Copria, 1994(2), pp. 274-288

Molecular Systematics and Radiation of the Haplochromine Cichlids (Teleostei: Perciformes) of Lake Malawi

PAUL MORAN, IRV KORNFIELD, AND PETER N. REINTHAL

Mitochondrial DNA (mtDNA) restriction fragment length polymorphisms were assayed among 40 species of Lake Malawi haplochromines (Cichlidae) including representatives ofecologically divergent genera. Six distinctive mtDNA lineages were distinguished, two of which were major clades, represented by a large number of species. The other four lineages were each represented by a single species with a divergent mtDNA haplotype. One of the two major clades was composed of the shallow-water, rock-dwelling mbuna species, whereas the other included a diverse array of sand-dwelling and pelagic species. A number of taxa, found to be firmly embedded within the mbuna clade, are quite distinct in mor­ phology and generally inhabit deeper, sediment-rich areas rather than the rocky habitats typical of other mbuna. The mbuna group is generally thought to be a monophyletic assemblage, but these results suggest that it is actually paraphy­ letic. In contrast to the high morphological diversity among Malawi haplochro­ mine species, mtDNA sequence divergence was found to be remarkably low. This finding underscores the unprecedented rapidity of speciation and evolutionary plasticity in this fish species flock.

eginning with the earliest collections and vented identification of sister-group relation­ B descriptions of Malawi haplochromines, ships (though many autapomorphic traits are taxonomists have been challenged and frus­ present). Second, an abundance of parallelism trated by attempts to reconstruct the phyloge­ has made it difficult to assure that shared traits netic history of this fauna. Two primary obsta­ are actually synapomorphic (Eccles and Tre­ cles have confounded this reconstruction. First, wavas, 1989). Early work on the Malawi ich­ a paucity of shared derived traits often pre- thyofauna revealed a large number of new spe­

© 1994 by the American Society of Ichthyologists and Herpetologists ~~~_____"'H,...... , ... .., th...... "N'~ ..h ... ,,__ .. ____ -_ .. l"'T.T_t.___ ....,~ ...... "·· MORAN ET AL.-RADIATION OF MALAWI CICHUDS 275 cies (Gunther, 1864; Boulenger, 1908; Regan, mitochondrial DNA (mtDNA) to examine an 1922). Christy made extensive collections in ecological cross-section of species. MtDNA 1925 and 1926, which Trewavas included in a analysis is particularly amenable to systematic synopsis of the Malawi haplochromines (1931, studies of closely related taxa (Wilson et aI., 1935). Based on these studies, it became clear 1985; Avise et aI., 1987; Moritz et aI., 1987) that Lake Malawi contained one of the most because of its primarily maternal, haploid, and species-rich vertebrate faunas known. Because nonrecombining mode of inheritance (but see of the lack of clear synapomorphic characters, Gyllensten et aI., 1991). Moreover, mtDNA a large number of the Malawi species were orig­ evolves more rapidly and shows greater sensi­ inally placed in the genus 'Haplochromis.' The tivity to historical biogeographic events than 1935 synopsis did not completely describe all does single copy nuclear DNA (Brown et aI., the members of 'Haj)lochromis' in Malawi, and 1979). This study examined 40 species repre­ it was widely recognized that this genus was senting 32 genera in Lake Malawi. These gen­ polyphyletic. The name ofthat species complex era included representatives of the most mor­ was subsequently changed to 'Cyrtocara' to re­ phologically divergent lineages, as well as seven flect its apparently discrete evolutionary path of the 10 mbuna genera and 11 of the newly relative to the Lake Victoria fauna in which described nonmbuna genera (Eccles and Tre­ Haplochromis was first described (Greenwood, wavas, 1989). Representatives of four genera 1979). from Lake Victoria and two from Lake Tan­ A recent monograph by Eccles and Trewavas ganyika were examined as outgroups. (1989) described 22 new genera in Lake Ma­ lawi, bringing to 21 the number ofgenera from the former 'Haplochrolllis' complex. The genus MATERIALS AND METHODS Cyrtocara is now monotypic containing only C. moorii. These new descriptions were based on Restriction enzyme analysis of mtDNA was phenotypic characters, principally melanin pat­ used to estimate genetic relationships among terning, which werejudged to be evolutionarily haplotypes. Estimates were made using both conserved. With these new descriptions, the to­ phenetic and cladistic techniques. Haplotypes tal number of haplochromine genera in Lake were defined by a unique composite of all re­ Malawi is now 49. striction profiles. MtDNA isolated from fresh The diverse and species-rich East African and frozen fish tissue was purified by density cichlid fauna presents an excellent model sys­ gradient ultracentrifugation (Lansman et aI., tem for critical examination of the processes of 1981; Dowling et aI., 1990). Restriction digests speciation and adaptive radiation (Futuyma, were carried out per manufacturers' instruc­ 1986; Greenwood, 1991; Kornfield, 1991). In tions (GIBCO BRL, Gaithersburg, Maryland; less than two million years, the haplochromine Boehringer Mannheim, Indianapolis, Indiana; cichlids of Lake Malawi have radiated to fill New England Biolabs, Beverly, Massachusetts); almost every conceivable trophic niche. This visualization offragments was achieved by rapid assemblage includes a diverse array of pelagic end-labeling (Drouin, 1980) and autoradiog­ planktivores, piscivores, detritivores, mollusci­ raphy. Fragment sizes were estimated by com­ vores, paedophages, scale eaters, and insecti­ parison to size standards included on each gel vores (Fryer and Iles, 1972). The species that [1 kilobase (kb) ladder, GIBeO BRL]. comprise these trophic groups fill niche space To identify a suite ofenzymes providing mul­ that might be occupied by 10 or more fish fam­ tiple restriction sites consistently resolvable by ilies in other great lakes of the world. A group agarose gel electrophoresis (0.5-2.0%), an ini­ of species of particular interest to evolutionary tial screening was conducted using 29 restric­ biologists is the rock-dwelling mbuna (Trewa­ tion endonucleases (data not shown). Subse­ vas, 1935; Ribbink et aI., 1983; Reinthal, 1990). quently, 55 individuals were examined using 18 This assemblage includes an estimated 200 spe­ restriction enzymes. Our final analysis included cies in 10 genera and has radiated over an ex­ 40 species representing 32 Malawi genera. This tremely short period of time (Kornfield, 1978). sample represented most of the morphologi­ The mbuna are morphologically distinct from cally divergent taxa in the lake, including seven the other haplochromines in Lake Malawi and of 10 mbuna genera and 13 of 15 nonmbuna are presumably monophyletic (Regan, 1922; genera recognized prior to the recent rede­ Trewavas, 1935). scription of the Malawi haplochromines (Eccles To provide additional perspective on the phy­ and Trewavas, 1989). In all, we have examined logenetic systematics of the Malawi ichthyofau­ representatives of over half (63%) of the 47 na, we have used restriction enzyme analysis of endemic cichlid genera in Lake Malawi. Collec­ 276 COPEIA, 1994, NO.2 tion locations and species are provided in Ma­ ping, we expect that the relationships presented terials Examined; restriction enzymes are listed here will be robust to further analysis, including in the Appendix. Voucher specimens have been site mapping and DNA sequencing. deposited with the American Museum of Nat­ As a phenetic measure of genetic distance, ural History (AMNH). sequence divergence was estimated among hap­ Cladistic analyses were conducted using Wag­ lotypes (nucleotide diversity, dij; Nei and Li, ner parsimony and strict consensus with the tree­ 1979; Nei, 1987). Phenetic relationships based bisection-reconnection algorithm of P A UP Ver. on sequence divergence estimates were visual­ 3.0 (Swofford, 1989). Characters for cladistic ized by clustering taxa with the neighbor-join­ analysis consisted of the presence or absence of ing algorithm (Saitou and Nei, 1987). Results individual restriction fragments. Replicated of UPGMA analysis (not shown) were broadly bootstrap analyses were used to evaluate the concordant with neighbor-joining. We have relative strength of cladistic groupings using chosen not to report these results and focus PHYLIP 3.1 (Felsenstein, 1988). The number rather on neighbor-joining because UPGMA has ofbootstrap replicates was limited to 200, which been shown to be less reliable than other meth­ required 36.1 h of mainframe CPU time (IBM ods of phylogenetic analysis (Hillis et aI., 1992). 3090). A limited number ofbootstrap replicates Because neither neighbor-joining nor parsi­ may limit the power of statistical inference mony analysis provide accurate portrayal of ge­ (Hedges, 1992). In spite of the controversy sur­ netic distance, principal coordinates analysis was rounding the use of bootstrapping and its sta­ performed (NTSYS Ver. 1.6, Rohlf, 1990). tisticallegitimacy, we feel that these results are Principal coordinates analysis maximizes the or­ useful as a relative measure of the strength of thogonal separation of haplotypes and balances specific groupings. the tendency of agglomerative methods to pro­ Two primary criticisms have been advanced duce clusters, regardless of the structure of ge­ against restriction fragment length polymor­ netic relationships. By employing both cluster­ phism (RFLP) analysis of mtDNA in favor of ing and ordination, groups common to both site mapping. First, the use offragments as char­ techniques were identified; such groups are like­ acters in cladistic analysis violates a fundamen­ ly to be particularly robust (Rohlf, 1990). Input tal Hennigian assumption, i.e., that the char­ data files for all of the above analyses (binary acter state changes are independent. It is argued code and d-value matrices) were generated us­ that single site changes are weighted more ing REAP (McElroy et aI., 1992). heavily than they should be, because one site The principal coordinates projection is pre­ change results in three fragment state changes sented with a minimum spanning tree (MST) (Swofford and Olsen, 1990). This is true in prin­ superimposed on the haplotypes. The MST is ciple; however, when a large number of char­ useful in that it can reveal local distortions in acter state changes separate major clades, the the principal coordinates projection by identi­ error induced at each state change becomes rel­ fying pairs of haplotypes which appear to be atively small. The critical issue in cladistic anal­ similar on the first three principal axes but are ysis is the ratio of synapomorphic characters to more distant if additional dimensions are in­ convergent characters (signal to noise). Because cluded (Rohlf, 1990). shared fragment states have equal weights whether they are synapomorphic or conver­ RESULTS gent, no directional bias is introduced. Ran­ domly distributed errors would not be expected From the 29 restriction enzymes originally to consistently give the same incorrect phylo­ screened, 18 were selected for their ability to genetic topology when a large number of char­ yield consistent cleavage and reliable labeling acters is examined. A second criticism of frag­ and to provide multiple restriction profiles with ment analysis is that unique fragments of the between one and 15 fragments. The mean num­ same size may be incorrectly scored as homol­ ber of restriction fragments per haplotype was ogous. This certainly happens although, when 95, representing an average of 510 bases sur­ taxa are closely related, and alternative restric­ veyed (approximately 3.14% ofthe 16,300 base tion profiles differ by only one or two sites at a pairs in the Malawi haplochromine mitochon­ given enzyme, the chance of error is minimal. drial genome). Some enzymes generated rela­ Moreover, when virtually every restriction pro­ tively few unique restriction profiles whereas file is visualized on the same ge I with every other others produced more than 30. All individuals, profile, the probability of such error is further including sympatric conspecifics, demonstrated diminished. Because we used the conservative unique haplotypes, each separated from others approaches of strict consensus and bootstrap­ by one or more restriction-site changes (Ap­ MORAN ET AL.-RADIATION OF MALAWI CICHLIDS 277 pendix). Where conspecifics differed by only one or two restriction-site changes, a single individ­ ual was selected arbitrarily to simplify analyses. Complete information on all haplotypes, in­ cluding the estimated sizes of restriction frag­ ments for each cleavage profile is available on request.

Genome size variation.-No cases ofmtDNA het­ eroplasmy were detected nor were genome size polymorphisms found within lakes. However, all four haplochromine species from Lake Vic­ toria were found to have an mtDNA genome approximately 1000 base pairs larger than the Malawi and Tanganyika genomes (17,300 base pairs, Fig. 1). Serranochromis robustus, a wide­ spread East African species occurring in Lake Malawi, had the same genome size as other Ma­ lawi taxa and the species of Lake Tanganyika. Variation in genome size causes inflated esti­ mates of genetic distance when calculated from fragment differences. This error occurs be­ ....2.0 cause fragment mobility shifts, resulting from the size difference, are interpreted as site changes produced by sequence divergence. Ge­ nome size variation also confounds cladistic analysis because fragments shared by all three groups (i.e., defined by the same restriction sites) appear to unite Malawi and Tanganyika species to the exclusion of those in Lake Victoria. For these reasons, and because full characterization ofthe genome size variation is beyond the scope ofthis study, the haplochromine species of Lake Victoria were excluded as outgroups in phylo­ genetic analyses of the Lake Malawi fauna. "'0.5 Phylogenetic structure.-Although genome size differences preclude comparisons of sequence divergence between Lake Victoria lineages and those of Lakes Tanganyika and Malawi based Fig. 1. SmaI digests of East African cichlid mt­ on restriction fragments, comparisons within DNAs. This autoradiograph demonstrates the differ­ lakes and comparisons between Malawi and ence in mtDNA genome size between Lake Malawi Tanganyika species are unbiased. The range of and Lake Tanganyika versus Lake Victoria haplo­ sequence divergence observed among species chromines. Cyphotilapia frontosa from Lake Tangan­ within Lake Victoria ranged from 1.0-1.3% yika and Copadichromis mloto from Lake Malawi share (data not shown). Divergence between the two a genome size of 16.3 kb. The mtDNA genome of Tanganyikan species was substantially larger Neochromis nigricans from Lake Victoria is approxi­ (5.4%) and was comparable to the divergence mately 1.0 kb larger. of 6.0% observed between Lakes Tanganyika and Malawi. Levels of mtDNA sequence diver­ species. One of the two included all the mbuna gence indicated that Serranochromis robustus was species examined, whereas the other contained equidistant from the Malawi and Tanganyika most of the nonmbuna haplochromines. These haplochromines (6.38% and 6.35%, respective­ two major groups are referred to as A (nonmbu­ ly). This relationship is further supported by na) and B (mbuna), after Meyer et al. (1990; A, Wagner parsimony analysis (see below). "sand dwellers"; B, "rock dwellers"). The level Six distinct lineages were found within the of divergence between these groups, as esti­ Malawi haplochromines (Figs. 2-5). Twoofthese mated by restriction analysis, was 3.3%, with 10 lineages contained a large number of diverse pairwise comparisons yielding estimates over 278 COPEIA, 1994, NO.2

..----A. maerocJeithrum "'-----A. "minutus" ~----A. nyassae A. "deep water" ~--A. jacobfreibergi "'------L. gossei C. obli~idens ~---L.fuelJebomi "'------J. sprengerae r------M auratus ~----p. genalutea Mbuna '------P. zebra 'BB', Thumbi Is. ~----p. /riden/iger L..-____P. heteropietus ....-----L.jreibergi L..-_____P. elongatus'slab' "'------P. elongatus .------P.zebra 'BB', Nkhata Bay '------P. zebra 'cobalt' ------P. zebra 'BB', Chilumba Pt. A. ealliptera I01' ty' ~------c. mloto IgO pIC ~------Diplotaxodon sp. lineages Arislochromis sp. T. macrosloma D. eompressieeps O. avatus HOXY~hus M Q!UlP nnus T. plac n T. holotaenia N. livingstoni N. polystigma Non·Mbuna &...------B. spectabilis L.....------C. rhoadesii ....------D.evelynae '------P. kirlcii r....------c. spi/orlrynchus ...------c.orthog!Jtllhus ...------c. mooni "'------P.m~a~ &...------C. eucinostomus ...... ------Rhamphochromissp. IOligotypic lineage ,------8.robustus INon-endemic ...------C.frontosa Tanganyika L.....------T. moorei I Fig. 2. Relationships among Malawi haplochromines based on estimated mtDNA sequence divergence (Nei and Li, 1979) produced by neighbor-joining (Saitou and Nei, 1987), two taxa from Lake Tanganyika were outgroups. The scale of values for the levels is arbitrary and only subset relationships are depicted. For full genus names, see Appendix. For relative genetic distances, see Figure 4.

4.0%. Meyer and co-workers found an average displacement loop, two tRNA genes, and the sequence divergence ofapproximately 2.5% be­ cytochrome B gene. The results presented here tween representatives of A and B haplotypes. show a strong indication of structuring within Their analysis of 803 base pairs from two dis­ the A and B lineages; both groups exhibit two crete mtDNA sequences included regions ofthe species clusters (Figs. 2-4). However, the phy­

. "-,.... ,.,., ~.-"'---'---"" .. --__~._·w_ .... ,_j.;WIUI>_~_...t' ______... ,_ ..... ___ MORAN ET AL.-RADIATION OF MALAWI CICHLIDS 279

A macrocleithrum A. "minutus" Anyassae A "deep water" A. jacobjreibergi L. gosse; L. jreibergi P. elongatus 94 P. eloogatus'slab' 1...-_...... P. zebra '88', Nkhata Bay P. zebra '88', Chilumba Pt. P.zebra 'cobalt' C. obliquidens 98 L. fuellebomi I sprengerae M. auralus P. genaJulea P. zebra 'BB', rhumbi Is. P. heteropictus P. tridentiger I...-----A. calliplera C. miolo Diplotaxodon sp. r---Aristochromis sp. T. macrosloma B. spectabilis LooT--C. rhoadesii H. oxyrhynchus 71 D. compressiceps M. anaphyrmus T. placodaz L.....---T. holotaenia N livings/ali N polys/igma O. ovatus 99 85 P. kirkii ...... --D. evel}tlae 91 1...----C. spilorlzynchus C. orlhognathus 86 C. eucinoslomus -...,--c. moorU P. subocularus I...-----Rhamphochromis sp. ....------s. robustus .....------IL__-C======c.jrontosa Tmoorei Fig. 3. Strict censuses cladogram based on Wagner parsimony. Forty-six trees of equal length (676 steps) were produced using tree-bisection-reconnection branch swapping (Swofford, 1989). The numbers at the nodes indicate the percentage oftimes the taxa in the indicated clade were united in 200 bootstrap resamplings (PHYLIP Ver. 3.1, Felsenstein, 1988). For full genus names, see Appendix. 280 COPEIA, 1994, NO.2

Mbuna Mbuna

Non-Mbuna ~ Non-Mbuna ~ ~

Fig. 4. Principal coordinates projection of se­ ~ quence divergence among haplotypes with a mini­ "...,'" mum spanning tree superimposed (Rohlf, 1990). Oli­ Fig. 5. Summary of relationships among Malawi gotypic lineages are shown as squares, outgroup taxa haplochromines, produced as a composite of cladistic as triangles. The filled triangles are Tanganyikan spe­ and phenetic analyses of mtDNA restriction frag­ cies. ments. This tree presents consensus results in a con­ servative manner, showing only the relationships sup­ ported by all methods of analysis. logenetic importance ofthese observations may well be compromised by retained ancestral poly­ morphisms (Moran and Kornfield, 1993). Rhamphochromis occupied a position external to Within the A and B lineages, remarkably low the A and B groupings, whereas the other oli­ sequence divergence was observed among hap­ gotypic lineages were united with the A group lotypes (1.7% and 1.0%, respectively). Six pairs to the exclusion of taxa in the B group (Figs. of individuals representing discrete genera dif­ 2-3). However, bootstrap results indicated that fered by less than 0.2%. Species as morpholog­ the unity of the oligotypic lineages to the A ically divergent as Dimidiochromis compressiceps lineage was not supported. Although support and Hemitilapia oxyrhynchus differed by as few as for the positioning of Rhamphochromis was not three restriction-site changes, or 0.26% se­ as strong as other relationships presented here, quence divergence; Cyathochromis obliquidens dif­ we have chosen to place it outside the major A fered from Labeotropheus fuelleborni by only 0.1 % and B groups (Fig. 5). Most of the bootstrap sequence divergence. replicates supported this interpretation (Fig. 3) Representatives of four taxa produced hap­ as did results of neighbor-joining (Fig. 2). lotypes that were equidistant from the A and B Six species in three nonmbuna genera (Eccles linages; these were Astatotilapia calliptera, Co­ and Trewavas, 1989) consistently occurred padichromis mloto, Rhamphochromis sp., and Di­ within the mbuna group in all analyses (Figs. 2­ plotaxodon sp. We refer to these taxa as oligo­ 4). Unlike most mbuna, the species Lethrinops typic lineages because they stood apart from the gossei, Alticorpus macrocleithrum, Aulonocara ja­ major A and B groupings and were only dis­ cobfreibergi, A. nyassae, and the undescribed spe­ tantly related to each other. Other haplotypes cies Aulonocara "minutus" generally inhabit may later be identified that are related to one deeper, sediment-rich areas and have distinctive or the other of these oligotypic linages, al­ morphologies. As an example of the unity of though it is unlikely that any of these four will these six species to the mbuna, the deep-water be shown to be part of a group with as many species Alticorpus macrocleithrum differed from species as A and B. Two patterns emerged for the nonmbuna haplochromines by at least 26 the relationships among the four species. First, restriction-site changes, yet differed from the Rhamphochromis was slightly more distant from mbuna by an average of seven sites. Moreover, the other three lineages than they were from some of the mbuna are substantially closer to each other (Fig. 4). Second, the remaining three the deep-water species than they are to other species formed a loose cluster that excluded the members of the mbuna. For example, Labeotro­ other endemic species examined. In both neigh­ pheus fuelleborni was at least 10 restriction sites bor-joining and Wagner parsimony analyses, divergent from Pseudotropheus zebra "cobalt," MORAN ET AL.-RADlATION OF MALAWI CICHLIDS 281 yet only about three sites from Aulonocara ja­ Alticorpus, show an average sequence diver­ cobfreibergi and as few as two sites from Aulono­ gence of only 1.0%. Sequence divergence was cara "deep water." Thus, the mbuna is a para­ only slightly greater among the nonmbuna hap­ phyletic group as currently recognized (Fryer lochromines (1.7%). The levels of sequence di­ and lies, 1972; Ribbink et ai., 1983; Eccles and vergence among Malawi genera are substan­ Trewavas, 1989). tially less than values commonly observed within Clustering and maximum parsimony tech­ species of other freshwater fishes and, in some niques provided broadly concordant results. cases, less than values within populations. For Both phenetic and cladistic analyses were con­ example, in the southeastern United States, the sistent in the separation of the A and B lineages sunfish species, Lepomis punctatus, showed pop­ and in the isolation of the four oligotypic lin­ ulation level divergence of 4.0% (Bermingham eages. Differences between analyses were lim­ and Avise, 1986). Third, several relatively di­ ited to relationships of taxa within major clades vergent oligotypic mtDNA lineages were rep­ and to the level of resolution of those relation­ resented by Rhalllphorhrolllis sp., Diplolaxodoll sp., ships. Bootstrap resampling of cladistic char­ A.slatolilapia mllijilPra, and Copadichrolllis 111[010. acters further supported the distinctiveness of These three primary results can be viewed both the A and B groups and the unity ofthe endemic in the context of their systematic significance haplochromines of Malawi (Fig. 3). The unity and also with respect to their implications for of the mbuna (B) group was supported in 98% morphological diversification. of the bootstrap resamplings, whereas the nonmbuna (A) group formed a monophyletic Mbuna are paraphyletic.-The molecular genet­ clade in 91 % of the replicates. When the oli­ ic data reported here provide new insight into gotypic lineages were excluded from the anal­ the systematic relationships of the Lake Malawi ysis, both lineages became monophyletic in 100% haplochromines. With respect to the bimodal of the replicates. (mbuna/nonmbuna) distribution ofhaplotypes, Sequence divergence among haplotypes pro­ the phylogenetic position of the genera Aulon­ jected on the first three principal axes revealed ocara, Alticorpus, and Lethrinops is unequivocal a pattern that further supported both clustering (Figs. 2-3). To enforce a topology placing any and parsimony techniques (Fig. 4). We provide one ofthese species into the nonmbuna A clade the results of ordination primarily because nei­ would require over 20 restriction-site reversals. ther neighbor-joining nor Wagner parsimony The unexpected affinity of these taxa with the analysis allow accurate interpretation ofrelative mbuna indicates that a revision is required in genetic distances. Moreover, the concordance the concept of the mbuna group if it is to be ofcladistic, clustering and ordination approach­ considered a monophyletic assemblage. It may es to phylogenetic analysis is consistent with the be appropriate to construct a super-generic tax­ idea that these results provide an accurate view onomic category to recognize formally the phy­ of the major phylogenetic relationships among logenetic systematic relationships among the Malawi haplochromines. A heuristic consensus Malawi haplochromines. Although these results tree for all methods of analysis is presented in alone cannot absolutely rule out a sister-group Figure 5 as an estimation ofthese relationships. relationship between the mbuna and the deep­ water species, we favor the explanation of par­

DISCUSSION aphyly because some mbuna are three times more divergent from each other than from one Three primary results emerged from this or more of the deep-water species (6-10 sites study. First, the distribution of sequence diver­ compared to 2-4 sites). gence among haplotypes was bimodal, forming the major clades A and B. Further, the B clade Taxonomic revisions are required.-Two addition­ (which included all the mbuna examined) con­ al phyletic observations have significant taxo­ tained a number of species previously thought nomic implications. First, Copadichromis mloto to be allied with other nonmbuna species. It does not appear closely related to C. eucinosto­ appeared that these species were actually em­ mus, suggesting that this genus is polyphyletic bedded within the mbuna rather than being a (Figs. 2-3). Additional members of this genus sister group. Therefore, the mbuna is a para­ need to be examined to determine whether they phyletic grouping. Second, remarkably low ge­ are close to C. mloto, C. eucinostomus, or distinct netic differentiation was observed among the from both of these species. Second, taxonomic ecologically diverse Malawi haplochromines. For revision is warranted for the genus Astatotilapia. example. all of the mbuna species, including Meyer et al. (1990) found that three Astatotilapia representatives of Aulonocara, Lethrinops, and species of Lake Victoria were more closely re­ 282 COPEIA, 1994, NO.2 lated to the endemic haplochromines of that An alternative less parsimonious explanation lake than to any of the Malawi taxa they ex­ is that deletion events occurred independently amined. Our results suggest that the congener in the lineage that gave rise to the Malawi hap­ A. calliptera is embedded within the Malawi flock lochromine radiation and in the lineage sup­ (Figs. 2-3). Although sequence divergence val­ porting the Tanganyikan radiation. This expla­ ues between Victoria and Malawi haplotypes nation is unlikely because of the position of were not estimated because of the difference in Serranochromis robustus (Figs. 2-4), which con­ mtDNA genome size, it is this very size differ­ tains the small mtDNA genome. Serranochromis ence that serves to unite A. calliptera with the robustus appears to have diverged from a com­ Malawi haplochromines (both showing the mon haplochromine ancestor at approximately smaller genome, whereas Victorian haplotypes the same time as the progenitor of the Malawi had the larger). Furthermore, A. calliptera was radiation and the two Tanganyikan genera ex­ less than 2.0% divergent from five ofthe mbuna amined here. IfSerranochromis and the Tangan­ species and less than 3.0% divergent from 26 yika species each form independent mtDNA lin­ others. Thus, these data strongly suggest that eages, then an additional deletion event would the genus Astatotilapia is polyphyletic. In addi­ also have been required. The independent de­ tion to the restriction data, this relationship has letion scenario is further discredited because been supported by subsequent mtDNA se­ the South African haplochromine, Pseudocreni­ quence analysis (A. Meyer, unpubl.). These re­ labrus philander, was found to have an mtDNA sults were not completely unexpected (Eccles genome size of 16,500 bp (de Villiers et aI., and Trewavas, 1989) because the genus Asta­ 1992). Additional characterization of the totilapia was defined only on the basis of shared mtDNA genome size differences observed here pleisiomorphic traits within the Victoria-Ed­ will require site mapping and sequencing. Such ward-Kivu flock (Greenwood, 1980). analyses are now in progress.

Autapom0 rphic mtDNA genome size in Lake Victoria Rapidity of Malawi haplochromine radiation.-The haplochromines.-The difference in genome size mtDNA results presented here provide further identified here could have been quite mislead­ evidence that the genetic divergence between ing. If these results were examined in the ab­ morphologically distinct taxa is minute. Se­ sence ofprevious molecular studies, it might be quence divergence among genera was found to concluded that the smaller mtDNA genome ob­ be quite low compared to other fish species (A v­ served in the cichlids of Lake Malawi and Lake ise et aI., 1987) including estimates among ti­ Tanganyika represents a synapomorphy uniting lapiine cichlids (Seyoum and Kornfield, 1992). these faunas to the exclusion of Lake Victoria Only the Victoria haplochromines are compa­ species. The pleisiomorphic condition may be rable to Malawi in having high levels of mor­ the larger (17.3 kb) genome size, as represented phological divergence while lacking propor­ by multiple mtDNA lineages in the tilapiine tional genetic divergence (Dorit, 1990; Meyer cichlid fishes (Seyoum, 1989; Seyoum and et aI., 1990). In Lake Malawi, the deep-water Kornfield, 1992). In contrast to the situation Lethrinops gossei is both ecologically and mor­ with Astatotilapia, a number ofindependent mo­ phologically distinct from the shallow-water, al­ lecular studies support the unity of the Malawi gae grazing Cyathochromis obliquidens, yet the two and Victorian cichlid faunas and clearly indicate species differ by less than 0.4% in mtDNA se­ that the Tanganyika cichlids fall outside this quence. These dramatic shifts in morphology group (Meyer et aI., 1990; Kornfield, 1991; with very little genetic divergence are indica­ Nishida, 1991). Given the broad and indepen­ tions of the evolutionary plasticity that char­ dent support for the relationships among the acterizes the haplochromine cichlids (Liem, haplochromine faunas ofthe three Great Lakes, 1973). This evolutionary plasticity underscores and assuming that the large genome size is ple­ one ofthe most interesting questions ofthe hap­ siomorphic, a deletion may have occurred in lochromine radiation in East Africa: how is such the mtDNA genome of the haplochromine an­ striking morphological diversity obtained with cestor relatively soon after its divergence from so little genetic divergence? The striking mor­ the tilapiine fishes. Subsequently, a duplication phological divergence and parallelism, evident or insertion restored the large genome size to in the phylogenetic position Alticorpus, Aulono­ the progenitor of the Victoria haplochromine cara, and Lethrinops indicates that morphologic lineage. Regardless, however, the larger ge­ divergence is a poor indicator of genetic dis­ nome size found in the Victorian haplochro­ tance and evolutionary time even within the mines appears to be an autapomorphic char­ closely related members of the Malawi fauna. acter. In general, parallelism effectively obscures MORAN ET AL.-RADIATlON OF MALAWI CICHLIDS 283 phylogenetic patterns. This situation presents Itmust be recognized that the use ofsequence acute problems for phylogenetic analyses based divergence for the estimation of time is highly on morphology alone, particularly for the de­ problematic (Moritz and Hillis, 1990). Rates of termination ofgeneric relationships. Indeed, the sequence divergence do not appear to be con­ designation of genera in the East African Lakes stant across distantly related taxa (Vawter and has been highly problematic (Greenwood, 1980; Brown, 1986). In the absence of four-point cal­ Hoogerhound, 1984). Eccles and Trewavas ibration in closely related taxa, conclusions (1989) emphasized the "problem of parallel­ drawn from estimated sequence divergence re­ ism" and indicated that, in Lake Malawi, abun­ garding time since reproductive isolation must dant parallelism, superimposed on an extensive be considered tentative. However, even in the radiation have "stretched to the limit" a cla­ absence ofcalibration, accurate estimates ofrel­ distic approach to phylogenetic reconstruction ative divergence times can be obtained among using purely anatomical data. Although in prin­ closely related taxa (Hillis and Moritz, 1990). ciple, parallelism is less of a problem with Among closely related taxa, the molecular ge­ mtDNA restriction data, the apparent rapidity netic processes which control the rate of se­ ofthe radiation in Malawi (Kornfield, 1978) has quence evolution are likely to be similar. It is, resulted in mtDNA analysis being stretched to therefore, reasonable to assume that the rate of its limit as well. Although higher order rela­ sequence divergence is nearly constant, and that tionships are clearly evident (e.g., between Ma­ the topology of our phylogenetic reconstruc­ lawi A and B lineages), the affinities of taxa tion is unlikely to have been influenced by rate within those major groups remain unclear be­ differences. Differing branch lengths in Figure cause of limited genetic divergence. 3 probably result from the stochastic distribu­ tion ofcharacter state changes rather than from Monophyly of Lake Malawi haplochrornines?-His­ real differences in the processes which underlie torically , there has been a consistent question rates of sequence evolution. regarding monophyly of the Lake Malawi hap­ An additional concern in the use of mtDNA lochromines and whether multiple coloniza­ for systematic studies is that stochastic extinc­ tions might explain some of the observed mor­ tion of mtDNA lineages may confound phylo­ phological diversity (Mayr, 1963; Fryer and lies, genetic reconstruction. Typically, this occurs in 1972; Greenwood, 1979). There is now consid­ groups of species that have been isolated re­ erable evidence that the endemic Malawi spe­ cently from a polymorphic ancestor. This phe­ cies are monophyletic with respect to the other nomenon may produce a gene tree which is sub­ African Great Lakes (Kornfield et aI., 1985; stantially different than the "true" species tree Meyer et aI., 1990; Kornfield, 1991). Our (Avise et aI., 1984; Nei, 1987). In such cases, it mtDNA restriction data are consistent with this is inappropriate to use mtDNA lineages as prox­ view because the taxa we examined as out­ ies for species. Only when sibling species reach groups from Lakes Victoria and Tanganyika a state of reciprocal monophyly can a single fell outside the Malawi flock. However, ifa rate DNA sequence (e.g., mtDNA) be used effec­ ofsequence divergence ofless than 1% per mil­ tively for phylogenetic reconstruction (Moran lion years is assumed (Bentzen et aI., 1989; E. and Kornfield, 1993). Bermingham, unpubl.), the level of sequence It is highly unlikely that the phylogenetic po­ divergence between the most distant mtDNA sition of Alticorpus, Aulonocara, and Lethrinops lineages within Lake Malawi (4.1 %) would sub­ suggested in this study resulted from either sto­ stantially predate the formation of the lake, es­ chastic lineage extinction or the retention of timated at 1-2 Ma (Banister and Clark, 1980). ancestral polymorphism. If these species had, Either Lake Malawi was colonized by a poly­ in fact, been isolated from the mbuna for as morphic ancestor, or the major lineages iden­ long a period of time as have the nonmbuna tified here had diverged before their invasion species, it would be expected that sequence di­ of the Lake. Thus, depending on how the vergence between the deep-water species and mtDNA "molecular clock" is calibrated (see be­ the mbuna would be greater than the diver­ low), the Malawi cichlids may not be monophy­ gence within the mbuna. Clearly, this is not the letic. Indeed, the possibility cannot be excluded case because divergence between many pairs of that a sister group to one or more ofthe Malawi mbuna haplotypes is over 1.0%; yet many spe­ lineages remains to be found outside the lake. cies of mbuna differ from the deep-water taxa However, even if these lineages existed prior to by less than 0.4%. This pattern could still be the formation of Lake Malawi, it is clear that explained by a series of very recent mtDNA the vast bulk of the endemic cichlid radiation lineage extinctions. Under this scenario, the occurred relatively recently within the lake. shared ancestor of the mbuna and nonmbuna 284 COPEIA, 1994, NO.2 would have been polymorphic for A and B The existence of the oligotypic mtDNA lin­ mtDNA lineages. The A lineage would have eages could also indicate some basic biological been lost in all the mbuna species, the B lineage difference between members of the clades that would have become extinct in all the nonmbuna have radiated and those that have not. For ex­ species except the deep-water taxa, and, finally, ample, Fryer and lies (1972) indicated that an­ the A lineage would have been lost in the deep­ other phyletically isolated taxon, Copadichromis water species. We reject this hypothesis as less mloto (Fig. 4) is the most pelagic of the utaka, parsimonious and suggest instead that rapid and the endemic, pelagic, plantivorous trophic striking morphological divergence character­ group. Perhaps this life history is less conducive izes the species of Alticorpus, Aulonocara, and to cladogenesis than is more stenotopic behav­ Lethrinops. ior. An alternative possibility is that one or more Phylogenetic reconstruction may also be ob­ of these taxa are relatively recent invaders of scured by hybridization that results in the trans­ Lake Malawi and have not yet begun to radiate. fer ofdivergent mtDNA genomes between pre­ If this were the case, it might be expected that viously isolated taxa. However, hybridization is sister groups to the oligotypic lineages could be unlikely to explain the phylogenetic position of discovered outside the lake. Alticorpus, Aulonocara, and Lethrinops; the first two species, in particular, have morphologies Further investigation.-In phylogenetic system­ that are distinct from the mbuna (Eccles and atics studies, tree topology may be dramatically Trewavas, 1989). The possession of such dis­ influenced by the inclusion of additional taxa. tinctive morphology along with mtDNA iden­ However, we expect the results presented here tical to the mbuna could occur ifthere had been to be relatively robust. We take this view based repeated unidirectional back-crossing. Howev­ on the fact that representatives ofthe most eco­ er, this possibility appears extremely remote, logically and morphologically divergent lin­ because (1) in situ hybridization is undocu­ eages in the lake have been examined. An ex­ mented among Malawi haplochromines, despite ception, however, might be the monotypic genus extensive study (Kornfield, 1991); and (2) all Lichnochromis, which was not examined. Lichno­ these species (except Aulonocara jacobfreibergi and rhrolllis acutiCfpS has a distinctive morphology A. nyassae) inhabit much deeper water and quite and might indeed carry a unique and divergent different habitat types than the mbuna. This mtDNA haplotype. Additional unique and di­ physical separation would have provided, at best, vergent lineages may be found, but it seems very limited opportunity to interbreed. unlikely that there are other major lineages that contain numbers ofdiscrete taxa comparable to Oligot)"pic lineages ma)" not hill'!' radiated.-The those observed in the A and B haplochromine fact that some divergent mtDN A haplotypes are groups. restricted to a few taxa (e.g., RhalllpllOrlirolllis, Although a large number of characters are Fig. 4) suggests that some lineages in Malawi available using mtDNA analysis, their utility is have radiated rapidly, producing large numbers limited because these characters are not inde­ of very diverse taxa (e.g., A and B mtDNA lin­ pendent. Because the mtDNA genome is in­ eages) while others have not. It is not clear, herited without recombination, mtDNA can be however, whether the contrast between the spe­ viewed as a single locus or tight linkage group cies-rich A and B lineages and the oligotypic with many alleles. Thus, if restricted to mt­ lineages represents a fundamental difference in DNA, higher resolution techniques, such as ex­ the temporal pattern ofradiation or whether it tensive sequencing, are unlikely to provide sub­ is simply the result of species extinction super­ stantially greater insight than is available imposed on homogeneous speciation rates through RFLP analysis. Because of the nonin­ (Stanley, 1979; Futuyma, 1987). For example, dependent nature of mtDNA polymorphism, an oligotypic mtDNA lineage such as that car­ further resolution ofphylogenetic relationships ried by Rhamphochromis may be restricted to a within the Malawi haplochromines will require few species simply because other species have additional independent molecular markers. Ap­ been lost to extinction. Rhamphochromis may have propriate target sequences might include non­ been part ofan earlier radiation which has since transcribed ribosomal DNA (Federoff, 1979; been pared down, leaving a few divergent hap­ Phillips et aI., 1989; Hillis and Dixon, 1991), lotypes. Under this scenario, species of Rham­ introns (IK, unpubl.) or single locus hypervari­ phochromis other than the one examined here able sequences (Nakamura et aI., 1987). These might be expected to exhibit additional mtDNA additional data will be required to obtain a more haplotypes as distinct as the oligotypic lineages complete view of the true cladistic relationships identified here. among the Malawi haplochromines. MORAN ET AL.-RADIATION OF MALAWI CICHLIDS 285

MATERIALS EXAMINED research was supported by the National Science Foundation (BSR 84-16131), the Smithsonian Mo!'t collections were deposited as voucher specimens at the Amer­ Oceanographic Sorting Center, and the Uni­ ican Museum of Natural History. For three individuals, the entire spec­ imen was used for mtDNA isolation. and there is no accession number. versity of Maine, Manuscript preparation was Where specimens were obtained through the aquarium trade, original supported by National Marine Fisheries Ser­ collection locations (where available) are in parentheses. Six species vice, Northwest Fisheries Science Center, marked with asterisks (*) are placed with the mbuna based on the results of mtDNA restriction analysis. Bootstrap resampling of cladistic char­ Coastal Zone and Estuarine Studies Division. acters indicated that these species were embedded within a monophy­ letic group containing all the mbuna species examined. Species are represented by a single individual unless otherwise noted. LITERATURE CITED

Lakl' Malawi mbuna.-A.lticorpu,\ macrorleithrwn-Maldeco Fishery, AMNH AVISE,J. C .. J. ARNOLD, R, M. BALL, E. BERMINGHAM, 98354; ,1ulonocara "deep water"-Maldeco Fishery. AMNH 98359; A. jacobfreibergi-Aquarium trade, AMNH 98364; A. Hminutus"-Monkey T. LAMB, J, E. NEIGEL, C, A. REEB, AND N. C. Bay. AMNH 98366; A 1'-)'assa,-Thumbi Island W .. AMNH 98357; SAUNDERS. 1987, Intraspecific phylogeography: the Cyathochrmnis obliquidms-Thumbi Island W.. AMNH 98353 (n ~ 2); mitochondrial bridge between population genetics Iodotropheus sprengerae-Aquarium trade ,(Chinyamwezi); Labfolropheus fudlrhorni-Aquarium trade (Mbenji Island). A"'NH 98345; Labido­ and systematics. Ann. Rev. Ecol. Syst. 18:489-522. rhrom;.s Jrfibngi-Thumhi Island W.; Lflhrinops gossfi-Monkey Bay, ---, J. E. NEIGEL, AND J. ARNOLD. 1984. Demo­ AMNH 92713 (n ~ 2); Mdanochromisaurahu-Aquarium trade (Mbenji graphic influences on mitochondrial DNA lineage Island). AMNH 98359;P,trotilapiag,nalUlRa-Thumbi Island W,. AMNH survivorship in animal populations. J. Mol. Evol. 98350; P. tridmtigPr-Nkhata Bay. AMNH 98370; pspudotrophrus flon­ gatus-Nkhata Bay. AMNH 98367; P. dongatus 'slab'-Thumbi Island 20:99-105. W.. AMNH 98358; P. hfteropictus-Thumbi Island W.. AMNH 92728; BANISTER, K. E., AND M, A. CLARKE. 1980, A revision P. ubra 'BB'-Chilumba Point. AMNH 98369; Nkhata Bay. AMNH of the large Barbus (Pisces, Cyprinidae) of Lake 98368; Thumbi Island W.. AMNH 98344; P. zebra 'cobalt'-Nkhata Bay. AMNH 98371. Malawi with a reconstruction of the history of the southern African Rift Valley lakes.]. Nat. Hist. 14: Lake Malawi nonmbuna.-Aristochromis chrisl)'i-Thumbi Island W., 483-543. AMNH 92466; ,1statotilapia calliptPTa-Thumbi Island W.. AMNH BENTZEN, P., G. C, BROWN, AND W. C, LEGGETT, 1989, 98356; Buccochromis spectabilis-Chimumbo Bay, AMNH 92645; Ca­ prichromis orthognathus-Chimumbo Bay, AMNH 98343; Champso­ Mitochondrial DNA polymorphism, population chromis spilorhynchus-Thumbi Island W.. AMNH 98347; Chilotilipia structure, and life history variation in American rhoadr.,ii-Chimumbo Bay. AMNH 92642; Copadichromis ,ucinosto­ shad (Alma sapidissima), Can. J. Fish. Aquat. Sci. 46: mus-Cape Maclear, AMNH 92666; C. mioto-Chimumbo Bay, AMNH 92717; Cyrtocaro mooni-Cape Maclear, AMNH 98351; Dimidiochromis 1446-1454. compressiaps-Aquarium trade, AMNH 98342; DiplotaxodrJil sp.-Mon­ BERMINGHAM, E., AND J. C. AVISE. 1986. Molecular key Bay. AMNH 92634; norimodu,\' l''Vely1UU'-Thumbi Island, AMNH zoogeography of freshwater fishes in the south­ 98348; Hemitilopia ox)'rhJnchus-Cape Maclear, AMNH 92628; .Wara'ui­ eastern United States. Genetics 113:939-965. chromis anaphrymus-Cape Maclt"ar, AMNH 92626; Nimvochromis liv­ ingstoni-Monkey Bay. AMNH 92631; N. polystigma-Cape Maclear. BOULENGER, G, A, 1908. Diagnoses of new fishes AMNH 92629; Otopharynx ova/us-Aquarium trade, AMNH 98365; discovered by Captain E. C. Rhoades in Lake Nyasa. Ploridochromis subncularus-Cape Maclear, AMNH 983:'>2; Protmnpias Ann, Mag, Nat. Hist. (8), 2:238-243. kirkil-Cape Made'f. AMNH 98355; Rhamphochromis sp.-Monkey Bay. BROWN, W. M., M. GEORGE, JR., AND A, C. WILSON, AMNH 92714; Srrranorhromis robustus-Monkey Bay. AMNH 92729; Tarniochromis holotaenio-Chimumbo Bay, AMNH 92647; TrematocTO­ 1979. Rapid evolution ofmitochondrial DNA. Proc, nus plocodon-Cape Maclear, AMNH 92704; Tyrann()chromis macros/o­ Natl. Acad, Sci. USA 76:1967-1971. Ina-Thumbi Island W.. AMNH 92721. Lake Tanganyika: Cyphotilapia DE VILLIERS, D. L, E. H, HARLEY, AND A.J. RIBBINK. fron/osa-Aquarium trade, AMNH 98366; Tropheu,\ moorei~Aquarium trade. Lake Victoria: NfOchromis nigricans-Aquarium trade, AMNH 1992, Mitochondrial DNA restriction enzyme vari­ 98363; Psammochrnmis riponianus-Aquarium trade, AMNH 98361; ation in allopatric populations of Pseudocrenilabrus Prognathochromis pellegrini-Aquarium trade, AMNH 98362; Pytochro­ philander (Pisces: Cichlidae). S. Afr. J. Sci, 88:96­ mis samragei-Aquarium trade, AMNH 98360. 99. DORIT, R. L 1990, The correlates of high diversity in Lake Victoria haplochromine cichlids: a neon­ ACKNOWLEDGMENTS tological perspective, p, 322-353. In: Causes ofevo­ lution: a paleontological perspective, R. M, Ross The cooperation and interest of the Malawi and W. D. Allmon (eds.). Univ. of Chicago Press, Department of Fisheries, Malawi Department Chicago, Illinois. of Parks, and the National Research Council of DOWLING, T, E., C. MORTIZ, AND J. D. PALMER. 1990. Malawi is gratefully acknowledged, S. Grant Nucleic acids II: restriction site analysis, p, 250­ (Salima, Malawi) provided continuous assis­ 317. In: Molecular systematics. D, M, Hillis and C. tance with fieldwork. We gratefully thank T. Moritz (eds,). Sinauer, Sunderland, Massachusetts. Dowling for introducing IK to mtDNA restric­ DROUIN, J. 1980. Cloning of human mitochondrial tion analysis and for conducting preliminary DNA in Escherichia coli. J. Mol. BioI. 140:15-34. work with African cichlids, J. Felsenstein and ECCLES D. E., AND E. TREWAVAS, 1989. Malawian cichlid fishes; the classification of some haplochro­ D. Swofford provided consultation regarding mine genera. Lake Fish Movies, Herten, Germany, data analysis, W. Glanz, D. McElroy, andJ. Rin­ FEDOROFF, N, V. 1979. On spacers. Cell 16:697-710, go kindly commented on drafts of this manu­ FELSENSTEIN, J. 1988. PHYLIP (Phylogenetic Infer­ script. Preliminary communication of the re­ ence Package, computer software) Ver. 3.1. Univ. sults of this research was made at the ASIH of Washington, Seattle. annual meeting in New York,June 1991. This FRYER, G., AND T. D, ILEs. 1972. The cichlid fishes 286 COPEIA, 1994, NO.2

of the Great Lakes of Africa. Oliver Boyd, Edin­ ment for the manipulation and phylogenetic anal­ burgh, Scotland. ysis of restriction data.]. Hered. 83: 157-158. FUTUYMA, D.]. 1986. Evolutionary biology, 2d ed. MEYER, A., T. D. KOCHER, P. BASASIBWAKI, AND A. Sinauer, Sunderland, Massachusetts. C. WILSON. 1990. Monophyletic origin of Lake ---. 1987. On the role of species in anagenesis. Victoria cichlid fishes suggested by mitochondrial Amer. Nat. 130:465-473. DNA sequences. Nature 347:550-553. GREENWOOD, P. H. 1979. Towards a phyletic clas­ MORAN, P., AND 1. KORNFlELD. 1993. Retention of sification of the genus 'Haplochrolllis' (Pisces, Cich­ an ancestral polymorphism in the mbuna species lidae) and related taxa. Part I. Bull. Br. Mus. Nat. flock (Teleostei: Cichlidae) of Lake Malawi. Mol. Hist. (Zool.) 35:265-322. BioI. Evol. 10:1015-1029. ---. 1980. Towards a phyletic classification of the MORITZ, C., AND D. M. HILLIS. 1990. Molecular sys­ genus 'H(lplocilrolllis' (Pisces, Cichlidae) and related tematics: context and controversies, p. 1-10. In: taxa. Part II; the species from Lakes Victoria, Na­ Molecular systematics. D. M. Hillis and C. Moritz bugabo, Edward, George and Kivu. Ibid. 39: 1-10I. (eds.). Sinauer, Sunderland, Massachusetts. --. 1991. Speciation, p. 86-102. In: Cichlid fish­ ---, T. E. DOWLING, AND W. M. BROWN. 1987. es. Behaviour, ecology and evolution. M. H. A. Evolution ofanimal mitochondrial DNA: relevance Keenleyside (ed.). Chapman and Hall, New York, for population biology and systematics. Ann. Rev. New York. Ecol. Syst. 18:269-292. GUNTHER, A. 1864. Report on a collection ofreptiles NAKAMURA, Y., M. LEPPERT, P. O'CONNELL, R. WOLFF, and fishes made by Dr. Kirk in the Zambesi and T. HOLM, M. CULVER, C. MARTIN, E. FUJIMOTO, Nyassa regions. Proc. Zool. Soc. London 1864:303­ M. HOFF, E. KUMLIN, AND R. WHITE. 1987. Vari­ 314. able number of tandem repeat (VNTR) markers GYLLENSTEN, U., D. WHARTON, A. JOSEFSSON, AND for human gene mapping. Science 23:1616-1622. A. C. WILSON. 1991. Paternal inheritance of mi­ NEI, M. 1987. Molecular evolutionary genetics. Co­ tochondrial DNA in mice. Nature 352:255-257. lumbia Univ. Press, New York, New York. HEDGES, S. B. 1992. The number of replications --, AND W.-H. Lr. 1979. Mathematical model needed for accurate estimation of the boostrap P for studying genetic variation in terms ofrestriction value in phylogenetic studies. Mol. BioI. Evol. 9:366­ endonucleases. Proc. Natl. Acad. Sci. USA 76:5269­ 369. 5273. HILLIS, D. M., AND M. T. DIXON. 1991. Ribosomal NISHIDA, M. 1991. Lake Tanganyika as an evolu­ DNA: molecular evolution and phylogenetic infer­ tionary reservoir of old lineages of East African ence. Quart. Rev. BioI. 66:411-453. cichlid fishes: inferences from allozyme data. Ex­ ---, AND C. MORITZ. 1990. An overview of ap­ perientia 47:974-979. plications ofmolecular systematics, p. 502-515. In: PHILLIPS, R. B., K. A. PLEYTE, L. M. VAN ERT, AND Molecular systematics. D. M. Hillis and C. Moritz S. E. HARTLEY. 1989. Evolution of nucleolar or­ (eds.). Sinauer, Sunderland, Massachusetts. ganizer regions and ribosomal RNA genes in Sal­ ---,].]. BULL, M. E. WHITE, M. R. BADGETT, AND velinus. Physiol. Ecol.Japan. Spec. Vol. 1:429-447. ].]. MOLINEUX. 1992. Experimental phylogenet­ REGAN, C. T. 1922. The cichlid fishes of Lake Nyasa. ics: generation ofa known phylogeny. Science 255: Proc. Zool. Soc. London 1921:675-727. 589-592. REINTHAL, P. N. 1990. The feeding habits ofa group HOOGERHOUND, R. J. C. 1984. A taxonomic recon­ of herbivorous rock-dwelling cichlid fishes (Cich­ sideration of the haplochromine genera Gauro­Iidae: Perciformes) from Lake Malawi, Africa. Env. chromis Greenwood, 1980 and Labidochromis Regan, BioI. Fishes 27:215-233. 1920 (Pisces, Cichlidae). Netherlands j. Zool. 34: RIBBINIK, A. j., A. C. MARSH, C. C. RIBBINK, AND B. j. SHARP. 1983. A preliminary survey of the cichlid 539-565. KORNFIELD, I. L. 1978. Evidence for rapid speciation fishes of rocky habitats in Lake Malawi. S. Afr. J. in African cichlid fishes. Experientia 34:335-336. Zool. 18:149-310. --. 1991. Genetics, p. 103-128. In: Cichlid fishes ROHLF, F.j. 1990. NTSYS: numerical taxonomy and behaviour, ecology and evolution. M. H. A. Keen­ multivariate analysis system Ver. 1.6. Exeter, Se­ leyside (ed.). Chapman and Hall, New York, New tauket, New York. York. SAITOU, N.,ANDM. NEI. 1987. Theneighbor-joining ---, K. R. McKAYE, AND T. KOCHER. 1985. Evi­ method: a new method for reconstructing phylo­ dence for the immigration hypothesis in the en­ genetic trees. Mol. BioI. Evol. 4:406-425. demic cichlid fauna of Tanganyika. Isozyme Bull. SEYOUM, S. 1989. Stock identification and the evo­ 18:76. lutionary relationships of the genera Oreochromis, LANSMAN, R. A., R. O. SHADE, j. F. SHAPIRA, AND j. Sarotherodon, and Tilapia (Pisces: Cichlidae) using t C. AVISE. 1981. The use of restriction endonu­ al10zyme analysis and restriction of mitochondrial c1eases to measure DNA sequence relatedness in DNA. Unpubl. Ph.D. diss., Univ. of Waterloo, Wa­ natural populations. j. Mol. Evol. 17:214-226. terloo, Ontario. LIEM, K. F. 1973. Evolutionary strategies and mor­ ---, AND 1. KORNFIELD. 1992. Taxonomic notes phological innovations: cichlid pharyngeal jaws. Syst. on the Oreochromis niloticus subspecies complex (Pi­ Zool. 22:425-441. sces: Cichlidae), with a description of a new sub­ MAYR, E. 1963. Animal species and evolution. Bel­ species. Can.j. Zool. 70:2161-2165. knap, Cambridge, Massachusetts. STANLEY, S. M. 1979. Macroevolution: pattern and McELROY, D., P. MORAN, E. BERMINGHAM, AND I. process. Freeman, San Francisco, California. KORNFIELD. 1992. REAP: an integrated environ­ SWOFFORD, D. L. 1989. PAUP: phylogenetic analysis MORAN ET AL.-RADIATION OF MALAWI CICHLIDS 287

using parsimony Ver. 3.0. Illinois Nat. Hist. Survey, HIGUCHI, S. R. PALUMBI, E. M. PRAGER, R. D. SAGE, Champaign, Illinois. AND M. STONEKING. 1985. Mitochondrial DNA and ---, AND G. J. OLSEN. 1990. Phylogeny recon­ two perspectives on evolutionary genetics. BioI. J. struction, p. 411-500. In: Molecular systematics. D. Linn. Soc. 26:375-400. M. Hillis and C. Moritz (eds.). Sinauer, Sunderland, Massachusetts. (PM, IK) DEPARTMENT OF ZOOLOGY AND CENTER TREWAVAS, E. 1931. A revision of the cichlid fishes FOR MARINE STUDIES, UNIVERSITY OF MAINE, of the genus Lethrinops, Regan. Ann. Mag. Nat. ORONO, MAINE 04469; AND (PNR) DE­ Hist. (10), 7:133-153. PARTMENT OF BIOLOGY, EASTERN MICHIGAN ---. 1935. A synopsis of the cichlid fishes of Lake UNIVERSITY, YPSILANTI, MICHIGAN 48197. Nyasa. Ibid. 10:65-118. VAWTER, L., AND W. M. BROWN. 1986. Nuclear and PRESENT ADDRESS: (PM) NMFSjCZES, mitochondrial DNA comparisons reveal extreme GENETICS, 2725 MONTLAKE BOULEVARD EAST, rate variation in the molecular clock. Science 234: SEATTLE, WASHINGTON 98112-2097. Sub­ 194-196. mitted 12 March 1992. Accepted 12 April WILSON, A. C., R. L. CANN, S. M. CARR, M. GEORGE, 1993. Section editor: D. G. Buth. U. B. GYLLENSTEN, K. M. HELM-BYCHOWSKI, R. G. 288 COPEIA, 1994, NO.2

ApPENDIX. HAPLOTYPE DESCRIPTION OF MTDNA GENOTYPES OBSERVED IN LAKE MALAWI ENDEMICS AND OTHER EAST AFRICAN CICHLlDS. Descriptions are based on the composite of observed restriction profiles. Restriction endonucleases used included (left to right): ApaI, AvaI, AvaIl, BamHI, Bell, BgLI, EstEll, DraI, EeoRV, HindI, HindIII, HpaI, Neil, SmaI, StyI, ThaI, Xbal, and Xhol. Six species marked with asterisks (*) were previously considered to be distinct from the mbuna. All individuals (including sympatric conspecifics) produced unique haplotypes. Taxon Haplotype description , Lake Malawi Mbuna Altieorpus maerocleithrum * D D C C C C C C K C C C E B DCA C Aulonocara "deep water"* D D C C C C C C C C C C E B D C C C A. jacobfreibergi* D D C C C C C C C C C C G B D C C C A. "minutus"* D D C C C C C C C C C C E B B C A C A. nyassae* D B ICC C C C C C C C E B D K M C Cyathochromis obliquidens J D DEC C C C C C C C E B D C J C Iodotropheus sprengerae D D C E C C C C C C G C E B D C C C Labeotropheus fuelleborni J D DEC C C C C C C C E B D C C C Labidochromis freibergi C C C C C C C B C 0 C B C C C C C C Lethrinops gossei D D G C C C C C C C C C E B D C C C Melanoehromis auratus F D E E D C C C C C C C B D C C C Petrotilapia genalutea N D M C D C C C C C C C E B 0 C C C P. tridentigeT F D F C D C C C E C C C E B D C C C Pseudotropheus elangatus C C C C E C C B C C C C C C C C C C P. elongatus 'slab' C C C C C C C A C C C C C C C C C C P. heteropictus F D M C D C C C C C D C E B D C C C P. zebra 'BB' Chilumba Point C C C C C C C B D C C C C C G C C C P. zebra 'BB' Nkhata Bay C C G C C C C B D C F C C C C C C C P. ubra 'BB' Thumbi Island E D M C D C C C C C C C C B 0 C C C P. zebra 'cobalt' G C H C C C C B o C C C F C C C C C Lake Malawi Nonmbuna Aristochromis sp. X E T 0 F B B D CAE AHCKAC D Astatotilapia caUiptera I G W F J A C C CBI DNBHBG C Buccochromis spectabilis H E V C F ABE CAE AHCEAC D Caprichromis orthagnathus A N o C C A E E CAE HQCFAC C Champsoehromis spilorhynchus P 0 C C A E C C F M E Q C F A C .0 Chilotilipia rhoadesii H E P 0 C B B C CAEADCEAC D Capadiehramis eucinostomus R N 5 C C A E F CAE HC CQAC C C. mloto B H A E D C C C C C C o L B H E F C Cyrtocara moorii R N 5 C C A E E CAE HHCPAC C Dimidiochromis compressiceps E Q D F A B D CAE AHCFAC D Diplotaxodon sp. D H y E A C C C C E D L BAD C Docimodus evelynae M E Z E F A E E FEE EMCEAE D Hemitilapia oxyrhynchus H F Q D F A B D CAE AHCEAC o Maraviehromis anaphyrmus E Q D F B B 0 CAE A K C J D C D Nimboehromis livingstoni E Q D F ABE CAE E PC E AC D N. polystigrna E J D F ABE CAE EVCEAC D Otopharynx ovatus I E o E F ABO CAE AHCOAC D Plaeidaehromis subocularus Q N K C C A E E CAE H R C F A C C Protomelas kirkii 1M2 E F ABE CAE AWBEAC D Rhamphoehramis sp. o 4 E H A D C I F E D 0 FCC C Serranochramis Tabustus T P L E L E C K J I N KS CTHK B Taeniaehromis holotaenia I E P D G D E F CAE FKCFAC D Trematoeranus plaeodon I E U D F B B E CAE AKCEDC D Tyrannoehromis maerostoma o E B D F ABE CAE AHCEAC D Lake Tanganyika Cyphatilapia frontosa Z A$CMIDL B L B J T CUI B E Tropheus moo rei Y Q$DCBIM K A J U E R J L E