Syst. Biol. 51(1):113–135, 2002

Phylogeny of the Flock and Its Relationship to the Central and East African Haplochromine Cichlid Fish Faunas

WALTER SALZBURGER,1,2 AXEL MEYER,3 SANJA BARIC,1,4 ERIK VERHEYEN,5 AND CHRISTIAN STURMBAUER1,6,7 1 Department of Zoology and Limnology, University of Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria 3 Department of Biology, University of Konstanz, 78457 Konstanz, Germany; E-mail: [email protected] 5Section and Biochemical Systematics, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium; E-mail: [email protected]

Abstract.—Lake Tanganyika, the oldest of the East African Great Lakes, harbors the ecologically, mor- phologically, and behaviorally most complex of all assemblages of cichlid Žshes, consisting of about 200 described species. The evolutionary old age of the cichlid assemblage, its extreme degree of mor- phological differentiation, the lack of species with intermediate morphologies, and the rapidity of lineage formation have made evolutionary reconstruction difŽcult. The number and origin of seeding lineages, particularly the possible contribution of riverine haplochromine to endemic lacus- trine lineages, remains unclear. Our phylogenetic analyses, based on mitochondrial DNA sequences of three gene segments of 49 species (25% of all described species, up to 2,400 bp each), yield ro- bust phylogenies that provide new insights into the Lake Tanganyika adaptive radiation as well as into the origin of the Central- and East-African haplochromine faunas. Our data suggest that eight ancient African lineages may have seeded the Tanganyikan cichlid radiation. One of these seeding lin- eages, probably comprising substrate spawning Lamprologus-like species, diversiŽed into six lineages that evolved mouthbrooding during the initial stage of the radiation. All analyzed haplochromines from surrounding rivers and lakes seem to have evolved within the radiating Tanganyikan lineages. Thus, our Žndings contradict the current hypothesis that ancestral riverine haplochromines colonized Lake Tanganyika to give rise to at least part of its spectacular endemic cichlid species assemblage. Instead, the early phases of the Tanganyikan radiation affected Central and East African rivers and lakes. The haplochromines may have evolved in the Tanganyikan basin before the lake became a hydrologically and ecologically closed system and then secondarily colonized surrounding rivers. Apparently, therefore, the current diversity of Central and East African haplochromines represents a relatively young and polyphyletic fauna that evolved from or in parallel to lineages now endemic to Lake Tanganyika. [Adaptive radiation; Cichlidae; Lake Tanganyika; mitochondrial DNA sequences; molecular phylogeny; species ock.]

The three East African Great Lakes— ciation (Rensch, 1933; Kocher et al., 1993; Victoria, Malawi, and Tanganyika (Fig. 1)— Meyer, 1993). The most obvious differences are major model systems for the study among these species ocks are their age of adaptive radiation (Boulenger, 1898; and complexity in terms of species num- Kosswig, 1947; Fryer and Iles, 1972; Meyer ber and degree of morphological specializa- et al., 1990; Nishida, 1991; Meyer, 1993; tion. With an age as great as 9 to 12 MY, Sturmbauer and Meyer, 1993; Sturmbauer the Lake Tanganyika species assemblage is et al., 1994; Kocher et al., 1995; Rossiter, 1995; by far the oldest. It is also morphologi- Verheyen et al., 1996; Sturmbauer, 1998). Each cally, ecologically, and behaviorally the most lake contains cichlid species ocks number- complex assemblage, comprising about 200 ing hundreds of endemic species that arose described species in 54 genera (Fryer and independently by way of intralacustrine spe- Iles, 1972; Greenwood, 1984; Poll, 1986). The ocks of Lakes Victoria and Malawi con- sist exclusively of maternal mouthbrood- 2 Current address: Department of Biology, Univer- ers, in which females incubate eggs and sity of Konstanz, 78457 Konstanz, Germany; E-mail: protect their fry against predation in their [email protected] buccal cavities (Eccles and Trewavas, 1989; 4 Current address: Research Center for Agriculture Barlow, 1991; Keenleyside, 1991). In con- and Forestry Laimburg, 39040 Auer/Ora, Italy; E-mail: trast, the Lake Tanganyika cichlid species as- [email protected] 6 Current address: Department of Zoology, University semblage encompasses a much greater di- of Graz, Universitaetsplatz 2, 8010 Graz, Austria; E-mail: versity of brood care patterns—not only [email protected] mouthbrooders of various styles but also 7 Address correspondence to this author. substrate spawners, which lay their eggs on 113 114 SYSTEMATIC BIOLOGY VOL. 51

Mondeguer, 1991). Its transformation from a riverine to a truly lacustrine environment may have already started about 20 MYA and proceeded in three major stages. The struc- ture of the lake basin suggests that a mean- dering river gave rise to at least three shal- low, swampy, protolakes (9–12 MYA), which progressively deepened to fuse Žnally into a single deep lake (5–6 MYA; Tiercelin and Mondeguer, 1991; Lezzar et al., 1996; Cohen et al., 1997).

The Cichlid Species Flock of Lake Tanganyika Although some other teleost families (such as catŽsh, sardines, and nile perches) evolved small species ocks in Lake Tanganyika, cichlid Žshes are by far the most diverse group. Both the smallest cichlid (Neolampro- logus brevis; 3 cm) and largest known ci- chlid species (Boulengerochromis microlepis; 80 cm) are endemic to this lake. Poll (1986) grouped the Tanganyikan cichlids into 12 tribes (see Table 1). Representatives of four tribes are also found elsewhere in . Among these, the Haplochromini— with about 1,700 species—represents the most species-rich tribe, but only two endemic FIGURE 1. Map of eastern Africa, showing the loca- species are in Lake Tanganyika (Ctenochromis tion of the Great Lakes and the major river systems. horei and C. benthicola), and seven other species occur in the lake and in sur- various substrates and protect their offspring rounding rivers and swamps (Greenwood, (Kuwamura, 1986; Keenleyside, 1991). Be- 1979, 1980; Poll, 1986). The Tilapiini, which cause cichlids from Lake Tanganyika exhibit form the second most species-rich taxo- both biparental forms of mouthbrooding, as nomic assemblage of cichlids, have a pan- well as highly specialized modes of mater- African distribution (Klett et al., 2002). Lake nal mouthbrooding, Goodwin et al. (1998) Tanganyika has two endemic tilapiine spe- suggested that mouthbrooding may have cies, Oreochromis tanganicae and B. microlepis. evolved more than once in these Žshes. A The Tylochromini are represented by a sin- similar behavioral diversity appears to have gle endemic species in the lake, all re- evolved among substrate breeding cichlids, maining species of this tribe being found for which the recruitment of brood care in West Africa (Stiassny, 1990). The tribe helpers, or dwarŽsm (allowing for breed- Lamprologini—with 84 described species, ing in snail shells), may serve as examples this is the most species-rich Tanganyikan (Taborsky et al., 1986). lineage—also contains Žve riverine species The long and complex geological history found in the Congo River system (Poll, of the Lake Tanganyika basin is character- 1986; Sturmbauer et al., 1994; Stiassny, 1997). ized by a dynamic basin morphology, as well Given that the most ancestral splits in the as by uctuations in the water level of the Lamprologini are formed by lake endemics, lake (Scholz and Rosendahl, 1988; Tiercelin Sturmbauer et al. (1994) suggested that the and Mondeguer, 1991; Lezzar et al., 1996; radiation of the Lamprologini started in Cohen et al., 1997). During the initial stages of the Tanganyikan basin before the ecosys- its formation, the lake interrupted the ow of tem was hydrologically and ecologically the ancient proto-Malagarazi–Congo River closed, whereas the riverine species left and progressed in step with the formation the lake secondarily. The remaining eight of the East African rift valley (Tiercelin and tribes include species that are exclusively 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 115

TABLE 1. Characterization of the studied species of Lake Tanganyika cichlids. Species names and tribe assignment follow the nomenclature of Poll (1986).

Tribe s/e(g) b Species (n) Control region cyt b ND2 Heterochromis multidensr (1) — AF428151 AF398214 8/8(2) mb Bathybates ferox/sp. (1) — AF428152 U07239a Cyprichromini 6/6(2) mb Cyprichromis jumbo (3) AF400699 AF42815 — C. leptosoma (>10) — AF428154 AF398224 Paracyprichromis brieni (2) AF400700 Z21776a AF398223 30/30(13) mb Asprotilapia leptura (3) AF400701 Z21758a — dewindti (3) AF400702 Z21759a — macrops (-) — Z21760a U07242 a descampsi (2) AF400703 Z21765a — lemairii (2) AF400704 Z21766a — Microdontochromis tenuidentata (2) AF400705 Z21769a — Ophthalmotilapi a ventralis (>10) — Z21771a U07257 a sima (1) AF400706 Z21772a U07270 a Eretmodini 4/4(3) mb Eretmodus cyanostictus (>10) AF400707 AF428155 AF398220 Spathodus erythrodon (3) AF400708 AF428156 AF398218 Tanganicodus irsacae (3) AF400709 Z21778a AF398219 Haplochromini 1,700/2(6) mb » Astatoreochromis alluaudir (1) AF213618 a AF428157 AF398234 Astatotilapia burtonir (1) AF400710 Z21773a AF317266 Cyclopharynx fwaer (1) AF400711 AF428158 — Orthochromis polyacanthusr (1) AF400712 AF428159 AF398231 Pseudocrenilabrus multicolorr (2) AF400713 AF428160 AF398233 Schwetzochromis malagarazensisr (2) AF400714 AF428161 AF398232 S. mazimeroensisr (2) AF400715 AF428162 — Lamprologini 84/79(7) sb Altolamprologus calvus (3) AF400716 Z29989a — A. compressiceps (2) — AF428163 AF398229 Julidochromis marlieri (3) AF400717 Z30077a AF398230 Lamprologus callipterus (2) AF400718 Z29992a AF398226 L. congoensisr (1) AF400719 Z29993a — L. mocquardir (1) AF400720 Z29995a AF398225 Neolamprologus brichardi (>10) AF400721 Z29997a AF398227 N. longior (1) AF400722 Z30000a — N. toae (1) AF400723 Z30002a — Telmatochromis bifrenatus (11) AF400724 Z30185a AF398228 13/13(8) mb Benthochromis tricoti (2) AF400725 AF428164 — permaxillaris (3) AF400726 AF428165 — G. pfefferi (2) AF400727 AF428166 U07248a Limnochromis auritus (1) AF400728 Z21775a AF398216 Triglachromis otostigma (1) AF400729 Z30004a AF398217 Perissodini 6/6(2) mb Perissodus microlepis (1) AF400730 AF428167 AF398222 Plecodus straeleni (2) AF400731 Z21777a AF398221 Tilapiini 250/2(2) » sb Boulengerochromis microlepis (1) — Z30076a U07240 a mb Oreochromis tanganicae (1) — Z12046a AF317240 a Trematocarini 8/8(2) mb Trematocara unimaculatum (3) — AF428168 AF317268a Tropheini 23/23(7) mb Cyphotilapia frontosa (7) AF400732 AF428169 U07247a labiatus (3) AF400733 AF428170 U07254a orthognathus (4) AF400734 AF428171 — curvifrons (2) AF400735 AF428172 — babaulti (2) AF400736 Z12045a — duboisi (6) AF400737 Z12041a — T. moorii (>10) — Z12037a U07267 a Tylochromini 20/1(1) mb » (2) — AF428173 AF398215

s, number of species in the tribe; e, number of species endemic to Lake Tanganyika; g, number of genera of the tribe in Lake Tanganyika; b, breeding style (mb mouthbrooding; sb substrate breeding); n, number of specimens sampled. aGenBank accession numbers ofDsequences published elsewhereD . rRiverine taxa. 116 SYSTEMATIC BIOLOGY VOL. 51 found in Lake Tanganyika. These tribes oc- Meyer, 1993; Kocher et al., 1995). Conse- cupy distinct niches or habitat types: The quently, such studies produced noncon- Bathybathini (8 species) and the Tremato- clusive results for the relationships among carini (8 species) inhabit the deep pelagic the tribes. In particular, the relationships zone, the Cyprichromini (6 species) are crus- of the substrate spawning Lamprologini tacean feeders of the shallow pelagic zone, to seven mouthbrooding tribes—the and the Limnochromini (13 species) form Eretmodini, Limnochromini, Perissodini, an assemblage of invertebrate feeders at the Ectodini, Cyprichromini, Haplochromini deep muddy bottom. Part of the Ectodini taxa and Tropheini—have remained unclear. A (22) are invertebrate feeders at sandy and polyphyletic origin of the Lake Tanganyika muddy shores (8). The remaining Ectodini species ock has been suggested, but neither species, the Tropheini (23 species), and the the geographic origin nor the identity of Eretmodini (4 species) dwellin rocky habitats relatives outside the lake is known for most and feed on invertebrates or graze algae. endemic lineages (Fryer and Iles, 1972; Poll, Finally, the bizarre scale eaters (Perissodini, 6 1986; Nishida, 1991, 1997). On the basis of species) feed mainly on scales of other Žshes, morphological characteristics, Fryer and Iles which they sometimes imitate by mimicry. (1972) suggested that the endemic mouth- brooding tribes have originated from river- Phylogenetic Relationships of Lake ine haplochromine colonizers. So far, the hy- Tanganyika Cichlids pothesis that ancestral haplochromines have The relatively old evolutionary age of the seeded the primary radiation of the maj- Tanganyikan cichlid species ock is mani- ority of the mouthbrooding lineages in Lake fested not only in the unparalleled degree of Tanganyika remains untested. Indeed, pre- morphological and ecological divergence vious phylogenetic analyses of Tanganyikan within and among the endemic lineages, mouthbrooding lineages never included a but also by the absence of morphologically representative sample of the haplochromine intermediate species that might reveal how taxa, which are potential sister groups of the lineages are interrelated (Greenwood, the lake endemics. Nishida (1991) combined 1984). Moreover, diversiŽcation has been the seven tribes Eretmodini, Limnochro- achieved mainly by allometric changes of mini, Perissodini, Ectodini, Cyprichromini, relatively few morphological structures, Haplochromini, and Tropheini into the rather than by true morphological inno- “H-lineage” (Fig. 2a), pointing to the pos- vations (Greenwood, 1984; Stiassny, 1991), sibility that they evolved within the lake thus posing considerable problems for from the same riverine ancestor or ancestors. morphology-based phylogenetic analyses. The study of Sturmbauer and Meyer (1993; Several molecular phylogenetic studies Fig. 2b), which included the seven mouth- have contributed to the current knowl- brooding tribes (the H-lineage), suggested edge about evolutionary relationships in a sister group relationship between the subgroups of Lake Tanganyika cichlids. haplochromine cichlid Astatotilapia burtoni Monophyly has been demonstrated so far and the endemic Tanganyikan tribe Tro- for only 5 of the 12 tribes, the Tropheini pheini. However, this representative of the (Sturmbauer and Meyer, 1992; Meyer et al., Haplochromini was not resolved as the most 1996; Sturmbauer et al., 1997, unpubl.; ancestral branch of the H-lineage. In the phy- Takahashi et al., 1998), the Lamprologini logeny of Kocher et al. (1995; Fig. 2c), based (Sturmbauer et al., 1994; Takahashi et al., on the mitochrondrial NADH dehydroge- 1998), the Eretmodini (Verheyen et al., 1996), nase subunit 2 gene (ND2), the mouthbrood- the Ectodini (Sturmbauer and Meyer, 1993; ing tribe Eretmodini was placed outside the Takahashi et al., 1998), and the Perissodini H-lineage as sister group to the substrate (Takahashi et al., 1998). Studies on the spawning Lamprologini. Five species of adaptive radiation of cichlids in Lake Tan- Haplochromini from Lake Malawi were ganyika agree that several lineages arose resolved as sister groups to the Tropheini. with dramatic speed immediately after the formation of a lacustrine habitat; this re- sponse is manifested in short branch lengths, Our Phylogenetic Approach which makes evolutionary reconstruction In this study we use DNA sequences of difŽcult (Nishida, 1991; Sturmbauer and three mitochondrial genes—1,047 bp of ND2, 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 117

described species of Lake Tanganyikan cich- lids are included in our analyses, represent- ing all 12 tribes and 37 of the 54 genera. Our choice of taxa is based on the previous results summarized above. Of the tribes Cyprichro- mini, Eretmodini, Tilapiini, and Tylochro- mini, all described genera are included in this survey. Representatives of the missing genera in the Ectodini (Žve genera miss- ing; Sturmbauer and Meyer, 1993; Takahashi et al., 1998), the Lamprologini (two genera missing; Sturmbauer et al., 1994; Takahashi et al., 1998), the Perissodini (two genera miss- ing; Takahashi et al., 1998), and the Tropheini (one missing; Sturmbauer et al., unpubl.) were already shown to be mem- bers of the corresponding tribes. The only en- demic haplochromine genus (Ctenochromis) was resolved within the Tropheini in a re- cent study (Sturmbauer et al., unpubl.). The monotypic genera Hemibates (tribe Bathy- batini) and Trematochromis (tribe Tremato- carini) are not included in our analyses be- cause they are not available from current collections. However, a close morphological and ecological similarity to the remainder of their tribes has been established (Poll, 1986). For the Limnochromini, no molecular-based phylogenetic hypotheses exist. Our dataset includes only four of the eight genera of this tribe, mostly because the adaptation of these four genera to deepwater habitats made sam- pling difŽcult. The two species of the genus Greenwoodochromis and the monotypic genus Baileychromis are known only from formalin- preserved specimens. Both are assumed to occur in depths >30–40 m, as do the mono- typic limnochromine genera Reganochromis and Tangachromis. Our Žrst goal was to identify the seeding lineages for the primary radiation on the ba- sis of a linearized tree. We also wanted to address the question of the phylogenetic re- lationships between the Tanganyikan radi- ation and riverine haplochromines, such as Cyclopharynx fwae from the upper reaches FIGURE 2. Recent phylogenetic hypothesis (re- of the Lualaba–Congo River and Schwet- drawn) of the relationships among the Lake Tanganyika zochromis malagarazensis and S. mazimeroensis cichlid species ock. TIL1, Tilapia-lineage 1 (Boulenge- rochromis microlepis); TIL 2, Tilapia lineage 2 (Oreochromis from the upper Malagarazi River. Both rivers tanganicae). were part of the ancient proto-Malagarazi– Congo river system before the lake was formed (see Fig. 1 for a map of East African 402 bp of cytochrome b (cyt b), and 974 bp of rivers and lakes). To test the hypothesis that the control region—to infer a phylogeny of riverine Haplochromini seeded the radiation the Lake Tanganyika cichlid species assem- of the mouthbrooding H-lineage (Fryer and blage. With 49 species, more than 25% of all Iles, 1972; Nishida, 1991), we also included 118 SYSTEMATIC BIOLOGY VOL. 51 in our analyses the riverine haplochromine quence (Astatotilapia burtoni) coding for gaps genera Orthochromis, Pseudocrenilabrus, Asta- were included in the control region dataset, totilapia, and Astatoreochromis. Consequently, yielding a total length of 974 bp. The longest all but one riverine haplochromine genera insertions consisted of two gaps. of this region (Thoracochromis) are included in this study. Finally, we tested the validity Phylogenetic Analyses of the generic and tribal classiŽcation of the Given the wide range of evolutionary ages Tanganyikan cichlids on the basis of the ob- among the species sequenced, we divided tained phylogenies. our analyses into two steps. The Žrst step, intended to situate the Tanganyikan cichlids MATERIALS AND METHODS in a broader phylogeographic context, was based on the two relatively slowly evolv- mtDNA from 49 species (of >150 speci- ing protein-coding genes, ND2 and cyt b. mens sampled) of cichlid Žshes from all parts This analysis was aimed at resolving the of Lake Tanganyika and from surrounding older cladogenetic events and identifying rivers was sequenced for as much as 2,423 bp the seeding lineages of the primary lacus- per specimen, and the results were com- trine radiation. The West African cichlid bined with available data from GenBank species H. multidens was used as outgroup. (Table 1). The West African species Hete- This choice was based on the Žndings of rochromis multidens was included as outgroup Farias et al. (2000; see also Farias et al., 1999; (Farias et al., 2000). Voucher specimens have Klett et al., 2002) (Fig. 2d), who identiŽed been deposited at the Department of Zool- Heterochromis as the most ancestral genus ogy and Limnology, University of Innsbruck, among the African cichlids, using a com- Austria, and at the Royal Africa Museum, bined data approach that included mitochon- Tervuren, Belgium. Total DNA was extracted drial and nuclear genes and morphological from ethanol-preserved Žn-clips or mus- traits. In a second step of analysis, we used cle tissue by using Chelex (as outlined in the more variable mitochondrial control re- Sturmbauer et al., 1998) or by digestion gion to resolve the more recent splits, declar- with proteinase K followed by sodium chlo- ing the eretmodine cichlids as new outgroup ride extraction and ethanol precipitation according to the resulting topologies of the (Bruford et al., 1998). Polymerase chain re- higher-level analysis. We also adopted a new action ampliŽcation, cloning, and DNA- approach, selecting sequences with similar sequencing were performed according to rates of evolution for tree construction. The standard methods (Meyer et al., 1990; Sturm- consideration of taxa with most similar evo- bauer and Meyer, 1993) by using an Air lutionary rates minimizes the detrimental ef- Thermo-Cycler (Idaho Tehnology) and the fect of rate variation on tree reconstruction Big Dye sequencing chemistry on an ABI (Hillis et al., 1994). 373A automated sequencer (Applied Biosys- tems). Published primers were used for amplifying ND2, cyt b, and the Žrst sec- Step 1: ND2 and Cyt b tion of the control region (Kocher et al., The Žrst analysis focused on identifying 1989, 1995; Meyer et al., 1990; Pa¨abo,¨ the seeding lineages and determining the 1990). For the second section of the con- most appropriate outgroup for the primary trol region we used published primers (Lee radiation of the species ock. To this end et al., 1995) and a newly designed prim- we combined ND2 sequences (1,047 bp) with er (5’-TAAGAGCCCACCATCAGTTGA-3’). 402 bp of cyt b from 33 species representing Sequences can be obtained from GenBank all 12 Tanganyikan tribes (Table 1). To test the under the accession numbers listed in Table 1. phylogenetic congruence of the two protein- Initial alignments were obtained by coding mitochondrial gene segments, we Clustal W (Thompson et al., 1994). The align- used the partition homogeneity test imple- ment of the control region was iteratively ad- mented in PAUP¤ 4.0b2a (Swofford, 2000), justed by eye, those alternative alignments with 300 replicates. We point out that the being preferred that resulted in shorter trees power of the partition homogeneity test has in parsimony analysis in which equal weight- been shown to be weak when one of the ings were used for all mutations (Levinton datasets compared is smaller than the other et al., 1996). As many as 28 indels per se- (Dolphin et al., 2000; Yoder et al., 2001), which 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 119 might well be the case when 1,047 bp of the Transitions at third positions of fourfold- ND2 gene is combined with 402 bp of cyt b. degenerate amino acids were excluded We then performed a likelihood mapping because of their degree of saturation, as analysis with Puzzle 4.0 (Strimmer and von depicted in the saturation plot in Fig. 3. In Haeseler, 1996, 1997) to visualize the strength an additional weighted MP analysis, C/T of the overall phylogenetic signal in the changes at Žrst positions of leucine codons dataset. This permutation method evaluates were treated as a Žfth base and such mu- the likelihood supports for the three alter- tations were down-weighted three times native topologies of multiple sets (10,000) of more than the nucleotide mutations that randomly chosen taxon quartets. Likelihood result in amino acid substitutions. The dif- frequencies of all evaluated quartet topolo- ferent MP topologies were compared by the gies are mapped on a triangle picturing a nonparametric two-tailed Wilcoxon signed barycentric coordinate system. The triangle rank test implemented in PAUP¤ 4.0b2a is partitioned into seven different zones: The (Swofford, 2000) (Table 2). To justify the use central region represents completely unre- of an appropriate substitution model for the solved topologies, the three corners represent ML analyses, we evaluated the ML scores of fully resolved topologies, and the three in- the unweighted MP topology by means of termediate regions between the corners rep- a likelihood-ratio test (Sullivan et al., 1997; resent partially resolved topologies. The re- Huelsenbeck and Crandall, 1997) (Table 3; sulting likelihood frequencies for each of see also Swofford et al., 1996). We tested the the regions provide information about the Jukes–Cantor (JC; Jukes and Cantor, 1969), strength and clarity of the phylogenetic sig- the Kimura-two-parameter (K2P; Kimura, nal in the dataset. To assess the degree of sat- 1980), the HKY, and the general time re- uration of transition and transversion mu- versible (GTR; Yang, 1994) models. The tations in each codon position of the two degrees of freedom for the likelihood-ratio protein-coding genes, we plotted the two tests were determined by the differences in types of mutations against the percentage of the number of model parameters between sequence divergence in 496 pairwise com- the different models being evaluated. The parisons (Fig. 3), not including the outgroup likelihood-ratio test indicated that the GTR H. multidens. Because of the degree of sat- substitution model signiŽcantly improved uration in transition mutations in third- the ln likelihood with respect to the HKY codon positions of both gene segments, model¡ (P < 0.001; Table 3). Moreover, the we decided not to include transitions in consideration of a 0 distribution correction third-codon positions of fourfold-degenerate greatly improved the ln likelihood. We amino acid sites. Transition mutations in the thus used the GTR ¡0 model of molec- remaining data turned out to be unsaturated ular evolution with Cestimated values for (Fig. 3). the gamma shape parameter and for the Phylogenetic trees were constructed with proportion of invariable sites for ML tree maximum parsimony (MP) and maximum construction, applying bootstrap analysis likelihood (ML) and using the computer with 500 pseudoreplicates under the fast program PAUP¤ 4.0b2a (Swofford, 2000). stepwise addition option in PAUP¤ 4.0b2a Pairwise distances were calculated by the (Swofford, 2000). ML scores of all topolo- Hasegawa—Kishino—Yano (HKY) model gies were evaluated by the nonparametric with a gamma distribution correction Shimodaira–Hasegawa test (Shimodaira (Hasegawa et al., 1985). MP topologies were and Hasegawa, 1999) under a resampling- obtained by the heuristic search option and estimated log-likelihood (Table 2; see also 50 replicates, applying bootstrap analyses Goldman et al., 2000; Buckley et al., 2001) as with 1,000 pseudoreplicates as the standard implemented in PAUP¤ 4.0b4a (Swofford, measure of conŽdence. An unweighted MP 2000). To estimate the support for distinct analysis was performed by including all internal branches critical for the interpre- of the sequence data. In the weighted MP tation of the evolutionary pathways, we analysis, all transition mutations at third performed a four-cluster likelihood map- positions were weighted 1:6 for ND2 and ping analysis with the program PUZZLE 4.0 1:3 for cyt b relative to transversions, based (Strimmer and von Haeseler, 1996, 1997). on the ML-estimated transition:transversion In this method, four clusters of taxa can be ratio of 6.42 for ND2 and 3.38 for cyt b. deŽned to calculate the relative likelihood 120 SYSTEMATIC BIOLOGY VOL. 51

FIGURE 3. Saturation plot of transition and transversion mutations (y-axis) versus percentage of sequence di- vergence (x-axis) of 496 pairwise comparisons for the two protein-coding genes ND2 and cyt b. The slope and the coefŽcient of determination (R2 ) are depicted for each type of substitution. 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 121

TABLE 2. Likelihood scores and MP tree lengths obtained from analyses under several weighting regimes for the dataset combining the protein-coding genes ND2 and cyt b and the dataset for the control region. The topologies were evaluated against the optimal topologies by using the Shimodaira–Hasegawa (S-H) and the nonparametric two-tailed Wilcoxon signed rank test (WSR) tests, implemented in PAUP¤ 4.0b4a (Swofford, 2000).

Maximum likelihood Maximum parsimony

Weighting strategy ln likelihood 1ML S-H-test Tree length 1MP WSR ¡ ND2 and cyt b MP, uwt 12,896.599 5.777 0.753 2,552 — — MP, wt 12,891.608 0.787 0.949 2,554 2 0.867 MP, 5th base, wt 12,912.648 21.826 0.150 2,568 16 0.131 ML 12,890.822 — — 2,552 0 1.000 Control region MP, uwt 13,167.613 12.791 0.453 1,409 — — MP, wt 13,167.151 12.328 0.468 1,413 4 0.382 MP, TV only 13,162.149 7.327 0.581 1,436 27 0.036 ML 13,154.822 — — 1,429 20 0.154

uwt, unweighted; wt, weighted; TV, transversions. frequencies for each of the three possible stancy is tested for all n 1 internal nodes topologies interrelating the four clusters. in a set of n sequences, excludin¡ g the out- Likelihood frequencies are mapped on a tri- group(s). Sequences that are evolving too angle partitioned into three regions, each rapidly or too slowly at a high signiŽcance deŽning the frequency for one of the three level (P 0:01) are excluded and the test alternative topologies. The sequences were is repeateDd until all remaining sequences grouped into four clusters according to the meet the signiŽcance level. Once hetero- optimal MP and ML (GTR 0) topology geneous sequences are eliminated, a tree (Fig. 4a). C for a given topology is constructed under We based the discrimination as to whether the assumption of rate constancy, which is a lineage was likely to have seeded the ra- termed a linearized tree. In our case, all se- diation or to have arisen during the radi- quences showed a clocklike behavior when ation itself on the use of a linearized tree. the HKY 0 model of molecular evolu- Therefore we performed the two-cluster test tion was applieC d and none of the taxa had of Takezaki et al. (1995), using the computer to be excluded for the construction of the program LINTRE available from the authors linearized tree. Because no fossil data are (Takezaki et al., 1995). The two-cluster test available from East African lake cichlids to examines whether the average distances calibrate a molecular clock, we decided to from any node in a given topology to the determine the sequence of the major clado- tips of the two clusters of taxa deŽned by genetic events on the basis of a relative time this node are signiŽcantly different. Rate con- scale.

TABLE 3. Likelihood-ratio test for different substitution models based on unweighted MP topologies for the dataset combining the protein-coding genes ND2 and cyt b and that for the control region (see text for details).

Substitution model ln likelihood Comparison 1ML P ¡ ND2 and cyt b JC 14,448.151 — — — K2P 13,355.641 JC vs. K2P 1,092.510 <0.001 HKY 13,152.666 K2P vs. HKY 202.975 <0.001 GTR 13,126.389 HKY vs. GTR 26.277 <0.001 GTR 0 12,910.103 GTR vs. GTR 0 194.727 <0.001 ControlCregion C JC 8,661.053 — — — K2P 8,413.037 JC vs. K2P 248.016 <0.001 HKY 8,287.339 K2P vs. HKY 128.698 <0.001 GTR 8,262.790 HKY vs. GTR 24.549 <0.001 GTR 0 8,084.431 GTR vs. GTR 0 202.908 <0.001 C C 122 SYSTEMATIC BIOLOGY VOL. 51 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 123

Step 2: Control Region TABLE 4. Species names and GenBank accession numbers of the control region sequences of the hap- In the second step of analyses we focused lochromine cichlids included from Lake Victoria and on the radiation itself, using the entire mito- Malawi. chondrial control region of 40 taxa. The tribes Trematocarini, Tylochromini, Bathybatini, Taxon Accession No. Reference and Tilapiini were excluded from this analy- Lake Victoria sis, because there was strong support for an- Haplochromis AF213523 Nagl et al., 2000 cestral position for them in the higher-level lividus Neochromis AF213544 Nagl et al., 2000 analysis (Fig. 4). Instead, we used the eretmo- nigricans dine cichlids Eretmodus cyanostictus, Tangan- Lake Malawi icodus irsacae, and Spathodus erythrodon as a Labeotropheus AF213623 Nagl et al., 2000 new outgroup. Again, a likelihood mapping trewavasae Lethrinops U12551 Lee et al., 1995 analysis was performed with PUZZLE 4.0 auritus (Strimmer and von Haeseler, 1996, 1997) to Pseudotropheus AF213622 Nagl et al., 2000 assess the strength of the overall phyloge- msobo netic signal in the dataset. A saturation plot Cyrtocara U12554 Lee et al., 1995 was constructed for the whole dataset. MP moorii and ML topologies were obtained by the same search parameters as in step 1, and pairwise distances were calculated by us- sequences from GenBank were included in ing the HKY 0 model (Hasegawa et al., the dataset for additional analyses (Table 4). 1985). In MP, gapC s were treated as Žfth base, To reduce the deterioration of the phylo- whereas in ML, gaps were treated as miss- genetic signal caused by increased levels of ing data. For MP analyses, various weighting homoplasy, the result of the arbitrary choice regimes were evaluated by means of the non- of taxa (Moritz et al., 1987), we tested the parametric two-tailed Wilcoxon signed rank relative rates of base substitutions in the test with PAUP¤ 4.0b2a (Swofford, 2000); sig- control region for 39 taxa. The DNA se- niŽcantly different ML trees were identiŽed quence of Astaoreochromis alluaudi obtained by the Shimodaira–Hasegawa test under a from GenBank was not included in this anal- resampling-estimated log-likelihood imple- ysis. We performed the branch length test of mented in the same program (Table 2). To Takezaki et al. (1995), using their computer identify the most appropriate substitution program LINTRE (Table 5). The measured model for ML analyses, we tested the JC, rate of molecular evolution has been shown K2P, HKY, and GTR models with and with- to be linear whenever the maximum per- out rate variation by means of a likelihood- centage of genetic divergence ( p-distance) ratio test (Table 3). Again, GTR 0 turned was <15% (Brown, 1983). After excluding out to be the appropriate modeCl and was 12 taxa according to the branch length test thus used. To link the Tanganyikan and river- (P < 0:01), the maximum p-value decreased ine Haplochromini to the species ocks of to 13.1%, compared with 18.4% for the com- Lakes Malawi and Victoria, six additional plete dataset. By excluding sequences with

FIGURE 4. (a) Depiction of ML (GTR 0) tree of Lake Tanganyika cichlids based on ND2 and cyt b DNA sequences (declaring H. multidens as outgroup). NumberC s above the branches (in italics) are ML bootstrap estimates; numbers below the branches are the corresponding MP bootstrap estimates (values <50% are not shown). MP groupings differing from ML are indicated with dotted lines. Names of the corresponding tribes are depicted on the left; the redeŽned H-lineage is highlighted with a gray box. Riverine taxa also are marked with a gray box, and taxa not shared with the control-region analyses are indicated with asterisks. (b, c) Results of the likelihood mapping analysis (Strimmer and von Haeseler, 1997), represented as triangles. Values at the corners in (c) indicate the percentages of fully resolved phylogenies for all possible quartets; values at the lateral sections of the triangle are the percentages of partially resolved quartet phylogenies; and the number in the center of the triangle represents the percentage of unresolved quartet topologies. (d, e) Four-cluster likelihood mapping analysis (Strimmer and von Haeseler, 1997). Sequences were split into four disjointed groups, each deŽning an internal branch under evaluation that is crucial for identiŽcation of the onset of the primary radiation of the Lake Tanganyika cichlid species ock. The internal branches under evaluation are identiŽed by roman numbers (I, II) on the phylogeny and at the corresponding triangle. The groupings are indicated by A, B, C, D, and E in (a). The corners of the triangle show the percentage of support for each of the three alternative topologies for the four clusters under evaluation. 124 SYSTEMATIC BIOLOGY VOL. 51

TABLE 5. Results of the branch-length test (Takezaki et al., 1995), obtained with the computer program LINTRE (E, taxa excluded from further analyses; R, taxa reincluded in an additional step).

z-value Taxon Control region cyt b Combined data set Altolamprologus calvus 2.60 0.18 0.49 Asprotilapia leptura 0.55 0.32 1.22 Astatotilapia burtoni 1.69 0.24 0.26 Aulonochromis dewindti 1.59 0.32 0.86 Benthochromis tricoti 3.01 0.71 E Cyclopharynx fwae 4.25 2.32 R Cyphotilapia frontosa 6.15 4.35 R Cyprichromis jumbo 5.58 0.94 E Ectodus descampsi 3.05 1.02 E Eretmodus cyanostictus outgroup outgroup outgroup Gnathochromis permaxillaris 4.40 1.25 E G. pfefferi 4.25 2.72 R Grammatotria lemairii 1.76 0.03 0.92 Julidochromis marlieri 1.70 0.15 0.09 Lamprologus callipterus 1.47 0.85 0.31 L. congoensis 0.64 0.54 0.41 L. mocquardi 1.10 0.39 0.04 Limnochromis auritus 0.01 1.93 1.54 Lobochilotes labiatus 2.50 0.87 0.02 Microdontochromis tenuidentata 1.09 0.18 0.88 Neolamprologus brichardi 0.64 1.60 0.22 N. longior 4.34 1.40 E N. toae 2.87 1.51 E Orthochromis polyacanthus 0.42 2.22 0.17 Paracyprichromis brieni 1.87 1.36 2.14 Perissodus microlepis 2.10 0.68 0.45 Plecodus straeleni 0.75 0.15 1.07 Petrochromis orthognathus 1.90 0.18 0.03 Pseudocrenilabrus multicolor 0.45 2.37 2.04 Pseudosimochromis curvifrons 1.10 0.94 0.01 Schwetzochromis malagarazensis 5.13 1.12 R S. mazimeroensis 4.34 0.98 E Simochromis babaulti 1.90 0.17 1.03 Spathodus erythrodon outgroup outgroup outgroup Tanganicodus irsacae outgroup outgroup outgroup Telmatochromis bifrenatus 1.22 0.90 0.62 Triglachromis otostigma 0.34 1.74 0.75 4.84 1.60 E Xenotilapia sima 2.53 0.25 1.19 uneven mutation rates, we were able to objec- rate of base substitution, the relative rates of tively reduce the number of sequences in our base substitutions of all remaining sequences dataset. This way, we retained only those rep- were not signiŽcantly different (Table 5). The resentatives of each tribe that met the crite- phylogenetic congruence of the two genes ria for obtaining optimum phylogenetic sig- was again tested by the partition homogene- nal for the branches interrelating the tribes. ity test with PAUP¤ 4.0b2a (Swofford, 2000). In this analysis, 27 sequences of the control We then performed MP and ML analyses, region were combined with 402 bp of the weighting transitions 1:2 relative to transver- cyt b gene, because most corresponding cyt b sions in MP (based on the ML estimate sequences were already available from ear- of the transition:transversion ratio of 1.6), lier studies (Sturmbauer and Meyer, 1993; and using the GTR 0 model for ML. Fi- Sturmbauer et al., 1994). In the cyt b gene nally, four species showinC g an uneven muta- segment, one sequence (from Cyphotilapia tion rate in the control region (Cyclopharynx frontosa) showed a markedly different rate fwae, Cyphotilapia frontosa, Gnathochromis pfef- of base substitution according to the branch feri, and Schwetzochromis malagarazensis) were length test (Table 5). In the combined dataset, reincluded in the MP analysis. Phylogenetic after excluding all sequences of an uneven placement of these species was achieved in 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 125 an additional phylogenetic analysis, in which and Trematocara unimaculatum, but their the consensus tree topology obtained with- branching order differed in the various out them was constrained. This procedure al- MP trees obtained. The three representatives lowed derivation of a more comprehensive, of the Eretmodini formed the next branch, albeit preliminary, phylogenetic hypothesis followed by the Lamprologini and the re- for the Lake Tanganyika species assemblage. maining representatives of the H-lineage. Cyphotilapia frontosa was consistently placed RESULTS outside the Tropheini, being resolved as a separate lineage, more closely related to the Step 1: ND2 and Cyt b Limnochromini; however, bootstrap support No variation in length was detected among for this was low. In accordance with previous ND2 and cyt b sequences. In ND2, 366 of the results (Kocher et al., 1995), Gnathochromis 1,047 nucleotide positions were parsimony- pfefferi, which is presently considered to be informative, 279 of which (76%) affected a member of the tribe Limnochromini (Poll, third-codon positions. The HKY 0 se- 1986), was placed among the Tropheini. The quence divergences in the ingroupCranged representatives of the Haplochromini were from 0.57786 (Tylochromis polylepis–Ortho- not resolved monophyletically: Although chromis polyacanthus) to 0.04795 (Neolamprolo- Schwetzochromis appeared to be a separate, gus brichardi–Telmatochromis bifrenatus). In cyt more ancestral lineage, more closely related b, 112 of the 402 positions were parsimony- to the Perissodini, the Astatotilapia invariably informative, and 105 of these (93%) occurred appeared as the sister genus to the Tropheini in third-codon positions. The HKY 0 dis- (as in previous analyses). tances were between 0.50696 (TylochromiC s In the optimal ML topology (depicted in polylepis–Pseudocrenilabrus multicolor) and Fig. 4a), Tylochromis polylepis was resolved 0.00514 (Tanganicodus irsacae–Spathodus ery- as the most ancestral lineage, followed throdon). The partition homogeneity test indi- by Oreochromis tanganicae. Boulengerochromis cated phylogenetic congruence between the microlepis, Bathybates ferox, and Trematocara two protein-coding genes (P 0:17). Like- unimaculatum branched next, with Boulenge- lihood mapping resulted in 97.1D % fully re- rochromis as the most ancestral split, albeit solved quartets, pointing to a strong phy- with low bootstrap support. The Eretmodini logenetic signal in the dataset (Fig. 4b,c). were resolved as sister group to the Lampro- The chi-square test of homogeneity of base logini and the remainder of the H-lineage. frequencies across taxa (as implemented in Again, Cyphotilapia frontosa was placed out- PAUP¤), evaluatinginformative sites only, re- side the Tropheini, and Gnathochromis pfef- 2 sulted in  (96) 121:076 (P 0:043) for ND2 feri clustered within the Tropheini. Schwet- 2 D D and  (96) 1:95 (P 1.000) for cyt b. Both zochromis malagarazensis was resolved as values doDnot reject homogeneitD y of base fre- a separate lineage of the Haplochromini, quencies at P 0:01. Astatotilapia as sister to the Tropheini. The UnweightedDMP analysis yielded a single four-cluster likelihood mapping analyses most-parsimonious tree [2,552 steps; consis- evaluating the likelihood supports for two tency index excluding uninformative sites crucial branches (labeled I and II in Fig. 4a) (CI; Kluge and Farris, 1969): 0.42; reten- yielded 58.8% (Fig. 4d) for branch I, 88.1% tion index (RI; Farris, 1989): 0.47], whereas (Fig. 4e) for branch II. This robust support for the weighted MP analysis yielded a tree of this particular branching order corroborates 2,554 steps. A topology with 2,568 steps the validity of the Eretmodini as outgroup was obtained from the weighted analy- for the analyses of the primary lacustrine sis that treated C/T changes at Žrst posi- radiation (branch I) as well as the place- tions of leucine codons as a Žfth base (see ment of the Lamprologini as sister group Table 2). The bootstrap supports for the un- to the remaining lineages of the H-lineage weighted parsimony analysis are depicted (branch II). These values correspond to the on the corresponding branches in Fig. 4a. bootstrapping values obtained with MP, al- In all MP topologies, Tylochromis polylepis though the bootstrap support for branch II was resolved as the most ancestral lineage was weaker in ML (Fig. 4a). The strong sup- of the Tanganyikan taxa. The next branches port for branch II in the four-cluster like- were occupied by Oreochromis tanganicae, lihood mapping with respect to ML itself Boulengerochromis microlepis, Bathybates ferox, might be the result of grouping the taxa into 126 SYSTEMATIC BIOLOGY VOL. 51

FIGURE 5. Linearized tree based on ND2 and cyt b DNA sequences constructed with the two-cluster test of the LINTRE program package (Takezaki et al., 1995), identifying two series of cladogenetic events in the Lake Tanganyika cichlid species ock. Riverine taxa are marked with a gray box; taxa not shared with the control-region analyses are indicated with asterisks. four clusters based on the optimal MP and netic events in the Lake Tanganyika cich- ML topology and of using four-cluster like- lids species ock (Fig. 5). These two se- lihood mapping. Each of the 10,000 taxon ries were delineated from clusters of very quartets evaluated consists of four randomly short branches, whereas gradual evolution- chosen representatives (one of each of the ary processes were indicated by longer inter- four clusters), which makes this method less nal branches. dependent on the selection of taxa. Because topologies combining representatives of two or more clusters on one of the four branches Step 2: Control Region are not valid, this method focuses on the In the second analysis we focused on branches interrelating the predeŽned clus- the Tanganyikan radiation itself. The tribes ters. Alternative arrangements of taxon clus- Trematocarini, Tylochromini, Bathybatini, ters are thus effectively excluded. and Tilapiini were excluded from this analy- The linearized tree, constructed with the sis, their ancestral position in the phylogeny LINTRE package (Takezaki et al., 1995), having already been strongly supported by clearly identiŽed two series of cladoge- the higher-level analysis (see Fig. 4). In the 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 127 control region, 294 of the 974 nucleotide Tropheini assemblage. The inclusion of six positions were parsimony-informative. The haplochromine taxa from lakes Victoria and HKY 0 distances were between 0.28028 Malawi (Fig. 6a) did not change the topolo- (NeolamprologuC s longior–Gnathochromis pfef- gies obtained without them. These taxa were feri) and 0.01217 (Lamprologus congoensis—L. consistently placed as sister group to the Tro- mocquardi). The chi-square test of homogene- pheini, with Astatotilapia burtoni sister to the ity of base frequencies across sequences re- two representatives from LakeVictoria. 2 sulted in  (135) 78:21 (P 0:999) for not The MP topology based on the reduced rejecting homogeneitD y of basD e frequencies. dataset (27 taxa; combining the control re- The likelihood mapping analysis revealed a gion with cyt b; partition homogeneity test: percentage of fully resolved quartet topolo- P 0:95), again identiŽed the Lamprologini gies to be 96.3% (Fig. 6b), pointing to an as Dsister group to the H-lineage (BS 55). excellent overall phylogenetic signal in the Within the H-lineage, the LimnochrominD i dataset. formed a clade, as did the Cyprichromini The unweighted MP analysis yielded with the Perissodini, and the Ectodini 216 most-parsimonious trees of 1,409 steps formed the sister group to the Tropheini/ in length. The Lamprologini were placed Haplochromini. The branching order among as sister group to the remaining endemic these clades was not resolved in the strict Tanganyikan and riverine lineages [boot- consensus topology. Phylogenetic placement strap support (BS) 31]. The branching or- of four taxa that were included after con- der of these endemiDc Tanganyikan and river- straining the strict MP consensus topology ine lineages was not resolved in the strict is depicted in Fig. 7a. In the ML tree, the MP topology. However, Cyphotilapia frontosa Ectodini occupied the most ancestral branch was consistently placed outside the Troheini, within the H-lineage. The Cyprichromini and as sister group to the Perissodini (BS 42); the Limochromini branched off next, sis- clustered withiDn the ter group to the Perissodini and Cyphoti- Tropheini; and a putative member of the lapia, and then the Haplochromini/Tropheini Limnochromini (Benthochromis tricoti) was assemblage. placed outside the Limnochromini, sister to Cyphotilapia and the Perissodini (BS 19). The weighted MP tree (1,413 steps; Dyield- DISCUSSION ing a better -ln likelihood than the un- A polyphyletic origin of the radiation of weighted MP trees; results not shown) re- Lake Tanganyika cichlid Žshes has long been solved the Lamprologini as sister group to suggested, given that 4 of the 12 tribes have the remainders of the H-lineage” (BS 47). A members that are found outside the lake clade comprising the LimnochrominiD, sister (Poll, 1986; Nishida, 1991). However, neither group to C. frontosa and the two representa- the geographic origin nor the identity of sis- tives of the Perissodini (BS 41), branched ter groups outside the lake is known for the off next. Then followed a cladD e comprising majority of the endemic lineages. Investiga- the Cyprichromini with Benthochromis tricoti tors have so far assumed that several en- (BS 18), followed by the Ectodini. Two demic mouthbrooding tribes originated from haplochrominD e lineages (Astatoreochromis riverine haplochromine colonizers (Fryer and Schwetzochromis; and Pseudocrenilabrus, and Iles, 1972; Nishida, 1991). Earlier stud- Cyclopharynx, and Orthochromis) were re- ies indeed demonstrated a close relation- solved as sister group to a clade compris- ship of haplochromine species to part of the ing Astatotilapia and the Tropheini (including Tanganyikan lineages (Meyer et al., 1991; Gnathochromis pfefferi) (BS 80). Nishida, 1991; Meyer, 1993; Sturmbauer and In the ML tree (not shown)D , the Lam- Meyer, 1993; Kocher et al., 1995). The anal- prologini were again placed outside the ysis presented here is the Žrst to include H-lineage. Within the H-lineage, the Ectodini several riverine haplochromines of the gen- and Schwetzochromis occupied the most an- era Orthochromis, Schwetzochromis, Cyclophar- cestral branch, followed by a clade compris- ynx, Pseudocrenilabrus, Astatotilapia, and As- ing the Cyprichromini (with Benthochromis) tatoreochromis (Greenwood, 1979, 1980; Poll, and the Limochromini, sister group to 1986), all of which are potential sister the Perissodini and Cyphotilapia. The next groups to lake endemics. The inclusion of branch was formed by the Haplochromini/ these taxa allowed us to test the current 128 SYSTEMATIC BIOLOGY VOL. 51 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 129 hypothesis that ancestral haplochromine col- Trematocarini and the Bathybatini as seed- onizers would have seeded the primary ra- ing lineages. Because both underwent radi- diation of most mouthbrooding lineages in ation in Lake Tanganyika and no relatives Lake Tanganyika. are known in extant river faunas, these en- demics may be the only surviving descen- Identity and Geographic Origin of Ancestral dants of two ancient African lineages. The Lineages Seeding the Tanganyika Radiation same may be true for the Eretmodini and the Lamprologini, which consistently branched Our evolutionary reconstruction, based on before the onset of the primary lacustrine ra- the linearized tree method (Takezaki et al., diation, and for the ancestor of the H-lineage 1995), identiŽed two series of cladogenetic (as above: sensu Nishida, 1991, but excluding events affecting the Lake Tanganyika cich- the Eretmodini). lid species assemblage (Fig. 5). The Žrst series of cladogenetic events involving the lineages that seeded the Lake Tanganyika The Primary Lacustrine Radiation and radiation (dashed box in Fig. 5) is likely the Evolution of Alternative Brood to have taken place at an early stage of Care Strategies the lake formation, when the protolake (or The second series of cladogenetic events lakes) were much shallower (see Tiercelin evident from the linearized tree analysis con- and Mondeguer, 1991). The second series cerns the primary lacustrine radiaton itself of cladogenetic events delineated from the (gray box in Fig. 5). The following lineages linearized tree (gray box in Fig. 5) appears are likely to have radiated in parallel: the to represent the primary radiation taking Lamprologini, the H-lineage (sensu Nishida, place in the lacustrine habitat. The branch- 1991, but excluding the Eretmodini), and ing pattern of the linearized tree suggests to possibly also the Eretmodini, Trematocarini, us that eight distinct lineages have seeded and Bathybatini. Earlier studies on the adap- the Tanganyika radiation: the Tylochromini, tive radiation of Lake Tanganyika cich- two lineages of the Tilapiini, the Bathybatini, lids have created conicting hypotheses for the Trematocarini, the Eretmodini, the an- the relationships of the nonmouthbrood- cestor(s) of the Lamprologini, and the an- ing Lamprologini to the H-lineage (Nishida, cestor(s) of the H-lineage (sensu Nishida, 1991; Sturmbauer and Meyer, 1993; Kocher 1991, but excluding the Eretmodini). The two et al., 1995). Our present analyses unam- species of the Tilapiini, Oreochromis tangan- biguously placed the Eretmodini as sepa- icae and Boulengerochromis microlepis, were rate seeding lineage from the other six. The resolved as separate lineages, in agreement Eretmodini comprise four described species with Klett et al. (2002). The Tanganyikan of biparental mouthbrooders (but most prob- representatives of the Tylochromini and ably include more species; see Ruber¨ et al., Tilapiini either were already part of the an- 1999). The remaining six lineages (which we cient fauna of the proto-Malagarazi–Congo redeŽne as H-lineage here) were placed as a River before the lake basin formed or entered sister group to the substrate spawning Lam- the lacustrine habitat more recently. The Žve prologini, but they all are mouthbrooders remaining seeding lineages radiated in step with a wide variety of strategies (Kuwamura, with the development of the current truly la- 1986; Keenleyside, 1991). All the Limnochro- custrine habitat. Our analyses identiŽed the mini and Perissodini are primitive biparental

FIGURE 6. (a) ML tree (GTR 0) of Lake Tanganyika cichlids and six additional haplochromine taxa from Lakes Victoria and Malawi, based on sequenceC s of the mitochondrial control region (declaring the Eretmodini as outgroup). Numbers above the branches are ML bootstrap estimates; numbers below the branches are the corresponding MP bootstrap estimates (values <50% are not shown). Riverine taxa are marked with a gray box, and taxa not shared with the ND2 and cyt b analyses are indicated with asterisks. (b) Results of the likelihoodmapping analysis (Strimmer and von Haeseler, 1997) of the control region dataset, represented as triangles. Values at the corners indicate the percentage of fully resolved quartet topologies, values at the lateral sections give the percentages of partially resolved topologies, and the number in the center of the triangle represents the percentage of unresolved trees. (c) Saturation plot of transition and transversion mutations (y-axis) against the percentage of sequence divergence (x-axis) of 742 pairwise comparisons for the control-region dataset. The slope and the coefŽcient of determination (R2 ) are depicted for transition and transversion mutations. 130 SYSTEMATIC BIOLOGY VOL. 51

FIGURE 7. Strict consensus tree of four most-parsimonious trees of 27 taxa based upon 895 bp of the mitochondrial control region combined with a 402-bp segment of cyt b, representing all lineages involved in the Tanganyika radiation. Three Eretmodine cichlids (Spathodus erythrodon, Tanganicodus irsacae, and Eretmodus cyanostictus) were declared to be outgroups, on the basis of the results of the higher-level analyses (Fig. 4). The four outlined taxa had a signiŽcantly different evolutionary rate and were thus reincluded in a separate analysis after constraining the topology that was obtained without them. Riverine taxa are marked with a gray box; taxa not shared with the ND2 and cyt b analyses are indicated with asterisks. ERE, Eretmodini; OG, outgroup; LIM, Limnochromini; C, Cyprichromini; PER, Perissodini; H, Haplochromini; Haplochr, Haplochromini. mouthbrooders. They form pairs to spawn, biparental mouthbrooders (Limnochromini, females lay as many as 300 eggs, and both Perissodini, and some Ectodini) and of tribes parents guard the fry. The Ectodini com- of more derived maternal mouthbrooders prise both biparental and maternal mouth- (Cyprichromini, Ectodini, Haplochromini, brooders, whereas the Cyprichromini are and Tropheini) at the base of the primary pelagic maternal mouthbrooders produc- lacustrine radiation, although the order of ing relatively high numbers of eggs. The their branching differed in the different Haplochromini and Tropheini are maternal analyses. This suggests that the diversiŽca- mouthbrooders, producing small numbers tion of these six mouthbrooding tribes was of eggs (10–50) and featuring a relatively connected to a single transition from sub- long time of buccal incubation. Our analy- strate breeding to mouthbrooding during the ses placed the origin of presumably ancestral primary lacustrine radiation. Subsequently, 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 131 each lineage must have evolved its particu- out instead to be a member of the Tropheini lar strategy of mouthbrooding in step with its (in agreement with Kocher et al., 1995). diversiŽcation, with some lineages remain- This result is also supported by observa- ing at relatively primitive styles and others tions of its breeding behavior, indicating that reaching highly derived styles (Barlow, 1991; G. permaxillaris is a biparental mouthbrooder Keenleyside, 1991). that forms breeding pairs (P. Henninger, pers. comm.), whereas G. pfefferi exhibits a Ecomorphological DiversiŽcation derived mode of maternal mouthbrooding. and Trophic Adaptations Another putative member of the Limnochro- mini, Benthochromis tricoti, was always placed A robust phylogenetic hypothesis is essen- tial in studying the evolutionary pathways outside this tribe; however, we have ana- lyzed only sequences of the control region of traits that allow coexistence of multiple for this species, and its exact position re- species. In cichlid Žshes, dental and pharyn- geal specializations of the jaw morphology, mains unresolved. Benthochromis tricoti was resolved as sister to Cyphotilapia frontosa as well as dietary specialization, are regarded and the Perissodini in the unweighted MP as the most important factors promoting co- existence (Fryer and Iles, 1972). In the rocky tree but was placed in close relationship to the Cyprichromini in the weighted MP tree littoral zone of Lake Tanganyika, for exam- and in the ML topology. Future studies in- ple, 50–60 cichlid species live in sympa- try or»close neighborship (Kawanabe et al., cluding additional genera assigned to the tribe Limnochromini might provide more 1997). The potential for coexistence is widely precise knowledge about the taxonomic in- seen in the small but distinct differences in ecology, morphology, and behavior of the tegrity of this tribe. Cyphotilapia frontosa, pre- viously considered a member of the Tro- different species. In terms of basic ecologi- pheini (Poll, 1986), was conŽrmed to be a cal segregation, many tribes occupy distinct niches and habitat types (discussed earlier). separate lineage (Kocher et al., 1995). We placed C. frontosa as sister to the Limnochro- Given the clear-cut ecomorphological dis- mini according to analyses of the protein- tinctions between all the Tanganyikan tribes (Greenwood, 1984), each major Tanganyikan coding genes ND2 and cyt b, although boot- strap support was reasonable only in the cichlid lineage must have undergone pro- ML analyses. In the analyses based on the gressive diversiŽcation within a range de- Žned by the initial ecological segregation mitochondrial control region, C. frontosa was resolved as sister to the Perissodini, but this during the very beginning of the primary ra- was not well supported in the bootstrap eval- diation. In terms of ecomorphological spe- uations. Its previous placement in the tribe cializations, the primary lacustrine radiation Tropheini was largely based on its Tropheus- apparently established a primary division like pharyngeal apophysis (Poll, 1956). Other into major ecological guilds within the scope features, however, such as its social orga- of the lake habitat. However, this observa- nization in swarms, its particular muscu- tion does not imply that particular trophic lar extension producing the hump on its specializations evolved in one tribe only. head, and its karyotype (J. Snoeks, pers. In terms of their trophic specialization, comm.), support its distinctness from the Tanganyikan cichlids can be divided into six Tropheini. We thus suggest placing the major trophic groups: epilithic algal feed- monotypic genus Cyphotilapia into a new ers, scale-eaters, piscivores, zoobenthos feed- tribe, to be named Cyphotilapiini. To resolve ers, planktivores, and omnivores (Yamaoka, the exact placement of C. frontosa in the rede- 1997). For example, epilithic algae grazers, Žned H-lineage, additional data are required. zooplankton–feeders, and zoobenthos feed- Previous research has already shown that ers are all found in the tribes Lamprologini, Astatoreochromis alluaudi is likely to be sis- Ectodini, and Tropheini. ter to several East African riverine con- geners and to the haplochromine species Taxonomic and Biogeographic Implications ocks of Lakes Victoria and Malawi (Meyer As a result of our analyses, the taxon- et al., 1990, 1991; Nagl et al., 2000). The omy of several species needs to be revised. genus Astatotilapia was shown to be more Gnathochromis pfefferi, considered a mem- closely related to the species ocks of Lakes ber of the Limnochromini as is its con- Victoria and Malawi than to Astatoreochromis. gener G. permaxillaris, consistently turned Whereas Astatotilapia calliptera clustered with 132 SYSTEMATIC BIOLOGY VOL. 51 the Lake Malawi cichlids, A. burtoni was understand the total impact of the primary placed as sister to the Lake Victoria ock lacustrine radiation in Lake Tanganyika to (Meyer et al., 1991). Furthermore, A. burtoni the radiation of this most species-rich assem- was identiŽed as the sister species of the en- blage of cichlid Žshes. demic Tanganyikan tribe Tropheini (Nishida, 1991; Sturmbauer and Meyer, 1993). In all our analyses, the riverine haplochromine Conclusions genera Astatoreochromis, Astatotilapia, Cy- The Žnding that riverine haplochromines clopharynx, Orthochromis, Pseudocrenilabrus, were not placed at the base of the pri- and Schwetzochromis do not form the most mary radiation of the redeŽned H-lineage basal branches of the primary lacustrine ra- leads us to several suggestions: (1) Riverine diation of the H-lineage. Moreover, indi- haplochromines may not have seeded the cations are strong that they do not repre- radiation but arose within the primary la- sent a monophyletic assemblage together custrine radiation of Lake Tanganyika it- with the Tropheini. In the analyses of the self; thus, the hypothesis that ancestral protein-coding genes ND2 and cyt b, the riverine haplochromines are the starting representatives of the genera Orthochromis, point for the primary lacustrine radiation of Pseudocrenilabrus, Astatoreochromis, and As- Lake Tanganyika is not supported. (2) Both tatotilapia clustered with the Tropheini, but the rapid lineage formation during the pri- Schwetzochromis malagarazensis was placed mary lacustrine radiation and the paral- outside the remaining haplochromines, sug- lel evolution of various reproductive strate- gesting that Schwetzochromis might be the gies suggest that this radiation gave rise sister group to the Perissodini (Fig. 4a). to six mouthbrooding lineages from a Only in the weighted MP tree of the con- Lamprologus-like substrate spawning ances- trol region dataset, with all sequences in- tor. (3) The ecomorphological diversiŽcation cluded, was the Haplochromini/Tropheini of the primary lacustrine radiation in Lake assemblage resolved as monophyletic, albeit Tanganyika was coupled with the evolution with low bootstrap support. The unweighted of mouthbrooding. MP analysis of the same dataset identi- The primary radiation of Lake Tanganyika Žed both Schwetzochromis and a clade com- most likely took place in a habitat con- prising the riverine haplochromines Asta- nected ecologically and hydrologically to toreochromis, Cyclopharynx, and Orthochromis rivers, permitting the ancestors of riverine as separate lineages. The ML tree and the haplochromines and lamprologines to leave MP analysis of the reduced dataset (with the protolake(s). Thus, the primary radiation reinclusion of four taxa after constraining was not restricted to the lacustrine zone itself the topology) also indicated the placement of but repeatedly enriched the cichlid fauna in Schwetzochromis outside the remaining hap- the rivers and other water bodies, even out- lochromines, in a sister group relationship side the present lake basin. This evolution- to the Ectodini. The inclusion of six hap- ary scenario is consistent with the geologi- lochromine species from Lakes Victoria and cal model of the origin of Lake Tanganyika Malawi in the control region dataset, to link (Tiercelin and Mondeguer, 1991), in which the primary lacustrine radiation in Lake Tan- the Žrst stage of the lake is described as ganyika to the East African Haplochromini a swampy meandering river that slowly is super-ock, led to the placement of these transformed into shallow protolakes. Im- six taxa as sister group to the Tropheini, portantly, we do not propose that all hap- with Astatotilapia burtoni sister to the rep- lochromine cichlids left the lake at an early resentatives of Lake Victoria (Fig. 6a). This stage of the radiation, to return at a later time outcome suggests that riverine East African to seed the evolution of the Tropheini. In- haplochromines of the genera Astatotilapia, deed, several modern haplochromines such Astatoreochromis, Cyclopharynx, Orthochromis, as Astatotilapia burtoni live in swamp and and Pseudocrenilabrus, as well as their rela- marsh areas of Lake Tanganyika (or other tives in Lakes Victoria and Malawi, evolved East African Lakes) as well as in several rivers from an ancestor they share with the endemic around the lake. The ancestor(s) of these Tanganyikan tribe Tropheini. Clearly, more haplochromines, which possibly inhabited sequence data and a more extensive sam- the same transition zone between rivers and ple of haplochromines are required to fully the lake habitat, could thus give rise to the 2002 SALZBURGER ET AL.—PHYLOGENY OF LAKE TANGANYIKA CICHLID SPECIES FLOCK 133 endemic Tanganyikan tribe Tropheini (at a BRUFORD, M. W., O. HANOTTE, J. F. Y. BROOKFIELD, later stage of the radiation) as well as to dif- AND T. BURKE. 1998. Multilocus and single-locus DNA Žngerprinting. Pages 287–336 in Molecular ge- ferent species ocks in other East African netic analysis of populations: A practical approach lakes (as is the case in Lakes Kivu, Victoria, (A. R. Hoelzel, ed.). Oxford Univ. Press, Oxford. and Malawi). BUCKLEY, T. R., C. SIMON, H. SHIMODAIRA, AND The Lake Tanganyika cichlid species as- G. K. CHAMBERS. 2001. Evaluating hypotheses semblage has so far been viewed as an on the origin and evolution of the New Zealand Alpine cicadas (Maoricicada) using multiple- evolutionary reservoir of old African lin- comparison tests of tree topology. Mol. Biol. Evol. 18: eages (Nishida, 1991). Alternatively, it may 223–234. be viewed as an evolutionary hotspot from COHEN, A. S., K.-E. LEZZAR, J. J. TIERCELIN, AND which modern African cichlid lineages arose, M. SORGEHAN. 1997. New palaeogeographic and lake-level reconstructions of Lake Tanganyika: Impli- spreading subsequently to rivers and lakes cations for tectonic, climatic and biological evolution in other African regions (Sturmbauer et al., in a rift lake. Basin Res. 9:107–132. 1994). Our new evolutionary hypothesis sug- DOLPHIN, K., R. BELS HAW, C. D. L. ORME, AND D. L. J. gests that both views may in fact be true QUICKE. 2000. Noise and incongruence: Interpreting because part of the species ock is de- results of the incongruence length difference test. Mol. Phyl. Evol. 17:401–406. rived from an ancient cichlid fauna that no ECCLES, D. H., AND E. TREWAVAS. 1989. Malawian cich- longer exists elsewhere in Africa. At the same lid Žshes: The classiŽcation of some haplochromine time, the Tanganyikan radiation precipitated genera. Lake Fish Movies, Herten, Germany. a major faunal change in the African cich- FARIAS, I. P., G. ORTI, AND A. MEYER. 2000. Total evi- lid fauna, through the origin and spread of dence: Molecules, morphology, and the phylogenetics of cichlid Žshes. Mol. Dev. Evol. 288:76–92. the most species-rich lineage of cichlids, the FARIAS, I. P., G. ORTI, I. SAMPAIO, H. SCHNEIDER, AND Haplochromini. A. MEYER. 1999. Mitochondrial DNA phylogeny of the family cichlidae: Monophyly and fast molecular evolution of the neotropical assemblage. J. Mol. Evol. ACKNOWLEDGMENTS 48:703–711. We thank L. Mwape, R. Shapola, C. Mwelwa, FARRIS, S. J. 1989. The retention index and the rescaled H. Mudenda, M. Nabuyanda, and the team at the consistency index. Cladistics 5:417–419. Mpulungu Station of the Ministery of Agriculture, Food FRYER, G., AND T. D. ILES. 1972. The cichlid Žshes of and Fisheries, Republic of Zambia, for their help during the Great Lakes of Afrika. T.H.F. Publikations, Inc., sample collection. We thank A. Brandstaetter, I. Farias, Neptune City, NJ. H. Hauffe, and V. Klett for providing unpublished DNA GOLDMAN, N., J. P. ANDERSON, AND A. G. RODRIGO. sequences. We are further grateful to P. Henninger for 2000. Likelihood-based test of topologies in phyloge- sharing his Žndings on cichlid breeding behavior and to netics. Syst. Biol. 49:652–670. R. Hanel, R. and G. Rieger, J. and W. Wieser, two anony- GOODWIN, N. B., S. BALSHINE-EARN, AND J. D. mous reviewers, associate editor M. W. Westneat, and REYNOLDS. 1998. Evolutionary transitions in parental C. Simon, editor of Systematic Biology, for valuable com- care in cichlid Žsh. Proc. R. Soc. London B 265:2265– ments on the manuscript. W.S. was supported by a DOC- 2272. fellowship of the Austrian Academy of Sciences. A.M. GREENWOOD, P. H. 1979. Towards a phyletic classiŽca- was supported by the U.S. National Science Founda- tion of the ‘genus’ Haplochromis (Pisces, Cichlidae) tion (grants BSR-9107838 and DEB-9615178), Deutsche and related taxa. Part 1. Bull. Br. Mus. Nat. Hist. (Zool.) Forschungsgemeinschaft, the University of Konstanz, 35:265–322. and the Fonds der Chemischen Industrie. C.S. and GREENWOOD, P. H. 1980. Towards a phyletic classiŽca- S.B. were supported by the Austrian Science Founda- tion of the ‘genus’ Haplochromis (Pisces, Cichlidae) tion (grant 12339-GEN) and the Jubilaumsfond¨ s der and related taxa. Part 2. Bull. Br. Mus. Nat. Hist. (Zool.) Osterreichische¨ n Nationalbank (grant 6058). E.V. was 39:1–99. supported by the Belgian Science Foundation (FWO-V GREENWOOD, P. H. 1984. African Cichlids and evolution- grant G0109.99) and a DWTC project (2000–2004). ary theories. Pages 141–154 in Evolution of Žsh species ocks (A. A. Echelle and I. KornŽeld, eds.). Univ. of Maine at Orono Press, Orono. HASEGAWA, M., H. KISHINO , AND T. YANO. 1985. REFERENCES Dating of the human-ape splitting by a molecular BARLOW, G. W. 1991. Mating systems in among cich- clock of mitochondrial DNA. J. Mol. Evol. 22:160– lid Žshes. 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