautt et al., 2020; Sowersby et
al., 2021). As for a sequence of divergence events, the evolution of
thick-lipped species of \emph{Labeobarbus} might be realized into three
steps: i) phenotypic divergence, ii) ecological (trophic) divergence,
iii) genetic divergence. At the last stage when all three types of
divergence (phenotypic, ecological, and genetic) begin to be detectable
a species status is achieved. Accordingly, the sympatric pair from the
Didessa River can be considered two different species. Notable
divergence in population genomic structure based on ddRAD SNPs in the
lack of sorting in mtDNA evidences for a rather recent speciation in the
case of the Didessa River radiation.

We detected a gradual increase of divergence among the population pairs
evolving in parallel. Similar situation of the sympatric differentiation
is reported for the cichlid genus \emph{Amphilophus} which demonstrates
a speciation continuum from incipient to fully completed (Torres-Dowdall
\& Meyer, 2021; Sowersby et al., 2021). These examples correspond to the
concept of speciation continuum that is considered a speciation as a
continuum of stages of reproductive (and other) isolations (Dr\selectlanguage{ngerman}ès \&
Mallet, 2002; Stankowski \& Ravinet, 2021; Bolnick et al., 2023). It is
noteworthy that such a continuum is found in
\emph{Labeobarbus}populations inhabiting the rivers while all previous
examples (among fishes) came from the lacustrine environment. Generally,
ecological speciation is more common among fishes under the lacustrine
conditions (reviewed in Seehausen \& Wagner, 2014). However, the
Ethiopian cyprinids (Levin et al., 2019; 2020; 2021a,b; Golubtsov et
al., 2021) and South American cichlids (Burress et al., 2018), as well
as the Kamchatka salmonids (Esin et al., 2021, 2022), demonstrate this
evolutionary pattern in rivers.

For a better understanding of the nature of numerously proliferated
polymorphisms of the mouth structure in \emph{Labeobarbus} we have to
study the evolutionary history of the Torinae lineages in the future.
Mouth polymorphisms in relation to lip size including state
`hyperthophied lips' is common within the closely-related polyploid
cyprinid lineages from the Middle East (e.g.,
\emph{Arabibarbus}Borkenhagen, 2014 and \emph{Carasobarbus} Karaman,
1971). This polymorphism is probably ancestral to both African and
Middle East Torinae being inherited from Southern and Southeastern
tetraploid Torinae lineages (e.g., \emph{Tor} Gray, 1834 and
\emph{Neolissochilus}Rainboth, 1985 -- Roberts \& Khaironizam, 2008;
Yang et. al, 2015, 2022). Thus, a proliferation of a repeated, and
predicted thick-lipped phenotype within \emph{Labeobarbus} lineage might
be due to an ancient genetic polymorphism that re-evolved under
particular ecological circumstances. Given this, the thick-lipped
phenotype of\emph{Labeobarbus} is obviously ``pre-adaptive'' upon
emergence\emph{de-novo} in various populations but not always
functional, i.e., not yet necessarily involved in trophic resource
partitioning according to our results (Figures 3B and 4). Nevertheless,
prevalence of invertebrates in a diet or elevated d15N values in the
thick-lipped ecomorph were discovered in all cases (including
non-significant differences). This finding may be interpreted as a
result of the generally adaptive value of the hypertrophied lips. In
those populations that could not demonstrate an adaptive value of the
hypertrophied lips in terms of trophic resource partitioning, the
thickened lips might serve as a latent ``adaptation in reserve'' or
``silent adaptation'' that can be recruited into invertebrate foraging
at appropriate environmental conditions possible due to being maintained
in the population in low frequency and due to simple genetic and
developmental programs that might also be facilitated by genetic
assimilation based on initially phenotypic plasticity (Meyer, 1987;
Schneider et al., 2014; Gunter et al., 2017).

\textbf{Conclusions}

We showed that repeated origin of sympatric ecomorphs differing in lip
size within the East African \emph{Labeobarbus} provides a nice example
for studying ecological speciation. The six pairs of sympatric ecomorphs
that have been equally divergent in phenotype (thick-lipped vs. common
lips) were at various stages of ecological and genetic differentiation.
This example of ecological speciation represents a continuum from
polymorphism in phenotype \emph{via} trophic resource partitioning (with
various strengths) to genetic divergence. This assumedly starts as an
intra-population strategy to broaden the food spectrum via
temporally/seasonally differential niche. However, in some
circumstances, polymorphism in lip size may be a major contributor to
trophic shift and consequently to divergent selection and ecological
speciation, for example in the Didessa River (see also example from
cichlids - Elmer et al., 2010).

\textbf{Supporting information}

This article contains 12 Supplementary Materials.

\textbf{Acknowledgments}

We are grateful to all members of the Joint Ethiopian--Russian
Biological Expedition (JERBE), who participated in our field operations
(S. E. Cherenkov, Y. Y. Dgebuadze, Genanaw Tesfaye, Fekadu Tefera, M. V.
Mina, I. S. Razgon, and S. A. Simanovsky), and especially to JERBE
coordinator Dr. A. A. Darkov for his permanent and invaluable aid. We
are grateful to S. E. Cherenkov for photographing the fish as well as to
Y. Y. Dgebuadze and M. V. Mina for discussion of unpublished results.
The study was supported by the Russian Science Foundation (grant no.
19-14-00218 - field work, morphology, diet, stable isotopes, and
partially genetics) and by Ministry of Science and Higher Education of
the Russian Federation (grant no. 075-15-2021-1069 -- genetics)
Financial support (to AM) from the DFG (German Science Foundation) is
acknowledged.

\textbf{Author Contributions}

BL, AK, ES, AT, ML, AG, FK, and AM designed and contributed to the
original concept of the studies. BL and AG collected most of the
specimens and related data, BL and ML obtained mtDNA data and prepared
DNA libraries for ddRAD, AK conducted morphologic, diet and stable
isotope analyses, AT conducted stable isotope analysis; ES conducted the
most of bioinformatics, and BL, AK, ES, AG, FK and AM finalized the
manuscript. All authors participated in project design, and read and
approved the final manuscript.

\section*{Data availability statement}

{\label{data-availability-statement}}

Morphologic data, data on diet and stable isotope compositioin, mtDNA
dataset (cytochrome \emph{b} ), and genotyping files (various sets of
SNPs) have been uploaded to
Dryad:~\emph{https://doi.org/10.5061/dryad.rbnzs7hgn}. Genetic (new
cytochrome \emph{b} sequences) and genomic data (raw reads) were
deposited to GenBank under Accession numbers OQ604627-OQ604649, and
Bioproject ID xxx (under preparation), respectively.

\textbf{References}

Aljanabi, S.M., \& Martinez, I. (1997). Universal and rapid
salt-extraction of high quality genomic DNA for PCR-based
techniques.\emph{Nucleic Acids Research} , \emph{25} , 4692--4693.
https://doi.org/10.1093/nar/25.22.4692

Andrews, S., \& Krueger, F. (2010). FastQC. A quality control tool for
high throughput sequence data (p. 370).

Bandelt, H.J., Forster, P., \& Röhl, A. (1999). Median-joining networks
for inferring intraspecific phylogenies. \emph{Molecular Biology and
Evolution} , \emph{16} , 37--48.
https://doi.org/10.1093/oxfordjournals.molbev.a026036

Banister, K.E. (1973). A revision of the large \emph{Barbus} (Pisces,
Cyprinidae) of East and Central Africa. Studies of African Cyprinidae.
Part II. \emph{Bulletin of the British Museum (Natural History) Zoology}
, \emph{26} , 3--148. https://doi.org/10.5962/bhl.part.204

Barluenga, M., Stölting, K.N., Salzburger, W., Muschick, M., \& Meyer,
A. (2006). Sympatric speciation in Nicaraguan crater lake cichlid
fish.\emph{Nature} , \emph{439} , 719--723.
https://doi.org/10.1038/nature04325

Baumgarten, L., Machado-Schiaffino, G., Henning, F., \& Meyer, A.
(2015). What big lips are good for: on the adaptive function of
repeatedly evolved hypertrophied lips of cichlid fishes.\emph{Biological
Journal of the Linnean Society} , \emph{115} , 448--455.
https://doi.org/10.1111/bij.12502

Beshera, K.A., \& Harris, P.M. (2014). Mitochondrial DNA phylogeography
of the \emph{Labeobarbus intermedius} complex (Pisces, Cyprinidae) from
Ethiopia. \emph{Journal of Fish Biology} , \emph{85} , 228--245.
https://doi.org/10.1111/jfb.12408

Binning, S.A., Chapman, L.J., \& Cosandey-Godin, A. (2009). Specialized
morphology for a generalist diet: evidence for Liem's paradox in a
cichlid fish. \emph{Journal of Fish Biology} , \emph{75} , 1683--1699.
https://doi.org/10.1111/j.1095-8649.2009.02421.x

Bolnick, D.I., Hund, A.K., Nosil, P., Peng, F., Ravinet, M., Stankowski,
S., Subramanian, S., Wolf, J.B.W., \& Yukilevich, R. (2023). A
multivariate view of the speciation continuum. \emph{Evolution}
,\emph{77} , 318--328. https://doi.org/10.1093/evolut/qpac004

Burress, E.D. (2016). Ecological diversification associated with the
pharyngeal jaw diversity of Neotropical cichlid fishes. \emph{Journal of
Animal Ecology} , \emph{85} , 302--313.
https://doi.org/10.1111/1365-2656.12457

Burress, E.D., Piálek, L., Casciotta, J.R., Almirón, A., Tan, M.,
Armbruster, J.W., \& \selectlanguage{polish}Ř\selectlanguage{ngerman}í\selectlanguage{polish}č\selectlanguage{english}an, O. (2018). Island- and lake-like parallel
adaptive radiations replicated in rivers. \emph{Proceedings of the Royal
Society B: Biological Sciences} , \emph{285} , 20171762.
https://doi.org/10.1098/rspb.2017.1762

Chen, S., Zhou, Y., Chen, Y., \& Gu, J. (2018). fastp: an ultra-fast
all-in-one FASTQ preprocessor. \emph{Bioinformatics} , \emph{34} ,
i884--i890. https://doi.org/10.1093/bioinformatics/bty560

Colombo, M., Diepeveen, E.T., Muschick, M., Santos, M.E., Indermaur, A.,
Boileau, N., Barluenga, M., \& Salzburger, W. (2013). The ecological and
genetic basis of convergent thick-lipped phenotypes in cichlid
fishes.\emph{Molecular Ecology} , \emph{22} , 670--684.
https://doi.org/10.1111/mec.12029

Comtois, D. (2018). Summarytools: Tools to Quickly and Neatly Summarize
Data. R Package Version 0.8. 72018. Available online:
https://CRAN.R-project.org/package=summarytools (accessed on 26 July
2022)

Decru, E., Snoeks, J., Walanga, A., \& Vreven, E.J.W.M.N. (2022).
Disentangling the diversity of the \emph{Labeobarbus} taxa
(Cypriniformes: Cyprinidae) from the Epulu Basin (DR Congo,
Africa).\emph{Diversity} , \emph{14} , 1022.
https://doi.org/10.3390/d14121022

Dr\selectlanguage{ngerman}ès, M., \& Mallet, J. (2002). Host races in plant--feeding insects and
their importance in sympatric speciation. \emph{Philosophical
Transactions of the Royal Society of London. Series B: Biological
Sciences} , \emph{357} , 471--492.
https://doi.org/10.1098/rstb.2002.1059

Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high
accuracy and high throughput. \emph{Nucleic Acids Research} , \emph{32}
, 1792--1797. https://doi.org/10.1093/nar/gkh340

Elmer, K.R., Lehtonen, T.K., Kautt, A.F., Harrod, C., \& Meyer, A.
(2010). Rapid sympatric ecological differentiation of crater lake
cichlid fishes within historic times. \emph{BMC Biology} , \emph{8} ,
60. https://doi.org/10.1186/1741-7007-8-60

Esin, E.V., Markevich, G.N., Melnik, N.O., Kapitanova, D.V., \& Shkil,
F.N. (2021). Natural toxic impact and thyroid signalling interplay
orchestrates riverine adaptive divergence of salmonid fish.\emph{Journal
of Animal Ecology} , \emph{90} , 1004--1019.
https://doi.org/10.1111/1365-2656.13429

Esin, E.V., Melnik, N.O., \& Markevich, G.N. (2022). Life history
variation as a source of diversity for endemic white charr (Salmonidae)
of the lower Kamchatka River. \emph{Journal of Fish Biology} ,\emph{101}
, 914--924. https://doi.org/10.1111/jfb.15149

Evanno, G., Regnaut, S., \& Goudet, J. (2005). Detecting the number of
clusters of individuals using the software STRUCTURE: a simulation
study. \emph{Molecular Ecology} , \emph{14} , 2611--2620.
https://doi.org/10.1111/j.1365-294X.2005.02553.x

Ewels, P., Magnusson, M., Lundin, S., \& Käller, M. (2016). MultiQC:
summarize analysis results for multiple tools and samples in a single
report. \emph{Bioinformatics} , \emph{32} , 3047--3048.
https://doi.org/10.1093/bioinformatics/btw354

Franchini, P., Fruciano, C., Spreitzer, M.L., Jones, J.C., Elmer, K.R.,
Henning, F., \& Meyer, A. (2014). Genomic architecture of ecologically
divergent body shape in a pair of sympatric crater lake cichlid
fishes.\emph{Molecular Ecology} , \emph{23} , 1828--1845.
https://doi.org/10.1111/mec.12590

Franchini, P., Monné Parera, D., Kautt, A.F., \& Meyer, A. (2017).
quaddRAD: a new high-multiplexing and PCR duplicate removal ddRAD
protocol produces novel evolutionary insights in a nonradiating cichlid
lineage. \emph{Molecular Ecology} , \emph{26} , 2783--2795.
https://doi.org/10.1111/mec.14077

Fricke, R., Eschmeyer, W.N., \& van der Laan, R. (eds.). (2022).
Eschmeyer's Catalog of Fishes: Genera, Species, References. Available
online:
http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatmain.asp
(accessed on 15 February 2022).

Fruciano, C., Franchini, P., Kovacova, V., Elmer, K.R., Henning, F., \&
Meyer, A. (2016). Genetic linkage of distinct adaptive traits in
sympatrically speciating crater lake cichlid fish. \emph{Nature
Communications} , \emph{7} , 12736. https://doi.org/10.1038/ncomms12736

Gibert, J.-M. (2017). The flexible stem hypothesis: evidence from
genetic data. \emph{Development Genes and Evolution} , \emph{227} ,
297--307. https://doi.org/10.1007/s00427-017-0589-0

Golcher-Benavides, J., \& Wagner, C.E. (2019). Playing out Liem's
paradox: opportunistic piscivory across Lake Tanganyikan
cichlids.\emph{The American Naturalist} , \emph{194} , 260--267.
https://doi.org/10.1086/704169

Golubtsov, A.S., \& Krysanov, E.Y. (1993). Karyological study of some
cyprinid species from Ethiopia. The ploidy differences between large and
small \emph{Barbus} of Africa. \emph{Journal of Fish Biology} ,\emph{42}
, 445--455. https://doi.org/10.1111/j.1095-8649.1993.tb00347.x

Golubtsov, A.S., Korostelev, N.B., \& Levin, B.A. (2021). Monsters with
a shortened vertebral column: A population phenomenon in radiating
fish\emph{Labeobarbus} (Cyprinidae). \emph{PLoS ONE} , \emph{16} ,
e0239639. https://doi.org/10.1371/journal.pone.0239639

Groenewald, A.A.V.J. (1957). A revision of the genera \emph{Barbus}
and\emph{Varicorhinus} (Pisces: Cyprinidae) in Transvaal. \emph{Annals
of the Transvaal Museum} , \emph{23} ,
263--330.\emph{https://doi.org/10.10520/AJA00411752\_1050}

Gunter, H.M., Schneider, R.F., Karner, I., Sturmbauer, C., \& Meyer, A.
(2017). Molecular investigation of genetic assimilation during the rapid
adaptive radiations of East African cichlid fishes.~\emph{Molecular
Ecology} ,~\emph{26} (23),
6634-6653.\emph{https://doi.org/10.1111/mec.14405}

Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q., \& Vinh, L.S.
(2018). UFBoot2: Improving the Ultrafast Bootstrap
Approximation.\emph{Molecular Biology and Evolution} , \emph{35} ,
518--522. https://doi.org/10.1093/molbev/msx281

Jackson, A.L., Parnell, A.C., Inger, R., \& Bearhop, S. (2011).
Comparing isotopic niche widths among and within communities: SIBER --
Stable Isotope Bayesian Ellipses in R. \emph{Journal of Animal Ecology}
,\emph{80} , 595--602. https://doi.org/10.1111/j.1365-2656.2011.01806.x

Jombart, T., \& Ahmed, I. (2011). adegenet 1.3-1: new tools for the
analysis of genome-wide SNP data. \emph{Bioinformatics} , \emph{27} ,
3070--3071. https://doi.org/10.1093/bioinformatics/btr521

Junker, J., Rick, J.A., McIntyre, P.B., Kimirei, I., Sweke, E.A.,
Mosille, J.B., Wehrli, B., Dinkel, C., Mwaiko, S., Seehausen, O., \&
Wagner, C.E. (2020). Structural genomic variation leads to genetic
differentiation in Lake Tanganyika's sardines. \emph{Molecular Ecology}
,\emph{29} , 3277--3298. https://doi.org/10.1111/mec.15559

Kassambara, A., \& Mundt, F. (2020). factoextra: Extract and Visualize
the Results of Multivariate Data Analyses. R package version 1.0.7.
2020. Available online: https://CRAN.R-project.org/package=factoextra
(accessed on 26 July 2022).

Kautt, A. F., Elmer, K. R., \& Meyer, A. (2012). Genomic signatures of
divergent selection and speciation patterns in a `natural experiment',
the young parallel radiations of Nicaraguan crater lake cichlid
fishes.~\emph{Molecular Ecology} ,~\emph{21} (19),
4770-4786.\emph{https://doi.org/10.1111/j.1365-294X.2012.05738.x}

Kautt, A. F., Kratochwil, C. F., Nater, A., Machado-Schiaffino, G.,
Olave, M., Henning, F., \ldots{} \& Meyer, A. (2020). Contrasting
signatures of genomic divergence during sympatric
speciation.~\emph{Nature} ,~\emph{588} (7836), 106-111.
https://doi.org/10.1038/s41586-020-2845-0

Kisekelwa, T., Snoeks, J., Zamba, A.I., Amzati, G.S., Isumbisho, M.,
Masilya, P.M., Lemmens, P., Vreven, E. (2021). Association
between\emph{Labeobarbus} spp. (Teleostei: Cyprinidae) and environmental
variables in the Luhoho basin (Eastern Congo River basin;
DRC).\emph{Journal of Fish Biology} , \emph{99} , 321--334.
https://doi.org/10.1111/jfb.14719

Komarova A.S., Rozanova O.L., \& Levin B.A. (2021). Trophic resource
partitioning by sympatric ecomorphs of \emph{Schizopygopsis}(Cyprinidae)
in a young Pamir Mountain lake: preliminary results.\emph{Ichthyological
Research} , \emph{68} , 191--197.
https://doi.org/10.1007/s10228-020-00773-3

Komarova, A.S., Golubtsov, A.S., \& Levin, B.A. (2022). Trophic
diversification out of ancestral specialization: an example from a
radiating African cyprinid fish (genus \emph{Garra} ). \emph{Diversity}
,\emph{14} , 629. https://doi.org/10.3390/d14080629

Kopelman, N.M., Mayzel, J., Jakobsson, M., Rosenberg, N.A., \& Mayrose,
I. (2015). Clumpak: a program for identifying clustering modes and
packaging population structure inferences across K. \emph{Molecular
Ecology Resources} , \emph{15} , 1179--1191.
https://doi.org/10.1111/1755-0998.12387

Kornfield, I., \& Carpenter, K.E. (1984). Cyprinids of Lake Lanao,
Philippines: taxonomic validity, evolutionary rates and speciation
scenarios. In A.E. Echelle \& I. Kornfield (Eds.), \emph{Evolution of
fish species flocks} (pp. 69--84). University of Maine Press, Orono.

Leigh, J.W., \& Bryant, D. (2015). POPART: full-feature software for
haplotype network construction. \emph{Methods in Ecology and Evolution}
,\emph{6} , 1110--1116. https://doi.org/10.1111/2041-210x.12410

Levin, B., Simonov, E., Franchini, P., Mugue, N., Golubtsov, A., \&
Meyer A. (2021a). Rapid adaptive radiation in a hillstream cyprinid fish
in the East African White Nile River basin. \emph{Molecular Ecology}
,\emph{30} , 5530--5550. https://doi.org/10.1111/mec.16130

Levin, B.A., Casal-López, M., Simonov, E., Dgebuadze, Y.Y., Mugue, N.S.,
Tiunov, A.V., Doadrio, I., \& Golubtsov, A.S. (2019). Adaptive radiation
of barbs of the genus \emph{Labeobarbus} (Cyprinidae) in an East African
river. \emph{Freshwater Biology} , \emph{64} , 1721--1736.
https://doi.org/10.1111/fwb.13364

Levin, B.A., Golubtsov, A.S., Dgebuadze, Y.Y., \& Mugue, N.S. (2013).
New evidence of homoplasy within the African genus
\emph{Varicorhinus}(Cyprinidae): An independent origin of specialized
scraping forms in the adjacent drainage systems of Ethiopia inferred
from mtDNA analysis.\emph{African Zoology} , \emph{48} , 400--406.
https://doi.org/10.1080/15627020.2013.11407609

Levin, B.A., Komarova, A.S., Rozanova, O.L., \& Golubtsov, A.S. (2021b).
Unexpected diversity of feeding modes among chisel-mouthed
Ethiopian\emph{Labeobarbus} (Cyprinidae). \emph{Water} , \emph{13} ,
2345. https://doi.org/10.3390/w13172345

Levin, B.A., Simonov, E., Dgebuadze, Y.Y., Levina, M., \& Golubtsov,
A.S. (2020). In the rivers: Multiple adaptive radiations of cyprinid
fishes (\emph{Labeobarbus} ) in Ethiopian Highlands. \emph{Scientific
Reports} , \emph{10} , 7192. https://doi.org/10.1038/s41598-020-64350-4.

Lewis, P.O. (2001). A likelihood approach to estimating phylogeny from
discrete morphological character data. \emph{Systematic Biology}
,\emph{50} , 913--925. https://doi.org/10.1080/106351501753462876

Li, H., \& Durbin, R. (2009). Fast and accurate short read alignment
with Burrows--Wheeler transform. \emph{Bioinformatics} , \emph{25} ,
1754--1760. https://doi.org/10.1093/bioinformatics/btp324

Liem, K.F. (1980). Adaptive significance of intra- and interspecific
differences in the feeding repertoires of cichlid fishes. \emph{American
Zoologist} , \emph{20} , 295--314. https://doi.org/10.1093/icb/20.1.295

Lysy, M., Stasko, A.D., \& Swanson, H.K. (2021). nicheROVER: Niche
Region and Niche Overlap Metrics for Multidimensional Ecological Niches.
R package version 1.1.0. 2021. Available online:
https://CRAN.R-project.org/package=nicheROVER (accessed on 26 July
2022).

Machado-Schiaffino, G., Kautt, A.F., Torres-Dowdall, J., Baumgarten, L.,
Henning, F., \& Meyer, A. (2017). Incipient speciation driven by
hypertrophied lips in Midas cichlid fishes? \emph{Molecular Ecology}
,\emph{26} , 2348--2362. https://doi.org/10.1111/mec.14029

Manousaki, T., Hull, P.M., Kusche, H., Machado-Schiaffino, G.,
Franchini, P., Harrod, C., Elmer, K.R., \& Meyer, A. (2013). Parsing
parallel evolution: ecological divergence and differential gene
expression in the adaptive radiations of thick-lipped Midas cichlid
fishes from Nicaragua. \emph{Molecular Ecology} , \emph{22} , 650--669.
https://doi.org/10.1111/mec.12034

Martin, C.H., \& Wainwright, P.C. (2011). Trophic novelty is linked to
exceptional rates of morphological diversification in two adaptive
radiations of \emph{Cyprinodon} pupfish. \emph{Evolution} , \emph{65} ,
2197--2212. https://doi.org/10.1111/j.1558-5646.2011.01294.x

Matthes, H. (1963). A comparative study of the feeding mechanisms of
some African Cyprinidae (Pisces, Cypriniformes). \emph{Bijdragen tot de
Dierkunde} , \emph{33} , 3--35.
https://doi.org/10.1163/26660644-03301001

Meyer, A. (1987). Phenotypic plasticity and heterochrony
in\emph{Cichlasoma managuense} (Pisces, Cichlidae) and their
implications for speciation in cichlid fishes. \emph{Evolution} ,
\emph{41} , 1357--1369.
https://doi.org/10.1111/j.1558-5646.1987.tb02473.x

Mina, M.V., Mironovsky, A.N., Dgebuadze, Y. (1996). Lake Tana large
barbs: phenetics, growth and diversification. \emph{Journal of Fish
Biology} , \emph{48} , 383--404.
https://doi.org/10.1111/j.1095-8649.1996.tb01435.x.

Mina, M.V., Mironovsky, A.N., Golubtsov, A.S., \& Dgebuadze, Y.Y.
(1998). II~--~Morphological diversity of ''large barbs'' from Lake Tana
and neighbouring areas: Homoplasies or synapomorphies? \emph{Italian
Journal of Zoology} , \emph{65} (S1), 9--14.
https://doi.org/10.1080/11250009809386789

Minh, B.Q., Schmidt, H.A., Chernomor, O., Schrempf, D., Woodhams, M.D.,
Von Haeseler, A., \& Lanfear, R. (2020). IQ-TREE 2: New models and
efficient methods for phylogenetic inference in the genomic
era.\emph{Molecular Biology and Evolution} , \emph{37} , 1530--1534.
doi.org/10.1093/molbev/msaa015

Mironovsky, A.N., Mina, M.V., \& Dgebuadze, Y.Y. (2019). Large African
barbs with hypertrophied lips and their relationship with generalized
forms of species of the genus \emph{Barbus} (\emph{Labeobarbusauctorum}
). \emph{Journal of Ichthyology} , \emph{59} , 327--335.
https://doi.org/10.1134/S0032945219030111

Muschick, M., Nosil, P., Roesti, M., Dittmann, M.T., Harmon, L., \&
Salzburger, W. (2014). Testing the stages model in the adaptive
radiation of cichlid fishes in East African Lake
Tanganyika.\emph{Proceedings of the Royal Society B: Biological
Sciences} ,\emph{281} , 20140605. https://doi.org/10.1098/rspb.2014.0605

Nagelkerke, L.A.J., Sibbing, F.A., van den Boogaart, J.G.M., Lammens,
E.H.R.R., \& Osse, J.W.M. (1994). The barbs (\emph{Barbus} spp.) of Lake
Tana: a forgotten species flock? \emph{Environmental Biology of Fishes}
,\emph{39} , 1--22. https://doi.org/10.1007/BF00004751

Natarajan, A.V., \& Jhingran, A.G. (1961). Index of preponderance -- A
method of grading the food elements in the stomach analysis of
fishes.\emph{Indian Journal of Fisheries} , \emph{8} , 54--59.

Oellermann, L.K., \& Skelton, P.H. (1990). Hexaploidy in yellowfish
species (\emph{Barbus} , Pisces, Cyprinidae) from southern
Africa.\emph{Journal of Fish Biology} , \emph{37} , 105--115.
https://doi.org/10.1111/j.1095-8649.1990.tb05932.x

Oliver, M.K., \& Arnegard, M.E. (2010). A new genus
for\emph{Melanochromis labrosus} , a problematic Lake Malawi cichlid
with hypertrophied lips (Teleostei: Cichlidae). \emph{Ichthyological
Exploration of Freshwaters} , \emph{21} , 209--232.

Palumbi, S.R. (1996). Nucleic acids II: The polymerase chain reaction.
In D.M. Hillis, C. Moritz, \& B.K. Mable (Eds.), \emph{Molecular
systematics} (pp. 205--247). Sinauer Associates.

Paris, J.R., Stevens, J.R., \& Catchen, J.M. (2017). Lost in parameter
space: a road map for stacks. \emph{Methods in Ecology and Evolution}
,\emph{8} , 1360--1373. https://doi.org/10.1111/2041-210X.12775

Perdices, A., \& Doadrio, I. (2001). The Molecular Systematics and
Biogeography of the European Cobitids Based on Mitochondrial DNA
Sequences. \emph{Molecular Phylogenetics and Evolution} , \emph{19} ,
468--478. https://doi.org/10.1006/mpev.2000.0900

Pina\selectlanguage{english}-Martins, F., Silva, D.N., Fino, J., \& Paulo, O.S. (2017).
Structure\_threader: an improved method for automation and
parallelization of programs structure, fastStructure and MavericK on
multicore CPU systems. \emph{Molecular Ecology Resources} , \emph{17} ,
e268--e274. https://doi.org/10.1111/1755-0998.12702

Popova, O.A., \& Reshetnikov, Y.S. (2011). On complex indices in
investigation of fish feeding. \emph{Journal of Ichthyology,} \emph{51}
, 686--691. https://doi.org/10.1134/S0032945211050171

Pritchard, J.K., Stephens, M., \& Donnelly, P. (2000). Inference of
population structure using multilocus genotype data. \emph{Genetics}
,\emph{155} , 945--959. https://doi.org/10.1093/genetics/155.2.945

Rambaut, A. (2014). ''FigTree 1.4. 2 software.''~\emph{Institute of
Evolutionary Biology, Univ. Edinburgh} .

Reich, D., Thangaraj, K., Patterson, N., Price, A.L., \& Singh, L.
(2009). Reconstructing Indian population history. \emph{Nature}
,\emph{461} , 489--494. https://doi.org/10.1038/nature08365

Ribbink, A.J., Marsh, B.A., Marsh, A.C., Ribbink, A.C. \& Sharp, B.J.
(1983). A preliminary survey of the cichlid fishes of rocky habitats in
Lake Malawi: results -- The Mbuna -- \emph{Pseudotropheus}
.\emph{African Zoology} , \emph{18} , 157--200.
https://doi.org/10.10520/AJA00445096\_1688

Roberts, T.R., \& Khaironizam, M.Z. (2008). Trophic polymorphism in the
Malaysian fish \emph{Neolissochilus soroides} and other Old World Barbs
(Teleostei, Cyprinidae). \emph{Natural History Bulletin of the Siam
Society} , \emph{56} , 25--53.

Robinson, B.W., Wilson, D.S. (1998). Optimal foraging, specialization,
and a solution to Liem's Paradox. \emph{The American Naturalist}
,\emph{151} , 223--235. https://doi.org/10.1086/286113

Rochette, N.C., Rivera-Colon, A.G., \& Catchen, J.M. (2019). Stacks 2:
Analytical methods for paired-end sequencing improve RADseq-based
population genomics. \emph{Molecular Ecology} , \emph{28} , 4737--4754.
https://doi.org/10.1111/mec.15253

RStudio Team. (2021). \emph{RStudio: Integrated Development for R} ;
RStudio, PBC: Boston, MA, USA. Available online: http://www.rstudio.com
(accessed on 26 July 2022).

Ruber, L., Verheyen, E., \& Meyer, A. (1999). Replicated evolution of
trophic specializations in an endemic cichlid fish lineage from Lake
Tanganyika. \emph{Proceedings of the National Academy of Sciences}
,\emph{96} , 10230--10235. https://doi.org/10.1073/pnas.96.18.10230

Rundle, H.D., Nagel, L., Boughman, J.W., \& Schluter, D. (2000). Natural
selection and parallel speciation in sympatric
sticklebacks.\emph{Science} , \emph{287} , 306--308.
https://doi.org/10.1126/science.287.5451.306

Savvaitova, K.A., Schanin, A.Yu., \& Maksimov, V.A. (1987). On
intraspecific speciation of \emph{Schizopygopsis
stoliczkai}Steindachner, 1866 from Pamir water basins. \emph{Moscow
University Biological Sciences Bulletin} , \emph{2} , 9--17. (In Russ.)

Schemmel, C. (1967). Vergleichende Untersuchungen an den
Hautsinnesorganen ober- und unterirdisch lebender
Astyanax-Formen.\emph{Z. Morphol. Tiere} , \emph{61} , 255--316.

Schluter, D. (2000). The ecology of adaptive radiation. New York: Oxford
University Press. 300 p.

Schneider, R.F., \& Meyer, A. (2017). How plasticity, genetic
assimilation and cryptic genetic variation may contribute to adaptive
radiations. \emph{Molecular Ecology} , \emph{26} , 330--350.
https://doi.org/10.1111/mec.13880

Schneider, R.F., Li, Y., Meyer, A., \& Gunter, H.M. (2014). Regulatory
gene networks that shape the development of adaptive phenotypic
plasticity in a cichlid fish. \emph{Molecular Ecology} , \emph{23} ,
4511--4526. https://doi.org/10.1111/mec.12851

Seehausen, O., \& Wagner, C.E. (2014). Speciation in Freshwater
Fishes.\emph{Annual Review of Ecology, Evolution, and Systematics} ,
\emph{45} , 621--651.
https://doi.org/10.1146/annurev-ecolsys-120213-091818

Sibbing, F.A., \& Nagelkerke, L.A.J. (2000). Resource partitioning by
Lake Tana barbs predicted from fish morphometrics and prey
characteristics. \emph{Reviews in Fish Biology and Fisheries} ,\emph{10}
, 393--437. https://doi.org/10.1023/A:1012270422092

Sibbing, F.A., Nagelkerke, L.A., Stet, R.J., \& Osse, J.W. (1998).
Speciation of endemic Lake Tana barbs (Cyprinidae, Ethiopia) driven by
trophic resource partitioning; a molecular and ecomorphological
approach. \emph{Aquatic Ecology} , \emph{32} , 217--227.
https://doi.org/10.1023/A:1009920522235

Skulason, S., \& Smith, T.B. (1995). Resource polymorphisms in
vertebrates. \emph{Trends in Ecology \& Evolution} , \emph{10} ,
366--370. https://doi.org/10.1016/S0169-5347(00)89135-1

Sowersby, W., Cerca, J., Wong, B.B.M., Lehtonen, T.K., Chapple, D.G.,
Leal-Cardin,~M., Barluenga, M., \& Ravinet, M. (2021). Pervasive
admixture and the spread of a large-lipped form in a cichlid fish
radiation. \emph{Molecular Ecology} , \emph{30} , 5551--5571.
https://doi.org/10.1111/mec.16139

Stankowski, S., \& Ravinet, M. (2021). Defining the speciation
continuum. \emph{Evolution} , \emph{75} , 1256--1273.
https://doi.org/10.1111/evo.14215

Stiassny, M.L.J., \& Meyer, A. (1999). Cichlids of the Rift
Lakes.\emph{Scientific American} , \emph{280} , 64--69.
https://doi.org/10.1038/scientificamerican0299-64

Stobie, C.S., Oosthuizen, C.J., Cunningham, M.J., \& Bloomer, P. (2018).
Exploring the phylogeography of a hexaploid freshwater fish by RAD
sequencing. \emph{Ecology and Evolution} , \emph{8} , 2326--2342.
https://doi.org/10.1002/ece3.3821

Sturmbauer, C., Mark, W., \& Dallinger, R. (1992). Ecophysiology of
Aufwuchs-eating cichlids in Lake Tanganyika: Niche separation by trophic
specialization. \emph{Environmental Biology of Fishes} , \emph{35} ,
283--290. https://doi.org/10.1007/BF00001895

Swanson, H.K., Lysy, M., Power, M., Stasko, A.D., Johnson, J.D., \&
Reist, J.D. (2015). A new probabilistic method for quantifying
n-dimensional ecological niches and niche overlap. \emph{Ecology}
,\emph{96} , 318--324. https://doi.org/10.1890/14-0235.1

Tamura, K., Stecher, G., Peterson, D., Filipski, A., \& Kumar, S.
(2013). MEGA6: Molecular Evolutionary Genetics Analysis Version
6.0.\emph{Molecular Biology and Evolution} , \emph{30} , 2725--2729.
https://doi.org/10.1093/molbev/mst197

Tang, Y., Horikoshi, M., \& Li, W. (2016). ''ggfortify: Unified
Interface to Visualize Statistical Result of Popular R Packages.''.
\emph{The R Journal} , \emph{8} , 478--489.

Torres-Dowdall, J., \& Meyer, A. (2021). Sympatric and Allopatric
Diversification in the Adaptive Radiations of Midas Cichlids in
Nicaraguan Lakes. In M.E. Abate \& D.L. Noakes (Eds.), \emph{The
Behavior, Ecology and Evolution of Cichlid Fishes. Fish \& Fisheries
Series} , vol 40. (pp. 175--216). Springer, Dordrecht.
https://doi.org/10.1007/978-94-024-2080-7\_6

Tsigenopoulos, C.S., Kasapidis, P., \& Berrebi, P. (2010). Phylogenetic
relationships of hexaploid large-sized barbs (genus \emph{Labeobarbus} ,
Cyprinidae) based on mtDNA data. \emph{Molecular Phylogenetics and
Evolution} , \emph{56} , 851--856.
https://doi.org/10.1016/j.ympev.2010.02.006

Vreven, E.J., Musschoot, T., Snoeks, J., \& Schliewen, U.K. (2016). The
African hexaploid Torini (Cypriniformes: Cyprinidae): Review of a
tumultuous history. \emph{Zoological Journal of the Linnean Society}
,\emph{177} , 231--305. https://doi.org/10.1111/zoj.12366

Wagner, C.E., McIntyre, P.B., Buels, K.S., Gilbert, D.M., \& Michel, E.
(2009). Diet predicts intestine length in Lake Tanganyika's cichlid
fishes. \emph{Functional Ecology} , \emph{23} , 1122--1131.
https://doi.org/10.1111/j.1365-2435.2009.01589.x

West-Eberhard M.J. 'Adaptive Radiation' // Developmental Plasticity and
Evolution (NY, 2003; online edn, Oxford Academic, 12 Nov. 2020). P.
564--597. https://doi.org/10.1093/oso/9780195122343.003.0036

Wickham, H. (2016). \emph{ggplot2: Elegant Graphics for Data Analysis} .
http://ggplot2.org

Wund, M.A., Baker, J.A., Clancy, B., Golub, J.L., \& Foster, S.A.
(2008). A Test of the ``Flexible Stem'' Model of Evolution: Ancestral
Plasticity, Genetic Accommodation, and Morphological Divergence in the
Threespine Stickleback Radiation. \emph{The American Naturalist}
,\emph{172} , 449--462. https://doi.org/10.1086/590966

Yang, L., Naylor, G.J.P., \& Mayden, R.L. (2022). Deciphering reticulate
evolution of the largest group of polyploid vertebrates, the subfamily
Cyprininae (Teleostei: Cypriniformes). \emph{Molecular Phylogenetics and
Evolution} , \emph{166} , 107323.
https://doi.org/10.1016/j.ympev.2021.107323

Yang, L., Sado, T., Hirt, M.V., Pasco-Viel, E., Arunachalam, M., Li, J.,
Wang, X., Freyhof, J., Saitoh, K., Simons, A.M., Miya, M., He, S., \&
Mayden, R.L. (2015). Phylogeny and polyploidy: Resolving the
classification of cyprinine fishes (Teleostei:
Cypriniformes).\emph{Molecular Phylogenetics and Evolution} , \emph{85}
, 97--116. https://doi.org/10.1016/j.ympev.2015.01.014

Data Accessibility and Benefit-Sharing~Section

We prepare our data to archive in a publicly accessible repository - in
Dryad and GenBank, in particular.

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