cichlid fishes have undergone spectacular radiations in different parts of the world . in particular , the species flocks of the east african great lakes are well - known examples for rapid evolution and speciation [ 15 ] .
tanganyika , malawi , and victoria  is inhabited by hundreds of mostly endemic cichlid species [ 6 , 7 ] .
notably , most of the diversity is found in the littoral habitat , whereas reduced species richness in the deep benthal and pelagial seems to be a common phenomenon in all east african great lakes [ 710 ] .
at least three factors may have contributed to this pattern : ( i ) reduced niche diversity in the pelagic and in deepwater benthic zones , ( ii ) a narrow ambient light spectrum consisting only of short - wavelength blue light and hence less promotive of diversification mechanisms contingent on color perception than the shallow clear - water habitats [ 1114 ] , and ( iii ) the absence of strong barriers to gene flow .
indeed , deepwater cichlid species often have lake - wide distributions with very low , if any , population genetic structure over large geographic distances [ 10 , 15 , 16 ] ( see also the lake tanganyika clupeid limnothrissa miodon   and the centropomid lates stappersii ) . on the other hand , high levels of genetic differentiation , sometimes accompanied by phenotypic divergence on small geographic scales , are characteristic for the species - rich guild of stenotopic rock - dwelling cichlid species [ 1931 ] .
however , allopatric diversification in the fragmented littoral zone was not necessarily accompanied by the evolution of pre- or postzygotic isolation , so that secondary contact imposed by lake level fluctuations has often led to hybridization or introgression between previously allopatric taxa [ 3235 ] . even today
, substrate breeders of the tribe lamprologini can be found in mixed - species pairs , and interspecific fertilizations occur in communally nesting , shell - breeding lamprologines .
indeed , phylogenetic analyses of predominantly littoral cichlid lineages revealed that interspecific hybridization has played , and still plays , an important role in the evolution of these fish [ 32 , 34 , 3641 ] .
thus , in the majority of recent molecular studies on species relationships within littoral tribes , especially when comparing mitochondrial and nuclear phylogenies , the explanation for the tree topologies involved the claim of introgression and hybridization between established lineages in addition to incomplete lineage sorting ( reviewed in ) .
we hypothesize that this is not the case in tribes composed of deepwater species . lacking the geographic structure introduced by littoral habitat heterogeneity
, deepwater species may still be spatially separated by distance , by segregation of breeding grounds , by variable hydrological conditions [ 16 , 43 , 44 ] , or by large - scale fragmentation of the lake basin during major droughts [ 45 , 46 ] .
generally , however , the potential barriers to gene flow for deepwater species are less insurmountable than those met by stenotopic littoral cichlids . specifically in lake
tanganyika , the evolution of stenotopy regarding depth , bottom type , or light intensity may have been prohibited by the seasonal upwelling of anoxic waters .
we postulate that given the high potential for gene flow , diversification of lineages will either be curtailed ( as suggested by the relative species paucity ) or be attended by strong reproductive isolation right from the start .
this would imply that introgression following cladogenesis occurred at much lower rates , if at all , in the deepwater species than in littoral cichlids .
we test this hypothesis in the deepwater cichlid tribe bathybatini by comparing phylogenetic trees based on mitochondrial sequence data with trees obtained with nuclear multilocus aflp data , an approach which has previously revealed hybridization in littoral cichlids and other contexts [ 34 , 36 , 37 , 39 , 40 , 4854 ] . moreover , in several studies , phylogenetic inference on species relationships has benefited from the use of multilocus genetic data , and we expect that the aflp data collected in the present study will also contribute to the resolution of intergeneric relationships within the bathybatini , which are still debated due to conflicting or ambiguous molecular and morphological evidence [ 46 , 5559 ] . the tribe bathybatini ( sensu takahashi ) comprises 17 species in three genera : ( 1 ) the genus bathybates comprises six large ( 3040  cm ) , piscivorous species preying mainly on pelagic freshwater clupeids ( b. fasciatus and b. leo ) , benthic cichlids ( b. graueri , b. vittatus , and b. ferox ) , or undefined prey ( the rare , elusive b. horni ) , in addition to the small ( 20  cm ) b. minor , which is a specialized clupeid hunter . in accordance with their trophic niches ,
coulter   distinguished three morphotypes among the bathybates species , the fast - swimming fusiform predators b. fasciatus , b. leo , and b. horni , the generalized shape of the benthic feeders b. graueri , b. vittatus , and b. ferox , and the small clupeid - mimicking b. minor , which mingles with its prey and accompanies the diurnal clupeid migrations . based on trawl net and gill net catches , b. minor were classified as pelagic , b. fasciatus and b. leo as chiefly bathypelagic and the remaining four species ( b. graueri , b. vittatus , b. ferox , and b. horni ) as chiefly benthic . except for b.
minor , which was never found below 70  m , bathybates species descend to depths of 150200  m. ( 2 ) the member of the monotypic genus hemibates , h. stenosoma , is an abundant benthic species on the muddy bottom of southern lake tanganyika feeding on fish and shrimps mainly at depths between 100 and 200  m .
( 3 ) the small - bodied ( < 15  cm ) species of the genus trematocara ( formerly assigned to the genera trematocara and telotrematocara , ) comprise nine benthic and bathypelagic species feeding on a variety on invertebrate prey , fish larvae , and phytoplankton .
they are found at maximum depths of 75 to 200  m . following the upward movement of zooplankton ,
some species release their fry in shallow areas , but overall , data on bathybatine breeding behaviour is anecdotal or lacking [ 43 , 60 ] .
the species are sexually dimorphic , with males of bathybates and hemibates exhibiting species - specific patterns of dark stripes , bars and dots on a silver background and egg - spots on the anal fins , and males of the silvery trematocara with dark dorsal fin markings .
all bathybatini have large eyes , which promote not only the detection of prey and predators but possibly also mate - recognition in the dark depths . in line with the latter , the monochromatic patterning of males may be viewed as adaptation to the short - wavelength dominated visual environment , in contrast to the colourful patterns of the mouthbrooding cichlids in the shallow , light - flooded littoral .
a recent phylogenetic study based on three mitochondrial genes supported the monophyly of bathybates as well as of the species therein and indicated a polytomy of three equidistant lineages representing bathybates , hemibates , and trematocara . within bathybates , b. minor appeared ancestral to a radiation of the six large species , which showed a basal split of b. graueri and low statistical support for the branching order of the remaining species ( figure 1 ) .
the short internal branches among the large bathybates species supported a rapid radiation at approximately 2.32.7 mya , coinciding with the rapid diversification of other lake tanganyika cichlids [ 28 , 45 , 62 ] .
competition and resource partitioning as well as potential geographic isolation during an extreme low - stand of the lake were proposed as promoters of bathybates speciation .
  this study is based on a total of 38 specimens , representing all seven bathybates species , hemibates stenosoma as well as trematocara unimaculata and t. macrostoma ( table 1 ) .
all specimens were obtained between 1999 and 2011 from local fishermen at lake tanganyika and identified to species by s. koblmller .
unfortunately , it was not possible to obtain a comprehensive taxon sampling for the genus trematocara as , because of their small size and hence low market value , these fish ( except for the largest species t. unimaculatum ) are not caught by local fishermen .
fin clips or white muscle tissue were preserved in ethanol and dna was isolated using a proteinase k digestion / high salt precipitation method .
the ten primer combinations used for selective amplification were ecori - aca / msei - caa , ecori - aca / msei - cag , ecori - aca / msei - cac , ecori - aca / msei - cat , ecori - act / msei - cat , ecori - act / msei - caa , ecori - act / msei - cag , ecori - act / msei - cac , ecori - acc / msei - caa , and ecori - acc / msei - cac .
selective amplification products were visualized using an abi 3130xl automated sequencer ( applied biosystems ) along with an internal size standard ( genescan-500 rox ; applied biosystems ) .
polymorphic positions were initially identified using genemapper 3.7 software ( applied biosystems ) in a range of 50500  bp . in order to adjust misaligned bins and avoid size homoplasy ,
bins containing ambiguous low intensity peaks in a large proportion of the samples and entire profiles with short read - lengths or very low peak heights were deleted .
these preprocessed , un - normalized peak - height data were analyzed with aflpscore 1.4a , which optimizes thresholds for locus retention and phenotype calling based on estimated error rates .
phenotype calling thresholds were set as absolute or relative depending on the number of retained loci and the achieved error rates obtained with each option .
20 replicate samples were included to calculate the mismatch error rate for all unique loci .
a neighbour joining ( nj ) tree based on nei and li 's distances   was constructed in paup 4.0b5 .
bootstrap values from 1000 pseudoreplicates were used as standard measure of confidence in the inferred tree topology . although accurate models for bayesian tree construction using aflp datasets do exist ) , the high demands for processing power make them unfeasible to use [ 67 , 68 ] .
thus , bayesian phylogenetic inference ( bi ) was conducted in mrbayes 3.1.2 , employing the restriction site model with the  noabsencesites  coding bias correction [ 68 , 70 ] .
the dirichlet prior for the state frequencies was set to ( 2.44 , 1.00 ) matching the actual 0/1 frequencies in the dataset .
posterior probabilities were obtained from metropolis - coupled markov chain monte carlo simulations ( 2 independent runs ; 10 chains with 8,000,000 generations each ; chain temperature : 0.2 ; sample frequency : 1,000 ; burn - in : 4,000,000 generations ) .
chain stationarity and run parameter convergence were checked in tracer 1.5 . to test for homoplasy excess introduced by hybridization
, we conducted a tree - based method as outlined by seehausen   by removing single species from the dataset and observing the change in bootstrap values in the nj tree ( see also [ 34 , 40 ] ) . in theory
, the inclusion of a hybrid taxon in a multilocus phylogeny introduces homoplasy with clades that contain its parental taxa .
hybrid taxa should be intermediate to the parental taxa since they carry a mosaic of parental characteristics .
thus , decreasing the amount of homoplasy in the dataset by removing the hybrid taxon should increase the bootstrap support for the clades that include the parental taxa or their descendants , whereas removing nonhybrid taxa should have no effect on the statistical support of other nodes ( figure 2 ) . testing for consistency between mtdna- and aflp - based tree topologies employed two different strategies . in a first test we evaluated whether our aflp - nj - topology can be explained by the mtdna data of koblmller et al . .
using the mitochondrial sequences , we inferred the log likelihood of the aflp - nj - topology data by constraining maximum likelihood ( ml ) tree search to a topology identical to the species tree suggested by the nj analyses of our aflp data , applying the substitution model used in this previous study ( hky+i+g ) . to test for significant differences between the unconstrained   and constrained mtdna topology we performed an ml - based shimodaira - hasegawa ( sh ) test   ( full optimization , 1,000 bootstrap replicates ) in paup . in a second test we evaluated by means of a bayes factors approach   whether the mtdna tree   can be explained by our aflp data , and whether the aflp - nj and bi trees differ significantly from each other .
we performed bi searches constraining the topology to that of the mtdna - topology   and the interspecific relationships implied by the aflp - nj - tree in mrbayes 3.1.2 employing the same settings as above .
bayes factor comparison  using the harmonic means of the likelihood throughout different runs [ 77 , 78]among the three alternative phylogenatic hypotheses was performed in tracer 1.5 .
values of 2  ln  bf ( two times the difference between the harmonic means of the two models ) > 10 are considered strong evidence for support of one model over another .
the final aflp dataset consisted of 659 unique loci with a mismatch error rate of roughly 3% , which falls within the acceptable limit for mismatch error rates as defined by .
both the nj and bi analysis yielded largely congruent and well - supported topologies with only minor differences between them ( figures 3(a ) and 3(b ) ) . whereas all species were monophyletic in the nj tree , bathybates graueri was not resolved as a monophylum in the bi tree , but as paraphylum including the well - supported clade of the other large bathybates species ( figure 3(b ) ) . despite this minor topological difference
, bayes factor comparison strongly supports the bi tree over the nj tree ( 2  ln  bf = 26.828 ; figure 3(c ) ) .
we note , however , that the two - state model implemented in mrbayes does not fully cover the complex genetic process of aflp evolution and thus provides accurate phylogenetic inference less likely than distance methods [ 67 , 79 , 80 ] .
hence , the observed differences between aflp and nj tree topologies might be attributed to this problem .
both the nj and bi analyses support the monophyly of all three genera with the genus trematocara representing the most ancestral branch ( figures 3(a ) and 3(b ) ) . within the genus bathybates ,
the small and morphologically most distinct member of the genus , b. minor , was sister taxon to the remaining large bathybates species .
branch lengths among the large bathybates species are rather short and some received rather low statistical support , indicating a period of rapid cladogenesis .
nevertheless , both nj and bi analyses revealed a largely consistent phylogenetic pattern within the large bathybates species . both sh - test and bayes factor comparison revealed significant differences between mtdna and aflp phylogenies ( sh - test : ln  l of 9505.385 versus 9585.142 for mtdna versus aflp - nj - topology - constraint , p < 0.001 ; bayes factors : 2  ln  bf of 88.576 between mtdna and aflp - bi - topology and 61.748 between mtdna and aflp - nj - topology , figure 3(c ) ) .
the homoplasy excess test provided no evidence for introgressive hybridization ( data not shown ) .
in the original classification of lake tanganyika cichlid tribes by poll , bathybates and hemibates were included in a tribe bathybatini as sister group to the trematocarini ( equivalent to the genus trematocara ) , a hypothesis supported both by lepidological   as well as allozyme data .
in contrast , based on morphological characteristics , stiassny   and takahashi   proposed a sister group relationship of bathybates and trematocara . currently , all three genera , bathybates , hemibates , and trematocara , are united in the tribe bathybatini .
a previous mtdna phylogenetic study remained equivocal with regard to the two competing morphological classifications and suggested that bathybates , hemibates , and trematocara diverged rapidly from their common ancestor . in the present aflp phylogeny , the length differences between the most ancestral branches favour poll 's original classification with trematocara as sister group to hemibates + bathybates [ 55 , 57 , 58 ] .
consistent with the mitochondrial phylogeny , the aflp data confirm the split between bathybates minor and the larger members of the genus bathybates with b. graueri as their most basal representative and identifies a period of rapid cladogenesis at the onset of the diversification of the large bathybates species .
however , mtdna and aflp phylogenies differ significantly with respect to the branching pattern among the remaining large bathybates species .
introgressive hybridization ( including the possibility of complete mtdna replacement ) and ancient incomplete lineage sorting are two alternative sources of topological disagreement between nuclear and mitochondrial trees [ 40 , 41 , 8183 ] , resulting in similar phylogenetic patterns that are difficult to resolve by strict hypothesis testing .
circumstantial inference can be based on the fact that lineage sorting is expected to lag behind rapid cladogenetic events , such that the rapid radiation of the large bathybates species predisposes this clade to mitonuclear phylogenetic incompatibilities without implying postcladogenetic introgression [ 72 , 73 ] .
likewise , monophyly of species in both mitochondrial and nuclear trees ( excepting the paraphyly of b. graueri in bayesian aflp tree ) and negative tests for homoplasy excess in the aflp data do not indicate the presence of hybrid taxa in the genus bathybates .
these findings support our hypothesis that deepwater species are less prone to introgressive hybridization and hybrid speciation than littoral species , but reservations arise on the one hand from the possibility that events of introgression were not detected in our samples and data and given the power of our analyses , and on the other hand from the small number of species in the phylogeny .
principally , rates of interspecific introgression may not differ between littoral and deepwater cichlids , but will nonetheless lead to higher incidences of introgression in the species - rich groups than in a less speciose clade .
if this was the case , the lack of a signal of introgression in bathybates and hemibates would be fully explained by the low diversification rate of the lineage and hence limited opportunity for interspecific hybridization , without implying the evolution of complete reproductive isolation early on during diversification .
the branching order of hemibates and the basal bathybates species , which is reconstructed congruently by mtdna and aflp markers , suggests repeated transitions between benthic and bathypelagic feeding mode ( ecological data from ) .
the basal h. stenosoma represents a benthic generalist feeding on shrimps and various species of fish .
the next split led to the specialized pelagic clupeid hunter b. minor , which mimics its prey in size and coloration and stages surprise attacks from within the sardine shoals .
then , b. graueri took a step back to the benthic habitat , specialized on cichlid prey and evolved a large body size .
the chronology of the following radiation of the large bathypelagic clupeid hunters and benthic cichlid hunters remains unresolved , but involved at least one transition from benthic to bathypelagic habitat preferences .
depth preferences may vary between these species , such that speciation may have involved both niche and spatial segregation . the apparent ecological differentiation among lineages
may have reduced the fitness of hybrids [ 8587 ] and may have promoted the evolution of a mate recognition system , perhaps based on the species - specific melanic patterns of male hemibates and bathybates .
the efficacy of monochromatic black , silvery , and white body and fin patterns in mediating assortative mating in the dark , short - wavelength dominated environment has recently been demonstrated for deepwater cichlid species of lake malawi .
these sympatric and morphologically similar species differ primarily in male nuptial patterns and their reproductive isolation is corroborated by genetic differentiation estimates
. however , there is increasing evidence that color pattern is not the only cue for mate recognition in cichlids fish and it is possible and likely that auditory   and olfactory cues   play a role in mediating assortative mating in deepwater species , too .
in concert with previous mitochondrial data , the present study provides an informative phylogeny of the species in the deepwater genera hemibates and bathybates . as far as the branching pattern can be resolved
the rapid radiation within bathybates mirrors a burst of speciation observed in several other cichlid tribes of lake tanganyika and reveals a congruent cladogenetic pattern across vastly different habitats , which suggests some kind of synchronization by environmental factors [ 27 , 45 , 46 , 62 ] .
consistent with the hypothesis that lineages evolving in the weakly structured deepwater habitat would develop stronger reproductive isolation than the allopatric lineages of the fragmented littoral , our data provided no evidence for the presence of hybrid taxa in the deepwater dwelling genus bathybates . in further support of the hypothesis , introgressive hybridization is also not indicated by the mitochondrial and aflp phylogenies of the lake tanganyika cichlid genus xenotilapia , which includes several deepwater - dwelling species comprising the prey of the benthic bathybates and hemibates . an increased sample size to evaluate this pattern
will be attained by the analyses of additional open - water and deepwater species , for example , the tribe limnochromini and the genus trematocara .
 hybridization among littoral cichlid species in lake tanganyika was inferred in several molecular phylogenetic studies . 
 the phenomenon is generally attributed to the lake level - induced shoreline and habitat changes . 
 these allow for allopatric divergence of geographically fragmented populations alternating with locally restricted secondary contact and introgression between incompletely isolated taxa . 
 in contrast , the deepwater habitat is characterized by weak geographic structure and a high potential for gene flow , which may explain the lower species richness of deepwater than littoral lineages . 
 for the same reason , divergent deepwater lineages should have evolved strong intrinsic reproductive isolation already in the incipient stages of diversification , and , consequently , hybridization among established lineages should have been less frequent than in littoral lineages . 
 we test this hypothesis in the endemic lake tanganyika deepwater cichlid tribe bathybatini by comparing phylogenetic trees of hemibates and bathybates species obtained with nuclear multilocus aflp data with a phylogeny based on mitochondrial sequences . 
 consistent with our hypothesis , largely congruent tree topologies and negative tests for introgression provided no evidence for introgressive hybridization between the deepwater taxa . 
 together , the nuclear and mitochondrial data established a well - supported phylogeny and suggested ecological segregation during speciation . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Summary and Conclusions