This article isprotected rights by All copyright. reserved. 10.1111/mec.13973doi: lead todifferences version between this andthe ofPl Version Record. been and throughtypesetting, proofreadingwhich may thecopyediting, process, pagination AcceptedThis article undergone has for and buthas accepted been not review publication fullpeer Running title: Fax: (718) 262 Phone: (650) 283 [email protected] ElizabethS. Alter *Corresponding author: Key words: diversification, Park West, New York, NY 10024 Article(5) Department ofIchthyology, American Museum of Natural History, 79th St (4) Street and Central Park West, New York, NY 10024 (3) 10016 Graduate(2) The Center of theCity University of New York, 365 Fifth Avenue, NewYork, NY Blvd, Jamaica NY (1) ElizabethS. Alter (1,2,3) divergence Genome Louis Calder Center The Department ofBiology,

Sackler Institute for Comparative Genom - wide SNP data reveal cryptic

in Phylogeography

- a rapids 2700

11451 - 5629

—

- * adapted clade of cichlids from Congo the River

, Jason Munshi Biological Field Station, Fordham University, Armonk, NY 10504 phylogeography York College/The City University of New York, 94

in rapids

- - South (4), Melanie adapted cichlids phyloge , fish, RAD ics, American Museum ofNatural History, 79th ographic

- Seq, African biogeography

L. J. Stiassny structure and and

easecite article this as ( 3,

microallopatric 5 ) -

reet and Central 20 Guy R. Brewer

This article isprotected rights by All copyright. reserved. underlying mechanisms shaping biogeography and evol morphological Winemiller radiations) 30 representing only development fish lower Congo River concern which represent I. Intro both below and above the species level. wellas the the lineage events were events, but diversification. hydrological and topographic barriers, indicating Species ranges appear to correspond to geographic regions broadly se unrecognized mitochondrial variation within and between taxa. Phylogenetic analyses reveal individuals across alldescribed species in the genus. genome w discret phylogenetic relationships within context of phylogeographic shaping some ofthe world's largest rapids. However, little is known about the evolutionary forces lowerThe Congo River (LCR) Abstract:

Accepted% ofthe fish species found Article importance of hydrolo diversity and e morphological characters among putative species. - duction Sub

specific patterning may be this diversity (Darwall

e regional

- no and ofthese around 25% areendemic to theLCR et al. analyzed double Saharan Africa harbors a numb likely

(Stiassny utility ofddRAD lineages clear assessments Mitonuclear discordance

threatened 2016) relation

endemism for a et al. about

ancient. A survey of female fin

(LCR) geography and hydrology evidence of , which include numerous endemic fishes

and instances of microallopatric divergence across little as as ~1.5 km . Because 2008; Thieme et al. 2% ofthetotal area of the Congo Basin,

ships ships in is ofparticular interest because it contains gical gical complexity in generating diversity in , knowledge of the

- hotspots 2011; Winemiller digest restriction is in the entire basin - Seq data in understanding diversification processes operating

admixture a freshwater biodiversity hotspot in Africa characterized by estimates of n African Teleogramma Teleogramma

acting to reinforce species cohesion. These analyses highlight

et al. of endemism and are subsequently ofconservation

indicates one or more

is prese 2005) fluvial system er ofhyperdiverse freshwater systems, many of . Previous studies have been unable to resolve

LCR fish

- true et al. associated , a small , asmall clade of

due to lack due tolack of variation in nuclear ( . Among these, the patterns exclusive ofthe Lake Tanganyikan cichli these features

nt in nuclear genomes, suggesting diversity ofthis region, as wellthe as

2016) W

diversity are e ution also

yet theis proposed for site major dam

sequencing (ddRAD) data from 53 . D hin

To To sample broadlymore across the espite (Lowenstein assessed within the system ts that ts . We investigated introgressive hybridization

rheophilic are likely important drivers of the LCR

a very based hydrologically complex previously undetected remarkably certain parated by major body shape

contains an estimated

almost entirely small footprint, et al. cichlids freshwater systems,

previously 2011; , remains

high high

, in the genes and

these levels of

and on d

. This article isprotected rights by All copyright. reserved. Accepted(including one species found just upstream) and placed in the diversification in LCR cichlids. unknown. energy Schwa allopatric diversification of endemic cichlid species of the most dynamic and diverse stretches of river on asEarth, a factor potentially driving studies have identified the unique hydrological complexity may represent an exception to this generalization. Recently, several 2006b have the relatively few between Articleconnectivity i investigating McKinn AfricanEast Great Lakes 2012; Seehausen 2006) of diversification tropical freshwater systems. conservation priorities, and can imp phylogeography of extremely generally rzer ; rapids rapids impede gene floweven at very small spatial scales (microallopatry) remains Koblmüller The genusThe Among fishes, cichlids have emerged as a model system for understanding mechanisms on 2012; Seehausen 2006; Wagner isolated

limited. et al.

chromidotilapiine divers n fluvial environments,

found weak structure

2012)

in these tropical freshwater systems drainages studies Teleogramma Applying the exceptionally diverse et al. ification processes in river . Whether this pattern extends .

However, i 2012 ( that have examined e.g.,

( molecular molecular methods to assess the diversity, evolutionary history and e.g.,

; Genner Th Egger

radiation

represents a represents useful case study for investigating patterns of Burridge is small clade of bathymetry n contrast to the well rove understanding ofprocesses underlying speciation in fish within drainages et al.

et al.

speciation via allopatry is thought to occur (Schwarzer

2015 et al.

et al. 2015; Keller LCR ichthyofauna local

ine and h

) 2008; Griffiths 2015; Tedesco 2013) . However, rivers characterized by extreme another in

rheophilic, cichlids. - scale (Markert ydrology ofthe

to other et al.

( , relatively little ( e.g., e.g.,

et al. - phylogeography studied lacustrine radiations

2015) Due to Due potentialhigh for in Kocher 2004; Santos & Salzburger the Kasai drainage Ready

rock et al. 2013; Kocher 2004; Pauers & LCR will , -

is is endemic to theLCR region

dwelling 2010; Schwarzer cichlid et al.

aid aid in LCR attention has focused on

2006a; Ready , which includes some

s focusing of , or whether

cichlids

river

et al.

(Fig. 1) ine

regional

, 2012)

et al. recently

primarily cichlids

. Below - et al. of channel high

the 2011; ,

and

- This article isprotected rights by All copyright. reserved. AcceptedAlter 2015) obamaorum association varies across the genus and fins, gas bladder reduction or loss, extremely dorso cataracts which theriver drops nearly 170in m elevation forming a series of massive ra Kinganga withfew surface rapids, and a lower section of some 95 km ending at andMatadi over rap by a section of about 130 km from just below Pool Malebo to the region of Manyanga, characterized Schwarzer divided the “rapids section” of the LCR into three Article2009; Oberg toappears haveprofound effects on flow dynamics changes in occasionally fringed grass or muddy, shorelines. habitats are almost entirely rocky and rock strewn, somewith intermittent sandy, or numerous changes intermittently narrow (<0.2 km) and wide (>2 km) gorges in a channel that undergoe upstream of Pool Malebo, the LCR channel is bedrock et al. submerged canyons, formed itdrops as 280m over 350 kmen route tothe Atlantic Ocean Pool Malebo, the river is characterized by a series of extremely high ids; an approximately 120 kmnavigable, middle section from above Manyanga to above relatively relatively small

2015; Jackson M . ost ost

bathymetry et al. , and the type species ofthe genus,

is restrictedis to deep water, rocky habitats just upstream of Pool Malebo et al. Teleogramma

2011) - ventrally depressed heads, elongate depressed bodies, hypertrophied pelvic

2009)

in direction in response to a hi et al. drop in elevation of over 80 m resulting in numerous stretches of surface .

even over ver These .

2009; Oberg Based onBased a combination of geomorphology and geology

species are notable for putatively rapids

regions, (Roberts & Stewart 1976) y short distances, and this highly irregular bed topography

et al. i though ndicated

2009; Robert 1946) Teleogramma gracile the degree

in Figure 1,broadly correspond to an upper In situ main ghly variable bedrock bathymetry

even in the absence of rapids

constrained and flows through a series of

regions measurements

of rheophilic adaptation and habitat

. Notably, the recently describe

( . see also In marked contrast to the river

- - Boulenger 1899,although ener adaptive features, such as have Alter gy rapids, eddies, and recorded dramatic

et al. pids and (Jackson (Robert 1946)

2015; . Littoral (Stiassny & s

(Alter et al. d T.

This article isprotected rights by All copyright. reserved. AcceptedDiscussion) nuclear data colonization and differentiation it plunges from Pool Malebo the towards Atlantic, suggests a pattern ofdownstream serial along the middle resolve key phylogenetic relationships within the genus. biogeographicThe array ofspecies been hampered by lack of genome designated herein as identify two cryptic lineages within extensive morphological and molecular variation across th kilometers downst distributional range, occurring just from below Pool Malebo to the Inga rapids some 275 river distributions and tributary (Kasai) Article just above Pool Malebo in the lower middle Congo, and one ( brichardi monomorphic. Of the five currently recognized species, three( than female fin patterning patterning sites, initiate courtship and exhibit strikingly differentiated caudal and dorsal pigmentationfin species are sexually dichromatic energy surface rapids. section of theLCR, where it i exhibiting a number of putatively rapids A thorough understanding ofthe evolutionary and adaptive history of Teleogramma ) appear to occur parapatrically along sections of theLCR, one ( (Sti (Schwarzer

(though it assny & Alter 2015; Wickler 1959) -

(Fig. 1) lower Congo and the persistent, high in T. depressa ream T

are pairbonding, highly territorial, cave and crevice spawners and most . cf

has been explo

et al. (Roberts & Stewart 1976) . Most species appear to have extremely narrow geographic . depressa ,

and s s usually associated with rocky outcrops but in the absence ofhigh

is the onlyis species thought to occupy a relatively broad 2011) belly . T (Wickler 1959) his hypothesis has not been tested for -

wide data,as traditional nuclear markers have failed to and ) T. depressa /fin . While aprevious study fo red in another genus ofLCR cichlids ( T.

- coloration during courtship, the sexes are essentially adaptive features, is found along the navigable middle

cf. cf. brichardi.

sensu and lato, these are provisionally . Females, rather than males, s . Although males attain larger adult sizes, other . However, preliminary observations suggest

- st is range, and in the present westudy

ream water velocities of theLCR, as T. monogramma T. depressa und strong support for the Teleogramma T. obamaorum , T. gracile Steatocranus ) occurs) in a distant Teleogramma elect spawning

and

with with ) occurs T. , see

has has This article isprotected rights by All copyright. reserved. and between molecular lineages. (c) quantifying the history ofintrogression; and (d) measuring morphological variation within assessing gene flow and connectivity between species and, where sam species boundaries and to data via double cichlids, we reconstructed population and phylogenetic relationships using genome phylog frequently exchange migrants to completely isolated species differentiation between taxa along of stages the species continuum, from populations that se share genes across species boundaries challenging for cichlid available, 2015) Similarly found across species these results were driven by variation in mitochondrial loci. Virtually no nuclear variation was thosewith found just below and above Pool Malebo ( monophyly ofdownstream LCR taxa ( quencing technologies now permit unprecedented resolution in assessing genomic Acceptedelucidate thephylogeographic Article

shows no nuclear variation between the two eographic Reconstructing the history ofdiversification , acomparison of data from three additional nuclear markers from T. depressa - digest restriction site associated sequencing (ddRAD

relationships and the evolutionary history ofthis unusual genus of rheophilic

using several traditionally employed phylogenetic markers and taxa, many ofwhich have radiated recently and may also continue to resolv T. brichardi ing

history of diversification in

phylogenetic relationships using genome . gracile

(Wagner

and

et al. depressa Teleogramma and identifying lineages brichardi

2013) ;

(Stiassny &(Stiassny Alter 2015) . However, high (Feder Teleogramma , obamaorum

species forspecies which datawere

et al. - Seq). pling allows, populations;

2012)

- Our object , by: ( - wide SNPs; (b) (Schwarzer throughput monogramma can can be notoriously . a) testing

(Li To exploreTo

compared -

et al. wide SNP ives were

et al.

2007) )

. This article isprotected rights by All copyright. reserved. Accepted clipsfin using the Gentra PureGene tissue extraction (Qiagen) protocol later and immediately preserved in http://entheros.amnh,org/db/emuwebamnh/index.php Museum ofNatural History (AMNH), available online at euthanasia. All specimens are cataloged and ameliorated by minimizing handling and through the use of anesthetics recommended guidelines for the use of fishes in res T. depressa analys T. depressa using ddRAD sequencing recognized Articlebeen able to sample multiple individuals from depressa and extent of in light grey remain reaches hydrological conditions. by a combination of A. Sample collection II. Methods transferred to 70%ethanol for i s All specimens Sampling

of 74 adult individuals (Figure 1). However, for most of the LCR, middle Congo, and Kasai , 10 , 11 Teleogramma

in the accompanying map overlap T. gracile T. gracile along along geographical ping

the LCR were euthanized prior to preservation in accordance withthe and library construction

numerous , , eight Despite intensive efforts over a ten , including five

species occurrence

T. monogramma where no sampling has been possible 95 T. monogramma

includ

% ethanol (Table S1). isolation,

stretches five long , ed

of and

T. brichardi

13 T. - term

we acknowledge cf.

limited river access, and extreme

and the voucher T. brichardi of the L stored in the ichthyology collection of theAmerican In all, we were able to sequence

, brichardi across much oftherange of

and five , and 10 , and storage

earch , 15 CR is . Weextracted total genomic DNA from T. obamaorum ,

T. obamaorum and T. 2 . logistically

(Nickum 2004) 0 cf. Tissue samples

T. cf. brichardi -

T

uncertainty regarding the

year period (2004 s brichardi . cf. pecimens

depressa . T

challenging . . , three hese regions are indicated

. totalThe amount of Geometric morphometric

fixed in formalin and (MS , and stress was

, and , were taken in the fi all currently 11 - Rivers Rivers 222) T. in 56 T. cf. depressa - T. gracile - cf. 2014) there stream

and individuals

depressa for we have

hindered

range

and , four , four , 10

s T. eld ,

This article isprotected rights by All copyright. reserved. ( 2012) uncalled bases, and truncate allreads to 92bp. We then used bowtie2 et al.(2013 likely genotype at that position. Full details ofthe models underlying Stacks are estimate ofthe sequencing error rate,and then uses a likelihood ra the likelihood of thetwo most common genotypes at each site given a maximum likelihood pipelines for both denovo and reference B. barcode and Illumina index. demultiplex the sequence for reads each sample based on the combination of sample Genome Center using paired purified using AMPure Illumina multiplexing indices pooled samples were fragments in the range 300 samples. S sample using two restriction enzymes ( outlined in samples with >500 ng total DNAwere used in the rest of the analyses. genomic DNA wasdetermined using a Qubit Oreochromis niloticus

AcceptedSNP Article

with default settings to align each sample’s paired end reads to the - calling population and genomic statistics First, weFirst, used the We preparedWe double specific barcodes and Illumina multiplexing read indices were ligated to individual amples

a (Peterson used - ), but we describe our general approach below. end sequen

(

Stacks 1.35 NYG with unique adapters C,

PCR et al. ) XP New York, NY reference genome, version OreN cing on an Illumina HiSeq 2500 across a single lane at the - process_ beads (Beckmanbeads Coulter Inc),quantified using qPCR, and sequenced

amplified using 2012) -

450 bp a using Pippin Prep system (Sage Sciences

- to digest RAD

(Peter filter MluCI .

radtag In brief, f son

and call SNPs ).

and

A custom script at the NYGC wasthen toused

were then pooled, - et al. - s S aligned data

eq libraries script to script filter Nla a Phusion polymerase kit and or each sample, 500 dsDNA

2012) III , New England Bioloabs Inc), and adapters with

from the demultiplexed sequence reads

. HS The finalThe pooled sample for (Catchen et al. 2013 Assay il1.1 reads with low 56 and each poolwas

individuals ( ( NCBI AssemblyNCBI Life Technologies - 1,000 ng ofDNA wasdigested tio test to identify the most

(Langmead & Salzberg

quality scores or using the proto primers t

Nile a ) . s Stacks calculates size

were then Inc). SizeInc). tilapia given in Catchen ), and only - hat hat introduce selected for New New York - specific - col selected using This article isprotected rights by All copyright. reserved. Acceptedsample are given in Table S2. notand thus comparable to the other samples we removed visualized the output using the Stacks webserver. Based on the results ofthe pipeline first run such as observed heterozygosity, nucleotide diversity, and pairwise TreeMix used parameter, i.e. the number of mismatches allowed between loci for a single individual (M = 3). overlap and were not aligned. and reverse generated reference and our study taxa Articlegenome may have limited ourability to identify SNPs duetoa lack of homology these two genera exceed 46 MYA subfamily ( 0.5), and had a minor allele frequency ofat least 0.05 that occurred in building the initial catalog ofloci, the ‘stack’ (m= 10) pipeline ref_map.pl GCA_000188235.2)

to produce We loadedWe For both reference Although ) with one SNP were used, de novo pipeline inpipeline Stacks toidentify ddRAD loci and SNPs. call Pseudocrenilabrinae)

reads for each individual into one fastq because file thetwo paired did reads not three

and the number of mismatches allowed when building the catalog (n = 2). After

six genotype outputin multi Teleogramma

SNP SNP calls in Stacks of the the RAD loci and individual data from Stacks into a MySQL database and

. out out of seven except for theminimum number of identical reads req A ll resulting SAM (Sequence Alignment/Map) files

from each locus ( 56 - aligned and

individuals because . We then reran the pipelines above as 53with individuals, For comparison with the reference The settings were the same and

taxa (p = 6), in ,

(Schwarzer they are distantly related and O

reochromis populations using de novo -- ple formats (i.e. write_single denovo_map.pl

et al. .

The mean and range ofloci obtained for each pipelines, the at least 50% ofindividuals they were genotyped at less than 2,500 are script script in Stacks

2011) both members of

(min_maf =0.05) _SNP), and_SNP), generated summary statistics . usingThus, the VCF, as above except for an additional .

First, First,

populations Genepop,

divergence time estimates for -

was used aligned we concatenated the D efault settings for the F ST

the same lar .

were run between allpopulations. PLINK,

analysis script script O. niloticus uired to create a

for taxon each to l filter between the in Stacks Structure through the ,

we also ge oci for those reference cichlid forward SNPs

was (r = , and

s ,

This article isprotected rights by All copyright. reserved. parameters assessed convergence and million generations, with thefirst 10% ofgenerations burn as 2015) (urates and v) were estimated from the data (Bouckaert 2000, (Bryant Species tree estimation was performed 11,459 SNPs (6 or fewer miss variable amounts of missing data on tree topologies, we conducted preliminary runs with geographicthe full range of representatives ofeach s with computationally intensive et al. 2012) removed)sites Phylogenomic analyses C. eight including two

Accepted ArticleddRAD

the highest numbers of reads and SNP genotypes 2012) T. monogramma

with and and We estimatedWe

et al. - seq d were (Chifman & Kubatko 2014)

θ

et al. implemented in BEASTv2.2.1 by examining likelihood plots and ESS values =0.001)

2012) T. brichardi : coalescent ata analyses

fixed at 2014) , using priors gamma with ashape parameter of2, and a scale parameter of . SNAPP , and 10 , and phylogenies using two methods based on the SNP

and we used a birth rate ( u pecies and, based on preliminary results, representatives from across = , 15

nature of - ensured that effective sample size ( 1.4501 based species tree estimation using the methods of T. employs ing genotypes) to 37,826 SNPs (10

T. T. obamaorum depressa cf .

and v= brichardi species tree estimation, we selected a subset of individuals

a Yule . First, we estimated t

( on bi sensu 0.8611 (Bouckaert

, three prior for - . base allelic SNP data (

Roberts & Stewart, 1976) . Runs were performed with a chain length of5 λ) = 0.00765 d on equations f T. cf

the species tree et al.

(n=25 .

depressa using Tracer v1.5

2014) he s he ; S2Table - ESS 37 in. We (Bryant pecies tree

or fewer missing genotypes). , , . 826 four four ) rom Because of the >200

determined the burn SNPs) and branch lengths

), with atleast two T. depressa (Drummond & Bouckaert et al. - .

only dataset for To assessTo the effects of using SNAPP

(Rambaut & all model 2012) in

SNAPP v. 1.1.4 (Bryant

, 11 . Mutation

(invariant T. gracile

et al. (Bryant

- in,

,

This article isprotected rights by All copyright. reserved. brichardi and PCA. Wealso conducted these ana components using Tracy submitted) analysis ( and Lange 2011) ADMIXTURE Teleogramma 20 estimates of parameters to estimate the most likely number of evolutionary clusters, K. evolutionary clusters and potential in evolutionarybased clustering approach in ADMIXTURE Population genomic analyses sampled 100,000 quartets using no using nonparametric bootstrapping. each, taxa (quartets) at a time (Chifman & Kubatko 2014) Maximum Clade Credibility tree and Drummond 2007)

Accepted Article replicates ofADMIXTURE for and Both For comparison ADMIXTUREwith results, we also conducted a To In addition to SNAPP, we estimatedalso a species tree using the program PCA, or reconstructs the species tree from quartets. investigate genetic differentiation between

on 53 T.

reference based on minimization of cross

individ (Patterson cf . .

brichardi Teleogramma .

TreeAnnotator (Rambaut and Drummond 2008) was used to find the uals described above - aligned - from the data matrix, calculates a singular decomposition value for

Widom testsWidom in LEA, and generated scatterplots using ggplot2 et al. , which is based on a coalescent model. This method to investigate fine

2006) and

individuals each value nparametric bootstrapping with 5 de novo

to estimate posterior probabilities. From our reduced datase ) trogression

lyses on a subset of17 individuals identified as using the LEA .

. The most likely values ofK were determined in SNP genotypes of Kfrom 1 We identified the number of significant principal - - validation error across replicate runs scale structure. These individuals were placed in . ADMIXTURE computes maximum likelihood

package in R (Frichot and Francois,

Uncertainty quantified is in this method Teleogramma – (Alexander et al. 2009)

15

were analyzed

using SNPs genotyped for the53 t of 25 individuals, we randomly taxa 00 replicates. principal components

, we using ADMIXTURE used theused model

subsampl to identify SVD

(Alexander

quartets T.

in R We ranWe es foures .

-

This article isprotected rights by All copyright. reserved. Accepted distances with theequal angle algorithm. (Huson & Bryant 2005) a used Ne treesimple withpopulations A and B, but rather may be amixture of A and B. script). wespp., used the different numbers of migration events. script that accompanies Treemix to calculate the percent variance explained by with models thenWe built ML trees that included from one to four migration events disequ SNPs independently, and using migration/ Articlephylogeny and subsequently models migration between branches to determine whether between lineages methods. First, we used Treemix Population introgression frequency ofat least 0.05. at least three to filter SNPs for these individuals above,as except were SNPs retained when they occurred in one of fiv i librium. The two trees were identical so further Treemix analysis used allo To determineTo In this test, the f3 (X; A,B) statistic is negative when a population doesX not form a In addition to ADMIXTURE, we assessed e groups based on sampl ighborNet algorithm admixture events improve the likelihood fit.

out offive sampling sites, 50% of individuals from each site

three ( N = 53 individuals) incompatible orambiguous phylogenetic signal within the dataset - . Split network p opulation test

(Bryant & Moulton 2004) windows (Pickrell & Pritchard 2012) ing locality . This method buildsfirst a maximum likelihood

relationships were estimated using uncorrected p

To (Reich

of 500 SNPs grouped together to account for linkage formally test for . We then the used

et al. gene flow

2009) We computed a basic ML tree using all

implemented in Splitstree (4.

included with Treemix ( between taxa using two

admixture

to assess ancestral admixture populations

between , and theused get_f() , and had a minor allele script from Stacks

Teleogramma f the SNPs. threepop tree

13.1) (ML) - based , we -

also

This article isprotected rights by All copyright. reserved. appropriate for secondary calibrations employed as here distribution priors are estimate for 2015) used Supplementary File 1) a As tree prior we used generations, and a burn v2.3 inference Uppsala University, Uppsal models of nucleotide evolution with theprogram (Biomatters Ltd., Aukland, NZ) and MUSCLE v3.5 Plainfield, NJ). Wealigned and forwardedited and reverse chroma for 10 min. 2 ND2Trp 72°C for 1 withmin, afinal extension at 72°C for10 min. fol using primers VF2_t1, based brichardi individual and NADH dehydrogenase subunit 2 (ND2) Mitochondrial data analyses min, 35min, cycles of 94°C for 55°C30 s, for 30 s, and 72°C for 1 min, with afinal extension at 72°C

Accepted Articlelowing amplification conditions: 94°C for2 min, 35 cycles of94°C for30 52°C s, for 40 s, and (Bouckaert the estimated

on the phylogenetic hypothesis of of 31.3 (24.3 We amplifiedWe and sequenced two mitochondrial markers: cytochrome oxidase I(COI

and ND2Met . Weused on the concatenated loci s ofalls putative

Successful amplifications were sequenced on an ABI 3730 at Genewiz, Inc (South Oreochromis lor

et al. Chromidotilapia - split 39.7) Myr

(Kocher 2014)

. FishF2_t1,

not always In order to estimate divergence times between mitochondrial the calibrated -

between in of25% Teleogramma a, Sweden

using 15 million generations with trees sampled every 1000 enzoi

et al. with a with

FishR2_t1, and FR1d_t1 Teleogramma † , using a lognormal distributed relaxed molecular clock optimal using Markov Chain Monte Carlo (MCMC) methods in

from the early Miocene 1995) sensu stricto

normal distribution (Posada 2008)

Yule

species (Schwarzer

with the following amplification conditions: 94°C for for (Heled & Drummond 20 (Kocher

use with

, as , as well as additionalseveral specimens of and

( (Edgar 2004) Chromidotilapia kingsleyae jM

odel Chromidotilapia

). Weperformed Bayesian phylogenetic et al. et al.

primary fossil calibrations, they

T

For the ND2

(Ivanova

(Ho & Phillips 2009) prior 1995) est (Evolutionary Biology Centre, 2015)

and non , based on the minimum age . We determined the best

. The COI across

tograms in Geneious R7.0 et al. 11) - cichlid fossils

from

marker, we used primers

the same (xml file available as

2007) marker amplifiedwas (Schwarzer ) anas outgroup

. with the with

We checkedWe representative . While normal clades, we

et al. can be - BEAST fit model T.

)

. This article isprotected rights by All copyright. reserved. ranged from 731K total of335.64 million reads A. III. Results available. examined, with theexception examination of female fin patterning a minimum of Analysis (PCA) in MorphoJ in order to summarize major axes of shape variation. shapes are obtained. Aligned shape coo configurations depends entirely on shape variation and consensus configurations or mean Analysis (GPA) in subsequently translated, scaled to unit centroid size and rotated with Generalized Procrustes configuration for each specimen was capture lens.Nikkor Shape variation assessedwas with aset of 8fixed landmarks and the landmark photographed on the left side witha Nikon Digital SLR camera and 60 f/2.8mm AF Micro shape in individuals of each putative species g E. Morphological analyses and effective sample size (ESS) >200 for all model parameters. convergence eometric

AcceptedSequencing and SNP calling Article Illumina sequencing of In the absence of evidence ofqualitative character differentiation between taxa,

mophometrics were used to compare variation in overall andhead anterior body

of parameters using Tracer v.1.5 MorphoJ –

14.3M, a with mean

(Klingenberg 2011)

after initial filtering for quality

of Teleogramma

T. cf. depressa rdinates were analyzed with Principal Component ± S D

or populatio d using tpsDIG2 v2.17 ddRAD libraries for 53 individuals resulted in a

= 6.33M ±

(Rambaut & Drummond 2007) for which only 4 female specimens were . After applying GPA, residual disparity among 10 female 2.95 n . Specimens were pinned flat,and

. Numbers of reads per individual M. Mean depth of coverage foreach specimens per taxon were

(Rohlf 2013)

to assess mixing

For qualitative and - This article isprotected rights by All copyright. reserved. sister Acceptedspecies, provides lineages recognized here gracile maximum support for missing data were consistent in all cases. Our final analysis Phylogenetic analysis analyses of the two pipelines, so we primarily present a Results for downstream population genomic analyses were qualitatively similar foroutput from small number ofindividuals, unless those individuals were unusually inbred data can yield accurate estimates of Articleprivate alleles), we expect that poten 0.079) comparable to results from other isolated fish populations, e.g. stickleback diversity in variation in sample size, the with exception of consistently lower heterozygosity and nucleotide for the 20.6Kfrom RAD individual ranged from 19X

sequencing statistics are provide tially tially have a large effect on some summary statistics (e.g., count to (Catchen , de novo The phylogeniesThe constructed Bayesianwith MCMC inference numberThe of nucleotide positions that were polymorphic in at least one taxon ranged T. depressa

indicating the remaining strong de novo – T. monogramma

24.2K for the reference pipeline

et al. support support for monophyly ofa clade containing all middle and lower Congo

that the sensu sensu stricto data in the supplementary material.

2013 all taxa

(Table S3 in current current species designations ( b

geographically isolated as as . ) –

W (Table (Table .

While differences in number of individuals across taxa could 74X (mean = 24.3X) for the reference T

hile large numbers of loci obtained as from . cf. , T. brichardi ).

the branching order of depressa All taxa exhibited similar levels ofgenetic variation despite 1 overall nucleotide diversity and het - ). aligned STACKS pipeline (Table d

Nucleotide diversity (0.067

in Table S2.

nalyses ofreference and )

as wellas a T . cf. Kasai River endemic,

brichardi s T. monogramma

for T. gracile

used two

(Figure 2 morphologically cryptic - 37 aligned data - - 0.131, Table 1) is

, and 826 aligned data. with - 1

based statistics such as genome ), and 16.7K from

erozygosity from even a ,

T. obamaorum SNPs T. monogramma ) T. obamaorum

. The analysis also different amounts of

(0.023

(e.g., herein, and shows - wide Additional - Nei 1978

RADseq

and

is not is , T. , –

is

20.2K ) . This article isprotected rights by All copyright. reserved. mastacembelid eels dates in cichlid radiations should be considered point molecular clock analyses are, ingeneral, sensitive to selection and placement ofcalibration these dates correspond to the ages of mitochondrial rather lineages than Myr recently remaining ~ between the the T. gracile nuclear tree across most clades variablewith bootstrap support Kinganga from strong support RapidesLes to Foulakari) group n As strongly supported, all analyses

Accepted4.7 Article LCR oted ( Myr s, clock models selected, and taxa sampled, and the thus age estimates presented here Banda Nyenge

95% HPDI: 1. obtained ; specifically, a group of individuals sampled from the upper From twoFrom mitochondrial loci (ND2 and weCOI), recovered

clade at ~

previously - within ( Inga region.

95% HPDI: 1.6 LCR

(Figure 1.4 brichardi

(Figure 3

. are consistent with divergence time clade Of t

Myr the

9 in he remaining

, - (Alter and vicinity are reciprocally monophyletic with e.g. T. depressa 3 not 8. ( ( Bayesian analyses ) tentative in light ofongoing debates about macroevolutionary timescales 95% HPDI: 0.

T. gracile SVDq mitochondrial lineage 8 : ). u (Friedman Myr all nlike the Divergence dating of mitochondrial clades indicates that the - form form

7.6 et al. specimens u ) artets recovered the same topology the as SNAPP analysis, but

. Myr

I , t i

a clade with + T. + 2015)

T. depressa (using different numbers ofSNPs) ( s s important to note that individuals originally designated as Figure S1 ) genome wide

et al. and that 4

cf. - 2.5

and two other cichlid clades depressa clade originally , but

2013; López Myr , Supporting Information ,

the T. diversification of with a markedly different topology than that of and the remainder of theLCR lineages occurred )

. cf.

The MRCA for the entire genus was dated as 5.3 geographically proximate nuclear

designated as depressa estimate - , Fernández and

signal , because of mitonuclear discordance, ,

T. brichardi T.

s

cf.

mitochondrial lineages in the for other LCR fishes, including , t T. depressa

brichardi he mitochondrialhe

et al. section well produced the same (Schwarzer ) T. depressa .

T. depressa

2013) falls outside the - supported relationships ) occurred much more species

T. brichardi of theLCR ( form a . However

et al.

from the from , . In addition, those those monophyletic signal

2011)

Kinsuka with with split , topology rest of collected the places

the

-

.

This article isprotected rights by All copyright. reserved. error and different sampling areas. While and higher pairs of patterns are reflected in pairwise brichardi cf. firstThe two PCs Information of thevariation (67.5%) wascaptured by the first four PCs (Figure statistics were significant ( depressa brichardi evolutionary clusters with to little no admixture, with the exception of gene flow between novo ADMIXTURE’s (ADMIXTURE) Pop et al. resulted events in inde thewithin last 5 Myr

Accepteddepressa Article ulation T

SNPs ( 2015; Schwarzer .

cf. cf. values for all other pairwise comparisons ( We alsoWe assessedWe population structure and migration using T.

in are clustered together, consistent phylogeneticwith results (Figure - brichardi and T.

groups the three left bank sampling sites together ( brichardi, T. the formation of theLCR's high

Figure (Figure

). PCA cf. cf. structure T. pendent clades have

, non CV errorCV was minimized for K = 5 for reference brichardi

applied cf. cf. separated outa patterns S2 brichardi individuals to determine population structure - 4c model based genetic PCA, . These studies have hypothesized that , Supporting Information ).

et al. cf. and migration

T ADMIXTURE (Figure . brichardi

depressa P

were broadly similar to 2011)

do not overlap but are close together (Figure < 0.01) for the first eight K 4a ll taxa except for

= 1 F . , likely

,b)

ST and

T

. values, nearly with all of the lowest values calculated for to a had the

. depressa

For K=5, T been driven by . cf. . cf. restricted - energy flow regime beginning roughly 5 Myr depressa lowest error CV

). Both analyses identified all taxa separate as and tree , and/or T.brichardi F T. ST the clustering set ofset samples containing brichardi

= 0.363

PCs are clearly separated along PC 4; - T based methods. the same geologicalthe same changes . cf.

for the genetic PCA analysis, but most -

T. similarly timed depressa , K – T - - cf. cf. T.

aligned SNPs and K = 6 for . model 0.605; Table brichardi

= brichardi cf. cf.

and identified by ADMIXTURE. 2 S brichardi had the second 3 - , Table S ( gene flow between based F We ST

(Kinsuka),

= 0.137 = 0.137 4d and

found that diversification S5 clustering

). 4a and

4 only

). T These These same , Supporting ). .

depressa

Tracy T – T . -

0.397) lowest CV depressa . T brichardi that .

cf - T.

Widom (Alter . -

de T T

and . cf. . - T

. This article isprotected rights by All copyright. reserved. admixture between any ofthe seven taxa werevalues positive with associated Z likelihood ofthe estimated tree model. T bank populations of the components corresponding to K= constant over time much higher extent of shared variation, and to populations for which equilibrium between alleles and linkage equilibrium between loci, resulting from with results ofthe full not significant, genetic structure across populations Supporting Information low water the distance between the two banks can be rapids are less prevalent locations (the Regina that the T. togethersites (Les Rapid brichardi .

cf. cf. Acceptedbrichar Article restricted dataset reveals brichardi T. brichardi Results from TREEMIX (Figure

massive . In contrast - di Kinsuka, and Mbudi/Ngombe ; and 2)

or Kintamo

at applying ADMIXTURE's perhaps ,

- rather than other cf. Mbudi/Ngombe and the right bank cluster cross dataset

nor T T. . ,

gracile some

- cf. cf. homogenous

bank

) Falls) due to limited samp

es, Foulakari and range between brichardi ADMIXTURE analysi 10

rapids betweenrapids left and right bank

-- the river

additional likely

> km downstream, in the region of Mbudi and Ngombe, surface T 5 . .

depressa

explain the majority ofgenetic struct play

across the genome. of , Republic of Congo brichardi -

channel 5 generative model, which From the scores , Democratic Republic of Congo T

) identified two migration events: 1) ed shared genetic variation 0.08 . cf.

(Table S7, Supporting Information)

a role in restricting gene flow between these . However, n le size brichardi - 0. s, s, >

narrows markedly, populations

17 in that some cf. 9.0 in thus allcases, indicating no evidence of three s).

across all sampled sites,

T

less than - (though some pairwise his pattern differs population test of admixture, a either event significantly the improved Whereas in the dataset,full the model ), with some admixture shown between

.

( We interpretWe t Figure S4

0.25 km s

brichardi assumes Hardy

at Kinsuka between to a such that

gene flow not has been

) ) and the two right bank re . .

ure underlying the data, This pattern suggests Values of Values

somewhat stricted dataset populations cluster T. brichardi his difference likewise indicating

and during periods of . T

F . ST - obamaorum Weinberg Les RapidesLes

values werevalues F ST from the

( ll f3

and left Table Table S6

as with

--

>

- a , This article isprotected rights by All copyright. reserved. Acceptedwhen compared to allother congeners gracile in the present context, the is presence in number Alter employed to distinguish between taxonomic assignments morphometric pattern needed to confirm this finding largestwas the largest known geographic distribution within the genus variable taxon, spanning the range ofshape variation exhibited by most other taxa, and also has detected exception of Articlevariation is associated with (Figure Morphology and between be that graph ( history ofhybridization presence of conflicting phylogenetic signal

2015

Fi among LCR species, between In addition to body shape M Finally, we 6 s

, scale counts etc.) are broadly overlapping in range T in th ) gure orphometric analysis . . ). As expected,As based

depressa for While most of these m T. obamaorum is S5 T.

analysis T , Supporting Information

cf. cf. . Morphometrics applied the brichardi

brichardi and

reflected in the PCA

. of individuals in the context of sexually reproducing organisms

T. Notably,

, cf. cf. of a the no and , analysis depressa .

o

Neighbor Despite lack of clear separation between lineages, significant separation of indicates markedly degree ofdorso n previous taxonomic description T ,

Teleogramma T various . eristic attributes (e.g. spinefin cf .

cf. .

. brichardi

. , and between the latterclade and

brich of additional specimens of other The N

high T. monogramma ) indicates the most conflicting phylogenetic signal reduced et combinations of

in the genome wide dataset is

low ardi algorithm

broadly levels , with lowerlevels of conflicting splits/ambiguity -

species ventral lateral line count

appears to be

number of pored scales along the lateralline of consistent with (Bryant & Moulton 2004) intra

depression (Roberts & Stewart 1976 genetically , T. obamaorum

meristic lineal lineal ; however, given that the

across the genus

the most morphologically

s

variation of 32 and ray

of these species, most recognized (Fig. 6b) features genetically , which can indicate a - 36 Teleogramma

T . The resulting splits and

number . large

across the genus gracile , but w

have , uniquely in ,

most noteworthymost lineages ,

to assess pore bearing ; Stiassny & determined

the overall also . s

ith ith , vertebral sample size

the taxa is be

w en

as

T. the

to

This article isprotected rights by All copyright. reserved. p Most river have considered both genome diversitygenetic IV. and an expansion of the these between have 7A a black margin dorsal fin. extentThe and thewidth of white activity) Teleogramma exception of the pattern are series other other LCR lineages. scales

Accepted- Article Discussion F). caudal fin distinguishable ine lineages examined

species ), polarity assessment of these alt c

in these three species contrasts with This seemingly species revious studies o hromidotilapiine ing are evident Riverine cichlids represent an important component of cichlid morphological and Despite

on the upper section of the caudal fin the fin patterning of T. obamaorum

,

patterning is characterized by a

are characterized by the presence of a and these are illustrated photographically and schematically in Figure 7.

on both the caudal and dorsal fin to date. As a

(Loh in order to investigate thefactors potentially responsible for differentiation. lack of

based on difference Unfortunately, as et al. in the posterior f s black clearly clearly , riverine

in possessing a single lateral line (versus with

2013; Brawand (Figure 7G), in which the sexes are monomorphic, allf /lineage T. gracile - margin wide data and quantitative morphological data in

diagnostic similarity cichlid Teleogramma over

specific patterning -

s most ernative configurations problematical.is (Figure 7E) and

diversification of

in

morpho

both the dorsal and caudal fins. female fin pattern a et al.

lateral linecount caudal regions range of marked

and

appears to characterize each 2014; Stiassny and Alter 2015) metric extend

is flag, co

white flagwhite of thedorsal fin and over the 46 highly unusual reduction

T. monogramma have - 60 ing is consistentis across allspecimens we features,

mbined thewith extent and width of

markedly , to varying degrees, revealed ing s

, we note (flushed red during reproductive

of thewidth of thewhite fla and coloration we an

among

interrupted relatively note smal

a (Figure 7F). In both of

strong

l that genetic er

cichlids lineage

scales .

, but few studies large Differences in

African similarity along

emale

dorsal field of lateral line ,

in all

and all - (Figure scale

With the the lineages of

g the This article isprotected rights by All copyright. reserved. Accepted(Showers 2011) entirely than previously thought, which is ofparticular concern given that habitatits overlaps region of Kinganga to Inga. stricto Figure Nyenge proximate LCR ( three geographically indicate that presence oftwo Species designations and cryptic lineages Article LCR development ofa mega history extreme hydrology ofthe LCR the riverine cichlid genus both densely sampled genome fine 2006b; rearrangements, flow reversals, allopatric divergence driven - (Showers 2011) scale, T s 1,s , . cf.

a species , Koblmüller Our SNP data strongly support most existing species designations, but also reveal of

with thearea ofthe LCR proposed for mega at

this group 2 brichardi phylogeographic structure at Kinsuka endemic, the boundary between the upper and middle sections ofthe LCR ) ,

T. depressa are distinct from, yet likely . cryptic lineages within the LCR clade.

restricted et al. ) .

.

comprise and genetically These findingsThese are

- dam (Grand Inga) that would massively impact the flow regime ofthe

(sensu Roberts(sensu & Stewart 1976) is non 2012 Teleogramma

These findings indicate that here primarily T. brichardi ; have -

a lineage that is resolved sister as to the Egger wide data and morphological analysis for allknown species in

in and

to likely

headwater distinct lineages. Individuals from the upper sections the of populations et al. by geological events , indicating highly of particular importance in light ofthe proposed

reciprocally monophyletic with (Figure shaped

2015

local

) capture s s 1, . , and

found In contrast, few studies have identified

that ized

- 2 dam development ). are continuing to shape, Specifically

I T. depressa s microallopatric in , within ndividuals collected a

(e.g., Ready the

resulting in lower section of theLCR in the - - channel monophyletic a , these genome

has a muchhas smaller range

et al.

in the Inga region geographically watershed processes

levels T. depressa

2006a; Ready

( T. round

.

the

cf. cf. Here, we present nd composed of - wide data depressa, driven by the evolutionary

sensu sensu Banda nearly

et al.

such the

This article isprotected rights by All copyright. reserved. with flowing, given its monophyletic clade. However, to relative congenerics, and species of sequential previous study of other LCR cichlid genera LCR g Teleogramma depressa these populations, recommend detailed taxonomic treatment to dispersal, we hypothesiz extent of differentiation. In light of these factors However, small sample suggests that these depressa ADMIXTURE results and distance between each of these cryptic lineages and related congeners, reflected as in cryptic lineages 6A) in female tail patterning ( Acceptedenome Article ,

appear to meristic T . cf Previous studies have been unable to resolve phylogenetic relationships within Although distinct - middle . wide data downstream LCR location

F . depressa

ST the downstream colonization not is supported. =0.27

the adoption of ,

or q middle using using have

section from their putative sister species. This in conjunction with 4 ualitative anatomical

thereby , analyzed here at

lineages play compared with described species, either -

the lower Con s sizes forsizes several ofthe taxa ed

of theLCR

F

upstream based on the single morphological criterion of discernible differences comparing ST

nuclear nuclear genetic a acknowledging their e thatthese lineages

the majority of may values the informal designations central

based on additional specimens the placement of go not yet

indicate that the

clade , with a

( (Figure 1) limit of its

T role in structuring diversification,

Figure . cf. cf. features have , have as expected onbased its distant brichardi vs or morphological distribution (Schwarzer LCR s

7B,C .

fully differentiated range To dateTo

, T. gracile endemic species are su may distinction from and

and 7A,D extreme preclude a definitive conclusion about the

and possibly by represent incipient species. given the presence of hydrological barriers T. yet

, Table S5Table T. monogramma T. T. et al. bordered by, and brichardi

cf. cf.

been found to differentiate these gracile outside ofthis clade is

bathymetry ) brichardi

2011) data

no other morphometric (Figure

(Stiassny & Alter in prep.), we , Supporting Information

has been

the related genetically (Stiassny & Alter 2015) F , ST

the simple null hypothesis of =0.13 T. depressa

and

but pported as

is resolvedis the as and hydrology ofthe , partially

T non 7 found only in

as . cf. t , he lower genetic

T. T. brichardi or morphologically was was -

cf. cf. contiguous depressa

at the interesting

P depressa vs

found in a overlapping forming ending a

slower slower for )

and . sister The The range a , T. T. .

This article isprotected rights by All copyright. reserved. mitonuclear discordance is evident between specie 2011), patterns in introgression is not as high in another as LCR cichlid examined, T divergence 2012a,b events in other cichlid taxa the mitochondrial and nuclear phylogenies has been lineages dating indicates for of agreement and a few areas of strong discord Mitonuclear discordance 10.4] Mya) are Mya), between centr 2011). Nanochromis ( other LCR cichlid genera that have been examined: downstream limit of rangeits S. . brichardi

Accepted Article aff. sp. T. gracile al Congo tributaries (5.3 [4.1 however, Divergence age estimates between When compared with the mitochondrial phylogeny, the nuclear tree shows several areas )

is old relative to subsequent . Teleogramma casuarius In the case of

introgressive hybridization and , the mitochondrial tree places , in which the olderlineage somewhat

age estimates an upstream lineage such as the Teleogramma

and split between the

found in

S. Tel

older. sp. sp.

eogramma and introgression.and (Koblmüller aff.

for ( Figure 1

central Congo show some similaritiesshow

casuarius central Congo - 6.7] Mya) are consistent

mtDNA mtDNA

, mitonuclear incongruence brichardi

) between

et al. . N. minor A similar complex pattern wasidentified in these

"brown pearl") are found in the lower LCR T. brichardi

T diversification events 2007; Nevado

. tributaries

obamaorum (

mitochondrial lineage Nanochromis

Steatocranus Figure 2 T.

is found in the middle LCR

depressa s found on either side ofPool Malebo. Steatocranus interpreted as a result of

outside the . In particular, in both genera, , 3

and

) .

with with our estimated divergence

. In addition to et al. and While the While complexity of

species and lineages the Steatocranus

, in which some older lineages T.

2009;

strongly LCR endemics (5.3 [1.9

gracile . LCR Such

and the remaining LCR and Schwarzer et 2011al. clade, and divergence incongruence between , N. minor

and their sister taxa suggests a

(Schwarzer et al. di

(Schwartzer et al.

hybridization ffer possibly ing

(8.36 [6.5

post the two

; and placement between -

-

8.8] from - ,

This article isprotected rights by All copyright. reserved. specific drift can (Alexander hypotheses method This may result fr apparent from ADMIXTURE results, which show there correspond to those forwhich we likelihood ofthe tree.However, brichardi maximum residuals across a residualfull notable that r these events lineages mitonuclear discordance ra internal branches radiation within expected to Lake Tanganikya (Takahashi ILSancient has primarily internode distances incomplete lineage sorting

Accepted Articlendomly across the tree

produce false negatives may may Despite evidence of An alternative explanation for conflicting mitochondrial and nuclear , does not explicitly fit a historical model, and

- rather than obamaorum to shape patterns across recently remain a weak signal of introgression in the nuclear genome

et al. such as be

remains in (Patterson esults from Treemix

distributed randomly across taxa also

Teleogramma 2009) , om and

no genetic drift and Hardy

been inferred from relatively old (eg.

ILS the limitations of ADMIXTURE

(Figure 5) particularly .

Similarly the nuclear data et al. . (Takahashi

as as but rather

(

ancient ILS past when one ofthepopulations has undergone strong population

et al. 2012)

shows little shows )

the lineages identified as having undergone migration , usually associated with rapid bursts of cladogenesis

, though hybridization in which mtDNA was transferred between , thethree

observe mitonuclear discordance, suggesting did did recover 2001; as . hybridization from m

et al. align with Despite these provisos patterns of mitonuclear discordance are not spread

Koblmüller based on ADMIXTURE and Treemix results

neither modeled event significantly impr covariance matrix 2001

evidence - diverged population test, while aformal test of admixture,

-

two episodes of migration ; geography Weinberg equilibrium in ancestral populat

Seehausen

(Maddison and Knowles 2006). no evidence of migration between these taxa.

to

of et al. relies on simplified population genetic taxa cichlid

detect detect ancient hybridization rapid itonuclear discordance, l ,

,

2010). In addition, rather than older divergences, we

2006 , ) overall speciation bursts, between cladogenesis with relatively long interpret ). As such,As ILS is expected our depressa .

the

analyses support a T

( hese events are not identified as likely signals

effects

- such as gracile in this casein this As theAs source ittle oved the

since the . is are It is

and short oftrace those and

of this

though ions -

in

This article isprotected rights by All copyright. reserved. region and between samples of with separated by a (right bank), whereas gene flow is higher at same that large rapids impede cross hydrological barriers. populations of assessment ofpopulation structu hydrological T populations in 2011) a majoras factor affecting the biogeography of species Hydrological complexity microallopatric and divergence although nuclear DNA (AFLPs) show no evidence of introgression which carry mitochondrial haplotypes closely related to those ofEast African haplochromines, clade of LCR endemic haplochromine cichlids (" (reviewed by Toews and Brelsford 2012) cichlids amounts of, orno detectable, nuclear introgression have been reported in a number of o scenario ofancient ngoing gene flow. Accepted . cf. Article fo

cross depressa , and und a high degree of isolation , located Previous studies (Nevado -

channel sites. h

ydrological features or ,

T. gracile T. Teleogramma considerably greater on either side ofthe

et al. cf

, perhaps . Such mitochondrial capture

brichardi T. depressa

Values ofValues 2009; Schwarzer ) have narrow geographic distributions

features regions of Likewise, LCR

asymmetric, introgression .

from the from majorityThe of - channel gene flow between Kinsuka (left bank) and RapidesLes

sensu strictosensu cichlids have identified F . W ST app re within

Markert (Table (Table S6 hile large

between individuals sampled at Kinganga and distance ear to be important in structuring five

our sampling not does permit a comprehensive et al. . rapids at Isangila

Notably,

each species, sites

et al.

, Supporting Information , but with from three locales 2012; Willis lineages Haplochromis or replacement sampled, also sep

( - 2010) bank sites mitochondrial capture out ( (Markert

T. the followed by population splits with

we analyzed genetic differentiation

brichardi et al. large intervening

and Fwamalo hydrological do

(Kinsuka/Mbudi "

demeusii find find 2014) events accompanied by low broadly in the lower section of theLCR

et al. ar (Schwarzer ,

genetic T. obamaorum ated by

) support the hypothesis 2010; Schwarzer and many othertaxa

both species and

and " correspond complexity of theLCR .

These rapids are was detected in a

structure

a series of rapids, H. -

et al. Ngombe) " fasciatus , the

T. depressa compared 2012) ing between Inga

other to et al. ), .

little

, ,

This article isprotected rights by All copyright. reserved. ecological conditions that (Alter cichlid genera colonization events have been supported in a number ofother biogeographic distribution. t yet relatively weak geographic neighbors characteristics with gracile onbased branch length phylogenetic position of T. depressa rapids,surface colonization by independent colonizations ofthe LCR by phylogenetic pattern we observe from genome not support our original hypothesis ofd of diversification accompanied by Histor water to extend across the entire channel notable due to the narrowness of theriver Acceptedhroughout the system Article rule outanrule alternative scenario in which T.

et al. y of shares with monogramma The The combination of genomic, , colonization diversificationand

2015) T.

cf. depressa,cf. ( T. followed by the subsequent

Steatocranus T. gracil support

gracile , perhaps resulting from watershed rearrangements and T.

obamaorum a strikingly similar fin patterning in the LCR , and experienced subsequent range

e when compared with LCR its congeners (Figure 2).Additionally, for T is , which is the only LCR species that . m gracile seemingly T. the node defining the phylogenetic position of While both scenarios remain speculative, multiple independent ay have

and

brichardi ( Nanochromis

T. ancient outside the and

occurred at least over thetwice last depressa morphological

more

T. and ownstream sequential diversification

Teleogramma monogramma

at introgression in channel

T. arrival similar to these Teleogramma ,

cf. cf. T. remaining , - (Schwarzer wide SNPs suggests the possibility oftwo

cf. cf. brichardi location of depressa at point this (0.3

and biogeographic

the ancestor of

these

by ancestor: an upstream an initial a (Stiassny

LCR clade similar lateralsimilar line configuration, and . This . This

s Teleogramma

arrived in the LCR once, shifts shifts et al. . ). However, upstream

occurs in regions

LCR scenario

2011) & Alter that led to the current

and fish clades, in -

the highly associated rapids 0.8km), causing white congeners ) and mastacembelid eels

data its relativelyits old age particularly given the ~5 ~5 Myr.

; however, thedata is is supported by 2015) T. gracile onset of suggests

for this genus with nowith large . In these

than cluding two Notably, favorable diversified , we cannot a to scenario its its the T t . he he .

do The

This article isprotected rights by All copyright. reserved. conditions allow, A courting female’s caudal finis Unlike most other chromidotilapines, forwhich observations are available, in colored belly toward the whilemale at the same time erecting and depressing herdorsal fin. tightlyheld against aquarium held Lamboj (2004) courtshi crevice spawners between clades. speculate that these lineage consistent (Fig hydrological barriers contemporary therefore discordance documented in this study, al. the LCR Teleogramma ( median ages 0.94 dditional studies, particularly mate choice assays

Accepted Article2015; Keller ure 6 - 4.48 Myr H

p over the last few T. ) ybridization has been . noteworthy

n Direct o differences in female brichardi or

of endemic LCR taxa

, with theexceptions of from species ) Teleogramma

and our ow et al.

A (Schwarzer

are clearly needed. N s noteds previously, and bservation of

mitochondrial the

, and 2013; Wagner . fanned and , even those W the females that we find

body the female contorts into an “S”or “U” shape directing hile million million years video of n observations confirm -

et al. specific patterns may most

( T. brichardi extensively fin fin reproduction prominently displayed

, rather than males, select spawning sites 2011) estimated for species courtship data, indicating the living in relatively

patterning et al. Teleogramma gracile .

onetheless these preliminary

has

are consistent with thedates we have estimated for

2013; Willis

are not strongly and

also close proximity documented in numerous cichlid behavior:

little and

in the Congo

in T

occurred in the past in Nanochromis .

cf. all genetically obamaorum evidence for ongoing

potentially

the following as wellas in

are pairbonding, territorial, cave and brichardi

et al. majority of https://youtu.be/8rA4i60YY0I

in front of

morphologically

2014) and in the absence ofobvious River ).

(1.6 , ecologic act to reinforce boundaries

After an initial approach with fins - - defined field observations w courtship

, and b ha - these 2.67 Myr)

the male (see Figure s behavioral

not been possible but ally Teleogramma

hybridization ased on the mitonuclear lineages diversified lineages , we do observe sequence , and differentiated and lineages Teleogramma

observations

initiate (Fig Steatocranus

her her intensely here here . ure 7 in

It (Ford

among ). is 8 of ) .

We We in et the

, in

This article isprotected rights by All copyright. reserved. continent providing important context for understanding the sources ofgenetic variation driving studies would aid our growing putatively adaptive features such dorso as identification of genomic regions that are linked to particular phenotypes, in particular evolutionarily distant tilapiines ( genome assembly of achromidotilapiine cichlid, as the closest genomes available now are from (islands), or evenly spread theacross genome example, divergence is between parapatric ecotypes limited to afew large genomic r opportunity to assess patterns ofgenomic differentiation along the speciation continuum. For As development on these lineages. al. microscales (<1.5 km), role Brawand anas important source of polymorphismsgenetic for lacu a whole,as especially in light ofrecent studies indicating standing genetic variation from rivers cichlids genomics Conclusions Teleogramma (Figurehere combination with the

2011)

Acceptedthis Article

in shaping genus includes divergences across a spectrum ofages, it presents an excellent

The dataThe presented here add to a growing number of studies exploring the population can can provide and - et al. and diversification patterns in wide diversification in this model group of vertebrates. 7

& future studies . are important for understanding the potential effects of proposed mega

)

2014 geographic structuring and hint

) that important insights into the complex evolutionary history ofAfrican cichlids .

apparently Our results are cons tail patterningtail may

knowledge

istent previouswith studies (Markert These resultsThese Oreochromis

, which indicate that hydrological barriers species/lineage specific female patterningfin documented river cichlids

- of div ventral depression patterns ofdiversification in

play a role in , ~46 Myr). ? also

Such studies would be facilitated by ersification mechanisms in highlight .

A more thorough understanding ofrive

Anothe strine radiations (Loh reinforcing

numerous

and swim bladder loss r potential area for isstudy

et al. species boundaries

areas for future studies. Teleogramma

2010; Schwarzer

river play an important et al. ine

a cichlids, egions

. - 2013 full full Such

dam even at

in ; et r This article isprotected rights by All copyright. reserved. AcceptedBouckaert RR (2010) DensiTree: making sense of sets ofphylogenetic trees. Bouckaert R, Heled J, Kühnert D Benjamini Y, Hochberg Y Alter Brown SE, B, Stiassny ML (2015) Molecular phylogenetics reveals convergent evolution in Alexander DH, Novembre J, Lange K(2009) Fast model References cited wrote an including specimen collection and all morphological data collection and analyses. All authors quality/filtering analyses and population genomic analyses. MLJS conducted field studies genomic data collec Author contributions: Articlegenerous gift from Ms. Janine Luke provided financial support for this study. the Sackler Institute for comments from four anonymous Axios reviewers greatly improved the manuscript. Edwards and Gisella Cacconne (YIBS) for guidance in constructing ddRAD libraries. Perez the project. the Sackler Institute for Comparative Genomics (AMNH) for logistical and financial support of photographs of courtship in captive held analys Arroyave and former AMNH colleagues, Robert Schelly, Hugo Escobar, Jake Lowenstein, and Jairo Monsembula Iyaba, Tobit Liyandja, Justin José Mbimbi Mayi Munene (University ofKinshasa) colleagues Victor of Congo) Marien Ngoubi, and the and Ministry ofAgriculture, D.R.Con Julien Punga Kumanenga, Jean Malekani Mukulire and Koffi Mulumba (University ofKinshasa, the Acknowledgments.

Democratic Republic of (York College/CUNY) e s, ands, 1372 evolutiona approach to multiple testing. lower Congo River spiny eels. unrelated individuals. d edited the manuscript. . . For companionship and assistance in the we field are grateful to our Congolese Chris Bianca Brown - 1373. Oliver Lucanus (Below Water, Montreal) kindly provided video footage and Martinez Davis)(UC provided much helpful input with geometric morphometric Mamonekene

ry analysis. For assistance with permits and logist

tion and conducted phylogenomic analyses. JMS

MLJS

Ministry ofScientific Research and Technological Innovation, Republic Comparative Genomics, Axelrod Research Curatorship, PSC

(1995) Controlling the False Discovery Rate: A practical and powerful

(York College/CUNY) and SEA Congo and the Republic ofCongo provided val PLoS computational biology Genome research and Armel Ibala , et al.

conceived of and designed the study. SEA carried out go) Journal ofthe Royal Statistical Society B

BMC Evolutionary Biology (2014) BEAST 2: a software platform for Bayesian ,

uable assistance in the lab. We and Valentin Serge Pangou (GERDIB, Teleogramma

19 , Rebecca Hersch (University ofMarien Ngouabi), Raoul , 1655 . - based estimation of ancestry in We thankWe George Amato and the ofstaff - ical support during collecting trips to 1664.

10

, e1003537. we extend our appreciation to

15

(SICB/AMNH) , 224. performed alldata are grateful to

Bioinformatics 57 University of

, and Guerry

289 - Helpful Funds from CUNY, and a – 300.

Dan

26

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This article isprotected rights by All copyright. reserved. observed heterozygosity per locus; nucleotide acrosssites the dataset; N calculated by STACKS for nucleotide positions that were polymorphic in at least one population. Table 1. Tables Figuresand T. monogramma T. T. obamaorum T. T. T. T = average number of individuals genotyped at each locus; T. brichardi T. . cf. Accepted Article depressa T. cf. T. gracile T. Taxon

brichardi depressa

Summary genetic diversity statistics for reference

12.5 8.9 4.7 7.5 3.4 2.5 1.4 N

24,115 20,673 22,054 23,468 22,567 24,207 20,769 Sites

%Poly π % Poly

= average nucleotide diversity. 0.202 0.109 0.198 0.147 0.144 0.275 0.109

= percentage ofpolymorphic loci;

0.132 0.108 0.114 0.155 0.124 0.135 0.098 H obs

- aligned data. These values were Sites 0.110 0.095 0.102 0.131 0.108 0.112 0.067 π

= number of

------0.039 0.019 0.019 0.044 0.024 0.041 0.057 F IS

H

obs polymorphic

= average This article isprotected rights by All copyright. reserved. Accepted Article Lenga, 8.Upstream Luozi, 9. Luozi, 10.Bulu, 11. Kinganga, 12.Inga. text are: 1. Kinsuka, 2.Les Rapides, 3.Mbudi, 4. Ngombe, 5.Foulakari, 6.Banda Nyenge, 7.Lenga boundarieswith ofthree main hydrobiological regions delineated. Locations mentioned in the green, an elevational profile oftheLCR from Pool Malebo (280m above level)sea to theAtlantic, have been found colored blocks. In distribution of species along the LCR color coded, and schematically represented by offset monogramma 1.Figure

(a) The lower Congo River (highlighted) and proximate Congo tributaries. Ranges of

(brown and stars),

in sampled regio light grey are regions where no sampling been has possible

T. obamaorum ns below the shown range of

(inverted green triangles) inset. (b)

T. depressa

, or in Pool Malebo . N o Teleogramma

. In T.

This article isprotected rights by All copyright. reserved. topologies recovere 5,000,000 MCMC steps) visualized using DENSITREE Underlying colored tree cloud of the last 4500 trees (sampled every 1000 generations of 2014) tree inferred in SNAPP Multispecies coalescent analysis using 37, 826 genome 2.Figure Accepted Article

is shown in black right Phylogenetic reconstruction of relationships between d.

(Bryant - ang

et al. led tree with posterior probabilities indicated at nodes.

2012)

as implemented in BEAST v2.2 (Bouckaert 2010) - wide SNPs. Maximum clade credibility

Teleogramma

shows the range of (Bouckaert

lineages. lineages.

et al.

This article isprotected rights by All copyright. reserved. Accepted Article the LCR showing approximate locations ofsampled sites inset at upper left. a with genes (COI and ND2) inferred using Bayesian inference in BEAST v2.2 3.Figure relaxed clock and using

Time - calibrated phylogeny of Chromidotilapia kingsleyae

Teleogramma

taxa usingconcatenated mitochondrial

as anas outgroup. Elevational profile of

(Bouckaert

et al.

2014)

This article isprotected rights by All copyright. reserved. Accepted Article aligned SNPs; d) scatterplot o scatterplot of PC1 vs.PC2 a geneticfrom principal component analysis (PCA) using reference (Alexander Likelihood estimation from reference 4.Figure

a) Ancestry proportions of

et al.

2009) , b) ADMIXTURE ance f PC1 vsPC4 from a genetic PCA.

Teleogramma - aligned SNP data forK = 5 in the program ADMIXTURE stry proportions from

specimens determinedas by

de novo SNPs for K = 6; c)

Maximum - This article isprotected rights by All copyright. reserved. mi 2012) 5.Figure

Accepted Article gration weight indicates the proportion of ancestry deriving from the migration edge.

with two migration edges. driftThe parameter proportional is to 2Ne generations and

Graph of admixture between 22 individuals fitted using TreeMix

(Pickrell & Pritchard

This article isprotected rights by All copyright. reserved. Accepted Article PC1 accounts for 49% of totalshape variance, and PC2 16%. landmarks in study, with deformation specimens sampled from acrossthe generic range: b) location of homologous lan 6.Figure a)

c) c) Scree plot of percentage variance explained by each PC as a % of totalvariance;

Principal Component Analysis of shape variance across s

implied by PC1 scores indicated by black bars subtending

7 4 adult

Teleogramma dmarks used

This article isprotected rights by All copyright. reserved. obamaorum b) female and male Fi gure 7.

Accepted Article T . cf.

brichardi

Variation in fin patterning across genetically .

, c) , c) Teleogramma brichardi T . cf.

depressa

, d) T. depressa . In panel below (only females shown): a) , e) , e) T. - gracile defined lineages. At top: aquarium

, f)

T. monogramma , g) T. brichardi T. - held , This article isprotected rights by All copyright. reserved. S1:Table Specimens and accession numbersAccepted Supporting Tables algorithm (Huson and Bryant 2006). Figure S5. Figure S4: Figure S3: Figure S2. correspond to SVD Figure S1: Supporting Figures Article Sup Oliver Lucanus). showing the characteristic display ofthe female presenting caudal fin to the (courtesymale of 8.Figure porting Information: quartets method ofphylogenetic analysis (Chifman and Kubatko 2014). Branch labels

Unrooted phylogenetic analysis computed in SplitsTree4 using the NeighborNet CV Error A pair of aquarium held Best supported topology of ADMIXTURE Percent variance in principal components using reference

proportion of

plots for ADMIXTURE analysis.

(see below)

(see below)

plot for

bootstrap values (500 bootstraps) T. brichardi

Telegramma brichardi

Teleogramma

and populations of

using theusing

photographed during courtship,

Single Root Decomposition

T. supporting each node

cf. cf. brichardi - aligned data .

.

This article isprotected rights by All copyright. reserved. Accepted discoveryfalse rate correction for multiple tests Articlereference S6:Table tests All values S5:Table S4:Table STACKS. S3:Table alignedreads to reference genome and mean coverage. S2:Table

(q=0.0476)

-

F Tracy Summary genetic diversity statistics for data analysed using the Number of reads, number ofvaria F in bold aligned pipeline ST ST

values calculatedvalues between calculatedvalues between taxa using the reference - Widom statistics for PCA using (Benjamini and Hochb were found to be significant after s

in STACKS . All values in bold were found to be significant after a erg 1995) T. brichardi ble loci genotyped in each individual, percent of reference

(q=0.0409) .

a false discovery rate

and populations of

- aligned data

(Benjamini and Hochberg 1995). - aligned pipeline

T.

correction for multiple cf. de novo brichardi s

pipeline in in STACKS using theusing

.